Author response:

The following is the authors’ response to the current reviews.

Reviewer #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #2 (Public review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The use of multiscale MD simulations allows for a detailed exploration of the system at different time and length scales. The combination of MD simulations and structural bioinformatics provides a comprehensive approach to validate the identified binding sites. Finally, the steered MD simulations offer quantitative insights into the binding strength between the membrane and bnAbs.

While the simulations and analyses provide qualitative insights into the binding interactions, they do not offer a quantitative assessment of energetics. The coarse-grained simulations exhibit artifacts and thus require careful analysis.

This study contributes to a deeper understanding of the molecular mechanisms underlying bnAb recognition of the HIV-1 envelope. The insights gained from this work could inform the design of more potent and broadly neutralizing antibodies.

Recommendations for the authors:

Reviewing Editor:

We recommend the authors remove the figure and section related to bnAb LN01, perform additional analysis (e.g., further expanding on the differences in antibody binding in the presence or absence of antigen), and present this as a separate manuscript in a follow-up study.

We consider the analysis of a bnAb with a transmembrane antigen and of LN01 as essential to the manuscript and novel results.  Study of LN01 provides many insights unique from the other MPER bnAbs in this study.  We agree further characterization of LN01 and bnAbs with transmembrane antigen or full-length Env are intriguing and necessary to complete the full mechanistic understanding of lipid-associated antibodies.  LN01 section in this paper is novel in the field and demonstrates the preliminary evidence motivating further work, which we agree are beyond the scope of this already long detailed study.

Reviewer #1 (Recommendations for the authors):

I appreciate the degree to which the authors responded to my previous points raised in the private review, including edits where I might have missed something in the manuscript or relevant literature. I imagine such a point-by-point response was quite onerous. Thank you also for balancing presentation/clarity with content/rigor considering the large information content of this manuscript; in silico results are inherently hard to present given the delicate balance between rigorous validation and novel information content. I apologize if I repeat points raised and addressed previously and commend the authors on their revised study, which is much improved in clarity; any additional revisions are of course entirely at your discretion.

"...now having more diversity in lipid headgroup chemistries" references the wrong figure-it should be: Figure 2-figure supplement 2A-C. The incorrect figure is also referenced again several sentences down: "...relevant CDR and framework surface loops..."

Thank you for pointing out this error. We have corrected figure references.

"One shared conformational difference observed for these bnAbs the higher cholesterol bilayers was slightly more extensive and broader interaction profiles as well as modestly deeper embedding of the relevant CDR and framework surfaces loops" please rephrase

Thank you for this suggestion.  We rephrased this for improved clarity and flow. 

"These results bolster the feasibility for using all-atom MD as an in silico platform to explore differential phospholipid affinity at these sites (i.e., specificity studies) and influence on antibody preferred conformation as membrane composition and lipid chemistry are systematically varied" Please tone down these speculations-you have demonstrated that simulations are robust to different headgroup chemistries but have not provided evidence for the exclusion of lipids that are known not to associate with these antibodies.

We rephrased this speculation to highlight the potential of this application. We also emphasize future studies that would be required to achieve this application in the following sentence.

“These results motivate use of all-atom MD as an in silico approach for exploring differential phospholipid affinity at these sites…”

Figure 2A: Specify which PDB entry corresponds to the displayed crystal structures in the main figure or caption.

We clarified these PDB entries in the figure caption. 

Check reference formatting in supplemental figures when generating VOR.

I am not sure how relevant this might be to the claims of Figure 2-figure supplement 3, but AlphaFold3-based phospholigand docking might provide an additional orthogonal approach if relevant ligand(s) are available for such analysis (particularly for the newly proposed 10E8 POPC complex).

Thank you for this suggestion.  AI/ML based prediction methods like AF3 and RoseTTAFold All-Atom (RFAA) are interesting new methods that have come since our initial submission.   We’ve decided these experiments are beyond the scope of this already long and detailed study. We have added a sentence suggesting use of these methods in future work.

"We next studied bnAb LN01 to interrogate differences" --> this transition still reads a bit unclear. Why shift gears and change antibodies? Also, while you do go into its interactions both +/- antigen, there's no lead into the simulation initialization with and without antigen to guide the reader into the comparisons you will draw in the figure. Also, the order of information presentation is a bit strange, where the rationale for choosing a single monomeric helix is brought up in the middle of the paragraph instead of at the beginning of the section. In the next paragraph, it goes back to the initialization of the membrane composition again, which feels a bit disorganized-I do appreciate the unique challenge of having to weave through so much quality data! In fact, if you were to conduct simulations of membrane + antigen vs. membrane + LN01 vs. membrane + LN01 + antigen, I am tempted to say that this could be removed from this manuscript and flow better as a paper in and of itself.

We thank the reviewer for the suggestion to improve the writing style.  We feel this section adds a lot of value to the manuscript, so we will keep it in the paper and improved the transition as well as rationale.  

We selected to study the additional antibody LN01 and the monomeric MPER-TM antigen conformation because of the existing structural evidence available without additional creative model building.  This rationale has been updated in the new text.  

We changd the order of information as suggested, moving the rationale for antigen fragment earlier in the paragraph followed by the background of the lipids sites from the crystal that can lead into simulation set-up.  We clarified the simulation initialization was similar for systems with and without antigen in the opening sentence of the paragraph

"previously observed snorkeling and hydration of TM Arg686" --> Is this R696 (numbering could be different based on the particular Env)?

Thank you for noting this typo, we have corrected the numbering.

Potential font color issue with Figure 3-Figure supplement 1 B and part of A text-could be fixed in typesetting.

The discussion reads very well. Is it possible to direct antibody maturation, even in an engineered context, towards membrane affinity without increasing immunogenic polyreactivity? This is mentioned very briefly and cited with ref 36, but I would be interested in the author's thoughts on this topic.

We thank the reviewer for the insightful idea to explore in future work.  Our conclusion alludes to possibly artificially evolving membrane affinity studied by MD, as done in vitro by Nieva and co-workers.  Because the hypothetical nature, we’ve chosen not to elaborate on those ideas from this manuscript.

Reviewer #2 (Recommendations for the authors):

To ensure reproducibility and facilitate further research, the authors should publicly deposit the code for running the MD simulations and analyses (e.g., on GitHub) along with the underlying data used in the study (e.g., on Zenodo.org).

We appreciate the consideration for open-source code and analysis. Representative code and simulation trajectories were uploaded to the following repositories:

https://github.com/cmaillie98/mper_bnAbs.git

https://zenodo.org/records/13830877

—-

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with a lipid composition similar to the native virion. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. Additional contacts and conformational restraints imposed by ectodomain regions of the envelope glycoprotein, however, remain unaddressed-the size of such simulations likely runs into technical limitations including sampling and compute time.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive. However, given the large amount of data presented within the manuscript, the text would benefit from clearer subsections that delineate discrete mechanistic discoveries, particularly for experimentalists interested in antibody discovery and design. One area the paper does not address involves the polyreactivity associated with membrane binding antibodies-MD simulations and/or pulling velocity experiments with model membranes of different compositions, with and without model antigens, would be needed. Finally, given the challenges in initializing these simulations and their limitations, the text regarding their generalized use for discovery, rather than mechanism, could be toned down.

Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #2 (Public Review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The conclusions from the paper are mostly well supported by the simulations, however, they remain very descriptive and the key findings should be better described and validated. In particular:

It has been shown that the lipid composition of HIV membrane is rich in cholesterol [1], which accounts for almost 50% molar ratio. The authors use a very different composition and should therefore provide a reference. It has been shown for 4E10 that the change in lipid composition affects dynamics of the binding. The robustness of the results to changes of the lipid composition should also be reported.

The real advantage of the multiscale approach (coarse grained (CG) simulation followed by a back-mapped all atom simulation) remains unclear. In most cases, the binding mode in the CG simulations seem to be an artifact.

The results reported in this study should be better compared to available experimental data. For example how does the approach angle compare to cryo-EM structure of the bnAbs engaging with the MPER region, e.g. [2-3]? How do these results from this study compare to previous molecular dynamics studies, e.g.[4-5]?

References<br /> (1) Brügger, Britta, et al. "The HIV lipidome: a raft with an unusual composition." Proceedings of the National Academy of Sciences 103.8 (2006): 2641-2646.<br /> (2) Rantalainen, Kimmo, et al. "HIV-1 envelope and MPER antibody structures in lipid assemblies." Cell Reports 31.4 (2020).<br /> (3) Yang, Shuang, et al. "Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack." Nature Communications 13.1 (2022): 6393.<br /> (4) Carravilla, Pablo, et al. "The bilayer collective properties govern the interaction of an HIV-1 antibody with the viral membrane." Biophysical Journal 118.1 (2020): 44-56.<br /> (5) Pinto, Dora, et al. "Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01." Cell host & microbe 26.5 (2019): 623-637.

Considering reviewer suggestions, we slightly reorganized the results section into specific sub-sections with headings and changed the order in which key results were presented to allow the subsequent analysis more accessible for readers.  Supplemental materials were redistributed into eLife format, having each supplemental item grouped to a corresponding main figure. Many slightly detail modifications were made to figures (mostly supplemental items) without changing their character, such as clearer axes labels or revised annotations within panels.

The major additions within the results sections based on the reviews were:

(1) An expanded the comparison between our simulation analyses to previous simulations and to existing cryo-EM structural evidence for MPER antibodies’ membrane orientation the context of full-length antigen, resulting in new supplemental figure panels.

(2) New atomistic simulations of 10E8, PGZL1, and 4E10 evaluating the phospholipid binding predictions in a different lipid composition more closely modeling HIV membranes.

Minor edits to the analyses and interpretations include:

(1) Outlining the geometric components contributing to variance in substates after clustering the atomistic 10E8, 4E10, and PGZL1 simulations.

(2) Better defining the variance and durability of membrane interactions within and across systems in the coarse grain methods section.

(3) Removed interpretations in the original results sections regarding polyreactivity and energetics for MPER bnAbs that were not explicitly supported by data.   

(4) More context of the prevenance of bnAb loop geometries in structural informatics section

(5) Rationale for the choice of the continuous helix MPER-TM conformation in LN01-antigen conformations, and citations to previous gp41 TM simulations.

(6) Removed language on the novelty of the coarse grain and steered pulling simulations as newly developed approaches; tempering the potential discriminating power and applications of those approaches, in light of their limitations.

The discussion was revised to provide more novel context of the results within the field, including discussing direct relevance of the simulation methods for evaluating immune tolerance mechanisms and into antibody engineering.   We have shared custom scripts used for molecular dynamics analysis on github (https://github.com/cmaillie98/mper_bnAbs.git) and uploaded trajectories to a public repository hosted on Zenodo (https://zenodo.org/records/13830877).

Recommendations for the authors:

Below, I provide an extensive list of minor edits associated with the text and figures for the authors to consider. I provide these with the hope of increasing the accessibility of the manuscript to broader audiences but leave changes to the discretion of the authors.

Text/clarity

Figure 1 main text

The main text discussing Figure 1 is disorganized, making the analysis difficult to follow. I would suggest the following: moving the sentence, "4E10 and PG2L1 are structurally homologous" immediately after the paragraph discussing the simulation initiation. Then, add a sentence that directly compares their experimental affinity, neutralization, and polyreactivity of 4E10 and PG2L1 (later, an unintroduced idea pops up, "These patterns may in part explain 4E10's greater polyreactivity"). Next, lead into the discussion of the MD simulation data with something to the effect of: "Given these similarities, we first compared mechanisms of membrane insertion between 4E10 and PG2L1 to bolster confidence in our predictions". Later, the sentence "Across 4E10 and PGZL1 simulations, the bound lipid phosphates"

We thank the reviewer for the suggestion and we have restructured the beginning of the results to implement this style: to first introduce then discuss the comparative PGZL1 & 4E10 results, i.e. Figure 1 plus associated supplements.

In the background and the introduction text leading up to Figure 1, CDR-H3 is discussed at length, however, the first figure focuses almost entirely on how CDR-H1 coordinates a lipid phosphate headgroup. Are there experimental mutations in this loop that do not affect affinity (e.g., to a soluble gp41 peptide), but do affect neutralization (like the WAWA mutation for CDR-H3, discussed later)?

We have altered the Introduction (para 2) and Results (4E10/PGZL1 sub-section) to give more balanced discussion of CDRs H1 & H3.  That includes referencing experimental data addressing the reviewer’s question; a PGZL1 clone H4K3 where mutations to CDRH1 were introduced and shown have minimal impact on affinity to MPER peptide via ELISA and BLI, but those mutant bnAbs had significantly reduced neutralization efficacy (PMC6879610).

The sentence "These phospholipid binding events were highly stable, typically persisting for hundreds of nanoseconds" should be moved down to immediately precede, "[However], in a PGZL1 simulation, we observed a". This would be a good place for a paragraph break following, "Thus, these bnABs constitutively", since this block of text is very long.

Similarly, the sentence and parts of the section, "Likewise, the interactions coordinating the lipid phosphate oxygens at CDR-H1" more appropriately belongs immediately before or after the sentence, "Our simulations uncover the CDR-lipid interactions that are the most feasible".

Thank you for the detailed guidance in reorganizing the Figure 1 results.  We followed the advice to directly compare 4E10 and PGZL1 results separately from 10E8, moving those sections of text appropriately.  New paragraph breaks were added to improve accessibility and flow of concepts throughout the Results.

In the sentence, "our simulations uncover CDR-lipid interactions that are the most feasible and biologically relevant in the context of a full [HIV] lipid bilayer... validation to which of the many possible ions" à have you confidently determined lipid binding and positioning outside of the site validated in figure 1? Which site(s) are these referencing? The next two sentences then introduce two new ideas on the loop backbone stability then lead into lipid exchange, which is a bit jarring.

We have adjusted the language concerning the putative ions/lipids electron density across the many PGZL1 and 4E10 crystal structures, and additionally make the explicit point that we confidently determined the lack of lipid binding outside of the site focused on in Figure 1.

“… both bnAbs showed strong hotspots for a lipid phosphate bound within the CDR-H1 loops, with minimal phospholipid or cholesterol ordering around the proteins elsewhere.  The simulated lipid phosphates bound within CDR-H1 have exceptional overlap with electron densities and atomic details of modelled headgroups from respective lipid-soaked co-crystal structures…”

Figure 2 main text

"We similarly investigated bnAb 10E8" - Please make this a separate subheader, the block text is very long up to this point.

Thank you for the suggestion. We introduced a sub-header to separate work on 10E8 all-atom simulations.

"we observed a POPC complexed with... modelled as headgroup phosphoglycerol anions..." - please cite the references within the text.

Thank you for pointing out this missing reference, we added the appropriate reference.

"One striking and novel observation" - please remove the phrase "striking" throughout, for following best practices in scientific writing (PMC10212555)-this is generally well-done throughout.

We removed “striking” from our text per your suggestion.

"This CDR-L1 site highlights... (>500 fold) across HIV strains" - How much do R29 and Y32 also contribute to antigen binding and the conformation of this loop? These mutants also decreased Kd by approximately 20X, and based on the co-crystal structure with the TM antigen (PDB: 4XCC), seem to play a more direct role in antigen contact. Additionally, these residues should be highlighted on a figure, otherwise it's difficult to understand why they are important for membrane association.

We thank the reviewer for deep engagement to these supporting experimental details.  The R29A+Y32A 10E8 mutant referenced in the text showed only 4-fold Kd increase, a modest change for an SPR binding experiment.  Whereas R29E+Y32E 10E8 mutant resulted in 40x Kd increase, the “20x” the reviewer refers to.  Both 10E8 mutants showed similar drastically reduced breadth and potency of over 2 orders of magnitude on average.

These mutated CDR-L1 residues are not directly involved in antigen contact and adopt the same loop helix conformation when antigen is bound.  A minor impact on antigen binding affinity could be due altering pre-organization of CDR loops upon losing interactions from the Tyr & Arg sidechains - particularly Tyr31 in contact with CDR-H3.

As per the suggestion, clearer annotated figure panel denoting these sidechains has been added to Figure 2-Figure Supplement 1 for 10E8 analysis.

"Structural searches querying... identified between 10^5 and 2*10^6..." - why is this value represented as such a large range? Does this depend on the parameters used for analysis? Please clarify.

Additionally, how prevalent are any random loop conformations compared to the ones you searched? It's otherwise difficult to attribute number of occurrences within the 2 A cutoff to biological significance, as this number is not put in context.

We appreciate the reviewers comment to contextualize the range and relative frequency of the bnAb loop conformations.   RMSD and length of loop are the key parameters, which can be controlled by searching reference loops of similar length.  The main point of the backbone-level searching is simply to imply the bnAb loops are not particularly rare when comparing loops of similar length.   

We did as was suggested and added comparison to random loops of the same length to the main text, including a new Supplementary Table 4.   

“…identified between 105 to 2∙106 geometrically similar sub-segments within natural proteins (<2 Å RMSD)40, reflecting they are relatively prevalent (not rare) in the protein universe, comparing well with frequency of other surface loops of similar length in antibodies (Supplementary Table 3).”

"We next examined the geometries" could start after its own new subheading. Moreover, while there's an emphasis on tilt for neutralization, there is not a figure clearly modelling the proposed Env tilt compared to the relatively planar bilayer. It would be helpful to have an additional panel somewhere that shows the orientation of the antibody (e.g., a representative pose) in the simulations relative to an appropriately curved membrane, Env, the binding conformation of the antibody to Env, and apo Env, given the tilting observed in PMID: 32348769 and theorized in PMC5338832. What additional conformational changes or tilting need to occur between the antibodies and Env to accomplish binding to their respective epitopes?

Thank you for outlining an interesting element to consider in our analysis of a multi-step binding mechanism for MPER antibodies. We added additional figure panels in the supplement to outline the similarities and differences between our simulations and Fabs with the inferred membranes in cryo-EM experiments of full-length HIV Env.  The simulated Fabs’ angles are very similar with only minor tilting to match the cryo-EM antibody-membrane geometries. 

We added Figure 1-figure supplement 1A & Figure 2-figure supplement 2A, and alter to text to reflect this:

“The primary difference is Env-bound Fabs in cryo-EM adopt slightly more shallow approach angles (~15_°_) relative to the bilayer normal.  The simulated bnAbs in isolation prefer orientations slightly more upright, but presenting CDRs at approximately the same depth and orientation.  Thus, these bnAbs appear pre-disposed in their membrane surface conformations, needing only a minor tilt to form the membrane-antibody-antigen neutralization complex.”   

Env tilt dynamics and membrane curvature of natural virions may reconcile some of these differences.  Recent in situ tomography of Full-length Env in pseudo-virions corroborates our approximation of flat bilayers over the short length scales around Env.

The sentence "we next examined the geometries" mentions "potential energy cost, if any, for reorienting...". However, there's no further discussions of geometry or energy cost within this section. Please rephrase, or move this figure to main and increase discussion associated with the various conformational ensembles, their geometry, and their phospholipid association.

As the reviewer highlights, the unbiased simulations and our analysis do not explicitly evaluate energetics.  We removed this phrase, and now only allude to the minimal energy barrier between the similar geometric conformations, relative to the tilting & access requirements for antigen binding mechanism.

“The apparent barrier for re-orientation is likely much less energetically constraining than shielding glycans and accessibility of MPER”

".. describing the spectrum of surface-bound conformations" cites the wrong figure.

Thank you for noticing this error; we correct the figure reference to (Figure 2-figure supplement 4).

Please comment on the significance of how global clustering (Fig. S5A-C) was similar for 4E10 and PGZL1, but different for 10E8 (e.g., blue, orange, and yellow clusters for 4E10 and PHZL1 versus cyan, red, and green clusters for 10E8). As the cyan cluster seems to be much closer in Euclidian space to the 4E10/PGZL1 clusters, it might warrant additional analysis. What do these clusters represent in terms of structure/conformation? How do these clusters differ in membrane insertion as in (A)?

We are grateful you identify analysis in the geometric clustering section that may be of interest to other readers. We have added additional supplementary table (Table 2) to detail the CDR loop membrane insertion and global Fab angles which describe each cluster, to demonstrate their similarities and differences.  We also better describe how global clustering was similar for 4E10 and PGZL1, but different for 10E8 in the relevant results section<br /> The cyan cluster is not close in structure to 4E10/PGZL1 clusters.  We note the original figure panel had an error.  The updated Figure 2-supplement 4B shows the correct Euclidian distance hierarchy with an early split between 4e10/pgzl1 and 10e8 clusters.

Figure 3 main text

The start of this section, "We next studied bnAb LN01...", is a good place for a new subheader.

We have added an additional subheader here: Antigen influence on membrane bound conformations and lipid binding sites for LN01

There should be a sentence in the main text defining the replicate setup and production MD run time. Is the apo and complex based on a published structure? How do you embed the MPER? Is the apo structure docked to membrane like in 4E10? The MD setup could also be better delineated within the methods.

The first two paragraphs in this section have been updated to clarify the relevant simulations configuration and Fab membrane docking prediction details. 

The procedure was the same for predicting an initial membrane insertion, albeit now we use the LN01-TM complex and the calculation will account for the membrane burial of the the TM domain and MPER fragment.  As mentioned, LN01 is predicted as inserted with CDR loops insert similarly with or without the TM-MPER fragment.  The geometry differs from PGZL1/4E10 and 10E8, denoted by the text.

Please comment on the oligomerization state of the antigen used in the MD simulation: how does the simulation differ from a crossed MPER as observed in an MPER antibody-bound Env cryo-EM structure (PMID: 32348769), a three-helix bundle (PMC7210310), or single transmembrane helix (PMC6121722)? How does the model MPER monomer embed in the membrane compared to simulations with a trimeric MPER (PMC6035291, PMID: 33882664)-namely, key arginine residues such as R696?

We thank the reviewer for pointing out critical underlying rationale for modeling this TM-MPER-LN01 complex which we have corrected in the revised draft. The range of potential conformations and display of MPER based on TM domain organization could easily be its own paper – we in fact have a manuscript in preparation on the topic.  

The updated text expands the rationale for choosing the monomeric uninterrupted helix form of the MPER-TM model antigen (para 1 of LN01 section). The alternative conformations we did not to explore are called out, with references provided by the reviewer.

The discussion qualified that the MPER presentation is likely oversimplified here, noting MPER display in the full-length Env trimer will vary in different conformational states or membrane environments. However, the only cryo-EM structures of full-length ENV with TM domains resolved have this continuous helix MPER-TM conformation – seen both within crossing TM dimers or dissociated TM monomers.

Are there additional analyses that can validate the dynamics of the MPER monomer in the membrane and relative to LN01? Such as key contacts you would expect to maintain over the duration of the MD simulation?

We also increased description of this TM domain’s behavior, dynamics (tilt, orientation, Arg696 snorkeling, and complex w LN01) to provide a clearer picture of the simulation results – which aligns with past MD of the gp41 TM domain as a monomer (para 2 of LN01 section).  As well, we noted key LN01-MPER contacts that were maintained.

How does the model MPER modulate membrane properties like lipid density and lipid proximities near LN01?

We checked and didn’t notice differences for the types of lipids (chol, etc) proximal to the MPER-TM or the CDR loops versus the bulk lipid bilayer distributions.  Due to the already long & detailed nature of this manuscript, we elect not to include discussion on this topic.

Supplemental figure 1H-I would be better positioned as a figure 3-associated supplemental figure.

We rearranged to follow the eLife format and have paired supplemental panels with their most relevant main figures.

Figure 3F/H reference a "loading site" but this site is defined much later in the text, which was confusing.

Thank you for pointing out this source of confusion, we rearranged our discussion to reflect the order in which we present data in figures.

What evidence suggests that lipids "quickly exchange from the Loading site into the X-ray site by diffusion"? I do not gather this from Figure S1H/I.

We have rearranged the loading side and x-ray site RMSD maps in Figure 3-Figure supplement 1 to better illustrate how a lipid exchanges between these sites.

Figure 4 main text

The authors assert that in the CG simulations, restraints, "[maintain] Fab tertiary and quaternary structure". However, backbone RMSD does not directly assert this claim-an additional analysis of the key interfacial residues between chains, or geometric analysis between the chains, would better support this claim.

Thank you for pointing this point.  We rephrased to add that the major sidechain contacts between heavy and light chain persist, in addition to backbone RMSD, to describe how these Fabs maintain the fold stably in CG representation. 

In several cases, CG models sample and then dissociate from the membrane. In the text, the authors mention, "course-grained models can distinguishing unfavorable and favorable membrane-bound conformations". Is there a particular orientation that causes/favors membrane association and dissociation? This analysis could look at conformations immediately preceding association and dissociation to give clues as to what orientation(s) favor each state.

Thank you for suggesting this interesting analysis.  Clustering analysis of associated states are presented in Figure 5, Figure 5-Figure Supplement 1, and Figure 6, which show all CDR and framework loop directed insertion.  This feature is currently described in the main text.  

We did not find strong correlation of specific orientations as “pre-dissociation” states or ineffective non-inserting “scanning” events.  We revised the key sentence to reflect the major take away – that non-CDR alternative conformations did not insert and most of those having CDRs inserted in a different manner than all-atom simulations also were prone to dissociate:

“Given that non-CDR directed and alternative CDR-embedded orientations readily dissociate, we conclude that course-grained models can distinguish unfavorable and favorable membrane-bound conformations to an extent that provides utility for characterizing antibody-bilayer interaction mechanisms.”

Figure 6 main text

"For 4E10, trajectories initiated from all three geometries..." only two geometries are shown for each antibody. Please include all three on the plot.

The plots include markers for all three geometries for 4E10, highlighted in stars or with letters on the density plots of angles sampled (Figure 6B,C)

"Aligning a full-length IgG... unlikely that two Fabs simultaneously..." Are there theoretical conformations in which two Fabs could simultaneously associate with membrane? If this was physiological or could be designed rationally, could an antibody benefit further from avidity?

Our modeling suggests the theoretical conformations having two Fabs on the membrane are infeasible.  It’s even less likely multiple Env antigens could be engaged by one IgG.  We have revised the text to express this more clearly.

Figure 7 main text

"An intermediate... showed a modest reduction in affinity..." what affinity does PGZL1 have for this antigen?

The preceding sentence for this information: “Mature PGZL1 has relatively high affinity to the MPER epitope peptide (Kd = 10 nM) and demonstrates great breadth and potency, neutralizing 84% of a 130 strain panel “

Figures

Figure 1

It would be helpful to have an additional panel at the top of this figure further zoomed out showing the orientation of the antibody (e.g., a representative pose) in the simulations relative to an appropriately curved membrane, Env, the binding conformation of the antibody to Env, and apo Env, given the tilting observed in PMID: 32348769 and theorized in PMC5338832. What additional conformational changes or tilting need to occur between the antibodies and Env to accomplish binding to their respective epitopes?

Thank you for the suggestion to include this analysis.  We have added to the text reflecting this information, as well as making new supplemental panels for 4E10 and 10E8 that we compare simulated 4E10 and 10E8 Fab conformations to cryoEM density maps with Fabs bound to full-length HIV Env. Figure 1-figure supplement 1A & Figure 2-figure supplement 2A

In Figure 1, space permitting, it would be helpful to annotate the distances between the phosphates and side chains (similarly, for Figure S1A).

To avoid the overloading the Main figure panels with text, those relevant distances are listed in the methods sections.  Those distances are used to define the “bound” lipid phosphate state.  Generally, we note the interactions are within hydrogen bonding distance.

Annotating "Replicate 1" and "Replicate 2" on the left side of Figure 1C/D would make this figure immediately intuitive.

We have added these labels.

Figure caption 1C: Please clarify the threshold/definition of a contact used to binarize "bound" versus "unbound" (for example, "mean distance cutoff of 2A between the phosphate oxygen and the COM of CDR-H1") [on further reading of the methods section, this criterion is quite involved and might benefit from: a sentence that includes "see methods"]. Additionally, C could use a sentence explaining the bar such as in E, "Phosphate binding is mapped to above each MD trajectory" Please define FR-H3 in the figure caption for E/F.

We have added these details to the figure caption.

Because Figure 1 is aggregated simulation time, it would be helpful to also represent the data as individual replicates or incorporate this information to calculate standard deviations/statistics (e.g., 1 microsecond max using the replicates to compute a standard deviation).

We believe the current quantification & display of data via sharing all trajectories is sufficient to convey the major point for how often each CDR-phosholipid binding site it occupied.  Further tracking and statistics of inter-atomic distances will likely be too tedious & add minimal value. There is some dynamics of the phosphate oxygens between the polar within the CDR site but our “bound” state definitions sufficiently describe the key participating interactions are made.

Figure 2

For A, it would be helpful to annotate the yellow and blue mesh on the figure itself.

We have defined the orange phosphate and blue choline densities.

Also, where are R29 and Y32 relative to this site? In the X-ray panels, Y38 is not shown, and the box delineating the zoom-in is almost imperceptible.

Thank you for this suggestion to include those amino acids which are referenced in the text as critical sites where mutation impacts function. To clarify, Y32 is the pdb numbering for residue Y38 in IMGT numbering. We have added a panel to Figure 2-Figure Supplement 1 having a cartoon graphic of 10E8 loop groove with sidechains & annotating R29 and Y38, staying consistent with out use of IMGT numbering in the manuscript.

Figure 3

It might read clearer to have "LN01+MPER-TM" and "LN01-Apo" in the middle of A/B and C/D, respectively, and a dotted line delineating the left and right side of the figure panels.

We have added these details to the figure for clarity for readers.

It would be helpful to show some critical interactions that are discussed in the text, such as the salt bridge with K31, by labeling these on the figure (e.g., in E-H).

We drafted figure panels with dashed lines to indicate those key interactions.  However, they became almost imperceptible and overloaded with annotations that distracted from the overall details.  For K31, the interaction occurs in LN01 crystal structures readers can refer to.

Why are axes cut off for J?

We corrected this.

Please re-define K/L plots as in Figure 1, and explain abbreviations.

We updated the figure caption to reflect these changes.

Figure 4

The caption for panel A states that the Fab begins in solvent 1-2 nm above the bilayer, but the main text states 0.5-2 nm.

We have reconciled this difference and listed the correct distances: 0.5-2nm.

Please label the y-axis as "Replicate" for relevant figure panels so that they are more immediately interpretable.

This label has been added.

A legend with "membrane-associated" and "non-associated" within the figure would be helpful. Additionally, the average percent membrane associated, with a standard deviation, should be shown (Similar to 1C, albeit with the statistics).

This legend has been added.  We also added the additional statistical metrics requested to strengthen our analysis.

The text references "10, 14, and 12 extended insertion events" for the three antibody-based simulations. How do you define "extended insertion events"? Would breaking this into average insertion time and standard deviation better highlight the association differences between MPER antibodies and controls, in addition to the variability due to difference random initialization?

We thank the reviewer for the insightful suggestion on how to better organize quantitative analysis to support the method. Supplemental Table 3 includes these numbers.

Figure 5

The analysis in Fig. S6C could be included here as a main figure.

The drafted revised figure adding S6C to Figure 5 made for too much information.  Likewise, putting this panel S6C separated it from the parent clustering data of S6B, so we decided to keep these figures separated.  The S6 figure is now Figure 5-figure supplement 1.

Figure 6

Please annotate membrane insertion on E as %.

These are phosphate binding RMSD/occupancy vs time.  The panels are now too small to annotate by %.  The qualitative presentation is sufficient at this stage.  The quantitative % are listed in-line within text when relevant to support assertions made. 

Please use the figure caption to explain why certain clusters (e.g., 10E8 cluster A, artifact, Fig. S6E) are not included in panel E.

We have added this information in the figure caption.

Figure 7

Please show all points on the box and whisker plots (panels E and F), and perform appropriate statistical tests to see if means are significantly different (these are mentioned in the text, but should be annotated on the graph and mentioned within the figure caption).

We have changed these plots to show all data points along with relevant statistical comparisons. The figure captions describe unpaired t-test statistical tests used.

Figure S1

G, H, and I do not belong here-they should be moved to accompany their relevant text section, which associates with Figure 3. It would be helpful to associate this with Figure 3 in the eLife format, "Figure 3-Supplemental Figure 1" or its equivalent.

It's very difficult to distinguish the green and blue circles on panel G.

We darkened the shading and added outline for better visualization

Subfigure I is missing a caption, could be included with H: "(H,I) Additional replicates for LN01+TM (H) and LN01 (I)".

We corrected this as suggested.

Why is H only 3 simulations and not 4? Does it not have a lipid in the x-ray site? Also, the caption states "(top, green)" and "(bottom, cyan)", but the green vs. cyan figures are organized on the left and right. Additional labels within the figure would help make this more intuitive.

If the point of H and I is to illustrate that POPC exchanges between the X-ray and loading sites, this is unclear from the figure. Consider clarifying these figures.

Thank you for describing the confusion in this figure, we have added labels to clarify.

Figure S2 (panels split between revised Figure 4 associated figure supplements)

The LN01 figures should likely follow later so that they can associate with Figure 3, despite being a similar analysis.

We corrected supplements to eLife format so supplements are associated with relevant main figures.

Figure S3 (panels split between revised Figure 1 & 2 associated figure supplements)

As hydrophobicity is discussed as a driving factor for residue insertion, it would be helpful to have a rolling hydrophobicity chart underneath each plot to make this claim obvious.

We prefer the current format, due to the worry of having too much information in these already data-rich panels.  As well, residues are not apolar but are deeply inserted.

Figure S4 (panels split between revised Figure 1 & 2 associated figure supplements)

It would be helpful to label the relevant loops on these figures.

We have labeled loops for clarity.

Do any of these loops have minor contacts with Env in the structure?

The 4E10 and PGZL1 CDRH-1 loop does not directly contact bound MPER peptides bound in crystal structures. 

FRL-3 and CDR-H1 in 10E8 do not contact the MPER peptide antigen component based on x-ray crystal structures.

Do motif contacts with lipid involve minor contacts with additional loops other than those displayed in this figure?

The phosphate-loop interactions in motifs used as query bait here are mediated solely by the backbone and side chain interactions of the loops displayed. We visually inspected most matches and did not see any “consensus” additional peripheral interactions common across each potential instance in the unrelated proteins.  The supplied Supplemental Table 2 contains the information if a reader wanted to conduct a detailed search. 

Why is there such a difference between the loop conformation adopted in the X-ray structure and that in the MD simulation, and why does this lead to the large observed differences in ligand-binding structure matches?

We thank the reviewer for carefully noting our error in labeling of CDR loop and framework region input queries. We revised the labeling to clarify the issue.

The is minimal structural difference between the loops in x-ray and MD.

Figure S5 (Figure 2-Figure supplement 4)

This figure is not colorblind friendly-it would be helpful to change to such a pallet as the data are interesting, but uninterpretable to some.

We have left this figure the same.

"Susbstates" - "Substates"

Corrected, thank you.

Panel B is uninterpretable-please break the axis so that the Euclidian distances can be represented accurately but the histograms can be interpreted.

We have adjusted axis for this plot to better illustrate the cluster thresholds.

The clusters in D-H should be analyzed in greater depth. What is the structural relevance of these clusters other than differences in phospholipid occupancy in (I)? Snapshots of representative poses for each cluster could help clarify these differences.

We have adjusted the text to describe the geometric differences in each of those clusters that result in the different exceptionally lower propensities for forming the key phospholipid interaction.  

The figure caption should make it clear that 3 μS of aggregate simulation time is being used here instead of 4 μS to start with unique tilt initializations. E.g., "unique starting membrane-bound conformations (0 degrees, -15 degrees, 15 degrees initialization relative to the docked pose)". Further, why was the particular 0-degree replicate chosen while the other was thrown out? Or was this information averaged? Why is the full 4 μS then used for D-I?

We thank the reviewer for noting these details.  We didn’t want to bias the differential between 10E8 and 4E10/PGZL1 by including the replicate simulations.  The analysis was mainly intended to achieve more coarse resolution distinction between 10E8 and the similar PGZL1/4E10.  

In the subsequent clustering of individual bnAb simulation groups, the replicate 0 degree simulations had sufficiently different geometric sampling and unique lipid binding behavior that we though it should be used (4 us total) to achieve finer conformational resolution for each bnAb.

Figure S6 (now Figure 5-Figure Supplement 1)

Please label the CDRs in C and provide a color key like in other figures. Also, please label the y-axes. This figure could move to main below 5B with the clusters "A,B,C" labeled on 5B.

We have added the axes labels and color key legend.  We retained a minimal CDR loop labeling scheme for the more throughput interaction profiles here where colored sections in the residue axes denote CDR loop regions.

Figure S7 (Figure 7 Figure Supplement 1)

Panels A and B would likely read better if swapped.

We have swapped these panels for a better flow.

For panel C, please display mean and standard deviation, and compare these values with an appropriate statistical test.

This is already displayed in main figure, we have removed it from supplement.

For E and F, please clarify from which trajectory(s) you are extracting this conformation from. Are these the global mean/representative poses? How do they compare to other geometrically distinct clusters?

The requested information was added to supplemental figure caption.  These are frames from 2 distinct time points selected phosphate bound frames from 0-degree tilt replicates for both 4E10 and 10E8, representing at least 2 distinct macroscopic substates differing in global light chain and heavy chain orientation towards the membrane. 

Table S2 (now Supplementary Table 3)

Please add details for the 13h11 simulation.

Additionally, please add average contact time and their standard deviation to the table, rather than just the aggregated total time. This will highlight the variability associated with the random initializations of each simulation.

We have added the details for 13h11 and the requested analysis (average aggregated time +/- standard deviation and average time per association event +- standard deviation) to supplement our summary statistics for this method.

Reviewer #2 (Recommendations For The Authors):

(1) The structure of the manuscript should be improved. For example, almost half of the introduction (three paragraphs) summarize the results. I found it hard to navigate all the data and specific interactions described in the result section. Furthermore, the claims at the end of several sections seem unsupported. Especially for the generalization of the approach. This should be moved to the discussion section. The discussion is pretty general and does not provide much context to the results presented in this study.

We have significantly reorganized the results section to improve the flow of the manuscript and accessibility for readers, especially the first sections of all-atom simulations. We also removed claims not directly supported by data from our results, and expanded on some of these concepts in the discussion to make some more novel context to the result.

(2) The author should cite more rigorously previous work and refrain from using the term "develop" to describe the simple use of a well established method. E.g. Several studies have investigated membrane protein interactions e.g. [1], membrane protein-bilayer self-assembly [2], steered molecular dynamics [3], etc.

Thank you for identifying relevant work for the simulations that set precedent for our novel application to antibody-membrane interactions.  We have removed language about development of simulation methods from the text and now better reference the precedent simulation methods used here.

(3) Have the authors considered estimating the PMF by combining the steered MD simulation through the application of Jarzynski's equality?

We performed from preliminary PMFs for Fab-membrane binding, but saw it was taking upward of 40 us to reach convergence.  Steered simulations focus on a key lipid may be easier.

Although PMFs are beyond the scope of this work, we added proposals & allusion to their utility as the next steps for more rigorous quantification of fab-membrane interactions.

Minor

(4) The term "integrative modeling" is usually used for computational pipelines which incorporate experimental data. Multiscale modeling would be more appropriate for this study.

We altered descriptions throughout the manuscript to reflect this comment.

(5) Units to report the force in the steered molecular dynamics are incorrect. They should be 98.

We changed axes and results to correctly report this unit.

(6) Labels for axes of several graphs are not missing.

We added labels to all axes of graphs, except for a few where stacked labels can be easily interpreted to save space and reduce complexity in figures.

(7) Figure 3 K & L is this really < 1% of total? The term "total" should also be clarified.

Thank you for pointing this out, we changed the % labels to be correct with axes from 0-100%. We clarified total in the figure caption.

(8) The font size in figures should be uniformized.

This suggestion has been applied

(9) Time needed for steered MD should be reported in CPUh and not hours (page 17).

We removed comments on explicit time measurements for our simulations.

(10) Version of Martini force field is missing in methods section

We used Martini 2.6 and added this to the methods.

References

(1) Prunotto, Alessio, et al. "Molecular bases of the membrane association mechanism potentiating antibiotic resistance by New Delhi metallo-β-lactamase 1." ACS infectious diseases 6.10 (2020): 2719-2731.

(2) Scott, Kathryn A., et al. "Coarse-grained MD simulations of membrane protein-bilayer self-assembly." Structure 16.4 (2008): 621-630.

(3) Izrailev, S., et al. "Computational molecular dynamics: challenges, methods, ideas. Chapter 1. Steered molecular dynamics." (1997).

Sermon-writing as an art form

There is a creative process in religious writing. It takes the humanity of being human to have the insight to write something that is moving.

Reviewer #1 (Public review):

This manuscript by Kleinman & Foster investigates the dependence of hippocampal replay on VTA activity. They recorded neural activity from the dorsal CA1 region of the hippocampus while chemogenetically silencing VTA dopamine neurons as rats completed laps on a linear track with reward delivery at each end. Reward amount changed across task epochs within a session on one end of the track. The authors report that VTA activity is necessary for an increase in sharp-wave rate to remain localized to the feeder that undergoes a change in reward magnitude, an effect that was especially pronounced in a novel environment. They follow up on this result with a second experiment in which reward magnitude varies unpredictably at one end of the linear track and report that changes in sharp-wave rate at the variable location reflect both the amount of reward rats just received there, in addition to a smaller modulation that is reminiscent of reward prediction error coding, in which the previous reward rats received at the variable location affects the magnitude of the subsequent change in sharp-wave rate that occurs on the present visit.

This work is technically innovative, combining neural recordings with chemogenetic inactivation. The question of how VTA activity affects replay in the hippocampus is interesting and important given that much of the work implicating hippocampal replay in memory consolidation and planning comes from reward-motivated behavioral tasks.

Comments on revisions:

Overall, I think the authors have done everything they could to address reviewer concerns, short of collecting more data. The more consistent statistical approach makes the paper easier to read and follow. It's helpful to have more details/rationale for the variability in CNO dose and timing. I think some of the results are still not fully convincing, especially the reward volatility experiment (which the authors also note requires additional validation). Given the small number of rats, the small effect sizes, and the complexity of the experimental manipulations, I still have concerns about whether these effects would hold with larger groups sizes.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1:

Chemogenetics validation

Little validation is provided for the chemogenetic manipulations. The authors report that animals were excluded due to lack of expression but do not quantify/document the extent of expression in the animals that were included in the study.

We thank the reviewer for raising this oversight. We have added additional examples of virus expression in sections from included and excluded animals in Figure 1 – Supplement 1. We also added additional comments on the extent of expression we observed in lines 92-95: “Post-experiment histology confirmed overlapping virus expression and TH-positive neurons in putative VTA near the injection site (-5.6 mm AP from bregma), as well as approximately 0.5 mm anterior and posterior (-5 to -6 mm AP).”

There's no independent verification that VTA was actually inhibited by the chemogenetic manipulation besides the experimental effects of interest.

While we did include animals expressing control virus to control for any effect of CNO administration itself, the reviewer is correct that we did not independently verify VTA neurons were inhibited. We have noted this limitation of the current study on lines 513-522 in the Discussion: “We did not directly measure the suppression of VTA neurons after CNO injection. Previous work in other brain areas found hM4Di activation suppressed firing rates to around 60% of baseline (Mahler et al., 2014; Chang et al., 2015), in addition to diminishing synaptic transmission even when spikes occurred (Stachniak et al., 2014). Combined with the incomplete expression of hM4Di in TH-positive neurons in our animals, we expect VTA activity was significantly but not completely suppressed. Because our results depend only on any degree of blunting differences in dopamine release at different reward locations, rather than the total absence of dopamine signaling, measuring the magnitude of suppression was not essential for our conclusions.”

The authors report a range of CNO doses. What determined the dose that each rat received? Was it constant for an individual rat? If not, how was the dose determined? The authors may wish to examine whether any of their CNO effects were dependent on dose.

The reviewer is completely correct that we omitted sufficient information regarding the dosage of CNO used in each animal and each session. We have included more details in the Methods lines 676-694, detailing both the doses and the rationale.

The authors tested the same animal multiple times per day with relatively little time between recording sessions. Can they be certain that the effect of CNO wore off between sessions? Might successive CNO injections in the same day have impacted neural activity in the VTA differently? Could the chemogenetic manipulation have grown stronger with each successive injection (or maybe weaker due to something like receptor desensitization)? The authors could test statistically whether the effects of CNO that they report do not depend on the number of CNO injections a rat received over a short period of time.

We thank the reviewer for bringing up the question of whether the order of sessions had an influence on the efficacy of CNO in inactivating VTA activity. To address this, we split our dataset in Experiment 1 into two based on what number session of the particular day it was: 1st sessions of the day vs. all subsequent sessions (2nd+ session of the day). Then, we examined the difference in sharp-wave ripple rate between the reward ends in Epoch 2, as in Figure 2D of the manuscript. Though the resulting number of sessions in each split of the dataset is too low to draw strong statistical conclusions, particularly for novel sessions, we see little evidence there is any systematic change in the effect of VTA inactivation as a function of session number in the day. We include this in the revised manuscript as Figure 2 – Supplement 3 and in the Results lines 255-258.

Motivational considerations

In a similar vein, running multiple sessions per day raises the possibility that rats' motivation was not constant across all data collection time points. The authors could test whether any measures of motivation (laps completed, running speed) changed across the sessions conducted within the same day.

We thank the reviewer for this suggestion. We examined behavioral measures of motivation across sessions conducted within the same day. First, we calculated how many total laps each animal completed each session as a function of the session number of the day. In individual animals, this ranged from -2.8 to 4.1 laps per additional session number (mean 2.01), with an average total laps per session of 43.2 laps. Second, we calculated the median running velocity per session, across both running directions and all epochs, and checked how it varied across session number of the day. Per additional session in the day, this ranged from -3.6 to 8.6 cm/s difference across animals (mean 2.7 cm/s), with an average running velocity of 34.1 cm/s in total. Taken together, while we found little behavioral evidence of strong motivational changes across session, our animals may have been slightly more motivated in later sessions in the day, which also corresponded to later in the light cycle and closer to the dark cycle. We mention this information in Results lines 255-258, related to Figure 2 – Supplement 3.

This is a particularly tricky issue, because my read of the methods is that saline sessions were only conducted as the first session of any recording day, which means there's a session order/time of day and potential motivational confound in comparing saline to CNO sessions.

We have clarified the ordering of CNO and saline sessions in the Methods lines 697-702. Briefly, we avoided running CNO sessions before saline sessions in the same day, but either could be the first session of a day. That is, saline -> saline, saline -> CNO, and CNO -> CNO were all valid orderings. On days with more than two sessions, any number of repeated saline and CNO sessions was permitted, provided that as soon as a CNO session occurred, any subsequent sessions were also CNO.

More generally, we shared this reviewer’s concern about potential confounds between drug and motivation. For novel sessions in Experiment 1, each animal had equal numbers of saline and CNO 1st and 2nd sessions of the day. For familiar sessions, animals had similar counts for 1st sessions of the day (experimental rats: 20 saline, 16 CNO; control rats: 17 saline, 15 CNO) but more CNO 2nd sessions of the day (experimental rats: 5 saline, 13 CNO; control rats: 5 saline, 10 CNO). There were occasionally 3rd or 4th sessions in a given day for some rats, and these were also approximately equal (experimental rat 2, 3rd sessions: 2 each of saline and CNO, 4th session: 1 saline; experimental rat 3 and 4, 3rd sessions: 1 each of saline and CNO; control rat 2, 3rd session: 1 saline).

Statistics, statistical power, and effect sizes

Throughout the manuscript, the authors employ a mixture of t-tests, ANOVAs, and mixed-effects models. Only the mixed effects models appropriately account for the fact that all of this data involves repeated measurements from the same subject. The t-tests are frequently doubly inappropriate because they both treat repeated measures as independent and are not corrected for multiple comparisons.

We thank the reviewer for pointing out these issues with our statistical analyses in places. We have made the following improvements:

Figure 1F-I, S1, reward end visit durations: We now use a linear mixed-effects model to analyze the difference in stopping period durations between epochs. For each session, we calculated the mean stopping duration for each reward end in each epoch, then modeled the difference between epochs as a function of drug and novelty, with animal-specific intercepts. For example, related to Figure 1F and also described in the Results, we modeled the stopping duration difference at the Unchanged reward end, Epoch 2 – Epoch 1, and found experimental rats had a significant intercept (Epoch 2 stops shorter than Epoch 1) and the drug × novelty interaction, while control rats had a significant intercept and novelty main effect. The other visit duration analysis shown in Figure 1 – Supplement 1 have similarly been updated.

Figure 2D-E, ripple rate difference between reward ends in Epoch 2: We now use a linear mixed-effects model to analyze the difference between ripple rates at the Incr. and Unch. reward ends in Epoch 2. For each session, we calculated the mean ripple rate at each end in Epoch 2, then modeled the difference as a function of drug and novelty, with animal-specific intercepts. With the full stopping periods, for experimental rats, there was a significant intercept (ripple rate at Incr. greater than Unch.) and the model with drug included performed significantly better than the one without it (AIC_nodrug – AIC_full = 5.22). Control rats had a significant intercept and effect of novelty (greater difference with novelty), and the model excluding drug terms performed better (AIC_nodrug – AIC_full = -3.54). Results with the trimmed stopping periods were similar. These analyses are described in Results lines 253-266.   

Figure 3D-E, ripple rate as a function of reward history: We now use a mixed-effects model that incorporates animal-specific intercepts. The results remained similar and have been updated in the text and legend.

Figure 4D-K, replay rates as a function of drug, novelty, and directionality: We now use mixed-effects models that incorporate animal-specific intercepts rather than three-way ANOVA. The results remained similar and have been updated in the text and legend.

The number of animals in these studies is on the lower end for this sort of work, raising questions about whether all of these results are statistically reliable and likely to generalize. This is particularly pronounced in the reward volatility experiment, where the number of rats in the experimental group is halved to just two. The results of this experiment are potentially very exciting, but the sample size makes this feel more like pilot data than a finished product.

We have added additional emphasis in the text that the experimental group results of CNO inactivation in the volatile reward task should be confirmed with future work, in Discussion line 529-533. Because these experiments were performed on familiar tracks, we see them as corroborating/complementing the results from Experiment 1. Although the analysis assumes VTA inactivation had no effect, our pooling of all Experiment 2 data to display in Figure 3 – Supplement 2 maximized our ability to analyze the effects of volatile reward deliveries on sharp-wave ripple rates, lending further support to the main results shown in Figure 3.

The effect sizes of the various manipulations appear to be relatively modest, and I wonder if the authors could help readers by contextualizing the magnitude of these results further. For instance, when VTA inactivation increases mis-localization of SWRs to the unchanged end of the track, roughly how many misplaced sharp-waves are occurring within a session, and what would their consequence be? On this particular behavioral task, it's not clear that the animals are doing worse in any way despite the mislocalization of sharp-waves. And it seems like the absolute number of extra sharp-waves that occur in some of these conditions would be quite small over the course of a session, so it would be helpful if the authors could speculate on how these differences might translate to meaningful changes in processes like consolidation, for instance.

We thank the reviewer for this helpful suggestion to give some context to the difference in sharp-wave ripple numbers and the functional consequence of these changes. We agree completely that this task is almost certainly too simple for animals to show any performance deficit from these changes. We chose this precisely so we could examine the consequences of VTA inactivation to the sharp-wave ripple response to reward changes per se, without any confound of performance or memory changes that could also conceivably alter sharp-wave ripples. We have added both more context about the magnitude and consequence of these sharp-wave ripple changes as well as comments about the choice of this particular task (Discussion lines 522-529).  

How directly is reward affecting sharp-wave rate?

Changes in reward magnitude on the authors' task cause rats to reallocate how much time they spent at each end. Coincident with this behavioral change, the authors identify changes in the sharp-wave rate, and the assumption is that changing reward is altering the sharp-wave rate. But it also seems possible that by inducing longer pauses, increased reward magnitude is affecting the hippocampal network state and creating an occasion for more sharp-waves to occur. It's possible that any manipulation so altering rats' behavior would similarly affect the sharp-wave rate.

For instance, in the volatility experiment, on trials when no reward is given sharp-wave rate looks like it is effectively zero. But this rate is somewhat hard to interpret. If rats hardly stopped moving on trials when no reward was given, and the hippocampus remained in a strong theta network state for the full duration of the rat's visit to the feeder, the lack of sharp-waves might not reflect something about reward processing so much as the fact that the rat's hippocampus didn't have the occasion to emit a sharp-wave. A better way to compute the sharp-wave rate might be to use not the entire visit duration in the denominator, but rather the total amount of time the hippocampus spends in a non-theta state during each visit. Another approach might be to include visit duration as a covariate with reward magnitude in some of the analyses. Increasing reward magnitude seems to increase visit duration, but these probably aren't perfectly correlated, so the authors might gain some leverage by showing that on the rare long visit to a low-reward end sharp-wave rate remains reliably low. This would help exclude the explanation that sharp-wave rate follows increases in reward magnitude simply because longer pauses allow a greater opportunity for the hippocampus to settle into a non-theta state.

We thank the reviewer for these important comments. We have better clarified the analysis of sharp-wave ripple rate in the Results (lines 172-173). To speak to the main concern of the reviewer, we do only consider times during “stopping periods” when the rat is actually stationary. That is, ripple rate for each visit is calculated as (# of ripples / total stationary time), rather than the full duration the rat is at the track end. With respect to including visit duration as a covariate, the Poisson model takes the total stationary time of each visit into account, so that it is effectively predicting the number of events (ripples) per unit of time (seconds) given the particular experimental variables (reward condition, drug condition, etc.). We have added additional clarification of this in the Methods (line 834-836).

The authors seem to acknowledge this issue to some extent, as a few analyses have the moments just after the rat's arrival at a feeder and just before departure trimmed out of consideration. But that assumes these sorts of non-theta states are only occurring at the very beginning and very end of visits when in fact rats might be doing all sorts of other things during visits that could affect the hippocampus network state and the propensity to observe sharp-waves.

We hope that with the clarification provided above, this control analysis helps remove any potential effects of approaching/leaving behavior or differences in movement at the reward end that could alter sharp-wave ripple rates. 

Minor issues

The title/abstract should reflect that only male animals were used in this study.

We have added this important information to the Abstract line 21.

The title refers to hippocampal replay, but for much of the paper the authors are measuring sharp-wave rate and not replay directly, so I would favor a more nuanced title.

We thank the reviewer for this suggestion. In the context of our work, we consider sharp-wave ripples as more-easily-detected markers for the occurrence of replay. Previous work from our lab (Ambrose et al., 2016) showed the effect of reward changes had very similar effects to both sharp-wave ripple rate and replay rate. We try to be explicit about viewing ripples as markers of replay content in both the Introduction and Discussion. Nevertheless, we do also demonstrate the title claim directly – by measuring replay and its spatial localization – therefore we feel comfortable with the title as it is.

Relatedly, the interpretation of the mislocalization of sharp-waves following VTA inactivation suggests that the hippocampus is perhaps representing information inappropriately/incorrectly for consolidation, as the increased rate is observed both for a location that has undergone a change in reward and one that has not. However, the authors are measuring replay rate, not replay content. It's entirely possible that the "mislocalized" replays at the unchanged end are, in fact, replaying information about the changed end of the track. A bit more nuance in the discussion of this effect would be helpful.

While we do show that replay content, in the form of reverse vs. forward replays, is altered with VTA inactivation, we take the reviewers point and completely agree. Especially in the context of the linear track, replays at either end could certainly be updating/consolidating information about both ends. We would argue our results suggest VTA is critical to localizing ripples and replay in more complex environments where this is not the case, but this is a hypothesis. We have added clarification and discussion of this point (Discussion lines 522-529).

However, in response to the reviewer’s comment, we have now also examined non-locally-initiated replays specifically to determine whether the increased ripple rate at the Unch. reward end in novel CNO sessions was likely due to more non-local replay, but found no significant increases in non-local replay at either reward end in either drug condition or novelty condition. We have included this result as Figure 4 – Supplement 3, and note it in the Results lines 487-488.

The authors use decoding accuracy during movement to determine which sessions should be included for decoding of replay direction. Details on cross-validation are omitted and would be appreciated. Also, the authors assume that sessions failed to meet inclusion criteria because of ensemble size, but this information is not reported anywhere directly. More info on the ensemble size of included/excluded sessions would be helpful.

We have added additional information about the run decoding procedure and related session inclusion criteria, as well as about recorded ensemble sizes (lines 417-421). Briefly, mean ensemble sizes were significantly smaller for excluded sessions (cell count, mean±sem; included sessions: 26.1±1.1, excluded sessions: 9.5±1.6; two-sample t-test, t(133)=5.3, p<10^-5). The average field size, defined as the number of spatial bins with greater than 1 hz firing rate, in excluded sessions was also larger (mean±sem, included sessions: 47.7±1.3, excluded sessions: 57.7±5.8; two-sample t-test, t(133)=-2.33, p<0.05), though the difference was less dramatic. Using a mixed effects model to predict position decoding error (as in Figure 4 – Supplement 2A) as a function of drug, novelty, cell count, and mean place field size, in both experimental and control groups cell count and field size were significant predictors: more cells and smaller average field size led to lower error. A similar model that instead predicted the fraction of running bins with correctly decoded running direction (as in Figure 4 – Supplement 2B), in neither group was field size significant, while cell count remained so: more cells led to more bins with running direction correctly classified. We include these analyses in the legend for the figure. With respect to cross validation of run decoding, because both the contribution of spikes in any single time bin to a neuron’s place field is extremely small and because we used run decoding accuracy simply to filter out sessions with poorer decoding, we did not use cross validation here.

For most of the paper, the authors detect sharp-waves using ripple power in the LFP, but for the analysis of replay direction, they use a different detection procedure based on the population firing rate of recorded neurons. Was there a reason for this switch? It's somewhat difficult to compare reported sharpwave/replay rates of the analyses given that different approaches were used.

We have added clarification for this change in detecting candidate events (lines 787-789). Briefly, sharp-wave ripples and spike density events are often but not always overlapping, such that there can be strong ripples with little spiking in the recorded ensemble or weak/absent ripples during vigorous spiking in the recorded ensemble. Because the decoding of replay content relies on spiking, our lab and others often use spike density or population burst events as candidate events. We have confirmed that the main results of Experiment 1 (e.g., Figure 2) remain the same if we use spike density events rather than sharp-wave ripples, but prefer to keep the use of sharp-wave ripples here for better comparison with Experiment 2 and to allow the inclusion of animals and sessions with low cell yield but clear ripples in the LFP.  

Reviewer #2 (Recommendations For The Authors):

Include additional histological data to confirm the extent of viral spread and precise tetrode placements. Providing detailed figures that clearly illustrate these aspects would strengthen the validity of the neural recordings and the specificity of the chemogenetic silencing.

We thank the reviewer for this suggestion and have added additional information regarding virus expression in Figure 1 – Supplement 1. We also added additional comments on the extent of expression we observed in lines 92-95: “Post-experiment histology confirmed overlapping virus expression and TH-positive neurons in putative VTA near the injection site (-5.6 mm AP from bregma), as well as approximately 0.5 mm anterior and posterior (-5 to -6 mm AP).”

While we do not show histological confirmation of hippocampal recording sites, the presence of sharp-wave ripples with upward deflections, presence of place cells, and recording coordinates and depth typical of dorsal CA1 made us confident in our recording location. We have noted these characteristics of our recordings in lines 128-131 in the Results: “Tetrodes were lowered to the pyramidal cell layer of dCA1, using the presence of sharp-wave ripples with upward deflections in the LFP, recording depth characteristic of dCA1, and spatially-restricted firing of place cells to confirm the recording location.”

Address the variability in CNO dosing and timing before recordings. It is recommended to standardize the dose and ensure a consistent timing interval between CNO administration and the start of recordings to minimize variability in the effects observed across different subjects. Instead of collecting new data, the authors could report the data for each animal, indicating the dose and interval between the injection and the recording.

We have further clarified the CNO dosing and timings in lines 676-702.

In Figure 1F, explicitly state whether the data represent averages across multiple sessions and confirm if these observations are primarily from the initial novel sessions. This clarification will help in accurately interpreting the effects of novelty on the measured neural activities.

We have changed the analyses shown in Figure 1F-I and Figure 1 – Supplement 1 thanks to the suggestions of Reviewer #1, but also more clearly spell out the analysis. Briefly, we average the durations for each condition within session (e.g., take the mean Unch. duration in Epoch 1), then perform the analysis across sessions. These data come from all sessions in Experiment 1, as described in lines 141-147, meaning there are around 2-3 times as many familiar sessions as novel sessions.

Reconsider the reporting of marginal p-values (e.g., p=0.055). If the results are borderline significant, either more data should be collected to robustly demonstrate the effects or a statistical discussion should be included to address the implications of these marginal findings.

We have removed the reporting of marginal p-values.

Ensure that the axes and scales are consistent across similar figures (specifically mentioned for Figure 2A) to prevent misinterpretation of the data. Consider showing the average across all animals in 2A, similar to 2B and 2C.

We have adjusted these axes to be consistent across all panels.

Add a legend to the heatmap in Figure 4A to facilitate understanding of the data presented.

We have added a heatmap to the figure and legend.

Provide a detailed examination and discussion of the apparent contradictions observed in control data, particularly where experimental conditions with saline show increased reverse replay in novel environments, which is absent in familiar sessions. See Figures 4E and 4I.

We thank the reviewer for noting that this feature of our data deserved discussion. We confirmed that the lack of an effect of reward on reverse replay rates in familiar sessions in control rats was due to generally low replay rates in these sessions. Replay rates have been observed to decrease as the familiarity of an environment or behavior increases, and the presence of the reward-related modulation of reverse replay in novel sessions in these animals is consistent with this observation. We now report in the Results lines 458-459 and 485-486 the low replay rates in this group in familiar sessions, and the likelihood that this is preventing any reward-related modulation from being detected.

Include a more detailed analysis of place cell properties, such as firing rates and field sizes, especially in novel environments where VTA inactivation appears to alter spatial coding. Decoding error is lower during CNO administration - does this mean place fields are smaller/more accurate? This analysis could offer deeper insights into the mechanisms by which dopamine influences hippocampal neural representations and memory processes.

We thank the reviewer for this helpful suggestion. We have expanded on our analysis of place field properties and decoding accuracy, describing properties of sessions with good enough decoding to be included compared to those that were excluded (lines 417-421). We also directly tested how decoding quality depended on several factors, including drug condition, novelty, number of cells recorded, and the average place field size of recorded cells (see legend for Figure 4 – Supplement 2). We found a small but significant effect of drug in experimental rats, but larger effects of number of recorded cells and average field size, that were also present in control animals.

Correct the typo on line 722 from "In ANOVA" to "An ANOVA".

We reworded this section and have corrected this error.

Reviewer #3 (Recommendations For The Authors):

The manuscript is clear and exciting. As a main criticism, I would have liked to see the effects on ripple duration not just the rate.

We thank the reviewer for this interesting idea. We performed a new analysis, similar to our analysis on SWR rate, probing the effect of our experimental manipulations on SWR duration in experimental rats. We have added the results in Figure 2 – Supplement 4, and note them in the main text lines 195-198: “SWR duration was reduced in novel sessions, consistent with replays becoming longer with increased familiarity  (Berners-Lee et al., 2021), as well as in Epoch 2, but was otherwise unaffected by reward or drug (Figure 2 – Supplement 4).”

I have a few other minor comments:

(1) I find it a little disturbing and counterintuitive that statistical differences are not always depicted in the figure graphs (for example Figures 2A-E). If the authors don't like to use the traditional *, ** or *** they could either just use one symbol to depict significance or simply depict the actual p values.

We thank the reviewer for this suggestion. We struggled with indicating significance values graphically in an intuitive way for interaction terms in the figures. We now added significance indicators in Figures 1F-I, added the significant model coefficients directly into Figure 2B-C, changed the analysis depicted in Figure 2D-E per Reviewer 1’s suggestions, and added significance indicators where previously missing in Figures 3 and 4.

(2) Related to the point above: in the page 7 legend D and E, it would be advantageous for clarity of the experimental results to also perform post-hoc analyses as depicted in the graphs, rather than just describe the p-value of the 3way ANOVA;

We thank the reviewer for this suggestion. Because the figure includes the mean and standard error of each condition, in addition to the significant effects of the mixed-effects model, we prefer the current format as it makes clearer the statistical tests that were performed while still allowing visual appreciation of differences between specific experimental conditions of interest to the reader.

(3) According to Figure 1H, the duration of the reward visits can go up to 15s (or more). Yet in Figure 2A only the first 10sec were analyzed. While I understand the rationale for using the initial 10 seconds where there is a lot more data, the results of graphs of Figures A to C will not have the same data/rate as Figures D-F where I assume the entire duration of the visit is taken into account.

A figure showing what happening to the ripple rate during the visits >10sec would help interpret the results of Figure 2.

We thank the reviewer for these interesting suggestions. We clarify now that all these analyses of Experiment 1 use only the first 10 s of each stopping period in Method line 758-764. However, examining the longer stopping periods is an excellent suggestion, and we re-analyzed the Experiment 1 dataset using up to the first 20 s of each stopping period. The main results (e.g., Figure 2) remain the same:

(1) Related to Figure 2B-C: For experimental rats, a mixed-effects generalized linear model predicting sharp-wave ripple rate as a function of reward end, block, drug, novelty, and interactions, had the following significant terms: drug (p<10^-5), novelty (p<10^-10), reward end × block (p<10^-10), and reward end × block × drug (p<0.05). The same model in control rats had significant terms: reward end (p<0.05), novelty (p<10^-4), reward end × block (p<10^-10).

(2) Related to Figure 2D-E: For experimental rats, we used a mixed-effects generalized linear model predicting the difference in sharp-wave ripple rate between the Incr. and Unch. reward ends in Epoch 2 as a function of novelty, drug, and their interaction. Model comparison found the full model performed better than a model removing the drug terms (AIC_nodrug – AIC_full = 2.94), while a model with only the intercept performed even worse (AIC_intercept – AIC_full = 13.76). For control rats, model comparison found the full model was equivalent to a model with only the intercept (AICintercept – AICfull = -0.36), with both modestly better than a model with no drug terms (AIC_nodrug – AIC_full = 1.38).

We have added a remark that results remain the same using this longer time window in Methods line 758-764.

Author response:

The following is the authors’ response to the previous reviews.

eLife Assessment

This study presents valuable findings on the potential of short-movie viewing fMRI protocol to explore the functional and topographical organization of the visual system in awake infants and toddlers. Although the data are compelling given the difficulty of studying this population, the evidence presented is incomplete and would be strengthened by additional analyses to support the authors' claims. This study will be of interest to cognitive neuroscientists and developmental psychologists, especially those interested in using fMRI to investigate brain organisation in pediatric and clinical populations with limited fMRI tolerance.

We are grateful for the thorough and thoughtful reviews. We have provided point-bypoint responses to the reviewers’ comments, but first, we summarize the major revisions here. We believe these revisions have substantially improved the clarity of the writing and impact of the results.

Regarding the framing of the paper, we have made the following major changes in response to the reviews:

(1) We have clarified that our goal in this paper was to show that movie data contains topographic, fine-grained details of the infant visual cortex. In the revision, we now state clearly that our results should not be taken as evidence that movies could replace retinotopy and have reworded parts of the manuscript that could mislead the reader in this regard.

(2) We have added extensive details to the (admittedly) complex methods to make them more approachable. An example of this change is that we have reorganized the figure explaining the Shared Response Modelling methods to divide the analytic steps more clearly.

(3) We have clarified the intermediate products contributing to the results by adding 6 supplementary figures that show the gradients for each IC or SRM movie and each infant participant.

In response to the reviews, we have conducted several major analyses to support our findings further:

(1) To verify that our analyses can identify fine-grained organization, we have manually traced and labeled adult data, and then performed the same analyses on them. The results from this additional dataset validate that these analyses can recover fine-grained organization of the visual cortex from movie data.

(2) To further explore how visual maps derived from movies compare to alternative methods, we performed an anatomical alignment control analysis. We show that high-quality maps can be predicted from other participants using anatomical alignment.

(3) To test the contribution of motion to the homotopy analyses, we regressed out the motion effects in these analyses. We found qualitatively similar results to our main analyses, suggesting motion did not play a substantial role.

(4) To test the contribution of data quantity to the homotopy analyses, we correlated the amount of movie data collected from each participant with the homotopy results. We did not find a relationship between data quantity and the homotopy results. 

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Ellis et al. investigated the functional and topographical organization of the visual cortex in infants and toddlers, as evidenced by movie-viewing data. They build directly on prior research that revealed topographic maps in infants who completed a retinotopy task, claiming that even a limited amount of rich, naturalistic movie-viewing data is sufficient to reveal this organization, within and across participants. Generating this evidence required methodological innovations to acquire high-quality fMRI data from awake infants (which have been described by this group, and elsewhere) and analytical creativity. The authors provide evidence for structured functional responses in infant visual cortex at multiple levels of analyses; homotopic brain regions (defined based on a retinotopy task) responded more similarly to one another than to other brain regions in visual cortex during movie-viewing; ICA applied to movie-viewing data revealed components that were identifiable as spatial frequency, and to a lesser degree, meridian maps, and shared response modeling analyses suggested that visual cortex responses were similar across infants/toddlers, as well as across infants/toddlers and adults. These results are suggestive of fairly mature functional response profiles in the visual cortex in infants/toddlers and highlight the potential of movie-viewing data for studying finer-grained aspects of functional brain responses, but further evidence is necessary to support their claims and the study motivation needs refining, in light of prior research.

Strengths:

- This study links the authors' prior evidence for retinotopic organization of visual cortex in human infants (Ellis et al., 2021) and research by others using movie-viewing fMRI experiments with adults to reveal retinotopic organization (Knapen, 2021).

- Awake infant fMRI data are rare, time-consuming, and expensive to collect; they are therefore of high value to the community. The raw and preprocessed fMRI and anatomical data analyzed will be made publicly available.

We are grateful to the reviewer for their clear and thoughtful description of the strengths of the paper, as well as their helpful outlining of areas we could improve.

Weaknesses:

- The Methods are at times difficult to understand and in some cases seem inappropriate for the conclusions drawn. For example, I believe that the movie-defined ICA components were validated using independent data from the retinotopy task, but this was a point of confusion among reviewers. 

We acknowledge the complexity of the methods and wish to clarify them as best as possible for the reviewers and the readers. We have extensively revised the methods and results sections to help avoid potential misunderstandings. For instance, we have revamped the figure and caption describing the SRM pipeline (Figure 5).

To answer the stated confusion directly, the ICA components were derived from the movie data and validated on the (completely independent) retinotopy data. There were no additional tasks. The following text in the paper explains this point:

“To assess the selected component maps, we correlated the gradients (described above) of the task-evoked and component maps. This test uses independent data: the components were defined based on movie data and validated against task-evoked retinotopic maps.” Pg. 11

In either case: more analyses should be done to support the conclusion that the components identified from the movie reproduce retinotopic maps (for example, by comparing the performance of movie-viewing maps to available alternatives (anatomical ROIs, group-defined ROIs). 

Before addressing this suggestion, we want to restate our conclusions: features of the retinotopic organization of infant visual cortex could be predicted from movie data. We did not conclude that movie data could ‘reproduce’ retinotopic maps in the sense that they would be a replacement. We recognize that this was not clear in our original manuscript and have clarified this point throughout, including in this section of the discussion:

“To be clear, we are not suggesting that movies work well enough to replace a retinotopy task when accurate maps are needed. For instance, even though ICA found components that were highly correlated with the spatial frequency map, we also selected some components that turned out to have lower correlations. Without knowing the ground truth from a retinotopy task, there would be no way to weed these out. Additionally, anatomical alignment (i.e., averaging the maps from other participants and anatomically aligning them to a held-out participant) resulted in maps that were highly similar to the ground truth. Indeed, we previously[23] found that adult-defined visual areas were moderately similar to infants. While functional alignment with adults can outperform anatomical alignment methods in similar analyses[27], here we find that functional alignment is inferior to anatomical alignment. Thus, if the goal is to define visual areas in an infant that lacks task-based retinotopy, anatomical alignment of other participants’ retinotopic maps is superior to using movie-based analyses, at least as we tested it.” Pg. 21

As per the reviewer’s suggestion and alluded to in the paragraph above, we have created anatomically aligned visual maps, providing an analogous test to the betweenparticipant analyses like SRM. We find that these maps are highly similar to the ground truth. We describe this result in a new section of the results:

“We performed an anatomical alignment analog of the functional alignment (SRM) approach. This analysis serves as a benchmark for predicting visual maps using taskbased data, rather than movie data, from other participants. For each infant participant, we aggregated all other infant or adult participants as a reference. The retinotopic maps from these reference participants were anatomically aligned to the standard surface template, and then averaged. These averages served as predictions of the maps in the test participant, akin to SRM, and were analyzed equivalently (i.e., correlating the gradients in the predicted map with the gradients in the task-based map). These correlations (Table S4) are significantly higher than for functional alignment (using infants to predict spatial frequency, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.44, CI=[0.32–0.58], p<.001; using infants to predict meridians, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.61, CI=[0.47–0.74], p<.001; using adults to predict spatial frequency, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.31, CI=[0.21–0.42], p<.001; using adults to predict meridians, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.49, CI=[0.39–0.60], p<.001). This suggests that even if SRM shows that movies can be used to produce retinotopic maps that are significantly similar to a participant, these maps are not as good as those that can be produced by anatomical alignment of the maps from other participants without any movie data.” Pg. 16–17

Also, the ROIs used for the homotopy analyses were defined based on the retinotopic task rather than based on movie-viewing data alone - leaving it unclear whether movie-viewing data alone can be used to recover functionally distinct regions within the visual cortex.

We agree with the reviewer that our approach does not test whether movie-viewing data alone can be used to recover functionally distinct regions. The goal of the homotopy analyses was to identify whether there was functional differentiation of visual areas in the infant brain while they watch movies. This was a novel question that provides positive evidence that these regions are functionally distinct. In subsequent analyses, we show that when these areas are defined anatomically, rather than functionally, they also show differentiated function (e.g., Figure 2). Nonetheless, our intention was not to use the homotopy analyses to define the regions. We have added text to clarify the goal and novelty of this analysis.

“Although these analyses cannot define visual maps, they test whether visual areas have different functional signatures.” Pg. 6

Additionally, even if the goal were to define areas based on homotopy, we believe the power of that analysis would be questionable. We would need to use a large amount of the movie data to define the areas, leaving a low-powered dataset to test whether their function is differentiated by these movie-based areas.

- The authors previously reported on retinotopic organization of the visual cortex in human infants (Ellis et al., 2021) and suggest that the feasibility of using movie-viewing experiments to recover these topographic maps is still in question. They point out that movies may not fully sample the stimulus parameters necessary for revealing topographic maps/areas in the visual cortex, or the time-resolution constraints of fMRI might limit the use of movie stimuli, or the rich, uncontrolled nature of movies might make them inferior to stimuli that are designed for retinotopic mapping, or might lead to variable attention between participants that makes measuring the structure of visual responses across individuals challenging. This motivation doesn't sufficiently highlight the importance or value of testing this question in infants. Further, it's unclear if/how this motivation takes into account prior research using movie-viewing fMRI experiments to reveal retinotopic organization in adults (e.g., Knapen, 2021). Given the evidence for retinotopic organization in infants and evidence for the use of movie-viewing experiments in adults, an alternative framing of the novel contribution of this study is that it tests whether retinotopic organization is measurable using a limited amount of movie-viewing data (i.e., a methodological stress test). The study motivation and discussion could be strengthened by more attention to relevant work with adults and/or more explanation of the importance of testing this question in infants (is the reason to test this question in infants purely methodological - i.e., as a way to negate the need for retinotopic tasks in subsequent research, given the time constraints of scanning human infants?).

We are grateful to the reviewer for giving us the opportunity to clarify the innovations of this research. We believe that this research contributes to our understanding of how infants process dynamic stimuli, demonstrates the viability and utility of movie experiments in infants, and highlights the potential for new movie-based analyses (e.g., SRM). We have now consolidated these motivations in the introduction to more clearly motivate this work:

“The primary goal of the current study is to investigate whether movie-watching data recapitulates the organization of visual cortex. Movies drive strong and naturalistic responses in sensory regions while minimizing task demands[12, 13, 24] and thus are a proxy for typical experience. In adults, movies and resting-state data have been used to characterize the visual cortex in a data-driven fashion[25–27]. Movies have been useful in awake infant fMRI for studying event segmentation[28], functional alignment[29], and brain networks[30]. However, this past work did not address the granularity and specificity of cortical organization that movies evoke. For example, movies evoke similar activity in infants in anatomically aligned visual areas[28], but it remains unclear whether responses to movie content differ between visual areas (e.g., is there more similarity of function within visual areas than between31). Moreover, it is unknown whether structure within visual areas, namely visual maps, contributes substantially to visual evoked activity. Additionally, we wish to test whether methods for functional alignment can be used with infants. Functional alignment finds a mapping between participants using functional activity – rather than anatomy – and in adults can improve signal-to-noise, enhance across participant prediction, and enable unique analyses[27, 32–34].” Pg. 3-4

Furthermore, the introduction culminates in the following statement on what the analyses will tell us about the nature of movie-driven activity in infants:

“These three analyses assess key indicators of the mature visual system: functional specialization between areas, organization within areas, and consistency between individuals.” Pg. 5

Furthermore, in the discussion we revisit these motivations and elaborate on them further:

[Regarding homotopy:] “This suggests that visual areas are functionally differentiated in infancy and that this function is shared across hemispheres[31].” Pg. 19

[Regarding ICA:] “This means that the retinotopic organization of the infant brain accounts for a detectable amount of variance in visual activity, otherwise components resembling these maps would not be discoverable.” Pg. 19–20

[Regarding SRM:] “This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45].” Pg. 21

Additionally, we have expanded our discussion of relevant work that uses similar methods such as the excellent research from Knapen (2021) and others:

“In adults, movies and resting-state data have been used to characterize the visual cortex in a data-driven fashion[25-27].” Pg. 4

“We next explored whether movies can reveal fine-grained organization within visual areas by using independent components analysis (ICA) to propose visual maps in individual infant brains[25,26,35,42,43].” Pg. 9

Reviewer #2 (Public Review):

Summary:

This manuscript shows evidence from a dataset with awake movie-watching in infants, that the infant brain contains areas with distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. However, substantial new analyses would be required to support the novel claim that movie-watching data in infants can be used to identify retinotopic areas or to capture within-area functional organization.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. These data position the authors to test their novel claim, that movie-watching data in infants can be used to identify retinotopic areas.

Weaknesses:

To claim that movie-watching data can identify retinotopic regions, the authors should provide evidence for two claims:

- Retinotopic areas defined based only on movie-watching data, predict retinotopic responses in independent retinotopy-task-driven data.

- Defining retinotopic areas based on the infant's own movie-watching response is more accurate than alternative approaches that don't require any movie-watching data, like anatomical parcellations or shared response activation from independent groups of participants.

We thank the reviewer for their comments. Before addressing their suggestions, we wish to clarify that we do not claim that movie data can be used to identify retinotopic areas, but instead that movie data captures components of the within and between visual area organization as defined by retinotopic mapping. We recognize that this was not clear in our original manuscript and have clarified this point throughout, including in this section of the discussion:

“To be clear, we are not suggesting that movies work well enough to replace a retinotopy task when accurate maps are needed. For instance, even though ICA found components that were highly correlated with the spatial frequency map, we also selected some components that turned out to have lower correlations. Without knowing the ground truth from a retinotopy task, there would be no way to weed these out. Additionally, anatomical alignment (i.e., averaging the maps from other participants and anatomically aligning them to a held-out participant) resulted in maps that were highly similar to the ground truth. Indeed, we previously[23] found that adult-defined visual areas were moderately similar to infants. While functional alignment with adults can outperform anatomical alignment methods in similar analyses[27], here we find that functional alignment with infants is inferior to anatomical alignment. Thus, if the goal is to define visual areas in an infant that lacks task-based retinotopy, anatomical alignment of other participants’ retinotopic maps is superior to using movie-based analyses, at least as we tested it.” Pg. 21

In response to the reviewer’s suggestion, we compare the maps identified by SRM to the averaged, anatomically aligned maps from infants. We find that these maps are highly similar to the task-based ground truth and we describe this result in a new section:

“We performed an anatomical alignment analog of the functional alignment (SRM) approach. This analysis serves as a benchmark for predicting visual maps using taskbased data, rather than movie data, from other participants. For each infant participant, we aggregated all other infant or adult participants as a reference. The retinotopic maps from these reference participants were anatomically aligned to the standard surface template, and then averaged. These averages served as predictions of the maps in the test participant, akin to SRM, and were analyzed equivalently (i.e., correlating the gradients in the predicted map with the gradients in the task-based map). These correlations (Table S4) are significantly higher than for functional alignment (using infants to predict spatial frequency, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.44, CI=[0.32–0.58], p<.001; using infants to predict meridians, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.61, CI=[0.47–0.74], p<.001; using adults to predict spatial frequency, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.31, CI=[0.21–0.42], p<.001; using adults to predict meridians, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.49, CI=[0.39–0.60], p<.001). This suggests that even if SRM shows that movies can be used to produce retinotopic maps that are significantly similar to a participant, these maps are not as good as those that can be produced by anatomical alignment of the maps from other participants without any movie data.” Pg. 16–17

Note that we do not compare the anatomically aligned maps with the ICA maps statistically. This is because these analyses are not comparable: ICA is run withinparticipant whereas anatomical alignment is necessarily between-participant — either infant or adults. Nonetheless, an interested reader can refer to the Table where we report the results of anatomical alignment and see that anatomical alignment outperforms ICA in terms of the correlation between the predicted and task-based maps.

Both of these analyses are possible, using the (valuable!) data that these authors have collected, but these are not the analyses that the authors have done so far. Instead, the authors report the inverse of (1): regions identified by the retinotopy task can be used to predict responses in the movies. The authors report one part of (2), shared responses from other participants can be used to predict individual infants' responses in the movies, but they do not test whether movie data from the same individual infant can be used to make better predictions of the retinotopy task data, than the shared response maps.

So to be clear, to support the claims of this paper, I recommend that the authors use the retinotopic task responses in each individual infant as the independent "Test" data, and compare the accuracy in predicting those responses, based on:

-  The same infant's movie-watching data, analysed with MELODIC, when blind experimenters select components for the SF and meridian boundaries with no access to the ground-truth retinotopy data.

-  Anatomical parcellations in the same infant.

-  Shared response maps from groups of other infants or adults.

-  (If possible, ICA of resting state data, in the same infant, or from independent groups of infants).

Or, possibly, combinations of these techniques.

If the infant's own movie-watching data leads to improved predictions of the infant's retinotopic task-driven response, relative to these existing alternatives that don't require movie-watching data from the same infant, then the authors' main claim will be supported.

These are excellent suggestions for additional analyses to test the suitability for moviebased maps to replace task-based maps. We hope it is now clear that it was never our intention to claim that movie-based data could replace task-based methods. We want to emphasize that the discoveries made in this paper — that movies evoke fine-grained organization in infant visual cortex — do not rely on movie-based maps being better than alternative methods for producing maps, such as the newly added anatomical alignment.

The proposed analysis above solves a critical problem with the analyses presented in the current manuscript: the data used to generate maps is identical to the data used to validate those maps. For the task-evoked maps, the same data are used to draw the lines along gradients and then test for gradient organization. For the component maps, the maps are manually selected to show the clearest gradients among many noisy options, and then the same data are tested for gradient organization. This is a double-dipping error. To fix this problem, the data must be split into independent train and test subsets.

We appreciate the reviewer’s concern; however, we believe it is a result of a miscommunication in our analytic strategy. We have now provided more details on the analyses to clarify how double-dipping was avoided. 

To summarize, a retinotopy task produced visual maps that were used to trace both area boundaries and gradients across the areas. These data were then fixed and unchanged, and we make no claims about the nature of these maps in this paper, other than to treat them as the ground truth to be used as a benchmark in our analyses. The movie data, which are collected independently from the same infant in the session, used the boundaries from the retinotopy task (in the case of homotopy) or were compared with the maps from the retinotopy task (in the case of ICA and SRM). In other words, the statement that “the data used to generate maps is identical to the data used to validate those maps” is incorrect because we generated the maps with a retinotopy task and validated the maps with the movie data. This means no double dipping occurred.

Perhaps a cause of the reviewer’s interpretation is that the gradients used in the analysis are not clearly described. We now provide this additional description:  “Using the same manually traced lines from the retinotopy task, we measured the intensity gradients in each component from the movie-watching data. We can then use the gradients of intensity in the retinotopy task-defined maps as a benchmark for comparison with the ICA-derived maps.” Pg. 10

Regarding the SRM analyses, we take great pains to avoid the possibility of data contamination. To emphasize how independent the SRM analysis is, the prediction of the retinotopic map from the test participant does not use their retinotopy data at all; in fact, the predicted maps could be made before that participant’s retinotopy data were ever collected. To make this prediction for a test participant, we need to learn the inversion of the SRM, but this only uses the movie data of the test participant. Hence, there is no double-dipping in the SRM analyses. We have elaborated on this point in the revision, and we remade the figure and its caption to clarify this point:

We also have updated the description of these results to emphasize how double-dipping was avoided:

“We then mapped the held-out participant's movie data into the learned shared space without changing the shared space (Figure 5c). In other words, the shared response model was learned and frozen before the held-out participant’s data was considered.

This approach has been used and validated in prior SRM studies[45].” Pg. 14

The reviewer suggests that manually choosing components from ICA is double-dipping. Although the reviewer is correct that the manual selection of components in ICA means that the components chosen ought to be good candidates, we are testing whether those choices were good by evaluating those components against the task-based maps that were not used for the ICA. Our statistical analyses evaluate whether the components chosen were better than the components that would have been chosen by random chance. Critically: all decisions about selecting the components happen before the components are compared to the retinotopic maps. Hence there is no double-dipping in the selection of components, as the choice of candidate ICA maps is not informed by the ground-truth retinotopic maps. We now clarify what the goal of this process is in the results:

“Success in this process requires that 1) retinotopic organization accounts for sufficient variance in visual activity to be identified by ICA and 2) experimenters can accurately identify these components.” Pg. 10

The reviewer also alludes to a concern that the researcher selecting the maps was not blind to the ground-truth retinotopic maps from participants and this could have influenced the results. In such a scenario, the researcher could have selected components that have the gradients of activity in the places that the infant has as ground truth. The researcher who made the selection of components (CTE) is one of the researchers who originally traced the areas in the participants approximately a year prior to the identification of ICs. The researcher selecting the components didn’t use the ground-truth retinotopic maps as reference, nor did they pay attention to the participant IDs when sorting the IC components. Indeed, they weren’t trying to find participant specific maps per se, but rather aimed to find good candidate retinotopic maps in general. In the case of the newly added adult analyses, the ICs were selected before the retinotopic mapping was reviewed or traced; hence, no knowledge about the participant-specific ground truth could have influenced the selection of ICs. Even with this process from adults, we find results of comparable strength as we found in infants, as shown below. Nonetheless, there is a possibility that this researcher’s previous experience of tracing the infant maps could have influenced their choice of components at the participant-specific level. If so, it was a small effect since the components the researcher selected were far from the best possible options (i.e., rankings of the selected components averaged in the 64th percentile for spatial frequency maps and the 68th percentile for meridian maps). We believe all reasonable steps were taken to mitigate bias in the selection of ICs.

Reviewer #3 (Public Review):

The manuscript reports data collected in awake toddlers recording BOLD while watching videos. The authors analyse the BOLD time series using two different statistical approaches, both very complex but do not require any a priori determination of the movie features or contents to be associated with regressors. The two main messages are that 1) toddlers have occipital visual areas very similar to adults, given that an SRM model derived from adult BOLD is consistent with the infant brains as well; 2) the retinotopic organization and the spatial frequency selectivity of the occipital maps derived by applying correlation analysis are consistent with the maps obtained by standard and conventional mapping.

Clearly, the data are important, and the author has achieved important and original results. However, the manuscript is totally unclear and very difficult to follow; the figures are not informative; the reader needs to trust the authors because no data to verify the output of the statistical analysis are presented (localization maps with proper statistics) nor so any validation of the statistical analysis provided. Indeed what I think that manuscript means, or better what I understood, may be very far from what the authors want to present, given how obscure the methods and the result presentation are.

In the present form, this reviewer considers that the manuscript needs to be totally rewritten, the results presented each technique with appropriate validation or comparison that the reader can evaluate.

We are grateful to the reviewer for the chance to improve the paper. We have broken their review into three parts: clarification of the methods, validation of the analyses, and enhancing the visualization.

Clarification of the methods

We acknowledge that the methods we employed are complex and uncommon in many fields of neuroimaging. That said, numerous papers have conducted these analyses on adults (Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Lu et al., 2017) and non-human primates (Arcaro & Livingstone, 2017; Moeller et al., 2009). We have redoubled our efforts in the revision to make the methods as clear as possible, expanding on the original text and providing intuitions where possible. These changes have been added throughout and are too vast in number to repeat here, especially without context, but we hope that readers will have an easier time following the analyses now. 

Additionally, we updated Figures 3 and 5 in which the main ICA and SRM analyses are described. For instance, in Figure 3’s caption we now add details about how the gradient analyses were performed on the components: 

“We used the same lines that were manually traced on the task-evoked map to assess the change in the component’s response. We found a monotonic trend within area from medial to lateral, just like we see in the ground truth.” Pg. 11

Regarding Figure 5, we reconsidered the best way to explain the SRM analyses and decided it would be helpful to partition the diagram into steps, reflecting the analytic process. These updates have been added to Figure 5, and the caption has been updated accordingly.

We hope that these changes have improved the clarity of the methods. For readers interested in learning more, we encourage them to either read the methods-focused papers that debut the analyses (e.g., Chen et al., 2015), read the papers applying the methods (e.g., Guntupalli et al., 2016), or read the annotated code we publicly release which implements these pipelines and can be used to replicate the findings.

Validation of the analyses

One of the requests the reviewer makes is to validate our analyses. Our initial approach was to lean on papers that have used these methods in adults or primates (e.g., Arcaro, & Livingstone, 2017; Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Moeller et al., 2009) where the underlying organization and neurophysiology is established. However, we have made changes to these methods that differ from their original usage (e.g., we used SRM rather than hyperalignment, we use meridian mapping rather than traveling wave retinotopy, we use movie-watching data rather than rest). Hence, the specifics of our design and pipeline warrant validation. 

To add further validation, we have rerun the main analyses on an adult sample. We collected 8 adult participants who completed the same retinotopy task and a large subset of the movies that infants saw. These participants were run under maximally similar conditions to infants (i.e., scanned using the same parameters and without the top of the head-coil) and were preprocessed using the same pipeline. Given that the relationship between adult visual maps and movie-driven (or resting-state) analyses has been shown in many studies (Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Lu et al., 2017), these adult data serve as a validation of our analysis pipeline. These adult participants were included in the original manuscript; however, they were previously only used to support the SRM analyses (i.e., can adults be used to predict infant visual maps). The adult results are described before any results with infants, as a way to engender confidence. Moreover, we have provided new supplementary figures of the adult results that we hope will be integrated with the article when viewing it online, such that it will be easy to compare infant and adult results, as per the reviewer’s request. 

As per the figures and captions below, the analyses were all successful with the adult participants: 1) Homotopic correlations are higher than correlations between comparable areas in other streams or areas that are more distant within stream. 2) A multidimensional scaling depiction of the data shows that areas in the dorsal and ventral stream are dissimilar. 3) Using independent components analysis on the movie data, we identified components that are highly correlated with the retinotopy task-based spatial frequency and meridian maps. 4) Using shared response modeling on the movie data, we predicted maps that are highly correlated with the retinotopy task-based spatial frequency and meridian maps.

These supplementary analyses are underpowered for between-group comparisons, so we do not statistically compare the results between infants and adults. Nonetheless, the pattern of adult results is comparable overall to the infant results. 

We believe these adult results provide a useful validation that the infant analyses we performed can recover fine-grained organization.

Enhancing the visualization

The reviewer raises an additional concern about the lack of visualization of the results. We recognize that the plots of the summary statistics do not provide information about the intermediate analyses. Indeed, we think the summary statistics can understate the degree of similarity between the components or predicted visual maps and the ground truth. Hence, we have added 6 new supplementary figures showing the intensity gradients for the following analyses: 1. spatial frequency prediction using ICA, 2. meridian prediction using ICA, 3. spatial frequency prediction using infant SRM, 4. meridian prediction using infant SRM, 5. spatial frequency prediction using adult SRM, and 6. meridian prediction using adult SRM.

We hope that these visualizations are helpful. It is possible that the reviewer wishes us to also visually present the raw maps from the ICA and SRM, akin to what we show in Figure 3A and 3B. We believe this is out of scope of this paper: of the 1140 components that were identified by ICA, we selected 36 for spatial frequency and 17 for meridian maps. We also created 20 predicted maps for spatial frequency and 20 predicted meridian maps using SRM. This would result in the depiction of 93 subfigures, requiring at least 15 new full-page supplementary figures to display with adequate resolution. Instead, we encourage the reader to access this content themselves: we have made the code to recreate the analyses publicly available, as well as both the raw and preprocessed data for these analyses, including the data for each of these selected maps.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) As mentioned in the public review, the authors should consider incorporating relevant adult fMRI research into the Introduction and explain the importance of testing this question in infants.

Our public response describes the several citations to relevant adult research we have added, and have provided further motivation for the project.

(2) The authors should conduct additional analyses to support their conclusion that movie data alone can generate accurate retinotopic maps (i.e., by comparing this approach to other available alternatives).

We have clarified in our public response that we did not wish to conclude that movie data alone can generate accurate retinotopic maps, and have made substantial edits to the text to emphasize this. Thus, because this claim is already not supported by our analyses, we do not think it is necessary to test it further.

(3) The authors should re-do the homotopy analyses using movie-defined ROIs (i.e., by splitting the movie-viewing data into independent folds for functional ROI definition and analyses).

As stated above, defining ROIs based on the movie content is not the intended goal of this project. Even if that were the general goal, we do not believe that it would be appropriate to run this specific analysis with the data we collected. Firstly, halving the data for ROI definition (e.g., using half the movie data to identify and trace areas, and then use those areas in the homotopy analysis to run on the other half of data) would qualitatively change the power of the analyses described here. Secondly, we would be unable to define areas beyond hV4/V3AB with confidence, since our retinotopic mapping only affords specification of early visual cortex. Thus we could not conduct the MDS analyses shown in Figure 2.

(4) If the authors agree that a primary contribution of this study and paper is to showcase what is possible to do with a limited amount of movie-viewing data, then they should make it clearer, sooner, how much usable movie data they have from infants. They could also consider conducting additional analyses to determine the minimum amount of fMRI data necessary to reveal the same detailed characteristics of functional responses in the visual cortex.

We agree it would be good to highlight the amount of movie data used. When the infant data is first introduced in the results section, we now state the durations:

“All available movies from each session were included (Table S2), with an average duration of 540.7s (range: 186--1116s).” Pg. 5

Additionally, we have added a homotopy analysis that describes the contribution of data quantity to the results observed. We compare the amount of data collected with the magnitude of same vs. different stream effect (Figure 1B) and within stream distance effect (Figure 1C). We find no effect of movie duration in the sample we tested, as reported below:

“We found no evidence that the variability in movie duration per participant correlated with this difference [of same stream vs. different stream] (r=0.08, p=.700).” Pg. 6-7

“There was no correlation between movie duration and the effect (Same > Adjacent: r=-0.01, p=.965, Adjacent > Distal: r=-0.09, p=.740).” Pg. 7

(5) If any of the methodological approaches are novel, the authors should make this clear. In particular, has the approach of visually inspecting and categorizing components generated from ICA and movie data been done before, in adults/other contexts?

The methods we employed are similar to others, as described in the public review.

However, changes were necessary to apply them to infant samples. For instance, Guntupalli et al. (2016) used hyperalignment to predict the visual maps of adult participants, whereas we use SRM. SRM and hyperalignment have the same goal — find a maximally aligned representation between participants based on brain function — but their implementation is different. The application of functional alignment to infants is novel, as is their use in movie data that is relatively short by comparison to standard adult data. Indeed, this is the most thorough demonstration that SRM — or any functional alignment procedure — can be usefully applied to infant data, awake or sleeping. We have clarified this point in the discussion.

“This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45], which may prove especially useful for infant fMRI[52].” Pg. 21

(6) The authors found that meridian maps were less identifiable from ICA and movie data and suggest that this may be because these maps are more susceptible to noise or gaze variability. If this is the case, you might predict that these maps are more identifiable in adult data. The authors could consider running additional analyses with their adult participants to better understand this result.

As described in the manuscript, we hypothesize that meridian maps are more difficult to identify than spatial frequency maps because meridian maps are a less smooth, more fine-grained map than spatial frequency. Indeed, it has previously been reported (Moeller et al., 2009) that similar procedures can result in meridian maps that are constituted by multiple independent components (e.g., a component sensitive to horizontal orientations, and a separate component sensitive to vertical components). Nonetheless, we have now conducted the ICA procedure on adult participants and again find it is easier to identify spatial frequency components compared to meridian maps, as reported in the public review.

Minor corrections:

(1) Typo: Figure 3 title: "Example retintopic task vs. ICA-based spatial frequency maps.".

Fixed

(2) Given the age range of the participants, consider using "infants and toddlers"? (Not to diminish the results at all; on the contrary, I think it is perhaps even more impressive to obtain awake fMRI data from ~1-2-year-olds). Example: Figure 3 legend: "A) Spatial frequency map of a 17.1-monthold infant.".

We agree with the reviewer that there is disagreement about the age range at which a child starts being considered a toddler. We have changed the terms in places where we refer to a toddler in particular (e.g., the figure caption the reviewer highlights) and added the phrase “infants and toddlers” in places where appropriate. Nonetheless, we have kept “infants” in some places, particularly those where we are comparing the sample to adults. Adding “and toddlers” could imply three samples being compared which would confuse the reader.

(3) Figure 6 legend: The following text should be omitted as there is no bar plot in this figure: "The bar plot is the average across participants. The error bar is the standard error across participants.".

Fixed

(4) Table S1 legend: Missing first single quote: Runs'.

Fixed

Reviewer #2 (Recommendations For The Authors):

I request that this paper cite more of the existing literature on the fMRI of human infants and toddlers using task-driven and resting-state data. For example, early studies by (first authors) Biagi, Dehaene-Lambertz, Cusack, and Fransson, and more recent studies by Chen, Cabral, Truzzi, Deen, and Kosakowski.

We have added several new citations of recent task-based and resting state studies to the second sentence of the main text:

“Despite the recent growth in infant fMRI[1-6], one of the most important obstacles facing this research is that infants are unable to maintain focus for long periods of time and struggle to complete traditional cognitive tasks[7].”

Reviewer #3 (Recommendations For The Authors):

In the following, I report some of my main perplexities, but many more may arise when the material is presented more clearly.

The age of the children varies from 5 months to about 2 years. While the developmental literature suggests that between 1 and 2 years children have a visual system nearly adult-like, below that age some areas may be very immature. I would split the sample and perhaps attempt to validate the adult SRM model with the youngest children (and those can be called infants).

We recognize the substantial age variability in our sample, which is why we report participant-specific data in our figures. While splitting up the data into age bins might reveal age effects, we do not think we can perform adequately powered null hypothesis testing of the age trend. In order to investigate the contribution of age, larger samples will be needed. That said, we can see from the data that we have reported that any effect of age is likely small. To elaborate: Figures 4 and 6 report the participant-specific data points and order the participants by age. There are no clear linear trends in these plots, thus there are no strong age effects.

More broadly, we do not think there is a principled way to divide the participants by age. The reviewer suggests that the visual system is immature before the first year of life and mature afterward; however, such claims are the exact motivation for the type of work we are doing here, and the verdict is still out. Indeed, the conclusion of our earlier work reporting retinotopy in infants (Ellis et al., 2021) suggests that the organization of the early visual cortex in infants as young as 5 months — the youngest infant in our sample — is surprisingly adult-like.

The title cannot refer to infants given the age span.

There is disagreement in the field about the age at which it is appropriate to refer to children as infants. In this paper, and in our prior work, we followed the practice of the most attended infant cognition conference and society, the International Congress of Infant Studies (ICIS), which considers infants as those aged between 0-3 years old, for the purposes of their conference. Indeed, we have never received this concern across dozens of prior reviews for previous papers covering a similar age range. That said, we understand the spirit of the reviewer’s comment and now refer to the sample as “infants and toddlers” and to older individuals in our sample as “toddlers” wherever it is appropriate (the younger individuals would fairly be considered “infants” under any definition).

Figure 1 is clear and an interesting approach. Please also show the average correlation maps on the cortical surface.

While we would like to create a figure as requested, we are unsure how to depict an area-by-area correlation map on the cortical surface. One option would be to generate a seed-based map in which we take an area and depict the correlation of that seed (e.g., vV1) with all other voxels. This approach would result in 8 maps for just the task-defined areas, and 17 maps for anatomically-defined areas. Hence, we believe this is out of scope of this paper, but an interested reader could easily generate these maps from the data we have released.

Figure 2 results are not easily interpretable. Ventral and dorsal V1-V3 areas represent upper or lower VF respectively. Higher dorsal and ventral areas represent both upper and lower VF, so we should predict an equal distance between the two streams. Again, how can we verify that it is not a result of some artifacts?

In adults, visual areas differ in their functional response properties along multiple dimensions, including spatial coding. The dorsal/ventral stream hypothesis is derived from the idea that areas in each stream support different functions, independent of spatial coding. The MDS analysis did not attempt to isolate the specific contribution of spatial representations of each area but instead tested the similarity of function that is evoked in naturalistic viewing. Other covariance-based analyses specifically isolate the contribution of spatial representations (Haak et al., 2013); however, they use a much more constrained analysis than what was implemented here. The fact that we find broad differentiation of dorsal and ventral visual areas in infants is consistent with adults (Haak & Beckman, 2018) and neonate non-human primates (Arcaro & Livingstone, 2017). 

Nonetheless, we recognize that we did not mention the differences in visual field properties across areas and what that means. If visual field properties alone drove the functional response then we would expect to see a clustering of areas based on the visual field they represent (e.g., hV4 and V3AB should have similar representations). Since we did not see that, and instead saw organization by visual stream, the result is interesting and thus warrants reporting. We now mention this difference in visual fields in the manuscript to highlight the surprising nature of the result.

“This separation between streams is striking when considering that it happens despite differences in visual field representations across areas: while dorsal V1 and ventral V1 represent the lower and upper visual field, respectively, V3A/B and hV4 both have full visual field maps. These visual field representations can be detected in adults[41]; however, they are often not the primary driver of function[39]. We see that in infants too: hV4 and V3A/B represent the same visual space yet have distinct functional profiles.” Pg. 8

The reviewer raises a concern that the MDS result may be spurious and caused by noise. Below, we present three reasons why we believe these results are not accounted for by artifacts but instead reflect real functional differentiation in the visual cortex. 

(1) Figure 2 is a visualization of the similarity matrix presented in Figure S1. In Figure S1, we report the significance testing we performed to confirm that the patterns differentiating dorsal and ventral streams — as well as adjacent areas from distal areas — are statistically reliable across participants. If an artifact accounted for the result then it would have to be a kind of systematic noise that is consistent across participants.

(2) One of the main sources of noise (both systematic and non-systematic) with infant fMRI is motion. Homotopy is a within-participant analysis that could be biased by motion. To assess whether motion accounts for the results, we took a conservative approach of regressing out the framewise motion (i.e., how much movement there is between fMRI volumes) from the comparisons of the functional activity in regions. Although the correlations numerically decreased with this procedure, they were qualitatively similar to the analysis that does not regress out motion:

“Additionally, if we control for motion in the correlation between areas --- in case motion transients drive consistent activity across areas --- then the effects described here are negligibly different (Figure S5).” Pg. 7

(3) We recognize that despite these analyses, it would be helpful to see what this pattern looks like in adults where we know more about the visual field properties and the function of dorsal and ventral streams. This has been done previously (e.g., Haak & Beckman, 2018), but we have now run those analyses on adults in our sample, as described in the public review. As with infants, there are reliable differences in the homotopy between streams (Figure S1). The MDS results show that the adult data was more complex than the infant data, since it was best described by 3 dimensions rather than 2. Nonetheless, there is a rotation of the MDS such that the structure of the ventral and dorsal streams is also dissociable. 

Figure 3 also raises several alternative interpretations. The spatial frequency component in B has strong activity ONLY at the extreme border of the VF and this is probably the origin of the strong correlation. I understand that it is only one subject, but this brings the need to show all subjects and to report the correlation. Also, it is important to show the putative average ICA for retinotopy and spatial frequencies across subjects and for adults. All methods should be validated on adults where we have clear data for retinotopy and spatial frequency.

The reviewer notes that the component in Figure 3 shows strong negative response in the periphery. It is often the case, as reported elsewhere (Moeller et al., 2009), that ICA extracts portions of visual maps. To make a full visual map would require combining components into a composite (e.g., a component that has a high response in the periphery and another component that has a high response in the fovea). If we were to claim that this component, or others like it, could replace the need for retinotopic mapping, then we would want to produce these composite maps; however, our conclusion in this project is that the topographic information of retinotopic maps manifest in individual components of ICA. For this purpose, the analysis we perform adequately assesses this topography.

Regarding the request to show the results for all subjects, we address this in the public response and repeat it here briefly: we have added 6 new figures to show results akin to Figure 3C and D. It is impractical to show the equivalent of Figure 3A and B for all participants, yet we do release the data necessary to see to visualize these maps easily.

Finally, the reviewer suggests that we validate the analyses on adult participants. As shown in Figure S3 and reported in the public response, we now run these analyses on adult participants and observe qualitatively similar results to infants.

How much was the variation in the presumed spatial frequency map? Is it consistent with the acuity range? 5-month-old infants should have an acuity of around 10c/deg, depending on the mean luminance of the scene.

The reviewer highlights an important weakness of conducting ICA: we cannot put units on the degree of variation we see in components. We now highlight this weakness in the discussion:

“Another limitation is that ICA does not provide a scale to the variation: although we find a correlation between gradients of spatial frequency in the ground truth and the selected component, we cannot use the component alone to infer the spatial frequency selectivity of any part of cortex. In other words, we cannot infer units of spatial frequency sensitivity from the components alone.” Pg. 20

Figure 5 pipeline is totally obscure. I presumed that I understood, but as it is it is useless. All methods should be clearly described, and the intermediate results should be illustrated in figures and appropriately discussed. Using such blind analyses in infants in principle may not be appropriate and this needs to be verified. Overall all these techniques rely on correlation activities that are all biased by head movement, eye movement, and probably the dummy sucking. All those movements need to be estimated and correlated with the variability of the results. It is a strong assumption that the techniques should work in infants, given the presence of movements.

We recognize that the SRM methods are complex. Given this feedback, we remade Figure 5 with explicit steps for the process and updated the caption (as reported in the public review).

Regarding the validation of these methods, we have added SRM analyses from adults and find comparable results. This means that using these methods on adults with comparable amounts of data as what we collected from infants can predict maps that are highly similar to the real maps. Even so, it is not a given that these methods are valid in infants. We present two considerations in this regard. 

First, as part of the SRM analyses reported in the manuscript, we show that control analyses are significantly worse than the real analyses (indicated by the lines on Figure 6). To clarify the control analysis: we break the mapping (i.e., flip the order of the data so that it is backwards) between the test participant and the training participants used to create the SRM. The fact that this control analysis is significantly worse indicates that SRM is learning meaningful representations that matter for retinotopy. 

Second, we believe that this paper is a validation of SRM for infants. Infant fMRI is a nascent field and SRM has the potential to increase the signal quality in this population. We hope that readers will see these analyses as a proof of concept that SRM can be used in their work with infants. We have stated this contribution in the paper now.

“Additionally, we wish to test whether methods for functional alignment can be used with infants. Functional alignment finds a mapping between participants using functional activity -- rather than anatomy -- and in adults can improve signal-to-noise, enhance across participant prediction, and enable unique analyses[27,32-34].” Pg. 4

“This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45].” Pg. 21

Regarding the reviewer’s concern that motion may bias the results, we wish to emphasize the nature of the analyses being conducted here: we are using data from a group of participants to predict the neural responses in a held-out participant. For motion to explain consistency between participants, the motion would need to be timelocked across participants. Even if motion was time-locked during movie watching, motion will impair the formation of an adequate model that can contain retinotopic information. Thus, motion should only hurt the ability for a shared response to be found that can be used for predicting retinotopic maps. Hence, the results we observed are despite motion and other sources of noise.

What is M??? is it simply the mean value??? If not, how it is estimated?

M is an abbreviation for mean. We have now expanded the abbreviation the first time we use it.

Figure 6 should be integrated with map activity where the individual area correlation should be illustrated. Probably fitting SMR adult works well for early cortical areas, but not for more ventral and associative, and the correlation should be evaluated for the different masks.

With the addition of plots showing the gradients for each participant and each movie (Figures S10–S13) we hope we have addressed this concern. We additionally want to clarify that the regions we tested in the analysis in Figure 6 are only the early visual areas V1, V2, V3, V3A/B, and hV4. The adult validation analyses show that SRM works well for predicting the visual maps in these areas. Nonetheless, it is an interesting question for future research with more extensive retinotopic mapping in infants to see if SRM can predict maps beyond extrastriate cortex.

Occipital masks have never been described or shown.

The occipital mask is from the MNI probabilistic structural atlas (Mazziotta et al., 2001), as reported in the original version and is shared with the public data release. We have added the additional detail that the probabilistic atlas is thresholded at 0% in order to be liberally inclusive. 

“We used the occipital mask from the MNI structural atlas[63] in standard space -- defined liberally to include any voxel with an above zero probability of being labelled as the occipital lobe -- and used the inverted transform to put it into native functional space.” Pg. 27–28

Methods lack the main explanation of the procedures and software description.

We hope that the additions we have made to address this reviewer’s concerns have provided better explanations for our procedures. Additionally, as part of the data and code release, we thoroughly explain all of the software needed to recreate the results we have observed here.

Summary of Tech Talk: Atomic Data and the Semantic Web

Introduction

• Speaker: Software developer and entrepreneur with experience in linked data and the semantic web.

"My name is [Hube], I'm a software developer and entrepreneur and I did a lot with linked data and the semantic web."

• Context: Focus on atomic data as an iteration on the semantic web and RDF concepts.

"Today I'm going to talk about the semantic web and atomic data which is an iteration on that."

Challenges with the Current Web and Semantic Web

• Centralization: Web has shifted from a decentralized network to a centralized model controlled by few actors.

"It has become way more centralized...and the nodes between individuals have kind of went missing over time."

• Non-interoperable APIs: Many systems are incompatible due to proprietary APIs.

"Even when developers create new types of applications, they often end up creating their totally own APIs."

• Initial Design Gaps: The web's machine readability and write capabilities were underdeveloped.

"The web is not machine-readable...The very first web browser actually had full read-write capabilities."

RDF and the Vision for a Semantic Web

• Concept: RDF uses triples (subject, predicate, object) to make data machine-readable and interoperable.

"The idea of RDF is that it really increases the quality of data on the web."

Challenges with RDF:

• Lack of native arrays or sequential data, making JSON more practical.

"RDF doesn't have any form of native arrays or sequential data."

• Subject-property uniqueness issues lead to complexity.

"A subject can have many triples with the same predicate...makes it very hard to store data in maps."

• Blank nodes and named graphs introduce high complexity.

"Blank nodes are this really complex thing...and there's also named graphs in RDF which provide just another layer of complexity."

Atomic Data: An Alternative Approach

• Vision: Combines RDF's interoperability, JSON's simplicity, and TypeScript's type safety.

"Atomic data is basically where all these three things are combined."

Key Features:

• Strict subset of RDF and JSON with tight coupling between schema and validation.

"Classes describe which properties are required and which ones are optional."

• Transactions standardize changes for versioning, history, and cryptographic verification.

"Every change is a transaction...every transaction is actually a resource."

• Fully resolvable URLs ensure semantic clarity and reusability.

"Every resource actually resolvable...you can use a local ID for resources not hosted globally."

• Extensibility for dynamic and interactive web applications (e.g., real-time collaboration, file handling).

"Static data is relatively easy but as we all know data changes over time."

Practical Implementations

• Atomic Server: Graph database in Rust for speed and robustness.

"A graph database...written in Rust and it's really fast."

Features:

• Full text search, authentication, and live synchronization.

"Embedded full text search index, queries, sorting, filtering, pagination, authorization."

• Front-end: Built with React for ease of use and collaboration.

"A graphical user interface that basically allows you to view all the data and add all data."

Community and Tools

• Libraries: JavaScript, TypeScript, and Rust libraries for integration.

"Atomic Lib Library which is a JavaScript TypeScript npm type of library."

• Open Source and Documentation: MIT-licensed projects with comprehensive guides.

"Everything shown is all MIT license...a very, very big documentation book."

• Community Engagement: Discussions on GitHub, Discord, and plans for W3C submission.

"There's a W3C community group for atomic data...most of the activity is on GitHub and Discord."

Conclusion and Vision

• Future of Atomic Data: Bridging the gaps of RDF with pragmatic, developer-friendly solutions.

"I think we should always use URLs that resolve."

• Call to Action: Developers encouraged to explore, contribute, and adopt atomic data.

"Thank you for listening, does anybody have a question?"

This summary encapsulates the core challenges and solutions proposed for evolving the web's interoperability through atomic data while highlighting the technical nuances and community-driven development efforts.

One thing I think is not explicitly stated here but which I think is really great is that the cells were grown in constant light in TAP media, meaning that they won't be synchronized/as homogenous, and therefore will likely capture a wider range of biology in the dataset than might otherwise be observed.

Another note is the use of the mat3-4 mutant strain, which as stated has the experimental benefit of a smaller size for vitrification (almost half the diameter of wild-type strains). If I recall correctly when we were looking for actin filaments in mat3-4 tomograms, they seemed much more infrequent than we would have expected based on how abundant filaments are when phalloidin staining CC-125 or CC-124 wild-type strains (https://doi.org/10.1091/mbc.E19-03-0141). And I recall we tried phalloidin staining the mat3-4s and had trouble doing so. So my suspicion is that there might be a few key differences in macromolecular composition in these mutant strains that might prevent some structures from resolving or being abundant enough to detect readily. It's not a criticism, but just worth noting I think for those trying to extrapolate from this extremely helpful resource.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Meissner et al describe an update on the collection of split-GAL4 lines generated by a consortium led by Janelia Research Campus. This follows the same experimental pipeline described before and presents as a significant increment to the present collection. This will strengthen the usefulness and relevance of "splits" as a standard tool for labs that already use this tool and attract more labs and researchers to use it.

Strengths:

This manuscript presents a solid step to establish Split-GAL4 lines as a relevant tool in the powerful Drosophila toolkit. Not only does the raw number of available lines contribute to the relevance of this tool in the "technical landscape" of genetic tools, but additional features of this effort contribute to the successful adoption. These include:

(1) A description of expression patterns in the adult and larvae, expanding the "audience" for these tools

(2) A classification of line combination according to quality levels, which provides a relevant criterion while deciding to use a particular set of "splits".

(3) Discrimination between male and female expression patterns, providing hints regarding the potential role of these gender-specific circuits.

(4) The search engine seems to be user-friendly, facilitating the retrieval of useful information.

Overall, the authors employed a pipeline that maximizes the potential of the Split-GAL4 collection to the scientific community.

Weaknesses:

The following aspects apply:

The use of split-GAL4 lines has improved tremendously the genetic toolkit of Drosophila and this manuscript is another step forward in establishing this tool in the genetic repertoire that laboratories use. Thus, this would be a perfect opportunity for the authors to review the current status of this tool, addressing its caveats and how to effectively implement it into the experimental pipeline.

(1) While the authors do bring up a series of relevant caveats that the community should be aware of while using split-GAL4 lines, the authors should take the opportunity to address some of the genetic issues that frequently arise while using the described genetic tools. This is particularly important for laboratories that lack the experience using split-GAL4 lines and wish to use them. Some of these issues are covertly brought up, but not entirely clarified.

First, why do the authors (wisely) rescreen the lines using UAS-CsChrimson-mVenus? One reason is that using another transgene (such as UAS-GFP) and/or another genomic locus can drive a different expression pattern or intensities. Although this is discussed, this should be made more explicit and the readers should be aware of this.

Second, it would be important to include a discussion regarding the potential of hemidriver lines to suffer from transvection effects whenever there is a genetic element in the same locus. These are serious issues that prevent a more reliable use of split-GAL4 lines that, once again, should be discussed.

We added additional explanatory text to the discussion.

(2) The authors simply mention that the goal of the manuscript is to "summarize the results obtained over the past decade.". A better explanation would be welcomed in order to understand the need of a dedicated manuscript to announce the availability of a new batch of lines when previous publications already described the Split-GAL4 lines. At the extreme, one might question why we need a manuscript for this when a simple footnote on Janelia's website would suffice.

We added an additional mention of the cell type split-GAL4 collection at the relevant section and added more emphasis on the curation process adding value to the final selections. We feel that the manuscript is useful to document the methods used for the contained analysis and datasets and gives a starting point to the reader to go through the many split-GAL4 publications and images.

Reviewer #2 (Public Review):

Summary: This manuscript describes the creation and curation of a collection of genetic driver lines that specifically label small numbers of neurons, often just a single to handful of cell types, in the central nervous system of the fruit fly, Drosophila melanogaster. The authors screened over 77,000 split hemidriver combinations to yield a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These genetic driver lines have already contributed to several important publications and will no doubt continue to do so. It is a truly valuable resource that represents the cooperation of several labs throughout the Drosophila community.

Strengths:

The authors have thoughtfully curated and documented the lines that they have created, so that they may be maximally useful to the greater community. This documentation includes confocal images of neurons labeled by each driver line and when possible, a list of cell types labeled by the genetic driver line and their identity in an EM connectome dataset. The authors have also made available some information from the other lines they created and tested but deemed not specific or strong enough to be included as part of the collection. This additional resource will be a valuable aid for those seeking to label cell types that may not be included in the main collection.

Weaknesses:

None, this is a valuable set of tools that took many years of effort by several labs. This collection will continue to facilitate important science for years to come.

We thank the reviewer for their positive feedback.

Reviewer #3 (Public Review):

Summary:

The manuscript by Meissner et al. describes a collection of 3060 Drosophila lines that can be used to genetically target very small numbers of brain cells. The collection is the product of over a decade of work by the FlyLight Project Team at the Janelia Research Campus and their collaborators. This painstaking work has used the intersectional split-Gal4 method to combine pairs of so-called hemidrivers into driver lines capable of highly refined expression, often targeting single cell types. Roughly one-third of the lines have been described and characterized in previous publications and others will be described in manuscripts still in preparation. They are brought together here with many new lines to form one high-quality collection of lines with exceptional selectivity of expression. As detailed in the manuscript, all of the lines described have been made publicly available accompanied by an online database of images and metadata that allow researchers to identify lines containing neurons of interest to them. Collectively, the lines include neurons in most regions of both the adult and larval nervous systems, and the imaging database is intended to eventually permit anatomical searching that can match cell types targeted by the lines to those identified at the EM level in emerging connectomes. In addition, the manuscript introduces a second, freely accessible database of raw imaging data for many lower quality, but still potentially useful, split-Gal4 driver lines made by the FlyLight Project Team.

Strengths:

Both the stock collection and the image databases are substantial and important resources that will be of obvious interest to neuroscientists conducting research in Drosophila. Although many researchers will already be aware of the basic resources generated at Janelia, the comprehensive description provided in this manuscript represents a useful summary of past and recent accomplishments of the FlyLight Team and their collaborators and will be very valuable to newcomers in the field. In addition, the new lines being made available and the effort to collect all lines that have been generated that have highly specific expression patterns is very useful to all.

Weaknesses:

The collection of lines presented here is obviously somewhat redundant in including lines from previously published collections. Potentially confusing is the fact that previously published split-Gal4 collections have also touted lines with highly selective expression, but only a fraction of those lines have been chosen for inclusion in the present manuscript. For example, the collection of Shuai et al. (2023) describes some 800 new lines, many with specificity for neurons with connectivity to the mushroom body, but only 168 of these lines were selected for inclusion here. This is presumably because of the more stringent criteria applied in selecting the lines described in this manuscript, but it would be useful to spell this out and explain what makes this collection different from those previously published (and those forthcoming).

We added more description of how this collection is focused on the best cell-type-specific lines across the CNS. An important requirement for inclusion was this degree of specificity across the CNS, while many prior publications had a greater emphasis on lines with a narrower focus of specificity.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Luckily for us, genetics is for the most part an exact science. However, there's still some "voodoo" in a lot of genetic combinations that the authors should disclose and be as clear as possible in the manuscript. This allows for the potential users to gauge expectations and devise a priori alternative plans.

We attempted to comprehensively cover the caveats inherent in our genetic targeting approach.

Minor points:

(1) The authors mention that fly age should be controlled as expression can vary. Is there any reference to support this claim?

We added a reference describing driver expression changes over development.

(2) There should be a citation for "Flies were typically 1-5 days old at dissection for the cell type collection rescreening, 1-8 days old for other non-MCFO crosses and 3-8 days old for MCFO".

We clarified that these descriptions were of our experimental preparations, not describing other citable work.

Reviewer #3 (Recommendations For The Authors):

General Points:

Overall, the manuscript is very clear, but there are a couple of points where more explicit information would be useful. One of these is with respect to the issue of selectivity of targeting. The cell type specificity of lines is often referred to, but cell types can range from single pairs of neurons to hundreds of indistinguishable neurons with similar morphology and function. It would be useful if the authors explained whether their use of the term "cell type" distinguishes cell type from cell number. It would also be useful if lines that target many neurons of a single cell type were identified.

We added further discussion of cell types vs. cell numbers. Our labeling strategy was not optimized for counting cell numbers labeled by each line. We believe EM studies are best positioned to comprehensively evaluate the number of cells making up each type.

The second point relates to vagueness about the intended schedule for providing resources that will match (or allow matching of) neurons to the connectome. For example, on pp. 5-6 it is stated that: "In the future all of the neurons in these lines will be uniquely identified and linked to neurons reconstructed in the electron microscopy volume of the larva" but no timeline is provided. Similarly, for the adult neurons it is stated on p. 4 that: "Anatomical searching for comparison to other light microscopy (LM) and EM data is being made available." A more explicit statement about what resources are and are not yet available, a timeline for full availability, and an indication of how many lines currently have been matched to EM data would be helpful.

During the review and revision period we have made progress on processing the images in the collection. We updated the text with the current status and anticipated timeline for completion.

Specific Points:

p. 4 "Although the lines used for these comparisons are not a random sample, the areas of greatest difference are in the vicinity of previously described sexual dimorphisms..." In the vicinity of is a very vague statement of localization. A couple of examples of what is meant here would be useful.

We added example images to Figure 3.

p. 5 "...may have specific expression outside our regions of interest." It's not clear what "our regions of interest" refers to here. Please clarify.

We clarified that we were referring to the regions studied in the publications listed in Table 1.

p. 5 "...lines that were sparse in VNC but dirty in the brain or SEZ..." A more quantitative descriptor than "dirty" would be helpful.

We unfortunately did not quantify the extent of undesired brain/SEZ expression, but attempted to clarify the statement.

p. 6 "...the images are being made instantly searchable for LM and EM comparisons at NeuronBridge..." Here again it is hard to know what is meant by "being made instantly searchable." How many have been made searchable and what is the bottleneck in making the rest searchable?

We updated the text as described above. The bottleneck has been available processing capacity for the hundreds of thousands of included images.

Figure 1 Supplemental File 2: The movie is beautiful, but it seems more useful as art than as a reference. Perhaps converting it to a pdf of searchable images for each line would make it more useful.

We replaced the movie with a searchable PDF.

Fig. 2(B) legend: "Other lines may have more than two types." It is not clear what "other lines" are being referred to.

As part of making the quality evaluation more robust, we scored lines for the clear presence of three or more cell types. We updated the text accordingly.

Fig. 2(C): Presumably the image shown is an example of variability in expression rather than weakness, but it is hard to know without a point of comparison. Perhaps show the expression patterns of other samples? Or describe briefly in the legend what other samples looked like?

We added Figure 2 - figure supplement 1 with examples of variable expression in a split-GAL4 line.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

1. General Statements [optional]

We thank the reviewers for their insightful comments regarding our study and for appreciating the range of experiments used, the depth of our study and the significance of our work. We also thank reviewers with expertise in evolutionary biology for highlighting the need for precise wrong of some parts of the manuscript and the need for balancing the various viewpoints on the current understanding of early metazoan evolution. A point-by-point response to each reviewer comment is given below. We believe that we can effectively address most reviewer comments in a revised version. The revised improved manuscript will be the first insightful study of intracellular signalling pathways in the context of early animal evolution. We thank the reviewer for noting that this study is highly impactful and can have a broader influence on the scientific community.

2. Description of the planned revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

__ Summary: The researchers identified PIP4K (phosphatidylinositol 5 phosphate 4-kinase) as a lipid kinase that is specific to metazoans. In order to determine its conserved function across metazoans, they compared PIP4K activity in both early-branching metazoans and bilaterian animals. Biochemical assays demonstrated a conserved catalytic activity between the sponge Amphimedon queenslandica (AqPIP4K) and human PIP4K. In in-vivo experiments, AqPIP4K was found to rescue the reduced cell size, growth, and development phenotype in larvae of null mutant in Drosophila PIP4K. Based on these findings, the authors suggest that the function of PIP4K was established in early metazoans to facilitate intercellular communication. The experiments were well designed, and a range of biochemical, in vitro, and in vivo experiments were conducted.__

__ That being said, there are some questions that require further discussion before we can fully accept the author's conclusion of an evolutionarily conserved function of PIP4K across metazoans.__

Major comments:

• The authors mentioned that PIP4K is metazoan-specific and involved in intercellular communication. How can we explain the presence of PIP4K in choanoflagellate genomes? Despite its high similarity with conserved domains and functionally important residues, experimental results with the PIP4K from Choanoflagellate (Monosiga brevicollis, MbPIP4K) such as Mass spectrometry-based kinase assay and mutant Drosophila PIP4K didn't show similar activity to sponge AqPIP4K. The authors suggested that "In the context of other ancient PIP4K it is possible that since choanoflagellates exist as both single-cell and a transient multicellular state and do not have the characteristics of metazoans, PIP4K does not play any important functional role in these." However, this explanation is not well justified; they need to provide a more detailed discussion on this. Response: PIP4K is found in the genome of the choanoflagellate, M.brevicollis. MbPIP4K has the requisite kinase domain and the critical residue in the activation loop (A381) required for PIP4K activity is also conserved with the Amphimedon enzyme. Despite this, MbPIP4K was unable to rescue the growth and cell size phenotype of dPIP4K mutants (dPIP4K29) unlike AqPIP4K.

We have previously published a comparison of the in vitro activity versus in vivo function for the three PIP4K enzymes in the human genome (Mathre et.al PMID: __30718367). While all three human PIP4K isoforms can functionally rescue the Drosophila dPIP4K mutant, there is a nearly 104-fold difference for in vitro activity between them with PIP4K2C showing almost no in vitro activity. __The difference in in vitro enzyme activity between MbPIP4K and AqPIP4K is similarly notable. We would however highlight that this is more likely a reflection of the limitations of the in vitro PIP4K activity.

However, while AqPIP4K can rescue function in vivo (rescue of fly mutant phenotypes) MbPIP4K could not when expressed in fly cells. This must imply that there are differences in the polypeptide sequences of AqPIP4K and MbPIP4K that allow the former but not the latter to couple to the Insulin PI3K signalling pathway in fly cells. Given that Amphimedon and Choanoflagellates are separated by 100-150 Mya in evolution, this is possible. Our data on expression of AqPIP4K and MbPIP4K in fly S2 cells shows that they do not have equivalent localization (Fig 2C). What are the differences in the two polypeptides that lead to this? We will perform a multiple sequence alignment using PIP4K sequences from multiple choanoflagellates and sponges to identify these differences.

We will include the results of this analysis and an appropriate discussion in the revised manuscript.

• Likewise, the PIP4K gene has been identified in cnidarians, which are a sister group to bilaterian animals. However, the Cnidaria HvPIP4K showed no activity in biochemical or functional assays. In comparison to sponges, cnidarians are relatively complex organisms, and I believe that PIP4K is highly important for intercellular communication, as it is in bilaterians. The authors attempted to explain this by suggesting that "Based on theories of parallel evolution between cnidarians and sponges during early metazoan evolution, it is possible that the PIP4K gene was retained functional in one lineage and not in other." However, I am not convinced by this statement.

Response: This is a really interesting and challenging question from the reviewer. We are aware that both sponges (Porifera) and Cnidaria are examples of primitive metazoans separated by 80-90 Mya of evolution, yet while AqPIP4K shows activity and can functionally rescue dPIP4K mutants, HvPIP4K cannot. What does this mean?

A key difference between sponges and cnidarians is that while cnidarians have a simple “nerve-net” like nervous system, sponges do not have such a mode of communication. Therefore, it is possible that PIP4K, which we propose works in the context of hormone-based communication, is functionally important in sponges.

We are of course aware and acknowledge that in a like for like experimental system (Drosophila cells) our data shows that the two proteins behave differently, be it in terms of in vitro activity or in vivo function. This must imply inherent differences in the two polypeptides.

What we propose to do is to compare available PIP4K sequences from multiple Porifera and Cnidaria genomes and try and understand differences in the protein sequence that might explain differences in function. These results and their implications will be included in the revised manuscript.

• Please provide details of the databases (Uniprot-KB, NCBI sequence database, Pfam) versions. After identifying the specific PIP4K protein in each species (e.g. AqPIP4K and HvPIP4K), have you considered performing a reciprocal blast against the human genome to see if you have a top hit to PIP4K? Hence, the main focus of the project is on PIP4K as a metazoan-specific protein. We need to include a wider representation of non-bilaterian animals, including multiple species from sponges, ctenophores, placozoans, and cnidarians. Additionally, please check if homologues of PIP4K are present in other unicellular holozoans besides choanoflagellates. Response: We will add the NCBI IDs for all the sequences. We have carried out reciprocal blast to human proteome and then classified the selected sequences as PIP4K, we will add the results in the supplementary for the same. We will add more species of sponges, ctenophores, placozoans, and cnidarians in our analysis of PIP4K sequences. We will also include an analysis of other unicellular holozoans where genome sequence is available.

• Authors suggested the identification of other components of the PI signaling pathway along with PIP4k in the sponge. What is the status of these PI signaling pathway genes in other non-bilaterians and choanoflagellates? Response: We will add the details of the same in the revised manuscript and agree that this will help enhance the interpretation of our results.

• Phylogenetic tree of all PIP4K sequences (Figure 1C): How authors can be certain that the identified PIP4K sequences (e.g. AqPIP4K, HvPIP4K, and MbPIP4K) are indeed PIP4K, especially when there are several closely related proteins? It is important to conduct phylogenetic analysis alongside other PIP sequences (such as PI3K, PI4K, PIP5K, and PIP4K). If this analysis is carried out, the identified AqPIP4K, HvPIP4K, and MbPIP4K should be grouped together with human PIP4K in the same cluster. Response: As described in the methods, we have searched all the individual genomes analyzed for all PIK and PIPK enzyme sequences. We have marked the domains (using Pfam and Interpro) on these sequences and eliminated other PIK and PIPK sequences (such as PI3K, PI4K, PIP5K) and selected only PIP4K. To additionally confirm the distinction between PIP5K and PIP4K, we have manually inspected each sequence to establish the identity of the A381 amino acid residue in the activation loop. The identity of the amino acid at this position in the activation loop has been experimentally demonstrated to be an essential feature of PIP4K (Kunze et.al PMID: 11733501) and we have also confirmed this independently in a recent study (Ghosh et.al PMID: 37316298).

We will perform the phylogenetic analysis of the phosphoinositide kinases in the format suggested by the reviewer and add it in the revision as a supporting evidence.

Minor comments:

• Line 157: Phylogenetic conservation of PIP4Ks: Please provide details about bootstrap analysis. Response: Will be added

• Line 230: symbol correction 30{degree sign}C Response: Will be done

• Line 429-430: "from early metazoans like Sponges, Cnidaria and Nematodes." Nematodes are not considered early metazoans. Response: Apologies for the typo. This will be corrected. We agree that nematodes are not early metazoans.

• Line 477-478: "However, interestingly, MbPIP4K::GFP localizes only at the plasma membrane in S2 cells (Figure 2C)." This part was not further discussed. Can you please elaborate on why MbPIP4K::GFP localizes only at the plasma membrane in S2 cells? Response: We have discussed this point specifically in response to major comment by the reviewer and it will be addressed as described.

• Line 598: "the earliest examples of metazoa, namely the coral A. queenslandica" A. queenslandica is a sponge, not coral. Response: Apologies for the error. We will correct it.

• Line 602: "Amphimedon and human enzyme, although separated by 50Mya years of evolution" I think it's 500 million years ago, not 50 million years ago. Response: This typo will be corrected.

• Line 612: "coordinated communication between the cells is the most likely function" the cell. Response: Will change the sentence accordingly

• Line 614: "intracellular phosphoinositide signalling the identity of the hormone" missing full stop punctuation. Response: Will change the sentence accordingly

• Line 802 - 804: "other by way of difference in colour. The sub clusters have been numbered (1- early metazoans, 2- Nematodes, 3- Arthropods, 4- Molluscs, 5- Vertebrates (isoform PIP4K2C), 6- Vertebrates (isoform PIP4K2A), 7- Vertebrates (isoform PIP4K2B)." In the Figure, I can't find numbers on the subclusters. Response: Will add the numbers in the figure.

• Line 805- 807: "Phylogenetic analysis of selected PIP4K sequences from model organisms of interest. PIP4K from A. queenslandica has been marked in rectangular box." The rectangular box is missing in the figure. Response: Will change the figure accordingly

• Figure 1C: full forms of species names are missing. Response: Will change the figure accordingly

Reviewer #1 (Significance (Required):

The data is presented well, and the authors used a wide range of assays to support their conclusion. The study is highly impactful and can have a broader influence on the scientific community, particularly in evolutionary molecular biology, development, and biochemistry.

The study provides interesting findings; however, the reasons for PIP4K not being functional in cnidarians as in sponges and why PIP4K is present in unicellular holozoans but not functional are unclear.

We thank the reviewer for appreciating the significance and impact of our study. The very helpful questions raised by the reviewer will help enhance the quality of our study even further. We will make every effort to address these queries.

Reviewer #2 (Evidence, reproducibility and clarity (Required):

The manuscript by Krishnan et al. uses molecular phylogenetics, in vitro kinase assays, heterologous expression assays in Drosophila S2 cells and mutant complementation assays in yeast to study the evolution and function of putative PIP4 kinase genes from a sponge, a cnidarian and a choanoflagellate. Based on these experiments, the authors conclude that PIP4K is metazoan-specific and that the sponge PIP4K has conserved functions in selectively phosphorylating PI5P.

The study is in principle of interest and it could all be valid data, but the large number of flaws in the data presentation and/or analysis just makes it hard to assess the quality and thus validity of the data and conclusions.

We thank the reviewer for appreciating the potential interest in our findings of PIP4K function in early metazoans. We thank them for noting the need for correcting data presentation and these will be done in the revision.

__ Major comments:__

Overall, the manuscript lacks scientific rigor in the analysis and representation of the results, and the validity of many of the conclusions is therefore difficult to assess.

Major problems are:

(i) The authors base their study on the evolution of PIP4K genes on a deeply flawed concept of animal evolution. On multiple occasions, including the title, the authors refer to extant species (e.g. Amphimedon) as 'early metazoan', 'regarded as the earliest evolved metazoan' (l. 46-7) or 'the earliest examples of metazoans' just to name a few. This reflects a 'ladder-like' view on evolution that suggests that extant sponges are identical to early 'steps' of animal evolution.

We thank the reviewer who is clearly vastly more experienced in the field of evolutionary biology for the possible imprecise/incorrect usage of the word “ancient metazoan”. As new entrants to this area of evolutionary biology, we have of course referred to the existing literature such as PMID: 20686567 to guide us. This paper describes the sequencing of the A. queenslandica genome. It is clear that there is perceived value in studying this sponge in the context of early animal evolution although we are aware of there are a multitude of sponges and not all of them may be of value in the study of early animal evolution. We will peruse the literature more carefully and revise the manuscript to provide a more balanced view of this very interesting but unresolved area.

Also, the author's interpretation that one cluster of genes 'contained the sequences from early metazoans like sponges, cnidaria and nematodes' is referring to an outdated idea of animal phylogeny where nematodes were thought to be ancestrally simple organisms grouped as 'Acoelomata'. This idea of animal phylogeny was however disproven by molecular phylogenetics since the 1990ies.

Response: We are aware that the field of animal classification is undergoing continuous evolution. While earlier classifications may have been based of the presence or otherwise of a coelom and/or other anatomical features, we are aware of the use of molecular phylogenetics.

The phylogeny presented in Fig 1C is based on the sequence relationships between the PIP4K sequences from various animal genomes. Any errors in the labelling of groups such as that highlighted by the reviewer will be revised or corrected after a careful consideration of extant views in the field, which are somewhat varied.

(ii) The description of taxa in the phylogenetic tree in Fig. 1B lacks any understanding of phylogenetic relationships between animals and other eukaryotic groups. What kind of taxa are 'invertebrates' or 'parasites'? And why would 'invertebrates' exclude cnidarians and sponges? Also, why is the outgroup of opisthokonts named 'Eukaryota'?? Are not all organisms represented on the tree eukaryotes?

Response: We apologize for this imprecision in labelling taxa. This will be corrected.

(iii) The methods part lacks any information about the type of analysis (ML, Bayesian, Parsimony?) used to perform the phylogenetic analysis shown in Fig. 1C. Also, the authors mention three distinct clusters (l.428) that are not labelled in the figure.

Response: We will update the methods to include the additional details requested by the reviewer. Fig 1C will be re-labelled.

(iv) The validity of the Western Blot is difficult to assess as the authors have cut away the MW markers. Without, it is for example difficult to assess the size differences visible between Hydra and Monosiga PIP4K-GFP proteins on Fig. 2B. Also, it has become standard practice to show the whole Western blot as supplementary data in order to assess the correct size of the bands and the specificity of the antibody. This is also missing from this manuscript.

Response: Cropped Western blots have been shown to facilitate figure preparation in the main manuscript. The complete uncropped Western blots, in all cases, will be shared as Source data as is the standard practice for multiple journals in the review Commons portfolio.

(v) The authors claim that AqPIP4K was able to convert PI3P into PI with very low efficiency (Figure 2E), but without further label in the figure or explanation, it remains unclear how the authors come to this conclusion.

Response: We regret the typo in line 500 of the manuscript we have stated that “Further,……… was able to convert PI3P into PI with very low efficiency (Figure 2E).” What we intended to write was “Further,……… was able to convert PI3P into PI (3,4) P2 with very low efficiency (Figure 2E).” The efficiency with which this reaction takes place is very low and has been reported by us (Ghosh et.al PMID: 31652444) and others (Zhang et.al PMID: 9211928). At the exposure of the TLC shown in Fig 2E the PI(3,4)P2 spot cannot been seen. Much longer exposures of the TLC plate will be needed to see the PI(3,4)P2 spot. This will be corrected in a revised version of the manuscript.

(vi) The box plots in Fig. 3C and D lack error bars and thus seem to be consisting of only single data points without replicates. Also, Fig. 3C is a quantification of Fig. 3B but it remains unclear what has been quantified and how. It is also unclear how %PIP2 was determined.

Response: For Fig 3C, the colony count has been done from three replicates and the average has been considered to calculate the % growth for each genotype. We will include error bars and clarify this in the revised figure legend. For Fig 3D, the PIP2/PIP ratio has been calculated from biological replicates and average has been represented in the graphs. The individual values can be provided as supplementary data.

(vii) Throughout Fig. 4, I do not understand the genotypes indicated on the x-axis of the plots and below the images. I read the figure legends and manuscript describing these results at least 3 times, but cannot figure out what it all means. On Fig. 4C, what is the wild-type situation?

Response: We apologize for the lack of precision in labelling the figures versus the figure legends. This will be corrected in the revision:

The genotypes are as follows

• w1118 (control) * Act-GAL4. This has been referred to as wild type in the figure legend and called Act-Gal4 in Fig4 panels A-E
• dPIP4K29 – This refers to the protein null strain of dPIP4K. This strain is the background in which all reconstitutions of PIP4K genes have been done.
• PIP4K transgene from A. queenslandica.
• AqPIP4KKD Kinase dead PIP4K transgene from * queenslandica. In panels A, B, D and E, Act-GAL4: dPIP4K29* indicates the genetic background in which either AqPIP4K or AqPIP4KKD has been reconstituted.

Reviewer #2 (Significance (Required)):

If validated and put in the right phylogenetic context, the study is potentially contributing to expanding our knowledge on the evolution of metazoan-specific features, especially the evolution of proteins involved in cell-cell signalling and growth control. My field of expertise is broadly in evo-devo, molecular phylogentics, developmental genetics and cell biology. The in vitro lipid analysis seems interesting and potentially valid but I do not have sufficient expertise to evaluate its validity.

We thank the reviewer for appreciating the novelty of our contribution and its potential to contribute to understanding the evolution of metazoan specific signalling systems, once appropriate corrections have been made. We also appreciate their positive comment on our in vitro experimental analysis. This paper is a big effort to not only perform phylogenetic analysis but address the emerging interpretations experimentally as much as possible.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary In this manuscript, the authors investigate the evolutionary origins of metazoan Phosphatidylinositol phosphates (PIPs) signaling by elucidating the sequence and function of the PIP4K enzyme, which is crucial for converting PI5P to PI(4,5)P2 through phosphorylation. The authors have described PIP4K-like sequences distributed throughout metazoans and choanoflagellates through an extensive sequence screening. With in vitro and in vivo functional assays, the authors have shown that the sponge A. queenslandica PIP4K (AqPIP4K) is functionally similar to its human counterpart and highlight the major discovery of this study - that PIP4K protein function dates back to as early as sponges.

We thank the reviewer for noting the major finding of our study and our efforts to experimentally validate, using multiple approaches, the findings of our detailed bioinformatics analysis of PIP4K gene distribution across the tree of life.

Major comments

There are two key limitations to this paper. Like the sponges, ctenophores are one of the earliest branching metazoans. They are not well addressed in the paper. Secondly, despite finding PIP4 homologs in choanoflagellates, the authors claim that PIP4 is metazoan-specific.

We thank the reviewer for highlighting these two points; we recognize that both of these are important to address, to the extent that it is possible to do so. These will be addressed using the approaches detailed in the response to reviewer 1 comments.

1. Line 46: A. queenslandica is the earliest branching metazoan. The phylogeny of sponges and ctenophores is not conclusively defined and hence, the statement must be rephrased. Despite the brief description of the evolution of metazoan lineage in the discussion section, ctenophores are missing from the phylogenetic tree. At least a sequence-level information PIP4K in ctenophores would strongly back the claims of the manuscript. Here is the link to the Mnemiopsis database. Response: We thank the reviewer for highlighting this point and pointing us to the Mnemiopsis database. We will most certainly analyse ctenophore genome sequences and add the ctenophore PIP4K sequence to the phylogeny, post analysis and the discussion will be modified to reflect the findings.

Mentioning that choanoflagellates contain homologs of PIP4K contradicts the statement that PIP4K is metazoan-specific. As per Fig 1E., the domain organization of PIP4K is conserved among choanoflagellates and metazoans. What is the percent sequence similarity to the query? This could answer why it doesn't show activity in Drosophila rescues - the system might simply not be compatible with the choanoflagellate homolog. The same may apply to the cnidarian homolog HvPIP4K. Further evidence is needed before concluding that MbPIP4K doesn't phosphorylate PIP5. It is additionally fascinating that MbPIP4K localizes at the plasma membrane unlike other homologs - this function might be choano-specific. Overall, PIP4K's possible origin in the choanoflagellate-metazoan common ancestor backs the current research that choanoflagellates indeed hold clues to understanding metazoan evolution. Further research is necessary before concluding (as in line 648) in the discussions section, where it is mentioned that "PIP4K does not play any important functional role in choanos".

Response: We thank the reviewer for highlighting the very interesting but incompletely understood facets of our study vis-à-vis choanoflagellates versus metazoans. The proposal for additional analysis is indeed interesting and we will carry out these analysis and revise the text accordingly.

__ Minor Comments__

1. A detailed comparison of the sequence of the hydra PIP4K might help understand why it may not have worked like the sponge PIP4K. The discussion on the cnidarian PIP4K evolution is not convincing. It may not have worked because of it being expressed in a non-natural system. Structure prediction and comparison of proteins from different early branching animals should be used. Response: Thank you for these suggestions to understand why the cnidarian PIP4K may not have been functional. We will perform the suggested analysis and incorporate the data into the revision.

78 - Multicellularity evolved many times. Maybe say 'first evolved metazoans'

Response: Thank you for the suggestion.

Line 598 A. queenslandica is not a coral, it's a sponge.

Response: Text will be changed accordingly

Line 612 'thcells' à 'the cells'

Response: Text will be changed.

Line 623 - full stop missing after metazoans.

Response: Text will be changed

Figure 1B - Classification should be consistent - C. elegans is a species name, whereas ctenophores and vertebrates belong to a different classification. Invertebrates is not a scientific group. The edges of the lines of the phylogenetic tree don't join and they need to be arranged correctly.

Response: The names in the phylogeny will be changed to maintain uniformity. The representation of the phylogeny will be changed as mentioned.

Figure 2B The full blot could be shown in the supplement.

Response: Full blot will be provided as source data on resubmission or included as supplementary based on the destination journal’s specification.

Optional

1. Heterologous overexpression does not always provide the full picture of the gene functionality. To make claims on the evolution of function, testing gene functions homologous systems can give a better picture. For example, performing in vitro kinase activity assays of MbPIP4K after overexpressing PIP4K in Monosiga brevicollis. would be a great. Data is missing also about the presence and function of ctenophore PIP4K. Overexpression of ctenophore-PIP4K in Drosophila for functional analyses could help in understanding the distribution/diversity of function of PIP4K in early animals. Response: We agree with the reviewer that heterologous expression may sometimes not replicate the biochemical environment of cells in the organism from which the gene being expressed was originally derived. Yet, heterologous expression experiments do sometimes provide an insight into properties solely dependent on the polypeptide with limited or no contribution from the cellular environment. In principle expressing PIP4K in M.brevicollis cells and then performing kinase assays would be a very good idea. However, we would like to highlight that till date there has been only one study where septins have been transfected in Choanoflagellates and their localization being observed. We are not set up to culture M. brevicollis and will be unable to do this for a revision of the current manuscript. However, we appreciate the importance of this experiment and will do this in collaboration with a choanoflagellate lab in a follow up study to this one.

Ctenophores like cnidarians have two main layers of cells that sandwich a middle layer of jelly-like material, while, more complex animals have three main cell layers and no intermediate jelly-like layer. Hence ctenophores and cnidarians have traditionally been labelled diploblastic. Studies have shown that ctenophores and unicellular eukaryotes share ancestral metazoan patterns of chromosomal arrangements, whereas sponges, bilaterians, and cnidarians share derived chromosomal rearrangements. Conserved syntenic characters unite sponges with bilaterians, cnidarians, and placozoans in a monophyletic clade while ctenophores are excluded from this clustering, placing ctenophores as the sister group to all other animals. Ctenophore PIP4K sequence can be identified and compared as discussed before to other PIP4K sequences used in this study.

Reviewer #3 (Significance (Required)):

Significance: This is the first study that addresses PIP signaling pathway in early metazoans. The findings of this manuscript contribute to the understanding of second-messenger signaling and its link with the origin and evolution of metazoan multicellularity. PIP signaling is crucial in different metazoan aspects such as cytoskeletal dynamics, neurotransmission, and vesicle trafficking, and hence, plays a critical role in metazoan multicellularity. Through this study, it was interesting to see that some components of the PIP signaling pathway are conserved in yeast, but some, such as the PIP4K protein evolved at the brink of metazoan evolution, highlighting the need for complexity in metazoans and their close relatives - the facultatively multicellular choanoflagellates. Since this is a crucial pathway in human biology and has medical significance due to its role in tumorigenesis and cancer cell migration, this study serves the audience in basic research such as evolutionary biology, and applied research such as human medicine. My field of expertise is molecular biology, cell biology and microbiology, with specific expertise on choanoflagellates. Therefore, it is exciting to see the homologs of PIP4K present in choanoflagellates.

__ Evidence, Reproducibility, and clarity:__

The authors have made a clear case of why PIP4K needs to be studied. They have thoroughly mapped PIP4K throughout the tree of life. The results are clear and reproducible. With the findings of this study, they have linked the PIP signalling cascade and metazoan evolution. Using the heterologous expression of sponge A. queensladica PIP4K, they have made compelling evidence that AqPIP4K functions in PIP5 phosphorylation, as seen in humans and Drosophila. However, it was not convincing why the hydra PIP4K was not functional. It was also not convincing why the PIP4K is metazoan-only when there is a conserved sequence (with conserved domain structure) present in choanoflagellates.

We thank the reviewer for appreciating the novelty and importance of our findings in multiple areas of basic biology related to early metazoans and basic biomedical sciences. We also note their comments on the clear and reproducible results presented. Points raised related to the lack of functionality of PIP4K from Hydra and choanoflagellates are noted and will be addressed as indicated in response to other reviewer comments.

Experiments/Analysis to be done

1. We will perform a multiple sequence alignment using PIP4K sequences from multiple choanoflagellates and sponges to identify these differences.
2. What we propose to do is to compare available PIP4K sequences from multiple Porifera and Cnidaria genomes and try and understand differences in the protein sequence that might explain differences in function.
3. We will add more species of sponges, ctenophores, placozoans, and cnidarians in our analysis of PIP4K sequences. We will also include an analysis of other unicellular holozoans where genome sequence is available.
4. We will perform the phylogenetic analysis of the phosphoinositide kinases in the format suggested by the reviewer and add it in the revision as a supporting evidence.
5. Structure prediction and comparison of proteins from different early branching animals should be used.
6. Uniformity of terminology and alignment with conventions in the field of animal taxonomy
7. NCBI ID of sequences to be added and include more non-bilaterian animals sequences in phylogeny- redo the phylogeny.
8. Check for PI signalling genes in choanoflagellates
9. More detailed description of phylogenetic analysis.
10. Add complete Western blot as source data.
11. *

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

• *

4. Description of analyses that authors prefer not to carry out

• Expression of PIP4K in choanoflagellates and in vitro kinase assays with lysates. It is beyond our technical ability to perform these experiments at this stage.

Reviewer #2 (Public review):

Summary:

Yonk and colleagues show that the posterior medial thalamus (POm), which is interconnected with sensory and motor systems, projects directly to major categories of neurons in the striatum, including direct and indirect pathway MSNs, and PV interneurons. Activity in POm-striatal neurons during a sensory-based learning task indicates a relationship between reward expectation and arousal. Inhibition of these neurons slows reaction to stimuli and overall learning. This circuit is positioned to feed salient event activation to the striatum to set the stage for effective learning and action selection.

Strengths:

The results are well presented and offer interesting insight into an understudied thalamostriatal circuit. In general, this work is important as part of a general need for an increased understanding of thalamostriatal circuits in complex learning and action selection processes, which have generally received less attention than corticostriatal systems.

Weaknesses:

There could be a stronger connection between the connectivity part of the data - showing that POm neurons context D1, D2, and PV neurons in striatum but with some different properties - and the functional side of the project. One wonders whether the POm neurons projecting to these subtypes or striatal neurons have unique signaling properties related to learning, or if there is a uniform, bulk signal sent to striatum. This is not a weakness per se, as it's reasonable for these questions to be answered in future papers.

All the in vivo activity-related conclusions stem from data from just 5 mice, which is a relatively small sample set. Optogenetic groups are also on the small side.

Comments on revisions:

The revision has a lot of thoughtful discussion added. I think overall the paper is more thorough and will also be a nice set up for a number of future research questions.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work aims to understand the role of thalamus POm in dorsal lateral striatum (DLS) projection in learning a sensorimotor associative task. The authors first confirm that POm forms "en passant" synapses with some of the DLS neuronal subtypes. They then perform a go/no-go associative task that consists of the mouse learning to discriminate between two different textures and to associate one of them with an action. During this task, they either record the activity of the POm to DLS axons using endoscopy or silence their activity. They report that POm axons in the DLS are activated around the sensory stimulus but that the activity is not modulated by the reward. Last, they showed that silencing the POm axons at the level of DLS slows down learning the task.

The authors show convincing evidence of projections from POm to DLS and that POm inputs to DLS code for whisking whatever the outcome of the task is. However, their results do not allow us to conclude if more neurons are recruited during the learning process or if the already activated fibres get activated more strongly. Last, because POm fibres in the DLS are also projecting to S1, silencing the POm fibres in the DLS could have affected inputs in S1 as well and therefore, the slowdown in acquiring the task is not necessarily specific to the POm to DLS pathway.

We thank the reviewer for these constructive comments. The points are addressed below.  

Strengths:

One of the main strengths of the paper is to go from slice electrophysiology to behaviour to get an in-depth characterization of one pathway. The authors did a comprehensive description of the POm projections to the DLS using transgenic mice to unambiguously identify the DLS neuronal population. They also used a carefully designed sensorimotor association task, and they exploited the results in depth.

It is a very nice effort to have measured the activity of the axons in the DLS not only after the mice have learned the task but throughout the learning process. It shows the progressive increase of activity of POm axons in the DLS, which could imply that there is a progressive strengthening of the pathway. The results show convincingly that POm axons in the DLS are not activated by the outcome of the task but by the whisker activity, and that this activity on average increases with learning.

Weaknesses:

One of the main targets of the striatum from thalamic input are the cholinergic neurons that weren't investigated here, is there information that could be provided?

This is true of the parafascicular (Pf) thalamic nucleus, which has been well studied in this context. However, there is much less known about the striatal projections of other thalamic nuclei, including POm, and their inputs to cholinergic neurons. Anatomical tracing evidence from Klug et al. (2018), which mapped brain-wide inputs to striatal cholinergic (ChAT) interneurons, suggests that Pf provides the majority of thalamic innervation of striatal ChAT neurons compared to other thalamic nuclei. Many other thalamic nuclei, including POm, showed very little of no labeling, suggesting weak innervation of ChAT interneurons. However, it is possible that these thalamic nuclei, including POm, do provide functional innervation of ChAT interneurons that is not sufficiently assessed by anatomical tracing. Understanding the innervation patterns of POm-striatal projections beyond the three cell types we have studied here would be an important area of further study.

It is interesting to know that the POm projects to all neuronal types in the DLS, but this information is not used further down the manuscript so the only take-home message of Figure 1 is that the axons that they image or silence in the DLS are indeed connected to DLS neurons and not just passing fibres. In this line, are these axons the same as the ones projecting to S1? If this is the case, why would we expect a different behaviour of the axon activity at the DLS level compared to S1?

Tracing of single POm axons by Ohno et al. (2012) indicated that POm axons form a branched collateral that innervates striatum, while the main axon continues in the rostral-dorsal direction to innervate cortex. We think it is reasonable, based on the morphology, that our optogenetic suppression experiment restricted the suppression of glutamate release to this branch and avoided the other branches of the axon that project to cortex. However, testing this would require monitoring S1 activity during the POm-striatal axon suppression, which we did not do in this study.

It is a very interesting question whether there could be different axon activity behavior in striatum versus S1. There is surprising evidence that POm synaptic terminals are different sizes in S1 and M1 and show different synaptic physiological properties depending on these cortical projection targets (Casas-Torremocha et al., 2022). Based on this, it is possible that POm-striatal synapses show distinct properties compared to cortex; however, this will need to be tested in future work.

The authors used endoscopy to measure the POm axons in the DLS activity, which makes it impossible to know if the progressive increase of POm response is due to an increase of activity from each individual neuron or if new neurons are progressively recruited in the process.

This is a good point. It would be necessary to perform chronic two-photon imaging of POm neurons (or chronic electrophysiological recordings) to determine whether the activity of individual neurons increased versus whether individual neuron activity levels remained similar but new neurons became active with learning. Even under baseline conditions, it is not known in detail what fraction of the population of POm neurons is active during sensory processing or behavior, highlighting how much is still to be discovered in this exciting area of neuroscience.

The picture presented in Figure 4 of the stimulation site is slightly concerning as there are hardly any fibres in neocortical layer 1 while there seems to be quite a lot of them in layer 4, suggesting that the animal here was injected in the VB. This is especially striking as the implantation and projection sites presented in Figures 1 and 2 are very clean and consistent with POm injection.

Although this image was selected to demonstrate the position of the POm injection site and optical fiber implant above striatal axons, the reviewer is correct that there appears to be mixed labeling of axons in L4 and L5a. In some cases, there was expression slightly outside the border of POm (see Fig. 1B, right), which might explain the cortical innervation pattern in this figure. While cortically bound VPM axons pass through the striatum, they do not form synaptic terminals until reaching the cortex (Hunnicutt et al., 2016). If, as may be the case, inhibitory opsins suppress release of neurotransmitter at synaptic terminals more effectively than action potential propagation in axons, it may be likely that optogenetic suppression of POm-striatal terminals is more effective than suppression of action potentials in off-target-labelled VPM axons of passage. Ideally, we could compare effects of suppression of POm-striatal synapses with POm-cortical synapses and VPM-cortical synapses, but this was outside the bandwidth of the present study.

Reviewer #2 (Public Review):

Summary:

Yonk and colleagues show that the posterior medial thalamus (POm), which is interconnected with sensory and motor systems, projects directly to major categories of neurons in the striatum, including direct and indirect pathway MSNs, and PV interneurons. Activity in POm-striatal neurons during a sensory-based learning task indicates a relationship between reward expectation and arousal. Inhibition of these neurons slows reaction to stimuli and overall learning. This circuit is positioned to feed salient event activation to the striatum to set the stage for effective learning and action selection.

Strengths:

The results are well presented and offer interesting insight into an understudied thalamostriatal circuit. In general, this work is important as part of a general need for an increased understanding of thalamostriatal circuits in complex learning and action selection processes, which have generally received less attention than corticostriatal systems.

Weaknesses:

There could be a stronger connection between the connectivity part of the data - showing that POm neurons context D1, D2, and PV neurons in the striatum but with some different properties - and the functional side of the project. One wonders whether the POm neurons projecting to these subtypes or striatal neurons have unique signaling properties related to learning, or if there is a uniform, bulk signal sent to the striatum. This is not a weakness per se, as it's reasonable for these questions to be answered in future papers.

We are very interested to understand the potentially distinct learning-related synaptic and circuit changes that potentially occur at the POm synapses with D1- and D2-SPNs and PV interneurons, and other striatal cell types. We agree that this would be an important topic for further investigation.

All the in vivo activity-related conclusions stem from data from just 5 mice, which is a relatively small sample set. Optogenetic groups are also on the small side.

We appreciate this point and agree that higher N can be important for observing robust effects. A factor of our experiments that helped reduce the number of animals used was the longitudinal design, with repeated measures in the same subjects. This allowed for the internal control of comparing learning effects in the same subject from naïve to expert stages and therefore increased robustness. Even with relatively small group sizes, results were statistically significant, suggesting that the use of more mice was unnecessary, which we considered consistent with best practice in the use of animals in research. We also note that our group sizes were consistent with other studies in the field.  

Reviewer #3 (Public Review):

Yonk and colleagues investigate the role of the thalamostriatal pathway. Specifically, they studied the interaction of the posterior thalamic nucleus (PO) and the dorsolateral striatum in the mouse. First, they characterize connectivity by recording DLS neurons in in-vitro slices and optogenetically activating PO terminals. PO is observed to establish depressing synapses onto D1 and D2 spiny neurons as well as PV neurons. Second, the image PO axons are imaged by fiber photometry in mice trained to discriminate textures. Initially, no trial-locked activity is observed, but as the mice learn PO develops responses timed to the audio cue that marks the start of the trial and precedes touch. PO does appear to encode the tactile stimulus type or outcome. Optogenetic suppression of PO terminals in striatum slow task acquisition. The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing.

A great strength of this paper is its timeliness. Thalamostriatal processing has received almost no attention in the past, and the field has become very interested in the possible functions of PO. Additionally, the experiments exploit multiple cutting-edge techniques.

There seem to be some technical/analytical weaknesses. The in vitro experiments appear to have some contamination of nearby thalamic nuclei by the virus delivering the opsin, which could change the interpretation. Some of the statistical analyses of these data also appear inappropriate. The correlative analysis of Pom activity in vivo, licking, and pupil could be more convincingly done.

The bigger weakness is conceptual - why should striatal circuitry need "priming" by the thalamus in order to process sensory stimuli? Why would such circuitry even be necessary? Why is a sensory signal from the cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? It is unclear from the experiments that the thalamostriatal pathway exists for priming sensory processing. In fact, the optogenetic suppression of the thalamostriatal pathway seems to speak against that idea.

We thank the reviewer for these constructive comments. The points are addressed below.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

Do POm neurons innervate CINs also? The connection between the PF thalamus and CINs is mentioned in a couple of places - one question is how unique are the input patterns for the POm versus adjacent sensorimotor thalamic regions, including the PF? This isn't a weakness per se but knowing the answer to that question would help in forming a more complete picture of how these different thalamostriatal circuits do or do not contribute uniquely to learning and action selection.

Anatomical tracing evidence from Klug et al. (2018), which mapped brain-wide inputs to striatal cholinergic (ChAT) interneurons, suggests that Pf provides the majority of thalamic innervation of striatal ChAT neurons compared to other thalamic nuclei. Many other thalamic nuclei, including POm, showed very little or no labeling, suggesting weak innervation of ChAT interneurons. However, it is possible that these thalamic nuclei, including POm, do provide functional innervation of ChAT interneurons that is not sufficiently assessed by anatomical tracing.

Another difference between Pf and other thalamic nuclei (likely including POm) comes from anatomical tracing evidence (Smith et al., 2014; PMID: 24523677) which indicates that Pf inputs form the majority of their synapses onto dendritic shafts of SPNs, while other thalamic nuclei form synapses onto dendritic spines. Understanding the innervation patterns of POm-striatal projections beyond the three cell types we have studied here, including ChAT neurons and subcellular localization, would be an important area of further study.

It would be useful to know to what extent these POm-striatum neurons are activated generally during movement, versus this discrimination task specifically.

We agree that distinguishing general movement-related activity from task-specific activity would be very useful. Earlier work (Petty et al., 2021) showed a close relationship between POm neuron activity, spontaneous (task-free) whisker movements, and pupil-indexed arousal in head-restrained mice. Oram et al. (2024; PMID: 39003286) recently recorded VPM and POm in freely moving mice during natural movements, finding that activity of both nuclei correlated with head and whisker movements. These studies indicate that POm is generally coactive with exploratory head and whisker movements.

During task performance, the situation may change with training and attentional effects. For example, Petty and Bruno (2024) (https://elifesciences.org/reviewed-preprints/97188) showed that POm activity correlates more closely with task demands than tactile or visual stimulus modality. Our data indicate that POm axonal signals are increased at trial start during anticipation of tactile stimulus delivery and through the sensory discrimination period, then decrease to baseline levels during licking and water reward collection (Fig. 3). Results of Petty and Bruno (2024) together with ours suggest that POm is particularly active during the context of behaviorally relevant task performance. Thus, we think it is likely that, while pupil dilation indexes general movement and arousal, POm activity is more specific to movement and arousal associated with task engagement and behavioral performance. We have strengthened this point in the Discussion.

Many of the data panels and text for legends/axes are quite small, and the stroke on line art is quite faint - overall figures could be improved from a readability standpoint.

We thank the reviewer for their careful attention to the figures. 

Reviewer #3 (Recommendations For The Authors):

Major

(1) Page 4, the Results regarding PSP and distance from injection site. The r-squared is the wrong thing to look at to test for a relationship. One should look at the p-value on the coefficient corresponding to the slope. The p-value is probably significant given the figures, in which case there may be a relationship contrary to what is stated. All the low r-squared value says is that, if there is a relationship, it does not explain a lot of the PSP variability.

We thank the reviewer for alerting us this oversight. We have included the p value (p = 0.0293) in the figure and legend, and indicated that the relationship is “small but significant”.

(2) Figure 1B suggests that the virus injections extend beyond POm and into other thalamic structures. Do any of the results change if the injections contaminating other nuclei are excluded from the analysis? I am not suggesting the authors change the figures/analyses. I am simply suggesting they double-check.

We selected for injections that were predominantly expressing in POm as determined by post-hoc histological analysis (see Fig. 1, right). As above, we think that axons of passage that do not form striatal synapses are less likely to be suppressed than axons with terminals; however, this would need to be determined in further experiments. Because the preponderance of expression is within POm, we think the results would be similar even with a stricter selection criterion. 

(3) The authors conclude that POm and licking are not correlated (bottom of page 6 pertaining to Figures 3A-F). The danger of these analyses is that they assume that GCaMP8 is a perfect linear reporter of POm spikes. The reliability of GCaMP8 has been quantified in some cell types, but not thalamic neurons, which have relatively higher firing rates.

The reviewer is correct that the relationship between GCaMP8 fluorescence changes and spiking has not been sufficiently characterized in thalamic neurons, and that this would be important to do.

What if the indicator is simply saturated late into the trial (after the average reaction time)? It would look like there is no response and one would conclude no correlation, but there could be a very strong correlation.

While saturation is worthy of concern, the signal dynamics here argue against this possibility. The reason is that the signal increased in the early part of the trial and decreased by the end. If saturation was an issue, this would have been apparent during the initial increase. When the signal decreased in amplitude at the end of the trial, this indicates that the signal is not saturated because it is returning from a point closer to its maximum (and is becoming less saturated).

Also, what happens between trials? Are the correlations the same, stronger, weaker? Ideally, the authors would analyze the data during and between trials.

Between trials the signal did not show further changes in baseline beyond what was displayed at the start and end of behavioral trials. There were no consistent increases or decreases in signals between trials, except perhaps during strong whisking bouts. This is anecdotal because we did not analyze between-trial data. However, it is interesting and important to note that signals increased dramatically in amplitude from naïve, early learning to expert behavioral performance (Fig. 3), highlighting that POm-axonal signals relate to behavioral engagement and performance rather than spontaneous behaviors.  

(4) Axonal activity could also appear more correlated with the pupil than licking because pupil dynamics are slow like the dynamics of calcium indicators. These kernels could artificially inflate the correlation. Ideally, the authors could consider these temporal effects. Perhaps they could deconvolve the temporal profiles of calcium and pupil before correlating? Or equivalently incorporate the profiles into their analysis?

We analyzed the lick probability histograms, which had a temporal profile similar to the calcium signals (Fig. 3D,E), ruling out concerns about effects of temporal effects on correlations. It is also worth noting that we observed changes in correlations between calcium signals and pupil with learning stage (Fig. 3I), even though the temporal profiles (signal dynamics) are not changing. Thus, temporal effects of the signals themselves are not the driver of correlations, but rather the changes in relative timing between calcium signals and pupil, as occur with learning.

(5) The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing. The data here support the first part. It is not clear that the data support the second part, largely because it is vague what "priming" of sensory processing or "a key role in the initial stages of action selection (p.9) even means here. Why would such circuitry even be necessary? Why is a sensory signal from the cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? Some conceptual proposals from the authors, even if speculative and not offered as a conclusion, would be helpful.

We appreciate these good points and have added further consideration and revision of the concept of priming and potential roles in an extensively revised Discussion section.

(6) The photometry shows that PO turns on about 2 seconds before the texture presentation. PO's activity seems locked to the auditory cue, not the texture (Figure 2). This means that the attempt to suppress the thalamostriatal pathway with JAWS (Figure 4) is rather late, isn't it? Some PO signals surely go through. This seems to contradict the idea of priming above. It would be good if the authors could factor this into their narrative. Perhaps labelling the time of the auditory cue in Figure 4C would also be helpful.

The start of texture presentation (movement of the texture panel toward the mouse) and auditory cue occur at the same time. To clarify this, we added a label “start tone” in Figure 4C and also in Figure 2C.

For optogenetic (JAWS) suppression, we intentionally chose a time window between start tone onset and texture presentation, because our photometry experiments showed that this was when the preponderance of the signal occurred. However, the reviewer is correct that our chosen optogenetic suppression (JAWS) onset occurs shortly after the photometry signal has already started, potentially leaving the early photometry signal un-suppressed. Our motivation for choosing a restricted time window surrounding the texture presentation time was 1) to minimize illumination and potential heating of brain tissue; 2) to target a time window that avoids the auditory cue but covers stimulus presentation. We did not want to extend the duration of the suppression to before the trial started, because this could produce task-non-specific effects, such as distraction or loss of attention before the start of the trial.

Even if some signal were getting through before suppression, we don’t think this contradicts the possibility of ‘priming’, because the process underlying priming would still be disrupted even if not totally suppressed. This would alter the temporal relationship between POm-striatal inputs and further corticostriatal inputs (from S1 and M1 cortex, for example). We have included further consideration of these points and possible relation to the priming concept in the Discussion.

Minor

(1) Page 5, "the sensitivity metric is artificially increased". What do you mean "artificially"? The mice are discriminating better. It is true that either a change in HR or FAR can cause the sensitivity metric to change, but there is nothing artificial or misleading about this.

We removed the word artificial and clarified our definition of behaviorally Expert in this context:

“Mice were considered Expert once they had reached ≥ 0.80 Hit Rate and ≤ 0.30 FA Rate for two consecutive sessions in lieu of a strict sensitivity (d’) threshold; we found this definition more intuitive because d’ is enhanced as Hit Rate and FA Rate approach their extremes (0 or 1)”

(2) Page 7, "Upon segmentation (Figure S4G-J)". Do you mean "segregation by trial outcome"?

Corrected.

(3) Page 9, "POm projections may have discrete target-specific functions, such that POm-striatal inputs may play a distinct role in sensorimotor behavior compared to POm-cortical inputs". Would POm-cortical inputs not also be sensorimotor? The somatosensory cortex contains a lot of corticostriatal cells. It also has various direct and indirect links to the motor cortex as well.

We have clarified the wording here to convey the possibility that POm signals could be received and processed differently by striatal versus cortical circuitry, and have moved this statement to later in the discussion for better elaboration.

(4) The Methods state that male and female mice were used. Why not say how many of each and whether or not there are any sex-specific differences?

We added the following information to the Methods:

The number of male and female mice were as follows, by experiment type: 6 male, 4 female (electrophysiology); 3 male, 2 female (fiber photometry); 4 male, 5 female (optogenetics). Data were not analyzed for sex differences.

Reviewer #2 (Public review):

Summary:

This paper aimed to determine the role EP sst+ neurons play in a probabilistic switching task.

Strengths:

- The in vivo recording of the EP sst+ neurons activity in the task is one of the strongest parts of this paper. Previous work had recorded from the EP-LHb population in rodents and primates in head fixed configurations, the recordings of this population in a freely moving context is a valuable addition to these studies and has highlighted more clearly that these neurons respond both at the time of choice and outcome.

- The use of a refined intersectional technique to record specifically the EP sst+ neurons is also an important strength of the paper. This is because previous work has shown that there are two genetically different types of glutamatergic EP neurons that project to the LHb. Previous work had not distinguished between these types in their recordings so the current results showing that the bidirectional value signaling is present in the EP sst+ population is valuable.

Weaknesses:

- One of the main weaknesses of the paper is to do with how the effect of the EP sst+ neurons on the behavior was assessed.

o All the manipulations (blocking synaptic release and blocking glutamatergic transmission) are chronic and more importantly the mice are given weeks of training after the manipulation before the behavioral effect is assessed. This means that as the authors point out in their discussion the mice will have time to adjust to the behavioral manipulation and compensate for the manipulations. The results do show that mice can adapt to these chronic manipulations and that the EP sst+ are not required to perform the task. What is unclear is whether the mice have compensated for the loss of EP sst+ neurons and whether they play a role in the task under normal conditions. Acute manipulations or chronic manipulations without additional training would be needed to assess this.

o Another weakness is that the effect of the manipulations was assessed in the 90/10 contingency version of the task. Under these contingencies, mice integrate past outcomes over fewer trials to determine their choice and animals act closer to a simple win-stay-lose switch strategy. Due to this it is unclear if the EP sst+ neurons would play a role in the task when they must integrate over a larger number of conditions in the less deterministic 70/30 version of the task. Indeed it is not clear that lesioning any other regions involved in evaluation of action outcomes such as VTA dopamine neurons, that encode reward prediction errors, would have any deficit when assessed in this way. Due to this, it's not clear if the mice have adapted to solve the task without evaluating action outcomes at all and are just acting in a more deterministic lose switch manner that would not presumably involve any of the circuitry in evaluating action outcomes.

- The authors conclude that they do not see any evidence for bidirectional prediction errors. It is not possible to conclude this. First, they see a large response in the EP sst+ neurons to the omission of an expected reward. This is what would be expected of a negative reward prediction error. There are much more specific well controlled tests for this that are commonplace in head-fixed and freely moving paradigms that could be tested to probe this. The authors do look at the effect of previous trials on the response and do not see strong consistent results, but this is not a strong formal test of what would be expected of a prediction error, either a positive or negative. The other way they assess this is by looking at the size of the responses in different recording sessions with different reward contingencies. They claim that the size of the reward expectation and prediction error should scale with the different reward probabilities. If all the reward probabilities were present in the same session this should be true as lots of others have shown for RPE. Because however this data was taken from different sessions it is not expected that the responses should scale, this is because reward prediction errors have been shown to adaptively scale to cover the range of values on offer (Tobler et al., Science 2005). A better test of positive prediction error would be to give a larger than expected reward on a subset of trials. Either way there is already evidence that responses reflect a negative prediction error in their data and more specific tests would be needed to formally rule in or out prediction error coding especially as previous recordings have shown it is present in previous primate and rodent recordings.

- There are a lot of variables in the GLM that occur extremely close in time such as the entry and exit of a port. If two variables occur closely in time and are always correlated it will be difficult if not impossible for a regression model to assign weights accurately to each event. This is not a large issue, but it is misleading to have regression kernels for port entry and exits unless the authors can show these are separable due to behavioral jitter or a lack of correlation under specific conditions, which does not seem to be the case.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript by Bell et. al. describes an analysis of the effects of removing one of two mutually exclusive splice exons at two distinct sites in the Drosophila CaV2 calcium channel Cacophony (Cac). The authors perform imaging and electrophysiology, along with some behavioral analysis of larval locomotion, to determine whether these alternatively spliced variants have the potential to diversify Cac function in presynaptic output at larval neuromuscular junctions. The author provided valuable insights into how alternative splicing at two sites in the calcium channel alters its function.

Strengths:

The authors find that both of the second alternatively spliced exons (I-IIA and I-IIB) that are found in the intracellular loop between the 1st and second set of transmembrane domains can support Cac function. However, loss of the I-IIB isoform (predicted to alter potential beta subunit interactions) results in 50% fewer channels at active zones and a decrease in neurotransmitter release and the ability to support presynaptic homeostatic potentiation. Overall, the study provides new insights into Cac diversity at two alternatively spliced sites within the protein, adding to our understanding of how regulation of presynaptic calcium channel function can be regulated by splicing.

Weaknesses:

The authors find that one splice isoform (IS4B) in the first S4 voltage sensor is essential for the protein's function in promoting neurotransmitter release, while the other isoform (IS4A) is dispensable. The authors conclude that IS4B is required to localize Cac channels to active zones. However, I find it more likely that IS4B is required for channel stability and leads to the protein being degraded, rather than any effect on active zone localization. More analysis would be required to establish that as the mechanism for the unique requirement for IS4B.

(1) We thank the reviewer for this important point. In fact, all three reviewers raised the same question, and the reviewing editor pointed out that caution or additional experiments were required to distinguish between IS4 splicing being important for cac channel localization versus channel stability/degradation. We provide multiple sets of experiments as well as text and figure revisions to strengthen our claim that the IS4B exon is required for cacophony channels to enter motoneuron presynaptic boutons and localize to active zones.

a. If IS4B was indeed required for cac channel stability (and not for localization to active zones) IS4A channels should be instable wherever they are. This is not the case because we have recorded somatodendritic cacophony currents from IS4A expressing adult motoneurons that were devoid of cac channels with the IS4B exon. Therefore, IS4A cac channels are not instable but underlie somatodendritic voltage dependent calcium currents in these motoneurons. These new data are now shown in the revised figure 3C and referred to in the text on page 7, line 42 to page 8 line 9.

b. Similarly, if IS4B was required for channel stability, it should not be present anywhere in the nervous system. We tested this by immunohistochemistry for GFP tagged IS4A channels in the larval CNS. Although IS4A channels are sparsely expressed, which is consistent with low expression levels seen in the Western blots (Fig. 1E), there are always defined and reproducible patterns of IS4A label in the larval brain lobes as well as in the anterior part of the VNC. This again shows that the absence of IS4A from presynaptic active zones is not caused by channel instability, because the channel is expressed in other parts of the nervous system. These data are shown in the new supplementary figure 1 and referred to in the text on page 15, lines 3 to 8.

c. As suggested in a similar context by reviewers 1 and 2, we now show enlargements of the presence of IS4B channels in presynaptic active zones as well as enlargements of the absence of IS4A channels in presynaptic active zones in the revised figures 2A-C and 3A. In these images, no IS4A label is detectable in active zones or anywhere else throughout the axon terminals, thus indicating that IS4B is required for expressing cac channels in the axon terminal boutons and localizing it to active zones. Text and figure legends have been adjusted accordingly.

d. Related to this, reviewer 1 also recommended to quantify the IS4A and ISB4 channel intensity and co-localization with the active zone marker brp (recommendation for authors). After following the reviewers’ suggestion to adjust the background values in IS4A and IS4B immunolabels to identical (revised Figs. 2A-C), it becomes obvious that IS4A channel are not detectable above background in presynaptic terminals or active zones, thus intensity is close to zero. We still calculated the Pearsons co-localization coefficient for both IS4 variants with the active zone marker brp. For IS4B channels the Pearson’s correlation coefficient is control like, just above 0.6, whereas for IS4A channels we do not find colocalization with brp (Pearson’s below 0.25). These new analyses are now shown in the revised figure 2D and referred to on page 6, lines 33 to 38.

e. Consistent with our finding that IS4B is required for cac channel localization to presynaptic active zones, upon removal of IS4B we find no evoked synaptic transmission (Fig. 2 in initial submission, now Fig. 3B).

Together these data are in line with a unique requirement of IS4B at presynaptic active zones (not excluding additional functions of IS4B), whereas IS4A containing cac isoforms are not found in presynaptic active zones and mediate different functions.

Reviewer #2 (Public Review):

This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones.

Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There are some outstanding questions regarding the mechanisms underlying the changes in Cac localization and function, and some additional suggestions are listed below for the authors to consider in strengthening this study. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

(2) We believe that the additional data on cac IS4A isoform localization and function as detailed above (response to public review 1) has strengthened the manuscript and answered some of the remaining questions the reviewer refers to. We are also grateful for the specific additional reviewer suggestions which we have addressed point-by-point and refer to below (section recommendations for authors).

Reviewer #3 (Public Review):

Summary:

Bell and colleagues studied how different splice isoforms of voltage-gated CaV2 calcium channels affect channel expression, localization, function, synaptic transmission, and locomotor behavior at the larval Drosophila neuromuscular junction. They reveal that one mutually exclusive exon located in the fourth transmembrane domain encoding the voltage sensor is essential for calcium channel expression, function, active zone localization, and synaptic transmission. Furthermore, a second mutually exclusive exon residing in an intracellular loop containing the binding sites for Caβ and G-protein βγ subunits promotes the expression and synaptic localization of around ~50% of CaV2 channels, thereby contributing to ~50% of synaptic transmission. This isoform enhances release probability, as evident from increased short-term depression, is vital for homeostatic potentiation of neurotransmitter release induced by glutamate receptor impairment, and promotes locomotion. The roles of the two other tested isoforms remain less clear.

Strengths:

The study is based on solid data that was obtained with a diverse set of approaches. Moreover, it generated valuable transgenic flies that will facilitate future research on the role of calcium channel splice isoforms in neural function.

Weaknesses:

(1) Based on the data shown in Figures 2A-C, and 2H, it is difficult to judge the localization of the cac isoforms. Could they analyze cac localization with regard to Brp localization (similar to Figure 3; the term "co-localization" should be avoided for confocal data), as well as cac and Brp fluorescence intensity in the different genotypes for the experiments shown in Figure 2 and 3 (Brp intensity appears lower in the dI-IIA example shown in Figure 3G)? Furthermore, heterozygous dIS4B imaging data (Figure 2C) should be quantified and compared to heterozygous cacsfGFP/+.

According to the reviewer’s suggestion, we have quantified cac localization relative to brp localization by computing the Pearson’s correlation coefficient for controls and IS4A as well as IS4B animals. These new data are shown in the revised Fig. 2D and referred to on page 6, lines 33-38. Furthermore, we now confirm control-like Pearson’s correlation coefficients for all exon out variants except ΔIS4B and show Pearson’s correlation coefficients for all genotypes side-by-side in the revised Fig. 4D (legend has been adjusted accordingly). In addition, in response to the recommendations to authors, we now provide selective enlargements for the co-labeling of Brp and each exon out variant in the revised figures 2-4. We have also adjusted the background in Fig. 2C (ΔIS4B) to match that in Figs. 2A and B (control and ΔIS4A). This allows a fair comparison of cac intensities following excision of IS4B versus excision of IS4A and control (see also Fig 3). Together, this demonstrates the absence of IS4A label in presynaptic active zones much clearer. As suggested, we have also quantified brp puncta intensity on m6/7 across homozygous exon excision mutants and found no differences (this is now stated for IS4A/IS4B in the results text on page 6, lines 37/38 and for I-IIA/I-IIB on page 8, lines 42-44.). We did not quantify the intensity of cacophony puncta upon excision of IS4B because the label revealed no significant difference from background (which can be seen much better in the images now), but the brp intensities remained control-like even upon excision of IS4B.

(2) They conclude that I-II splicing is not required for cac localization (p. 13). However, cac channel number is reduced in dI-IIB. Could the channels be mis-localized (e.g., in the soma/axon)? What is their definition of localization? Could cac be also mis-localized in dIS4B? Furthermore, the Western Blots indicate a prominent decrease in cac levels in dIS4B/+ and dI-IIB (Figure 1D). How do the decreased protein levels seen in both genotypes fit to a "localization" defect? Could decreased cac expression levels explain the phenotypes alone?

We have now precisely defined what we mean by cac localization, namely the selective label of cac channels in presynaptic active zones that are defined as brp puncta, but no cac label elsewhere in the presynaptic bouton (page 6, lines 18 to 20). On the level of CLSM microscopy this corresponds to overlapping cac puncta and brp puncta, but no cac label elsewhere in the bouton. Based on the additional analysis and data sets outlined in our response 1 (see above) we conclude that excision of IS4B does not cause channel mislocalization because we find reproducible expression patterns elsewhere in the nervous system as well as somatodendritic cac current in ΔIS4B (for detail see above). Therefore, the isoforms containing the mutually exclusive IS4A exon are expressed and mediate other functions, but cannot substitute IS4B containing isoforms at the presynaptic AZ. In fact, our Western blots are in line with reduced cac expression if all isoforms that mediate evoked release are missing, again indicating that the presynapse specific cac isoforms cannot be replaced by other cac isoforms. This is also in line with the sparse expression of IS4A throughout the CNS as seen in the new supplementary figure 1 (for detail see above).

(3) Cac-IS4B is required for Cav2 expression, active zone localization, and synaptic transmission. Similarly, loss of cac-I-IIB reduces calcium channel expression and number. Hence, the major phenotype of the tested splice isoforms is the loss of/a reduction in Cav2 channel number. What is the physiological role of these isoforms? Is the idea that channel numbers can be regulated by splicing? Is there any data from other systems relating channel number regulation to splicing (vs. transcription or post-transcriptional regulation)?

Our data are not consistent with the idea that splicing regulates channel numbers. Rather, splicing can be used to generate channels with specific properties that match the demand at the site of expression. For the IS4 exon pair we find differences in activation voltage between IS4A and IS4B channels (revised Fig. 3C), with IS4B being required for sustained HVA current. IS4A does not localize to presynaptic active zones at the NMJ and is only sparsely expressed elsewhere in the NS (new supplementary Fig. 1). By contrast, IS4B is abundantly expressed in many neuropils. Therefore, taking out IS4B takes out the more abundant IS4 isoform. This is consistent with different expression levels for IS4 isoforms that have different functions, but we do not find evidence for splicing regulating expression levels per se.

Similarly, the I-II mutually exclusive exon pair differs markedly in the presence or absence of G-protein βγ binding sites that play a role in acute channel regulation as well the conservation of the sequence for β-subunit binding (see page 5, lines 9-17). Channel number reduction in active zones occurs specifically if expression of the cac channels with the G_βγ-binding site as well as the more conserved β-subunit binding is prohibited by excision of the I-IIB exon (see Fig. 5F). Vice versa, excision of I-IIA does not result in reduced channel numbers. This scenario is consistent with the hypothesis that conserved β-subunit binding affects channel number in the active zone (see page 17, lines 3 to 6 and lines 33-36), but we have no evidence that I-II splicing per se affects channel number.

(4) Although not supported by statistics, and as appreciated by the authors (p. 14), there is a slight increase in PSC amplitude in dIS4A mutants (Figure 2). Similarly, PSC amplitudes appear slightly larger (Figure 3J), and cac fluorescence intensity is slightly higher (Figure 3H) in dI-IIA mutants. Furthermore, cac intensity and PSC amplitude distributions appear larger in dI-IIA mutants (Figures 3H, J), suggesting a correlation between cac levels and release. Can they exclude that IS4A and/or I-IIA negatively regulate release? I suggest increasing the sample size for Canton S to assess whether dIS4A mutant PSCs differ from controls (Figure 2E). Experiments at lower extracellular calcium may help reveal potential increases in PSC amplitude in the two genotypes (but are not required). A potential increase in PSC amplitude in either isoform would be very interesting because it would suggest that cac splicing could negatively regulate release.

There are several possibilities to explain this, but as none of the effects is statistically significant, we prefer to not investigate this in further depth. However, given that we cannot find IS4A in presynaptic active zones (revised figures 2C and 3A plus the new enlargements 2Ci and 3Ai, revised text page 6, lines 22 to 24 and 29 to 31, and page 7, second paragraph, same as public response 1D) IS4A channels cannot have a direct negative effect on release probability. Nonetheless, given that IS4A containing cac isoforms mediate functions in other neuronal compartments (see revised Fig. 3C) it may regulate release indirectly by affecting e.g. action potential shape. Moreover, in response to the more detailed suggestions to authors we provide new data that give additional insight.

(5) They provide compelling evidence that IS4A is required for the amplitude of somatic sustained HVA calcium currents. However, the evidence for effects on biophysical properties and activation voltage (p. 13) is less convincing. Is the phenotype confined to the sustained phase, or are other aspects of the current also affected (Figure 2J)? Could they also show the quantification of further parameters, such as CaV2 peak current density, charge density, as well as inactivation kinetics for the two genotypes? I also suggest plotting peaknormalized HVA current density and conductance (G/Gmax) as a function of Vm. Could a decrease in current density due to decreased channel expression be the only phenotype? How would changes in the sustained phase translate into altered synaptic transmission in response to AP stimulation?

Most importantly, sustained HVA current is abolished upon excision of IS4B (not IS4A, we think the reviewer accidentally mixed up the genotype) and presynaptic active zones at the NMJ contain only cac isoforms with the IS4B exon. This indicates that the cac isoforms that mediate evoked release encode HVA channels. The somatodendritic currents shown in the revised figure 3C (previously 2J) that remain upon excision of IS4B are mediated by IS4A containing cac isoforms. Please note that these never localize to the presynaptic active zone, and thus do not contribute to evoked release. Therefore, the interpretation is that specifically sustained HVA current encoded by IS4B cac isoforms is required for synaptic transmission. Reduced cac current density due to decreased channel expression is not the cause for impaired evoked release upon IS4B excision, but instead, the cause is the absence of any cac channels in active zones. IS4B-containing cac isoforms encode sustained HVA current, and we speculate that this might be a well suited current to minimize cacophony channel inactivation in the presynaptic active zone. Given that HVA current shows fast voltage dependent activation and fast inactivation upon repolarization, it is useful at large intraburst firing frequencies as observed during crawling (Kadas et al., 2017) without excessive cac inactivation (see page 15, Kadas, lines 16 to 20).

However, we agree with the reviewer that a deeper electrophysiological analysis of splice isoform specific cac currents will be instructive. We have now added traces of control and ΔIS4B from a holding potential of -90 mv (revised Fig. 3C, bottom traces and revised text on page 7, line 43 to page 8, lines 1 to 10), and these are also consistent with IS4B mediating sustained HVA cac current. However, further analysis of activation and inactivation voltages and kinetics suffers form space clamp issues in recordings from the somata of such complex neurons (DLM motoneurons of the adult fly contain roughly 6000 µm of dendrites with over 4000 branches, Ryglewski et al., 2017, Neuron 93(3):632-645). Therefore, we will analyze the currents in a heterologous expression system and present these data to the scientific community as a separate study at a later time point.

(6) Why was the STED data analysis confined to the same optical section, and not to max. intensity z-projections? How many and which optical sections were considered for each active zone? What were the criteria for choosing the optical sections? Was synapse orientation considered for the nearest neighbor Cac - Brp cluster distance analysis? How do the nearest-neighbor distances compare between "planar" and "side-view" Brp puncta?

Maximum intensity z-projections would be imprecise because they can artificially suggest close proximity of label that is close by in x and y but far away in z. Therefore, the analysis was executed in xy-direction of various planes of entire 3D image stacks. We considered active zones of different orientations (Figs. 5C, D) to account for all planes. In fact, we searched the entire z-stacks until we found active zones of all orientations within the same boutons, as shown in figures 5C1-C6. The same active zone orientations were analyzed for all exon-out mutants with cac localization in active zones. The distance between cac and brp did not change if viewed from the side or any other orientation. We now explain this in more clarity in the results text on page 9, lines 23/24.

(7) Cac clusters localize to the Brp center (e.g., Liu et al., 2011). They conclude that Cav2 localization within Brp is not affected in the cac variants (p. 8). However, their analysis is not informative regarding a potential offset between the central cac cluster and the Brp "ring". Did they/could they analyze cac localization with regard to Brp ring center localization of planar synapses, as well as Brp-ring dimensions?

In the top views (planar) we did not find any clear offset in cac orientation to brp between genotypes. In such planar synapses (top views, Fig. 5D, left row) we did not find any difference in Brp ring dimensions. We did not quantify brp ring dimensions rigorously, because this study focusses on cac splice isoform-specific localization and function. Possible effects of different cac isoforms on brp-ring dimensions or other aspects of scaffold structure are not central to our study, in particular given that brp puncta are clearly present even if cac is absent from the synapse (Fig. 3A), indicating that cac is not instructive for the formation of the brp scaffold.

(8) Given the accelerated PSC decay/ decreased half width in dI-IIA (Fig. 5Q), I recommend reporting PSC charge in Figure 3, and PPR charge in Figures 5A-D. The charge-based PPRs of dI-IIA mutants likely resemble WT more closely than the amplitude-based PPR. In addition, miniature PSC decay kinetics should be reported, as they may contribute to altered decay kinetics. How could faster cac inactivation kinetics in response to single AP stimulation result in a decreased PSC half-width? Is there any evidence for an effect of calcium current inactivation on PSC kinetics? On a similar note, is there any evidence that AP waveform changes accelerate PSC kinetics? PSC decay kinetics are mainly determined by GluR decay kinetics/desensitization. The arguments supporting the role of cac splice isoforms in PSC kinetics outlined in the discussion section are not convincing and should be revised.

We agree that reporting charge in figure 3 is informative and do so in the revised text. Since the result (no significant difference in the PSCs between between CS, cac^GFP, ^ΔI-IIA, and transheterozygous I-IIA/I-IIB, but significantly smaller values in ΔI-IIB) remained unchanged no matter whether charge or amplitude were analyzed, we decided to leave the figure as is and report the additional analysis in the text (page 8, lines 40 to 42). This way, both types of analysis are reported. Please note that EPSC amplitude is slightly but not significantly increased upon excision of I-IIA (Fig. 4J), whereas EPSC half amplitude width is significantly smaller (Fig. 5Q, now revised Fig 6R). Together, a tendency of increased EPSC amplitudes and smaller half amplitude width result in statistically insignificant changes in EPSC in ∆I-IIA (now discussed on page 15, lines 37 to 40). We also understand the reviewer’s concern attributing altered EPSC kinetics to presynaptic cac channel properties. We have toned down our interpretation in the discussion and list possible alterations in presynaptic AP shape or cac channel kinetics as alternative explanations (not conclusions; see revised discussion on page 15, line 40 to page 16, line 2). Moreover, we have quantified postsynaptic GluRIIA abundance to test whether altered PSC kinetics are caused by altered GluRIIA expression. In our opinion, the latter is more instructive than mini decay kinetic analysis because this depends strongly on the distance of the recording electrode to the actual site of transmission in these large muscle cells. Although we find no difference in GluRIIA expression levels we now clearly state that we cannot exclude other changes in GluR receptor fields, which of course, could also explain altered PSC kinetics. We have updated the discussion on page 16, lines 2/3 accordingly.

(9) Paired-pulse ratios (PPRs): On how many sweeps are the PPRs based? In which sequence were the intervals applied? Are PPR values based on the average of the second over the first PSC amplitudes of all sweeps, or on the PPRs of each sweep and then averaged? The latter calculation may result in spurious facilitation, and thus to the large PPRs seen in dI-IIB mutants (Kim & Alger, 2001; doi: 10.1523/JNEUROSCI.21-2409608.2001).

We agree that the PP protocol and analyses had to be described more precisely in the methods and have done so on page 23, lines 31 to 37 in the methods. Mean PPR values are based on the PPRs of each sweep and then averaged. We are aware of the study of Kim and Alger 2001 and have re-analyzed the PP data in both ways outlined by the reviewer. We get identical results with either analyses method. Spurious facilitation is thus not an issue in our data. We now explain this in the methods section along with the PPR protocol. The large spread seen in dI-IIB is indeed caused by reduced calcium influx into active zones with fewer channels, as anticipated by the reviewer (see next point).

(10) Could the dI-IIB phenotype be simply explained by a decrease in channel number/ release probability? To test this, I propose investigating PPRs and short-term dynamics during train stimulation at lower extracellular Ca2+ concentration in WT. The Ca2+ concentration could be titrated such that the first PSC amplitude is similar between WT and dI-IIB mutants. This experiment would test if the increased PPR/depression variability is a secondary consequence of a decrease in Ca2+ influx, or specific to the splice isoform.

In fact, the interpretation that decreased PSC amplitude upon I-IIB excision is caused mainly by reduced channel number is precisely our interpretation (see discussion page 14, last paragraph to page 15, first paragraph in the original submission, now page 16, second paragraph paragraph). In addition, we are grateful for the reviewer’s suggestion to triturate the external calcium such that the first PSC amplitude in matches in ∆I-IIB and control. This experiment tests whether altered short term plasticity is solely a function of altered channel number or whether additional causes, such as altered channel properties, also play into this. We triturated the first pulse amplitude in ∆I-IIB to match control and find that paired pulse ratio and the variance thereof are not different anymore. Therefore, the differences observed in identical external calcium can be fully explained by altered channel numbers. This additional dataset is shown in the revised figures 6D and E and referred to in the results section on page 10, lines 14 to 25 and the discussion on page16, lines 36 to 38.

(11) How were the depression kinetics analyzed? How many trains were used for each cell, and how do the tau values depend on the first PSC amplitude? Time constants in the range of a few (5-10) milliseconds are not informative for train stimulations with a frequency of 1 or 10 Hz (the unit is missing in Figure 5H). Also, the data shown in Figures 5E-K suggest slower time constants than 5-10 ms. Together, are the data indeed consistent with the idea that dIIIB does not only affect cac channel number, but also PPR/depression variability (p. 9)?

For each animal the amplitudes of all subsequent PSCs in each train were plotted over time and fitted with a single exponential. For depression at 1 and 10 Hz, we used one train per animal, and 5-6 animals per genotype (as reflected in the data points in Figs. 6I, M). This is now explained in more detail in the revised methods section (page 23, lines 39 to 41). The tau values are not affected by the amplitude of the first PSC. First, we carefully re-fitted new and previously presented depression data and find that the taus for depression at low stimulation frequencies (1 and 10Hz) are not affected by exon excisions at the I-II site. We thank the reviewer for detecting our error in units and tau values in the previous figure panels 5H and L (this has now been corrected in the revised figure panels 6I and M). Given that PSC amplitude upon I-IIB excision is significantly smaller than in controls and following I-IIA excision, we suspected that the time course of depression at low stimulation frequency is not significantly affected by the amount of calcium influx during the first PSC. To further test this, we followed the reviewer ’s suggestion and re-measured depression at 1 and 10 Hz for cac-GFP controls and for delta I-IIB in a higher external calcium concentration (1.8 mM), so that the first PSC was increased in amplitude in both genotypes (1.8 mM external calcium triturates the PSC amplitude in delta I-IIB to match that of controls measured in 0.5 mM external calcium, see revised Figs. 6H, L). Neither in control, nor in delta I-IIB did this affect the time course of synaptic depression (see revised Figs. 6I, M). This indicates that at low stimulation frequencies (1 and 10Hz) the time course of depression is not affected by mean quantal content. This is consistent with the paired pulse ratio at 100 ms interpulse interval shown in figures 6A-D. However, for synaptic depression at 1 Hz stimulation the variability of the data is higher for delta I-IIB (independent of external calcium concentration, see rev. Fig. 6I), which might also be due to reduced channel number in this genotype. Taken together, the data are in line with the idea that altered cac channel numbers in active zones are sufficient to explain all effects that we observe upon I-IIB excision on PPRs and synaptic depression at low stimulation frequencies. This is now clarified in the revised text on page 12, lines 3 to 7.

(12) The GFP-tagged I-IIA and mEOS4b-tagged I-IIB cac puncta shown in Figure 6N appear larger than the Brp puncta. Endogenously tagged cac puncta are typically smaller than Brp puncta (Gratz et al., 2019). Also, the I-IIA and I-IIB fluorescence sometimes appear to be partially non-overlapping. First, I suggest adding panels that show all three channels merged. Second, could they analyze the area and area overlap of I-IIA and I-IIB with regard to each other and to Brp, and compare it to cac-GFP? Any speculation as to how the different tags could affect localization? Finally, I recommend moving the dI-IIA and dI-IIB localization data shown in Figure 6N to an earlier figure (Figure 1 or Figure 3).

We now show panels with the two I-II cac isoforms merged in the revised figure 7H (previously 6N). We also tested merging all three labels as suggested, but found this not instructive for the reader. We thank the reviewer for pointing out that the Brp puncta appeared smaller than the cac puncta in some panels. We carefully went through the data and found that the Brp puncta are not systematically smaller than the cac puncta. Please note that punctum size can appear quite differently, depending on different staining qualities as well as different laser intensities and different point spread in different imaging channels. The purpose of this figure was not to analyze punctum size and labeling intensity, but instead, to demonstrate that I-IIA and I-IIB are both present in most active zones, but some active zones show only I-IIB labeling, as quantified in figure 7I. We did not follow the suggestion to conduct additional co-localization analyses and compare it with cac-GFP controls, because Pearson co-localization coefficients for cac-GFP and all exon-out variants analyzed, including delta I-IIA and delta I-IIB are presented in the revised figure 4D. Moreover, delta I-IIA and delta I-IIB show similar Manders 1 and 2 co-localization coefficients with Brp (see Figs. 4E, F). We do not want to speculate whether the different tags have any effect on localization precision. Artificial differences in localization precision can also be suggested by different antibodies, but we know from our STED analyses with identical tags and antibodies for all isoforms that I-IIA and I-IIB co-localize identically with Brp (see Figs. 5A-E). Finally, we prefer to not move the figure because we believe it is informative to show our finding that active zones usually contain both splice I-II variants together with the finding that only I-IIB is required for PHP.

Recommendations for the authors:

Reviewing Editor Comments:

We thank you for your submission. All three reviewers urge caution in interpreting the S4 splice variant playing a role specifically in Cac localization, as opposed to just leading to instability and degradation. There are other issues with the electrophysiological experiments, a need for improved imaging and analyses, and some areas of interpretation detailed in the reviews.

We agree that additional data was required to conclude that IS4 splicing plays a specific role in cac channel localization and is not just leading to channel instability and degradation. As outlined in detail in our response to reviewer 1, comment 1, we conducted several sets of experiments to support our interpretation. First, electrophysiological experiments show that upon removal of IS4B, which eliminates synaptic transmission at the larval NMJ and cac positive label in presynaptic active zones, somatodendritic cac current is reliably recorded (new data in revised figure 3C). This is not in line with a channel instability or degradation effect, but instead with IS4B containing isoforms being required and sufficient for evoked release from NMJ motor terminals, whereas IS4A isoforms are not sufficient for evoked release from axon terminals, but IS4A isoforms alone can mediate a distinct component of somatodendritic calcium current. Second, immunohostochemical analyses reveal that IS4A, which is not present in NMJ presynaptic active zones, is expressed sparsely, but in reproducible patterns in the larval brain lobes and in specific regions of the anterior VNC parts (new supplementary figure 1). Again, the absence of a IS4A-containing cac isoform from presynaptic active zones but their simultaneous presence in other parts of the nervous system is in accord with isoform specific localization, but not with general channel isoform instability. Third, enlargements of NMJ boutons with brp positive presynaptic active zones confirm the absence of IS4A and the presence of IS4B in active zones (these enlargements are now shown in the revised figures 2A-C, 3A, and 4A-C). Fourth, as suggested we have quantified the Pearson co-localization of IS4 isoforms with Brp in presynaptic active zones (revised Fig. 2D). This confirms quantitatively similar co-localization of IS4B and control with Brp, but no co-localization of IS4A with Brp. In fact, the labeling intensity of IS4A in presynaptic active zones is quantitatively not significantly different from background, no IS4A label is detected anywhere in the axon terminals at the NMJ, but we find IS4 label in the CNS. Together, these data strongly support our interpretation that the IS4 splice site plays a distinct role in cac channel localization. Figure legends as well as results and discussion section have been modified accordingly (the respective page and line numbers are listed in our-point-by-point responses).

In addition, we have carefully addressed all other public comments as well as all other recommendations for authors by providing multiple new data sets, new image analyses, and revising text. Addressing the insightful comments of all three reviewers and the reviewing editor has greatly helped to make the manuscript better.

Reviewer #1 (Recommendations For The Authors):

The conclusion that the IS4B exon controls Cac localization to active zones versus simply being required for channel abundance is not well supported. The authors need to either mention both possibilities or provide stronger support for the active zone localization model if they want to emphasize this point.

We agree and have included several additional data sets as outlined in our response to point 1 of reviewer 1 and to the reviewing editor (see above). These new data strongly support our interpretation that the IS4B exon controls Cac localization to active zones and is not simply required for channel abundance. The additions to the figures and accompanying text (including the respective figure panel, page, and line numbers) are listed in the point-bypoint responses to the reviewers’ public suggestions.

Figure 2C staining for Cac localization in the delta 4B line is difficult to compare to the others, as the background staining is so high (muscles are green for example). As such, it is hard to determine whether the arrows in C are just background.

We had over-emphasized the green label to show that there really is no cacophony label in active zones. However, we agree that this hampered image interpretation. Thus, we have adjusted brightness such that it matches the other genotypes (see new figure panel 2C, and figure 3A, bottom). Revising the figure as suggested by the reviewer shows much more clearly that IS4B puncta are detected exclusively in presynaptic active zones, whereas IS4A channels are not detectable in active zones or anywhere else in the axon terminal boutons. Quantification of IS4A label in brp positive active zones confirms that labeling intensity is not significantly above background (page 6, lines 29 to 31 and page 7, lines 19 to 21). Therefore, IS4A is not detectable in active zones at the NMJ.

It seems more likely that the removal of the 4B exon simply destabilizes the protein and causes it to be degraded (as suggested by the Western), rather than mislocalizing it away from active zones. It's hard to imagine how some residue changes in the S4 voltage sensor would control active zone localization to begin with. The authors should note that the alternative explanation is that the protein is just degraded when the 4B exon is removed.

Based on additional data and analyses, we disagree with the interpretation that removal of IS4B disrupts protein integrity and present multiple lines of evidence that support sparse expression of IS4A channels (ΔIS4B). As outlined in our response to reviewer 1 and to the reviewing editor, we show (1) in new immunohistochemical stainings (new supplementary figure 1) that upon removal of IS4B, sparse label is detectable in the VNC and the brain lobes (for detail see above). (2) In our new figure 3C, we show cacophony-mediated somatodendritic calcium currents recorded from adult flight motoneurons in a control situation and upon removal of IS4B that leaves only IS4A channels. This clearly demonstrates that IS4A underlies a substantial component of the HVA somatodendritic calcium current, although it is absence from axon terminals. This is in line with isoform specific functions at different locations, but not with IS4A instability/degradation. (3) We do not agree with the reviewer’s interpretation of the Western Blot data in figure 1E (formerly figure 1D). Together with our immunohistochemical data that show sparse cacophony IS4A expression, we think that the faint band upon removal of IS4B in a heterozygous background (that reduces labeled channels even further) reflects the sparseness of IS4A expression. This sparseness is not due to channel instability, but to IS4A functions that are less abundant than the ubiquitously expressed cac^IS4B channels at presynaptic active zones of fast chemical synapses (see page 15, lines 24 to 29).

If they really want to claim the 4B exon governs active zone localization, much higher quality imaging is required (with enlarged views of individual boutons and their AZs, rather than the low-quality full NMJ imaging provided). Similarly, higher resolution imaging of Cac localization at Muscle 12 (Figure 2H) boutons would be very useful, as the current images are blurry and hard to interpret. Figure 6N shows beautiful high-resolution Cac and Brp imaging in single boutons for the I-II exon manipulations - the authors should do the same for the 4B line. For all immuno in Figure 2, it is important to quantify Cac intensity as well. There is no quantification provided, just a sample image. The authors should provide quantification as they do for the delta I-II exons in Figure 3.

We did as suggested and added figure panels to figure 2A-C and to new figures 3A (formerly part of figure 2 and 4A-C (formerly figure 3) showing magnified label at the NMJ AZs to better judge on cacophony expression after exon excision. These data are now referred to in the results section on page 6, lines 22 to 24, page 7, lines 18 to 21 and page 8, lines 17/18.

As suggested, we now also provide quantification of co-localization with brp puncta as Pearson’s correlation coefficient for control, IS4B, and IS4A in the new figure panel 2D (text on page 6, lines 34 to 38). This further underscores control-like active zone localization of IS4B but no significant active zone localization of IS4A. As suggested, we quantified now also the intensity of IS4B label in active zones, and it was not different from control (see revised figure 4H and text on page 8, lines 38/39). We did not quantify the intensity of IS4A label, because it was not over background (text, page 6, lines 30/31).

Reviewer #2 (Recommendations For The Authors):

(1a) Questions about the engineered Cac splice isoform alleles:

The authors using CRISPR gene editing to selectively remove the entire alternatively spliced exons of interest. Do the authors know what happens to the cac transcript with the deleted exon? Is the deleted exon just skipped and spliced to the next exon? Or does the transcript instead undergo nonsense-mediated decay?

We do not believe that there is nonsense mediated mRNA decay, because for all exon excisions the respective mRNA and protein are made. Protein has been detected on the level of Western blotting and immunocytochemistry. Therefore, we are certain that the mRNA is viable for each exon excision (and we have confirmed this for low abundance cac protein isoforms by rt-PCR), but only subsets of cac isoforms can be made from mRNAs that are lacking specific exons. However, we can not make any statements as to whether the lack of specific protein isoforms exerts feedback on mRNA stability, the rate of transcription and translation, or other unknown effects.

(1b) While it is clear that the IS4 exons encode part of the voltage sensor in the first repeat, are there studies in Drosophila to support the putative Ca-beta and G-protein beta-gamma binding sites in the I-II loop? Or are these inferred from Mammalian studies?

To the best of our knowledge, there are no studies in Drosophila that unambiguously show Caβ and Gβγ binding sites in the I-II loop of cacophony. However, sequence analysis strongly suggests that I-IIB contains both, a Caβ as well as a Gβγ binding site (AID: α-interacting domain) because the binding motif QXXER is present. In mouse Cav2.1 and Ca_v2.2 channels the sequence is QQIER, while in Drosophila cacophony I-IIB it is QQLER. In the alternative IIIA, this motif is not present, strongly suggesting that G_βγ subunits cannot interact at the AID. However, as already suggested by Smith et al. (1998), based on sequence analysis, Ca_β should still be able to bind, although possibly with a lower affinity. We agree that this information should be given to the reader and have revised the text accordingly on page 5, lines 9 to 17.

(1c) The authors assert that splicing of Cav2/cac in flies is a means to encode diversity, as mammals obviously have 4 Cav2 genes vs 1 in flies. However, as the authors likely know, mammalian Cav2 channels also have various splice isoforms encoded in each of the 4 Cav2 genes. The authors should discuss in more detail what is known about the splicing of individual mammalian Cav2 channels and whether there are any homologous properties in mammalian channels controlled by alternative splicing.

We agree and now provide a more comprehensive discussion of vertebrate Ca_v2 splicing and its impact on channel function. In line to what we report in Drosophila, properties like G_βγ binding and activation voltage can also be affected by alternative splicing in vertebrate Ca_v2 channel, through the exon patterns are quite different from Drosophila. We integrated this part on page 14, first paragraph) in the revised discussion. The respective text is below for the reviewer’s convenience:

“However, alternative splicing increases functional diversity also in mammalian Ca_v2 channels. Although the mutually exclusive splice site in the S4 segment of the first homologous repeat (IS4) is not present in vertebrate Cav channels, alternative splicing in the extracellular linker region between S3 and S4 is at a position to potentially change voltage sensor properties (Bezanilla 2002). Alternative splice sites in rat Ca_v2.1 exon 24 (homologous repeat III) and in exon 31 (homologous repeat IV) within the S3-S4 loop modulate channel pharmacology, such as differences in the sensitivity of Ca_v2.1 to Agatoxin. Alternative splicing is thus a potential cause for the different pharmacological profiles of P- and Q-channels (both Ca_v2.1; Bourinet et al. 1999). Moreover, the intracellular loop connecting homologous repeats I and II is encoded by 3-5 exons and provides strong interaction with G_βγ-subunits (Herlitze et al. 1996). In Ca_v2.1 channels, binding to G_βγ subunits is potentially modulated by alternative splicing of exon 10 (Bourinet et al. 1999). Moreover, whole cell currents of splice forms α1A-a (no Valine at position 421) and α1A-b (with Valine) represent alternative variants for the I-II intracellular loop in rat Ca_v2.1 and Ca_v2.2 channels. While α1A-a exhibits fast inactivation and more negative activation, α1A-b has delayed inactivation and a positive shift in the IV-curve (Bourinet et al. 1999). This is phenotypically similar to what we find for the mutually exclusive exons at the IS4 site, in which IS4B mediates high voltage activated cacophony currents while IS4A channels activate at more negative potentials and show transient current (Fig. 3; see also Ryglewski et al. 2012). Furthermore, altered Ca_β interaction have been shown for splice isoforms in loop III (Bourinet et al. 1999), similar to what we suspect for the I-II site in cacophony. Finally, in mammalian VGCCs, the C-terminus presents a large splicing hub affecting channel function as well as coupling distance to other proteins. Taken together, Ca_v2  channel diversity is greatly enhanced by alternative splicing also in vertebrates, but the specific two mutually exclusive exon pairs investigated here are not present in vertebrate Ca_v2 genes.”

(1d) In Figure 1, it would be helpful to see the entire cac genomic locus with all introns/exons and the 4 specific exons targeted for deletion.

We agree and have changed figure 1 accordingly.

(2a) Cav2.IS4B deletion alleles:

More work is necessary to explain the localization of Cac controlled by the IS4B exon. First, can the authors determine whether actual Cac channels are present at NMJ boutons? The authors seem to indicate that in the IS4B deletion mutants, some Cac (GFP) signal remains in a diffuse pattern across NMJ boutons. However, from the imaging of wild-type Cac-GFP (and previous studies), there is no Cac signal outside of active zones defined by the BRP signal. It would benefit the study to a) take additional, higher resolution images of the remaining Cac signal at NMJs in IS4B deletion mutants, and b) comment on whether the apparent remaining signal in these mutants is only observed in the absence of IS4Bcontaining Cac channels, or if the IS4A-positive channels are normally observed (but perhaps mis-localized?).

We have conducted additional analyses to show convincingly that IS4A channels (that remain upon IS4B deletion) are absent from presynaptic active zone. Please see also responses to reviewers 1 and 3. By adjusting the background values in of CLSM images to identical values in control, delta IS4A, and delta IS4B, as well as by providing selective enlargements as suggested, the figure panels 2C, Ci and 3A now show much clearer, that upon deletion of IS4B no cac label remains in active zones or anywhere else in the axon terminal boutons (see text on page 6, lines 22 to 24). This is further confirmed by quantification showing the in IS4B mutants cac labeling intensity in active zones is not above background (see text on page 6, lines 27 to 31). We never intended to indicate that there was cac signal outside of active zones defined by the brp signal, and we now carefully went through the text to not indicate this possibility unintentionally anywhere in the manuscript.

(2b) Do the authors know whether any presynaptic Ca2+ influx is contributed by IS4Apositive Cac channels at boutons, given the potential diffuse localization? There are various approaches for doing presynaptic Ca2+ imaging that could provide insight into this question.

We agree that this is an interesting question. However, based on the revisions made, we now show with more clarity that IS4A channels are absent from the presynaptic terminal at the NMJ. IS4A labeling intensities within active zones and anywhere else in the axon terminals are not different from background (see text on page 6, lines 27 to 31 and revised Figs. 2C, Ci, and 3A with new selective enlargements in response to comments of both other reviewers). This is in line with our finding that evoked synaptic transmission from NMJ axon terminals to muscle cells is mostly absent upon excision of IS4B (see Fig. 3B). The very small amplitude EPSC (below 5 % of the normal amplitude of evoked EPSCs) that can still be recorded in the absence of IS4B is similar to what is observed in cac null mutant junctions and is mediated by calcium influx through another voltage gated calcium channels, a Ca_v1 homolog named Dmca1D, as we have previously published (Krick et al., 2021, PNAS 118(28):e2106621118. Gathering additional support for the absence of IS4A from presynaptic terminals by calcium imaging experiments would suffer significantly from the presence of additional types of VGCCs in presynaptic terminals (for sure Dmca1D (Krick et al., 2021) and potentially also the Ca_v3 homolog DmαG or Dm-α1T). Such experiments would require mosaic null mutants for cac and DmαG channels in a mosaic IS4B excision mutant, which, if feasible at all, would be very hard and time consuming to generate. In the light of the additional clarification that IS4A is not located in NMJ axon terminal boutons, as shown by additional labeling intensity analysis, revised figures with selective enlargement, and revised text, we feel confident to state that IS4A is not sufficient for evoked SV release.

(2c) Mechanistically, how are amino acid changes in one of the voltage sensing domains in Cac related to trafficking/stabilization/localization of Cac to AZs?

This is an exciting question that has occupied our discussions a lot. Some sorting mechanism must exist that recognizes the correct protein isoforms, just as sorting and transport mechanisms exist that transport other synaptic proteins to the synapse. We do not think that the few amino acid changes in the voltage sensor are directly involved in protein targeting. We rather believe that the cacophony variants that happen to contain this specific voltage sensor are selected for transport out to the synapse. There are possibilities to achieve this cell biological, but we have not further addressed potential mechanisms because we do not want enter the realms of speculation.

(3) How are auxiliary subunits impacted in the Cac isoform mutants?

Recent work by Kate O'Connor-Giles has shown that both Stj and Ca-Beta subunits localize to active zones along with Cac at the Drosophila NMJ. Endogenously tagged Stj and CaBeta alleles are now available, so it would be of interest to determine if Stj and particular Cabeta levels or localization change in the various Cac isoform alleles. This would be particularly interesting given the putative binding site for Ca-beta encoded in the I-II linker.

We agree that the synthesis of the work of Kate O'Connor-Giles group and our study open up new avenues to explore exciting hypotheses about differential coupling of specific cacophony splice isoforms with distinct accessory proteins such as Caβ and α₂δ subunits. However, this requires numerous full sets of additional experiments and is beyond the scope of this study.

(4a) Interpretation of short-term plasticity in the I-IIB exon deletion:

The changes in short-term plasticity presented in Figure 5 are interpreted as an additional phenotype due to the loss of the I-IIB exon, but it seems this might be entirely explained simply due to the reduced Cac levels. Reduced Cac levels at active zones will obviously reduce Ca2+ influx and neurotransmitter release. This may be really the only phenotype/function of the I-IIB exon. Hence, to determine whether loss of the I-IIB exon encodes any functions in short-term plasticity, separate from reduced Cac levels, the authors should compare short-term plasticity in I-IIB loss alleles compared to wild type with starting EPSC amplitudes are equal (for example by reducing extracellular Ca2+ levels in wild type to achieve the same levels at in Cac I-IIB exon deleted alleles). Reduced release probability, simply by reduced Ca2+ influx (either by reduced Cac abundance or extracellular Ca2+) should result in more variability in transmission, so I am not sure there is any particular function of the I-IIB exon in maintaining transmission variability beyond controlling Cac abundance at active zones.

For two reasons we are particularly grateful for this comment. First, it shows us that we needed to explain much clearer that our interpretation is that changes in paired pulse ratios (PPRs) and in depression at low stimulation frequencies are a causal consequence of lower channel numbers upon I-IIB exon deletion, precisely as pointed out by the reviewer. We have carefully revised the text accordingly on page 10, lines 14-25, page 11, lines 3-7 and 22-28; page 16, lines 36-38. Second, the experiment suggested by the reviewer is superb to provide additional evidence that the cause of altered PPRs is in fact reduced channel number, but not altered channel properties. Accordingly, we have conducted additional TEVC recordings in elevated external calcium (1.8 mM) so that the single PSC amplitudes in I-IIB excision animals match those of controls in 0.5 mM extracellular calcium. This makes the amplitudes and the variance of PPR for all interpulse intervals tested control-like (see revised Figs. 6D, E). This strongly indicates that differences observed in PPRs as well as the variance thereof were caused by the amount of calcium influx during the first EPSC, and thus by different channel numbers in active zones.

(4b) Another point about the data in Figure 5: If "behaviorally relevant" motor neuron stimulation and recordings are the goal, the authors should also record under physiological Ca2+ conditions (1.8 mM), rather than the highly reduced Ca2+ levels (0.5 mM) they are using in their protocols.

Although we doubt that the effective extracellular calcium concentration that determines the electromotoric force for calcium to enter the ensheathed motoneuron terminals in vivo during crawling is known, we followed the reviewer’s suggestion partly and have repeated the high frequency stimulation trains for ΔI-IIB in 1.8 mM calcium. As for short-term plasticity this brings the charge conducted to values as observed in control and in ΔI-IIA in 0.5 mM calcium. Therefore, all difference observed in previous figure 5 (now revised figure 6) can be accounted to different channel numbers in presynaptic active zones. This is now explained on page 11, lines 19-28. For controls recordings at high frequency stimulation in higher external calcium (e.g. 2 mM) have previously been published and show significant synaptic depression (e.g. Krick et al., 2021, PNAS). Given that in the exon out variants we do not expect any differences except from those caused by different channel numbers, we did not repeat these experiments for control and ΔI-IIA.

(5a) Mechanism of Cac's role in PHP :

As the authors likely know, mutations in Cac were previously reported to disrupt PHP expression (see Frank et al., 2006 Neuron). Inexplicably, this finding and publication were not cited anywhere in this manuscript (this paper should also be cited when introducing PhTx, as it was the first to characterize PhTx as a means of acutely inducing PHP). In the Frank et al. paper (and in several subsequent studies), PHP was shown to be blocked in mutations in Cac, namely the CacS allele. This allele, like the I-IIB excision allele, reduces baseline transmission presumably due to reduced Ca2+ influx through Cac. The authors should at a minimum discuss these previous findings and how they relate to what they find in Figure 6 regarding the block in PHP in the Cac I-IIB excision allele.

We thank the reviewer for pointing this out and apologize for this oversight. We agree that it is imperative to cite the 2006 paper by Frank et al. when introducing PhTx mediated PHP as well as when discussing cac the effects of cac mutants on PHP together with other published work. We have revised the text accordingly on page 12, lines 9-11 and 21-23 and on page 17, lines 29-33.

In terms of data presentation in Fig. 6, as is typical in the field, the authors should normalize their mEPSC/QC data as a percentage of baseline (+PhTx/-PhTx). This makes it easier to see the reduction in mEPSC values (the "homeostatic pressure" on the system) and then the homeostatic enhancement in QC. Similarly, in Fig. 6M, the authors should show both mEPSC and QC as a percentage of baseline (wild type or non-GluRIIA mutant background).

We agree and have changed figure presentation accordingly. Figure 7 (formerly figure 6) was updated as was the accompanying results text on page 12, lines 23-40.

(6) Cac I-IIA and I-IIB excision allele colocalization at AZs:

These are very nice and important experiments shown in Figures 6N and O, which I suggest the authors consider analyzing in further detail. Most significantly:

(6i) The authors nicely show that most AZs have a mix of both Cac IIA and IIB isoforms. Using simple intensity analysis, can the authors say anything about whether there is a consistent stoichiometric ratio of IIA vs IIB at single AZs? It is difficult to extract actual numbers of IIA vs IIB at individual AZs without having both isoforms labeled mEOS4b, but as a rough estimate can the authors say whether the immunofluorescence intensity of IIA:IIB is similar across each AZ? Or is there broad heterogeneity, with some AZs having low vs high ratios of each isoform (as the authors suggest across proximal to distal NMJ AZs)?

We agree and have conducted experiments and analyses to provide these data. We measured the cac puncta fluorescence intensities for heterozygous cac^sfGFP/cac, cacIIIA^sfGFP/cacI-IIB, and cacI-IIB^sfGFP/cacI-IIA animals. We preferred this strategy, because intensity was always measured from cac puncta with the same GFP tag. Next, we normalized all values to the intensities obtained in active zones from heterozygous cac^sfGFP/cac controls and then plotted the intensities of I-IIA versus I-IIB containing active zones side by side. Across junctions and animals, we find a consistent ratio 2:1 in the relative intensities of I-IIB and I-IIA, thus indicating on average roughly twice as many I-IIB as compared to I-IIA channels across active zones. This is consistent with the counts in our STED analysis (see Fig. 5F). These new data are shown in the new figure panel 7J and referred to on page 13, lines 10-16 in the revised text.

(6ii) Intensity analysis of Cac IIA vs IIB after PHP: Previous studies have shown Cac abundance increases at NMJ AZs after PHP. Can the authors determine whether both Cac IIA vs IIB isoforms increase after PHP or whether just one isoform is targeted for this enhancement?

We already show that PHP is not possible in the absence of I-IIB channels (see figure 7). However, we agree that it is an interesting question to test whether I-IIA channel are added in the presence of I-IIB channels during PHP, but we consider this a detail beyond the scope of this study.

Minor points:

(1) Including line numbers in the manuscript would help to make reviewing easier.

We agree and now provide line numbers.

(2) Several typos (abstract "The By contrast", etc).

We carefully double checked for typos.

(3) Throughout the manuscript, the authors refer to Cac alleles and channels as "Cav2", which is unconventional in the field. Unless there is a compelling reason to deviate, I suggest the authors stick to referring to "Cac" (i.e. cacdIS4B, etc) rather than Cav2. The authors make clear in the introduction that Cac is the sole fly Cav2 channel, so there shouldn't be a need to constantly reinforce that cac=Cav2.

We agree and have changed all fly Ca_v2 reference to cac.

(4) In some figures/text the authors use "PSC" to refer to "postsynaptic current", while in others (i.e. Figure 6) they switch to the more conventional terms of mEPSC or EPSC. I suggest the authors stick to a common convention (mEPSC and EPSC).

We have changed PSC to EPSC throughout.

Reviewer #3 (Recommendations For The Authors):

(1) The abstract could focus more on the results at the expense of the background.

We agree and have deleted the second introductory background sentence and added information on PPRs and depression during low frequency stimulation.

(2) What does "strict" active zone localization refer to? Could they please define the term strict?

Strict active zone localization means that cac puncta are detected in active zones but no cac label above background is found anywhere else throughout the presynaptic terminal, now defined on page 6, lines 27-29.

(3) Single boutons/zoomed versions of the confocal images shown in Figures 2A-C, 2H, and 3A-C would be very helpful.

We have provided these panels as suggested (see above and revised figures 2-4). Figure 3 is now figure 4.

(4) The authors cite Ghelani et al. (2023) for increased cac levels during homeostatic plasticity. I recommend citing earlier work making similar observations (Gratz et al., 2019; DOI: 10.1523/JNEUROSCI.3068-18.2019), and linking them to increased presynaptic calcium influx (Müller & Davis, 2012; DOI: 10.1016/j.cub.2012.04.018).

We agree and have added Gratz et al. 2019 and Davis and Müller 2012 to the results section on page 12, lines 17/18 and lines 21-23, in the discussion on page 17, lines 29-33.

(5) The data shown in Figure 3 does not directly support the conclusion of altered release probability in dI-IIB. I therefore suggest changing the legend's title.

We have reworded to “Excisions at the I-II exon do not affect active zone cacophony localization but can alter cacsfGFP label intensity in active zones and PSC amplitude” as this is reflecting the data shown in the figure panels more directly.

(6) It would be helpful to specify "adult flight muscle" in Figure 2J.

We agree that it is helpful to specify in the figure (now revised figure 3C) that the voltage clamp recordings of somatodendritic calcium current were conducted in adult flight motoneurons and have revised the headline of figure panel 3C and the legend accordingly. Please note, these are not muscle cells but central neurons.

(7) Do dIS4B/Cav2null MNs indeed show an inward or outward current at -90 to -70 mV/-40 and -50 mV, or is this an analysis artifact?

No, this is due to baseline fluctuations as typical for voltage clamp in central neurons with more than 6000 µm dendritic length and more than 4000 dendritic branches.

(8) Loss of several presynaptic proteins, including Brp (Kittel et al., 2006), and RBP (Liu et al., 2011), induce changes in GluR field size (without apparent changes in miniature amplitude). The statement regarding the Cav2 isoform and possible effects on GluR number (p. 8) should be revised accordingly.

We understand and have done two things. First, we measured the intensity of GluRIIA immunolabel in ΔI-IIA, ΔI-IIB, and controls and found no differences. Second, we reworded the statement. It now reads on page 9, lines 1-6: “It seems unlikely that presynaptic cac channel isoform type affects glutamate receptor types or numbers, because the amplitude of spontaneous miniature postsynaptic currents (mEPSCs, Fig. 4K) and the labeling intensity of postsynaptic GluRIIA receptors are not significantly different between controls, I-IIA, and I-IIB junctions (see suppl. Fig. 2, p = 0.48, ordinary one-way ANOVA, mean and SD intensity values are 61.0 ± 6.9 (control), 55.8 ± 8.5 (∆I-IIA), 61.1 ± 17.3 (∆I-IIB)). However, we cannot exclude altered GluRIIB numbers and have not quantified GluR receptor field sizes.”

(9) The statement relating miniature frequency to RRP size is unclear (p. 8). Is there any evidence for a correlation between miniature frequency to RRP size? Could the authors please clarify?

We agree that this statement requires caution. Although there is some published evidence for a correlation of RRP size and mini frequency (Neuron, 2009 61(3):412-24. doi: 10.1016/j.neuron.2008.12.029 and Journal of Neuroscience 44 (18) e1253232024; doi: 10.1523/JNEUROSCI.1253-23.2024), which we now refer to on page 9, it is not clear whether this is true for all synapses and how linear such a relationship may be. Therefore, we have revised the text on page 9, lines 6-9. It now reads: “Similarly, the frequency of miniature postsynaptic currents (mEPSCs) remains unaltered. Since mEPSCs frequency has been related to RRP size at some synapses (Pan et al., 2009; Ralowicz et al., 2024) this indicates unaltered RRP size upon I-IIB excision, but we have not directly measured RRP size.”

(10) Please define the "strict top view" of synapses (p. 8).

Top view is what this reviewer referred to as “planar view” in the public review points 6 and 7. In our responses to these public review points we now also define “strict top view”, see page 9, lines 17-19.

(11) Two papers are cited regarding a linear relationship between calcium channel number and release probability (p. 15). Many more papers could be cited to demonstrate a supralinear relationship (e.g., Dodge & Rahaminoff, 1967; Weyhersmüller et al., 2011 doi: 10.1523/JNEUROSCI.6698-10.2011). The data of the present study were collected at an extracellular calcium concentration of 0.5 mM, whereas Meideiros et al. (2023) used 1.5 mM. The relationship between calcium and release is supra-linear around 0.5 mM extracellular calcium (Weyhersmüller et al. 2011). This should be discussed/the statements be revised. Also, the reference to Meideiros et al. (2023) should be included in the reference list.

We have now updated the Medeiros reference (updated version of that paper appeared in eLife in 2024) in the text and reference list. We agree that the relationship of the calcium concentration and P_r can also be non-linear and refer to this on page 16, lines 26-32, but the point we want to make is to relate defined changes in calcium channel number (not calcium influx) as assessed by multiple methods (CLSM intensity measures and sptPALM channel counting) to release probability. We now also clearly state that we measured at 0.5 mM external calcium (page 16, lines 27/28) whereas Medeiros et al. 2024 measured at 1.5 mM calcium (page 16, lines 31/32).

(12) Figure 6: Quantal content does not have any units - please remove "n vesicles".

We have revised this figure in response to reviewer 2 (comment 5) and quantal content is now expressed as percent baseline, thus without units (see revised figure 7).

(13) Figure 6C should be auto-scaled from zero.

This has been fixed by revising that figure in response to reviewer 2 (comment 5)

(14) The data supporting the statement on impaired motor behavior and reduced vitality of adult IS4A should be either shown, or the statement should be removed (p. 13). Any hypotheses as to why IS4A is important for behavior and or viability?

As suggested, we have removed that statement.

(15) They do not provide any data supporting the statement that changes in PSC decay kinetics "counteract" the increase in PSC amplitude (p. 14). The sentence should be changed accordingly.

We agree and have down toned. It now reads on page 16, lines 7-9: “During repetitive firing, the median increase of PSC amplitude by ~10 % is potentially counteracted by the significant decrease in PSC half amplitude width by ~25 %...”.

(16) How do they explain the net locomotion speed increase in dI    -IIA larvae? Although the overall charge transfer is not affected during the stimulus protocols used, could the accelerated PSC decay affect PSP summation (I would actually expect a decrease in summation/slower speed)? Independent of the voltage-clamp data, is muscle input resistance changed in dI-IIA mutants?

Muscle input resistance is not altered in I-II mutants. We refer to potential causes of the locomotion effects of I-IIA excision in the discussion. On page 16, lines 12 to 21 it reads: “there is no difference in charge transfer from the motoneuron axon terminal to the postsynaptic muscle cell between ∆I-IIA and control. Surprisingly, crawling is significantly affected by the removal of I-IIA, in that the animals show a significantly increased mean crawling speed but no significant change in the number of stops. Given that the presynaptic function at the NMJ is not strongly altered upon I-IIA excision, and that I-IIA likely mediates also Ca_v2 functions outside presynaptic AZs (see above) and in other neuron types than motoneurons, and that the muscle calcium current is mediated by Ca_v1>/i> and Ca_v3, the effects of I-IIA excision of increasing crawling speed is unlikely caused by altered pre- or postsynaptic function at the NMJ. We judge it more likely that excision of I-IIA has multiple effects on sensory and pre-motor processing, but identification of these functions is beyond the scope of this study.”

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in the vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for a thorough biomarker review to determine optimal dosing in future studies.

Strengths:

This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making sharing this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.

Weaknesses:

No particular weaknesses. The study was carefully performed and limitations are discussed.

I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?

The authors thank the reviewer for the positive response. Regarding the ELISA assays used to measure the array of complement proteins described, these were extensively validated for the following parameters: specificity, intra-assay and inter-assay precision, accuracy, stability, reference range, and parallelism. All assays were validated in plasma, vitreous and aqueous humour. Due to the limited volume and availability of ocular fluids from individuals in the study, validation in vitreous and aqueous matrices was performed using a pool of several samples from post-mortem donors. At the time this study was initiated, the Millipore Luminex complement panels and the Quidel C3a and Ba EIA were the most sensitive assays and the only commercially available options capable of measuring the proteins of interest in the context of limited vitreous and aqueous humor sample. The concentrations measured were observed at similar ranges as those published in the literature using assays in distinct patient populations e.g. in (Mandava et al, Invest Ophthalmol Vis Sci, 2020).

Measurements from vitreous and aqueous from subject samples were deemed reportable if they were within the quantifiable ranges defined for these sample types during the validation (coefficient of variation of 20%, or 30% when results were below the lower limit of quantification but above limit of detection). Notably, given the limited amount of biomarker data due to small sample size, we share results from outlier biomarker measurements, to illustrate the heterogeneity in sample quality. We further publish plasma sample biomarker results in supplemental table 5 wherein complement protein concentrations can be observed and compared to normal ranges in the literature.

Adding confidence to the robustness of our assays was the observation that some of the complement proteins quantified by standard assay (e.g. plate and bead-based ELISAs) were also measured by the OLINK assay, and there was a general trend observed for positive correlation between results from both assays for FI levels post-treatment. However, we did not provide detailed correlative statistical analyses for further complement proteins as OLINK findings were deemed highly exploratory and hypothesis generating, and because the OLINK assay produced normalised results which are challenging to directly compare to ELISA results that were absolute.

Reviewer #2 (Public Review):

Summary:

The results presented demonstrate that AAV2-CFI gene therapy delivers long-term and marginally higher FI protein in vitreous humor that results in a concomitant reduction in the FB activation product Ba. However, the lack of clinical efficacy in the phase I/II study, possibly due to lower in vitro potency when compared to currently approved pegcetacoplan, raises important considerations for the utility of this therapeutic approach. Despite the early termination of the PPY988 clinical development program, the study achieved significant milestones, including the implementation of subretinal gene therapy delivery in older adults, complement biomarker comparison between serial vitreous humor and aqueous humor samples and vitreous humor proteomic assessment via Olink.

Strengths:

Long-term augmentation of FI protein in vitreous humor over 96 weeks and reduction of FB breakdown product Ba in vitreous humor suggests modulation of the complement system. Developed a novel in vitro assay suggesting FI's ability to reduce C3 convertase activity is weaker than pegcetacoplan and FH and may suggest a higher dose of FI will be required for clinical efficacy. Warn of the poor correlation between vitreous humor and aqueous humor biomarkers and suggest aqueous humor may not be a reliable proxy for vitreous humor with regard to complement activation/inhibition studies.

Weaknesses:

The vitrectomy required for the subretinal route of administration causes a long-term loss of total protein and may influence the interpretation of complement biomarker results even with normalization. The modified in vitro assay of complement activation suggests a several hundred-fold increase in FI protein is required to significantly affect C3a levels. Interestingly, the in vitro assay demonstrates 100% inhibition of C3a with pegcetacoplan and FH therapeutics, but only a 50% reduction with FI even at the highest concentrations tested. This observation suggests FI may not be rate-limiting for negative complement regulation under the in vitro conditions tested and potentially in the eye. It is unclear if pharmacokinetic and pharmacodynamic properties in aqueous humor and vitreous humor compartments are reliable predictors of FI level/activity after subretinal delivery AAV2-CFI gene therapy.

The authors thank the reviewer for the positive response and we agree that a limitation of the biomarker strategy for ocular gene therapy delivered to the retinal tissues is inferring PK/PD from vitreous and aqueous samples, which are the fluid sample compartments accessible from subjects available to measure molecular treatment response. We agree that these compartments may not accurately represent sub-retinal and tissue level complement turnover. In the discussion, line 508, we state: ‘Overall, the data suggests that fully functional FI is being secreted into the VH, but the regulatory effects on the level of Ba may be representative of convertase formation in the VH and not the macula retina/RPE nor the choroid. To validate this hypothesis, one approach would be to conduct vitreal sampling using an effective drug targeting C3 for GA in a larger cohort’.

However, the observation of elevation of FI in VH (and AH) post treatment, and changes in levels of downstream complement proteins that align with prior knowledge of control of alternative pathway activation, is compelling evidence that these measurements reflect modest but direct consequences of an FI-gene therapy that was delivered to the subretinal space. We add to the discussion, line 479: ‘the findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment.’

Furthermore, the observation of moderately raised FI levels in modelled VH post treatment being insufficient to control CS activation in vitro accords with the lack of clinical response observed at phase II. We note that measuring FI and complement biomarkers in retinal tissues from treated eyes at post-mortem would be one way to explore the PK/PD effects from AAV2-FI gene therapy.

Reviewer #3 (Public Review):

Summary:

The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of time points, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delivered FI was much less efficacious than Pegceptacoplan (FDA-approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.

Strengths:

The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.

Weaknesses:

Perhaps the conclusions drawn regarding the lack of observed efficacy are not fully justified. The authors focus on the hypothesis that not enough FI was synthesised in these patients receiving the PPY988 gene therapy, suggesting a delivery/transduction/expression issue. But beyond rare CFI genetic variants, most genetic associations with AMD imply that it is a FI-cofactor disease. A hypothesis supported by the authors' own experiments when they supplement their artificial VH matrix with FH and achieve a significantly greater breakdown of C3b than achieved with PPY988 treatment alone. Justification around doubling FI levels driving complement turnover refers to studies conducted in blood, which has an entirely different complement protein profile than VH. In Supplemental Table 5 we see there is approx. 10-fold more FH than FI (533ug/ml vs 50ug/ml respectively) so increasing FI levels will have a direct effect. Yet in Supplemental Table 3 we see there is more FI than FH in VH (608ng/ml vs 466ng/ml respectively). Therefore, adding more FI without more co-factors would have a very limited effect. Surely this demonstrates that the study was delivering the wrong payload, i.e. FI, which hit a natural ceiling of endogenous co-factors within the eye?

See response to reviewer 3’s review after reviewer 3 recommendations section below.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

The authors present strong evidence using validated complement biomarker assays and comprehensive proteomic profiling that support their findings. The presentation of complement biomarker data in vitreous humor and aqueous humor after FI augmentation is presented in a clear and concise format. The direct comparison of complement biomarkers in vitreous humor and aqueous humor from the same patients and demonstrating similarities and differences is important for the nascent complement gene therapy field. Developing a novel in vitro complement model and comparing pegcetacoplan, FH, and FI inhibitors provides the field with a valuable assay to benchmark other complement therapeutics. As currently designed, the in vitro assay supports why FI augmentation did not contribute to clinical success. It also suggests that non-physiological concentrations of FI protein (over 100 µg/mL) maximally inhibit C3a signal by ~50%, whereas both pegcetacoplan and FH reduce the signal by 100%. Does this suggest that CFI is not an appropriate therapeutic target to control complement overactivation in the eye?

We agree with the reviewer that the new data from the novel in vitro assay coupled with the clinical findings from the phase II gene therapy trial does now suggest FI is less attractive as a therapeutic target for controlling complement activation in the retinal tissues of subjects with Geographic Atrophy.

Reviewer #3 (Recommendations For The Authors):

I think the authors have done a great job collecting and analysing these clinical samples and elucidating the baseline complement protein profile in both the AH and VH. I only have minimal suggested changes.

Perhaps a more direct discussion around the limitations of adding more FI into environments where there is no excess of FI-cofactors present? And a discussion around the limitations of VH (and VA for that matter) biomarker sampling for a disease that primarily affects the neurosensory retina and outer blood/retinal barrier: perhaps the landscape of complement proteins is different yet again (although, admittedly, impossible to sample in a patient)? Finally, would it not have been better to perform complement activation experiments using the VH of treated patients directly rather than creating an artificial VH matrix (there may, or may not, be a couple of things in human VH that directly affect complement turnover...)?

We thank the reviewer for the supportive comments. This study is the first to describe FI and FH levels and respective ratios in vitreous humour (plus aqueous and plasma) from GA subjects, before and after sub-retinal gene therapy. It is compelling to observe that in the VH the levels of FI are greater than FH, the primary fluid phase co-factor for FI enzymatic activity. This new information does indeed argue against further FI supplementation (using gene therapy) being of added benefit to controlling the complement system in the broader population in individuals with Geographic Atrophy. We note that at the start of the clinical development of GT005/PPY988 AAV2-FI gene therapy, there was limited information on FI and FH levels in AMD in ocular fluids to inform the pharmacodynamics of complement activation. Now, by running the FOCUS phase I clinical trial and measuring the complement biomarker data using validated assays we have added to our understanding on the levels and ratio of FI to FH and other complement proteins in a larger number of GA subjects’ ocular samples.  We report the levels of complement proteins measured in ocular and systemic samples, to show the ranges and also the differences in ratios between the different matrices.   

Regarding the statement that FI supplementation could likely be ineffective due to limited FH cofactor; FH is not the only co-factor that FI may partner with at cell surfaces to become enzymatically active (others include MCP (CD46) and CR1 (CD35), although the latter is known to be of limited expression in the eye), as such, it is certainly true that other proteins may be present in the tissue altering the kinetics of FI’s activity after sub-retinal gene-therapy. In addition, the ratio between FI and FH detected in the VH may not be the same as in retinal tissue. As such, we agree that drawing insights from biomarkers in the VH may not fully reflect the disease processes and treatment response at the retinal cell layers, but it is the closest fluid sample available to sample tissue released soluble proteins. We acknowledge that VH biomarkers will not fully capture retinal disease processes and treatment responses, but due to their proximity, will reflect retina-released soluble proteins. The findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment. We agree modelling different inhibitor effects on complement activation directly using subject’s vitreous would be informative, but this was not possible due to the limitations of very small sample volume.

We add several sentences to the discussion regarding the points above. Line 473: ‘Notably, that FI does not reduce C3a breakdown to baseline even at supermolecular concentrations suggests cofactor limitation that might be more pronounced in VH given FH is not in excess of FI as is the case in blood 27. Moreover, there are additional cell-bound cofactors for FI that may be present in retinal tissue that are not present in the VH and could further alter the kinetics of the assay, such as MCP (CD46) albeit with disease related changes observed 37. However, the findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment.’

Minor comments:

Line 237: Missing parenthesis at the end of the sentence

Manuscript updated.

Line 435: Missing secondary parenthesis after .....Figure 3A)......

Manuscript updated.

Line 536: I don't think suggesting the addition of FHR proteins into the neurosensory retina/VH is such a good idea

The reference to FHRs has been clarified in the manuscript, line 558. The authors note that FHR dimerization domains have been engineered to dimerize Factor H constructs increasing half-life and potency for drugs currently in development.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Chlamydia spp. has a biphasic developmental cycle consisting of an extracellular, infectious form called an elementary body (EB) and an intracellular, replicative form known as a reticular body (RB). The structural stability of EBs is maintained by extensive cross-linking of outer membrane proteins while the outer membrane proteins of RBs are in a reduced state. The overall redox state of EBs is more oxidized than RBs. The authors propose that the redox state may be a controlling factor in the developmental cycle. To test this, alkyl hydroperoxide reductase subunit C (ahpC) was overexpressed or knocked down to examine effects on developmental gene expression. KD of ahpC induced increased expression of EB-specific genes and accelerated EB production. Conversely, overexpression of ahpC delayed differentiation to EBs. The results suggest that chlamydial redox state may play a role in differentiation.

Strengths:

Uses modern genetic tools to explore the difficult area of temporal gene expression throughout the chlamydial developmental cycle.

Weaknesses:

The environmental signals triggering ahpC expression/activity are not determined.

Thank you for your comments. Our data and those of others have shown that ahpC is expressed as a mid-developmental cycle gene (i.e., when RBs predominate in the population). This is true of most chlamydial genes, and the factors that determine developmental expression are not fully understood. As we noted in the Discussion, Chlamydia lacks AhpF/D orthologs, so it is not clear how AhpC activity is regulated. Related to determining environmental signals that trigger activity of AhpC, then this is a non-trivial issue in an obligate intracellular bacterium. Our assumption is that AhpC is constitutively active and that the increasing metabolic production of ROS eventually overcomes the innate (and stochastic) activity of AhpC to handle it, hence the threshold hypothesis. Importantly, the stochasticity is consistent with what we know about secondary differentiation in Chlamydia. We have tried to clarify these points in the Discussion.

Reviewer #2 (Public Review):

The factors that influence the differentiation of EBs and RBs during Chlamydial development are not clearly understood. A previous study had shown a redox oscillation during the Chlamydial developmental cycle. Based on this observation, the authors hypothesize that the bacterial redox state may play a role in regulating the differentiation in Chlamydia. To test their hypothesis, they make knock-down and overexpression strains of the major ROS regulator, ahpC. They show that the knock-down of ahpC leads to a significant increase in ROS levels leading to an increase in the production of elementary bodies and overexpression leads to a decrease in EB production likely caused by a decrease in oxidation. From their observations, they present an interesting model wherein an increase in oxidation favors the production of EBs.

Major concern:

In the absence of proper redox potential measurements, it is not clear if what they observe is a general oxidative stress response, especially when the knock-down of ahpC leads to a significant increase in ROS levels. Direct redox potential measurement in the ahpC overexpression and knock-down cells is required to support the model. This can be done using the roGFP-based measurements mentioned in the Wang et al. 2014 study cited by the authors.

Thank you for this suggestion. It is definitely something that we are looking to implement. However, our current vectors don’t allow for roGFP2 in combination with inducible expression of a gene of interest. We will need to completely redesign our vectors, and, in Chlamydia, the validation of such new vectors together with ahpC OE or KD may literally take a year or longer.

In lieu of this, we used the CellRox redox reactive dye to image live chlamydiae during normal growth or ahpC KD. During ahpC KD, these organisms are clearly much brighter than the control, uninduced conditions. These data are included as new Figure 5 to go along with the data we previously reported from the plate reader measurements. These data also clearly indicate that the readings we observed are from Chlamydia and not the host cell.

As far as a general oxidative stress response, Chlamydia lacks any transcriptional regulators akin to OxyR. The response we’ve measured, earlier expression of genes associated with secondary differentiation, would be an odd stress response not consistent with a focused program to respond to oxidative stress. We added new RNAseq data further showing this effect of a global earlier increase in late gene transcripts.

Reviewer #3 (Public Review):

Summary:

The study reports clearly on the role of the AhpC protein as an antioxidant factor in Chlamydia trachomatis and speculates on the role of AhpC as an indirect regulator of developmental transcription induced by redox stress in this differentiating obligate intracellular bacterium.

Strengths:

The question posed and the concluding model about redox-dependent differentiation in chlamydia is interesting and highly relevant. This work fits with other propositions in which redox changes have been reported during bacterial developmental cycles, potentially as triggers, but have not been cited (examples PMID: 2865432, PMID: 32090198, PMID: 26063575). Here, AhpC over-expression is shown to protect Chlamydia towards redox stress imposed by H2O2, CHP, TBHP, and PN, while CRISPRi-mediated depletion of AhpC curbed intracellular replication and resulted in increased ROS levels and sensitivity to oxidizing agents. Importantly, the addition of ROS scavengers mitigated the growth defect caused by AhpC depletion. These results clearly establish the role of AhpC affects the redox state and growth in Ct (with the complicated KO genetics and complementation that are very nicely done).

Weaknesses:

However, with respect to the most important implication and claims of this work, the role of redox in controlling the chlamydial developmental cycle rather than simply being a correlation/passenger effect, I am less convinced about the impact of this work. First, the study is largely observational and does not resolve how this redox control of the cell cycle could be achieved, whereas in the case of _Caulobacte_r, a clear molecular link between DNA replication and redox has been proposed. How would progressive oxidation in RBs eventually trigger the secondary developmental genes to induce EB differentiation? Is there an OxyR homolog that could elicit this change and why would the oxidation stress in RBs gradually accumulate during growth despite the presence of AhpC? In other words, the role of AhpC is simply to delay or dampen the redox stress response until the trigger kicks in, again, what is the trigger? Is this caused by increasing oxidative respiration of RBs in the inclusion? But what determines the redox threshold?

Thank you for your comments. As the reviewer notes, our work clearly demonstrates that AhpC acts as an antioxidant in Chlamydia trachomatis. Further, we have shown that transcription of the late cycle genes is altered upon altered activity of AhpC, which acts as a proof of concept that redox is (one of) the key factor(s) controlling developmental cycle progression in Chlamydia. Our new RNAseq data indicate that a broad swath of well characterized late genes is activated, which contradicts the argument that what we’ve measured is a stress response (unless activation of late genes in Chlamydia is generally a stress response (not the case based on other models of stress) – in which case we would not be able to differentiate between these effects). We hypothesize that ROS production from the metabolic activities of RBs serves as a signal to trigger secondary differentiation from RBs to EBs. How this exact threshold is determined is currently unknown as Chlamydia does not have any annotated homolog for OxyR. It is beyond the scope of the present manuscript to identify and then characterize what specific factor(s) control(s) this response. We fully anticipate that multiple factors are likely impacted by increasing oxidation, so dissecting the exact contributions of any one factor will represent (a) potential separate manuscript(s). Nonetheless, this remains an overarching goal of the lab yet remains challenging given the obligate intracellular nature of Chlamydia. Strategies that would work in a model system, like Caulobacter, that can be grown in axenic media are not easily implemented in Chlamydia.

As we noted above in another response, ahpC is transcribed as a mid-cycle gene with a peak of transcription corresponding to the RB phase of growth. We hypothesize that the gradual accumulation of ROS from metabolic activity will eventually supercede the ability of AhpC to detoxify it. This would result in any given RB asynchronously and stochastically passing this threshold (and triggering EB formation), which is consistent with what we know about secondary differentiation in Chlamydia.

I also find the experiment with Pen treatment to have little predictive power. The fact that transcription just proceeds when division is blocked is not unprecedented. This also happens during the Caulobacter cell cycle when FtsZ is depleted for most developmental genes, except for those that are activated upon completion of the asymmetric cell division and that is dependent on the completion of compartmentalization. This is a smaller subset of developmental genes in caulobacter, but if there is a similar subset that depends on division on chlamydia and if these are affected by redox as well, then the argument about the interplay between developmental transcription and redox becomes much stronger and the link more intriguing. Another possibility to strengthen the study is to show that redox-regulated genes are under the direct control of chlamydial developmental regulators such as Euo, HctA, or others and at least show dual regulation by these inputs -perhaps the feed occurs through the same path.

Comparisons to other model systems are generally of limited value with Chlamydia. All chlamydial cell division genes are mid-cycle (RB-specific) genes, just like ahpC. There is no evidence of a redox-responsive transcription factor (whether EUO, HctA, or another) that activates or represses a subset of genes in Chlamydia. Similarly, there is no evidence that redox directly and specifically impacts transcription of cell division genes based on our new RNAseq data. The types of experiments suggested are not easily implemented in Chlamydia, but we would certainly like to be able to do them.

As it pertains to penicillin specifically, we and others have shown that treating chlamydiae with Pen blocks secondary differentiation (meaning late genes are not transcribed). Effectively, Pen treatment freezes the organism in an RB state with continued transcription of RB genes. What we have shown is that, even during Pen treatment (which blocks late gene transcription), ahpC KD can overcome this block, which shows that elevated oxidation is able to trigger late gene expression even when the organisms are phenotypically blocked from progressing to EBs. The comparison from our perspective to Caulobacter is of limited value.

This redox-transcription shortcoming is also reflected in the discussion where most are about the effects and molecular mitigation of redox stress in various systems, but there is little discussion on its link with developmental transcription in bacteria in general and chlamydia.

We have edited the Discussion to include a broader description of the results and included additional citations as suggested by the reviewer (PMID: 32090198, PMID: 26063575). However, we found one suggested article (PMID: 2865432) is not relevant to our study, so we didn’t cite it in our present manuscript. There may have been a typo, so feel free to provide us the correct PMID that can be cited.

Reviewer #1 (Recommendations For The Authors):

(1) Line 146. A minor point, but inclusion-forming units directly measure infectious EBs. In some cases, the particle-to-infectivity ratio approaches unity. I don't believe IFUs are a "proxy".

Following reviewers comment we have modified the statement.

(2) Figure 2E. Results are normalized to uninduced. The actual number of IFUs in the uninduced should be provided.

In the revised version of the manuscript, we have provided actual number of IFUs at 24 and 48 hpi in the uninduced condition of both ahpC OE and EV.

(3) Figures 3B&D. The shades of gray are not possible to distinguish. I'd suggest color or direct labeling.

Following reviewer’s suggestion, in the latest version of the manuscript we have replaced gray shaded graphs with RGB colored graphs for better visualization and understanding.

(4) Lines 217-224, Figure 4. Is it possible to get micrographs of the reporter retention in chlamydiae to demonstrate that it is chlamydial ROS levels that are being measured and not cellular?

Following reviewer’s comment, we performed live-cell microscopy using uninfected HeLa cells and ahpC KD in the uninduced and induced conditions at 24 and 40 hpi. We have created new Fig. 5A with the quantitative ROS measurement graph done by the plate reader (old figure 4 E) and these new 24 hpi/40 hpi microscopy images (Fig 5B and S4).

(5) The Discussion is overly long and redundant. Large portions of the discussion are simply a rehash of the Results listing by figure number the relevant conclusions.

Following reviewer’s suggestion, the discussion is modified.

Reviewer #2 (Recommendations For The Authors):

(1) In Figure 2, ahpC is significantly overexpressed at 14 hpi. An IFA as in 2B for 14hpi will be useful. This will help to understand how quick the effect of ahpC overexpression is on development.

We have added 14 hpi IFA of ahpC and EV as part of Fig 2B.

(2) In Figure 2E, is there a reason that there is no increase in recoverable IFUs between 24h and 48h for the EV?

The graph in 2E is % of uninduced. For more clarity, we have mentioned absolute IFUs of uninduced samples in the revised manuscript and IFU level at 48 hpi IFU is higher than the 24 hpi.

(3) In Figure 3, Can relative levels of RB vs EB measured? This will provide a convincing case for the production of more EBs even when only less/more RBs are present. The same stands for Figure 4.

We assumed that the comment is for Fig. 2 not the Fig. 3 and following reviewer’s constructive suggestion, we have attempted to resolve the issue. We normalized log10 IFUs/ml with log10 gDNA for 24 hpi and added as figure 2F and 4E. This may resolve the reviewer’s concern about the levels of RBs and EBs.

(4) A colour-coded Figure 3B and D, instead of various shades of grey, will be easy for the reader to interpret.

Agreed with the reviewer. For better visualization and understanding of the data, we have replaced gray shaded graphs with RGB colored graphs in the latest version of the manuscript.

Reviewer #3 (Recommendations For The Authors):

Other comments:

(1) The first paragraph of the discussion should be deleted. It's not very useful or revealing and just delivers self-citations.

Following reviewer’s suggestion, we rewrote the discussion.

(2) The first paragraph of the results section does not present results. It's an intro.

We incorporated this information into the Intro as suggested.

(3) Has the redox difference between RBs and EBs been experimentally verified by these authors as depicted and claimed in Figure 1A with the cell-permeable, fluorogenic dye CellROX Deep Red for example? It is important to confirm this for EBs and RBs in this setup.

The difference between redox status of RBs and EBs is studied and established before by previous studies such as Wang et al., 2014.

(4) l77. Obligate intracellular alpha-proteobacteria also differentiate ... not only chlamydiae.

We have modified the sentence.

(5) l127. Is the redox state altered upon ahpC overexpression?

The ahpC overexpression strain showed hyper resistance for the tested oxidizing agents (including the highest concentration tested) indicating highly reduced conditions as a result of higher activity of AhpC.

Reviewer #1 (Public review):

Summary:

Carter et al. present the eduWOSM imaging platform, a promising development in open-source microscopy for educational purposes. The paper outlines the construction and setup of this versatile microscope, demonstrating its capabilities through three key examples: single fluorophore tracking of tubulin heterodimers in gliding microtubules, 4D deconvolution imaging and tracking of chromosome movements in dividing human cells, and automated single-particle tracking in vitro and in live cells, with motion classified into sub-diffusive, diffusive, and super-diffusive behaviors.

The paper is well-written and could be strengthened by providing more empirical data on its performance, addressing potential limitations, and offering detailed insights into its educational impact. The project holds great potential and more discussion on long-term support and broader applications would provide a more comprehensive view of its relevance in different contexts.

Strengths:

(1) The eduWOSM addresses a crucial need in education, providing research-quality imaging at a lower cost (<$10k). The fact that it is open-source adds significant value, enabling broad accessibility even in under resourced areas.<br /> (2) There is availability of extensive resources, including a dedicated website, YouTube channel, and comprehensive tutorial guides to help users replicate the microscope.<br /> (3) The compact, portable, and stable design makes it easy to build multiple systems for use in diverse environments, including crowded labs and classrooms. This is further enhanced by the fact multiple kind of imaging experiments can be run on the system, from live imaging to super-resolution imaging.<br /> (4) The paper highlights the user-friendly nature of the platform, with the imaging examples in the paper being acquired by undergrad students.

Weaknesses:

(1) The paper mentions the microscope is suitable not just for education but even for research purposes. This claim needs validation through quantitative comparison to existing research-grade microscopes in terms of resolution, signal-to-noise ratio, and other key metrics. Adding more rigorous comparisons would solidify its credibility for research use, which would immensely increase the potential of the microscope.<br /> (2) The open-source microscope field is crowded with various options catering to hobby, educational, and research purposes (e.g., openFLexure, Flamingo, Octopi, etc.). The paper would benefit from discussing whether any aspects set the eduWOSM platform apart or fulfill specific roles that other microscopes do not.<br /> (3) While the eduWOSM platform is designed to be user-friendly, the paper would benefit from discussing whether the microscope can be successfully built and operated by users without direct help from the authors. It's important to know if someone with basic technical knowledge, relying solely on the provided resources (website, YouTube tutorials, and documentation), can independently assemble, calibrate, and operate the eduWOSM.<br /> (4) Ensuring long-term support and maintenance of the platform is crucial. The paper would benefit from addressing how the eduWOSM developers plan to support updates, improvements, or troubleshooting.

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• AQUI la informacion es CORRECTA
• el problema es la traduccion al español!!!

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review): 

Summary: 

The authors introduced their previous paper with the concise statement that "the relationships between lineage-specific attributes and genotypic differences of tumors are not understood" (Chen et al., JEM 2019, PMID: 30737256). For example, it is not clear why combined loss of RB1 and TP53 is required for tumorigenesis in SCLC or other aggressive neuroendocrine (NE) cancers, or why the oncogenic mutations in KRAS or EGFR that drive NSCLC tumorigenesis are found so infrequently in SCLC. This is the main question addressed by the previous and current papers. 

One approach to this question is to identify a discrete set of genetic/biochemical manipulations that are sufficient to transform non-malignant human cells into SCLC-like tumors. One group reported the transformation of primary human bronchial epithelial cells into NE tumors through a complex lentiviral cocktail involving the inactivation of pRB and p53 and activation of AKT, cMYC, and BCL2 (PARCB) (Park et al., Science 2018, PMID: 30287662). The cocktail previously reported by Chen and colleagues to transform human pluripotent stem-cell (hPSC)-derived lung progenitors (LPs) into NE xenografts was more concise: DAPT to inactivate NOTCH signaling combined with shRNAs against RB1 and TP53. However, the resulting RP xenografts lacked important characteristics of SCLC. Unlike SCLC, these tumors proliferated slowly and did not metastasize, and although small subpopulations expressed MYC or MYCL, none expressed NEUROD1. 

MYC is frequently amplified or expressed at high levels in SCLC, and here, the authors have tested whether inducible expression of MYC could increase the resemblance of their hPSC-derived NE tumors to SCLC. These RPM cells (or RPM T58A with stabilized cMYC) engrafted more consistently and grew more rapidly than RP cells, and unlike RP cells, formed liver metastases when injected into the renal capsule. Gene expression analyses revealed that RPM tumor subpopulations expressed NEUROD1, ASCL1, and/or YAP1. 

The hPSC-derived RPM model is a major advance over the previous RP model. This may become a powerful tool for understanding SCLC tumorigenesis and progression and for discovering gene dependencies and molecular targets for novel therapies. However, the specific role of cMYC in this model needs to be clarified. 

cMYC can drive proliferation, tumorigenesis, or apoptosis in a variety of lineages depending on concurrent mutations. For example, in the Park et al., study, normal human prostate cells could be reprogrammed to form adenocarcinoma-like tumors by activation of cMYC and AKT alone, without manipulation of TP53 or RB1. In their previous manuscript, the authors carefully showed the role of each molecular manipulation in NE tumorigenesis. DAPT was required for NE differentiation of LPs to PNECs, shRB1 was required for expansion of the PNECs, and shTP53 was required for xenograft formation. cMYC expression could influence each of these steps, and importantly, could render some steps dispensable. For example, shRB1 was previously necessary to expand the DAPT-induced PNECs, as neither shTP53 nor activation of KRAS or EGFR had no effect on this population, but perhaps cMYC overexpression could expand PNECs even in the presence of pRB, or even induce LPs to become PNECs without DAPT. Similarly, both shRB1 and shTP53 were necessary for xenograft formation, but maybe not if cMYC is overexpressed. If a molecular hallmark of SCLC, such as loss of RB1 or TP53, has become dispensable with the addition of cMYC, this information is critically important in interpreting this as a model of SCLC tumorigenesis.  

The reviewer’s suggestion may be possible; indeed, in a recent report from our group (Gardner EE, et al., Science 2024) we have shown, using genetically engineered mouse modeling coupled with lineage tracing, that the cMyc oncogene can selectively expand Ascl1+ PNECs in the lung.

We agree with the reviewer that not having a better understanding of the individual components necessary and/or sufficient to transform hESC-derived LPs is an important shortcoming of this current work. However, we would like to stress three important points about the comments:  1) tumors were reviewed and the histological diagnoses were certified by a practicing pulmonary pathologist at WCM (our co-author, C. Zhang); 2 )the observed  transcriptional programs were consistent with primary human SCLC; and 3) RB1-proficient SCLC is now recognized as a rare presentation of SCLC (Febrese-Aldana CA, et al., Clin. Can. Res. 2022. PMID: 35792876).

To interpret the role of cMYC expression in hPSC-derived RPM tumors, we need to know what this manipulation does without manipulation of pRB, p53, or NOTCH, alone or in combination. Seven relevant combinations should be presented in this manuscript: (1) cMYC alone in LPs, (2) cMYC + DAPT, (3) cMYC + shRB1, (4) cMYC + DAPT + shRB1, (5) cMYC + shTP53, (6) cMYC + DAPT + shTP53, and (7) cMYC + shRB1 + shTP53. Wildtype cMYC is sufficient; further exploration with the T58A mutant would not be necessary. 

We respectfully disagree that an interrogation of the differences between the phenotypes produced by wildtype and Myc(T58A) would not be informative. (Our view is confirmed by the second reviewer; see below.)    It is well established that Myc gene or protein dosage can have profound effects on in vivo phenotypes (Murphy DJ, et al., Cancer Cell 2008. PMID: 19061836). The “RPM” model of variant SCLC developed by Trudy Oliver’s lab relied on the conditional T58A point mutant of cMyc, originally made by Rob Wechsler-Reya. While we do not discuss the differences between Myc and Myc(T58A), it is nonetheless important to present our results with both the WT and mutant MYC constructs, as we are aware of others actively investigating differences between them in GEMM models of SCLC tumor development.

We agree with the reviewer about the virtues of trying to identify the effects of individual gene manipulations; indeed our original paper (Chen et al., J. Expt. Med. 2019), describing the RUES2derived model of SCLC did just that, carefully dissecting events required to transform LPs towards a SCLC-like state. The central  purpose of the current study was to determine the effects of adding cMyc on the behavior of weakly tumorigenic SCLC-like cells cMyc.  Presenting data with these two alleles to seek effects of different doses of MYC protein seems reasonable.

This reviewer considers that there should be a presentation of the effects of these combinations on LP differentiation to PNECs, expansion of PNECs as well as other lung cells, xenograft formation and histology, and xenograft growth rate and capacity for metastasis. If this could be clarified experimentally, and the results discussed in the context of other similar approaches such as the Park et al., paper, this study would be a major addition to the field.  

Reviewer #2 (Public Review): 

Summary: 

Chen et al use human embryonic stem cells (ESCs) to determine the impact of wildtype MYC and a point mutant stable form of MYC (MYC-T58A) in the transformation of induced pulmonary neuroendocrine cells (PNEC) in the context of RB1/P53 (RP) loss (tumor suppressors that are nearly universally lost in small cell lung cancer (SCLC)). Upon transplant into immune-deficient mice, they find that RP-MYC and RP-MYC-T58A cells grow more rapidly, and are more likely to be metastatic when transplanted into the kidney capsule, than RP controls. Through single-cell RNA sequencing and immunostaining approaches, they find that these RPM tumors and their metastases express NEUROD1, which is a transcription factor whose expression marks a distinct molecular state of SCLC. While MYC is already known to promote aggressive NEUROD1+ SCLC in other models, these data demonstrate its capacity in a human setting that provides a rationale for further use of the ESC-based model going forward. Overall, these findings provide a minor advance over the previous characterization of this ESC-based model of SCLC published in Chen et al, J Exp Med, 2019. 

We consider the findings more than a “minor” advance in the development of the model, since any useful model for SCLC would need to form aggressive and metastatic tumors.

The major conclusion of the paper is generally well supported, but some minor conclusions are inadequate and require important controls and more careful analysis. 

Strengths:

(1) Both MYC and MYC-T58A yield similar results when RP-MYC and RP-MYCT58A PNEC ESCs are injected subcutaneously, or into the renal capsule, of immune-deficient mice, leading to the conclusion that MYC promotes faster growth and more metastases than RP controls. 

(2) Consistent with numerous prior studies in mice with a neuroendocrine (NE) cell of origin (Mollaoglu et al, Cancer Cell, 2017; Ireland et al, Cancer Cell, 2020; Olsen et al, Genes Dev, 2021), MYC appears sufficient in the context of RB/P53 loss to induce the NEUROD1 state. Prior studies also show that MYC can convert human ASCL1+ neuroendocrine SCLC cell lines to a NEUROD1 state (Patel et al, Sci Advances, 2021); this study for the first time demonstrates that RB/P53/MYC from a human neuroendocrine cell of origin is sufficient to transform a NE state to aggressive NEUROD1+ SCLC. This finding provides a solid rationale for using the human ESC system to better understand the function of human oncogenes and tumor suppressors from a neuroendocrine origin. 

Weaknesses:

(1) There is a major concern about the conclusion that MYC "yields a larger neuroendocrine compartment" related to Figures 4C and 4G, which is inadequately supported and likely inaccurate. There is overwhelming published data that while MYC can promote NEUROD1, it also tends to correlate with reduced ASCL1 and reduced NE fate (Mollaoglu et al, Cancer Cell, 2017; Zhang et al, TLCR, 2018; Ireland et al, Cancer Cell, 2020; Patel et al, Sci Advances, 2021). Most importantly, there is a lack of in vivo RP tumor controls to make the proper comparison to judge MYC's impact on neuroendocrine identity. RPM tumors are largely neuroendocrine compared to in vitro conditions, but since RP control tumors (in vivo) are missing, it is impossible to determine whether MYC promotes more or less neuroendocrine fate than RP controls. It is not appropriate to compare RPM tumors to in vitro RP cells when it comes to cell fate. Upon inspection of the sample identity in S1B, the fibroblast and basal-like cells appear to only grow in vitro and are not well represented in vivo; it is, therefore, unclear whether these are transformed or even lack RB/P53 or express MYC. Indeed, a close inspection of Figure S1B shows that RPM tumor cells have little ASCL1 expression, consistent with lower NE fate than expected in control RP tumors. 

We would like to clarify two points related to the conclusions that we draw about MYC’s ability to promote an increase in the neuroendocrine fraction in hESC-derived cultures:  1) The comparisons in Figures 4C were made between cells isolated in culture following the standard 50 day differentiation protocol, where, following generation of LPs around day 25, MYC was added to the other factors previously shown to enrich for a PNEC phenotype (shRB1, shTP53, and DAPT). Therefore, the argument that MYC increased the frequency of “neuroendocrine cells” (which we define by a gene expression signature) is a reasonable conclusion in the system we are using; and 2) following injection of these cells into immunocompromised mice, an ASCL1-low / NEUROD1-high presentation is noted (Supplemental Figures 1F-G). In the few metastases that we were able use to sequence bulk RNA, there is an even more pronounced increase in expression of NEUROD1 with a decrease in ASCL1.

Some confusion may have arisen from our previous characterization of neuroendocrine (NE) cells using either ASCL1 or NEUROD1 as markers. To clarify, we have now designated cells positive for ASCL1 as classical NE cells and those positive for NEUROD1 as the NE variant. According to this revised classification, our findings indicate that MYC expression leads to an increase in the NEUROD1+ NE variant and a decrease in ASCL1+ classical NE cells. This adjustment has been reflected on the results section titled, “Inoculation of the renal capsule facilitates metastasis of the RUES2-derived RPM tumors” of the manuscript.  

From the limited samples in hand, we compared the expression of ASCL1 and NEUROD1 in the weakly tumorigenic hESC RP cells after successful primary engraftment into immunocompromised mice. As expected, the RP tumors were distinguished by the lack of expression of NEUROD1, compared to levels observed in the RPM tumors.

In addition, since MYC appears to require Notch signaling to induce  NE fate (cf Ireland et al), the presence of DAPT in culture could enrich for NE fate despite MYC's presence. It's important to clarify in the legend of Fig 4A which samples are used in the scRNA-seq data and whether they were derived from in vitro or in vivo conditions (as such, Supplementary Figure S1B should be provided in the main figure). Given their conclusion is confusing and challenges robustly supported data in other models, it is critical to resolve this issue properly. I suspect when properly resolved, MYC actually consistently does reduce NE fate compared to RP controls, even though tumors are still relatively NE compared to completely distinct cellular identities such as fibroblasts.

We have clarified the source of tumor sequencing data and the platform (single cell or bulk) in Figure 4 and Supplemental Figure 1. To reiterate – the RNA sequencing results from paired metastatic and primary tumors from the RPM model are derived from bulk RNA;  the single cell RNA data in RP or RPM datasets are from cells in culture.  These distinctions are clarified in the legend to Supplemental Figure 1.

(2) The rigor of the conclusions in Figure 1 would be strengthened by comparing an equivalent number of RP animals in the renal capsule assay, which is n = 6 compared to n = 11-14 in the MYC conditions.

As we did not perform a power calculation to determine a sample size required to draw a level of statistical significance from our conclusions, this comment is not entirely accurate. Our statistical rigor was limited by the availability of samples from the RP tumor model.

(3) Statistical analysis is not provided for Figures 2A-2B, and while the results are compelling, may be strengthened by additional samples due to the variability observed. 

We acknowledge that the cohorts are relatively small but we have added statistical comparisons in Figure 2B. 

(4a) Related to Figure 3, primary tumors and liver metastases from RPM or RPM-T58A-expressing cells express NEUROD1 by immunohistochemistry (IHC) but the putative negative controls (RP) are not shown, and there is no assessment of variability from tumor to tumor, ie, this is not quantified across multiple animals. 

The results of H&E and IF staining for ASCL1, NEUROD1, CGRP, and CD56 in negative control (RP tumors) are presented in the updated Figure 3F-G.

(4b) Relatedly, MYC has been shown to be able to push cells beyond NEUROD1 to a double-negative or YAP1+ state (Mollaoglu et al, Cancer Cell, 2017; Ireland et al, Cancer Cell, 2020), but the authors do not assess subtype markers by IHC. They do show subtype markers by mRNA levels in Fig 4B, and since there is expression of ASCL1, and potentially expression of YAP1 and POU2F3, it would be valuable to examine the protein levels by IHC in control RP vs. RPM samples.

YAP1 positive SCLC is still somewhat controversial, so it is not clear what value staining for YAP1 offers beyond showing the well-established markers, ASCL1 and NEUROD1.  

(5) Given that MYC has been shown to function distinctly from MYCL in SCLC models, it would have raised the impact and value of the study if MYC was compared to MYCL or MYCL fusions in this context since generally, SCLC expresses a MYC family member. However, it is quite possible that the control RP cells do express MYCL, and as such, it would be useful to show. 

We now include Supplemental Figure S2 to illustrate four important points raised by this reviewer and others:  1) expression of MYC family members in the merged dataset (RP and RPM) is low or undetectable in the basal/fibroblast cultures; 2) MYC does have a weak correlation with EGFP in the neuroendocrine cluster when either WT MYC or T58A MYC is overexpressed; 3) MYCL and MYCN are detectable, but at low levels compared to CMYC; and 4) Expression of  ASCL1 is anticorrelated with MYC expression across the merged single cell datasets using RP and RPM models.

Reviewer #3 (Public Review): 

Summary: 

The authors continue their study of the experimental model of small cell lung cancer (SCLC) they created from human embryonic stem cells (hESCs) using a protocol for differentiating the hESCs into pulmonary lineages followed by NOTCH signaling inactivation with DAPT, and then knockdown of TP53 and RB1 (RP models) with DOX inducible shRNAs. To this published model, they now add DOX-controlled activation of expression of a MYC or T58A MYC transgenes (RPM and RPMT58A models) and study the impact of this on xenograft tumor growth and metastases. Their major findings are that the addition of MYC increased dramatically subcutaneous tumor growth and also the growth of tumors implanted into the renal capsule. In addition, they only found liver and occasional lung metastases with renal capsule implantation. Molecular studies including scRNAseq showed that tumor lines with MYC or T58A MYC led surprisingly to more neuroendocrine differentiation, and (not surprisingly) that MYC expression was most highly correlated with NEUROD1 expression. Of interest, many of the hESCs with RPM/RPMT58A expressed ASCL1. Of note, even in the renal capsule RPM/RPMT58A models only 6/12 and 4/9 mice developed metastases (mainly liver with one lung metastasis) and a few mice of each type did not even develop a renal sub capsule tumor. The authors start their Discussion by concluding: " In this report, we show that the addition of an efficiently expressed transgene encoding normal or mutant human cMYC can convert weakly tumorigenic human PNEC cells, derived from a human ESC line and depleted of tumor suppressors RB1 and TP53, into highly malignant, metastatic SCLC-like cancers after implantation into the renal capsule of immunodeficient mice.". 

Strengths: 

The in vivo study of a human preclinical model of SCLC demonstrates the important role of c-Myc in the development of a malignant phenotype and metastases. Also the role of c-Myc in selecting for expression of NEUROD1 lineage oncogene expression. 

Weaknesses: 

There are no data on results from an orthotopic (pulmonary) implantation on generation of metastases; no comparative study of other myc family members (MYCL, MYCN); no indication of analyses of other common metastatic sites found in SCLC (e.g. brain, adrenal gland, lymph nodes, bone marrow); no studies of response to standard platin-etoposide doublet chemotherapy; no data on the status of NEUROD1 and ASCL1 expression in the individual metastatic lesions they identified. 

We have acknowledged from the outset that our study has significant limitations, as noted by this reviewer, and we explained in our initial letter of response why we need to present this limited, but still consequential, story at this time. 

In particular, while we have attempted orthotopic transplantations of RPM tumor cells into NSG mice (by tail vein or intra-pulmonary injection, or intra-tracheal instillation of tumor cells), these methods were not successful in colonizing the lung. Additionally, we have compared the efficacy of platinum/etoposide to that of removing DOX in established RPM subcutaneous tumors, but we chose not to include these data as we lacked a chemotherapy responsive tumor model, and thus could not say with confidence that the chemotherapeutic agants were active and that the RPM models were truly resistant to standard SCLC chemotherapy. In a discussion about other metastatic sites, we have now included the following text: 

“In animals administered DOX, histological examinations showed that approximately half developed metastases in distant organs, including the liver or lung (Figure 1D). No metastases were observed in the bone, brain, or lymph nodes. For a more detailed assessment, future studies could employ more sensitive imaging methods, such as luciferase imaging.”

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors): 

Technical points related to Major Weakness #1: 

For Figure 4: Cells were enriched for EGFP-high cells only, under the hypothesis that cells with lower EGFP may have silenced expression of the integrated vector. Since EGFP is expressed only in the shRB1 construct, selection for high EGFP may inadvertently alter/exclude heterogeneity within the transformed population for the other transgenes (shP53, shMYC/MYC-T58A). Can authors include data to show the expression of MYC/MYC T58A in EGFP-high v -med v-low cells? MYC levels may alter the NEdifferentiation status of tumor cells. 

Please now refer to Supplemental Figure S2.

Related to the appropriateness of the methods for Figure 4C, the authors state, "We performed differential cluster abundance analysis after accounting for the fraction of cells that were EGFP+". If only EGFP+ cells were accounted for in the analysis for 4C, the majority of RP cells in the "Neuroendocrine differentiated" cluster would not be included in the analysis (according to EGFP expression in Fig S1A-B), and therefore inappropriately reduce NE identity compared to RPM samples that have higher levels of EGFP. 

There is no consideration or analysis of cell cycling/proliferation until after the conclusion is stated. Yet, increased proliferation of MYC-high vs MYC-low cultures would enhance selection for more tumors (termed "NE-diff") than non-tumors (basal/fibroblast) in 2D cultures. 

The expression of MYC itself isn't assessed for this analysis but assumed, and whether higher levels of MYC/MYC-T58A may be present in EGFP+ tumor cells that are in the NE-low populations isn't clear. Can MYC-T58A/HA also be included in the reference genome? 

We did not include an HA tag in our reference transcriptome. For [some] answers to this and other related questions, please refer to Supplemental Figure S2.

Reviewer #3 (Recommendations For The Authors): 

(1) The experiments are all technically well done and clearly presented and represent a logical extension exploring the role of c-Myc in the hESC experimental model system. 

We appreciate this supportive comment!

(2) It is of great interest that both the initial RP model only forms "benign" tumors and that with the addition of a strong oncogene like c-myc, where expression is known to be associated with a very bad prognosis in SCLC, that while one gets tumor formation there are still occasional mice both for subcutaneous and renal capsule test sites that don't get tumors even with the injection of 500,000 RPM/RPMT58A cells. In addition, of the mice that do form tumors, only ~50% exhibit metastases from the renal sub-capsule site. The authors need to comment on this further in their Discussion. To me, this illustrates both how incredibly resistant/difficult it is to form metastases, thus indicating the need for other pathways to be activated to achieve such spread, and also represents an opportunity for further functional genomic tests using their preclinical model to systematically attack this problem. Obvious candidate genes are those recently identified in genetically engineered mouse models (GEMMs) related to neuronal behavior. In addition, we already know that full-fledged patient-derived SCLC when injected subcutaneously into immune-deprived mice don't exhibit metastases - thus, while the hESC RPM result is not surprising, it indicates to me the power of their model (logs less complicated genetically than a patient SCLC) to sort through a mechanism that would allow metastases to develop from subcutaneous sites. The authors can point these things out in their Discussion section to provide a "roadmap" for future research. 

Although we remain mindful of the relatively small cohorts we have studied, the thrust of Reviewer #3’s comments is now included in the Discussion. And there is, of course, a lot more to do, and it has taken several years already to get to this point. Additional information about the prolonged gestation of this project and about the difficulties of doing more in the near future was described in our initial response to reviewers/Editor, included near the start of this letter.    

(3) I will state the obvious that this paper would be much more valuable if they had compared and contrasted at least one of the myc family members (MYCL or MYCN) with the CMYC findings whatever the results would be. Most SCLC patients develop metastases, and most of their tumors don't express high levels of CMYC (and often use MYCL). In any event, as the authors Discuss, this will be an important next stage to test.

We have acknowledged and explained the limitations of the work in several ways. Further, we were unaware of the relationship between metastases and the expression of MYC and MYCL1 noted by the reviewer; we will look for confirmation of this association in any future studies, although we have not encountered it in current literature.

(4) Their assays for metastases involved looking for anatomically "gross" lesions. While that is fine, particularly given that the "gross" lesions they show in figures are actually pretty small, we still need to know if they performed straightforward autopsies on mice and looked for other well-known sites of metastases in SCLC patients besides liver and lung - namely lymph nodes, adrenal, bone marrow, and brain. I would guess these would probably not show metastatic growth but with the current report, we don't know if these were looked for or not. Again, while this could be a "negative" result, the paper's value would be increased by these simple data. Let's assume no metastases are seen, then the authors could further strengthen the case for the value of their hESC model in systematically exploring with functional genomics the requirements to achieve metastases to these other sites.

We have included descriptions of what we found and didn’t find at other potential sites of metastasis in the results section, with the following sentences: 

“In animals administered DOX, histological examinations showed that approximately half developed metastases in distant organs, including the liver or lung (Figure 1D). No metastases were observed in the bone, brain, or lymph nodes. For a more detailed assessment, future studies could employ more sensitive imaging methods, such as luciferase imaging.”

(5) Related to this, we have no idea if the mice that developed liver metastases (or the one mouse with lung metastasis) had more than one metastatic site. They will know this and should report it. Again, my guess is that these were isolated metastases in each mouse. Again, they can indicate the value of their model in searching for programs that would increase the number of the various organs. 

We appreciate the suggestion. We observed that one of the mice developed metastatic tumors in both the liver and lungs. This information has been incorporated into the Results section.

(6) While renal capsule implantation for testing growth and metastatic behavior is reasonable and based on substantial literature using this site for implantation of patient tumor specimens, what would have increased the value of the paper is knowing the results from orthotopic (lung implantation). Whatever the results were (they occurred or did not occur) they will be important to know. I understand the "future experiments" argument, but in reading the manuscript this jumped out at me as an obvious thing for the authors to try. 

We conducted orthotopic implantation several ways, including via intra-tracheal instillation of 0.5 million RP or RPM cells in PBS per mouse. However, none of the subjects (0/5 mice) developed tumor-like growths and the number of animals used was small. Further, this outcome could be attributed to biological or physical factors. For instance, the conducting airway is coated with secretory cells producing protective mucins and may not have retained the 0.5 million cells. This is one example that may have hindered effective colonization. Future adjustments, such as increasing the number of cells, embedding them in Matrigel, or damaging the airway to denude secretory cells and trigger regeneration might alter the outcomes. These ideas might guide future work to strengthen the utility of the models.

(7) Another obvious piece of data that would have improved the value of this manuscript would be to know whether the RPM tumors responded to platin-etoposide chemotherapy. Such data was not presented in their first RP hESC notch inhibition paper (which we now know generated what the authors call "benign" tumors). While I realize chemotherapy responses represent other types of experiments, as the authors point out one of the main reasons they developed their new human model was for therapy testing. Two papers in and we are all still asking - does their model respond or not respond dramatically to platin-etoposide therapy? Whatever the results are they are a vital next step in considering the use of their model. 

Please see the comments above regarding our decision not to include data from a clinical trial that lacked appropriate controls.

(8) The finding of RPM cells that expressed NEUROD1, ASCL1, or both was interesting. From the way the data were presented, I don't have a clear idea which of these lineage oncogenes the metastatic lesions from ~11 different mice expressed. Whatever the result is it would be useful to know - all NEUROD1, some ASCL1, some mixed etc.

Based on the bulk RNA-sequencing of a few metastatic sites (Figure 4H), what we can demonstrate is that all sites were NEUROD1 and expressed low or no detectable  ASCL1.

(9) While several H&E histologic images were presented, even when I enlarged them to 400% I couldn't clearly see most of them. For future reference, I think it would be important to have several high-quality images of the RP, RPM, RPMT58A subcutaneous tumors, sub-renal capsule tumors, and liver and lung metastatic lesions. If there is heterogeneity in the primary tumors or the metastases it would be important to show this. The quality of the images they have in the pdf file is suboptimal. If they have already provided higher-quality images - great. If not, I think in the long run as people come back to this paper, it will help both the field and the authors to have really great images of their tumors and metastases. 

We have attempted to improve the quality of the embedded images. Digital resolution is a tradeoff with data size – higher resolution images are always available upon request, but may not be suitable  for generation of figures in a manuscript viewed on-line.

Lingopie vs lingQ: is the difference between them in meaning and translation for multiple words, or context?

Does Lingopie really lack this obviously needed multi-word ability?

new comment

Author response:

The following is the authors’ response to the previous reviews.

We have carefully addressed all the reviewers' suggestions, and detailed responses are provided at the end of this letter. In summary:

• We conducted two additional replicates of the study to obtain more robust and reliable data.

• The Introduction has been revised for greater clarity and conciseness.

• The Results section was shortened and reorganized to highlight the key findings more effectively.

• The Discussion was modified according to the reviewers' suggestions, with a focus on reorganization and conciseness.

We hope you find this revised version of the manuscript satisfactory.

Reviewer #1 (Public Review):

Summary:

This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.

Strengths:

Host blood meal source, temperature, and photoperiod were all examined together.

Weaknesses:

The study was conducted in laboratory conditions with a local population of Cx. quinquefasciatus from Argentina. I'm not sure if there is any evidence for a seasonal shift in the host use pattern in Cx. quinquefasciatus populations from the southern latitudes.

Comments on the revision: 

Overall, I am not quite convinced about the possible shift in host use in the Argentinian populations of Cx. quinquefasciatus. The evidence from the papers that the authors cite is not strong enough to derive this conclusion. Therefore, I think that the introduction and discussion parts where they talk about host shift in Cx. quinquefasciatus should be removed completely as it misleads the readers. I suggest limiting the manuscript to talking only about the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quinquefasciatus. 

As mentioned in the previous revision, we agree on the reviewer observation about the lack of evidence on seasonal shift in the host use pattern in Cx. quinquefasciatus populations from Argentina. We include this topic in the discussion.

Additionally, we also added a paragraph in the discussion section to include the limitations of our study and conclusions. One of them is the fact that our results are based on controlled conditions experiments. Future studies are needed to elucidate if the same trend is found in the field.

Reviewer #1 (Recommendations for the authors): 

Abstract

Line 73: shift in feeding behavior

Accepted as suggested. 

Discussion

Line 258: addressed that Accepted as suggested.

Line 263: blood is nutritionally richer

Accepted as suggested.

Reviewer #2 (Public Review): 

Summary:

Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness on birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used a generalized linear model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite from that hypothesized. The authors have done a very good job of addressing many of the reviewer concerns, with several exception that continue to cause concern about the conclusions of the study. 

Strengths:

(1) Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.

(2) The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field. 

(3) The manuscript has become a lot clearer and easier to read with the revisions - thank you to the authors for working hard to make many of the suggested changes. 

Weaknesses:

(1) The authors have decided not to follow the suggestion of conducting experimental replicates of the study. This is understandable given the significant investment of resources and time necessary, however, it leaves the study lacking support. Experimental replication is an important feature of a strong study and helps to provide confidence that the observed patterns are real and replicable. Without replication, I continue to lack confidence in the conclusions of the study. 

We included replicates as suggested.  

(2) The authors have included some additional discussion about the counterintuitive nature of their results, but the paragraph discussing this in the discussion was confusing. I believe that this should be revised. This is a key point of the paper and needs to be clear to the reader.

Revised as suggested. 

(3) There should be more discussion of the host switching observed in the two studies conducted in Argentina referenced by the authors. Since host switching is the foundation for the hypothesis tested in this paper, it is important to fully explain what is currently known in Argentina. 

Accepted as suggested.

(4) In some cases, the explanations of referenced papers are not entirely accurate. For example, when referencing Erram et al 2022, I think the authors misrepresented the paper's discussion regarding pre-diuresis- Erram et al. are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility (rather than leading to higher fecundity on birds, as stated in this manuscript). The study performed by Erram et al. also didn't prove this phenomenon, they just suggest it as a possible mechanism to explain their results, so that should be made clear when referencing the paper. 

Changed as suggested.

(5) In some cases, the conclusions continue to be too strongly worded for the evidence available. For example, lines 322-324: I don't think the data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness. 

Redaction was modified as suggested to tight our discussion with results.

(6) There is limited mention of the caveat that this experiment performed with simulated seasonality that does not perfectly replicate seasonality in the field. I think this caveat should be discussed in the discussion (e.g. that humidity is held constant).

This topic is now included in the discussion as suggested. 

Reviewer #2 (Recommendations for the authors): 

59-60: These terms should end with -phagic instead of -philic. These papers study blood feeding patterns, not preference. I understand that the Janssen papers calls it "mammalophilic" in their title, but this was an incorrect use of the term in their paper. There are some review papers that explain the difference in this terminology if it's helpful.

Accepted as suggested. 

73: edit to "in" feeding behavior 

Accepted as suggested.

77-78: Given that the premise of your study is based on the phenomenon of host switching, I suggest that you expand your discussion of these two papers. What did they observe? Which hosts did they switch from / to and how dramatic was the shift?

Accepted as suggested. 

79: replace acknowledged with experienced 

Accepted as suggested.

79-80: the way that this is written is misleading. It suggests that Spinsanti showed that seasonal variation in SLEV could be attributed to a host shift, which isn't true. This citation should come before the comma and then you should use more cautious language in the second half. E.g which MIGHT be possible to attribute to .... 

Accepted as suggested.

80-82: this is not convincing. Even if the Robin isn't in Argentina, Argentina does have migrating birds, so couldn't this be the case for other species of birds? Do any of the birds observed in previous blood meal analyses in Argentina migrate? If so, couldn't this hypothesis indeed play a role? 

A paragraph about this topic was added to the discussion as suggested.

90: hypotheses for what? The fall peak in cases? Or host switching? 

Changed to be clearer.

98: where was this mentioned before? I think "as mentioned before" can be removed. 

Accepted as suggested.

101: edit to "whether an interaction effect exists" 

Accepted as suggested.

104: edit to "We hypothesize that..." 

Accepted as suggested.

106: reported host USE changes, not host PREFERENCE changes, right? 

All the terminology was change to host pattern and not preference to avoid confusion.

200: Briefly reading Carsey and Harden, it looks like the methodology was developed for social science. Is there anything you can cite to show this applied to other types of data? If not, I think this requires more explanation in your MS. 

This was removed as replicates were included.

237-239: I think it is best not to make a definitive statement about greater/higher if it isn't statistically significant; I suggest modifying the sentences to state that the differences you are listing were not significantly different up front rather than at the end, otherwise if people aren't reading carefully, they may get the wrong impression. 

Accepted as suggested.

245: you only use the term MS-I once before and I forgot what it meant since it wasn't repeated, so I had to search back through with command-F. I suggest writing this out rather than using the acronym. 

Accepted as suggested.

249: edit to: "an interaction exists between the effect of..." 

Accepted as suggested.

253-254: greater compared to what? 

Change for clearness. 258-260: edit for grammar 

Accepted as suggested.

260-262: edit for grammar; e.g. "However, this assumption lacks solid evidence; there is a scarcity of studies regarding nutritional quality of avian blood and its impact on mosquito fitness." 

Accepted as suggested.

263: edit: blood is nutritionally... 

Accepted as suggested.

264-267: This doesn't sound like an accurate interpretation of what the paper suggests regarding pre-diuresis in their discussion - they are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility. They also don't show this, they just suggest it as a possible mechanism to explain their results. 

This topic was removed given the restructuring of discussion.

253-269: You should tie this paragraph back to your results to explicitly compare/contrast your findings with the previous literature. 

Accepted as suggested.

270-282: This paragraph would be a good place to explain the caveat of working in the laboratory - for example, humidity was the same across the two seasons which I'm guessing isn't the case in the field in Argentina. You can discuss what aspects of laboratory season simulation do not accurately replicate field conditions and how this can impact your findings. You said in your response to the reviewers that you weren't interested in measuring other variables (which is fair, and not expected!), but the beauty of the discussion section is to be able to think about how your experimental design might impact your results - one possibility is that your season simulation may not have produced the results produced by true seasonal shifts. 

Accepted as suggested.

279-281: You say your experiment was conducted within the optimal range, which would suggest that both summer and autumn were within that range, but then you only talk about summer as optimal in the following sentence. 

Changed for clearness.

281-282: You should clarify this sentence - state what the interaction has an effect on. 

Accepted as suggested.

283-291: I appreciate that your discussion now acknowledges the small sample size and the questions that remain unanswered due to the results being opposite to that of the hypothesis, but this paragraph lacks some details and in places doesn't make sense. 

I think you need to emphasize which groups had small sample size and which conclusions that might impact. I also think you need to explain why the sample size was substantially smaller for some groups (e.g. did they refuse to feed on the mouse in the autumn?). I appreciate that sample sizes are hard to keep high across many groups and two gonotrophic periods, but unfortunately, that is why fitness experiments are so hard to do and by their nature, take a long time. I understand that other papers have even lower sample size, but I was not asked to review those papers and would have had the same critique of them. I don't believe that creating simulated data via a Monte Carlo approach can make up for generating real data. As I understand it from your explanation, you are parametrizing the Monte Carlo simulations with your original data, which was small to begin with for autumn mouse. Using this simulation doesn't seem like a satisfactory replacement for an experimental replicate in my opinion. I maintain that at least a second replicate is necessary to see whether the patterns that you have observed hold. 

The performing of a power analysis and addition of more replicates tried to solve the issue of sample size. More about this critic is added in the discussion. The simulation approach was totally removed.

Regarding the directionality of the interaction effect, I think this warrants more discussion. Lines 287-291 don't make sense to me. You suggest that feeding on birds in the autumn may confer a reproductive advantage when conditions are more challenging. But then why wouldn't they preferentially feed on birds in the autumn, rather than mammals? I suggest rewriting this paragraph to make it clearer. 

Accepted as suggested.

297: earlier mentioned treatments? Do you mean compared to the first gonotrophic cycle? This isn't clear. 

Changed for clearness.

302-303: Did you clarify whether you are allowed to reference unpublished data in eLife? 

This was removed to follow the guidelines of eLife.

316-317: "it becomes apparent" sounds awkward, I suggest rewording and also explaining how this conclusion was made. 

Accepted as suggested.

322-324: I think that this statement is too strongly worded. I don't think your data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness. Please modify this and make your conclusions more cautious and closely linked to what you actually demonstrated. 

Accepted as suggested.

325: change will perform to would have 

Accepted as suggested.

326: add to the sentence: "and vice versa in the summer" 

Accepted as suggested.

330: possible explanations, not explaining scenarios. 

Accepted as suggested.

517: I think you should repeat the abbreviation definitions in the caption to make it easier for readers, otherwise they have to flip back and forth which can be difficult depending on formatting.

Accepted as suggested. 

In general, I think that your captions need more information. I think the best captions explain the figure relatively thoroughly such that the reader can look at the figure and caption and understand without reading the paper in depth. (e.g. the statistical test used).

Data availability: The eLife author instructions do say that data must be made available, so there should be a statement on data availability in your MS. I also suggest you make the code available.

Accepted as suggested.

RF LV2 host Lauren's pronunciation: I am right about: bien, faire, etc. She is Québecois.

Speaker’s Mixed Feelings on Language Models

Key Quote: “The second controversial statement is language models suck or rather AI sucks and specifically the way our culture has been using it.”

The speaker acknowledges that while language models are groundbreaking (“language models are pretty neat”), there are serious ethical, social, and environmental concerns, which creates a personal and professional dilemma.

Motivation: Making AI Good

Key Quote: “How can we make the tech actually good if it comes with all these trade-offs...so let’s make it as good as it can possibly be but how?”

The talk’s central goal is to explore how to refine language model technology to maximize its societal benefits and minimize harms.

RDF Overview and History

Key Quote: “rdf is an attempt to tackle some of the hardest problems of knowledge representation and reasoning...from the same group of people that put together all the internet specifications the w3c.”

RDF (Resource Description Framework) emerged post-internet boom, aiming to provide a universal system for knowledge representation, rooted in symbolic AI traditions and overseen by the W3C.

Why RDF Fell Out of Favor

Key Quote: “One is rdf XML which is one of the initial formats...this is a verbose complex format it’s just honestly not great.”

Early technical choices and heavy enterprise solutions contributed to RDF’s reputation as being cumbersome and outdated, even though “under the hood” it remains robust and conceptually sound.

RDF’s Elegant Core: Resources and Triples

Key Quote: “It’s the resource description framework so let’s talk about resources first...a resource is anything in the world that you can talk about.”

RDF structures knowledge as “triples” (subject, predicate, object) linked by unique identifiers (IRIs), enabling precise, context-rich data representation.

Federation and Union of Data

Key Quote: “An rdf data set is a set in the closure sense or the mathematical sense...we can also safely union sets.”

By standardizing each piece of data (triples + IRIs), RDF allows combining multiple datasets (federation) without losing context or creating duplication conflicts.

Inference, Logic, and Schemas

Key Quote: “This is all about entailment...given a set of triples I can derive other triples from them conceptually.”

RDF includes logical rules for automatically deriving new facts (entailment) and validating data, reflecting decades of research in symbolic AI and formal logic.

Language Models: Context and Probabilistic Reasoning

Key Quote: “All a model is is a pure function that predicts the next token...with some constants in it.”

Modern language models leverage the Transformer architecture for predicting tokens and exhibit capabilities in semantics, grammar, and even “fact patterns,” though they remain probabilistic approximations.

Challenges of Using Language Models

Key Quote: “All of AI programming...is putting the right stuff into the model to try to get it to get out the stuff that you want.”

Because re-training an LLM is expensive, practitioners focus on techniques like prompt engineering, retrieval-augmented generation, query generation, and tool use to shape the model’s output.

Core Problem: Integrating Data and Language

Key Quote: “We write programs that work between them a lot...But how do I get my data to meet my language?”

The speaker emphasizes the need for a mechanism that unifies formal data representation and natural language capabilities, highlighting RDF as the bridge.

RDF as the Bridge Between LLMs and Data

Key Quote: “We should be putting rdf data in our prompts and when we are asking to get kind of more structured data out of models we should be asking for it in rdf format.”

RDF’s subject-predicate-object structure aligns naturally with the grammar captured by language models, enabling more precise input/output handling and reducing ambiguity.

RDF Queries for Tool Use

Key Quote: “If my rdf implementation supports reasoning...the language model is asking a different question who is Luke a descendant of.”

By combining RDF’s inference with LLM queries, complex or open-ended questions (“who am I?” or genealogical lookups) can be answered reliably, without forcing the language model to handle all logic internally.

Combining Symbolic and Neural Approaches

Key Quote: “Neuorsymbolic AI is...any research that is trying to combine the abstract fuzzy neural network with hard concrete logical symbols.”

RDF can serve as the symbolic layer, and the language model as the neural layer—together addressing knowledge gaps that purely logical or purely neural methods struggle with alone.

Conclusion and Practical Use Cases

Key Quote: “I think it’s a tool to actually automate a lot of the dishes and the laundry of working with data...particularly on the data side.”

The speaker envisions AI not to replace creative work or coding but to handle the “tedious chores” of data management, with RDF acting as a structured, logic-friendly foundation to harness LLMs effectively.

As hyenas are scavengers, and we might interpret Clay as prey, Clay must be a carrioned prey. He's already been hunted, and he suffers from inferiority complex. Clay believes that white people are the superiors. Now, it's time for scavengers. Scavengers harass him on the subway, on the street, on social media, just like Lula did, so they dig up what's left of the hunt.

What about Bitcoin Lightning Network State Channel transactions, those are regional right?

Author response:

The following is the authors’ response to the original reviews.

The revised manuscript contains new results and additional text. Major revisions:

(1) Additional simulations and analyses of networks with different biophysical parameters and with identical time constants for E and I neurons (Methods, Supplementary Fig. 5).

(2) Additional simulations and analyses of networks with modifications of connectivity parameters to further analyze effects of E/I assemblies on manifold geometry (Supplementary Fig. 6).

(3) Analysis of synaptic current components (Figure 3 D-F; to analyze mechanism of modest amplification in Tuned networks). 

(4) More detailed explanation of pattern completion analysis (Results).

(5) Analysis of classification performance of Scaled networks (Supplementary Fig.8).

(6) Additional analysis (Figure 5D-F) and discussion (particularly section “Computational functions of networks with E/I assemblies”) of functional benefits of continuous representations in networks with E-I assemblies. 

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing. 

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks. 

Strengths: 

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models. 

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation. 

(2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.  (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model. 

Weaknesses: 

Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model. 

(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper. 

We agree that further mechanistic insights would be of interest and addressed this issue at different levels:

(1) Biophysical parameters: to determine whether network behavior depends on specific choices of biophysical parameters in E and I neurons we equalized biophysical parameters across neuron types. The main observations are unchanged, suggesting that the observed effects depend primarily on network connectivity (see also response to comment [2]).

(2) Mechanism of modest amplification in E/I assemblies: analyzing the different components of the synaptic currents demonstrate that the modest amplification of activity in Tuned networks results from an “imperfect” balance of recurrent excitation and inhibition within assemblies (see new Figures 3D-F and text p.7). Hence, E/I co-tuning substantially reduces the net amplification in Tuned networks as compared to Scaled networks, thus preventing discrete attractor dynamics and stabilizing network activity, but a modest amplification still occurs, consistent with biological observations.

(3) Representational geometry: to obtain insights into the network mechanisms underlying effects of E/I assemblies on the geometry of population activity we tested the hypothesis that geometrical changes depend, at least in part, on the modest amplification of activity within E/I assemblies (see Supplementary Figure 6). We changed model parameters to either prevent the modest amplification in Tuned networks (increasing I-to-E connectivity within assemblies) or introduce a modest amplification in subsets of neurons by other mechanisms (concentration-dependent increase in the excitability of pseudo-assembly neurons; Scaled I networks with reduced connectivity within assemblies). Manipulations that introduced a modest, input-dependent amplification in neuronal subsets had geometrical effects similar to those observed in Tuned networks, whereas manipulations that prevented a modest amplification abolished these effects (Supplementary Figure 6). Note however that these manipulations generated different firing rate distributions. These results provide a starting point for more detailed analyses of the relationship between network connectivity and representational geometry (see p.12).

In summary, our additional analyses indicate that effects of E/I assemblies on representational geometry depend primarily on network connectivity, rather than specific biophysical parameters, and that the resulting modest amplification of activity within assemblies makes an important contribution. Further analyses may reveal more specific relationships between E/I assemblies and representational geometry, but such analyses are beyond the scope of this study.

(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.  

We thank the reviewer for raising this point. We chose a relatively slow time constant for excitatory synapses because experimental data indicate that excitatory synaptic currents in Dp and piriform cortex contain a prominent NMDA component. Nevertheless, to assess whether network behavior depends on specific choices of biophysical parameters in E and I neurons, we have performed additional simulations with equal synaptic time constants and equal biophysical parameters for all neurons. Each neuron also received the same number of inputs from each population (see revised Methods). Results were similar to those observed previously (Supplementary Fig.5 and p.9 of main text). We therefore conclude that the main effects observed in Tuned networks cannot be explained by differences in biophysical parameters between E and I neurons but is primarily a consequence of network connectivity.

(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning. 

One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function. 

In the previous manuscript, the analysis of potential computational benefits other than pattern classification was limited and the discussion of this issue was condensed into a single itemized paragraph to avoid excessive speculation. Although a thorough analysis of potential computational benefits exceeds the scope of a single paper, we agree with the reviewer that this issue is of interest and therefore added additional analyses and discussion.

In the initial manuscript we analyzed pattern classification primarily to investigate whether Tuned networks can support this function at all, given that they do not exhibit discrete attractor states. We found this to be the case, which we consider a first important result.

Furthermore, we found that precise balance of E/I assemblies can protect networks against catastrophic firing rate instabilities when assemblies are added sequentially, as in continual learning. Results from these simulations are now described and discussed in more detail (see Results p.11 and Discussion p.13).

In the revised manuscript, we now also examine additional potential benefits of Tuned networks and discuss them in more detail (see new Figure 5D-F and text p.11). One hypothesis is that continuous representations provide a distance metric between a given input and relevant (learned) stimuli. To address this hypothesis, we (1) performed regression analysis and (2) trained support vector machines (SVMs) to predict the concentration of a given odor in a mixture based on population activity. In both cases, Tuned E+I networks outperformed Scaled and _rand n_etworks in predicting the concentration of learned odors across a wide range mixtures (Figure 5D-F).  E/I assemblies therefore support the quantification of learned odors within mixtures or, more generally, assessments of how strongly a (potentially complex) input is related to relevant odors stored in memory. Such a metric assessment of stimulus quality is not well supported by discrete attractor networks because inputs are mapped onto discrete network states.

The observation that Tuned networks do not map inputs onto discrete outputs indicates that such networks do not classify inputs as distinct items. Nonetheless, the observed geometrical modifications of continuous representations support the classification of learned inputs or the assessment of metric relationships by hypothetical readout neurons. Geometrical modifications of odor representations may therefore serve as one of multiple steps in multi-layer computations for pattern classification (and/or other computations). In this scenario, the transformation of odor representations in Dp may be seen as related to transformations of representations between different layers in artificial networks, which collectively perform a given task (notwithstanding obvious structural and mechanistic differences between artificial and biological networks). In other words, geometrical transformations of representations in Tuned networks may overrepresent learned (relevant) information at the expense of other information and thereby support further learning processes in other brain areas. An obvious corollary of this scenario is that Dp does not perform odor classification per se based on inputs from the olfactory bulb but reformats representations of odor space based on experience to support computational tasks as part of a larger system. This scenario is now explicitly discussed (p.14).

Reviewer #2 (Public Review): 

Summary: 

The authors conducted a comparative analysis of four networks, varying in the presence of excitatory assemblies and the architecture of inhibitory cell assembly connectivity. They found that co-tuned E-I assemblies provide network stability and a continuous representation of input patterns (on locally constrained manifolds), contrasting with networks with global inhibition that result in attractor networks. 

Strengths: 

The findings presented in this paper are very interesting and cutting-edge. The manuscript effectively conveys the message and presents a creative way to represent high-dimensional inputs and network responses. Particularly, the result regarding the projection of input patterns onto local manifolds and continuous representation of input/memory is very Intriguing and novel. Both computational and experimental neuroscientists would find value in reading the paper. 

Weaknesses: 

that have continuous representations. This could also be shown in Figure 5B, along with the performance of the random and tuned E-I networks. The latter networks have the advantage of providing network stability compared to the Scaled I network, but at the cost of reduced network salience and, therefore, reduced input decodability. The authors may consider designing a decoder to quantify and compare the classification performance of all four networks. 

We have now quantified classification by networks with discrete attractor dynamics (Scaled) along with other networks. However, because the neuronal covariance matrix for such networks is low rank and not invertible, pattern classification cannot be analyzed by QDA as in Figure 5B. We therefore classified patterns from the odor subspace by template matching, assigning test patterns to one of the four classes based on correlations (see Supplementary Figure 8). As expected, Scaled networks performed well, but they did not outperform Tuned networks. Moreover, the performance of Scaled networks, but not Tuned networks, depended on the order in which odors were presented to the network. This hysteresis effect is a direct consequence of persistent attractor states and decreased the general classification performance of Scaled networks (see Supplementary Figure 8 for details). These results confirm the prediction that networks with discrete attractor states can efficiently classify inputs, but also reveal disadvantages arising from attractor dynamics. Moreover, the results indicate that the classification performance of Tuned networks is also high under the given task conditions, which simulate a biologically realistic scenario.

We would also like to emphasize that classification may not be the only task, and perhaps not even a main task, of Dp/piriform cortex or other memory networks with E/I assemblies. Conceivably, other computations could include metric assessments of inputs relative to learned inputs or additional learning-related computations. Please see our response to comment (3) of reviewer 1 for a further discussion of this issue. 

Networks featuring E/I assemblies could potentially represent multistable attractors by exploring the parameter space for their reciprocal connectivity and connectivity with the rest of the network. However, for co-tuned E-I networks, the scope for achieving multistability is relatively constrained compared to networks employing global or lateral inhibition between assemblies. It would be good if the authors mentioned this in the discussion. Also, the fact that reciprocal inhibition increases network stability has been shown before and should be cited in the statements addressing network stability (e.g., some of the citations in the manuscript, including Rost et al. 2018, Lagzi & Fairhall 2022, and Vogels et al. 2011 have shown this).  

We thank the reviewer for this comment. We now explicitly discuss multistability (see p. 12) and refer to additional references in the statements addressing network stability.

Providing raster plots of the pDp network for familiar and novel inputs would help with understanding the claims regarding continuous versus discrete representation of inputs, allowing readers to visualize the activity patterns of the four different networks. (similar to Figure 1B). 

We thank the reviewer for this suggestion. We have added raster plots of responses to both familiar and novel inputs in the revised manuscript (Figure 2D and Supplementary Figure 4A).

Reviewer #3 (Public Review): 

Summary: 

This work investigates the computational consequences of assemblies containing both excitatory and inhibitory neurons (E/I assembly) in a model with parameters constrained by experimental data from the telencephalic area Dp of zebrafish. The authors show how this precise E/I balance shapes the geometry of neuronal dynamics in comparison to unstructured networks and networks with more global inhibitory balance. Specifically, E/I assemblies lead to the activity being locally restricted onto manifolds - a dynamical structure in between high-dimensional representations in unstructured networks and discrete attractors in networks with global inhibitory balance. Furthermore, E/I assemblies lead to smoother representations of mixtures of stimuli while those stimuli can still be reliably classified, and allow for more robust learning of additional stimuli. 

Strengths: 

Since experimental studies do suggest that E/I balance is very precise and E/I assemblies exist, it is important to study the consequences of those connectivity structures on network dynamics. The authors convincingly show that E/I assemblies lead to different geometries of stimulus representation compared to unstructured networks and networks with global inhibition. This finding might open the door for future studies for exploring the functional advantage of these locally defined manifolds, and how other network properties allow to shape those manifolds. 

The authors also make sure that their spiking model is well-constrained by experimental data from the zebrafish pDp. Both spontaneous and odor stimulus triggered spiking activity is within the range of experimental measurements. But the model is also general enough to be potentially applied to findings in other animal models and brain regions. 

Weaknesses: 

I find the point about pattern completion a bit confusing. In Fig. 3 the authors argue that only the Scaled I network can lead to pattern completion for morphed inputs since the output correlations are higher than the input correlations. For me, this sounds less like the network can perform pattern completion but it can nonlinearly increase the output correlations. Furthermore, in Suppl. Fig. 3 the authors show that activating half the assembly does lead to pattern completion in the sense that also non-activated assembly cells become highly active and that this pattern completion can be seen for Scaled I, Tuned E+I, and Tuned I networks. These two results seem a bit contradictory to me and require further clarification, and the authors might want to clarify how exactly they define pattern completion. 

We believe that this comment concerns a semantic misunderstanding and apologize for any lack of clarity. We added a definition of pattern completion in the text: “…the retrieval of the whole memory from noisy or corrupted versions of the learned input.”. Pattern completion may be assessed using different procedures. In computational studies, it is often analyzed by delivering input to a subset of the assembly neurons which store a given memory (partial activation). Under these conditions, we find recruitment of the entire assembly in all structured networks, as demonstrated in Supplementary Figure 3. However, these conditions are unlikely to occur during odor presentation because the majority of neurons do not receive any input.

Another more biologically motivated approach to assess pattern completion is to gradually modify a realistic odor input into a learned input, thereby gradually increasing the overlap between the two inputs. This approach had been used previously in experimental studies (references added to the text p.6). In the presence of assemblies, recurrent connectivity is expected to recruit assembly neurons (and thus retrieve the stored pattern) more efficiently as the learned pattern is approached. This should result in a nonlinear increase in the similarity between the evoked and the learned activity pattern. This signature was prominent in Scaled networks but not in Tuned or rand networks. Obviously, the underlying procedure is different from the partial activation of the assembly described above because input patterns target many neurons (including neurons outside assemblies) and exhibit a biologically realistic distribution of activity. However, this approach has also been referred to as “pattern completion” in the neuroscience literature, which may be the source of semantic confusion here. To clarify the difference between these approaches we have now revised the text and explicitly described each procedure in more detail (see p.6). 

The authors argue that Tuned E+I networks have several advantages over Scaled I networks. While I agree with the authors that in some cases adding this localized E/I balance is beneficial, I believe that a more rigorous comparison between Tuned E+I networks and Scaled I networks is needed: quantification of variance (Fig. 4G) and angle distributions (Fig. 4H) should also be shown for the Scaled I network. Similarly in Fig. 5, what is the Mahalanobis distance for Scaled I networks and how well can the Scaled I network be classified compared to the Tuned E+I network? I suspect that the Scaled I network will actually be better at classifying odors compared to the E+I network. The authors might want to speculate about the benefit of having networks with both sources of inhibition (local and global) and hence being able to switch between locally defined manifolds and discrete attractor states. 

We agree that a more rigorous comparison of Tuned and Scaled networks would be of interest. We have added the variance analysis (Fig 4G) and angle distributions (Fig. 4H) for both Tuned I and Scaled networks. However, the Mahalanobis distances and Quadratic Discriminant Analysis cannot be applied to Scaled networks because their neuronal covariance matrix is low rank and not invertible_. To nevertheless compare these networks, we performed template matching by assigning test patterns to one of the four odor classes based on correlations to template patterns (Supplementary Figure 8; see also response to the first comment of reviewer 2). Interestingly, _Scaled networks performed well at classification but did not outperform Tuned networks, and exhibited disadvantages arising from attractor dynamics (Supplementary Figure 8; see also response to the first comment of reviewer 2). Furthermore, in further analyses we found that continuous representational manifolds support metric assessments of inputs relative to learned odors, which cannot be achieved by discrete representations. These results are now shown in Figure 5D-E and discussed explicitly in the text on p.11 (see also response to comment 3 of reviewer 1).

We preferred not to add a sentence in the Discussion about benefits of networks having both sources of inhibition_,_ as we find this a bit too speculative.

At a few points in the manuscript, the authors use statements without actually providing evidence in terms of a Figure. Often the authors themselves acknowledge this, by adding the term "not shown" to the end of the sentence. I believe it will be helpful to the reader to be provided with figures or panels in support of the statements.  

Thank you for this comment. We have provided additional data figures to support the following statements:

“d_M was again increased upon learning, particularly between learned odors and reference classes representing other odors (Supplementary Figure 9)”

“decreasing amplification in assemblies of Scaled networks changed transformations towards the intermediate behavior, albeit with broader firing rate distributions than in Tuned networks (Supplementary Figure 6 B)”  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing. 

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks. 

The paper is generally well-written, the figures are informative and of good quality, and multiple approaches and metrics have been used to test and support the main results of the paper. 

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models. 

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation. 

(2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.   (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model. 

Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model. 

(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper. 

Precise balancing of excitation and inhibition in subnetworks would lead to the cancellation of specific dynamical modes responsible for the amplification of responses (hence, deviating from the attractor dynamics with an unstable specific mode). What is the key difference in the specific E/I networks here (tuned I or/and tuned E+I) which make them stand between random and attractor networks? Excitatory and inhibitory neurons have different parameters in the model (Table 1). Time constants of inhibitory and excitatory synapses are also different (P. 13). Are these parameters causing networks to be effectively more excitation dominated (hence deviating from a random spectrum which would be expected from a precisely balanced E/I network, with exactly the same parameters of E and I neurons)? It is necessary to analyse the network models, describe the key mechanism for their amplification, and pinpoint the key differences between E and I neurons which are crucial for this. 

To address these comments we performed additional simulations and analyses at different levels. Please see our reply to comment (1) of the public review (reviewer 1) for a detailed description. We thank the reviewer for these constructive comments.

(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.  

We thank the reviewer for this comment. We have now carried out additional simulations with equal time constants for all neurons. Please see our reply to the public review for more details (comment 2 of reviewer 1).

(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning. 

One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function.

Please see our reply to the public review (comment 3 of reviewer 1).

Specific comments: 

Abstract: "resulting in continuous representations that reflected both relatedness of inputs and *an individual's experience*" 

It didn't become apparent from the text or the model where the role of "individual's experience" component (or "internal representations" - in the next line) was introduced or shown (apart from a couple of lines in the Discussion) 

We consider the scenario that that assemblies are the outcome of an experience-dependent plasticity process. To clarify this, we have now made a small addition to the text: “Biological memory networks are thought to store information by experience-dependent changes in the synaptic connectivity between assemblies of neurons.”.

P. 2: "The resulting state of "precise" synaptic balance stabilizes firing rates because inhomogeneities or fluctuations in excitation are tracked by correlated inhibition" 

It is not clear what the "inhomogeneities" specifically refers to - they can be temporal, or they can refer to the quenched noise of connectivity, for instance. Please clarify what you mean. 

The statement has been modified to be more precise: “…“precise” synaptic balance stabilizes firing rates because inhomogeneities in excitation across the population or temporal variations in excitation are tracked by correlated inhibition…”.

P. 3 (and Methods): When odour stimulus is simulated in the OB, the activity of a fraction of mitral cells is increased (10% to 15 Hz) - but also a fraction of mitral cells is suppressed (5% to 2 Hz). What is the biological motivation or reference for this? It is not provided. Is it needed for the results? Also, it is not explained how the suppressed 5% are chosen (e.g. randomly, without any relation to the increased cells?). 

We thank the reviewer for this comment. These changes in activity directly reflect experimental observations. We apologize that we forgot to include the references reporting these observations (Friedrich and Laurent, 2001 and 2004); this is now fixed.

In our simulation, OB neurons do not interact with each other, and the suppressed 5% were indeed randomly selected. We changed the text in Methods accordingly to read: “An additional 75 randomly selected mitral cells were inhibited” 

P. 4, L. 1-2: "... sparsely connected integrate-and-fire neurons with conductance-based synapses (connection probability {less than or equal to}5%)." 

Specify the connection probability of specific subtypes (EE, EI, IE, II).  

We now refer to the Methods section, where this information can be found. 

“... conductance-based synapses (connection probability ≤5%, Methods)”  

P. 4, L. 6-7: "Population activity was odor-specific and activity patterns evoked by uncorrelated OB inputs remained uncorrelated in Dp (Figure 1H)" 

What would happen to correlated OB inputs (e.g. as a result of mixture of two overlapping odours) in this baseline state of the network (before memories being introduced to it)? It would be good to know this, as it sheds light on the initial operating regime of the network in terms of E/I balance and decorrelation of inputs.  

This information was present in the original manuscript at (Figure 3) but we improved the writing to further clarify this issue: “ (…) we morphed a novel odor into a learned odor (Figure 3A), or a learned odor into another learned odor (Supplementary Figure 3B), and quantified the similarity between morphed and learned odors by the Pearson correlation of the OB activity patterns (input correlation). We then compared input correlations to the corresponding pattern correlations among E neurons in Dp (output correlation). In rand networks, output correlations increased linearly with input correlations but did not exceed them (Figure 3B and Supplementary Figure 3B)”

P. 4, L. 12-13: "Shuffling spike times of inhibitory neurons resulted in runaway activity with a probability of ~80%, .."   Where is this shown? 

(There are other occasions too in the paper where references to the supporting figures are missing). 

We now provide the statistics: “Shuffling spike times of inhibitory neurons resulted in runaway activity with a probability of 0.79 ± 0.20”

P. 4: "In each network, we created 15 assemblies representing uncorrelated odors. As a consequence, ~30% of E neurons were part of an assembly ..." 

15 x 100 / 4000 = 37.5% - so it's closer to 40% than 30%. Unless there is some overlap? 

Yes: despite odors being uncorrelated and connectivity being random, some neurons (6 % of E neurons) belong to more than one assembly.

P. 4: "When a reached a critical value of ~6, networks became unstable and generated runaway activity (Figure 2B)." 

Can this transition point be calculated or estimated from the network parameters, and linked to the underlying mechanisms causing it? 

We thank the reviewer for this interesting question. The unstability arises when inhibitions fails to counterbalance efficiently the increased recurrent excitation within Dp. The transition point is difficult to estimate, as it can depend on several parameters, including the probability of E to E connections, their strength, assembly size, and others. We have therefore not attempted to estimate it analytically.

P. 4: "Hence, non-specific scaling of inhibition resulted in a divergence of firing rates that exhausted the dynamic range of individual neurons in the population, implying that homeostatic   global inhibition is insufficient to maintain a stable firing rate distribution." 

I don't think this is justified based on the results and figures presented here (Fig. 2E) - the interpretation is a bit strong and biased towards the conclusions the authors want to draw. 

To more clearly illustrate the finding that in Scaled networks, assembly neurons are highly active (close to maximal realistic firing rates) whereas non-assembly neurons are nearly silent we have now added Supplementary Fig. 2B. Moreover, we have toned down the text: “Hence, non-specific scaling of inhibition resulted in a large and biologically unrealistic divergence of firing rates (Supplementary Figure 2B) that nearly exhausted the dynamic range of individual neurons in the population, indicating that homeostatic global inhibition is insufficient to maintain a stable firing rate distribution”

P. 5, third paragraph: Description of Figure 2I, inset is needed, either in the text or caption. 

The inset is now referred to in the text: ”we projected synaptic conductances of each neuron onto a line representing the E/I ratio expected in a balanced network (“balanced axis”) and onto an orthogonal line (“counter-balanced axis”; Figure 2I inset, Methods).”

P. 5, last paragraph: another example of writing about results without showing/referring to the corresponding figures: 

"In rand networks, firing rates increased after stimulus onset and rapidly returned to a low baseline after stimulus offset. Correlations between activity patterns evoked by the same odor at different time points and in different trials were positive but substantially lower than unity, indicating high variability ..." 

And the continuation with similar lack of references on P. 6: 

"Scaled networks responded to learned odors with persistent firing of assembly neurons and high pattern correlations across trials and time, implying attractor dynamics (Hopfield, 1982; Khona and Fiete, 2022), whereas Tuned networks exhibited transient responses and modest pattern correlations similar to rand networks." 

Please go through the Results and fix the references to the corresponding figures on all instances. 

We thank the reviewer for pointing out these overlooked figure references, which are now fixed.

P. 8: "These observations further support the conclusion that E/I assemblies locally constrain neuronal dynamics onto manifolds." 

As discussed in the general major points, mechanistic explanation in terms of how the interaction of E/I dynamics leads to this is missing. 

As discussed in the reply to the public review (comment 3 of reviewer 1), we have now provided more mechanistic analyses of our observations.

P. 9: "Hence, E/I assemblies enhanced the classification of inputs related to learned patterns."   The effect seems to be very small. Also, any explanation for why for low test-target correlation the effect is negative (random doing better than tuned E/I)? 

The size of the effect (plearned – pnovel = 0.074; difference of means; Figure 5C) may appear small in terms of absolute probability, but it is substantial relative to the maximum possible increase (1 – p_novel =  0.133; Figure 5C). The fact that for low test-target correlations the effect is negative is a direct consequence of the positive effect for high test-target correlations and the presence of 2 learned odors in the 4-way forced choice task. 

P. 9: "In Scaled I networks, creating two additional memories resulted in a substantial increase   in firing rates, particularly in response to the learned and related odors"   Where is this shown? Please refer to the figure. 

We thank the reviewer again for pointing this out. We forgot to include a reference to the relevant figure which has now been added in the revised manuscript (Figure 6C).

P. 10: "The resulting Tuned networks reproduced additional experimental observations that were not used as constraints including irregular firing patterns, lower output than input correlations, and the absence of persistent activity" 

It is difficult to present these as "additional experimental observations", as all of them are negative, and can exist in random networks too - hence cannot be used as biological evidence in favour of specific E/I networks when compared to random networks. 

We agree with the reviewer that these additional experimental observations cannot be used as biological evidence favouring Tuned E+I networks over random networks. We here just wanted to point out that additional observations which we did not take into account to fit the model are not invalidating the existence of E-I assemblies in biological networks. As assemblies tend to result in persistent activity in other types of networks, we feel that this observation is worth pointing out.

Methods: 

P. 13: Describe the parameters of Eq. 2 after the equation. 

Done.

P. 13: "The time constants of inhibitory and excitatory synapses were 10 ms and 30 ms, respectively." 

What is the (biological) justification for the choice of these parameters? 

How would varying them affect the main results (e.g. local manifolds)? 

We chose a relatively slow time constant for excitatory synapses because experimental data indicate that excitatory synaptic currents in Dp and piriform cortex contain a prominent NMDA component. We have now also simulated networks with equal time constants for excitatory and inhibitory synapses and equal biophysical parameters for excitatory and inhibitory neurons, which did not affect the main results (see also reply to the public review: comment 2 of reviewer 1).

P. 14: "Care was also taken to ensure that the variation in the number of output connections was low across neurons"   How exactly?

More detailed explanations have now been added in the Methods section: “connections of a presynaptic neuron y to postsynaptic neurons x were randomly deleted when their total number exceeded the average number of output connections by ≥5%, or added when they were lower by ≥5%.“

Reviewer #2 (Recommendations For The Authors): 

Congratulations on the great and interesting work! The results were nicely presented and the idea of continuous encoding on manifolds is very interesting. To improve the quality of the paper, in addition to the major points raised in the public review, here are some more detailed comments for the paper: 

(1) Generally, citations have to improve. Spiking networks with excitatory assemblies and different architectures of inhibitory populations have been studied before, and the claim about improved network stability in co-tuned E-I networks has been made in the following papers that need to be correctly cited: 

• Vogels TP, Sprekeler H, Zenke F, Clopath C, Gerstner W. 2011. Inhibitory Plasticity Balances Excitation and Inhibition in Sensory Pathways and Memory Networks. Science 334:1-7. doi:10.1126/science.1212991 (mentions that emerging precise balance on the synaptic weights can result in the overall network stability) 

• Lagzi F, Bustos MC, Oswald AM, Doiron B. 2021. Assembly formation is stabilized by Parvalbumin neurons and accelerated by Somatostatin neurons. bioRxiv doi: https://doi.org/10.1101/2021.09.06.459211 (among other things, contrasts stability and competition which arises from multistable networks with global inhibition and reciprocal inhibition)   • Rost T, Deger M, Nawrot MP. 2018. Winnerless competition in clustered balanced networks: inhibitory assemblies do the trick. Biol Cybern 112:81-98. doi:10.1007/s00422-017-0737-7 (compares different architectures of inhibition and their effects on network dynamics) 

• Lagzi F, Fairhall A. 2022. Tuned inhibitory firing rate and connection weights as emergent network properties. bioRxiv 2022.04.12.488114. doi:10.1101/2022.04.12.488114 (here, see the eigenvalue and UMAP analysis for a network with global inhibition and E/I assemblies) 

Additionally, there are lots of pioneering work about tracking of excitatory synaptic inputs by inhibitory populations, that are missing in references. Also, experimental work that show existence of cell assemblies in the brain are largely missing. On the other hand, some references that do not fit the focus of the statements have been incorrectly cited. 

The authors may consider referencing the following more pertinent studies on spiking networks to support the statement regarding attractor dynamics in the first paragraph in the Introduction (the current citations of Hopfield and Kohonen are for rate-based networks): 

• Wong, K.-F., & Wang, X.-J. (2006). A recurrent network mechanism of time integration in perceptual decisions. Journal of Neuroscience, 26(4), 1314-1328. https://doi.org/10.1523/JNEUROSCI.3733-05.2006 

• Wang, X.-J. (2008). Decision making in recurrent neuronal circuits. Neuron, 60(2), 215-234. https://doi.org/10.1016/j.neuron.2008.09.034  

• F. Lagzi, & S. Rotter. (2015). Dynamics of competition between subnetworks of spiking neuronal networks in the balanced state. PloS One. 

• Goldman-Rakic, P. S. (1995). Cellular basis of working memory. Neuron, 14(3), 477-485. 

• Rost T, Deger M, Nawrot MP. 2018. Winnerless competition in clustered balanced networks: inhibitory assemblies do the trick. Biol Cybern 112:81-98. doi:10.1007/s00422-017-0737-7. 

• Amit DJ, Tsodyks M (1991) Quantitative study of attractor neural network retrieving at low spike rates: I. substrate-spikes, rates and neuronal gain. Network 2:259-273. 

• Mazzucato, L., Fontanini, A., & La Camera, G. (2015). Dynamics of Multistable States during Ongoing and Evoked Cortical Activity. Journal of Neuroscience, 35(21), 8214-8231. 

We thank the reviewer for the references suggestions. We have carefully reviewed the reference list and made the following changes, which we hope address the reviewer’s concerns:

(1) We adjusted References about network stability in co-tuned E-I networks.

(2) We added the Lagzi & Rotter (2015), Amit et al. (1991), Mazzucato et al. (2015) and GoldmanRakic (1995) papers in the Introduction as studies on attractor dynamics in spiking neural networks. We preferred to omit the two X.J Wang papers, as they describe attractors in decision making rather than memory processes.

(3) We added the Ko et al. 2011 paper as experimental evidence for assemblies in the brain. In our view, there are few experimental studies showing the existence of cell assemblies in the brain, which we distinguish from cell ensembles, group of coactive neurons. 

(4) We also included Hennequin 2018, Brunel 2000, Lagzi et al. 2021 and Eckmann et al. 2024, which we had not cited in the initial manuscript.

(5) We removed the Wiechert et al. 2010 reference as it does not support the statement about geometry-preserving transformation by random networks.

(2) The gist of the paper is about how the architecture of inhibition (reciprocal vs. global in this case) can determine network stability and salient responses (related to multistable attractors and variations) for classification purposes. It would improve the narrative of the paper if this point is raised in the Introduction and Discussion section. Also see a relevant paper that addresses this point here: 

Lagzi F, Bustos MC, Oswald AM, Doiron B. 2021. Assembly formation is stabilized by Parvalbumin neurons and accelerated by Somatostatin neurons. bioRxiv doi: https://doi.org/10.1101/2021.09.06.459211 

Classification has long been proposed to be a function of piriform cortex and autoassociative memory networks in general, and we consider it important. However, the computational function of Dp or piriform cortex is still poorly understood, and we do not focus only on odor classification as a possibility. In fact, continuous representational manifolds also support other functions such as the quantification of distance relationships of an input to previously memorized stimuli, or multi-layer network computations (including classification). In the revised manuscript, we have performed additional analyses to explore these notions in more detail, as explained above (response to public reviews, comment 3 of reviewer 1). Furthermore, we have now expanded the discussion of potential computational functions of Tuned networks and explicitly discuss classification but also other potential functions. 

(3) A plot for the values of the inhibitory conductances in Figure 1 would complete the analysis for that section. 

In Figure 1, we decided to only show the conductances that we use to fit our model, namely the afferent and total synaptic conductances. As the values of the inhibitory conductances can be derived from panel E, we refrained from plotting them separately for the sake of simplicity. 

(4) How did the authors calculate correlations between activity patterns as a function of time in Figure 2E, bottom row? Does the color represent correlation coefficient (which should not be time dependent) or is it a correlation function? This should be explained in the Methods section. 

The color represents the Pearson correlation coefficient between activity patterns within a narrow time window (100 ms). We updated the Figure legend to clarify this: “Mean correlation between activity patterns evoked by a learned odor at different time points during odor presentation. Correlation coefficients were calculated between pairs of activity vectors composed of the mean firing rates of E neurons in 100 ms time bins. Activity vectors were taken from the same or different trials, except for the diagonal, where only patterns from different trials were considered.”

(5) Figure 3 needs more clarification (both in the main text and the figure caption). It is not clear what the axes are exactly, and why the network responses for familiar and novel inputs are different. The gray shaded area in panel B needs more explanation as well.  

We thank the reviewer for the comment. We have improved Figure 3A, the figure caption, as well as the text (see p.6). We hope that the figure is now clearer.

(6) The "scaled I" network, known for representing input patterns in discrete attractors, should exhibit clear separation between network responses in the 2D PC space in the PCA plots. However, Figure 4D and Figure 6D do not reflect this, as all network responses are overlapped. Can the authors explain the overlap in Figure 4D? 

In Figure 4D, activity of Scaled networks is distributed between three subregions in state space that are separated by the first 2 PCs. Two of them indeed correspond to attractor states representing the two learned odors while the third represents inputs that are not associated with these attractor states. To clarify this, please see also the density plot in Figure 4E. The few datapoints between these three subregions are likely outliers generated by the sequential change in inputs, as described in Supplementary Figure 8C.

(7) The reason for writing about the ISN networks is not clear. Co-tuned E-I assemblies do not necessarily have to operate in this regime. Also, the results of the paper do not rely on any of the properties of ISNs, but they are more general. Authors should either show the paradoxical effect associated with ISN (i.e., if increasing input to I neurons decreases their responses) or show ISN properties using stability analysis (See computational research conducted at the Allen Institute, namely Millman et al. 2020, eLife ). Currently, the paper reads as if being in the ISN regime is a necessary requirement, which is not true. Also, the arguments do not connect with the rest of the paper and never show up again. Since we know it is not a requirement, there is no need to have those few sentences in the Results section. Also, the choice of alpha=5.0 is extreme, and therefore, it would help to judge the biological realism if the raster plots for Figs 2-6 are shown.

We have toned down the part on ISN and reduced it to one sentence for readers who might be interested in knowing whether activity is inhibition-stabilized or not. We have also added the reference to the Tsodyks et al. 1997 paper from which we derive our stability analysis. The text now reads “Hence, pDp_sim entered a balanced state during odor stimulation (Figure 1D, E) with recurrent input dominating over afferent input, as observed in pDp (Rupprecht and Friedrich, 2018). Shuffling spike times of inhibitory neurons resulted in runaway activity with a probability of 0.79 ± 0.20, demonstrating that activity was inhibition-stabilized (Sadeh and Clopath, 2020b, Tsodyks et al., 1997).”  

We have now also added the raster plots as suggested by the reviewer (see Figure 2D, Supplementary Figure 1 G, Supplementary Figure 4). We thank the reviewer for this comment.

(8) In the abstract, authors mention "fast pattern classification" and "continual learning," but in the paper, those issues have not been addressed. The study does not include any synaptic plasticity. 

Concerning “continual learning” we agree that we do not simulate the learning process itself. However, Figure 6 show results of a simulation where two additional patterns were stored in a network that already contained assemblies representing other odors. We consider this a crude way of exploring the end result of a “continual learning” process. “Fast pattern classification” is mentioned because activity in balanced networks can follow fluctuating inputs with high temporal resolution, while networks with stable attractor states tend to be slow. This is likely to account for the occurrence of hysteresis effects in Scaled but not Tuned networks as shown in Supplementary

Fig. 8.

(9) In the Introduction, the first sentence in the second paragraph reads: "... when neurons receive strong excitatory and inhibitory synaptic input ...". The word strong should be changed to "weak".

Also, see the pioneering work of Brunel 2000. 

In classical balanced networks, strong excitatory inputs are counterbalanced by strong inhibitory inputs, leading to a fluctuation-driven regime. We have added Brunel 2000.

(10) In the second paragraph of the introduction, the authors refer to studies about structural co-tuning (e.g., where "precise" synaptic balance is mentioned, and Vogels et al. 2011 should be cited there) and functional co-tuning (which is, in fact, different than tracking of excitation by inhibition, but the authors refer to that as co-tuning). It makes it easier to understand which studies talk about structural co-tuning and which ones are about functional co-tuning. The paper by Znamenski 2018, which showed both structural and functional tuning in experiments, is missing here. 

We added the citation to the now published paper by Znamenskyi et al. (2024).  

(11) The third paragraph in the Introduction misses some references that address network dynamics that are shaped by the inhibitory architecture in E/I assemblies in spiking networks, like Rost et al 2018 and Lagzi et al 2021. 

These references have been added.

(12) The last sentence of the fourth paragraph in the Introduction implies that functional co-tuning is due to structural co-tuning, which is not necessarily true. While structural co-tuning results in functional co-tuning, functional co-tuning does not require structural co-tuning because it could arise from shared correlated input or heterogeneity in synaptic connections from E to I cells.  

We generally agree with the reviewer, but we are uncertain which sentence the reviewer refers to.

We assume the reviewer refers to the last sentence of the second (rather than the fourth paragraph), which explicitly mentions the “…structural basis of E/I co-tuning…”. If so, we consider this sentence still correct because the “structural basis” refers not specifically to E/I assemblies, but also includes any other connectivity that may produce co-tuning, including the connectivity underlying the alternative possibilities mentioned by the reviewer (shared correlated input or heterogeneity of synaptic connections).

(13) In order to ensure that the comparison between network dynamics is legit, authors should mention up front that for all networks, the average firing rates for the excitatory cells were kept at 1 Hz, and the background input was identical for all E and I cells across different networks.

We slightly revised the text to make this more clear “We (…) uniformly scaled I-to-E connection weights by a factor of χ until E population firing rates in response to learned odors matched the corresponding firing rates in rand networks, i.e., 1 Hz”

(14) In the last paragraph on page 5, my understanding was that an individual odor could target different cells within an assembly in different trials to generate trial to trail variability. If this is correct, this needs to be mentioned clearly. 

This is not correct, an odor consists of 150 activated mitral cells with defined firing rates. As now mentioned in the Methods, “Spikes were then generated from a Poisson distribution, and this process was repeated to create trial-to-trial variability.”

(15) The last paragraph on page 6 mentions that the four OB activity patterns were uncorrelated but if they were designed as in Figure 4A, dues to the existing overlap between the patterns, they cannot be uncorrelated. 

This appears to be a misunderstanding. We mention in the text (and show in Figure 4B) that the four odors which “… were assigned to the corners of a square…” are uncorrelated.  The intermediate odors are of course not uncorrelated. We slightly modified the corresponding paragraph (now on page 7) to clarify this: “The subspace consisted of a set of OB activity patterns representing four uncorrelated pure odors and mixtures of these pure odors. Pure odors were assigned to the corners of a square and mixtures were generated by selecting active mitral cells from each of the pure odors with probabilities depending on the relative distances from the corners (Figure 4A, Methods).”

(16) The notion of "learned" and "novel" odors may be misleading as there was no plasticity in the network to acquire an input representation. It would be beneficial for the authors to clarify that by "learned," they imply the presence of the corresponding E assembly for the odor in the network, with the input solely impacting that assembly. Conversely, for "novel" inputs, the input does not target a predefined assembly. In Figure 2 and Figure 4, it would be especially helpful to have the spiking raster plots of some sample E and I cells.  

As suggested by the reviewer, we have modified the existing spiking raster plots in Figure 2, such that they include examples of responses to both learned and novel odors. We added spiking raster plots showing responses of I neurons to the same odors in Supplementary Figure 1F, as well as spiking raster plots of E neurons in Supplementary Figure 4A. To clarify the usage of “learned” and “novel”, we have added a sentence in the Results section: “We thus refer to an odor as “learned” when a network contains a corresponding assembly, and as “novel” when no such assembly is present.”.

(17) In the last paragraph of page 8, can the authors explain where the asymmetry comes from? 

As mentioned in the text, the asymmetry comes from the difference in the covariance structure of different classes. To clarify, we have rephrased the sentence defining the Mahalanobis distance: 

“This measure quantifies the distance between the pattern and the class center, taking into account covariation of neuronal activity within the class. In bidirectional comparisons between patterns from different classes, the mean dM may be asymmetric if neural covariance differs between classes.”

(18) The first paragraph of page 9: random networks are not expected to perform pattern classification, but just pattern representation. It would have been better if the authors compared Scaled I network with E/I co-tuned network. Regardless of the expected poorer performance of the E/I co-tuned networks, the result would have been interesting. 

Please see our reply to the public review (reviewer 2).

(19) Second paragraph on page 9, the authors should provide statistical significance test analysis for the statement "... was significantly higher ...". 

We have performed a Wilcoxon signed-rank test, and reported the p-value in the revised manuscript (p < 0.01). 

(20) The last sentence in the first paragraph on page 11 is not clear. What do the authors mean by "linearize input-output functions", and how does it support their claim? 

We have now amended this sentence to clarify what we mean: “…linearize the relationship between the mean input and output firing rates of neuronal populations…”.

(21) In the first sentence of the last paragraph on page 11, the authors mentioned “high variability”, but it is not clear compared with which of the other 3 networks they observed high variability.

Structurally co-tuned E/I networks are expected to diminish network-level variability. 

“High variability” refers to the variability of spike trains, which is now mentioned explicity in the text. We hope this more precise statement clarifies this point.

(22) Methods section, page 14: "firing rates decreased with a time constant of 1, 2 or 4 s". How did they decrease? Was it an implementation algorithm? The time scale of input presentation is 2 s and it overlaps with the decay time constant (particularly with the one with 4 s decrease).  

Firing rates decreased exponentially. We have added this information in the Methods section.

Reviewer #3 (Recommendations For The Authors): 

In the following, I suggest minor corrections to each section which I believe can improve the manuscript. 

- There was no github link to the code in the manuscript. The code should be made available with a link to github in the final manuscript. 

The code can be found here: https://github.com/clairemb90/pDp-model. The link has been added in the Methods section.

Figure 1: 

- Fig. 1A: call it pDp not Dp. Please check if this name is consistent in every figure and the text. 

Thank you for catching this. Now corrected in Figure 1, Figure 2 and in the text.

- The authors write: "Hence, pDpsim entered an inhibition-stabilized balanced state (Sadeh and Clopath, 2020b) during odor stimulation (Figure 1D, E)." and then later "Shuffling spike times of inhibitory neurons resulted in runaway activity with a probability of ~80%, demonstrating that activity was indeed inhibition-stabilized. These results were robust against parameter variations (Methods)." I would suggest moving the second sentence before the first sentence, because the fact that the network is in the ISN regime follows from the shuffled spike timing result. 

Also, I'd suggest showing this as a supplementary figure. 

We thank the reviewer for this comment. We have removed “inhibition-stabilized” in the first sentence as there is no strong evidence of this in Rupprecht and Friedrich, 2018. And removed “indeed” in the second sentence. We also provided more detailed statistics. The text now reads “Hence, pDpsim entered a balanced state during odor stimulation (Figure 1D, E) with recurrent input dominating over afferent input, as observed in pDp (Rupprecht and Friedrich, 2018). Shuffling spike times of inhibitory neurons resulted in runaway activity with a probability of 0.79 ± 0.20, demonstrating that activity was inhibition-stabilized (Sadeh and Clopath, 2020b).”

Figure 2: 

- "... Scaled I networks (Figure 2H." Missing ) 

Corrected.

- The authors write "Unlike in Scaled I networks, mean firing rates evoked by novel odors were indistinguishable from those evoked by learned odors and from mean firing rates in rand networks (Figure 2F)." 

Why is this something you want to see? Isn't it that novel stimuli usually lead to high responses? Eg in the paper Schulz et al., 2021 (eLife) which is also cited by the authors it is shown that novel responses have high onset firing rates. I suggest clarifying this (same in the context of Fig. 3C). 

In Dp and piriform cortex, firing rates evoked by learned odors are not substantially different from firing rates evoked by novel odors. While small differences between responses to learned versus novel odors cannot be excluded, substantial learning-related differences in firing rates, as observed in other brain areas, have not been described in Dp or piriform cortex. We added references in the last paragraph of p.5. Note that the paper by Schulz et al. (2021) models a different type of circuit.  

- Fig. 2B: Indicate in figure caption that this is the case "Scaled I" 

This is not exactly the case “Scaled I”, as the parameter 𝝌𝝌 (increased I to E strength) is set to 1.

- Suppl Fig. 2I: Is E&F ever used in the manuscript? I couldn't find a reference. I suggest removing it if not needed. 

Suppl. Fig 2I E&F is now Suppl Fig.1G&H. We now refer to it in the text: “Activity of networks with E assemblies could not be stabilized around 1 Hz by increasing connectivity from subsets of I neurons receiving dense feed-forward input from activated mitral cells (Supplementary Figure 1GH; Sadeh and Clopath, 2020).”

Figure 3: 

- As mentioned in my comment in the public review section, I find the arguments about pattern completion a little bit confusing. For me it's not clear why an increase of output correlations over input correlations is considered "pattern completion" (this is not to say that I don't find the nonlinear increase of output correlations interesting). For me, to test pattern completion with second-order statistics one would need to do a similar separation as in Suppl Fig. 3, ie measuring the pairwise correlation at cells in the assembly L that get direct input from L OB with cells in the assembly L that do not get direct input from OB. If the pairwise correlations of assembly cells which do not get direct input from OB increase in correlations, I would consider this as pattern completion (similar to the argument that increase in firing rate in cells which are not directly driven by OB are considered a sign of pattern completion). 

Also, for me it now seems like that there are contradictory results, in Fig. 3 only Scaled I can lead to pattern completion while in the context of Suppl. Fig. 3 the authors write "We found that assemblies were recruited by partial inputs in all structured pDpsim networks (Scaled and Tuned) without a significant increase in the overall population activity (Supplementary Figure 3A)."   I suggest clarifying what the authors exactly mean by pattern completion, why the increase of output correlations above input correlations can be considered as pattern completion, and why the results differs when looking at firing rates versus correlations. 

Please see our reply to the public review (reviewer 3).

- I actually would suggest adding Suppl. Fig. 3 to the main figure. It shows a more intuitive form of pattern completion and in the text there is a lot of back and forth between Fig. 3 and Suppl. Fig. 3 

We feel that the additional explanations and panels in Fig.3 should clarify this issue and therefore prefer to keep Supplementary Figure 3 as part of the Supplementary Figures for simplicity.  

- In the whole section "We next explored effects of assemblies ... prevented strong recurrent amplification within E/I assemblies." the authors could provide a link to the respective panel in Fig. 2 after each statement. This would help the reader follow your arguments. 

We thank the reviewer for pointing this out. The references to the appropriate panels have been added. 

- Fig. 3A: I guess the x-axis has been shifted upwards? Should be at zero. 

We have modified the x-axis to make it consistent with panels B and C.  

- Fig. 3B: In the figure caption, the dotted line is described as the novel odor but it is actually the unit line. The dashed lines represent the reference to the novel odor. 

Fixed.

- Fig. 3C: The " is missing for Pseudo-Assembly N

Fixed.

- "...or a learned odor into another learned odor." Have here a ref to the Supplementary Figure 3B.

Added.

Figure 4:   

- "This geometry was largely maintained in the output of rand networks, consistent with the notion that random networks tend to preserve similarity relationships between input patterns (Babadi and Sompolinsky, 2014; Marr, 1969; Schaffer et al., 2018; Wiechert et al., 2010)." I suggest adding here reference to Fig. 4D (left). 

Added.

- Please add a definition of E/I assemblies. How do the authors define E/I assemblies? I think they consider both, Tuned I and Tuned E+I as E/I assemblies? In Suppl. Fig. 2I E it looks like tuned feedforward input is defined as E/I assemblies. 

We thank the reviewer for pointing this out. E/I assemblies are groups of E and I neurons with enhanced connectivity. In other words, in E/I assemblies, connectivity is enhanced not only between subsets of E neurons, but also between these E neurons and a subset of I neurons. This is now clarified in the text: “We first selected the 25 I neurons that received the largest number of connections from the 100 E neurons of an assembly. To generate E/I assemblies, the connectivity between these two sets of neurons was then enhanced by two procedures.”. We removed “E/I assemblies” in Suppl. Fig.2, where the term was not used correctly, and apologize for the confusion.

- Suppl. Fig. 4: Could the authors please define what they mean by "Loadings" 

The loadings indicate the contribution of each neuron to each principal component, see adjusted legend of Suppl. Fig. 4: “G. Loading plot: contribution of neurons to the first two PCs of a rand and a Tuned E+I network (Figure 4D).”

- Fig. 4F: The authors might want to normalize the participation ratio by the number of neurons (see e.g. Dahmen et al., 2023 bioRxiv, "relative PR"), so the PR is bound between 0 and 1 and the dependence on N is removed. 

We thank the reviewer for the suggestion, but we prefer to use the non-normalized PR as we find it more easily interpretable (e.g. number of attractor states in Scaled networks).

- Fig. 4G&H: as mentioned in the public review, I'd add the case of Scaled I to be able to compare it to the Tuned E+I case. 

As already mentioned in the public review, we thank the reviewer for this suggestion, which we have implemented.

- Figure caption Fig. 4H "Similar results were obtained in the full-dimensional space." I suggest showing this as a supplemental panel. 

Since this only adds little information, we have chosen not to include it as a supplemental panel to avoid overloading the paper with figures.

Figure 5: 

- As mentioned in the public review, I suggest that the authors add the Scaled I case to Fig. 5 (it's shown in all figures and also in Fig. 6 again). I guess for Scaled I the separation between L and M will be very good? 

Please see our reply to the public review (reviewer 3).

- Fig. 5A&B: I am a bit confused about which neurons are drawn to calculate the Mahalanobis distance. In Fig. 5A, the schematic indicates that the vector B from which the neurons are drawn is distinct from the distribution Q. For the example of odor L, the distribution Q consists of pure odor L with odors that have little mixtures with the other odors. But the vector v for odor L seems to be drawn only from odors that have slightly higher mixtures (as shown in the schematic in Fig. 5A). Is there a reason to choose the vector v from different odors than the distribution Q? 

The distribution Q and the vector v consist of activity patterns across the same neurons in response to different odors. The reason to choose a different odor for v was to avoid having this test datapoint being included in the distribution Q. We also wanted Q to be the same for all test datapoints. 

What does "drawn from whole population" mean? Does this mean that the vectors are drawn from any neuron in pDp? If yes, then I don't understand how the authors can distinguish between different odors (L,M,O,N) on the y-axis. Or does "whole population" mean that the vector is drawn across all assemblies as shown in the schematic in Fig. 5A and the case "neurons drawn from (pseudo-) assembly" means that the authors choose only one specific assembly? In any case, the description here is a bit confusing, I think it would help the reader to clarify those terms better.  

Yes, “drawn from whole population” means that we randomly draw 80 neurons from the 4000 E neurons in pDp. The y-axis means that we use the activity patterns of these neurons evoked by one of the 4 odors (L, M, N, O) as reference. We have modified the Figure legend to clarify this: “d_M was computed based on the activity patterns of 80 E neurons drawn from the four (pseudo-) assemblies (top) or from the whole population of 4000 E neurons (bottom). Average of 50 draws.”

- Suppl Fig. 5A: In the schematic the distance is called d_E(\bar{Q},\bar{V}) while the colorbar has d_E(\bar{Q},\bar{Q}) with the Qs in different color. The green Q should be a V. 

We thank the reviewer for spotting this mistake, it is now fixed.

- Fig. 5: Could the authors comment on the fact that a random network seems to be very good in classifying patterns on it's own. Maybe in the Discussion? 

The task shown in Figure 5 is a relatively easy one, a forced-choice between four classes which are uncorrelated. In Supplementary Figure 9, we now show classification for correlated classes, which is already much harder.

Figure 6: 

- Is the correlation induced by creating mixtures like in the other Figures? Please clarify how the correlations were induced. 

We clarified this point in the Methods section: “The pixel at each vertex corresponded to one pure odor with 150 activated and 75 inhibited mitral cells (…) and the remaining pixels corresponded to mixtures. In the case of correlated pure odors (Figure 6), adjacent pure odors shared half of their activated and half of their inhibited cells.”. An explicit reference to the Methods section has also been added to the figure legend.

- Fig. 6C (right): why don't we see the clear separation in PC space as shown in Fig. 4? Is this related to the existence of correlations? Please clarify. 

Yes. The assemblies corresponding to the correlated odors X and Y overlap significantly, and therefore responses to these odors cannot be well separated, especially for Scaled networks. We added the overlap quantification in the Results section to make this clear. “These two additional assemblies had on average 16% of neurons in common due to the similarity of the odors.”

- "Furthermore, in this regime of higher pattern similarity, dM was again increased upon learning, particularly between learned odors and reference classes representing other odors (not shown)." Please show this (maybe as a supplemental figure). 

We now show the data in Supplementary Figure 9.

Discussion: 

- The authors write: "We found that transformations became more discrete map-like when amplification within assemblies was increased and precision of synaptic balance was reduced. Likewise, decreasing amplification in assemblies of Scaled networks changed transformations towards the intermediate behavior, albeit with broader firing rate distributions than in Tuned networks (not shown)." 

Where do I see the first point? I guess when I compare in Fig. 4D the case of Scaled I vs Tuned E+I, but the sentence above sounds like the authors showed this in a more step-wise way eg by changing the strength of \alpha or \beta (as defined in Fig. 1). 

Also I think if the authors want to make the point that decreasing amplification in assemblies changes transformation with a different rate distribution in scaled vs tuned networks, the authors should show it (eg adding a supplemental figure). 

The first point is indeed supported by data from different figures. Please note that the revised manuscript now contains further simulations that reinforce this statement, particularly those shown in Supplementary Figure 6, and that this point is now discussed more extensively in the Discussion. We hope that these revisions clarify this general point.

The data showing effects of decreasing amplification in assemblies is now shown in Supplementary Figure 6 (Scaled[adjust])

- I suggest adding the citation Znamenskiy et al., 2024 (Neuron; https://doi.org/10.1016/j.neuron.2023.12.013), which shows that excitatory and inhibitory (PV) neurons with functional similarities are indeed strongly connected in mouse V1, suggesting the existence of E/I assembly structure also in mammals.

Done.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This provocative manuscript from presents valuable comparisons of the morphologies of Archaean bacterial microfossils to those of microbes transformed under environmental conditions that mimic those present on Earth during the same Eon, although the evidence in support of the conclusions is currently incomplete. The reasons include that taphonomy is not presently considered, and a greater diversity of experimental environmental conditions is not evaluated -- which is important because we ultimately do not know much about Earth's early environments. The authors may want to reframe their conclusions to reflect this work as a first step towards an interpretation of some microfossils as 'proto-cells,' and less so as providing strong support for this hypothesis. 

Regarding the taphonomic alterations: The editor and reviewers are correct in pointing out this issue. Taphonomic alteration of the microfossils attains special significance in the case of microorganisms, as they lack rigid structures and are prone to morphological alterations during or after their fossilization. We are acutely aware of this issue and have conducted long-term experiments (lasting two years) to observe how cells die, decay, and get preserved. A large section of the manuscript (pages 11 to 20) and a substantial portion of the supplementary information is dedicated to understanding the taphonomic alterations. To the best of our knowledge, these are among the longest experiments done to understand the taphonomic alterations of the cells within laboratory conditions. 

Recent reports by Orange et al. (1,2)  showed that under favorable environmental conditions, cells could be fossilized rather rapidly with little morphological modifications. We observed a similar phenomenon in this work. Cells in our study underwent rapid encrustation with cations from the growth media. We have analyzed the morphological changes over a period of 18 months. After 18 months, the softer biofilms got encrusted entirely in salt and turned solid (Fig. ). Despite this transformation, morphologically intact cells could still be observed within these structures. This suggests that the cells inhabiting Archaean coastal marine environments could undergo rather rapid encrustation, and their morphological features could be preserved in the geological record with little taphonomic alteration.    

Regarding the environmental conditions: We are in total agreement with the reviewers that much is unknown about Archaean geology and its environmental conditions. Like the present-day Earth, Archaean Earth certainly had regions that greatly differed in their environmental conditions—volcanic freshwater ponds, brines, mildly halophilic coastal marine environments, and geothermal and hydrothermal vents, to name a few. Our experimental design focuses on one environment we have a relatively good understanding of rather than the rest of the planet, of which we know little. Below, we list our reasons for restricting to coastal marine environments and studying cells under mildly halophilic experimental conditions.  

(1) Very little continental crust from Haden and early Archaean Eon exists on the presentday Earth. Much of our geochemical understanding of this time period was a result of studying the Pilbara Iron Formations and the Barberton Greenstone Belt. Geological investigations suggest that these sites were coastal marine environments. The salinity of coastal marine environments is higher than that of open oceans due to the greater water evaporation within these environments. Moreover, brines were discovered within pillow basalts within the Barberton greenstone belt, suggesting that the salinity within these sites is higher or similar to marine environments. 

(2) We are not certain about the environmental conditions that could have supported the origin of life. However, all currently known Archaean microfossils were reported from coastal marine environments (3.8-2.4Ga). This suggests that proto-life likely flourished in mildly halophilic environments, similar to the experimental conditions employed in our study. 

(3) The chemical analysis of Archaean microfossils also suggests that they lived in saltrich environments, as most, if not all, microfossils are closely associated, often encrusted in a thin layer of salt.  

However, we concur with the reviewers that our interpretations should be reassessed if Archaean microfossils that greatly differ from the currently known microfossils are to be discovered or if new microfossils are to be reported from environments other than coastal marine sites.

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

Microfossils from the Paleoarchean Eon represent the oldest evidence of life, but their nature has been strongly debated among scientists. To resolve this, the authors reconstructed the lifecycles of Archaean organisms by transforming a Gram-positive bacterium into a primitive lipid vesicle-like state and simulating early Earth conditions. They successfully replicated all morphologies and life cycles of Archaean microfossils and studied cell degradation processes over several years, finding that encrustation with minerals like salt preserved these cells as fossilized organic carbon. Their findings suggest that microfossils from 3.8 to 2.5 billion years ago were likely liposome-like protocells with energy conservation pathways but without regulated morphology. 

Strengths: 

The authors have crafted a compelling narrative about the morphological similarities between microfossils from various sites and proliferating wall-deficient bacterial cells, providing detailed comparisons that have never been demonstrated in this detail before. The extensive number of supporting figures is impressive, highlighting numerous similarities. While conclusively proving that these microfossils are proliferating protocells morphologically akin to those studied here is challenging, we applaud this effort as the first detailed comparison between microfossils and morphologically primitive cells. 

Weaknesses: 

Although the species used in this study closely resembles the fossils morphologically, it would be beneficial to provide a clearer explanation for its selection. The literature indicates that many bacteria, if not all, can be rendered cell wall-deficient, making the rationale for choosing this specific species somewhat unclear. While this manuscript includes clear morphological comparisons, we believe the authors do not adequately address the limitations of using modern bacterial species in their study. All contemporary bacteria have undergone extensive evolutionary changes, developing complex and intertwined genetic pathways unlike those of early life forms. Consequently, comparing existing bacteria with fossilized life forms is largely hypothetical, a point that should be more thoroughly emphasized in the discussion. 

Another weak aspect of the study is the absence of any quantitative data. While we understand that obtaining such data for microfossils may be challenging, it would be helpful to present the frequencies of different proliferative events observed in the bacterium used. Additionally, reflecting on the chemical factors in early life that might cause these distinct proliferation modes would provide valuable context. 

Regarding our choice of using modern organisms or this particular bacterial species: 

Based on current scientific knowledge, it is logical to infer that cellular life originated as protocells; nevertheless, there has been no direct geological evidence for the existence of such cells on early Earth. Hence, protocells remain an entirely theoretical concept. Moreover, protocells are considered to have been far more primitive than present-day cells. Surprisingly, this lack of sophistication was the biggest challenge in understanding protocells. Designing experiments in which cells are primitive (but not as primitive as non-living lipid vesicles) and still retain a functional resemblance to a living cell does pose some practical challenges. Laboratory experiments with substitute (proxy) protocells almost always come with some limitations. Although not a perfect proxy, we believe protocells and protoplasts share certain characteristics. Having said that, we would like to reemphasize that protoplasts are not protocells. Our reasons for using protoplasts as model organisms and working with this bacterial species (Exiguobacterium Strain-Molly) are based on several scientific and practical criteria listed below.

(1) Irrespective of cell physiology and intracellular complexity, we believe that protoplasts and protocells share certain similarities in the biophysical properties of their cytoplasm. We explained our reasoning in the manuscript introduction and in our previous manuscripts (Kanaparthi et al., 2024 & Kanaparthi et al., 2023). In short, to be classified as a cell, even a protocell should possess minimal biosynthetic pathways, a physiological mechanism of harvesting free energy from the surrounding (energy-yielding pathways), and a means of replicating its genetic material and transferring it to the daughter cells. These minimal physiological processes could incorporate considerable cytoplasmic complexity. Hence, the biophysical properties of the protocell cytoplasm could have resembled those of the cytoplasm of protoplasts, irrespective of the genomic complexity. 

(2) Irrespective of their physiology, protoplasts exhibit several key similarities to protocells, such as their inherent inability to regulate their morphology or reproduction. This similarity was pointed out in previous studies (3). Despite possessing all the necessary genetic information, protoplasts undergo reproduction through simple physiochemical processes independent of canonical molecular biological processes. This method of reproduction is considered to have been erratic and rather primitive, akin to the theoretical propositions on protocells. Although protoplasts are fully evolved cells with considerable physiological complexity, the above-mentioned biophysical similarities suggest that the protoplast life cycle could morphologically resemble that of protocells (in no other aspect except for their morphology and reproduction).  

(3) Physiologically or genomically different species of Gram-positive protoplasts are shown to exhibit similar morphologies. This suggests that when Gram-positive bacteria lose their cell wall and turn into a protoplast,  they reproduce in a similar manner independent of physiological or genome-based differences. As morphology and only morphology is key to our study, at least from the scope of this study, intracellular complexity is not a key consideration. 

(4) This specific strain was isolated from submerged freshwater springs in the Dead Sea. This isolate and members of this bacterial genus are known to have been well acclimatized to growing in a wide range of salt concentrations and in different salt species. This is important for our study (this and previous manuscript), in which cells must be grown not only at high salt concentrations (1-15%) but in different salts like NaCl, MgCl₂, and KCl. 

(5) Our initial interest in this isolate was due to its ability to reduce iron at high salt concentrations. Given that most spherical microfossils are found in Archaean-banded iron formations covered in pyrite, this suggests that these microfossils could have been reducing oxidized iron species like Fe(III). Nevertheless, over the course of our study, we realized the complexities of live cell staining and imaging under anoxic conditions. Given that the scope of the manuscript is restricted only to comparing the morphologies, not the physiology, we abandoned the idea of growing cells under anoxic conditions.  

Based on these observations, cell physiology may not be a key consideration, at least within the scope of studying microfossil morphology. However, we want to emphasize again that “We do not claim present-day protoplasts are protocells.”  

Regarding the absence of quantitative data:

We are unsure what the reviewer meant by the absence of quantitative data. Is it from the cell size/reproductive pathways perspective or from a microfossil/ecological perspective? At the risk of being portrayed in a bad light, we admit that we did not present quantitative data from either of these perspectives. In our defense, this was not due to our lack of effort but due to the practical limitations imposed by our model organism. 

If the reviewer means the quantitative data regarding cell sizes and morphology: In our previous work, we studied the relationship between protoplast morphology, growth rate, and environmental conditions. In that study, we proposed that the growth rate is one factor that regulates protoplast morphology. Nevertheless, we did not observe uniformity in the sizes of the cells. This lack of uniformity was not just between the replicates but even among the cells grown within the same culture flask or the cells within the same microscopic field. Moreover, cells are often observed to be reproducing either by forming internal or external or by both these processes at the same time. The size and morphological differences among cells within a growth stage could be explained by the physiological and growth rate heterogenicity among cells. 

Bacterial growth curves and their partition into different stages (lag, log & stationary), in general, represent the growth dynamics of an entire bacterial population. Nevertheless, averaging the data obscures the behavior of individual cells (4,5). It is known that genetically identical cells within a single bacterial population could exhibit considerable cell-to-cell variation in gene expression (6,7) and growth rates (8). The reason for such stochastic behavior among monoclonal cells has not been well understood. In the case of normal cells, morphological manifestation of these variations is restricted by a rigid cell wall. Given the absence of a cell wall in protoplasts, we assume such cell-to-cell variations in growth rate is manifested in cell morphology. This makes it challenging to quantitatively determine variations in cell sizes or the size increase in a statically robust manner, even in monoclonal cells. 

Although this lack of uniformity in cell sizes should not be perceived as a limitation, this behavior is consistently observed among microfossils. Spherical microfossils of similar morphology but different sizes were reported from different microfossil sites (9,10). In this regard, both protoplasts and microfossils are very similar. 

If the reviewer means the quantitative data from an ecological perspective: 

Based on the elemental composition and the isotopic signatures of the organic carbon, we can deduce if these structures are of biological origin or not. However, any further interpretation of this data to annotate these microfossils to a particular physiology group is fraught with errors. Hence, we refrain from making any inferences about the physiology and ecological function of these microfossils. This lack of clarity on the physiology of microfossils reduces the chance of quantitative studies on their ecological functions. Moreover, we would like to re-emphasize that the scope of this work is restricted to morphological comparison and is not targeted at understanding the ecological function of these microfossils. This narrow objective also limits the nature of the quantitative data we could present.

Moreover, developing a quantitative understanding of some phenomena could be technically challenging. Many theories on the origin of life, like chemical evolution, started with the qualitative observation that lightning could mediate the synthesis of biologically relevant organic carbon. Our quantitative understanding of this process is still being explored and debated even to this day.     

Reviewer #2 (Public Review): 

Summary: 

In summary, the manuscript describes life-cycle-related morphologies of primitive vesiclelike states (Em-P) produced in the laboratory from the Gram-positive bacterium Exiguobacterium Strain-Molly) under assumed Archean environmental conditions. Em-P morphologies (life cycles) are controlled by the "native environment". In order to mimic Archean environmental conditions, soy broth supplemented with Dead Sea salt was used to cultivate Em-Ps. The manuscript compares Archean microfossils and biofilms from selected photos with those laboratory morphologies. The photos derive from publications on various stratigraphic sections of Paleo- to Neoarchean ages. Based on the similarity of morphologies of microfossils and Em-Ps, the manuscript concludes that all Archean microfossils are in fact not prokaryotes, but merely "sacks of cytoplasm". 

Strengths: 

The approach of the authors to recognize the possibility that "real" cells were not around in the Archean time is appealing. The manuscript reflects the very hard work by the authors composing the Em-Ps used for comparison and selecting the appropriate photo material of fossils. 

Weaknesses: 

While the basic idea is very interesting, the manuscript includes flaws and falls short in presenting supportive data. The manuscript makes too simplistic assumptions on the "Archean paleoenvironment". First, like in our modern world, the environmental conditions during the Archean time were not globally the same. Second, we do not know much about the Archean paleoenvironment due to the immense lack of rock records. More so, the Archean stratigraphic sections from where the fossil material derived record different paleoenvironments: shelf to tidal flat and lacustrine settings, so differences must have been significant. Finally, the Archean spanned 2.500 billion years and it is unlikely that environmental conditions remained the same. Diurnal or seasonal variations are not considered. Sediment types are not considered. Due to these reasons, the laboratory model of an Archean paleoenvironment and the life therein is too simplistic. Another aspect is that eucaryote cells are described from Archean rocks, so it seems unlikely that prokaryotes were not around at the same time. Considering other fossil evidence preserved in Archean rocks except for microfossils, the many early Archean microbialites that show baffling and trapping cannot be explained without the presence of "real cells". With respect to lithology: chert is a rock predominantly composed of silica, not salt. The formation of Em-Ps in the "salty" laboratory set-up seems therefore not a good fit to evaluate chert fossils. Formation of structures in sediment is one step. The second step is their preservation. However, the second aspect of taphonomy is largely excluded in the manuscript, and the role of fossilization (lithification) of Em-Ps is not discussed. This is important because Archean rock successions are known for their tectonic and hydrothermal overprint, as well as recrystallization over time. Some of the comparisons of laboratory morphologies with fossil microfossils and biofilms are incorrect because scales differ by magnitudes. In general, one has to recognize that prokaryote cell morphologies do not offer many variations. It is possible to arrive at the morphologies described in various ways including abiotic ones. 

Regarding the simplistic presumptions on the Archaean Eon environmental conditions, we provided a detailed explanation of this issue in our response to the eLife evaluation. In short, we agree with the reviewer that little is known about the Archaean Eon environmental conditions at a planetary scale. Hence, we restricted our study to one particular environment of which we had a comparatively good understanding. The Archaean Eon spanned 2.5 billion years. However, most of the microfossil sites we discussed in the manuscript are older than 3 billion years, with one exception (2.4 billion years old Turee Creek microfossils). We presume that conditions within this niche (coastal marine) environment could not have changed greatly until 2Ga, after which there have been major changes in the ocean salt composition and salinities.

In the manuscript, we discussed extensively the reasons for restricting our study to these particular environmental conditions. Further explanations of these choices are presented in our response to the eLife evaluation (also see our previous manuscript). In short, the fact that all known microfossils are restricted to coastal marine environments justifies the experimental conditions employed in our study. Nevertheless, we agree with the reviewer that all lab-based studies involve some extent of simplification. This gap/mismatch is even wider when it comes to studies involving origin or early life on Earth.

We are not arguing that prokaryotes are not around at this time. The key message of the manuscript is that they are present, but they have not developed intracellular mechanisms to regulate their morphology and remained primitive in this aspect.  

The sizes of the microfossils and cells from our study were similar in most cases. However, we agree with the reviewer that they deviated considerably in some cases, for example, S70, S73, and S83. These size variations are limited to sedimentary structures like laminations rather than cells. These differences should be expected as we try to replicate the real-life morphologies of biofilms that could have extended over large swats of natural environments in a 2ml volume chamber slide. More specifically, in Fig. S70, there is a considerable size mismatch. But, in Fig. S73, the sizes were comparable between A & C (of course, the size of our reproduction did not match B). In the case of Fig. S83, we do not see a huge size mismatch.      

Reviewer #1 (Recommendations For The Authors): 

We would like to provide several suggestions for changes in text and additions to data analysis. 

39-41: It has been stated that reconstructing the lifecycle is the only way of understanding the nature of these microfossils. First of all, I would rephrase this to 'the most promising way', as there are always multiple approaches to comparing phenomena. 

We agree with the reviewer's suggestion. The suggested changes have been made (line 41). 

125: Please rephrase "under the environmental condition of early Earth" to "under experimental conditions possibly resembling the conditions of the Paleoarchean Eon". Now it sounds like the exact environmental conditions have been produced, which has already been debated in the discussion. 

We agree with the reviewer's suggestion. The suggested changes have been made (line 127). 

125: Please mention the fold change in size, the original size in numbers, and whether this change is statistically significant. 

In the above sections of this document, we explained our reservations about presenting the exact number.

128: Have you found a difference in the relative percentages of modes of reproduction? In other words, is there a difference in percentage between forming internal daughter cells or a string of external daughter cells? 

We explained our reservations about presenting the exact number above. But this has been extensively discussed in our accompaining manuscript. We want to reemphasize that the scope of this manuscript is restricted to comparing morphologies rather than providing a mechanistic explanation of the reproduction process. 

151: A similar model for endocytosis has already been described in proliferating wall-less cells (Kapteijn et al., 2023). In the discussion, please compare your results with the observations made in that paper. 

This is an oversight on our part. The manuscript suggested by the reviewer has now been added (line 154 & 155).  

163: Please use another word for uncanny. We suggest using 'strong resemblance'. 

We changed this according to the reviewers' suggestion (line 168). 

433: Please elaborate on why the results are not shown. This sounds like a statement that should be substantiated further. 

To observe growth and simultaneously image the cells, we conducted these experiments in chamber slides (2ml volume). Over time, we observed cells growing and breaking out of the salt crust (Fig. S86, S87 & Movie 22) and a gradual increase in the turbidity of the media. Although not quantitative, this is a qualitative indication of growth. We did not take precise measurements for several reasons. This sample is precious; it took us almost two years to solidify the biofilm completely, as shown in Fig. S84A. Hence, it was in limited supply, which prevented us from inoculating these salt crusts into large volumes of fresh media. Given a long period of starvation, these cells often exhibited a long lag phase (several days), and there wasn't enough volume to do OD measurements over time. 

We also crushed the solidified biofilm with a sterile spatula before transferring it into the chamber slide with growth media. This resulted in debris in the form of small solid particles, which interfered with our OD measurements. These practical considerations made it challenging to determine the growth precisely. Despite these challenges, we measured an OD of 4 in some chamber slides after two weeks of incubation. Given that these measurements were done haphazardly, we chose not to present this data. 

456: Could you please double-check whether the description is correct for the figure? 8C and 8D are part of Figure 8B, but this is stated otherwise in the description. 

We thank the reviewer for pointing it out. It has now been rectified (line 461-472).

Reviewer #2 (Recommendations For The Authors): 

We thank Reviewer #2  for carefully reading the manuscript and such an elaborate list of questions. The revisions suggested have definitely improved the quality of the manuscript. Here, we would like to address some of the questions that came up repeatedly below. One frequently asked question is regarding the letters denoting the individual figures within the images. For comparison purposes, we often reproduced previously published images. To maintain a consistent figure style, we often have to block the previous denotations with an opaque square and give a new letter. 

The second question that appeared repeatedly below is the missing scale bars in some of the images within a figure. We often did not include a scale bar in the images when this image is an enlarged section of another image within the same figure.     

Title: Please consider being more precise in the title. Microfossils are only one fossil group of "oldest life". Perhaps better: "On the nature of some microfossils in Archean rocks". (see also Line 37).  

Authors’ response: The title conveys a broader message without quantitative insinuations. If our manuscript had been titled "On the nature of all known Archaean microfossils,” we should have agreed with the reviewer's suggestion and changed it to "On the nature of some microfossils in Archean rocks". As it is not, we respectfully decline to make this modification.     

Abstract:  

Line 41: "one way", not "the only way" 

We agree with the reviewer’s comment, and necessary changes have been made (line 41).  

Introduction: 

Line 58f: "oldest sedimentary rock successions", not "oldest known rock formations". There are rocks of much older ages, but those are not well preserved due to metamorphic overprint, or the rocks are igneous to begin with. Minor issue: please note that "formations" are used as stratigraphic units, not so much to describe a rock succession in the field. 

We agree with the reviewer’s comment and have made necessary changes (line 58).

Line 67: Microfossils are widely accepted as evidence of life. Please rephrase. 

We agree with the reviewer’s comment, and necessary changes have been made.

Line 71 - 74: perhaps add a sentence of information here.

We agree with the reviewer’s comment, and necessary changes have been made (line 71).

Line 76: which "chemical and mineralogical considerations"? 

This has been rephrased to “Apart from the chemical and δ¹³C-biomass composition” (line 76).

Line 84ff: This is a somewhat sweeping statement. Please remember that there are microbialites in such rocks that require already a high level of biofilm organization. The existence of cyanobacteria-type microbes in the Archean is also increasingly considered. 

We are aware of literature that labeled the clusters of Archaean microfossils as biofilms and layered structures as microbialites or stromatolite-like structures. However, the use of these terms is increasingly being discouraged. A more recent consensus among researchers suggests annotating these structures simply as sedimentary structures, as microbially induced sedimentary structures (MISS). 

We respectfully disagree with the reviewer’s comment that Archaean microfossils exhibit a high level of biofilm organization. We are not aware of any studies that have conducted such comprehensive research on the architecture of Archaean biofilms. We are not even certain if these clusters of Archaean cells could even be labeled as biofilms in the true sense of the term. We presently lack an exact definition of a biofilm. In our study, we do see sedimentation and bacteria and their encapsulation in cell debris. From a broader perspective, any such aggregation of cells enclosed in cell debris could be annotated as a biofilm. However, more in-depth studies show that biofilm is not a random but a highly organized structure. Different bacterial species have different biofilm architectures and chemical composition. The multispecies biofilms in natural environments are even more complex. We do agree with the reviewer that these structures could broadly be labeled as biofilms, but we presently lack a good, if any, understanding of the Archaean biofilm architecture. 

Regarding the annotation of microfossils as cyanobacteria, we respectfully disagree with the reviewer. This is not a new concept. Many of the Archaean microfossils were annotated as cyanobacteria at the time of their discovery. This annotation is not without controversy. With the advent of genome-based studies, researchers are increasingly moving away from this school of thought.  

Line 101ff: The conditions on early Earth are unknown - there are many varying opinions. Perhaps simply state that this laboratory model simulates an Archean Earth environment of these conditions outlined. 

This is a good idea. We thank the reviewer for this suggestion, and we made appropriate changes. 

Line 112: manuscript to be replaced by "paper"? 

This change has been made (line 114).

Line 116: "spanned years" - how many years? 

We now added the number of years in the brackets (line 118).

Results: 

Line 125: see comment for 101ff. 

we made appropriate changes. 

Figure 1: Caption: Please write out ICV the first time this abbreviation is used. Images: Note that some lettering appears to not fit their white labels underneath. (G, H, I, J0, and M). 

We apologize; this is an oversight on our part. We now spell complete expansion of ICV, the first time we used this abbreviation. 

We took these images from previously published work (references in the figure legend), so we must block out the previous figure captions. This is necessary to maintain a uniform style throughout the manuscript. 

Line 152ff.: here would be a great opportunity to show in a graph the size variations of modern ICVs and to compare the variations with those in the fossil material. 

In the above sections of this document, we explained our reservations about presenting the exact number.

Line 159f.: Fig.1K - what is to see here? Maybe a close-up or - better - a small sketch would help? 

Fig. 1K shows the surface depressions formed during the vesicle formation. The surface characteristics of EM-P and microfossils is very similar.   

Line 161f.: reference?  

The paragraph spanning lines 159 to 172 discusses the morphological similarities between EM-P and SPF microfossils. We rechecked the reference no 35 (Delarue 2019). This is the correct reference. We do not see a mistake if the reviewer meant the reference to the figures.    

Line 164ff.: A question may be asked, how many fossils of the Strelley Pool population would look similar to the "modeled" ones. Questions may rise in which way the environmental conditions control such morphology variations. Perhaps more details? 

This relationship between the environmental conditions and the morphology is discussed extensively in our previous work (11).  

Line 193: what is meant by "similar discontinuous distribution of organic carbon"?

This statement highlights similarities between EM-P and microfossils. The distribution of cytoplasm within the cells is not uniform. There are regions with and devoid of cytoplasm, which is quite unusual for bacteria. Some previous studies argued that this could indicate that these organic structures are of abiotic origin. Here, we show that EMP-like cells could exhibit such a patchy distribution of cytoplasm within the cell.    

Line 218 - 291: The observations are very nice, however, the figures of fossil material in Figures 3 A, B, and C appear not to conform. Perhaps use D, E and I to K. Also, S48 does not show features as described here (see below).  

We did not completely understand the reviewer’s question. As mentioned in the figure legend, both the microfossils and the cells exhibit string with spherical daughter cells within them. Moreover, there are also other similarities like the presence of hollow spherical structures devoid of organic carbon. We also saw several mistakes in the Fig. S48 legend. We have rectified them, and we thank the reviewer for pointing them out.   

Line 293f: Title with "." at end?

This change has been made.

Line 298: predominantly in chert. In clastic material preservation of cells and pores is unlikely due to the common lack of in situ entombment by silica. 

We rephrased this entire paragraph to better convey our message. Either way, we are not arguing that hollow pore spaces exist. As the reviewer mentioned, they will, of course, be filled up with silica. In this entire paragraph, we did not refer to hollow spaces. So, we are not entirely sure what the question was.     

Line 324, 328-349: Please see below comments on the supplementary figures 51-62. Some of the interpretations of morphologies may be incorrect. 

Please find our response to the reviewer’s comments on individual figures below.  

Figure 5 A to D look interesting, however E to J appear to be unconvincing. What is the grey frame in D (not the white insert). 

The grey color is just the background that was added during the 3D rendering process.  

Figure 6 does not appear to be convincing. - Erase? 

We did not understand the reviewer’s reservations regarding this figure. Images A-F within the figure show the gradual transformation of cells into honeycomb-like structures, and images G-J show such structures from the Archaean that are closely associated with microfossils. Moreover, we did not come up with this terminology (honeycomb-like). Previous manuscripts proposed it.  

Line 379ff: S66 and 69, please see my comments below. Microfossils "were often discovered" in layers of organic carbon. 

Please see our response below.   

Line 393-403: Laminae? There are many ways to arrive at C-rich laminae, especially, if the material was compressed during burial. Basically, any type of biofilm would appear as laminae, if compressed. The appearance of thin layers is a mere coincidence. Note that the scale difference in S70, S73, as well as S83, is way too high (cm versus μm!) to allow any such sweeping conclusions. What are α- and β- laminations, the one described by Tice et al.? The arguments are not convincing.

We propose that cells be compressed to form laminae. We answered this question above about the differences in the scale bars. Yes, we are referring to α- and β- laminations described by Tice et al.       

Figure 7: This is an interesting figure, but what are the arguments for B and C, the fossil material, being a membrane? Debris cannot be distinguished with certainty at this scale in the insert of C. B could also be a shriveled-up set of trichomes.  

We agree with the reviewer that debris cannot be definitely differentiated. Traditionally, annotations given to microfossil structures such as biofilm, intact cells, or laminations were all based on morphological similarities with existing structures observed in microorganisms. Given that the structures observed in our study are very similar to the microfossil structures, it is logical to make such inferences. Scales in A & B match perfectly well. The structure in C is much larger, but, as we mentioned in reply to one of the reviewer’s earlier questions, some of the structures from natural environments could not be reproduced at scale in lab experiments. Working in a 2 ml chamber slides does impose some restrictions.   

Figure 8: The figure does not show any honeycomb patterns. The "gaps" in the Moodies laminae are known as lenticular particles in biofilms. They form by desiccated and shriveledup biofilm that mineralizes in situ. Sometimes also entrapped gases induce precipitation. Note also that the modelled material shows a kind of skin around the blobs that are not present in the Moodies material.  

We agree that entrapped gas bubbles could have formed lenticular gaps. In the manuscript, we did not discount this possibility. However, if that is the case, one should explain why we often find clumps of organic carbon within these gaps. As we presented a step-by-step transformation of parallel layers of cells into laminations, which also had similar lenticular gaps, we believe this is a more plausible way such structures could have formed. In the end, there could have been more than one way such structures could have been formed. 

We do see the honeycomb pattern in the hollow gaps. Often, the 3D-rendering of the STED images obscures some details. Hence, in the figure legend, we referred to the supplementary figures also show the sequence of steps involved in the formation of such a pattern.      

Line 405-417: During deposition of clastic sediment any hollow space would be compressed during burial and settling. It is rare that additional pore space (except between the graingrain-contacts) remains visible, especially after consolidation. The exception would be if very early silicification took place filling in any pore space. What about EPS being replaced by mineralic substance? The arguments are not convincing. 

We are suggesting that EPS or cell debris is rapidly encrusted by cations from the surrounding environment and gets solidified into rigid structures. This makes it possible for the structures to be preserved in the fossil record. We believe that hollow structures like the lenticular gaps will be filled up with silica. 

We do not agree with the reviewer’s comment that all biological structures will be compressed. If this is true, there should be no intact microfossils in the Archaean sedimentary structures, which is definitely not the case.      

Line 419-430: Lithification takes place within the sediment and therefore is commonly controlled by the chemistry of pore water and chemical compounds that derive from the dissolution of minerals close by. Another aspect to consider is whether "desiccation cracks" on that small scale may be artefacts related to sample preparation (?).  

We agree that desiccation cracks could have formed during the sample preparation for SEM imaging, as this involves drying the biofilms. However, we observed that the sample we used for SEM is a completely solidified biofilm (Fig. S84), so we expect little change in its morphology during drying. Moreover, visible cracks and pointy edges were also observed in wet samples, as shown in Fig. S87.        

Line 432 - 439: Please see comments on the supplementary material below.

Please find our response to the reviewer’s comments on individual figures below.  

Discussion:  

Line 477f: "all known microfossil morphologies" - is this a correct statement? Also, would the Archean world provide only one kind of "EM-P type"? Morphologies of prokaryote cells (spherical, rod-shaped, filamentous) in general are very simple, and any researcher of Precambrian material will appreciate the difficulties in concluding on taxonomy. There are papers that investigate putative microfossils in chert as features related to life cycles. Microfossil-papers commonly appear not to be controversial give and take some specific cases.  

We made a mistake in using the term “all known microfossil morphologies.” We have now changed it to “all known spherical microfossils” from this statement (line 483). However, we do not agree with the statement that microfossil manuscripts tend not to be controversial. Assigning taxonomy to microfossils is anything but controversial. This has been intensely debated among the scientific community.     

Line 494-496: This statement should be in the Introduction.

We agree with the reviewer’s comment. In an earlier version of the manuscript this statement was in the introduction. To put this statement in its proper context, it needs to be associated with a discussion about the importance of morphology in the identification of microfossils. The present version of the manuscript do not permit moving an entire paragraph into the introduction. Hence, we think making this statement in the discussion section is appropriate. 

Line 484ff. The discussion on biogenicity of microfossils is long-standing (e.g., biogenicity criteria by Buick 1990 and other papers), and nothing new. In paleontology, modern prokaryotes may serve as models but everyone working on Archean microfossils will agree that these cannot correspond to modern groups. An example is fossil "cyanobacteria" that is thought to have been around already in the early Archean. While morphologically very similar to modern cyanobacteria, their genetic information certainly differed - how much will perhaps remain undisclosed by material of that high age.  

Yes, we agree with the reviewer that there has been a longstanding conflict on the topic of biogenicity of microfossils. However, we have never come across manuscripts suggesting that modern microorganisms should only be used as models. If at all, there have been numerous manuscripts suggesting that these microfossils represent cyanobacteria, streptomycetes, and methanotrophs. Regarding the annotation of microfossils as cyanobacteria, we addressed this issue in one of the previous questions raised by the reviewer.    

Line 498ff: Can the variation of morphology and sizes of the EM-Ps be demonstrated statistically? Line 505ff are very speculative statements. Relabeling of what could be vesicles as "microfossils" appears inappropriate. Contrary to what is stated in the manuscript, the morphologies of the Dresser Formation vesicles do not resemble the S3 to S14 spheroids from the Strelley Pool, the Waterfall, and Mt Goldsworthy sites listed in the manuscript. The spindle-shaped vesicles in Wacey et al are not addressed by this manuscript. What roles in mineral and element composition would have played diagenetic alteration and the extreme hydrothermal overprint and weathering typical for Dresser material? S59, S60 do not show what is stated, and the material derives from the Barberton Greenstone Belt, not the Pilbara.

Please see the comments below regarding the supplementary images. 

We did not observe huge variations in the cell morphology. Morphologies, in most cases, were restricted to spherical cells with intracellular vesicles or filamentous extensions. Regarding the sizes of the cells, we see some variations. However, we are reluctant to provide exact numbers. We have presented our reasons above.

We respectfully disagree with the reviewer’s comments. We see quite some similarities between Dresser formation microfossils and our cells. Not just the similarities, we have provided step-by-step transformation of cells that resulted in these morphologies. We fail to see what exactly is the speculation here. The argument that they should be classified as abiotic structures is based on the opinion that cells do form such structures. We clearly show here that they can, and these biological structures resemble Dresser formation microfossils more closely than the abiotic structures. 

Regarding the figures S3-S14. We think they are morphologically very similar. Often, it's not just comparing both images or making exact reproductions (which is not possible). We should focus on reproducing the distinctive morphological features of these microfossils.  

We agree with the reviewer that we did not reproduce all the structures reported by Wacey’s original manuscript, such as spherical structures. We are currently preparing another manuscript to address the filamentous microfossils. These spindle-like structures will be addressed in this subsequent work. 

We agree with the reviewer, we often have difficulties differentiating between cells and vesicles. This is not a problem in the early stages of growth. During the log phase, a significant volume of the cell consists of the cytoplasm, with hollow vesicles constituting only a minor volume (Fig. 1B or S1A). During the later growth stages (Fig. 1E7F or S11), cells were almost hollow, with numerous daughter cells within them. These cells often resemble hollow vesicles rather than cells. However, given these are biologically formed structures, and one could argue that these vesicles are still alive as there is still a minimal amount of cytoplasm (Fig. S27). Hence, we should consider them as cells until they break apart to release daughter cells. 

Regarding Figures S59 and S60, we did not claim either of these microfossils is from Pilbara Iron Formations. The legend of Figure S59 clearly states that these structures are from Buck Reef Chert, originally reported by Tice et al., 2006 (Figure 16 in the original manuscript). The legend of Figure S60 says these structures were originally reported by Barlow et al., 2018, from the Turee Creek Formation. 

Line 546f and 552: The sites including microfossils in the Archean represent different paleoenvironments ranging from marine to terrestrial to lacustrine. References 6 and 66 are well-developed studies focusing on specific stratigraphic successions, but cannot include information covering other Archean worlds of the over 2.5 Ga years Archean time.  

All the Archaean microfossils reported to date are from volcanic coastal marine environments. We are aware that there are rocky terrestrial environments, but no microfossils have been reported from these sites. We are unaware of any Archaean microfossils reported from freshwater environments. 

Line 570ff: The statements may represent a hypothesis, but the data presented are too preliminary to substantiate the assumptions.

We believe this is a correct inference from an evolutionary, genomic, and now from a morphological perspective. 

Figures:  

Please check all text and supplementary figures, whether scale bars are of different styles within the figure (minor quibble). 

S3 (no scale in C, D); S4, S5: Note that scale bars are of different styles. 

We believe we addressed this issue above. 

S6 D: depressions here are well visible - perhaps exchange with a photo in the main text? Note that scale bars are of different styles.  

We agree that depressions are well visible in E. The same image of EM-P cell in E is also present in Fig. 1D in the main text.   

S7: Scale bars should all be of the same style, if anyhow possible. Scale in D? 

We believe we addressed this issue above. 

S9: F appears to be distorted. Is the fossil like this? The figure would need additional indicators (arrows) pointing toward what the reader needs to see - not clear in this version. More explanation in the figure caption could be offered. 

We rechecked the figure from the original publication to check if by mistake the figure was distorted during the assembly of this image. We can assure you that this is not the case. We are not sure what further could be said in the figure legend.     

S13: What is shown in the inserts of D and E that is also visible in A and B? Here a sketch of the steps would help. 

We did not understand the question.  

S14: Scale in A, B? 

We believe we addressed this issue above. 

S15: Scales in A, E, C, D 

We believe we addressed this issue above. 

S16: scales in D, E, G, H, I, J?  

We believe we addressed this issue above. 

S17: "I" appears squeezed, is that so? If morphology is an important message, perhaps reduce the entire figure so it fits the layout. Note that labels A, B, C, and D are displaced. 

As shown in several subsequent figures, the hollow spherical vesicles are compressed first into honeycomb-like structures, and they often undergo further compression to form lamination-like structures. Such images often give the impression that the entire figure is squashed, but this is not the case. If one examines the figure closely, you could see perfectly spherical vesicles together with laterally sqeezed structures. Regarding the figure labels, we addressed this issue above. 

S18: The filamentous feature in C could also be the grain boundaries of the crystals. Can this be excluded as an interpretation? Are there microfossils with the cell membranes? That would be an excellent contribution to this figure. Note that scale bars are of different styles.

If this is a one-off observation, we could have arrived at the reviewer's opinion. But spherical cells in a “string of beads” configuration were frequently reported from several sites, to be discounted as mere interpretation.    

S19: The morphologies in A - insert appear to be similar to E - insert in the lower left corner. The chain of cells in A may look similar to the morphologies in E - insert upper right of the image. B - what is to see here? D - the inclusions do not appear spherical (?). Does C look similar to the cluster with the arrow in the lower part of image E? Note that scale bars are of different styles (minor quibble). A, B, C, and D appear compressed. Perhaps reduce the size of the overall image?  

The structures highlighted (yellow box) in C are similar to the highlighted regions in E—the agglomeration of hollow vesicles. It is hard to get understand this similarity in one figure. The similarities are apparent when one sees the Movie 4 and Fig. S12, clearly showing the spherical daughter cells within the hollow vesicle. We now added the movie reference to the figure legend.    

S20: A appears not to contribute much. The lineations in B appear to be diagenetic. However, C is suitable. Perhaps use only C, D, E? 

We believe too many unrecognizable structures are being labeled as diagenetic. Nevertheless, we do not subscribe to the notion that these are too lenient interpretations. These interpretations are justified as such structures have not been reported from live cells. This is the first study to report that cells could form such structures. As we now reproduced these structures, an alternate interpretation that these are organic structures derived from microfossils should be entertained. 

S 21: Note that scale bars are of different styles.  

We believe we addressed this issue above. 

S22: Perhaps add an arrow in F, where the cell opened, and add "see arrow" in the caption? Is this the same situation as shown in C (white arrow)? What is shown by the white arrow in A? Note that scale bars are of different styles.

We did the necessary changes.  

S23: In the caption and main text, please replace "&" with "and" (please check also the other figure captions, e.g. S24). Note that scale bars are of different styles. What is shown in F? A, D - what is shown here?

We replaced “&” with “and.”  

S24: Note that scale bars are of different styles. Note that Wacey et al. describe the vesicles as abiotic not as "microfossils"; please correct in figure caption [same also S26; 25; 28].

We are aware of Prof. Dr. Wacey’s interpretations. We discuss it at length in the discussion section our manuscript. Based on the similarities between the Dresser formation structures and structures formed by EM-P, we contest that these are abiotic structures.  

S25: Appears compressed; note different scale bars. 

We believe we addressed this issue above. 

S28: The label in B is still in the upper right corner; scale in D? What is to see in rectangles (blue and red) in A, B? In fossil material, this could be anything. 

These figures are taken from a previous manuscript cited in the figure legend. We could not erase or modify these figures.  

S33: "L"ewis; G appears a bit too diffuse - erase? Note that scale bars are of different styles.

We believe we addressed this issue above. 

S34: This figure appears unconvincing. Erase? 

There are considerable similarities between the microfossils and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we can address his reservations.    

S35: It would be more convincing to show only the morphological similarities between the cell clusters. B and C are too blurry to distinguish much. Scales in D to F and in sketches? A appears compressed (?). 

We rechecked the original manuscript to see if image A was distorted while making this figure, but this is not the case. Regarding B & C, cells in this image are faint as they are hollow vesicles and, by nature, do not generate too much contrast when imaged with a phase-contrast microscope. There are some limitations on how much we can improve the contrast. We now added scale bars for D-I. Similarly, faint hollow vesicles can be seen in Fig. S21 C & D, and Fig. 3H.  

S36: Very nice; in B no purple arrow is visible. Note that scale bars are of different styles. S37 and S36 are very much the same - fuse, perhaps?  

We are sorry for the confusion. There are purple arrows in Fig. S37B-D. 

S38: this is a more unconvincing figure - erase? 

Unconvincing in wahy sense. There are considerable similarities between the microfossils and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we can address his reservations.

S39: white rectangle in A? Arrow in A? Note that scale bars are of different styles.

These are some of the unavoidable remnants from the image from the original publication. 

S40: in F: CM, V = ?; Note that scale bars are of different style. 

It’s an oversite on our part. We now added the definitions to the figure legaend. We thank the reviewer for pointing it out.  

S41: Rectangles in D, E, F, G can be deleted? Scales and labels missing in photos lower right. 

Those rectangles are added by the image processing software to the 3Drendered images. Regarding the missing scale bars in H & I they are the magnified regions of F. The scale bar is already present in F.   

S42: appears compressed. G could be trimmed. Labels too small; scale in G? 

This is a curled-up folded membrane. We needed to lower the resolution of some images to restrict the size of the supplement to journal size restrictions. It is not possible to present 85 figures in high resolution. But we assure you that the image is not laterally compressed in any manner.   

S43: This figure appears to be unconvincing. Reducing to pairing B, C, D with L, K? Spherical inclusions in B? Scales in E to G? Similar in S44: A, B, E only? Note that scale bars are of different styles. 

Figures I to K are important. They show not just the morphological similarities but also the sequence of steps through which such structures are formed. We addressed the issue of the scale bars above.  

S45: A, B, and C appear to show live or subrecent material. How was this isolated of a rock? Note that scale bars are of different styles.  

It is common to treat rocks with acids to dissolve them and then retrieve organic structures within them. This technique is becoming increasingly common. The procedure is quite extensively discussed in the original manuscript. We don’t see much differences in the scale bars of microfossils and EM-P cells, they are quite similar. 

S46: A: what is to see here? Note that scale bars are of different styles. 

There are considerable similarities between the folded fabric like organic structures with spherical inclusions and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we can address his reservations.    

S47: Perhaps enlarge B and erase A. Note that scale bars are of different styles. 

S48: Image B appears to show the fossil material - is the figure caption inconsistent? There are no aggregations visible in the boxes in A. H is described in the figure caption but missing in the figure. Overall, F and G do not appear to mirror anything in A to E (which may be fossil material?). 

S51; S52 B, C, E; S53: these figures appear unconvincing - erase? 

Unconvincing in what sense? The structures from our study are very similar to the microfossils.   

S54: North "Pole; scale bars in A to C =? 

These figures were borrowed from an earlier publication referenced in the figure legend. That is the reason for the differences in the styles of scale bars.  

S55: D and E appear not to contribute anything. Perhaps add arrow(s) and more explanation? Check the spelling in the caption, please. 

D & E show morphological similarities between cells from our study and microfossils (A).   

S56: Hexagonal morphologies may also be a consequence of diagenesis. Overall, perhaps erase this figure?  

I certainly agree that could be one of the reasons for the hexagonal morphologies. Such geometric polygonal morphologies have not been observed in living organisms. Nevertheless, as you can see from the figure, such morphologies could also be formed by living organisms. Hence, this alternate interpretation should not be discounted.   

S57: The figure caption needs improvement. Please add more description. What show arrows in A, what are the numbers in A? What is the relation between the image attached to the right side of A? Is this a close-up? Note that scale bars are of different styles. 

We expanded a bit on our original description of the figure. However, we request the reviewer to keep in mind that the parts of the figure are taken from previous publication. We are not at liberty to modifiy them, like removing the arrows. This imposes some constrains. 

S58: There are no honeycomb-shaped features visible. What is to see here? Erase this figure? 

Clearly, one can see spherical and polygonal shapes within the Archaean organic structures and mat-like structures formed by EM-P.  

S59 and S60: What is to see here? - Erase? 

Clearly, one can see spherical and polygonal shapes within the Archaean organic structures and mat-like structures formed by EM-P in Fig. S59. Further disintegration of these honeycomb shaped mats into filamentous struructures with spherical cells attached to them can be seen in both Archaean organic structures and structures formed by EM-P.   

S61: This figure appears to be unconvincing. B and F may be a good pairing. Note that scale bars are of different styles.  

There are considerable similarities between the microfossils and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we might be able to address his reservations.     

S62: This figure appears to be unconvincing - erase?

There are considerable similarities between the microfossils and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we might be able to address his reservations.     

S66: This figure is unconvincing - erase? 

There are considerable similarities between the microfossils and structures formed by EM-P. If the reviewer expands a bit on what he finds unconvincing, we might be able to address his reservations.    

S68: Scale in B, D, and E? 

Image B is just a magnified image of a small portion of image A. Hence, there is no need for an additional scale bar. The same is true for images D and E. 

S69: This figure appears to be unconvincing, at least the fossil part. Filamentous features are visible in fossil material as well, but nothing else. 

We are not sure what filamentous features the reviewer is referring to. Both the figures show morphologically similar spherical cells covered in membrane debris.    

S70 [as well as S82]: Good thinking here, but scales differ by magnitudes (cm to μm). Erase this figure? Very similar to Figure S73: Insert in C has which scale in comparison to B? Note that scale bars are of different styles.  

We realize the scale bars are of different sizes. In our defense, our experiments are conducted in 1ml volume chamber slides. We don’t have the luxury of doing these experiments on a scale similar to the natural environments. The size differences are to be expected. 

S71: Scale in E? 

Image E is just a magnified image of a small portion of image D. Hence, we believe a scale bar is unnecessary. 

S72: Scale in insert?  

The insert is just a magnified region of A & C

S75: This figure appears to be unconvincing. This is clastic sediment, not chert. Lenticular gaps would collapse during burial by subsequent sediment. - Erase? 

Regarding the similarities, we see similar lenticular gaps within the parallel layers of organic carbon in both microfossils, and structures formed by EM-P.

S76: A, C, D do not look similar to B - erase? Similar to S79, also with respect to the differences in scale. Erase? 

Regarding the similarities, we see similar lenticular gaps within the parallel layers of organic carbon in both microfossils, and structures formed by EM-P. We believe we addressed the issue of scale bars above. 

S80: A appears to be diagenetic, not primary. Erase? 

These two structures share too many resemblances to ignore or discount just as diagenic structures - Raised filamentous structures originate out of parallel layers of organic carbon (laminations), with spherical cells within this filamentous organic carbon.  

S85: What role would diagenesis play here? This figure appears unconvincing. Erase?

We do believe that diagenesis plays a major role in microfossil preservation. However, we also do not suscribe to the notion that we should by default assign diagenesis to all microfossil features. Our study shows that there could be an alternate explanation to some of the observations.  

S86 and S87: These appear unconvincing. What is to see here? Erase? 

The morphological similarities between these two structures. Stellarshaped organic structures with strings of spherical daughter cells growing out of them.  

S88: Does this image suggest the preservation of "salt" in organic material once preserved in chert?  

That is one inference we conclude from this observation. Crystaline NaCl was previously reported from within the microfossil cells.    

S89: What is to see here? Spherical phenomena in different materials? 

At present, the presence of honeycomb-like structures is often considered to have been an indication of volcanic pumice. We meant to show that biofilms of living organisms could result in honeycomb-shaped patterns similar to volcanic pumice.

References 

Please check the spelling in the references. 

We found a few references that required corrention. We now rectified them. 

References  

(1) Orange F, Westall F, Disnar JR, Prieur D, Bienvenu N, Le Romancer M, et al. Experimental silicification of the extremophilic archaea pyrococcus abyssi and methanocaldococcus jannaschii: Applications in the search for evidence of life in early earth and extraterrestrial rocks. Geobiology. 2009;7(4). 

(2) Orange F, Disnar JR, Westall F, Prieur D, Baillif P. Metal cation binding by the hyperthermophilic microorganism, Archaea Methanocaldococcus Jannaschii, and its effects on silicification. Palaeontology. 2011;54(5). 

(3) Errington J. L-form bacteria, cell walls and the origins of life. Open Biol. 2013;3(1):120143. 

(4) Cooper S. Distinguishing between linear and exponential cell growth during the division cycle: Single-cell studies, cell-culture studies, and the object of cell-cycle research. Theor Biol Med Model. 2006; 

(5) Mitchison JM. Single cell studies of the cell cycle and some models. Theor Biol Med Model. 2005; 

(6) Kærn M, Elston TC, Blake WJ, Collins JJ. Stochasticity in gene expression: From theories to phenotypes. Nat Rev Genet. 2005; 

(7) Elowitz MB, Levine AJ, Siggia ED, Swain PS. Stochastic gene expression in a single cell. Science. 2002; 

(8) Strovas TJ, Sauter LM, Guo X, Lidstrom ME. Cell-to-cell heterogeneity in growth rate and gene expression in Methylobacterium extorquens AM1. J Bacteriol. 2007; 

(9) Knoll AH, Barghoorn ES. Archean microfossils showing cell division from the Swaziland System of South Africa. Science. 1977;198(4315):396–8. 

(10) Sugitani K, Grey K, Allwood A, Nagaoka T, Mimura K, Minami M, et al. Diverse microstructures from Archaean chert from the Mount Goldsworthy–Mount Grant area, Pilbara Craton, Western Australia: microfossils, dubiofossils, or pseudofossils? Precambrian Res. 2007;158(3–4):228–62. 

(11) Kanaparthi D, Lampe M, Krohn JH, Zhu B, Hildebrand F, Boesen T, et al. The reproduction process of Gram-positive protocells. Sci Rep. 2024 Mar 25;14(1):7075.

Author response:

The following is the authors’ response to the original reviews.

Reviewer 1:

We thank the reviewer for their comments and suggestions. We have made several edits to the paper to address these comments, including the addition of several new control experiments, corrections to mislabeled figures in Fig 2, and other additions to improve the clarity of several figures.

This work is missing several controls that are necessary to substantiate their claims. My most important concern is that the optogenetic screen for neurons that alter pathogenic lawn occupancy does not have an accompanying control on non-pathogenic OP50 bacteria. Hence, it remains unclear whether these neuronal inhibition experiments lead to pathogen-specific or generalized lawn-leaving alterations. For strains that show statistical differences between - and + ATR conditions, the authors should perform follow-up validation experiments on non-pathogenic OP50 lawns to ensure that the observed effect is PA14-specific. Similarly, neuronal inhibition experiments in Figures 5E and H are only performed with naïve animals on PA14 - we need to see the latency to re-entry on OP50 as well, to make general conclusions about these neurons' role in pathogen-specific avoidance.

We have added data from new control experiments to Fig. S1 (subfigures B, C) for both exit and re-entry dynamics on OP50. We find that inhibition of neurons produces different effects on both lawn entry and exit on PA14 compared to OP50. We observed that inhibition of neurons failed to change the re-entry dynamics for any of the lines which showed delayed latency to re-entry on PA14. Our results suggest that the neural control of re-entry dynamics we see are PA14 specific.

My second major concern is regarding the calcium imaging experiments of candidate neurons involved in lawn re-entry behavior. Although the data shows that AIY, AVK, and SIA/SIB neurons all show reduced activity following pathogen exposure, the authors do not relate these activity changes to changes in behavior. Given the well-established links between these cells and forward locomotion, it is essential to not only report differences in activity but also in the relationship between this activity and locomotory behavior. If animals are paused outside of the pathogen lawn, these neurons may show low activity simply because the animals are not moving forward. Other forward-modulated neurons may also show this pattern of reduced activity if the animals remain paused. Given that the authors have recorded neural activity before and after contact with pathogenic bacteria in freely moving animals, they should also provide an analysis of the relationship between proximity to the lawn and the activity of these neurons.

In response, we added an additional supplementary figure S7 to illustrate the role of each neuron in navigational control and added text to the discussion to better explain the role of each neuron type in the regulation of re-entry, in light of our previously published work on SIA in speed control.

This work is missing methodological descriptions that are necessary for the correct interpretation of the results shown here. Figure 2 suggests that the determination of statistical significance across the optogenetic inhibition screen will be found in the Methods, but this information is not to be found there. At various points in the text, authors refer to "exit rate", "rate constant", and "entry rate". These metrics seem derived from an averaged measurement across many individual animals in one lawn evacuation assay plate. However "latency to re-entry" is only defined on a per-animal basis in the lawn re-exposure assay. These differences should be clearly stated in the methods section to avoid confusion and to ensure that statistics are computed correctly.

Additional details have been added to the methods section to provide more in depth information on the statistical analysis performed. In brief, the latency to re-entry is calculated in the same way across all assays – re-entry events across replicate experiments for a given experimental condition are aggregated together and used to calculate relevant statistics.

This work also contains mislabeled graphs and incorrect correspondence with the text, which make it difficult to follow the authors 'claims. The text suggests that Pdop-2::Arch3 and Pmpz-1::Arch3 show increased exit rates, whereas Figure 2 shows that Pflp-4::Arch3 but not Pmpz-1::Arch3 has increased exit rate. The authors should also make a greater effort to correctly and clearly label which type of behavioral experiment is used to generate each figure and describe the differences in experimental design in the main text, figure legends, and methods. Figure 2E depicts trajectories of animals leaving a lawn over a 2.5-minute interval but it is unclear when this time window occurs within the 18-hour lawn leaving assay. Likewise, Figure 2H depicts a 30-minute time window which has an unclear relationship to the overall time course of lawn leaving. This figure legend is also mislabeled as "Infected/Healthy", whereas it should be labeled "-/+ ATR".

In Figures 2C and F, the x-axis labels are in a different order, making it difficult to compare between the 2 plots. Promoter names should be italicized. What does the red ring mean in Figure 2A? Figure 2 legend incorrectly states that four lines showed statistically significant changes for the Exist rate constant - only 2 lines are significant according to the figure.

We thank the reviewer for identifying this embarrassing error. Figure 2C and F were flipped, and we have corrected this, we are sorry for the error. Promoter names have been italicized, and we have added additional text in the captions that the red ring is a ring light for background illumination of the worms. In addition, we have corrected the error in the figure legends from “Infected/Healthy” to “+/- ATR”.

Lines in figure 2C and 2F are ordered by significance rather than keeping the same order in both. Majority feedback from colleagues suggested that this ordering was preferred.

This work raises the interesting possibility that different sets of neurons control lawn exit and lawn re-entry behaviors following pathogen exposure. However, the authors never directly test this claim. To rigorously show this, the authors would need to show that lawn-exit-promoting neurons (CEPs, HSNs, RIAs, RIDs, SIAs) are dispensable for lawn re-entry behavior and that lawn re-entry promoting neurons (AVK, SIA, AIY, MI) are dispensable for lawn exit behavior in pathogen-exposed animals.

We agree with the reviewer’s comments that there is insufficient evidence to show a complete decoupling of lawn exit and lawn re-entry. However, we note that our screen results show that only 1 line (dop-2) shows changes in both exit and re-entry dynamics upon neural inhibition (Fig. 2). This seems to suggest that at least some degree of neural control of re-entry is decoupled from exit.

Please label graph axes with units in Figure 1 - instead of "Exit Rate" make it #exits per worm per hour, and make it more clear that Figures 1C and E have a different kind of assay than Figures 1A, B and D. There should be more consistency between the meaning of "pre/post" and "naive/infected/healthy" - and how many hours constitutes post.

We have edited Figure 1 and made additions to the captions of figure 1 to make both points clearer. We have also standardized our language for subsequent figures (such as figure 5) to provide less ambiguity in pre/post and naïve/infected/healthy.

Figure 5 - it should be made more clear when the stimulation/inhibition occurred in these experiments and how long they were recorded/analyzed.

We have added additional details to the figure captions to make it clearer when the data was collected.

Workspaces and code have been added under a data availability section in the manuscript.

Reviewer 2:

However, the paper's main weakness lies in its lack of a detailed mechanism explaining how the delayed reentry process directly influences the actual locomotor output that results in avoidance. The term 'delayed reentry' is used as a dynamic metric for quantifying the screening, yet the causal link between this metric and the mechanistic output remains unclear. Despite this, the study is well-structured, with comprehensive control experiments, and is very well constructed.

We thank the reviewer for their comments and suggestions. We have added additional data and details to our work to cover these weaknesses, as can be seen in our responses to the suggestions below.

(1) A key issue in the manuscript is the mechanistic link between the delayed process and locomotor output. AIY is identified as a crucial neuron in this process, but the specifics of how AIY influences this delay are not clear. For instance, does AIY decrease the reversal rate, causing animals to get into long-range search when they leave the bacterial lawn? Is there any relationship between pdf-2 expression and reversal rates? Given that AIY typically promotes long-range motion when activated, the suppression of this function and its implications on motion warrants further clarification.

We have included additional data to explain how AIY might be able to regulate lawn entry behaviors and have added more to the discussion to explain how neural suppression might lead to changes in the behavior (new figure S7). Both AIY and SIA dynamics have been linked to worm navigation. In previous work (Lee 2019), we have demonstrated that SIA can control locomotory speed. Inhibition of SIA decreases locomotory speed, and as a result may serve to drive the increased latency of re-entry.

AIY’s role in navigation has been previously established (Zhaoyu 2014), but we have added an additional supplementary figure and edited our discussion to further illustrate this point. As can be seen in the new figure S7, AIY neural activity undergoes a transition after removal from a bacterial lawn, going from low activity to high activity. This activity increase is correlated with a transition from a high reversal rate local search state to a long range search state characterized by longer runs. Inhibition of AIY during this long range search state increased the reversal rate resulting in a higher rate of re-orientations. This might serve as a part of the mechanistic explanation for AIY’s role in preventing lawn re-entry, as inhibition dramatically increased the rate of re-orientation, preventing worms from making directed runs into the bacterial lawn. However, there is an additional effect of the inhibition of AIY, not seen during food search. Inhibition of AIY in the context of a pathogenic bacterial lawn leads to stalling at the edge. Therefore, re-entry AIY could have an additional role in governing the animals movement, post exposure, upon contact with a pathogenic lawn.

(2) I recommend including supplementary videos to visually demonstrate the process. These videos might help others identify aspects of the mechanism that are currently missing or unclear in the text.

(4) The authors mention that the worms "left the lawn," but the images suggest that the worms do not stray far and remain around the perimeter. Providing videos could help clarify this observation and strengthen the argument by visually connecting these points

Additional supplementary videos (1-3) taken at several stages of lawn evacuation have been added to visually demonstrate the process.

(3) Regarding the control experiments (Figure 1E-G), the manuscript describes testing animals picked from a PA14-seeded plate and retesting them on different plates. It's crucial to clarify the differences between these plates. Specifically, the region just outside the lawn should be considered, as it is not empty and worms can spread bacteria around. Testing animals on a new plate with a pristine proximity region might introduce variables that affect their behavior.

We have reworded the paper to make it clearer that these new conditions on a fresh PA14 lawn represent a different type of assay from the lawn evacuation assay. Fresh PA14 plates will indeed have a pristine proximity region compared to plates where the worms have spread the bacteria.

These experiments were done to test if the evacuation effect is purely due to aversive signals left on the lawn or attractive signals left outside of the lawn. Given that worms are known to be able to leave compounds such as ascarosides to communicate with each other, we wanted to test that this lawn re-entry defect was not simply the result of deposited pheromones. Without any other method to remove such compounds, we relied on using fresh PA14 lawns instead to test this. We have updated the manuscript to clarify this point.

(5) The manuscript notes that the PA14 strain was grown without shaking. Typically, growing this strain without agitation leads to biofilm formation. Clarifying whether there is a link between biofilm formation and avoidance behavior would add depth to the understanding of the experimental conditions and their impact on the observed behaviors.

As the reviewer has noted, growth of PA14 without shaking might indeed lead to biofilm formation. This does represent a legitimate concern, as evidence from previous work has suggested that biofilm formation could be linked to pathogen avoidance as worms make use of mechanosensation to avoid pathogenic bacteria (Chang et al. 2011).  However, we do not observe substantial formation of biofilm in our cultured bacteria, likely since our growth time might be insufficient for sufficient biofilm formation to occur. We also note that our evacuation dynamics appear to be of similar timescale to results reported in previous work which used different growth conditions. As such, we believe that our growth conditions thus represent similar conditions as to those historically used in the lawn evacuation literature.

Reviewer 3:

Weaknesses:

My only concern is that the authors should be more careful about describing their "compressed sensing-based approach". Authors often cite their previous Nature Methods paper, but should explain more because this method is critical for this manuscript. Also, this analysis is based on the hypothesis that only a small number of neurons are responsible for a given behavior. Authors should explain more about how to determine scarcity parameters, for example.

We have added more details to our paper outlining some of the details involved in our compressed sensing approach. We go into more detail about how we chose sparsity parameters and note that our discovered neurons for re-entry appear to be robust over choice of sparsity parameters. These additional details can be found in both the paper body and the methods section.

Line 45: This paragraph tries to mention that there should be "small sets of neurons" that can play key roles in integrating previous information to influence subsequent behavior. Is it valid as an assumption in the nervous systems?

We want to clarify that what is important is not that there are ‘small sets of neurons’, but rather that these key neurons make up a small fraction of the total number of neurons in the nervous system. More correctly: the compressed sensing approach identifies information bottlenecks in the neural circuits, and the assumption is that the number of neurons in these bottlenecks are small. This is the underlying sparsity assumption being made here that allows us to utilize a compressed sensing based approach to identify these neurons. We have reworded this section to make it clear that what is important is not that the total number of neurons is small, but that they must be a small fraction of the total number of neurons in the nervous system.

Line 125: "These approaches…" Authors repeatedly mentioned this statement to emphasize that their compressed sensing-based approach is the best choice. Are you really sure?

We agree that there are several approaches that might allow for faster screening of the nervous system. For example, many studies approach the problem by looking at neurons with synapses onto a neuron already known to be implicated in the behavior or find neurons that express a key gene known to regulate the behavior of interest. These approaches utilize prior information to greatly reduce the pool of candidate neurons needed to be screened.

In the absence of such prior information, we believe that our compressed sensing based approach allows a rapid way to perform an unbiased interrogation of the entire nervous system to identify key neurons at bottlenecks of neural circuits. Once these key neurons are identified, neurons upstream and downstream of these key neurons can be investigated in the future.  This approach gives us the added advantage of being able to identify neurons that do not connect to neurons that are already implicated in the behavior, or that don’t have clear genetic signatures in the behavior of interest. Our approach further allows for screening of neurons with no clear single genetic marker without the next to utilize intersectional genetic strategies.  We should not use the phrase “best choice” which might not be justified. We have reworded these statements, and we believe that compressed sensing based methods provide a complementary approach to those in the literature.

Line 42: If authors refer to mushroom bodies and human hippocampus in relation to the significance of their work, authors should go back to these references in the Discussion and explain how their work is important.

We thank the reviewer for this feedback, and we have added to our discussion to expand upon these points.

Line 151: "the accelerated pathogen avoidance" Accelerated pathogen avoidance does not necessarily indicate the existence of the neural mechanism that inhibits the association of pathogenicity with microbe-specific cues (during early stages: first two hours).

We agree with the reviewer’s statements that these results alone do not indicate the presence of an early avoidance mechanism. Other evidence for early avoidance mechanisms exists as seen in two choice assay experiments (Zhang 2005), and our results do seem to support this. However, we agree that early neural inhibition is insufficient evidence towards such a mechanism. We have thus removed this statement for accuracy.

Inspirational

All these "rules" are really just guidance/suggestions... I highly recommend you try out the thing you would imagine to work and see how it goes. If it works for you, then great. If not, try something else. What works for someone else isn't necessarily going to work for you. How do you think these things came about? They really weren't invented, but slight variations on a pre-existing theme that someone customized for their needs.

It's called a "zettelkasten practice" for a reason. After you've been at it for a few months, write up your experience and let us know how it all worked out. What worked well? What didn't? Speculate on the reasons why...

reply to u/King_PenguinOs at https://old.reddit.com/r/Zettelkasten/comments/1hklaii/getting_started_with_zettelkasten/

Introduction & Problem Statement

Quoted sentence: “The problem is that datomic users can’t reason about transactor performance and our objective today is that everyone should be able to leave being able to answer two questions: where is all the time going and what can I do about it?”

Summary: The speaker highlights a key challenge: Datomic users struggle to understand transactor performance. The goal of this presentation is to enable them to identify performance bottlenecks and find actionable solutions.

Datomic Architecture Overview

Quoted sentence: “We then have another component called the transactor and the transactor is an appliance which processes transactions safely one at a time… sometimes given enough load transactions will cue.”

Summary: Datomic consists of peer processes co-located with user applications for fast reads and a single-threaded transactor responsible for safely processing write transactions, which can become a bottleneck under heavy load.

Queuing Theory Basics

Quoted sentence: “As the utilization of any queuing system increases, the residence time trends toward infinity.”

Summary: The talk introduces queuing theory to explain how increased transaction arrival rates and high resource utilization cause wait times to grow dramatically. Key metrics—service time, utilization, throughput, and response time—are all crucial to analyzing performance.

Impact of High Utilization on Latency

Quoted sentence: “We can determine the max utilization in order to achieve a response time that we could tolerate.”

Summary: By controlling or reducing service time (S) in Datomic’s transactor, one can keep overall utilization manageable and thus keep transaction latencies within acceptable limits.

Working Set Model Explanation

Quoted sentence: “The working set model is effectively the smallest collection of information that must be present in main memory to ensure efficient execution of your program.”

Summary: The speaker references a classic 1968 MIT paper to illustrate that performance is fundamentally affected by how much necessary data (segments) are in memory. Excessive page traffic (moving segments from external storage to memory) slows down transaction processing.

Performance Benchmark: 10-Billion-Datum Database

Quoted sentence: “We’re going to…flood this system with 5,000 TPS and it’s going to be completely saturated… so this is just a way for us to ensure that we’re absolutely saturating this system.”

Summary: A large-scale test environment demonstrates how Datomic transactor performance responds under heavy load, revealing that as data size grows, the transactor becomes increasingly IO-bound.

Identifying IO Bottlenecks

Quoted sentence: “If we wanted to know what can we do about it we would look at the TX stats chart and we can see…most of the time is spent resolving transaction identities.”

Summary: Transaction resolution and de-duplication dominate overall processing time once the database is large. Reducing disk or network IO through smarter caching and smaller working sets is key to boosting throughput.

Caching Hierarchy in Datomic

Quoted sentence: “We have…a valache which is an NVMe SSD…Light Years faster than a network hop especially across availability zones.”

Summary: Datomic’s multi-tier caching structure (object cache, in-flight lookup, val cache, external storage) significantly impacts latency, showing that local NVMe-based caching outperforms remote storage solutions.

Strategies to Improve Transactor Performance

Quoted sentence: “If you wanted to improve transactor performance you could fiddle with some knobs… deploy faster Hardware… or semantic improvements in your application.”

Summary: Tuning existing settings is a short-term fix. More powerful hardware (e.g., NVMe val cache) or re-thinking application design (e.g., minimal uniqueness checks, fewer data collisions, smaller transactions) often yields far bigger gains.

Semantic Optimizations & Sequential Identifiers

Quoted sentence: “Why do sequential identifiers actually work this way? … if you’re using a squid… you only need to check against the random part of competing squid identifiers within a slide time window of one second.”

Summary: The talk stresses that using sequential or time-ordered IDs (like squids) can drastically reduce random lookups and maintain a small, hot working set in memory, leading to significant transaction throughput improvements.

Platform-Level Improvements

Quoted sentence: “An interesting consequence of this… is that as the utilization increases we end up getting more time waiting in the Q which then gives us more time to prefetch the IO which then reduces the utilization.”

Summary: Recent Datomic enhancements, such as “datomic hints” and advanced prefetching, exploit queue wait time to fetch needed segments preemptively, alleviating IO stalls in the critical apply phase and thereby driving down utilization and latency.

Datomic Hints & Segment Prefetch

Quoted sentence: “We can begin to prefetch some of the reads… purely for side effects to pre-populate the cache with the appropriate data… it can reduce the IO inside the apply thread.”

Summary: A notable new feature allows peers to send “hints” about which segments the transaction will touch, letting the transactor load them while transactions are queued. This cuts down service time dramatically.

Conclusion

Quoted sentence: “With that I think we’ve run through our agenda… so thank you.”

Summary: The talk ends by reiterating how queueing theory, the working set model, semantic changes, and new Datomic features like segment prefetch collectively empower engineers to tackle transactor bottlenecks at scale.

The chat room with a bot posting RSS links – the hidden comment section.

Reviewer #1 (Public review):

Summary:

The authors in this manuscript performed scRNA-seq on a cohort of 15 early-stage cervical cancer patients with a mixture of adeno- and squamous cell carcinoma, HPV status, and several samples that were upstaged at the time of surgery. From their analyses they identified differential cell populations in both immune and tumour subsets related to stage, HPV status, and whether a sample was adenocarcinoma or squamous cell. Putative microenvironmental signaling was explored as a potential explanation for their differential cell populations. Through these analyses the authors also identified SLC26A3 as a potential biomarker for later stage/lymph node metastasis which was verified by IHC and IF. The dataset is likely useful for the community. The accuracy and clarity have been improved from the previous version, and additional immunofluorescence supporting the existence of their proposed cluster is now present. That said, there remain some issues with the strength of some claims (particularly in the abstract and results sections) and some of the cell type definitions.

Strengths

The dataset could be useful for the community<br /> SLC26A3 could potentially be a useful marker to predict lymph node metastasis with further study

Weaknesses

Casual language is used in the abstract around immunosuppressive microenvironment and signal cross-talk between Epi_10_CYSTM1 cluster and Tregs. The data show localization that supports a possible interaction and probable cytokines, but functional experiments would be needed to establish causality.

In the description of the single cell data processing there is no mention of batch effect correction. Given that many patients were analyzed, and no mention was made of pooling or deconvolution, it must be assumed these were run separately which invariably leads to batch effects. Given the good overlays across patients some batch correction must have been performed. How was batch effect correction performed?

While statistics were added to the clinical correlates, it would appear that single variables are being assessed one at a time by chi-squared analysis. This ignores the higher order structure of the data and the correlations between some variables resulting in potentially spurious findings. This is compounded as some categories had below 5 observations violating the assumptions of a chi-squared test.

The description of all analytical steps remains quite truncated. While the inclusion of annotated code is useful, a full description of which tools were used, with which settings, and why each were chosen, is a minimum needed to properly interpret the results. This is as important in a mainly analytical paper as the experimental parameters.

Validation of the clustering results remains a problem. The only details provided are that FindClusters was used. This depends on a manual choice of multiple parameters including the k-nearest neighbours included, whether Louvain or Leiden clustering is used, the resolution parameter, and others (how many variable genes/PCs etc...). Why were these parameters selected, how do you know that you're not over or under-clustering.

The cluster Epi_10_CYSTM1 remains somewhat problematic. None of the additional data supports its existence outside of the single patient who has cells from that population. Additionally, it falls well outside of any of the other Epithelial cells to the point that drawing it as part of a differentiation order doesn't even make sense. Indeed, most of the upregulated pathways in this cluster appear to be related to class II antigen presentation which would fit better with a dendritic cell/macrophage than an epithelial cell. While the IF at the end does support the existence of the cluster, numbers are still very limited, and this doesn't have data on the antigen presenting function. At the least a strong disclaimer should be included in the text that this population is essentially exclusive to one sample in the scRNA data.

The linkage between the cluster types and IHC for prediction of lymph node metastasis is tenuous. Most of the strongly cluster associated markers were not predictive despite their clusters being theoretically enriched. This inability to recognize the clusters in additional samples using alternative methods does not give confidence that these clusters are robust. SLC26A3 being associated with upstaging may very well be a useful marker, however, given the lack of association of the other markers, it may be premature to say this is due to the same Epi_10_CYSTM1 cluster.

There are multiple issues in the classification of T cells and neutrophils. In the analysis of T cell subset, all CD4+ T cells are currently scored as Tregs, what happened to the T-helper cells? Additionally, Activated T and Cytotoxic T both seem to contain CD8+ cells, but all their populations have equivalent expression of the activation marker CD69. Moreover, the "Cytotoxic" ones also express TIGIT, HAVCR2 and LAG3 which are generally exhaustion markers. For neutrophils, several obviously different clusters have been grouped together (Neu_1 containing two diametrically opposite cell clouds being an obvious example).

Again in the CellChat section of the results causal language is being repeatedly used. These are just possible interactions, not validated ones. While the co-localization in the provided IF images certainly supports the co-localization, this still is only correlative and doesn't prove causality.

Minor Issues<br /> The sentence "However, due to the low morbidity of ADC, in-depth investigations are insufficient" could be misinterpreted. Morbidity generally refers to the severity or health burden rather than the frequency of cases, though it's true in some studies prevalence is used for the overall impact of the disease on a population and referred to as morbidity. In this instance though, "incidence" or "prevalence" would be clearer word choices.

The previous rebuttal states that clusters/cell type calls were refined to eliminate issues such as epithelial cells creeping into the T cell cluster, however, the cell %s have not been altered according to the change tracking. Shouldn't all the %s have been altered even if only slightly?

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

This work makes several contributions: (1) a method for the self-supervised segmentation of cells in 3D microscopy images, (2) an cell-segmented dataset comprising six volumes from a mesoSPIM sample of a mouse brain, and (3) a napari plugin to apply and train the proposed method.

First, thanks for acknowledging our contributions of a new tool, new dataset, and new software.

(1) Method

This work presents itself as a generalizable method contribution with a wide scope: self-supervised 3D cell segmentation in microscopy images. My main critique is that there is almost no evidence for the proposed method to have that wide of a scope. Instead, the paper is more akin to a case report that shows that a particular self-supervised method is good enough to segment cells in two datasets with specific properties.

First, thanks for acknowledging our contributions of a new tool, new dataset, and new software. We agree we focus on lightsheet microscopy data, therefore to narrow the scope we have changed the title to “CellSeg3D: self-supervised 3D cell segmentation for light-sheet microscopy”.

To support the claim that their method "address[es] the inherent complexity of quantifying cells in 3D volumes", the method should be evaluated in a comprehensive study including different kinds of light and electron microscopy images, different markers, and resolutions to cover the diversity of microscopy images that both title and abstract are alluding to.

You have selectively dropped the last part of that sentence that is key: “.... 3D volumes, often in cleared neural tissue” – which is what we tackle. The next sentence goes on to say: “We offer a new 3D mesoSPIM dataset and show that CellSeg3D can match state-of-the-art supervised methods.” Thus, we literally make it clear our claims are on MesoSPIM and cleared data.

The main dataset used here (a mesoSPIM dataset of a whole mouse brain) features well-isolated cells that are easily distinguishable from the background. Otsu thresholding followed by a connected component analysis already segments most of those cells correctly.

This is not the case, as all the other leading methods we fairly benchmark cannot solve the task without deep learning (i.e., no method is at an F1-Score of 1).

The proposed method relies on an intensity-based segmentation method (a soft version of a normalized cut) and has at least five free parameters (radius, intensity, and spatial sigma for SoftNCut, as well as a morphological closing radius, and a merge threshold for touching cells in the post-processing). Given the benefit of tweaking parameters (like thresholds, morphological operation radii, and expected object sizes), it would be illuminating to know how other non-learning-based methods will compare on this dataset, especially if given the same treatment of segmentation post-processing that the proposed method receives. After inspecting the WNet3D predictions (using the napari plugin) on the used datasets I find them almost identical to the raw intensity values, casting doubt as to whether the high segmentation accuracy is really due to the self-supervised learning or instead a function of the post-processing pipeline after thresholding.

First, thanks for testing our tool, and glad it works for you. The deep learning methods we use cannot “solve” this dataset, and we also have a F1-Score (dice) of ~0.8 with our self-supervised method. We don’t see the value in applying non-learning methods; this is unnecessary and beyond the scope of this work.

I suggest the following baselines be included to better understand how much of the segmentation accuracy is due to parameter tweaking on the considered datasets versus a novel method contribution:

*  comparison to thresholding (with the same post-processing as the proposed method) * comparison to a normalized cut segmentation (with the same post-processing as the proposed method)

*  comparison to references 8 and 9.

Ref 8 and 9 don’t have readily usable (https://github.com/LiangHann/USAR) or even shared code (https://github.com/Kaiseem/AD-GAN), so re-implementing this work is well beyond the bounds of this paper. We benchmarked Cellpose, StartDist, SegResNets, and a transformer – SwinURNet. Moreover, models in the MONAI package can be used. Note, to our knowledge the transformer results also are a new contribution that the Reviewer does not acknowledge.

I further strongly encourage the authors to discuss the limitations of their method. From what I understand, the proposed method works only on well-separated objects (due to the semantic segmentation bottleneck), is based on contrastive FG/BG intensity values (due to the SoftNCut loss), and requires tuning of a few parameters (which might be challenging if no ground-truth is available).

We added text on limitations. Thanks for this suggestion.

(2) Dataset

I commend the authors for providing ground-truth labels for more than 2500 cells. I would appreciate it if the Methods section could mention how exactly the cells were labelled. I found a good overlap between the ground truth and Otsu thresholding of the intensity images. Was the ground truth generated by proofreading an initial automatic segmentation, or entirely done by hand? If the former, which method was used to generate the initial segmentation, and are there any concerns that the ground truth might be biased towards a given segmentation method?

In the already submitted version, we have a 5-page DataSet card that fully answers your questions. They are ALL labeled by hand, without any semi-automatic process.

In our main text we even stated “Using whole-brain data from mice we cropped small regions and human annotated in 3D 2,632 neurons that were endogenously labeled by TPH2-tdTomato” - clearly mentioning it is human-annotated.

(3) Napari plugin

The plugin is well-documented and works by following the installation instructions.

Great, thanks for the positive feedback.

However, I was not able to recreate the segmentations reported in the paper with the default settings for the pre-trained WNet3D: segments are generally too large and there are a lot of false positives. Both the prediction and the final instance segmentation also show substantial border artifacts, possibly due to a block-wise processing scheme.

Your review here does not match your comments above; above you said it was working well, such that you doubt the GT is real and the data is too easy as it was perfectly easy to threshold with non-learning methods.

You would need to share more details on what you tried. We suggest following our code; namely, we provide the full experimental code and processing for every figure, as was noted in our original submission: https://github.com/C-Achard/cellseg3d-figures.

Reviewer #2 (Public Review):

Summary:

The authors propose a new method for self-supervised learning of 3d semantic segmentation for fluorescence microscopy. It is based on a WNet architecture (Encoder / Decoder using a UNet for each of these components) that reconstructs the image data after binarization in the bottleneck with a soft n-cuts clustering. They annotate a new dataset for nucleus segmentation in mesoSPIM imaging and train their model on this dataset. They create a napari plugin that provides access to this model and provides additional functionality for training of own models (both supervised and self-supervised), data labeling, and instance segmentation via post-processing of the semantic model predictions. This plugin also provides access to models trained on the contributed dataset in a supervised fashion.

Strengths:

(1) The idea behind the self-supervised learning loss is interesting.

(2) The paper addresses an important challenge. Data annotation is very time-consuming for 3d microscopy data, so a self-supervised method that yields similar results to supervised segmentation would provide massive benefits.

Thank you for highlighting the strengths of our work and new contributions.

Weaknesses:

The experiments presented by the authors do not adequately support the claims made in the paper. There are several shortcomings in the design of the experiment, presentation of the results, and reproducibility.

We address your concerns and misunderstandings below.

Major weaknesses:

(1) The main experiments are conducted on the new mesoSPIM dataset, which contains quite small nuclei, much smaller than the pretraining datasets of CellPose and StarDist. I assume that this is one of the main reasons why these well-established methods don't work for this dataset.

StarDist is not pretrained, we trained it from scratch as we did for WNet3D. We retrained Cellpose and reported the results both with their pretrained model and our best-retrained model. This is documented in Figure 1 and Suppl. Figure 1. We also want to push back and say that they both work very well on this data. In fact, our main claim is not that we beat them, it is that we can match them with a self-supervised method.

Limiting method comparison to only this dataset may create a misleading impression that CellSeg3D is superior for all kinds of 3D nucleus segmentation tasks, whereas this might only hold for small nuclei.

The GT dataset we labeled has nuclei that are normal brain-cell sized. Moreover in Figure 2 we show very different samples with both dense and noisy (c-FOS) labeling.

We also clearly do not claim this is superior for all tasks, from our text: “First, we benchmark our methods against Cellpose and StarDist, two leading supervised cell segmentation packages with user-friendly workflows, and show our methods match or outperform them in 3D instance segmentation on mesoSPIM-acquired volumes" – we explicitly do NOT claim beyond the scope of the benchmark. Moreover we state: "We found that WNet3D could be as good or better than the fully supervised models, especially in the low data regime, on this dataset at semantic and instance segmentation" – again noting on this dataset. Again, we only claimed we can be as good as these methods with an unsupervised approach, and in the low-GT data regime we can excel.

Further, additional preprocessing of the mesoSPIM images may improve results for StarDist and CellPose (see the first point in minor weaknesses). Note: having a method that works better for small nuclei would be an important contribution. But I doubt that the claims hold for larger and or more crowded nuclei as the current version of the paper implies.

Figure 2 benchmarks our method on larger and denser nuclei, but we do not intend to claim this is a universal tool. It was specifically designed for light-sheet (brain) data, and we have adjusted the title to be more clear. But we also show in Figure 2 it works well on more dense and noisy samples, hinting that it could be a promising approach. But we agree, as-is, it’s unlikely to be good for extremely dense samples like in electron microscopy, which we never claim it would be.

With regards to preprocessing, we respectfully disagree. We trained StarDist (and asked the main developer of StarDist, Martin Weigert, to check our work and he is acknowledged in the paper) and it does very well. Cellpose we also retrained and optimized and we show it works as-well-as leading transformer and CNN-based approaches. Again, we only claimed we can be as good as these methods with an unsupervised approach.

The contribution of the paper would be much stronger if a **fair** comparison with StarDist / CellPose was also done on the additional datasets from Figure 2.

We appreciate that more datasets would be ideal, but we always feel it’s best for the authors of tools to benchmark their own tools on data. We only compared others in Figure 1 to the new dataset we provide so people get a sense of the quality of the data too; there we did extensive searches for best parameters for those tools. So while we think it would be nice, we will leave it to those authors to be most fair. We also narrowed the scope of our claims to mesoSPIM data (added light-sheet to the title), which none of the other examples in Figure 2 are.

(2) The experimental setup for the additional datasets seems to be unrealistic. In general, the description of these experiments is quite short and so the exact strategy is unclear from the text. However, you write the following: "The channel containing the foreground was then thresholded and the Voronoi-Otsu algorithm used to generate instance labels (for Platynereis data), with hyperparameters based on the Dice metric with the ground truth." I.e., the hyperparameters for the post-processing are found based on the ground truth. From the description it is unclear whether this is done a) on the part of the data that is then also used to compute metrics or b) on a separate validation split that is not used to compute metrics. If a) this is not a valid experimental setup and amounts to training on your test set. If b) this is ok from an experimental point of view, but likely still significantly overestimates the quality of predictions that can be achieved by manual tuning of these hyperparameters by a user that is not themselves a developer of this plugin or an absolute expert in classical image analysis, see also 3.

We apologize for this confusion; we have now expanded the methods to clarify the setup is now b; you can see what we exactly did as well in the figure notebook: https://c-achard.github.io/cellseg3d-figures/fig2-b-c-extra-datasets/self-supervised-ext ra.html#threshold-predictions.

For clarity, we additionally link each individual notebook now in the Methods.

(3) I cannot reproduce any of the results using the plugin. I tried to reproduce some of the results from the paper qualitatively: First I downloaded one of the volumes from the mesoSPIM dataset (c5image) and applied the WNet3D to it. The prediction looks ok, however the value range is quite close (Average BG intensity ~0.4, FG intensity 0.6-0.7). I try to apply the instance segmentation using "Convert to instance labels" from "Utilities". Using "Voronoi-Otsu" does not work due to an error in pyClesperanto ("clGetPlatformIDs failed: PLATFORM_NOT_FOUND_KHR"). Segmentation via "Connected Components" and "Watershed" requires extensive manual tuning to get a somewhat decent result, which is still far from perfect.

We are sorry to hear of the installation issue; pyClesperanto is a dependency that would be required to reproduce the images (sounds like you had this issue; https://forum.image.sc/t/pyclesperanto-prototype-doesnt-work/45724 ) We added to our docs now explicitly the fix:https://github.com/AdaptiveMotorControlLab/CellSeg3D/pull/90. We recommend checking the reproduction notebooks (which were linked in initial submission): https://c-achard.github.io/cellseg3d-figures/intro.html.

Then I tried to reproduce the results for the Mouse Skull Nuclei Dataset from EmbedSeg. The results look like a denoised version of the input image, not a semantic segmentation. I was skeptical from the beginning that the method would transfer without retraining, due to the very different morphology of nuclei (much larger and elongated). None of the available segmentation methods yield a good result, the best I can achieve is a strong over-segmentation with watersheds.

We are surprised to hear this; did you follow the following notebook which directly produces the steps to create this figure? (This was linked in preprint): https://c-achard.github.io/cellseg3d-figures/fig2-c-extra-datasets/self-supervised-extra .html

We also expanded the methods to include the exact values from the notebook into the text.

Minor weaknesses:

(1) CellPose can work better if images are resized so that the median object size in new images matches the training data. For CellPose the cyto2 model should do this automatically. It would be important to report if this was done, and if not would be advisable to check if this can improve results.

We reported this value in Figure 1 and found it to work poorly, that is why we retrained Cellpose and found good performance results (also reported in Figure 1). Resizing GB to TB volumes for mesoSPIM data is otherwise not practical, so simply retraining seems the preferable option, which is what we did.

(2) It is a bit confusing that F1-Score and Dice Score are used interchangeably to evaluate results. The dice score only evaluates semantic predictions, whereas F1-Score evaluates the actual instance segmentation results. I would advise to only use F1-Score, which is the more appropriate metric. For Figure 1f either the mean F1 score over thresholds or F1 @ 0.5 could be reported. Furthermore, I would advise adopting the recommendations on metric reporting from https://www.nature.com/articles/s41592-023-01942-8.

We are using the common metrics in the field for instance and semantic segmentation, and report them in the methods. In Figure 2f we actually report the “Dice” as defined in StarDist (as we stated in the Methods). Note, their implementation is functionally equivalent to F1-Score of an IoU >= 0, so we simply changed this label in the figure now for clarity. We agree this clarifies for the expert readers what was done, and we expanded the methods to be more clear about metrics.

We added a link to the paper you mention as well.

(3) A more conceptual limitation is that the (self-supervised) method is limited to intensity-based segmentation, and so will not be able to work for cases where structures cannot be distinguished based on intensity only. It is further unclear how well it can separate crowded nuclei. While some object separation can be achieved by morphological operations this is generally limited for crowded segmentation tasks and the main motivation behind the segmentation objective used in StarDist, CellPose, and other instance segmentation methods. This limitation is only superficially acknowledged in "Note that WNet3D uses brightness to detect objects [...]" but should be discussed in more depth. Note: this limitation does not mean at all that the underlying contribution is not significant, but I think it is important to address this in more detail so that potential users know where the method is applicable and where it isn't.

We agree, and we added a new section specifically on limitations. Thanks for raising this good point. Thus, while self-supervision comes at the saving of hundreds of manual labor, it comes at the cost of more limited regimes it can work on. Hence why we don’t claim this should replace excellent methods like Cellpose or Stardist, but rather complement them and can be used on mesoSPIM samples, as we show here.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) One of the listed contributions is "adding the SoftNCuts loss". This is not true, reference 10 already introduced that loss.

“Our changes include a conversion to a fully 3D architecture and adding the SoftNCuts loss” - we dropped the common and added the word “AND” to note that we added the 3D version of the SoftNCuts loss TO the 3D architecture, which 10 did not do.

(2) "Typically, these methods use a multi-step approach" to segment 3D from 2D: this is only true for CellPose, StarDist does real 3D.

That is why we preface with “typically” which implies not always.

(3) "see Methods, Figure 1c, c)" is missing an opening in parentheses.

(4) K is not introduced in equation (1) (presumably the number of classes, which seems to be 2 for all experiments considered).

k actually was introduced just below equation 1 as the number of classes. We added the note that k was set to 2.

(5) X is not introduced in equation (2) (presumably the spatial position of a voxel).

Sorry for this oversight. We add that $X$ is the spatial position of the voxel.

Reviewer #2 (Recommendations For The Authors):

To improve the paper the weaknesses mentioned above should be addressed:

(1) Compare to StarDist and/or CellPose on further datasets, esp. using pre-trained CellPose, to see if the claims of competitive performance with state-of-the-art approaches hold up for the case of different nucleus morphologies. The EmbedSeg datasets from Figure 2 c are well suited for this. In the current form, the claims are too broad and not supported if thorough experiments are performed on a single dataset with a very specific morphology. Note: even if the method is not fully competitive with CellPose / StarDist on these Datasets it holds merit since a segmentation method that works for small nuclei as in the mesoSPIM dataset and works self-supervised is very valuable.

(2) Clarify how the best instance segmentation hyperparameters are found. If you indeed optimize these on the same part of the dataset used for evaluating metrics then the current experimental set-up is invalid. If this is not the case I would still rethink if this is a good way to report the results since it does not seem to reflect user experience. I found it not possible to find good hyperparameters for either of the two segmentation approaches I tried (see also next point) so I think these numbers are too optimistic.

(3) Improve the instance segmentation part of the plugin: either provide troubleshooting for how to install pyClesperanto correctly to use the voronoi-based instance segmentation or implement it based on more standard functionality like skimage / scipy. Provide more guidance for finding good hyperparameters for the segmentation task.

(4) Make sure image resizing is done correctly when using pre-trained CellPose models and report on this.

(5) Report F1 Scores only (unless there is a compelling reason to also report Dice).

(6) Address the limitations of the method in more detail.

On a positive note: all data and code are available and easy to download/install. A minor comment: it would be very helpful to have line numbers for reviewing a revised version.

All comments are also addressed in the public reviews.

for - quote - curiosity is not just an intellectual tool - from TED Talk - Can curiosity heal division? - Scott Shigeoka - 2024 Dec

quote - curiosity is more than a tool - (see below) - Curiosity is not just this intellectual tool, - u t's also this heart-centered force that we can bring into our life, and - I think it's a practice we really need right now in our country and in the world. - It also reminds us to look for the good in our lives and not just focus on the bad. - It reminds us to look for what’s uniting our communities and our country and - not to just focus on what's fracturing and dividing us. - It also tells us to prioritize the questions that we're asking, as an important step to problem-solving, because - we can't just focus on the answers,

for - adjacency - improvisation - conversation - from TED Talk YouTube - Everything is Improvisation - Reggie Watts

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP-deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:

The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, which could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:

The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

We thank the reviewer for the thoughtful feedback. In our mind, the two parts of the paper are inherently related as they both focus on proteasome-mediated degradation of TEADs. We first demonstrated that TEAD can be turned over by the ubiquitin proteasome system under endogenous conditions and identified a PARylation-dependent E3 ligase RNF146 as a major regulator of TEAD stability. Intriguingly, we observed that the four TEAD paralogs show different levels of polyubiquitination with some of them being highly stable in cells. These observations raised the question of whether the activity of the ubiquitin-proteasome system could be further enhanced pharmacologically to effectively target TEADs. We then tackled this question by providing a proof-of-concept demonstration of engineered heterobifunctional protein degraders can effectively degrade TEADs in cells and can be exploited as a therapeutic strategy for treating Hippo-dependent cancers.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation were difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

We appreciate the suggestions from the reviewer and have updated the figures with high resolution images. We also corrected typos and grammatical errors in the text.

Reviewer #2 (Public Review):

The paper is made of two parts. One deals with RNF146, the other with the development of compounds that may cause TEAD degradation. The two parts are rather unrelated to each other.

The main limit of this work is the lack of evidence that TEAD factors are in fact regulated by the proteasome and ubiquitylation under endogenous conditions. Also lacking is the demonstration that TEADs are labile proteins to the extent that such quantitative regulation at the level of stability can impact on YAP-TAZ biology. Without these two elements, the relevance and physiological significance of all these data is lacking.

As for the development of new inhibitors of TEAD, this is potentially very interesting but underdeveloped in this manuscript. Irrespectively, if TEAD is stable, these molecules are likely lead compounds of interest. If TEAD is unstable, as entertained in the first part of the paper, then these molecules are likely marginal.

We thank the reviewer for evaluating our manuscript. As the reviewer pointed out, the paper aimed to address 1) whether TEAD is being regulated by the proteasome and ubiquitination under endogenous conditions, and 2) whether TEAD can be inhibited through pharmacologically-induced degradation. First, we demonstrated that TEAD is ubiquitinated in cells and mapped the lysine residues that are poly-ubiquitinated (Fig. 1). Next, we identified RNF146 as a major E3 ligase that ubiquitinates TEADs and reduces their stability. Third, we show that RNF146-mediated TEAD ubiquitination is functionally important: RNF146 suppresses TEAD activity, and RNF146 genetically interacts with Hippo pathway components in fruit flies. Furthermore, as we showed in Fig. S2H, RNF-146 does not affect TEAD1 and TEAD4 to the same extent. Across all four cell lines evaluated, TEAD1 is more stable than TEAD4, raising the question of whether more consistent degradation of different TEAD paralogues could be achieved. To this end, we demonstrated that while the TEAD family of proteins is labile under endogenous conditions, more complete degradation of the TEAD proteins could be achieved using a heterobifunctional CRBN degrader. We further characterized these TEAD degraders in a series of cellular and genomic assays to demonstrate their cellular activity, selectivity, and inhibitory effects against YAP/TAZ target genes. We believe these degrader compounds would be of great interest to the Hippo community. We have revised the main text to clarify these points.

Here are a few other specific observations:

(1) The effect of MG is shown in a convoluted way, by MS. What about endogenous TEAD protein stability?

We thank the reviewer for the question. The MS experiment shown in Figure 1 is a standard KGG experiment, where we used MS to map ubiquitination sites on TEADs. The graphical representation of the process is included in Fig. 1C, and the details of the procedure are included in the Methods section. Fig. 1D shows the different KGG peptides detected with or without MG-132 treatment. Fig. 1E shows the quantified abundance of each of the peptides across the four conditions indicated at the bottom of the plot. Regarding endogenous TEAD stability, ​​we conducted cycloheximide chase experiments to assess the stability of endogenously expressed TEAD isoforms upon RNF146 knockdown (Fig. S2G and S2H). Using isoform-specific antibodies, we demonstrated that siRNF146 significantly stabilized TEAD4 in multiple cell lines, including H226, PATU-8902, Detroit-562, and OVCAR-8 (Fig. S2G, S2H, and S2I), supporting the notion that RNF146 is a negative regulator of TEAD stability. Notably, the effect of siRNF146 on TEAD1 stability was less pronounced, and TEAD1 is more stable than TEAD4 across all four cell lines. These results are consistent with the lower level of ubiquitination of TEAD1 (Fig. 1A) and are corroborated by various biochemical, molecular, and genetic characterizations (Fig. 3A-C and S3E).

(2) The relevance of siRNF on YAP target genes of Fig.2D is not statistically significant.

We thank the reviewer for this comment. We have now removed the statistically significant claim.

(3) All assays are with protein overexpression and Ub-laddering

We thank the reviewer for the comment. To examine the ubiquitination level of TEAD proteins, we adopted an in vivo ubiquitination assay as described in our Materials and Methods section. To our knowledge, this assay is very standard in the ubiquitination field. Furthermore, as mentioned above, we have included in our revised manuscript cycloheximide chase experiments to assess the stability of endogenously expressed TEAD isoforms upon RNF146 knockdown (Fig. S2G and S2H). In addition to the overexpression system, we also assessed endogenously expressed TEAD using isoform-specific antibodies. We demonstrated that siRNF146 firmly stabilized TEAD4 in multiple cell lines, including H226, PATU-8902, Detroit-562, and OVCAR-8 (Fig. S2G with quantification and t-test), supporting the notion that RNF146 is a negative regulator of TEAD stability.

(4) An inconsistency exists on the only biological validation (only by overexpression) on the fly eye size. RNF gain in Fig4C is doing the opposite of what is expected from what is portrayed here as a YAP/TEAD inhibitor: RNF gain is shown to INCREASE eye size, phenocopying a Hippo loss of function phenotype. According to the model proposed, RNF addition should reduce eye size. The authors stated that " This is in contrast to the anti-growth effect of RNF-146 in the Hpo loss-of-function background and indicates RNF146 may regulate other genes/pathways controlling eye sizes besides its role as a negative regulator of Sd/yki activity". This raises questions on what the authors are really studying: why, according to the authors, these caveats should occur on the controls, and not when they study Hpo mutants?

We thank the reviewer for the comment. We acknowledge the complexity of the fly phenotype compared to tumor growth. TEAD (Sd) isn’t the only substrate of RNF146 in the fly. For instance, RNF146 is known to positively regulate Wnt signaling by degrading Axin. Previous studies have shown that activation of the Wnt signaling pathway by removal of the negative regulator Axin from clones of cells results in an overgrowth phenotype (Legent and Treisman, 2008). The overgrowth phenotype that we observed with overexpressing RNF146 only, therefore, likely is due to the role of RNF146 in regulating other signaling pathways. Importantly, we showed that upon Hippo loss of function, overexpression of RNF146 can rescue the Hippo overgrowth phenotype (Fig 4B). This differential outcome of RNF146 expression in wildtype versus Hippo-deficient flies indicates that the genetic interactions between RNF146 and Hippo pathway components altered the phenotypic outcome, and the phenotype we get with RNF146 overexpression in a Hippo loss of function background is not simply due to additive effects of functional loss of either component alone.

Complementary to these overexpression data, we showed that knockdown of RNF146 increased the eye size further (Fig. S4A, B) in Hippo loss of function background, further supporting the role of RNF146 as a negative regulator of the overall pro-growth signals induced by yki upon Hippo loss of function.

(5) The role of TEAD inactivation on YAP function is already well known. Disappointingly, no prior literature is cited. In any case, this is a mere control.

We thank the reviewer for the suggestion. We have cited several published reviews that touch upon this aspect of the TEAD-YAP function, including Calses et al., 2019; Dey et al., 2020; Halder and Johnson, 2011; Wang et al., 2018. We are open to your suggestions on additional citations.

(6) The second part of the paper on the Development and Screening of pan-TEAD lipid pocket degraders is interesting but unconnected to the above. The degradation pathway it involves has nothing to do with the enzyme described in the first figures.

We thank the reviewer for the comment. We acknowledge that our paper broadly covers two aspects. We believe that they are inherently connected as they both address ubiquitin/proteasome-mediated TEAD degradation and the functional consequences of TEAD degradation. Given the increasing interest in targeting TEAD/YAP/TAZ in cancers, we think the pharmacological approaches to enhance TEAD degradation using orthogonal E3 ligases provide an important toolbox to understand how this pathway can be regulated under both physiological and pathological conditions. While RNF146 appears to be a major E3 ligase responsible for TEAD turnover under physiological conditions, we showed that the four TEAD paralogs have different poly-ubiquitination levels (Fig. 1A), and are differentially labile in cells (Fig. S2G-I). These observations raised the question of whether the activity of the ubiquitination-proteasome system could be further enhanced to allow more complete removal of TEADs. To this end, we demonstrated that E3 ligases that do not regulate TEAD under endogenous conditions can be leveraged pharmacologically to achieve deep TEAD degradation, thus providing a proof of concept that TEADs can be targeted simultaneously using such approaches. Finally, in addition to establishing the basic biological concept linking RNF146 to TEAD degradation, the compounds we engineered will serve as valuable chemical tools for future studies of TEAD biology and the Hippo pathway in cancers and beyond.

(7) The role of CIDE on YAP accessibility to Chromatin is superficially executed. Key controls are missing along with the connection with mechanisms and prior knowledge of TEAD, YAP, chromatin, and other TEAD inhibitors, just to mention a few.

We used ATAC-seq to assess chromatin accessibility comparing cells treated with DMSO and two different concentrations of compound D. We acknowledge there are small molecule inhibitors of TEADs that can modulate accessibility of YAP binding sites. Potential mechanistic differences between TEAD degraders versus TEAD small molecule inhibitions will be a future area of investigation.

(8) The physiological relevance and the mechanistic interpretation of what should be in the ATAC seq in ovcar cells is missing.

We showed in Fig. 7A-D the dose response of OVCAR cells to the TEAD degraders. As evident from those experiments, TEAD degraders inhibit the proliferation of OVCAR cells as expected from their dependencies on the TEAD/YAP/TAZ transcription complex. In the ATAC-seq experiment, we showed that the canonical TEAD/YAP/TAZ target genes ANKRD1 and CCN1 have reduced chromatin accessibility at their promoter/enhancer regions (Fig. 8C). By unbiased motif and pathway analyses, we show that TEAD binding sites and YAP signatures are most significantly downregulated in OVCAR-8 cells (Fig. 8D-E). These results are incorporated into the results section of the manuscript.

Reviewer #3 (Public Review):

Summary

Pham, Pahuja, Hagenbeek, et al. have conducted a comprehensive range of assays to biochemically and genetically determine TEAD degradation through RNF146 ubiquitination. Additionally, they designed a PROTAC protein degrader system to regulate the Hippo pathway through TEAD degradation. Overall, the data appears robust. However, the manuscript lacks detailed methodological descriptions, which should be addressed and improved before publication. For instance, the methods used to analyze the K48 ubiquitination site on TEAD and the gene expression analysis of Hippo Signaling are unclear. Furthermore, the multiple proteomics, RNA-seq, and ATAC-seq data must be made publicly available upon publication to ensure reproducibility. Most of the main figures are of low resolution, which needs addressing.

We thank the reviewer for evaluating our manuscript. All of the data will be uploaded to public databases. We apologize for the low figure resolution and have updated the figures in the revised manuscript. We also expanded the methods section with more details.

Strengths:

- A broad range of assays was used to robustly determine the role of RNF146 in TEAD degradation.

- Development of novel PROTAC for degrading TEAD.

Weaknesses:

- An orthogonal approach is needed (e.g., PARP1 inhibitor) to demonstrate PARP1's dependency in TEAD ubiquitination.

We thank the reviewer for the suggestion. We had attempted to assess the effect of PARP inhibitors (including veliparib and olaparib) on TEAD ubiquitination, but the data is relatively complex to interpret. Besides inhibiting PARP1/2 catalytic activities, these PARP inhibitors also trap PARP on chromatin. Hence, these inhibitors could induce other cellular changes in addition to inhibiting the catalytic activities of PARP1/2. Given these potential pitfalls, we decided not to include these inconclusive data. Even though the experiments with PARP inhibitors were inconclusive, our study supports that TEAD2 and TEAD4 are PARylated in cells using an anti-PAR antibody (Fig. 3B). Furthermore, we show that mutation of the D70 PARsylation site to alanine greatly abolished TEAD4 ubiquitination in cells, suggesting PARylation is important for TEAD4 ubiquitination. In addition, PARP1 depletion by siRNA and CRISPR guide RNA reduced TEAD2 and TEAD4 ubiquitination levels, indicating PARP1 is one of the PARPs responsible for TEAD PARylation in cells.

- The data from Table 2 is unclear in illustrating the association of identified K48 ubiquitination with TEAD4, especially since the experiments were presumably to be conducted on whole cell lysates with KGG enrichment. This raises the possibility that the K48 ubiquitination could originate from other proteins. Alternatively, if the authors performed immunoprecipitation on TEAD followed by mass spectrometry, this should be explicitly described in the text and materials and methods section.

We thank the reviewer for this question. The experiment was an IP-mass spectrometry study in a TEAD4 amplified cell line model (PATU-8902) after IP with a pan-TEAD antibody. Here, we observed K48 ubiquitin and other ubiquitin linkages as shown in the Supplementary Table S2 of the original submission. Although it is possible that the IP wash steps could be more stringent, we did enrich for TEAD protein prior to mass spectrometry. While the ubiquitin linkage signals may come mainly from TEAD protein (mainly TEAD4), we recognized that some signals may come from other proteins. Given the caveat, we have now removed the table from our paper and updated the text accordingly.

- Figure 2D: The methodology for measuring the Hippo signature is unclear, as is the case for Figures 7E and F regarding the analysis of Hippo target genes.

We apologize for the lack of clarification. In short, we previously developed the Hippo signature using machine learning and chemogenomics as described previously (Pham et al. Cancer Discovery 2021). In the revised version of the manuscript, we added the methodology for measuring the Hippo signature and cited our previous publication where we developed the Hippo signature.

- Figure S3F requires quantification with additional replicates for validation.

We thank the reviewer for the suggestion. We added the quantification for the blot and indicated the replication in the figure legend. Note that Figure S3F is now S3G.

- There is a misleading claim in the discussion stating "TEAD PARylation by PAR-family members (Figure 3)"; however, the demonstration is only for PARP1, which should be corrected.

We apologize for the statement. We observed both PARP1 and PARP9 in our TEAD IP-mass spec (now Figure S3E), which suggest both PARP-family members could be invovled. Nonetheless, we primarily focus on PARP1, which is widely expressed aross cell line models and present in higher abundance. Thus, our study only experimentally validated PARP1's role in regulating TEAD.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

General comments:

(1) Please provide a smoother transition and well-defined connection between the first and second parts of the manuscript. The manuscript reads as two papers that were combined into one, without much attempt to disguise the fact.

We thank the reviewer for the suggestion. We have added a transition paragraph to smoothen the transition. We acknowledge that our paper broadly covers two aspects. However, they both touch upon TEAD ubiquitination and degradation. In the first part of the manuscript, we described TEAD biology and showed that TEADs are post-translationally modified and subsequently regulated through PARylation-dependent RNF146-mediated ubiquitination. In the second part, we highlighted our abilities to leverage the PROTAC system for degrading such labile oncogenic proteins like TEADs. In addition to the biological concept, the compounds we engineered will serve as valuable chemical tools for future studies of TEAD biology and the Hippo pathway in cancers and beyond.

(2) To confirm the proteasome mechanism of action, viability assays should be conducted with a CRBN KO.

We thank the reviewer for the comment. In Figure 6E, we measured TEAD protein levels under CRBN knockdown and observed an expected change in TEAD stability. This observation and the other data presented in Figure 6 suggest that TEAD proteins are targeted for proteasomal degradation under compound D treatment.

(3) As a control, sgPARP1 or PARP1 inhibitors should be used to confirm TEAD PARylation reduction.

We thank the reviewer for the suggestion. We had attempted to assess the effect of PARP inhibitors (including veliparib and olaparib) on TEAD ubiquitination, but the data is relatively complex to interpret. Besides inhibiting PARP1/2 catalytic activities, PARP inhibitors also trap PARP on chromatin. Hence, these inhibitors could induce other cellular changes in addition to inhibit the catalytic activities of PARP1/2. Given these pitfalls, we decided not to include these inconclusive data. Even though the experiments with PARP inhibitors were inconclusive, our study supports that TEAD2 and TEAD4 are PARylated in cells using an anti-PAR antibody (Fig. 3B). Furthermore, we show that mutation of the D70 PARsylation site to alanine greatly abolished TEAD4 ubiquitination in cells, suggesting PARylation is important for TEAD4 ubiquitination. In addition, PARP1 depletion by siRNA and CRISPR guide RNA reduced TEAD2 and TEAD4 ubiquitination levels, indicating PARP1 is one of the PARPs responsible for TEAD PARylation in cells.

(4) MS data looks convincing but an FDR of 1% should be applied - this is the accepted standard in the proteomics field. Please research the data with the more stringent filter.

We thank the reviewer for the suggestion. Our IP-MS experiment comparing siNTC versus siYAP1/WWTR1 in Patu-8902 cells did not have replicates and FDR could not be derived. Therefore, we listed the raw data in Supplemental Table 3 without showing statistics. To validate the putative interactions identified by IP-MS, we performed IP-Western experiments to confirm that TEAD4 interacts with PARP1 (Figure 3A). It is important to note that in addition to our report, the interaction between PARP1 and TEADs has been observed in other publications (Calses et al., 2023; Yang et al., 2017). We have included more details of the IP-MS experiment reported in Supplemental Table 3 in the revised manuscript and cited previous work reporting TEAD-PARP1 interaction.

(5) Proofread the manuscript more thoroughly for typos and grammatical errors.

We thank the reviewer for raising this issue and have addressed it in the revision.

(6) Improve figure clarity (e.g., clearly labeling graph axes).

We apologize for the oversight. The revised manuscript contains high resolution figures.

Specific points:

Generally, the manuscript could use additional proofreading for grammar and clarity. It would not be practical to list all, but some representative examples are listed below:

Run-on: "They act through an event-driven mechanism instead of conventional occupancy-driven pharmacology, in addition, target protein degradation removes all functions of the target protein and may also lead to destabilization of entire multidomain protein complexes."

Typo: "Compound D exhibits significant inhibition of cell proliferation and downstream signaling compared to compound A, a reversible TEAD lipid pocket binder that lack the ubiquitin ligase binding moiety."

Typo: "Thus, we sought to deplete TEAD proteins by directly target them for ubiquitination and proteasomal degradation via pharmacologically inducing interactions between TEAD and other abundantly expressed and PARylation-independent E3 ligases."

Typo: "Compound A is a close in analog of Compound B as described previously (Holden et al., 2020)."

We have revised the manuscript and corrected the typos and grammatical errors listed above and beyond.

Specific comments on the figures are listed below:

Figure 2:

• Figures 2B and 2C should be separated into separate panels for clarity.

We have updated the Figures 2B and 2C as suggested.

• Figure 2C - "To further assess the function of RNF146, we depleted RNF146 by either sgRNA or siRNA." This should say either CRISPR-Cas9 KO or siRNA-mediated knockdown.

We thank the reviewer for the suggestion. We revised the text to address this issue.

• Figure 2D - y-axis is not labeled well/clearly. Additionally, there are different resolutions for the p-values on the graph (the top p-value is slightly clearer than the other two, suggesting either a different font was used or the value was pasted on top of a picture of the graph at a different resolution).

We updated the figures according to the suggestions.

• Figure S2A - "We identified three ubiquitin ligases - RNF146, TRAF3, and PH5A - as potential negative regulators for the Hippos pathway from the primary screen using the luciferase reporter." However, the siPHF5A data appears to decrease luciferase levels whereas siRNF146 and siTRAF3 increase it.

We thank the reviewer for catching this error. We removed PH5A from this list.

Figure 3:

• Figure 3A - label more clearly. Is this an endogenous TEAD4 co-IP?

We thank the reviewer for the suggestion. The experiment was an IP-mass spectrometry study in a TEAD4 amplified cell line model (PATU-8902) with pan-TEAD antibody. We have included the details to in the figure legends. Figure 3A is now Figure S3E in the revised manuscript.

• Figure 3C - why are the dark and light exposures not matching/corresponding? In the dark exposure, there are two particularly dark bands, the darkest of which is at the top of the gel. However, this darkest band disappears in the light exposure gel. Additionally, the last lane is marked as +TEAD2 and +TEAD4. Not sure if this is a typo, and meant to be only +TEAD4? Seems a bit strange to have a double TEAD lane.

We thank the reviewer for this comment and apologize for the oversight. There was a typo in the label. The light exposure image was from a replicate run instead of the same run, therefore the lanes didn’t all match up. We have removed the light exposure panel to resolve the confusion. (Figure 3B).

Figure 5:

• Figure 5B - why is shTEAD1-4/Sucrose a much higher tumor volume than shNTC/Sucrose negative control? Additionally, should the legend say "sNTC/Sucrose" as it does or "shNTC/Sucrose"?

The labels for shTEAD1-4/Sucrose and shNTC/Sucrose are correct. We do not understand why there is a slight increase in tumor volume for shTEAD1-4/Sucrose and suspect that is due to the considerable variation in the experiment. This slight change, however, doesn’t influence our observation of tumor regression in shTEAD1-4 under the Doxycycline treatment.

"sNTC/Sucrose" is a typo. We apologize for the oversight and have revised the figure.

• Figure 5E - cited in text after Figures 6 and 7.

We have updated the text accordingly.

Figure 6:

• Figure 6B - it is very interesting how this clearly shows the Hook effect for Compound D, but it's a bit harder to see for compound E that the compound degrades pan-TEAD. Would it be possible to quantify the blots to reinforce claims about protein degradation here?

We thank the reviewer for the question. There may seem to be some hook effect across the three concentrations of compound D treatment in Fig. 6B.  However, in Fig. 6C-E, we observed pretty consistent TEAD degradation levels across a variety of concentrations. In addition, these experiments have been repeated in multiple cell lines with consistent results. We respectfully argue that more detailed investigation of the hook effect is beyond the scope of our study.

Figure 7:

• Figure 7F - this heat map is extremely difficult to interpret. Are there any interesting clusters? What are the darker/lighter bands for Compound D compared to DMSO control?

We thank the reviewer for the comment and apologize for the lack of information on the figure. These are genes from a Hippo signature derived from our earlier work (Pham et al. Cancer Discovery). As a result of degrading TEAD when treating the cells with Compound D, we observed an expected downregulation of most of these genes compared to compound A.

Figure 8:

• Figure 8B - these two pie charts are also difficult to interpret. Perhaps try to present the data in a form other than encircling pie charts?

We thank the reviewer for the suggestion. However, this is a very descriptive pie chart, we used this format to save space.

• Figure 8C - what is GNE-6915? Is this Compound D?

Yes, this is compound D. The text is updated accordingly.

Reviewer #3 (Recommendations For The Authors):

Figure 3A would benefit from explicitly stating the conditions within the figure, rather than referring to the legend. This clarity is also needed for Figure 8C, indicating whether the treatment was with compound D or GNE-6915.

We thank the reviewer for the suggestion. We have added the details to the figures and made the suggested edits.

Standardize the terms "ubiquitination" and "ubiquitylation" throughout the paper for consistency.

We now use the term “ubiquitination” throughout the manuscript.

The statement "In this study, we show that the activity of TEAD transcription factors can be post-transcriptionally regulated via the ubiquitin/proteasome system" should be corrected to "post-translationally regulated."

We have update the manuscript accordingly.

There is an additional exclamation mark above Figure 5E that should be removed.

We have revised Figure 5E.

Reviewer #1 (Public review):

Summary:

The authors used multiple approaches to study salt effects in liquid-liquid phase separation (LLPS). Results on both wild-type Caprin1 and mutants and on different types of salts contribute to a comprehensive understanding.

Strengths:

The main strength of this work is the thoroughness of investigation. This aspect is highlighted by the multiple approaches used in the study, and reinforced by the multiple protein variants and different salts studied.

Weaknesses:

(1) The multiple computational approaches are a strength, but they're cruder than explicit-solvent all-atom molecular dynamics (MD) simulations and may miss subtle effects of salts. In particular, all-atom MD simulations demonstrate that high salt strengthens pi-types of interactions (ref. 42 and MacAinsh et al, https://www.biorxiv.org/content/10.1101/2024.05.26.596000v3).<br /> (2) The paper can be improved by distilling the various results into a simple set of conclusions. By example, based on salt effects revealed by all-atom MD simulations, MacAinsh et al. presented a sequence-based predictor for classes of salt dependence. Wild-type Caprin1 fits right into the "high net charge" class, with a high net charge and a high aromatic content, showing no LLPS at 0 NaCl and an increasing tendency of LLPS with increasing NaCl. In contrast, pY-Caprin1 belongs to the "screening" class, with a high level of charged residues and showing a decreasing tendency of LLLPS.<br /> (3) Mechanistic interpretations can be further simplified or clarified. (i) Reentrant salt effects (e.g., Fig. 4a) are reported but no simple explanation seems to have been provided. Fig. 4a,b look very similar to what has been reported as strong-attraction promotor and weak-attraction suppressor, respectively (ref. 50; see also PMC5928213 Fig. 2d,b). According to the latter two studies, the "reentrant" behavior of a strong-attraction promotor, CL- in the present case, is due to Cl-mediated attraction at low to medium [NaCl] and repulsion between Cl- ions at high salt. Do the authors agree with this explanation? If not, could they provide another simple physical explanation? (ii) The authors attributed the promotional effect of Cl- to counterion-bridged interchain contacts, based on a single instance. There is another simple explanation, i.e., neutralization of the net charge on Caprin1. The authors should analyze their simulation results to distinguish net charge neutralization and interchain bridging; see MacAinsh et al.<br /> (4) The authors presented ATP-Mg both as a single ion and as two separate ions; there is no explanation of which of the two versions reflects reality. When presenting ATP-Mg as a single ion, it's as though it forms a salt with Na+. I assume NaCl, ATP, and MgCl2 were used in the experiment. Why is Cl- not considered? Related to this point, it looks ATP is just another salt ion studied and much of the Results section is on NaCl, so the emphasis of ATP ("Diverse Roles of ATP" in the title is somewhat misleading.

Comments on revisions:

This revision addressed all my previous comments.

for - Buddhism - Tibetan - Kagyu and Nyingma schools don't make a big deal out of Tukdam - nondual awareness can emerge with other techniques - key insight - Buddhism - Tibetan - Clear light meditation at time of death - Tukdam - a physiological technique - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - key insight - Buddhism - TIbetan - Clear light meditation - Tukdam at time of death - no longer capable of thinking, seeing, hearing, etc - all that's left is naked consciousness without even subject-object from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

This is a playful twist on the idiom "the shoe fits," but it's a little twisted in that it's the "shoe" on the hand. It feels like a moment of acceptance-like the speaker is saying, "Well, if this is how things are. I'll just roll with it."

The speaker admits that things are tough, maybe tougher than you think, but still pulls back to the mantra 'it's alright'. It's almost like saying, "Yeah, life's tough, but you just have to deal with it and keep going."

The speaker admits that things are tough, maybe tougher than you think, but still pulls back to the mantra: it’s alright. It’s almost like saying, “Yeah, life’s tough, but you just have to deal with it and keep going."

This is a playful twist on the idiom “the shoe fits,” but it’s a little twisted in that it’s the “shoe” on the hand. It feels like a moment of acceptance—like the speaker is saying, “Well, if this is how things are, I’ll just roll with it."

Author response:

(1) General Statements

We thank all three reviewers for their constructive comments and suggestions. We also thank reviewers #2 and #3 for considering our work to be timely and of interest to the field, not only for basic researchers, but also for translational scientists and industry. We are now providing additional results to further support our hypothesis and hope that all reviewers will find that our manuscript is now ready for publication. 

(2) Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)): 

The manuscript by Coquel et al. investigates the effects of BKC and IBC, two compounds found in Psoralea corylifolia in DNA replication and the response to DNA damage, and explores their potential use in cancer treatment. These compounds have been previously shown to affect different cellular pathways and the authors use transformed cancer cells of different origins and a non-transformed cell line to question if their combination is toxic in cancer versus non-cancer cells. They propose that BKC inhibits DNA polymerases while IBC targets CHK2. Their results show that both compounds do affect DNA replication, inducing replication stress and affecting double strand break repair. They also show that their combined use increases their toxicity in a synergistic manner. 

However, there are some major conclusions that are still not very well supported by the data: first, the differential effect on cancer and non-transformed cells; second, the direct link of BKC to the inhibition of DNA polymerases; and third, it is unclear if CHK2 is the relevant target for IBC in this context. 

Regarding these points the authors should address the following issues: 

(1) Most of the experiments use BJ fibroblasts as a control cell line. In order to evaluate if these compounds are preferentially toxic for cancer cells, the use of more than one non-transformed cell line is necessary. In addition, BJ cells are fibroblasts while most of the cancer cell lines employed are of epithelial origin. The authors could use MCF10 and RPE cells (both of epithelial origin) as control cell lines to complement the results and better support this claim. 

We have now monitored the effect of IBC and BKC on the proliferation of MCF-7, MCF-10A and RPE-1 cells using the WST-1 assay and obtained similar results as for BJ and MCF-7 cells. These results are now included in the revised manuscript as Fig. S1A and S1B.

(2) In order to explore what are the targets of BKC and IBC Cellular Thermal Shift Assays (CETSA) could be used. Either by doing an unbiased mass spectrometry analysis of proteins stabilized by these compounds or by a direct analysis of candidate proteins by western blot (a similar approach has been used for IBC to show that it inhibits SIRT2 in Ren et al., 2024 Phytotherapy Res).

We thank this Reviewer for suggesting the use of the CETSA assay. We have now performed  CETSA on MCF-7 cells and found that IBC stabilizes CHK2 but not CHK1, to the same extent as the commercial CHK2 inhibitor BML-277 used here as a positive control. These results are now shown in new Fig. 4G and 4H.

(3) For BKC in vitro polymerase assays could be carried out to show the direct inhibition of the DNA polymerase delta, for instance. 

We have used high-speed Xenopus egg extracts to replicate ssDNA in vitro (Fig. S2C). This assay differs from the in vitro replication assay using low-speed Xenopus egg extracts (Fig. 2H) in that it only monitors elongation by replicative DNA polymerases (Pol δ and ε) and not earlier steps such as origin licensing and activation. The combined use of both low-speed and highspeed extracts strongly supports the view that BKC inhibits replicative DNA polymerases. 

To confirm this result, we have also used CETSA to monitor BKC binding to different subunits of DNA Polδ and Polε in MCF-7 cells and in Xenopus egg extracts (Fig. 3C-D Fig. S3). We found that BKC binds POLD1 and POLE, the catalytic subunits of Pol δ and ε respectively, but not the accessory subunit POLD3 nor PCNA. Together with our docking results and DNA fiber experiments, these data strongly support the view that BKC is a potent inhibitor of DNA Pol and Pol. 

(4) In addition, the authors could analyze the integrity of replication forks by PCNA immunofluorescence analysis. The colocalization of PCNA and POLD or POLE subunits could also support the role of DNA polymerases as targets of BKC. 

Our molecular docking results also show that BKC occupies the catalytic sites of DNA Pol δ and ε, which may not affect their subcellular localization and/or PCNA binding. Since our DNA replication assays, CETSA and DNA fiber analyses strongly support the view that BKC inhibits replicative DNA polymerases, we have not performed this additional experiment.

(5) In the case of IBC and the inhibition of CHK2, the authors should check the effect of IBC on the phosphorylation of BRCA1 on S988. The changes in CHK2 phosphorylation in Figure 3B are not convincing. The experiment should be repeated and the average of at least three experiments needs to be quantified. 

We now provide evidence that IBC inhibits BRCA1 phosphorylation on S988. Western blots and quantification for three biological replicates are shown in Fig. 4C and Fig. S4H. Densitometric quantification of CHK2 phosphorylation on S516 from 3 biological replicates, along with statistical analysis, is now shown in Fig. S4G.

(6) To prove that CHK2 is the relevant target for IBC the authors could test if ATM and CHK2 knockout cells are more resistant to this compound, since it would prevent the phosphorylation of CHK2. 

We have performed siRNA transfection targeting CHK2. The transfected cells died after 72 hours in culture, so we have been unable to determine whether CHK2-KD cells have increased resistance to IBC.  

In addition to these experiments, I would suggest some other major improvements in the manuscript: 

(1) The concentration of both compounds should be provided in molar units throughout the paper.

Thanks for pointing this out, we now use molar units throughout the paper.

(2) The authors do not clearly indicate the concentration that is employed in the different experiments, making it difficult to assess the results. For instance, Figure 2 does not include the concentration in the legend or in the text. Time and concentration need to be clearly shown for each experiment. 

The experimental conditions and inhibitor concentrations are now clearly indicated for each experiment.

(3) Some experiments are only repeated once (fiber assays) or twice (cell cycle analysis by flow cytometry). These experiments need to be repeated 3 times and the proper statistical analysis performed (comparison of the medians). 

Superplots with biological replicates for all DNA fiber assays are now displayed. The number of biological replicates is now indicated in the legends and appropriate statistical analyses are used.

Other minor points or suggestions: 

(1) Analyzing fork asymmetry would further support the direct effect of BKC on DNA polymerases. 

The effect of BKC on fork asymmetry is now shown in Fig. 2F. 

(2) A dose dependent analysis of BKC on the speed of DNA replication would also support this point. 

Superplots of DNA fiber assays showing the effect of different concentrations of BKC on fork speed from three biological replicates are now included in Fig. 2E.

(3) Page 7: BKC reduces fork speed ...two-fold. This sentence is not very clear, it would be better to say that speed is half of the control. 

This sentence was changed to “BKC reduced fork speed by a factor of two relative to untreated cells”.

(4) Figure 4G and S4D show contradictory results regarding the induction of Rad51 foci by IBC treatment. This needs to be clarified. 

Figure 4G and S4D (now Fig. 5G and S5D) do not show contradictory results. In both cases, IBC treatment impaired the induction of RAD51 foci by IR or bleomycin.  

(5) Page 12, Figure S5C is called for but it does not exist (probably meaning Figure S5B). 

We apologize for this error, which has now been corrected.  

Reviewer #1 (Significance): 

The work by Coquel et al. aims at elucidating the use of BKC and IBC as a combined therapy to induce cell death in cancer cells by targeting DNA replication and CHK2. Both BKC and IBC have been previously shown to affect the proliferation of cancer cells. BKC has been shown to induce S phase arrest in an ATR dependent manner in MCF7 cells (Li et al., 2016 Front Pharm), while IBC induces cell death in MDA-MB-231 cells (Wu et al., 2022 Molecules). In this regard, the more interesting contribution of the manuscript is the potential identification of the targets of these compounds in cancer cells. The inhibition of CHK2 by IBC is quite compelling although it needs to be further proven. In contrast, the hypothesis that BKC inhibits DNA polymerases remains highly speculative. The results offer a limited advance in the knowledge of the mechanism of action of these two compounds. Focusing on the action of IBC on CHK2 would increase the impact of the results. In this sense a very recent report has been published showing that IBC inhibits SIRT2 (Ren et al., 2024 Phyto Res), showing that IBC can affect multiple enzymes and processes. This should be taken into account for a further analysis of its mechanism of action. 

In addition to the identification of the targets of BKC and IBC, the authors also focus on their combination for cancer treatment. This is based on the idea that blocking the DSB repair and inducing replication stress at the same time is an efficient approach to induce cancer cell death. This is not a new concept, since the loss of ATM sensitizes cancer cells to the inhibition of the replication stress response and several combination therapies have been put forward with the idea of generating replication stress and preventing the subsequent repair of the double strand breaks induced in these cells. Thus, the novelty here is limited, especially considering that the effect of BKC on DNA replication has already been described. Further, since its mechanism of action is unclear, it is difficult to ascribe the observed synergy to the speculated hypothesis. A deeper analysis of IBC as a CHK2 inhibitor would be more interesting, and the potential combination with other chemotherapy agents such as replication stress inhibitors, HU or DNA damaging agents. Also, the lack of a good control of non-transformed cells also reduces the relevance of the work. 

In its current state, the interest of the manuscript is limited. The mechanistical advance is not strong enough and is not completely supported by the data, and the use of these compounds as a combination therapy does not provide new insights in cancer treatment. In my opinion, focusing on the inhibition of CHK2 by IBC and its potential use would broaden the impact of the results beyond the mere analysis of the action of these compounds. 

We thank this reviewer for his/her constructive and insightful comments. We have followed his/her advice and focused our analysis on the action of IBC on CHK2. Using CETSA, we confirmed that IBC binds CHK2 to the same extent as BML-277 inhibitor, but does not bind CHK1. We also show that IBC inhibits BRCA1 phosphorylation on S988 and CHK2 phosphorylation on S516. Together with the results presented in the initial version of the manuscript, these data support the view that CHK2 is a key IBC target. We have also applied CETSA to DNA polymerases and confirmed that BKC directly targets DNA Polδ and ε. Although it is unlikely that IBC and BKC will ever be used in combination therapies, the synergistic effect that we measured on cancer cells in vivo and in vitro indicates that IBC sensitizes cancer cells to endogenous replication stress and to exogenous sources of DNA damage, which could be used to replace BKC in combination therapies. For instance, our data indicate that IBC can be used in combination with drugs such as etoposide, doxorubicin or cyclophosphamide to potentiate their effect on drug-resistant lymphoma cell lines (DLBCL). As requested by this Reviewer, we have modified the discussion section to put more emphasis on IBC and CHK2 inhibitors and we hope that he/she will now find this revised version suitable for publication.

Reviewer #2 (Evidence, reproducibility and clarity): 

In the manuscript by Coquel et al., the authors report their findings on the effect of 2 natural compounds from Psoralea corylofolia plant extracts on cancer cells. They show that these compounds, bakuchiol (BKC) and isobavachalcone (IBC), inhibit proliferation of cancer cells and tumor development in xenografted mice, particularly when used in combination. They further show that BKC inhibited DNA polymerases and induced replication stress, and show evidence that IBC inhibits Chk2 kinase activity and downstream double-strand break repair. Based on their findings, the authors conclude that Chk2 inhibition and DNA replication inhibition represent a potential synergistic strategy to selecting target cancer cells. 

Major: 

(1) The data showing IBC is a Chk2 inhibitor is weak and more rigorous investigation is needed to establish this compound as a Chk2 inhibitor. 

As indicate in our response to Reviewer #1, we have now analyzed the binding of IBC to CHK2 using the Cellular Thermal Shift Assay (CETSA) in MCF-7 cells. Our data clearly show that IBC binds to CHK2 but not CHK1. These results are now shown in Fig. 4G and 4H.

For one, the authors mention they screened 43 cell cycle-related kinases in vitro, but only show data for 8 kinases in their kinase activity screens. Of these 8 kinases, Chk2 is the most strongly inhibited, but there are no data shown for the other 35 kinases. 

Data for all the protein kinases tested in the in vitro assay are now presented in Fig. S4D and S4E.  

Additionally, the purpose of the CHK2 mutants should be discussed in the text. 

The CHK2(I157T) mutation is linked to an increased risk of breast and colorectal cancers. CHK2(R145W) is associated with Li-Fraumeni Syndrome. Both mutations do not affect the basal kinase activity of CHK2. This information is now indicated in the legend of Fig. S4D. 

Secondly, the western blot in Fig 3B, appears to show a very modest effect of IBC on Chk2 autophosphorylation and not that different from the effect of IBC on Akt phosphorylation in Fig S3a. Yet, the authors claim that IBC inhibits Chk2 but not Akt. To strengthen these blots, a known Chk2 inhibitor, such as the one shown in Fig 4 (BML-277) should be included as a positive control for pChk2 similarly to what was shown for Akt with MK-2206. 

We have now replaced the western blot in Fig. 3B (now Fig. 4B) with another biological replicate. Quantifications and statistical analyses of biological replicates are shown in Fig. S4G. Overall, we observed a 50% reduction of CHK2 auto-phosphorylation in MCF7 cells treated with IBC, and a 20% reduction in AKT phosphorylation (Fig. S4A). There was no additional reduction in AKT phosphorylation when cells were treated with IBC in combination with MK-2206, compared to cells treated with MK-2206 alone. We now include the CHK2 inhibitor BML-277 as a positive control alongside with IBC to monitor CHK2 and CHK1 auto-phosphorylation in Fig. 4B, S4G, 4D and S4I, respectively.

Western blots showing a loss of phosphorylation of additional Chk2 targets is also needed. The manuscript mentions Brca1 S988 as a Chk2 substrate important for DSB repair. Showing the effect of IBC on this phosphorylation site would strengthen the conclusions. 

We now provide evidence that IBC inhibits BRCA1 phosphorylation at S988. Western blots and quantification for three biological replicates are shown in Fig. 4C and S4H. 

(2) The authors claim that the combination of IBC and BKC inhibit cell growth in a synergistic manner and that the "effect is more pronounce on cancer cells than on non-cancer cells." However, only 1 non-malignant cell line was used, and it was a fibroblast line. To make this claim, the authors need to show the effect in additional non-malignant cells, preferably with epithelial cell types. 

We have now monitored cell proliferation using the WST-1 assay in two additional non-malignant cell lines, namely MCF-10A and RPE-1 cells. Cells were treated with IBC/BKC and their growth was compared to that of MCF-7 cells. These experiments yielded similar results to those obtained with BJ fibroblasts. These new data are now included in the revised version as Fig. S1A and S1B. 

Minor: 

(1) Densitometry data for all western blots should be shown with mean+/- stdev of independent western blots. 

Densitometry data for all western blots with biological replicates are now shown in supplementary figures.

(2) In Figure 1B the statistical test used to analyze cell number was not stated. 

The statistical test is now indicated in Fig. 1B.

(3) In Figure 2A, the DAPI image for BKC is the merged image and should be replaced with just DAPI. 

This error has now been corrected.

(4) In Figure 2B, the y-axis label says "yH2AX foci (MFI)". MFI and foci are not the same thing, and for yH2AX, the signal is often not focal. MFI of yH2AX is an appropriate measurement for replication stress, it's just not appropriate to equate MFI to foci. 

We apologize for this labeling error, which has now been corrected.

(5) For the 53BP1 MFI and Rad51 MFI shown in Fig 4 and Fig S4, it is more appropriate to show the number of foci/cell as these are better indicators of breaks and repair sites. MFI is influenced by expression levels of the proteins and not necessarily the break/repair. 

The numbers of 53BP1 and RAD51 foci are now shown.

(6) The data in Figures 5B and 5C are very difficult to read. Perhaps color-coat the lines/symbols. 

We have now colored the graph to increase its readability. 

Reviewer #2 (Significance): 

The findings reported in this manuscript are timely, of interest to the field, and are mostly wellsupported by the experimental data. However, there are a few concerns that need to be addressed. 

We are grateful to Reviewer #2 for his positive assessment of our manuscript. We hope that we have adequately addressed all of his/her specific concerns and that he/she will agree with the need to put more emphasis on IBC and CHK2 inhibition as requested by Reviewer #1.

Reviewer #3 (Evidence, reproducibility and clarity): 

The manuscript: "Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth" successfully demonstrates that the compounds BKC and IBC found in Psoralea corylifolia act synergistically to inhibit cancer cell proliferation, using a wide range of well-chosen methodologies. Moreover, the authors characterized the mechanisms of action of both drugs, which result in inhibition of cell proliferation. The use of multiple cell lines and the mice models makes the study robust and complete. The manuscript presents a well written study that offers new insights and contributions to the field. 

A few suggestions to improve the study: 

(1) Given that both compounds BKC and IBC have already been previously described in the literature, it would be helpful for the reader to have them described better at the beginning of the study. 

Thanks for pointing this out. We have now better described BKC and IBC at the beginning of the results section, as well as in the discussion. We agree that this could be helpful to readers.

(2) Addition of western blot quantifications over the number of experimental repeats is important specifically for Fig. 2C and Fig. 3C where partial effect of treatment on a signal level is reported. 

The densitometry analysis of data shown in Fig. 2C and biological replicates are now shown in Fig. S2B. Quantification for Fig. 3C (now Fig. 4D) is shown in Fig. S4I.

(3) The quantification of mean intensity for 53BP1 and RAD51 foci should be exchanged with the quantification of number of foci per cell. While the quantification of gH2AX signal intensity is a correct representation of induction of this signal upon damage, foci formed by protein recruitment to DNA damage sites should be quantified by counting the number of foci, rather than signal in the whole cell/nucleus. These proteins exist before damage and are re-located in response to the damage. 

Quantification of 53BP1 and RAD51 foci is now expressed as the number of foci per cell. 

(4) Materials & Methods section is missing the methods for the experiment described in Fig. 1B. In summary, after addressing our few concerns, we believe the manuscript should be accepted for publication. 

The WST-1 assay used for cell number quantification is included in “Reagents” in Material & Methods section.

Reviewer #3 (Significance):

The manuscript presents a well written study that offers new insights and contributions to the field. Although the inhibitors described have been known in science, the authors present convincingly their mode of action, which is either better characterized (for BKC) or inhibiting a different than previously suggested enzyme (for IBC). Authors also nicely pinpoint and explain the narrow window of concentrations when these two compounds act synergistically rather than additively. The analyses in multiple cell lines, mouse models and in combination with other cancer treatments, makes this study of interest not only for fundamental researchers but also for translational scientists and industry.

My field of expertise: DNA replication and replication stress across model systems. 

We are grateful to Reviewer #3 for his/her very positive assessment of our work and we hope that he/she will find this revised version suitable for publication.

The interview design really stood out to me here. Starting with a question about times participants felt unheard or uncomfortable allows them to share their personal experiences in a way that feels validating. Following that up with, “What would you change?” is such a thoughtful way to shift the focus from frustration to actionable solutions. It’s empowering and gives participants a voice in improving care, rather than just reliving negative experiences. Plus, having two researchers there—one leading and one clarifying—shows a commitment to making sure nothing important is missed.

This is a true and unbaised statement that just shows truth.

This really shows how important shared decision-making is for building trust between patients and providers, especially for transgender and intersex patients. The fear that asking questions might lead to losing care speaks to a deeper issue of vulnerability and lack of control. It’s a reminder that providers need to create a safe, open space where patients feel comfortable speaking up and making informed choices about their care without fear of judgment or consequences.

curse of lisp

for - new insight - second cause of human separation - after settling down, it was WRITING! intriguing! - from - Emergence Magazine - interview - An Ethics of Wild Mind - David Hinton - adjacency - sense of separation - first - settling down - human place - second - writing - from - Emergence Magazine - interview - An Ethics of Wild Mind - David Hinton

adjacency - between - sense of separation - first - settling down - human place - second - transition from oral to written language - adjacency relationship - Interesting that I was just reading an article on language and perception from the General Semantics organization: General Semantics and non-verbal awareness - The claim is that the transition from oral language to written language created the feeling of interiority and of a separate "soul". - This is definitely worth exploring!

explore claim - the transition from oral language traditions to writing led us to form the sense of interiority and of a "soul" separate from the body - This claim, if we can validate it, can have profound implications - Writing definitely led us to create much more complex words but we were able to do much more efficient timebinding - transmitting knowledge from one generation to the next. - We didn't have to depend on just a few elders to pass the knowledge on. With the invention of the printing press, written language got an exponential acceleration in intergenerational knowledge transmission. - This had a huge feedback effect on the oral language itself, increase the number of words and meanings exponentially. - There are complex recipes for everything and written words allow us to capture the complex recipes or instructions in ways that would overwhelm oral traditions.

to - article - General Semantics and Non-Verbal Awareness - https://hyp.is/BePQhLvTEe-wYD_MPM9N3Q/www.time-binding.org/Article-Database

for - adjacency - paleolithic hunter-gatherer - to neolithic agricultural village - dawn of agriculture - village - cultivated fields around it - created a human space - the village - thus began the - great separation - Emergence Magazine - interview - An Ethics of Wild Mind - David Hinton

adjacency - between - paleolithic hunter-gatherer - to neolithic agricultural village - dawn of agriculture village - cultivated fields around it - settling down - birth of the human space - the village - thus began - the great separation - adjacency relationship - He connects two important ideas together, the transition from - always-moving, never settling down paleolithic hunter-gatherer to - settled-down neolithic agricultural farmers - The key connection is that this transition from moving around and mobile to stationary is the beginning of our separation from nature - John Ikerd talks about the same thing in his article on the "three great separations". He identifies agriculture as the first of three major cultural separation events that led to our modern form of alienation - The development of a human place had humble beginnings but today, these places are "human-made worlds" that are foreign to any other species. - The act of settling down in one fixed space gave us a place we can continually build upon, accrue and most importantly, begin and continue timebinding - After all, a library is a fixed place, it doesn't move. It would be very difficult to maintain were it always moving.

to - article - In These Times - The Three “Great Separations” that Unravelled Our Connection to Earth and Each Other - John Ikerd - https://hyp.is/CEzS6Bd_Ee6l6KswKZEGkw/inthesetimes.com/article/industrial-agricultural-revolution-planet-earth-david-korten - timebinding - Alfred Korzyski

Author response:

The following is the authors’ response to the original reviews.

Overall authors’ response

We would like to thank the 3 reviewers for a thorough critique of our manuscript, and acknowledging the novelty and importance of our studies, in particular the relevance to collagenrelated pathologies such as idiopathic pulmonary fibrosis and chronic skin wound. We appreciate that there are shortcomings in these studies, as highlighted by reviewers; we have rewritten parts of our manuscript to clarify any misunderstandings, and conducted additional experiments to address concerns raised by reviewers (please see below red text within each response), which have been incorporated into our revised manuscript (modified text highlighted in yellow in revised manuscript). We believe that the revision had made our manuscript stronger in support of our original conclusions. 

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors describe that the endocytic pathway is crucial for ColI fibrillogenesis. ColI is endocytosed by fibroblasts, prior to exocytosis and formation of fibrils, which can include a mixture of endogenous/nascent ColI chains and exogenous ColI. ColI uptake and fibrillogenesis are regulated by circadian rhythm as described by the authors in 2020, thanks to the dependence of this pathway on circadian-clock-regulated protein VPS33B. Cells are capable of forming fibrils with recently endocytosed ColI when nascent chains are not available. Previously identified VPS33B is demonstrated not to have a role in endocytosis of ColI, but to play a role in fibril formation, which the authors demonstrate by showing the loss of fibril formation in VPS33B KO, and an excess of insoluble fibrils - along-side a decrease in soluble ColI secretion - in VPS33B overexpression conditions. A VPS33B binding protein VIPAS39 is also shown to be required for fibrillogenesis and to colocalise with ColI. The authors thus conclude that ColI is internalised into endosomal structures within the cell, and that ColI, VPS33B, and VIPA39 are co-trafficked to the site of fibrillogenesis, where along with ITGA11, which by mass spectrometric analysis is shown to be regulated by VPS33B levels, ColI fibrils are formed. Interestingly, in involved human skin sections from idiopathic pulmonary fibrosis (IPF) patients, ITGA11 and VPS33B expression is increased compared to healthy tissue, while in patient-derived fibroblasts, uptake of fluorescently-labelled ColI is also increased. This suggests that there may be a significant contribution of endocytosis-dependent fibrillogenesis in the formation of fibrotic and chronic wound-healing diseases in humans. 

Strengths: 

This is an interesting paper that contributes an exciting novel understanding of the formation of fibrotic disease, which despite its high occurrence, still has no robust therapeutic options. The precise mechanisms of fibrillogenesis are also not well understood, so a study devoted to this complex and key mechanism is well appreciated. The dependence of fibrillogenesis on VPS33B and VIPA39 is convincing and robust, while the distinction between soluble ColI secretion and insoluble fibrillar ColI is interesting and informative. 

Weaknesses: 

There are a number of limitations to this study in its current state. Inhibition of ColI uptake is performed using Dyngo4a, which although proposed as an inhibitor of Clathrin-dependent endocytosis is known to be quite un-specific. This may not be a problem however, as the endocytic mechanism for ColI also does not seem to be well defined in the literature, in fact, the principle mechanism described in the papers referred to by the authors is that of phagocytosis.

We thank the reviewer for pointing this out. Macropinocytosis or phagocytosis could be modelled using high molecular weight dextran, and we have used fluorescently-labelled dextran to investigate potential co-localisation with exogenous collagen to investigate the involvement of these mechanisms in addition to endocytosis, and showed very little co-localisation (revised Figure S2B, lines 123-126). Further, we have performed a competition experiment where unlabelled collagen was added in excess at the same time as labelled collagen and showed that excess unlabelled collagen led to a retention of labelled collagen at the cell periphery (revised Figure S2C, lines 126-129). This is suggestive of collagen-I uptake utilises a different pathway to dextran (i.e. fluid-phase endocytosis) and is a receptor-mediated process.  

It would be interesting to explore this important part of the mechanism further, especially in relation to the intracellular destination of ColI.

We agree with the reviewer that the intracellular destination of ColI is very interesting, which is what the current Chang lab is investigating, although we believe the research findings fall out of scope for the revised manuscript here. However, we have included additional immunofluorescence data to support that collagen is indeed taken up into endosomal compartments using GFP-tagged Rab5 constructs (revised Figure 1D, Figure S6A).

The circadian regulation does not appear as robust as the authors' last paper, however, there could be a larger lag between endocytosis of ColI and realisation of fibrils.

The authors state that the endocytic pathway is the mechanism of trafficking and that they show ColI, VPS33B, and VIPA39 are co-trafficked. However, the only link that is put forward to the endosomes is rather tenuously through VPS33B/VIPA39.

We would like to clarify that we meant the post-Golgi compartment. We did not mean VPS33b/VIPAS39 as an endosome marker; however as we see collagen entering the cell in intracellular compartments, which is then recycled, we take that as convention, the endosome would be involved. This is further supported that we see some colocalisation with the classic Rab5 endosome marker.

There is no direct demonstration of ColI localisation to endosomes (ie. immunofluorescence), and this is overstated throughout the text.

We appreciate the comment and have modified overstatements in the revised manuscript as appropriate. As stated above, we have included additional immunofluorescence data to support that collagen is indeed taken up into endosomal compartments.

Demonstrating the intracellular trafficking and localisation of ColI, and its actual relationship to VPS33B and VIPA39, followed by ITGA11, would broaden the relevance of this paper significantly to incorporate the field of protein trafficking. Finally, the "self-formation" of ColI fibrils is discussed in relation to the literature and the concentration of fluorescently-tagged ColI, however as the key message of the paper is the fibrillogenesis from exocytosed colI, I do not feel like it is demonstrated to leave no doubt. Specific inhibition of intracellular trafficking steps, or following the progressive formation of ColI fibrils over time by immunofluorescence would demonstrate without any further doubt that ColI must be endocytosed first, to form fibrils as a secondary step, rather than externally-added ColI being incorporated directly to fibrils, independent of cellular uptake.

We appreciate the concern raised here. This is precisely why we trypsinised and replated cells as part of the workflow, so we can make sure that there is no residual exogenous collagen which is not endocytosed being incorporated onto pre-existing fibrils. We have new data using flow imaging, which showed that cells that don’t endocytose exogenous collagen has accumulation of said collagen at the periphery of the cells, which is greatly reduced after trypsinisation. This new data is in a more detailed methodology-based study which is under preparation, which will allow future studies to further dissect the collagen intracellular trafficking process, and thus is not included in the revised manuscript. 

Reviewer #2 (Public Review): 

Summary: 

In this manuscript, the authors describe a mechanism, by which fluorescently-labelled Collagen type

I is taken up by cells via endocytosis and then incorporated into newly synthesized fibers via an ITGA11 and VPS33B-dependent mechanism. The authors claim the existence of this collagen recycling mechanism and link it to fibrotic diseases such as IPF and chronic wounds. 

Strengths: 

he manuscript is well-written, and experimentally contains a broad variation of assays to support their conclusions. Also, the authors added data of IPF patient-derived fibroblasts, patient-derived lung samples, and patient-derived samples of chronic wounds that highlight a potential in vivo disease correlation of their findings. 

The authors were also analyzing the membrane topology of VPS33B and could unravel a likely 'hairpin' like conformation in the ER membrane. 

Weaknesses: 

Experimental evidence is missing that supports the non-degradative endocytosis of the labeled collagen.

We thank the reviewer for raising this. We would like to clarify that we do not think that all endocytosed collagen-I is recycled, but rather sorted in the endosome which determines the fate of endocytosed collagen. Interestingly, results from Kadler’s group has shown that blocking lysosome function (through chloroqine and bafilomycin) significantly reduced endogenous collagen fibril formation (https://www.biorxiv.org/content/10.1101/2024.05.09.593302v1), suggesting a nondegradative role for lysosome in fibrillogenesis.   

The authors show and mention in the text that the endocytosis inhibitor Dyngo®4a shows an effect on collagen secretion. It is not clear to me how specific this readout is if the inhibitor affects more than endocytosis. This issue was unfortunately not further discussed.

We thank the reviewer for this comment and have included in discussion the specificity of Dyngo4a (revised manuscript lines 383392). The ponceau stain suggests that Dyngo4a treatment did not affect global secretion and thus the effects are specific to collagen-I (Fig 2B).

The authors use commercial rat tail collagen, it is unclear to me which state the collagen is in when it's endocytosed. Is it fully assembled as collagen fiber or are those single heterotrimers or homotrimers?

We apologise for the confusion and will clarify in our revision. These would be single helical trimers from acid-extracted rat tail collagen. We have performed additional light scattering and CD spectra to confirm the molecular weight and helicity, and confirm that adding fluorescent tags did not alter the readout. We have included this in the revised manuscript (revised Figure S1A-C, manuscript lines 82-86).    

The Cy-labeled collagen is clearly incorporated into new fibers, but I'm not sure whether the collagen is needed to be endocytosed to be incorporated into the fibers or if that is happening in the extracellular space mediated by the cells.

We appreciate the concern raised here, which is also raised by reviewer 1. As answered above, this is why we trypsinised and replated cells as part of the workflow, so we can make sure that there is no residual exogenous collagen being incorporated onto pre-existing fibrils. We also have new data using flow imaging, which shows that cells that don’t endocytose exogenous collagen has accumulation of said collagen at the periphery of the cells, which is greatly reduced after trypsinisation. This new data is in a methodology-based manuscript which is under preparation, thus will not be included in the revised manuscript.  

In general for the collagen blots, due to the lack of molecular weight markers, what chain/form of collagen type I are you showing here?

Apologies for the lack of molecular weight markers, it was an oversight by the authors and have been included in the revised figures.  

Besides the VPS33B siRNA transfected cells the authors also use CRISPR/Cas9-generated KO. The KO cells do not seem to be a clean system, as there is still a lot of mRNA produced. Were the clones sequenced to verify the KO on a genomic level?

Yes, the clones were verified and used in our previous paper on circadian control of collagen homeostasis. There are instances where despite knockout at the protein level, mRNA is still persistent; however these transcripts are likely then directed to degradation through nonsense-mediated mRNA decay. To fully understand this mechanism is beyond the scope of this paper. 

For the siRNA transfection, a control blot for efficiency would be great to estimate the effect size. To me it is not clear where the endocytosed collagen and VPS33B eventually meet in the cells and whether they interact. Or is ITGA11 required to mediate this process, in case VPS33B is not reaching the lumen?

This is an interesting question. We have conducted experiments with Col1-GFP11 containing conditioned media incubated with VPS33b-barrell in the revised paper, which showed that they interact within the cell and not at the cell periphery (revised Figure 6G, lines 293-296), again highlighting that VPS33b is not involved in the endocytosis step but interacts with endocytosed collagen-I intracellularly. We have attempted colocliasation studies using the split GFP approach with VPS33B and ITGA11 to investigate where they interact, but as the ITGA11 construct we used did not localise to the cell surface as expected, we are not confident that this system is appropriate for investigating how/if VPS33B interacts with ITGA11, and there are simply no good antibody for VPS33B for staining. 

The authors show an upregulation of ITGA11 and VPS33B in IPF patients-derived fibroblasts, which can be correlated to an increased level of ColI uptake, however, it is not clear whether this increased uptake in those cells is due to the elevated levels of VPS33B and/or ITGA11.

We would like to clarify here that we do not think collagen-I uptake is due to VPS33B and/or ITGA11, as siITGA11 and VPS33B in fibroblasts showed no consistent changes in uptake as determined by flow cytometry, which was included in the original manuscript (now revised Figure 6H, 7I). VPS33B and ITGA11 are involved in the ‘outward’ arm of recycled collagen-I, i.e. directing to fibrillogenesis route. We agree that the inclusion of additional functional studies using IPF patient-derived patient fibroblasts would add to the manuscript, and have performed siRNA against VPS33B and ITGA11 on IPF fibroblasts, and demonstrated a late of endocytic recycling events (revised Figure 8D, S6B, lines 351-353).  

Reviewer #3 (Public Review): 

Summary: 

Chang et al. investigated the mechanisms governing collagen fibrillogenesis, firstly demonstrating that cells within tail tendons are able to uptake exogenous collagen and use this to synthesize new collagen-1 fibrils. Using an endocytic inhibitor, the authors next showed that endocytosis was required for collagen fibrillogenesis and that this process occurs in a circadian rhythmic manner. Using knockdown and overexpression assays, it was then demonstrated that collagen fibril formation is controlled by vacuolar protein sorting 33b (VPS33b), and this VPS33b-dependent fibrillogenesis is mediated via Integrin alpha-11 (ITGA11). Finally, the authors demonstrated increased expression of VPS33b and ITGA11 at the gene level in fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), and greater expression of these proteins in both lung samples from IPF patients and in chronic skin wounds, indicating that endocytic recycling is disrupted in fibrotic diseases. 

Strengths: 

The authors have performed a comprehensive functional analysis of the regulators of endocytic recycling of collagen, providing compelling evidence that VPS33b and ITGA11 are crucial regulators of this process. 

Weaknesses: 

Throughout the study, several different cell types have been used (immortalised tail tendon fibroblasts, NIHT3T cells, and HEK293T cells). In general, it is not clear which cells have been used for a particular experiment, and the rationale for using these different cell types is not explained. In addition, some experimental details are missing from the methods.

We thank the reviewer for pointing out the lack of clarity, and have filled in missing information in the methods. HEK293T cells were used for virus production for the VPSoe system, and we have clarified the cell types used in figure legends (predominantly iTTF). We have also provided justification when NIH3T3 cells were used (revised lines 290-291).    

There is also a lack of functional studies in patient-derived IPF fibroblasts which means the link between endocytic recycling of collagen and the role of VPS33b and ITGA11 cannot be fully established.

We thank the reviewer for this comment, which was also raised by reviewer 2 above. We agree that the inclusion of additional functional studies using IPF patient-derived patient fibroblasts would add to the manuscript and have performed siRNA against VPS33B and ITGA11 on IPF fibroblasts, and demonstrated a late of endocytic recycling events (revised Figure 8D, S6B, lines 351-353).  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

The authors inhibit Clathrin-dependent endocytosis with dyngo4a. It is well known that this inhibitor is not highly specific for this pathway. It is also not explained why the authors only inhibit the Clathrin uptake pathway, and not pinocytosis or Clathrin-independent endocytosis too. The authors refer to papers that describe pinocytosis for collagen endocytosis.

We thank the reviewer for raising this question. Based on the fact that inhibition of clathrin-dependent pathway does not completely abrogate endocytosis of collagen-I, we anticipate that other pathways are involved in mediating collagen-I uptake, although additional data suggested this is unlikely through fluid-phase endocytosis, and is receptor mediated (revised Figure S2B, C).  

Where does the ColI go in the cell? Depending on the uptake pathway, it is likely to pass through endocytic carriers to endosomes, where it may be recycled to the PM or degraded. From the start, the authors describe the ColI as being in vesicular structures, however, the imaging data that this is based on is not co-labelled with anything to determine the potential structure/localisation. This is not done at any point in the paper, until IF is shown of ColI with VIPA39, however without the relevant controls, this IF is unconvincing, as the general pattern of ColI and VIPA39 as an endosomal marker are not classically recognisable. Additionally, VPS33B is described as a late endosome/lysosome marker, which would have different connotations on ColI trafficking or destination than other types of endosomes.

We thank the reviewer for pointing out the weaknesses in our original IF. We have included new confocal images showing labelled collagen co-localisation with GFP-tagged Rab5 through transient transfection, which is a more traditional endosome marker (revised Figure 1D, Figure S6A).  

We are currently characterising the compartments to where ColI is trafficked to, which is being prepared as part of a methodology-based manuscript. We believe that this characterisation would be too detailed to be included in a revised version of this manuscript. The Kadler lab also have data suggesting that the lysosome is involved in collagen fibrillogenesis instead of its canonical degradation function, which is in another submitted manuscript (https://www.researchsquare.com/article/rs-1336021/v1). It was not included in this manuscript due to our focus (i.e. endocytic-recycling).   

In Figure 5H, the pattern of Cy5-ColI staining looks like it could even be ER/Golgi in the VPSKO zoom panel, but in the absence of co-labelling, we cannot conclude anything. In order for the authors to conclude that ColI is within the endosomes, co-labelled If should be performed to demonstrate ColIendosomal colocalization. Likewise for the role of VPS33B in ColI fibrillogenesis: dependence of the process is demonstrated, but the relationship is not defined. This could be clarified using IF. This would also support the authors' statements of co-trafficking between ColI, VPS33B, and VIPA39, which as the paper stands, is not demonstrated.

We would like to clarify that our hypothesis is that the endosome controls how collagen is being deposited outside the cell, i.e. whether it’s protomeric secretion or fibrillogenesis, and that the decision of whether an endocytosed collagen is recycled or degraded lies in this compartment. The reviewer is correct that it may not be just the endosome that endocytosed collagen-I ends up in, as we have new data suggesting involvement of other intracellular compartment, although the detailed mechanism is beyond the scope of this manuscript. Nonetheless, we have included new data showing co-localisation of endocytosed collagen with Rab5 in this revised manuscript (revised Figure 1D, Figure S6A).  

The basis of this paper is that endocytosis of ColI must occur before re-exocytosis as fibrillar ColI. The authors show this through pulse-chase experiments, with a trypsinisation step to remove any externally bound ColI. The authors also show nice time progression by flow cytometry, but it would truly demonstrate this point if they showed 0 timepoint, or low timepoint of IF to show progressive lengthening of ColI fibrils. This is used early on in Figure 1D, although the presentation here is not very clear. This is especially important as the authors address the self-seeding capabilities of Collagen in cell-free conditions in Figure 1F.

We would like to thank the reviewer for this suggestion.  From previous endogenously tagged collagen data, we know that the appearance of collagen fibrils is rather rapid, thus it may not be a gradual lengthening as expected, but rather a depletion of endocytosed collagen in the initial seeding/growth step (please see https://www.researchsquare.com/article/rs-1336021/v1). We have included an image of replated fibroblasts after 18 hours showing no appearance of extracellular collagen, endogenous or otherwise (revised Figures S2A, line 110).  

Finally, although the involvement of ITGA11 is interesting, it is not well described, and its role is not well demonstrated. This could likely be clarified by an additional introduction to ITGA11 and its role in collagen exocytosis/fibrillogenesis.

We would like to thank the reviewer for pointing this out and have included additional sentences to specifically introduce ITGA11 and its role in fibrillogenesis (see lines 320, 321; 446-450).  

Specific points: 

Line 73: You haven't compared reuse vs production, so you can't say that reuse is central rather than production. They may be both as important or production still may be the most crucial, maybe it depends on cell/collagen type. Using the ColI KD or CHX to block nascent synthesis, you could directly compare the impact of both.

We would like to clarify that we are not referring to reuse/recycling here. We meant that production of collagen (i.e. single hetero/homotrimer molecules within the cell) is not as crucial as the utilisation (i.e. are these being secreted as protomers, or assembled into fibrils) of these building blocks by the cells, which was supported by our finding that production (as suggested by mRNA levels) of IPF fibroblasts are similar to that in control fibroblasts (now revised Figure 8A). We have conducted ColI siRNA to block nascent synthesis in the original manuscript and showed that fibroblasts can efficiently make new fibrils by recycling exogenous collagen (Figure 3B, C), although we appreciate that siRNA may not completely inhibit endogenous production. Thus, we have also included new data using collagen-I knockout cells to support our hypothesis that without endogenous production, fibroblasts can still effectively make collagen fibrils if they can reuse what is available in the extracellular space (revised Figure 4, Figure S3C, D; lines 178-199).  

Lines 83-87: The rationale for this experiment is not clear. Cy3-ColI is added, taken up into cells, and incorporated into fibrils coming from cells. 5FAM-ColI is added at a later stage, then at 2 days (when incorporation is demonstrated in Fig 1B), it is also incorporated into cells as expected. Why does this comment on ColI not being degraded any more than Cy3-ColI alone?

We believe that the pulse chase experiment using the differently tagged collagen demonstrated a dimension of dynamics that is not demonstrated with Cy3-ColI alone. In this case, Cy3-ColI was initially added, and removed after 3 days; 5FAM-ColI is then added and incubated for 2 more days. Thus after 5 days since the initial pulse, the Cy3-ColI persisted and was not degraded. We would like to apologise for causing this confusion, and have clarified in the revised manuscript (lines 542-549; Figure S1D figure legend).  

Figure 1A: I would like to see a negative control: either dark colI or no Cy3-Col, or timescale. Is B quantified from these images?

We thank the reviewer for this comment. We have added the nocollagen control image in our revision (revised Figure S1D). 1B is not quantified from the ex vivo tendon experiments, but rather the in vitro cell culture experiments (i.e. those from 1D-1F, although they are all from independent experiments).  

Figure 1B: in iTTF cells (immortalised tendon cells) Corrected to max: What does that mean?

As there are variations between individual experiments (e.g. changes in the amount of collagen added due to pipetting) we have normalised to the maximum value obtained in each individual experiments so that we can display all biological repeats within the same graph.  

Figure 1C: You can't say ColI is in vesicular structures from this, they are spots, yes, but that could also be in Golgi/ER (unlikely to be cytosolic but not impossible).

We appreciate this comment and have change the wording accordingly and call them intracellular/punctate structures.

Figure 1D: Not the best presentation: The cell mask has structures: what are these? It's not clear if this is a single cell, would be better with a defined marker (endocytic marker, lysosome etc). Instead of a low-resolution 3D view, it would be clearer with normal confocal XY and zooms of "vesicular structures" using appropriate markers as 3D reconstructions I think it could be removed.

This is a single cell and the cell mask is staining plasma membrane. We didn’t use defined marker as we wanted to visualise the whole intracellular cell compartment. We appreciate that further proof is needed to verify the location of the endocytosed collagen, and have included additional confocal imaging data to support the localisation of collagen into Rab5 positive intracellular compartments (revised Figure 1D, Figure S6B).  

Figure 1 E/F: Cy3 is only visible in extracellular structure, not also intracellular. Why? Would be useful to see the time points of incorporation at the end of the pulse, then at an early point into the chase, to demonstrate 1) Cy3-ColI uptake into cells and progressive incorporation rather than potential direct binding of ColI-Cy3 to ECM, or other non-specific factors. Showing the image at 0t would demonstrate an absence of external labelled colI and therefore its appearance later could be presumed that it had been internalised before.

As the cells were trypsinized and replated after one hour labelled collagen feeding to ensure we are only tracking endocytosed collagen, t=0 in this case would be cells that are unattached. We have included t=18hr images post replate instead to show baseline level of collagen (revised Figures S2A, line 110).

Figure S1A: yellow box: doesn't show only Cy3-ColI, there is red and yellow in the central cell, and large yellow blobs in the cell above. These images do not support this claim, including the Fiber Zoom box. They should also be shown in single channels to demonstrate the authors' points better.

Apologies for the confusion – this is to show that newly added FAM5 Collagen is also co-localising with previously endocytosed Cy3-ColI, i.e. the Cy3-ColI is persisting rather than being degraded.  

Line 92: endocytosed into distinct structures: These images are very vague, but I don't think you can call them distinct structures, all you can say from this is that they are spots.

We have changed the wording to ‘distinct puncta’.  

It is not clear why the authors use Cy3, Cy5, and 5FAM labelled colI. A brief explanation would be useful.

Apologies for the confusion, we initially included our justification (to show that the fluorescence labels do not change the way collagen is internalised) but removed it in the final manuscript due to length. We have added the justification (revised line 101-102).   

Figure 1F: It would be useful to see a quantification of the Cy3 channel here: I agree with the conclusions, and find the 0.5 ug/ml condition more convincing than 0.1 actually, although there is some feint Cy3 in cell-free samples there seems to be quite a big increase in the presence of cells, and this would look more convincing if quantified.

We thank the reviewer for this suggestion and have included quantification in the revised manuscript (revised Figure 1G-I).  

Figure 2B: Dyng is not an abbreviation of Dyng. Standardise Dyng/Dyngo/Dyngo4a. WB is soluble colI and represents little (if any) insoluble col. IF is more or less the other way round. How do they compare this?

Thank you for pointing out the inconsistencies, we have corrected this in the revised manuscript. We took the conditioned media from the same experiment where cells are fixed for IF and carried out Western blot analyses. The IF showed some collagen still present, albeit significantly reduced. This is in agreement with the western blot results (i.e. Dyng4a inhibits both soluble and insoluble forms of collagen deposition).  

Figure 2C: not an image series. Quant: no cells/independent exps and STATS?

Apologies for the missing experimental details in figure legends, it should say ‘representative of N=3 experiments’. We are not sure what the reviewer meant by Figure 2C not being an image series, as we meant it to be an image series of the individual fluorescence channels. We have changed this terminology to avoid confusion, and have included statistical analyses in the methods section. The statistical analyses of the fibril quantification is next to the fluorescence images.  

Figures 2D/E: The authors show that internalised ColI peaks at 20h and decreases to 60h, Fibers peak at 40h. How is this measured? ECM removed? Why would there be less in the cells, degradation? Whats the synchronisation?

We apologise for omitting the synchronisation method in methods section, and have included in our revised manuscript (revised lines 542-544). This is through dexamethasone addition (and removal after 1hr incubation) as standard. The internalised Col-I is measured using Cy3ColI so the cells would have both nascent and external collagen. Total intracellular collagen at the different time points would likely be higher than represented as a result, but here we are demonstrating that internalisation is a rhythmic event using the external labelled collagen. Fibers are measured using standard IF and then fibril counting.  

Please note that we are only overlaying the two graphs to form our hypothesis that endocytosis may be used for accumulation of collagen protomers that then allows for efficient fibrillogenesis. They are not directly comparable as the quantification are of different things (internalised Cy3-ColI, total collagen fibrils). We have clarified this in our discussion (revised lines 399-401).  

Discussion: Where does the ColI go? Solubilised? Degraded? Taken up by other cells? 

The inverse correlation is not very tight. In fact, at 38h where fiber count peaks, Cy3-ColI also peaks (esp in normalised data, Figure S2D).

We thank the reviewer for this comment and have reworded our main text to reflect this, and included additional discussion in our revised manuscript (revised lines 401-404).  

Line 123: What is the turnover rate of Fibrils? Don't know for how long the transcription has been done, or when this would affect the fibril number. You have the quant for Fn1, where is the quant for ColI?

We have included the quantification of collagen-I in original Figure 2A. We appreciate that it might cause confusion in Figure 2C (as we co-stained ColI and Fn1 in the same experiment) we have removed the collagen-I panel from the revised Figure 2C. We know from previous results that the number of fibrils fluctuate over 24hour period, although the turnover of one specific fibril is unlikely going to be 24 hours (https://www.biorxiv.org/content/10.1101/331496v2)

Line 124: no accumulation of col in extracellular space, but you don't know how much endogenous colI (or other endogenous ECM proteins) they're taking up as it isn't measured here. If the author wants to comment on this, should use either exogenous col to monitor take up and resection or block transcription/translation to show fibril formation endo/exocytosis independent of endogenous synthesis.

This experiment has been done in the original manuscript – siCol1a1 experiment was done with two rounds of siRNA, first round is normal transfection followed by reverse transfection onto fresh coverslips (this will ensure no prior ECM is being deposited, see Figure 3). However we appreciate that there may still be low levels of endogenous collagen-I, and thus have included new data using collagen-I knock-out fibroblasts to strengthen our findings (revised Figure 4).  

Line 142: Why is fibronectin synthesis also decreased in Col KD? This is clear in the image but no explanation/reference is given.

Due to the dynamic and complex nature of ECM, it is unsurprising if there is a knockon effect when knocking down one matrix protein. However, we have quantified the amount of fibronectin fibril deposited by scr and siCol1a1 fibroblasts, and showed that there was in fact no significant change between the two treatments (revised Figure 3A).

Figure 3A: Need labels for which colour/protein is shown. Needs quantifying, especially as the Fn1 decrease is not so obvious here, it is consistent between Figure 3A and 2C?

We have provided quantification in the revision (revised Figure 3A). Figure 3A and 2C are two separate experiments (one is Dyngo treatment and one is siCol1a1), and neither showed significant changes in fibronectin fibril areas.   

Figure 3B: Line 151: the text states that "The observation of fibrillar Cy3 signals in siCol1a1 cells showed that the cells can repurpose collagen into fibrils without the requirement for intrinsic collagen-I production (red arrow Figure 3B), however, there is clearly endogenous colI here too (along the fiber and also strongly at each end). Does the ColI antibody recognise the exogenous ColI?

In our hands the ColI antibody does not recognise exogenous ColI, as the cell-free Cy3-ColI images were also stained with ColI antibody to ensure the two experimental conditions were treated exactly the same.

This conclusion could only be made in the true absence of collagen: either in knock-out cells, or where collagen production/trafficking has been blocked (ie knockout of ColI chaperone or ERES block), or in a cell type that produces collagens but not ColI. Alternatively, if there are any fibrils seen that are completely negative, they should be shown in the figure and quantified (number of Cy3-ColI+-ColI+ vs Cy3-ColI+-ColI-).

We thank the reviewer for this suggestion. We have included new data from collagen knock-out fibroblasts in this revision (revised Figure 4).  

Figure S4A: the quality of this blot isn't very high, the result is not very clear and the high intensity (unspecific?) band below confounds the interpretation. In the author's previous paper (NCB 2020) the blots for VPS33B were much clearer, as is Fig S4D. It would be nice to include a clearer blot, maybe from the other repeats.

This is the only blot that we used to select which knockout clones to use for our previous paper, which is why the quality is not as high. Knockout clones were all verified with additional western blots, and we do not think that endogenous VPS33b is expressed at high levels (also verified by MS analyses).  Fig S4D is overexpression of VPS33b, which is much easier to detect.  

Figure S4D: This blot is much clearer, it would be useful to include a high gain to show the VPS33B band in CT to be able to understand the true increase.

From the qPCR data one can see that the increase at mRNA is 20+ fold increase; we’ve always had problems trying to detect endogenous VPS33b using western blot or mass spectrometry analysis.  

Figure 4A: The fibrils here in the CT are not obvious, and the difference between CT and KOs is not appreciable. Would this be clearer shown at a lower magnification, with zooms where needed? Or immunogold labelling/CLEM to label the ColI?

It is not trivial to carry out immunogold labelling/CLEM. These are cell-derived matrices in culture and thus lower magnification may not show as many collagen fibrils as one would expect. We are not confident that lower magnification will provide more information as the characteristic D-banded collagen pattern will be lost.  

Line 167/Figure 4B: It looks like there is more internal ColI in KO, but the images are not good enough to tell. This could be better shown by flow cytometry.

We have previously seen that VPSKO leads to accumulation of collagen-I in intracellular punctas (NCB2020) which is also seen here. Flow cytometry data for internalisation of external collagen is already included in original Figure 5G (revised Figure 6H).  

Again you mention intercellular vesicles, but based on these images, it is not possible to conclude this. These large spots could be aggregation elsewhere in the cell. Specific localisation should be shown by co-labelled IF/confocal, or it could be nicely shown by EM + fluorescent element (CLEM / Immunogold), or these statements removed from the text.

We appreciate that the term ‘vesicles’ is very defined in the trafficking field, and have changed it to ‘intracellular compartments’.  

Line 173-174 / Figure 4E: Why do you think the matrix mass is not increased in VPSoe by the approach shown in E when there is seemingly a huge increase by IF? E must also measure other ECM matrix proteins, which do you expect to be secreted by these cells? Could this confound the data if they too are affected by VPSoe?

IF is showing specifically collagen-I. Hydroxyproline detects multiple collagens, and shows a trend of increase (although not significant due to one outlier). Matrix mass is a very generic measurement of total ECM deposited based on decellularized ECM weight. The reviewer is correct that VPSoe may also affect other ECM deposition, however here we are focussing specifically with its effect on collagen-I. How VPSoe changes other types of ECM deposition would be something that could be addressed in future studies and is not within scope of this manuscript.   

Are the results in E paired?

Individual values between control and VPSoe in each separate experiments are paired.  

Figure 4F: Is quantification from IF shown in D? Specify which kind of microscopy it is based on.

Quantification is based on fibril counting using standard fluorescence microscopy, as used in our previous paper. D is independent of F, as F is specifically looking at synchronised circadian effects, and D (and elsewhere) we are looking at global collagen deposition effects, irrespective of what time of day the cells are in.  

Figure S5F: What do the yellow/red spots in the blots represent?

We apologise for the initial unclear description of what the yellow/magenta circles depict in relation to the phosphoimages of the radiolabelled cell free translation products displayed in Supplementary Figure 5, panels F, G and I. These circles indicate non-glycosylated (yellow) and N-glycosylated (magenta) species respectively, as is now clearly descried in the revised manuscript.

Figure 5 title: You can't conclude this from these images, need confocal and PM or cytosolic marker.

We have changed the title to ‘VPS33B co-trafficks with collagen-I”. There is no good commercial VPS33b antibody for immunofluorescence staining, which is why we used the split GFP approach in this paper, and the images were acquired using confocal imaging (Olympus SpinSR system).  

Figure 5E: The authors describe that ColI is in endosomes throughout most of the paper, and this is based on the involvement of VPS33B in the colI pathway. VPS33B is thought to be at the late endosome/lysosome. However, these images do not look like classic endosomes or lysosomes, or other normal organelle IF phenotypes. The fluorescent intensity looks saturated, and it is difficult to conclude anything from these images. It is unclear where in the cell the largest blob in the zoom would be localised and in which cell. I would suggest that this image is replaced and proper controls included (IgG controls and single channels) as well as using different markers for other potential intracellular structures.

We appreciate the reviewers comment with regards to the classification of VPS33b localisation in the endosome compartment. We did not mean to use VPS33b as an endosome marker, as the focus of our studies are the function of VPS33b in directing endogenous or exogenous collagen to fibrillogenesis. With live imaging we could see endocytosed collagen moving in intracellular compartments, and have conducted additional staining to show co-localisation with Rab5 (revised Figure 1), which we take to indicate, through convention, that it is occupying an endosome compartment. We have included single channel images in the revised manuscript (revised Figure 6E).

Line 255/ Figure 5G: no consistent change in uptake. Why are the results so varied in the KO and oe, here and in Fig 4C/E? N=4, what does that mean? 4 cells? 4 independent exps?

In all cases, “N” represents independent biological experiments in this manuscript. Thus “N=4” in this case is 4 independent biological experiments, with at least 10,000 cells analysed per experiment. 

We don’t know why there is a variation in response, however that is also why we concluded that it is unlikely that VPS33B is directly involved with collagen uptake. We have changed 5G (now revised Figure 5H) to a paired line graph for better representation.  

Figure 5H shows the uptake of Cy5ColI. At this resolution, VP2ko looks like the col is ER, in one of the cells in the zoom, it looks like it is at Golgi. I think that the uptake route of ColI needs to be better defined, as there is no way to tell here where the colI goes. ColI being recycled/degraded would be most likely. But this figure looks like that might not be the case. It is also not clear where the zooms come from, they should be indicated with dashed boxes in the lower mag image

We thank the reviewer for this comment, and agree that we need to define the uptake route of ColI. This is currently being assembled as a methodology manuscript, and how ColI is being recycled/degraded is one major research area of the Chang lab. 

We have added dashed boxes in the lower mag images to indicate where the zooms derived from, and we would also like to thank the reviewer for pointing this out as we realised we have accidentally cropped the image to a slightly different area for the VPSko image, and have now corrected this.  

Line 257: Based on this data, it could be trafficking through the cell as well as into the extracellular space.

We think that VPS33B is involved in trafficking collagen through the cell to plasma membrane but not secreted, as based on our split-GFP experiment we never observed extracellular GFP signal, which suggests VPS33b is not deposited extracellularly.

Line 259: "highlighting the role in recycling col to fibril formation sites" is an overstatement based on the data shown here, there is no data on colI trafficking or its regulation

We respectfully disagree that we have not shown data on col-I trafficking or regulation by VPS33b – split GFP highlighted cotrafficking to the plasma membrane, and we have shown a clear relationship between VPS33b and collagen-I fibril formation, with minimal changes to collagen-I mRNA levels. We acknowledge that we have not shown specifically the location of VPS33b at fibrillogenic sites and have modified this statement in revised manuscript (revised line 302).  

Line 262: "Having identified VPS33B as specifically driving collagen-I fibril formation" is also an overstatement.

We refer here the data that VPS33b is not controlling collagen-I secretion (as demonstrated by the CM westerns) and specifically fibrillogenesis. We have clarified this in the revised text (revised line 304).  

Line 286: It would be useful to have a brief intro to PLOD3.

We have included a brief intro to PLOD3 in the introduction, as well as the results highlighted by the reviewer, in our revised manuscript (revised line 54-58).  

Line 289/290: There could be other explanations for disruption to exo-endocytosis when disrupting col trafficking. Is VPS33B controlling exocytosis in general? Why should it be specific to col? Likewise with siITGA11 KD? Hypothesis for ITGA11 and fibrillogenesis?

The relationship between ITGA11 and collagen fibrillogenesis is currently in a manuscript by Donald Gullberg and Cedric Zeltz, under revision at Matrix Biology (see reference 63 in revised manuscript). We do not think that VPS33b is controlling exocytosis in general, which is supported by the minimal change in ponceau stain of the western blots in the manuscript. Previously it has been shown that VPS33B co-trafficks with PLOD3, a collagen-I modifier.  

Figure 6I: Why only quant Scr + siITGA11, not in VPSoe? It looks like there is still an increase in intracellular or fibril formation in VPSoe + siITGA11, which would be a key result to discuss.

We would like to clarify that 6I (now revised Figure 7I) is on the endocytosis of exogenous collagen-I, not quantification of Figure 6H.  

Line 307: Discuss fibrillogenic sites, what are they?

As we have not shown direct evidence of VPS33B delivering endocytosed collagen at the site of fibrillogenesis, we have decided to alter the text to avoid overstatement, as suggested from previous reviewers’ comments.  

Figure 8: What does pentachrome label?

Pentachrome staining allows for simultaneous staining of multiple species: collagen in red, sulphated mucopolysaccharides in violet, red blood cells in yellow, muscle in orange, nuclei in green.

Line 326: "In this study we have identified the endosome as a major protagonist in..." This is an overstatement and cant be drawn from this data.

We have modified this statement to “In this study we have identified an endocytic recycling mechanism for type I collagen fibrillogenesis that is under circadian regulation”

Line 330/331: "Collagen-I co-traffics with VPS33B in a VIPAS-containing endosomal compartment that directs collagen-I to sites of fibril assembly," This is also an overstatement that cannot be drawn from this data.

We have modified this statement to “Collagen-I co-traffics with VPS33B to the plasma membrane for fibrillogenesis”.  

Line 340: again, the demonstration of the involvement of the endocytic pathway is very limited.

We have provided new evidence in the revised manuscript that support the involvement of classical endosomal compartments.  

Line 366: You cant conclude this, you have not manipulated these proteins to show a functional effect or modulation of fibrillogenesis, it could still be a secondary effect.

We have provided new evidence in the revised manuscript that supports this conclusion. 

Line 569: "Unless otherwise stated, incubation and washes were done at room temperature." Which incubations? Specify if this is just post-fixation during the EM prep or during cell culture.

This is specific to the EM preparation and we have clarified in the revised manuscript (revised line 663).  

Small text alterations:

Overall we would like to thank the reviewer for highlighting these errors and mistakes in our manuscript, and have corrected them in our revised manuscript.  

Figure 1E: Fluoro image series? This is only one image.

We wrote this to mean single channel images, we have corrected the terminology.  

Line 111: Ref for Dyngo4a?

We have included this in the revised manuscript  

Line 121: introduction/abbreviation definition for Fn1? Instead it is on Line 140.

Thank you for highlighting this, we have corrected this in revised manuscript.  

Figure S2C: Alignment of labels cleaves x-axis.

We thank the reviewer for catching this and have corrected this with our revised manuscript.  

Figure S4F and G should be inverted to mention sequentially in the text.

We thank the reviewer for catching this and have corrected this in our revised manuscript.  

Line 182: Figure 4J should be G.

We thank the reviewer for catching this and have corrected this in our revised manuscript.

Line 209: typo: N-glycosylated.

We have corrected this typo in our revised manuscript.

Fig 6E: Very big as a figure element compared to others.

We have made this smaller in the revised manuscript to fit better with rest of the figure.  

Line 313: Figure 7E not F.

Thank you for spotting this, we have corrected it.  

Line 555: Typo: Scraped.

We have corrected this typo in our revised manuscript.

Line 562: missing )

We have corrected this typo in our revised manuscript.

Standardise

We thank the reviewer for spotting the mistakes below and have corrected in our revised manuscript.  

Legends: Include numbers of repeats and STATs throughout. 

Terminology: Dyng etc. 

Scale bars: some included as editable lines, some with size on top, small/large etc.

In certain cases we have positioned the scale bars in different regions of the figures to ensure no obscuring of the images.

VPS33b v B. 

Reviewer #2 (Recommendations For The Authors):  

The authors can improve the experimental part of the manuscript the following: 

-  For all the western blots please include molecular weight markers.

We thank the reviewer for noticing this omission and have included molecular weight markers in the revised manuscript.  

- Performing immunofluorescence and western blot analysis of endocytosed collagen -/+ inhibitors for lysosomal degradation (BafA1 or E64d+PepstatinA) in order to exclude endocytosis for degradation.

We thank the reviewer for this comment, another paper from the lab has identified lysosome to be involved in collagen fibrillogenesis (https://www.biorxiv.org/content/10.1101/2024.05.09.593302v1), thus  

- Figure out how Dyngo4a is affecting Col1 secretion in the first place? Does it interfere with the secretory pathway. Alternatively, use a different model to block endocytosis (e.g. siRNA Dynamin).

We thank the reviewer for raising this. The Dyngo CM blot for total ponceau stain (revised Figure 2B) showed minimal changes, which suggest that global secretion is not affected.  

- Further characterization of the VPS33B / collagen vesicles by immunofluorescence containing markers for early, late, and recycling endosomes. Block endocytic recycling by depletion of either Rabs or e.g. EHD1.

There are no good VPS33b antibody for staining. We have included images of GFP-tagged Rab5 co-localisation with labelled collagen-I (revised Figure 1D, Figure S6B).  

- Further clarify the status of the VPS33B knockouts e.g. by sequencing. also provide a readout of the siRNA KD, besides the mRNA levels, since there the difference is not striking.

The knockout cell lines were characterised previously in our 2020 paper, which is referred to in our revised manuscript. We have always had issues detecting endogenous VPS33b due to reagents limitations, which is why we resorted to mRNA as the key readout.  

- Doing siRNA knockdowns and endocytosis inhibition in the IPF fibroblasts to further strengthen the link between elevated expression of VPS33B/ ITGA11 and increased collagen uptake.

We thank the reviewer for suggesting these experiments. Due to limitations of the patient-derived fibroblasts (cell numbers and passage numbers) we had to prioritise experiments, and thus have performed siRNA against VPS33B and ITGA11 in the IPF fibroblasts. We showed that in both cases the amount of recycled labelled-collagen in collagen fibrils is significantly reduced (revised Figure 8D).  

Reviewer #3 (Recommendations For The Authors): 

Major points 

(1) Choice of cells: Please provide a rationale for why each cell line was used, and make sure that it is clear throughout the manuscript which cell line was used for each particular experiment. The HEK293T cell line is also missing from the reagent table.

We thank the reviewer for pointing out this omission, and have clarified in our revised manuscript which cell lines were used in each experiment. We used HEK293T to generate lentiviruses as described in the methods section.  

(2) Missing information from methods. Experimental details are missing from the methods in several places, making it difficult for someone to replicate an experiment. For example, no details are given in the methods describing the explant culture of murine tail tendons (described in results lines 78100), and there are no details on how the skin samples were obtained or stained. Further, no ethical approval details are provided for the use of human skin tissue.

We apologise for leaving the ethical approval details and skin sample collection out, this was an oversight and will be included in the revised manuscript. We have also included the method to how murine tail tendons were cultured ex vivo (revised lines 527-531, 546-553).  

(3) Functional studies in patient-derived cells. To fully establish the role of VPS33b and ITGA11 in fibrotic diseases, functional studies including the knockdown/overexpression of these genes could be performed to establish if the same response is seen as in non-diseased cells.

We agree that this will add much to the paper, and have performed siRNA against VPS33B and ITGA11 in the IPF fibroblasts. We showed that in both cases the amount of recycled labelled-collagen in collagen fibrils is significantly reduced (revised Figure 8D).

Minor Points

We thank the reviewer for pointing out these mistakes, and have corrected and included additional details in the revised manuscript.  

(1) Lines 51-52. Wording of this sentence is unclear, please rephrase. 

(2) Line 182. Should this be Fig 4G rather than J? 

(3) Line 209. Correct spelling of glycosylated. 

(4) Line 463. Incomplete brackets and details missing? 

(5) Line 590. Correct tense - was rather than are. 

(6) Line 593. Specify centrifugation speed. 

(7) Line 619. Nuclei rather than nucleus. 

(8) Ln 650. Statistical analysis - was normality tested? 

(9) Figure 1e - Difficult to read labels for coll/DAPI.

The primary virtue of this approach is that it is extremely simple. However, using return codes has a number of drawbacks which can quickly become apparent when used in non-trivial cases:

First, return values can be cryptic -- if a function returns -1, is it trying to indicate an error, or is that actually a valid return value? It’s often hard to tell without digging into the guts of the function or consulting documentation.

Second, functions can only return one value, so what happens when you need to return both a function result and a possible error code? Consider the following function:

double divide(int x, int y) { return static_cast<double>(x)/y; } This function is in desperate need of some error handling, because it will crash if the user passes in 0 for parameter y. However, it also needs to return the result of x/y. How can it do both? The most common answer is that either the result or the error handling will have to be passed back as a reference parameter, which makes for ugly code that is less convenient to use. For example:

include <iostream>

double divide(int x, int y, bool& outSuccess) { if (y == 0) { outSuccess = false; return 0.0; }

outSuccess = true;
return static_cast<double>(x)/y;

}

int main() { bool success {}; // we must now pass in a bool value to see if the call was successful double result { divide(5, 3, success) };

if (!success) // and check it before we use the result
    std::cerr << "An error occurred" << std::endl;
else
    std::cout << "The answer is " << result << '\n';

} Third, in sequences of code where many things can go wrong, error codes have to be checked constantly. Consider the following snippet of code that involves parsing a text file for values that are supposed to be there:

std::ifstream setupIni { "setup.ini" }; // open setup.ini for reading // If the file couldn't be opened (e.g. because it was missing) return some error enum if (!setupIni) return ERROR_OPENING_FILE;

// Now read a bunch of values from a file if (!readIntegerFromFile(setupIni, m_firstParameter)) // try to read an integer from the file return ERROR_READING_VALUE; // Return enum value indicating value couldn't be read

if (!readDoubleFromFile(setupIni, m_secondParameter)) // try to read a double from the file return ERROR_READING_VALUE;

if (!readFloatFromFile(setupIni, m_thirdParameter)) // try to read a float from the file return ERROR_READING_VALUE; We haven’t covered file access yet, so don’t worry if you don’t understand how the above works -- just note the fact that every call requires an error-check and return back to the caller. Now imagine if there were twenty parameters of differing types -- you’re essentially checking for an error and returning ERROR_READING_VALUE twenty times! All of this error checking and returning values makes determining what the function is trying to do much harder to discern.

Fourth, return codes do not mix with constructors very well. What happens if you’re creating an object and something inside the constructor goes catastrophically wrong? Constructors have no return type to pass back a status indicator, and passing one back via a reference parameter is messy and must be explicitly checked. Furthermore, even if you do this, the object will still be created and then has to be dealt with or disposed of.

Finally, when an error code is returned to the caller, the caller may not always be equipped to handle the error. If the caller doesn’t want to handle the error, it either has to ignore it (in which case it will be lost forever), or return the error up the stack to the function that called it. This can be messy and lead to many of the same issues noted above.

for - (online) communities - potential devolution of - from - YouTube - situational assessment - Luigi Mangione - - the Stoa

for - definition - vibe shift - Some kind of event that changes the vibe. It is pre-cognitive and pre-narrative - Alison P Davis wrote about "Vibe Shift" in The Cut - Youtube - from The STOA - Situational Assessment - Luigi Mangione

They just said that it's centro-symmetric (which I believe means that it has inversion symmetry, Why do they now say that it breaks inversion symmetry?

for - to - synchronicity - same quote mentioned in - YouTube I watched yesterday - prenatal and perinatal healing happens in layers - Kate White - Third is related to the subject of prenatal and perinatal psychology - trauma suffered by the fetus while still in the womb it the newly born can be remembered somatically by the body and carried on into later life - As adults, we can carry on these old patterns of behaviours that were adaptive responses rooted in the initial trauma but which no longer exists - It's a form of post traumatic stress disorder where the body stop carries the memories - https://hyp.is/go?url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DUcgWsFqPe7Q&group=world

Yes! Yes! The contrast isn't as strong as it could be because Eugen has been trying with his product decisions to follow the same dream (with some really good effects and some bad) but if you lookst Darius Kazemi's stuff this becomes so, so crisp.

for - prenatal and perinatal psychology - is not just mental health - it's holistic mind body practice - somatic memories are alive in our body right now - Youtube - Prenatal and Perinatal Healing Happens in Layers - Kate White

I feel like there may or may not be a very specific narrative being pushed here. One that I don't think I am smart enough or as informed about to make any big statements here. It's just a hunch though.

What I believe is being said here is that inequality is not a result of technology, but rather a refusal to adopt said technology will be what creates the inequality. An argument over free will vs determinism.

It is always important to look at both sides of the coin when looking at any complicated subject. We do not live in a world that is merely black and white. A level of nuance is important to get anywhere in life. It kind of reminds me of discussions in class we had about activism when we read that section in Digital Keywords. While activism in the digital age has lead to many great outcomes, it has had the consequence of leading to means that have little to no effect (in some cases it can even be a detriment...). This doesn't undermine the good that comes from it though, it just means it's complicated. Just like AI. It's kind of really complicated.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews

Reviewer #1 (Public Review): 

(1) Although the theory is based on memory, it also is based on spatially-selective cells.

Not all cells in the hippocampus fulfill the criteria of place/HD/border/grid cells, and place a role in memory. E.g., Tonegawa, Buszaki labs' work does not focus on only those cells, and there are certainly a lot of non-pure spatial cells in monkeys (Martinez-Trujillo) and humans (iEEG). Does the author mainly focus on saying that "spatial cells" are memory, but do not account for non-spatial memory cells? This seems to be an incomplete account of memory - which is fine, but the way the model is set up suggests that *all* memory is, place (what/where), and non-spatial attributes ("grid") - but cells that don't fulfil these criteria in MTL (Diehl et al., 2017, Neuron; non-grid cells; Schaeffer et al., 2022, ICML; Luo et al., 2024, bioRxiv) certainly contribute to memory, and even navigation. This is also related to the question of whether these cell definitions matter at all (Luo et al., 2024). The authors note "However, this memory conjunction view of the MTL must be reconciled with the rodent electrophysiology finding that most cells in MTL appear to have receptive fields related to some aspect of spatial navigation (Boccara et al., 2010; Grieves & Jeffery, 2017). The paucity of non-spatial cells in MTL could be explained if grid cells have been mischaracterized as spatial." Is the author mainly talking about rodent work?

There is a new section in the introduction that deals with these issues, titled ‘Why Model the Rodent Navigation Literature with a Memory Model?’ That section reads:

“Spatial navigation is inherently a memory problem – learning the spatial arrangement of a new enclosure requires memory for the conjunction of what and where. This has long been realized and in the introduction to ‘Hippocampus as a Cognitive Map’, O’Keefe and Nadel (1978) wrote “We shall argue that the hippocampus is the core of a neural memory system providing an objective spatial framework within which the items and events of an organism's experience are located and interrelated” (emphasis added). Furthermore, in the last chapter of their book, they extended cognitive map theory to human memory for non-spatial characteristics. However, in the decades since the development of cognitive map theory, the rodent spatial navigation and human memory literatures have progressed somewhat independently.

The ideas proposed in this model are an attempt to reunify these literatures by returning to the original claim that spatial navigation is inherently a memory problem. The goal of the current study is to explain the rodent spatial navigation literature using a memory model that has the potential to also explain the human memory literature. In contrast, most grid cell models (Bellmund et al., 2016; Bush et al., 2015; Castro & Aguiar, 2014; Hasselmo, 2009; Mhatre et al., 2012; Solstad et al., 2006; Sorscher et al., 2023; Stepanyuk, 2015; Widloski & Fiete, 2014) are domain specific models of spatial navigation and as such, they do not lend themselves to explanations of human memory. Thus, the reason to prefer this model is parsimony. Rather than needing to develop a theory of memory that is separate from a theory of spatial navigation, it might be possible to address both literatures with a unified account.

This study does not attempt to falsify other theories of grid cells. Instead, this model reaches a radically different interpretation regarding the function of grid cells; an interpretation that emerges from viewing spatial navigation as a memory problem. All other grid cell models assume that an entorhinal grid cell displaying a spatially arranged grid of firing fields serves the function of spatial coding (i.e., spatial grid cells exist to support a spatial metric). In contrast, the proposed memory model of grid cells assumes that the hexagonal tiling reflects the need to keep memories separate from each other to minimize confusion and confabulation – the grid pattern is the byproduct of pattern separation between memories rather than the basis of a spatial code. 

It is now understood that grid-like firing fields can occur for non-spatial twodimensional spaces. For instance, human entorhinal cortex exhibits grid-like responses to video morph trajectories in a two-dimensional bird neck-length versus bird leg-length space (Constantinescu et al., 2016). As a general theory of learning and memory, the proposed memory model of grid cells is easily extended to explain these results (e.g., relabeling the border cell inputs in the model as neck-length and leg-length inputs). However, there are other grid cell models that can explain both spatial grid cells as well as non-spatial grid-like responses (Mok & Love, 2019; Rodríguez-Domínguez & Caplan, 2019; Stachenfeld et al., 2017; Wei et al., 2015). Similar to this memory model of grid cells, these models are also positioned to explain both the rodent spatial navigation and human memory literatures. Nevertheless, there is a key difference between this model and other grid cell models that generalize to non-spatial representations. Specifically, these other models assume that grid cells exhibiting spatial receptive fields serve the function of identifying positions in the environment (i.e., their function is spatial). As such, these models do not explain why most of the input to rodent hippocampus appears to be spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). This memory model of grid cells provides an answer to the apparent paucity of nonspatial cell types in rodent MTL by proposing that grid cells with spatial receptive fields have been misclassified as spatial (they are what cells rather than where cells) and that place cells are fundamentally memory cells that conjoin what and where.”

(2) Related to the last point, how about non-grid multi-field mEC cells? In theory, these also should be the same; but the author only presents perfect-look grid cells. In empirical work, clearly, this is not the case, and many mEC cells are multi-field non-grid cells (Diehl et al., 2017). Does the model find these cells? Do they play a different role? As noted by the author "Because the non-spatial attributes are constant throughout the two-dimensional surface, this results in an array of discrete memory locations that are approximately hexagonal (as explained in the Model Methods, an "online" memory consolidation process employing pattern separation rapidly turns an approximately hexagonal array into one that is precisely hexagonal). " If they are indeed all precisely hexagonal, does that mean the model doesn't have non-grid spatial cells? 

Grid cells with irregular firing fields are now considered in the discussion with the following paragraphs

“According to this model, hexagonally arranged grid cells should be the exception rather than the rule when considering more naturalistic environments. In a more ecologically valid situation, such as with landmarks, varied sounds, food sources, threats, and interactions with conspecifics, there may still be remembered locations were events occurred or remembered properties can be found, but because the non-spatial properties are non-uniform in the environment, the arrangement of memory feedback will be irregular, reflecting the varied nature of the environment. This may explain the finding that even in a situation where there are regular hexagonal grid cells, there are often irregular non-grid cells that have a reliable multi-location firing field, but the arrangement of the firing fields is irregular (Diehl et al., 2017). For instance, even when navigating in an enclosure that has uniform properties as dictated by experimental procedures, they may be other properties that were not well-controlled (e.g., a view of exterior lighting in some locations but not others), and these uncontrolled properties may produce an irregular grid (i.e., because the uncontrolled properties are reliably associated with some locations but not others, hippocampal memory feedback triggers retrieval of those properties in the associations locations).

In this memory model, there are other situations in which an irregular but reliable multilocation grid may occur, even when everything is well controlled. In the reported simulations, when the hippocampal place cells were based on variation in X/Y (as defined by Border cells), nothing else changed as a function of location, and the model rapidly produced a precise hexagonal arrangement of hippocampal place cell memories. When head direction was included (i.e., real-world variation in X, Y, and head direction), the model still produced a hexagonal arrangement as per face-centered cubic packing of memories, but this precise arrangement was slower to emerge, with place cells continuing to shift their positions until the borders of the enclosure were sufficiently well learned from multiple viewpoints. If there is real-world variation in four or more dimensions, as is likely the case in a more ecologically valid situation, it will be even harder for place cell memories to settle on a precise regular lattice. Furthermore, in the case of four dimensions, mathematicians studying the “sphere packing problem” recently concluded that densest packing is irregular (Campos et al., 2023). This may explain why the multifield grid cells for freely flying bats have a systematic minimum distance between firing fields, but their arrangement is globally irregular (Ginosar et al., 2021). Assuming that the memories encoded by a bat include not just the three real-world dimensions of variation, but also head direction, the grid will likely be irregular even under optimal conditions of laboratory control.”

(3) Theoretical reasons for why the model is put together this way, and why grid cells must be coding a non-spatial attribute: Is this account more data-driven (fits the data so formulated this way), or is it theoretical - there is a reason why place, border, grid cells are formulated to be like this. For example, is it an efficient way to code these variables? It can be both, like how the BVC model makes theoretical sense that you can use boundaries to determine a specific location (and so place cell), but also works (creates realistic place cells). 

The motivation for this model is now articulated in the new section, quoted above, titled ‘Why Model the Rodent Navigation Literature with a Memory Model?’ Regarding the assumption that border cells provide a spatial metric, this assumption is made for the same reasons as in the BVC model. Regarding this, the text said: “These assumptions regarding border cells are based on the boundary vector cell (BVC) model of Barry et al. (2006). As in the BVC model, combinations of border cells encode where each memory occurred in the realworld X/Y plane.”. A new sentence is added to model methods, stating: “This assumption is made because border cells provide an efficient representation of Euclidean space (e.g., if the animal knows how far it is from different walls of the enclosure, this already available information can be used to calculate location).”

But in this case, the purpose of grid cell coding a non-spatial attribute, and having some kind of system where it doesn't fire at all locations seems a little arbitrary. If it's not encoding a spatial attribute, it doesn't have to have a spatial field. For example, it could fire in the whole arena - which some cells do (and don't pass the criteria of spatial cells as they are not spatially "selective" to another location, related to above).  

Some cells have a constant high firing rate, but they are the exception rather than the rule. More typically, cells habituate in the presence of ongoing excitatory drive and by doing so become sensitive to fluctuations in excitatory drive. Habituation is advantageous both in terms of metabolic cost and in terms of function (i.e., sensitivity to change). This is now explained in the following paragraph:

“In theory, a cell representing a non-spatial attribute found at all locations of an enclosure (aka, a grid cell in the context of this model), could fire constantly within the enclosure. However, in practice, cells habituate and rapidly reduce their firing rate by an order of magnitude when their preferred stimulus is presented without cessation (Abbott et al., 1997; Tsodyks & Markram, 1997). After habituation, the firing rate of the cell fluctuates with minor variation in the strength of the excitatory drive. In other words, habituation allows the cell to become sensitive to changes in the excitatory drive (Huber & O’Reilly, 2003). Thus, if there is stronger top-down memory feedback in some locations as compared to others, the cell will fire at a higher rate in those remembered locations rather than in all locations even though the attribute is found at all locations. In brief when faced with constant excitatory drive, the cell accommodates, and becomes sensitive to change in the magnitude of the excitatory drive. In the model simulation, this dynamic adaptation is captured by supposing that cells fire 5% of the time on-average across the simulation, regardless of their excitatory inputs.”

(4) Why are grid cells given such a large role for encoding non-spatial attributes? If anything, shouldn't it be lateral EC or perirhinal cortex? Of course, they both could, but there is less reason to think this, at least for rodent mEC.  

This is a good point and the following paragraph has been added to the introduction to explain that lateral EC is likely part of the explanation. But even when including lateral EC, it still appears that most of the input to hippocampus is spatial.

“One possible answer to the apparent lack of non-spatial cells in MTL is to highlight the role of the lateral entorhinal cortex (LEC) as the source of non-spatial what information for memory encoding (Deshmukh & Knierim, 2011). LEC can be contrasted with mEC, which appears to only provide where information (Boccara et al., 2010a; Diehl et al., 2017). Although it is generally true that LEC is involved in non-spatial processing, there is evidence that LEC provides some forms of spatial information (Knierim et al., 2014). The kind of non-spatial information provided by LEC appears to be in relation to objects (Connor & Knierim, 2017; Wilson et al., 2013). However, in a typical rodent spatial navigation study there are no objects within the enclosure. Thus, although the distinction between mEC and LEC is likely part of the explanation, it is still the case that rodent entorhinal input to hippocampus appears to heavily favor spatial information.”

(5) Clarification: why do place cells and grid cells differ in terms of stability in the model? Place cells are not stable initially but grid cells come out immediately. They seem directly connected so a bit unclear why; especially if place cell feedback leads to grid cell fields. There is an explanation in the text - based on grid cells coding the on-average memories, but these should be based on place cell inputs as well. So how is it that place fields are unstable then grid fields do not move at all? I wonder if a set of images or videos (gifs) showing the differences in spatial learning would be nice and clarify this point.  

In this revision, I provide a new video focused on learning of place cell memories that include head direction. This second video is in relation to the results reported in Figure 9. The short answer is that the grid fields for the non-spatial cell are based on the average across several view-dependent memories (i.e., across several place cells that have head direction sensitivity) and the average is reliable even if the place cells are unstable. The text of this explanation now reads:

“Why was the grid immediately apparent for the non-spatial attribute cell whereas the grid took considerable prior experience for the head direction cells? The answer relates to memory consolidation and the shifting nature of the hippocampal place cells. Head direction cells only produced a reliable grid once the hippocampal place cells (aka, memory cells) assumed stable locations. During the first few sessions, the hippocampal place cells were shifting their positions owing to pattern separation and consolidation. But once the place cells stabilized, they provided reliable top-down memory feedback to the head direction cells in some places but not others, thus producing a reliable grid arrangement to the firing maps of the head direction cells. In other words, for the head direction cells, the grid only appeared once the place cells stabilized. This slow stabilization of place fields is a known property (Bostock et al., 1991; Frank et al., 2004).

In the simulation, the place cells did not stabilize until a sufficient number of place cells were created (Figure 9C). Specifically, these additional memories were located immediately outside the enclosure, around all borders (Figure 9D). These “outside the box” memories served to constrain the interior place cells, locking them in position despite ongoing consolidation. This dynamic can be seen in a movie showing a representative simulation. The movie shows the positions of the head direction sensitive place cells during initial learning, and then during additional sessions of prior experience as the movie speeds up (see link in Figure 9 capture).

Why did the non-spatial grid cell (k) produce a grid immediately, before the place cells stabilized? As discussed in relation to Figure 8, the non-spatial grid cell is the projection through the 3D volume of real-world coordinates that includes X, Y, and head direction. Each grid field of a non-spatial grid cell reflects feedback from several place cells that each have a different head direction sensitivity (see for instance the allocentric pairs of memories illustrated in Figure 8C and 8D). Thus, each grid field is the average across several memories that entail different viewpoints and this averaging across memories provides stability even if the individual memories are not yet stable. This average of unstable memories produces a blurry sort of grid pattern without any prior experience.

A final piece of the puzzle relies on the same mechanism that caused the grid pattern to align with the borders as reported in the results of Figures 6 and 7. Specifically, there are some “sticky” locations with ongoing consolidation because the connection weights are bounded. Because weights cannot go below their minimum or above their maximum, it is slightly more difficult for consolidation to push or pull connection weights over the peak value or under the minimum value of the tuning curve. Thus, the place cells tend to linger in locations that correspond to the peak or trough of a border cell. There are multiple peak and trough locations but for the parameter values in this simulation, the grid pattern seen in Figure 9C shows the set of peak/trough locations that satisfy the desired spacing between memories. Thus, the average across memories shows a reliable grid field at these locations even though the memories are unstable.”

(6) Other predictions. Clearly, the model makes many interesting (and quite specific!) predictions. But does it make some known simple predictions? 

• More place cells at rewarded (or more visited) locations. Some empirical researchers seem to think this is not as obvious as it seems (e.g., Duvellle et al., 2019; JoN; Nyberg et al., 2021, Neuron Review).  

• Grid cell field moves toward reward (Butler et al., 2019; Boccera et al., 2019).  

• Grid cells deform in trapezoid (Krupic et al., 2015) and change in environments like mazes (Derikman et al., 2014).  

Thank you for these suggestions and I have added the following paragraph to the discussion:

“In terms of the animal’s internal state, all locations in the enclosure may be viewed as equally aversive and unrewarding, which is a memorable characteristic of the enclosure. Reward, or lack thereof, is arguably one of the most important nonspatial characteristics and application of this model to reward might explain the existence of goal-related activity in place cells (Hok et al., 2007; although see Duvelle et al., 2019), reflecting the need to remember rewarding locations for goal directed behavior. Furthermore, if place cell memories for a rewarding location activate entorhinal grid cells, this may explain the finding that grid cells remap in an enclosure with a rewarded location such that firing fields are attracted to that location (Boccara et al., 2019; Butler et al., 2019). Studies that introduce reward into the enclosure are an important first step in terms of examining what happens to grid cells when the animal is placed in a more varied environment.”

Regarding the changes in shape of the environment, this was discussed in the section of the paper that reads “As seen in Figure 12, because all but one of the place cells was exterior when the simulated animal was constrained to a narrow passage, the hippocampal place cell memories were no longer arranged in a hexagonal grid. This disruption of the grid array for narrow passages might explain the finding that the grid pattern (of grid cells) is disrupted in the thin corner of a trapezoid (Krupic et al., 2015) and disrupted when a previously open enclosure is converted to a hairpin maze by insertion of additional walls within the enclosure (Derdikman et al., 2009).” This particular section of the paper now appears in the Appendix and Figure 12 is now Appendix Figure 2.

Reviewer #2 (Public Review): 

The manuscript describes a new framework for thinking about the place and grid cell system in the hippocampus and entorhinal cortex in which these cells are fundamentally involved in supporting non-spatial information coding. If this framework were shown to be correct, it could have high impact because it would suggest a completely new way of thinking about the mammalian memory system in which this system is non-spatial. Although this idea is intriguing and thought-provoking, a very significant caveat is that the paper does not provide evidence that specifically supports its framework and rules out the alternate interpretations. Thus, although the work provides interesting new ideas, it leaves the reader with more questions than answers because it does not rule out any earlier ideas. 

Basically, the strongest claim in the paper, that grid cells are inherently non-spatial, cannot be specifically evaluated versus existing frameworks on the basis of the evidence that is shown here. If, for example, the author had provided behavioral experiments showing that human memory encoding/retrieval performance shifts in relation to the predictions of the model following changes in the environment, it would have been potentially exciting because it could potentially support the author's reconceptualization of this system. But in its current form, the paper merely shows that a new type of model is capable of explaining the existing findings. There is not adequate data or results to show that the new model is a significantly better fit to the data compared to earlier models, which limits the impact of the work. In fact, there are some key data points in which the earlier models seem to better fit the data.  

Overall, I would be more convinced that the findings from the paper are impactful if the author showed specific animal memory behavioral results that were only supported by their memory model but not by a purely spatial model. Perhaps the author could run new experiments to show that there are specific patterns of human or animal behavior that are only explained by their memory model and not by earlier models. But in its current form, I cannot rule out the existing frameworks and I believe some of the claims in this regard are overstated. 

As previously detailed in Box 1 and as explained in the text in several places, the model provides an explanation of several findings that remain unexplained by other theories (see “Results Uniquely Explained by the Memory Model”). But more generally this is a good point, and the initial draft failed to fully articulate why a researcher might choose this model to guide future empirical investigations. A new section in the introduction that deals with these issues, titled ‘Why Model the Rodent Navigation Literature with a Memory Model?’ That section reads:

“Spatial navigation is inherently a memory problem – learning the spatial arrangement of a new enclosure requires memory for the conjunction of what and where. This has long been realized and in the introduction to ‘Hippocampus as a Cognitive Map’, O’Keefe and Nadel (1978) wrote “We shall argue that the hippocampus is the core of a neural memory system providing an objective spatial framework within which the items and events of an organism's experience are located and interrelated” (emphasis added). Furthermore, in the last chapter of their book, they extended cognitive map theory to human memory for non-spatial characteristics. However, in the decades since the development of cognitive map theory, the rodent spatial navigation and human memory literatures have progressed somewhat independently.

The ideas proposed in this model are an attempt to reunify these literatures by returning to the original claim that spatial navigation is inherently a memory problem. The goal of the current study is to explain the rodent spatial navigation literature using a memory model that has the potential to also explain the human memory literature. In contrast, most grid cell models (Bellmund et al., 2016; Bush et al., 2015; Castro & Aguiar, 2014; Hasselmo, 2009; Mhatre et al., 2012; Solstad et al., 2006; Sorscher et al., 2023; Stepanyuk, 2015; Widloski & Fiete, 2014) are domain specific models of spatial navigation and as such, they do not lend themselves to explanations of human memory. Thus, the reason to prefer this model is parsimony. Rather than needing to develop a theory of memory that is separate from a theory of spatial navigation, it might be possible to address both literatures with a unified account.

This study does not attempt to falsify other theories of grid cells. Instead, this model reaches a radically different interpretation regarding the function of grid cells; an interpretation that emerges from viewing spatial navigation as a memory problem. All other grid cell models assume that an entorhinal grid cell displaying a spatially arranged grid of firing fields serves the function of spatial coding (i.e., spatial grid cells exist to support a spatial metric). In contrast, the proposed memory model of grid cells assumes that the hexagonal tiling reflects the need to keep memories separate from each other to minimize confusion and confabulation – the grid pattern is the byproduct of pattern separation between memories rather than the basis of a spatial code. 

It is now understood that grid-like firing fields can occur for non-spatial twodimensional spaces. For instance, human entorhinal cortex exhibits grid-like responses to video morph trajectories in a two-dimensional bird neck-length versus bird leg-length space (Constantinescu et al., 2016). As a general theory of learning and memory, the proposed memory model of grid cells is easily extended to explain these results (e.g., relabeling the border cell inputs in the model as neck-length and leg-length inputs). However, there are other grid cell models that can explain both spatial grid cells as well as non-spatial grid-like responses (Mok & Love, 2019; Rodríguez-Domínguez & Caplan, 2019; Stachenfeld et al., 2017; Wei et al., 2015). Similar to this memory model of grid cells, these models are also positioned to explain both the rodent spatial navigation and human memory literatures. Nevertheless, there is a key difference between this model and other grid cell models that generalize to non-spatial representations. Specifically, these other models assume that grid cells exhibiting spatial receptive fields serve the function of identifying positions in the environment (i.e., their function is spatial). As such, these models do not explain why most of the input to rodent hippocampus appears to be spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). This memory model of grid cells provides an answer to the apparent paucity of nonspatial cell types in rodent MTL by proposing that grid cells with spatial receptive fields have been misclassified as spatial (they are what cells rather than where cells) and that place cells are fundamentally memory cells that conjoin what and where.”

- The paper does not fully take into account all the findings regarding grid cells, some of which very clearly show spatial processing in this system. For example, findings on grid-bydirection cells (e.g., Sargolini et al. 2006) would seem to suggest that the entorhinal grid system is very specifically spatial and related to path integration. Why would grid-bydirection cells be present and intertwined with grid cells in the author's memory-related reconceptualization? It seems to me that the existence of grid-by-direction cells is strong evidence that at least part of this network is specifically spatial.

Head by direction grid cells were a key part of the reported results. These grid cells naturally arise in the model as the animal forms memories (aka, hippocampal place cells) that conjoin location (as defined by border cells), head direction at the time of memory formation, and one or more non-spatial properties found at that location. In this revision, I have attempted to better explain how including head direction in hippocampal memories naturally gives rise to these cell types. The introduction to the head direction module simulations now reads:

“According to this memory model of spatial navigation, place cells are the conjunction of location, as defined by border cells, and one or more properties that are remembered to exist at that location. Such memories could, for instance, allow an animal to remember the location of a food cache (Payne et al., 2021). The next set of simulations investigates behavior of the model when one of the to-be-remembered properties is head direction at the time when the memory was formed (e.g., the direction of a pathway leading to a food cache). Indicating that head direction is an important part of place cell representations, early work on place cells in mazes found strong sensitivity to head direction, such that the place field is found in one direction of travel but not the other (McNaughton et al., 1983; Muller et al., 1994). Place cells can exhibit a less extreme version of head direction sensitivity in open field recordings (Rubin et al., 2014), but the nature of the sensitivity is more complicated, depending on location of the animal relative to the place field center (Jercog et al., 2019).

It is possible that some place cell memories do not receive head direction input, as was the case for the simulations reported in Figures 6/7 – in those simulations, place cells were entirely insensitive to head direction, owing to a lack of input from head direction cells. However, removal of head direction input to hippocampus affects place cell responses (Calton et al., 2003) and grid cell responses (Winter et al., 2015), suggesting that head direction is a key component of the circuit. Furthermore, if place cells represent episodic memories, it seems natural that they should include head direction (i.e., viewpoint at the time of memory formation).

In the simulations reported next, head direction is simply another property that is conjoined in a hippocampal place cell memory. In this case, a head direction cell should become a head direction conjunctive grid cell (i.e., a grid cell, but only when the animal is heading in a particular direction), owing to memory feedback from the hexagonal array of hippocampal place cell memories. When including head direction, the real-world dimensions of variation are across three dimensions (X, Y, and head direction) rather than two, and consolidation will cause the place cells to arrange in a three-dimensional volume. The simulation reported below demonstrates that this situation provides a “grid module”.”

- I am also concerned that the paper does not do enough to address findings regarding how the elliptical shape of grid fields shifts when boundaries of an environment compress in one direction or change shape/angles (Lever et al., & Krupic et al). Those studies show compression in grid fields based on boundary position, and I don't see how the authors' model would explain these findings.  

This finding was covered in the original submission: “For instance, perhaps one egocentric/allocentric pair of mEC grid modules is based on head direction (viewpoint) in remembered positions relative to the enclosure borders whereas a different egocentric/allocentric pair is based on head direction in remembered positions relative to landmarks exterior to the enclosure. This might explain why a deformation of the enclosure (moving in one of the walls to form a rectangle rather than a square) caused some of the grid modules but not others to undergo a deformation of the grid pattern in response to the deformation of the enclosure wall (see also Barry et al., 2007). More specifically, if there is one set of non-orthogonal dimensions for enclosure borders and the movement of one wall is too modest as to cause avoid global remapping, this would deform the grid modules based the enclosure border cells. At the same time, if other grid modules are based on exterior properties (e.g., perhaps border cells in relation to the experimental room rather than the enclosure), then those grid modules would be unperturbed by moving the enclosure wall.”

I apologize for being unclear in describing how the model might explain this result. The paragraph has been rewritten and now reads:

“Consider the possibility that one mEC grid modules is based on head direction (viewpoint) in remembered positions relative to the enclosure borders (e.g., learning the properties of the enclosure, such as the metal surface) while a different grid module is based on head direction in remembered positions relative to landmarks exterior to the enclosure (e.g., learning the properties of the experimental room, such as the sound of electronics that the animal is subject to at all locations). This might explain why a deformation of the enclosure (moving one of the walls to form a rectangle rather than a square) caused some of the grid modules but not others to undergo a deformation of the grid pattern in response to the deformation of the enclosure wall (see also Barry et al., 2007). More specifically, suppose that the movement of one wall is modest and after moving the wall, the animal views the enclosure as being the same enclosure, albeit slightly modified (e.g., when a home is partially renovated, it is still considered the same home). In this case, the set of non-orthogonal dimensions associated with enclosure borders would still be associated with the now-changed borders and any memories in reference to this border-determined space would adjust their positions accordingly in real-world coordinates (i.e., the place cells would subtly shift their positions owing to this deformation of the borders, producing a corresponding deformation of the grid). At the same time, there may be other sets of memories that are in relation to dimensions exterior to the enclosure. Because these exterior properties are unchanged, any place cells and grid cells associated with the exterior-oriented memories would be unchanged by moving the enclosure wall.”

- Are findings regarding speed modulation of grid cells problematic for the paper's memory results? 

- A further issue is that the paper does not seem to adequately address developmental findings related to the timecourses of the emergence of different cell types. In their simulation, researchers demonstrate the immediate emergence of grid fields in a novel environment, while noting that the stabilization of place cell positions takes time. However, these simulation findings contradict previous empirical developmental studies (Langston et al., 2010). Those studies showed that head direction cells show the earliest development of spatial response, followed by the appearance of place cells at a similar developmental stage. In contrast, grid cells emerge later in this developmental sequence. The gradual improvement in spatial stability in firing patterns likely plays a crucial role in the developmental trajectory of grid cells. Contrary to the model simulation, grid cells emerge later than place cells and head direction cells, yet they also hold significance in spatial mapping. 

- The model simulations suggest that certain grid patterns are acquired more gradually than others. For instance, egocentric grid cells require the stabilization of place cell memories amidst ongoing consolidation, while allocentric grid cells tend to reflect average place field positions. However, these findings seemingly conflict with empirical studies, particularly those on the conjunctive representation of distance and direction in the earliest grid cells. Previous studies show no significant differences were found in grid cells and grid cells with directional correlates across these age groups, relative to adults (Wills et al., 2012). This indicates that the combined representation of distance and direction in single mEC cells is present from the earliest ages at which grid cells emerge. 

These are good points and they have been addressed in a new section of the introduction titled ‘The Scope of the Proposed Model’. That section reads:

“The reported simulations explain why most mEC cell types in the rodent literature appear to be spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). Assuming that rodents can form non-spatial memories, rodent hippocampus must receive non-spatial input from entorhinal cortex. These simulations suggest that characterization of the rodent mEC cortex as primarily spatial might be incorrect if most grid cells (except perhaps head direction conjunctive grid cells) have been mischaracterized as spatial. Other literatures with other species find non-spatial representations in MTL (Gulli et al., 2020; Quiroga et al., 2005; Wixted et al., 2014) and non-spatial hippocampal memory encoding has been found in rodents (Liu et al., 2012; McEchron & Disterhoft, 1999). The proposed memory model is compatible with these results – the ideas contained in this model could be applied to nonspatial memory representations. However, surveys of cell types in rodent entorhinal cortex seem to indicate that most cells are spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). How can the rodent hippocampus encode nonspatial memories if most of its input is spatial? The goal of the reported simulations is to explain the apparent paucity of non-spatial cells in rodent entorhinal cortex by proposing that grid cells have been misclassified as spatial (see also Luo et al., 2024).

Given the simplicity of the proposed model, there are important findings that the model cannot address -- it is not that the model makes the wrong predictions but rather that it makes no predictions. The role of running speed (Kraus et al., 2015) is one such variable for which the model makes no predictions. Similarly, because the model is a rate-coded model rather than a model of oscillating spiking neurons, it makes no predictions regarding theta oscillations (Buzsáki & Moser, 2013). The model is an account of learning and memory for an adult animal, and it makes no predictions regarding the developmental (Langston et al., 2010; Muessig et al., 2015; Wills et al., 2012) or evolutionary (Rodrıguez et al., 2002) time course of different cell types. This model contains several purely spatial representations such as border cells, head direction cells, and head direction conjunctive grid cells and it may be that these purely spatial cell types emerged first, followed by the evolution and/or development of non-spatial cell types. However, this does not invalidate the model. Instead, this is a model for an adult animal that has both episodic memory capabilities and spatial navigation capabilities, irrespective of the order in which these capabilities emerged.

This model has the potential to explain context effects in memory (Godden & Baddeley, 1975; Gulli et al., 2020; Howard et al., 2005). According to this model, different grid cells represent different non-spatial characteristics and place cells represent the combination of these “context” factors and location. In the simulation, just one grid cell is simulated but the same results would emerge when simulating hundreds of different non-spatial inputs provided that all of the simulated non-spatial inputs exist throughout the recording session. However, there is evidence that hippocampus can explicitly represent the passage of time (Eichenbaum, 2014), and time is assuredly an important factor in defining episodic memory (Bright et al., 2020). Thus, although the current model addresses unique combinations of what and where, it is left to future work to incorporate representations of when in the memory model.”

Reviewer #3 (Public Review): 

A crucial assumption of the model is that the content of experience must be constant in space. It's difficult to imagine a real-world example that satisfies this assumption. Odors and sounds are used as examples. While they are often more spatially diffuse than an objects on the ground, odors and sounds have sources that are readily detectable. Animals can easily navigate to a food source or to a vocalizing conspecific. This assumption is especially problematic because it predicts that all grid cells should become silent when their preferred non-spatial attribute (e.g. a specific odor) is missing. I'm not aware of any experimental data showing that grid cells become silent. On the contrary, grid cells are known to remain active across all contexts that have been tested, including across sleep/wake states. Unlike place cells, grid cells do not seem to turn off. Since grid cells are active in all contexts, their preferred attribute must also be present in all contexts, and therefore they would not convey any information about the specific content of an experience.  

These are good points and in this revision I have attempted to explain that there is a great deal of contextual similarity across all recording sessions. One paragraph in the discussion now reads

“In a typical rodent spatial navigation study, the non-spatial attributes are wellcontrolled, existing at all locations regardless of the enclosure used during testing (hence, a grid cell in one enclosure will be a grid cell in a different enclosure). Because labs adopt standard procedures, the surfaces, odors (e.g., from cleaning), external lighting, time of day, human handler, electronic apparatus, hunger/thirst state, etc. might be the same for all recording sessions. Additionally, the animal is not allowed to interact with other animals during recording and this isolation may be an unusual and highly salient property of all recording sessions. Notably, the animal is always attached to wires during recording. The internal state of the animal (fear, aloneness, the noise of electronics, etc.) is likely similar across all recording situations and attributes of this internal state are likely represented in the hippocampus and entorhinal input to hippocampus. According to this model, hippocampal place cells are “marking” all locations in the enclosure as places where these things tend to happen.”

The proposed novelty of this theory is that other models all assume that grid cells encode space. This isn't quite true of models based on continuous attractor networks, the discussion of which is notably absent. More specifically, these models focus on the importance of intrinsic dynamics within the entorhinal cortex in generating the grid pattern. While this firing pattern is aligned to space during navigation and therefore can be used as a representation of that space, the neural dynamics are preserved even during sleep. Similarly, it is because the grid pattern does not strictly encode physical space that gridlike signals are also observed in relation to other two-dimensional continuous variables. 

These models were briefly discussed in the general discussion section and in this revision they are further discussed in the introduction in a new section, titled ‘Why Model the Rodent Navigation Literature with a Memory Model?’ That section reads:

“Spatial navigation is inherently a memory problem – learning the spatial arrangement of a new enclosure requires memory for the conjunction of what and where. This has long been realized and in the introduction to ‘Hippocampus as a Cognitive Map’, O’Keefe and Nadel (1978) wrote “We shall argue that the hippocampus is the core of a neural memory system providing an objective spatial framework within which the items and events of an organism's experience are located and interrelated” (emphasis added). Furthermore, in the last chapter of their book, they extended cognitive map theory to human memory for non-spatial characteristics. However, in the decades since the development of cognitive map theory, the rodent spatial navigation and human memory literatures have progressed somewhat independently.

The ideas proposed in this model are an attempt to reunify these literatures by returning to the original claim that spatial navigation is inherently a memory problem. The goal of the current study is to explain the rodent spatial navigation literature using a memory model that has the potential to also explain the human memory literature. In contrast, most grid cell models (Bellmund et al., 2016; Bush et al., 2015; Castro & Aguiar, 2014; Hasselmo, 2009; Mhatre et al., 2012; Solstad et al., 2006; Sorscher et al., 2023; Stepanyuk, 2015; Widloski & Fiete, 2014) are domain specific models of spatial navigation and as such, they do not lend themselves to explanations of human memory. Thus, the reason to prefer this model is parsimony. Rather than needing to develop a theory of memory that is separate from a theory of spatial navigation, it might be possible to address both literatures with a unified account.

This study does not attempt to falsify other theories of grid cells. Instead, this model reaches a radically different interpretation regarding the function of grid cells; an interpretation that emerges from viewing spatial navigation as a memory problem. All other grid cell models assume that an entorhinal grid cell displaying a spatially arranged grid of firing fields serves the function of spatial coding (i.e., spatial grid cells exist to support a spatial metric). In contrast, the proposed memory model of grid cells assumes that the hexagonal tiling reflects the need to keep memories separate from each other to minimize confusion and confabulation – the grid pattern is the byproduct of pattern separation between memories rather than the basis of a spatial code. 

It is now understood that grid-like firing fields can occur for non-spatial two dimensional spaces. For instance, human entorhinal cortex exhibits grid-like responses to video morph trajectories in a two-dimensional bird neck-length versus bird leg-length space (Constantinescu et al., 2016). As a general theory of learning and memory, the proposed memory model of grid cells is easily extended to explain these results (e.g., relabeling the border cell inputs in the model as neck-length and leg-length inputs). However, there are other grid cell models that can explain both spatial grid cells as well as non-spatial grid-like responses (Mok & Love, 2019; Rodríguez-Domínguez & Caplan, 2019; Stachenfeld et al., 2017; Wei et al., 2015). Similar to this memory model of grid cells, these models are also positioned to explain both the rodent spatial navigation and human memory literatures. Nevertheless, there is a key difference between this model and other grid cell models that generalize to non-spatial representations. Specifically, these other models assume that grid cells exhibiting spatial receptive fields serve the function of identifying positions in the environment (i.e., their function is spatial). As such, these models do not explain why most of the input to rodent hippocampus appears to be spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). This memory model of grid cells provides an answer to the apparent paucity of nonspatial cell types in rodent MTL by proposing that grid cells with spatial receptive fields have been misclassified as spatial (they are what cells rather than where cells) and that place cells are fundamentally memory cells that conjoin what and where.”

The use of border cells or boundary vector cells as the main (or only) source of spatial information in the hippocampus is not well supported by experimental data. Border cells in the entorhinal cortex are not active in the center of an environment. Boundary-vector cells can fire farther away from the walls but are not found in the entorhinal cortex. They are located in the subiculum, a major output of the hippocampus. While the entorhinalhippocampal circuit is a loop, the route from boundary-vector cells to place cells is much less clear than from grid cells. Moreover, both border cells and boundary-vector cells (which are conflated in this paper) comprise a small population of neurons compared to grid cells.

AUTHOR RESPONSE: The model can be built without assuming between-border cells (early simulations with the model did not make this assumption). Regarding this issue, the text reads “Unlike the BVC model, the boundary cell representation is sparsely populated using a basis set of three cells for each of the three dimensions (i.e., 9 cells in total), such that for each of the three non-orthogonal orientations, one cell captures one border, another the opposite border, and the third cell captures positions between the opposing borders (Solstad et al., 2008). However, this is not a core assumption, and it is possible to configure the model with border cell configurations that contain two opponent border cells per dimension, without needing to assume that any cells prefer positions between the borders (with the current parameters, the model predicts there will be two border cells for each between-border cell). Similarly, it is possible to configure the model with more than 3 cells for each dimension (i.e., multiple cells representing positions between the borders).” The Solstad paper found a few cells that responded in positions between borders, but perhaps not as many as 1 out of 3 cells, such as this particular model simulation predicts. If the paucity of between-border cells is a crucial data point, the model can be reconfigured with opponent-border cells without any between border cells. The reason that 3 border cells were used rather than 2 opponent border cells was for simplicity. Because 3 head direction cells were used to capture the face-centered cubic packing of memories, the simulation also used 3 border cells per dimensions to allow a common linear sum metric when conjoining dimensions to form memories. If the border dimensions used 2 cells while head direction used 3 cells, a dimensional weighting scheme would be needed to allow this mixing of “apples and oranges” in terms of distances in the 3D space that includes head direction.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

Specific questions/clarifications:  

(1) Assumption of population-based vs single unit link to biological cells: At the start, the author assumes that each unit here can be associated with a population: "the simulated activation values can be thought of as proportional to the average firing rate of an ensemble of neurons with similar inputs and outputs (O'Reilly & Munakata, 2000)." But is a 'grid cell' found here a single cell or an average of many cells? Does this mean the model assumes many cells that have different fields that are averaged, which become a grid-like unit in the model? But in biology, these are single cells? Or does it mean a grid response is an average of the place cell inputs? 

I apologize for being unclear about this. The grid cells in the model are equivalent to real single cells except that the simulation uses a ratecoded cell rather than a spiking cell. The averaging that was mentioned in the paper is across identically behaving spiking cells rather than across cells with different grid field arrangements. To better explain this, I have added the following text:

“For instance, consider a set of several thousand spiking grid cells that are identical in terms of their firing fields. At any moment, some of these identically-behaving cells will produce an action potential while others do not (i.e., the cells are not perfectly synchronized), but a snapshot of their behavior can be extracted by calculating average firing rate across the ensemble. The simulated cells in the model represent this average firing rate of identically-behaving ensembles of spiking neurons.” 

This is a mathematical short-cut to avoid simulating many spiking neurons. Because this model was compared to real spike rate maps, this real-valued average firing rate is down-sampled to produce spikes by finding the locations that produced the top 5% of real-valued activation values across the simulation.

(2) It is not clear to me why they are circular border cells/basis sets.  

In the initial submission, there was a brief paragraph describing this assumption. In this revision, that paragraph has been expanded and modified for greater clarity. It now reads:

“Because head direction is necessarily a circular dimension, it was assumed that all dimensions are circular (a circular dimension is approximately linear for nearby locations). This assumption of circular dimensions was made to keep the model relatively simple, making it easier to combine dimensions and allowing application of the same processes for all dimensions. For instance, the model requires a weight normalization process to ensure that the pattern of weights for each dimension corresponds to a possible input value along that dimension. However, the normalization for a linear dimension is necessarily different than for a circular dimension. Because the neural tuning functions were assumed to be sine waves, normalization requires that the sum of squared weights add up to a constant value. For a linear dimension, this sum of squares rule only applies to the subset of cells that are relevant to a particular value along the dimension whereas for a circular dimension, this sum of squares rule is over the entire set of cells that represent the dimension (i.e., weight normalization is easier to implement with circular dimensions). Although all dimensions were assumed to be circular for reasons of mathematical convenience and parsimony, circular dimensions may relate to the finding that human observers have difficultly re-orienting themselves in a room depending on the degree of rotational symmetry of the room (Kelly et al., 2008). In addition, this simplifying assumption allows the model to capture the finding that the population of grid cells lies on a torus (Gardner et al., 2022), although I note that the model was developed before this result was known.”

(3) Why is it 3 components? I realise that the number doesn't matter too much, but I believe more is better, so is it just for simplicity? 

In this revision, additional text has been added to explain this assumption: “To keep the model simple, the same number of cells was assumed for all dimensions and all dimensions were assumed to be circular (head direction is necessarily circular and because one dimension needed to be circular, all dimensions were assumed to be circular). Three cells per dimensions was chosen because this provides a sparse population code of each dimension, with few border cells responding between borders, with few border cells responding between borders, while allowing three separate phases of grid cells within a grid cell module (in the model, a grid cell module arises from combination of a third dimension, such as head direction, with the real-world X/Y dimensions defined by border cells).”

As a reminder, the text explaining the sparse coding of border cells reads: “However, this is not a core assumption, and it is possible to configure the model with border cell configurations that contain two opponent border cells per dimension, without needing to assume that any cells prefer positions between the borders (with the current parameters, the model predicts there will be two border cells for each between-border cell). Similarly, it is possible to configure the model with more than 3 cells for each dimension (i.e., multiple cells representing positions between the borders).”

The model can work with just two opponent cells or with more than three cells per basis set. In different simulations, I have explored these possibilities. Three was chosen because it is a convenient way to highlight the face-centered cubic packing of memories that tends to occur (FCP produces 3 alternating layers of hexagonally arranged firing fields). Thus, each of the three head direction cells captures a different layer of the FCP arrangement. A more realistic simulation might combine 6 different head direction cells tiling the head direction dimension with opponent border cells (just 2 cells for each border dimensions). Such a combination would produce responses at borders, but no responses between borders and, at the same time, the head direction cells would still reveal the FCP arrangement. However, it is not easy to find the right parameters for such a mix-and-match simulation in which different dimensions have different numbers of tuning functions (e.g., some dimensions having 2 cells while others have 3 or 6 and some dimensions being linear while others are circular). When all of the dimensions are of the same type, the simple sum that arises from multiplying the input by the weight values gives rise to Euclidean distance (see Figure 3B). With a mix-and-match model of different dimension-types, it should be possible to adjust the sum to nevertheless produce a monotonic function with Euclidean distance although I leave this to future work. To keep things simple, I assumed that all dimensions are of the same type (circular, with 3 cells per dimension).  

(4) Confusion due to the border cells/box was unclear to me. "If the period of the circular border cells was the same as the width of the box, then a memory pushed outside the box on one side would appear on the opposite side of the box, in which case the partial grid field on one side should match up with its remainder on the other side. This would entail complete confusion between opposite sides of the box, and the representation of the box would be a torus (donut-shaped) rather than a flat two-dimensional surface. To reduce confusion ..." Is this confusion of the model? Of the animal?  

This would be confusion of the animal (e.g., a memory field overlapping with one border would also appear at the opposite border in the corresponding location). At one point in model development, I made the assumption that one side of the box wraps to the other side, and I asked Trygve Solstad to run some analyses of real data to see if cells actually wrap around in this manner. He did not find any evidence of this, and so I decided to include outsidethe-box representational area which, as it turned out, allowed the model to capture other behaviors as detailed in the paper.

This section of the paper now reads:

“The cosine tuning curves of the simulated border cells represent distance from the border on both sides of the border (i.e., firing rate increases as the animal approaches the border from either the inside or the outside of the enclosure). Experimental procedures do not allow the animal to experience locations immediately outside the enclosure, but these locations remain an important part of the hypothetic representation, particularly when considering the modification of memories through consolidation (i.e., a memory created inside the enclosure might be moved to a location outside the enclosure). This symmetry about the border cell’s preferred location is needed to maintain an unbiased representation, with a constant sum of squares for the border cell inputs (see methods section). Rather than using linear dimensions, all dimensions were assumed to be circular to keep the model relatively simple. This assumption was made because head direction is necessarily a circular dimension and by having all dimensions be circular, it is easy to combine dimensions in a consistent manner to produce multidimensional hippocampal place cell memories. Thus, the border cells define a torus (or more accurately a three-torus) of possible locations. This provides a hypothetical space of locations that could be represented.

In light of the assumption to represent border cells with a circular dimension, when a memory is pushed outside the East wall of the enclosure, it would necessarily be moved to the West wall of the enclosure if the period of the circular dimension was equal to the width of the enclosure. If this were true, then the partial grid field on one side of the enclosure would match up with its remainder on the other side. Such a situation would cause the animal to become completely confused regarding opposite sides of the enclosure (a location on the West wall would be indistinguishable from the corresponding location on the East wall). To reduce confusion between opposite sides of the enclosure, the width of the enclosure in which the animal navigated (Figure 5) was assumed to be half as wide as the full period of the border cells. In other words, although the space of possible representations was a three-torus, it was assumed that the real-world twodimensional enclosure encompassed a section of the torus (e.g., a square piece of tape stuck onto the surface of a donut). The torus is better thought of as “playing field” in which different sizes and shapes of enclosure can be represented (i.e., different sizes and shapes of tape placed on the donut). Furthermore, this assumption provides representational space that is outside the box without such locations wrapping around to the opposite side of the box.”

(5) Figure 3 - This result seems to be related to whether you use Euclidean or city-block distance. If you use Euclidean distances in two dimensions wouldn't this work out fine?  

Euclidean distance was the metric used in the analysis of the two-dimensional simulation, but this did not work out. To make this clear, I have changed the label on the x-axes to read “Euclidean distance” for both the two- and three-dimensional simulations. The two-dimensional simulation produced city block behavior rather than Euclidean behavior because memory retrieval is the sum of the two dimensions, as is standard in neural networks, rather than the Euclidian distance formula, which would require that memory retrieval be the square root of the sum of squares of the two dimensions. One way to address this problem with the two-dimensional simulation would be to use a specific Euclidean-mimicking activation function rather than a simple sum of dimensions. The very first model I developed used such an activation function as applied to opponent border cells with just two dimensions (so 4 cells in total – left/right and top/down). This produced Euclidean behavior, but the activation function was implausible and did not generalize to simulations that also included head direction. In contrast, with three non-orthogonal dimensions, the simple sum of dimensions is approximately Euclidean.

(6) Final sentence of the Discussion: "However, unlike the present model, these models still assume that entorhinal grid cells represent space rather than a non-spatial attribute." I am not sure if the authors of the cited papers will agree with this. They consider the spatial cases, but most argue they can treat non-spatial features as well. What the author might mean is that they assume non-spatial features are in some metric space that, in a way, is spatial. However, I am not sure if the author would argue that non-spatial features cannot be encoded metrically (e.g., Euclidean distance based on the similarity of odours). 

In this section, when referring to “entorhinal grid cells” I was specifically referring to traditional grid cells in a rodent spatial navigation experiment. I did not mean to imply that these other theories cannot explain nonspatial grid fields, such as in the two-dimensional bird space grid cells found with humans. The way in which the proposed memory model and these other models differ is in terms of what they assume regarding the function of grid cells that exhibit spatial grid fields. In this revision, I have changed this text to read:

“These models can capture some of the grid cell results presented in the current simulations, including extension to non-spatial grid-like responses (e.g., grid field that cover a two-dimensional neck/leg length bird space). Furthermore, these models may be able to explain memory phenomena similar to the model proposed in this study. However, unlike the proposed model, these models assume that the function of entorhinal grid cells that exhibit spatial X/Y grid fields during navigation is to represent space. In contrast, the memory model proposed in this study assume that the function of spatial X/Y grid cells is to represent a non-spatial attribute; the only reason they exhibit a spatial X/Y grid is because memories of that non-spatial attribute are arranged in a hexagonal grid owing to the uncluttered/unvarying nature of the enclosure. Thus, these model do not explain why most of the input to rodent hippocampus appears to be spatial (Boccara et al., 2010b; Diehl et al., 2017; Grieves & Jeffery, 2017) whereas the proposed model can explain this situation as reflecting the miss-classification of grid cells with a spatial arrangement as providing spatial input to hippocampus.”

(7) It would be interesting to see videos/gifs of the model learning, and an idea of how many steps of trials it takes (is it capturing real-time rodent cell firing whilst foraging, or is it more abstracted, taking more trials). 

The short answer is “yes”, the model is capturing real-time rodent cell firing while foraging. This is particularly true when simulating place cell memories in the absence of head direction information, as was shown in a video provided in the initial submission in relation to Figure 4. In this revision, I have provided a second video of learning when simulating place cell memories that include head direction. This second video is in relation to the results reported in Figure 9. This shows that even when learning a three-dimensional real-world space (X, Y, and head direction), the model rapidly produces an on-average hexagonal arrangement of place cells memories owing to the slight tendency of the place cell memories to linger in some locations as compared to others during consolidation. More specifically, they are more likely to linger in the locations that are the intersections of the peaks and/or troughs of the border cells and it is this tendency that supports the immediate appearance of grid cells. However, because the place cell memories are still shifting, head direction conjunctive grid cells are slower to emerge (the head direction conjunctive grid cells require stabilization of the place cells). The video then speeds up the learning process to so how place cells eventually stabilize after sufficient learning of the borders of the enclosure from different head/view directions.

(8) One question is whether all the results have to be presented in the main text. It was difficult to see which key predictions fit the data and do so better than a spatial/navigation account. 

Thank you for this suggestion. To make the paper more readable and easier for different readers with different interests to choose different aspects of the results to read, the second half of the results have been put in an appendix. More specifically, the second half of the results concerned place cells rather than grid cells. Thus, in this revision, the main text concerns grid cell results and the appendix concerns place cell results.

Reviewer #3 (Recommendations For The Authors):  

The title could usefully be shortened to focus on the main argument that observed firing patterns could be consistent with mapping memories instead of space. It's a stretch to argue that memory is the primary role when no such data is presented (i.e., there is no comparison of competing models). 

This is a good point (I do not present evidence that conclusively indicates the function of MTL). This original title was chosen to make clear how this account is a radical departure from other accounts of grid cells. The revised title highlights that: 1) a memory model can also explain rodent single cell recording data during navigation; and 2) grid cell may not be non-spatial. The revised title is: “A Memory Model of Rodent Spatial Navigation: Place Cells are Memories Arranged in a Grid and Grid Cells are Non-spatial”

When arguing that the main role of the hippocampus is memory, I strongly suggest engaging with the work of people like Howard Eichenbaum who spent the better part of their career arguing the same (e.g. DOI:10.1152/jn.00005.2017.)  

Thank you for pointing out this important oversight. Early in introduction, I now write: “The proposal that hippocampus represents the multimodal conjunctions that define an episode is not new (Marr et al., 1991; Sutherland & Rudy, 1989) and neither is the proposal that hippocampal memory supports spatial/navigation ability (Eichenbaum, 2017). This view of the hippocampus is consistent with “feature in place” results (O’Keefe & Krupic, 2021) in which hippocampal cells respond to the conjunction of a non-spatial attribute affixed to a specific location, rather than responding more generically to any instance of a non-spatial attribute. In other words, the what/where conjunction is unique. Furthermore, the uniqueness of the what/where conjunction may be the fundamental building block of spatial memory and navigation. In reviewing the hippocampal literature, Howard Eichenbaum (2017) concludes that ‘the hippocampal system is not dedicated to spatial cognition and navigation, but organizes experiences in memory, for which spatial mapping and navigation are both a metaphor for and a prominent application of relational memory organization.’”

With a focus on episodic memory, there should be a mention of the temporal component of memory. While it may rightfully be beyond the scope of this model, it's confusing to omit time completely from the discussion. 

This issue and several others are now addressed in a new section in the introduction titled ‘The Scope of the Proposed Model’. That section reads:

“The reported simulations explain why most mEC cell types in the rodent literature appear to be spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). Assuming that rodents can form non-spatial memories, rodent hippocampus must receive non-spatial input from entorhinal cortex. These simulations suggest that characterization of the rodent mEC cortex as primarily spatial might be incorrect if most grid cells (except perhaps head direction conjunctive grid cells) have been mischaracterized as spatial. Other literatures with other species find non-spatial representations in MTL (Gulli et al., 2020; Quiroga et al., 2005; Wixted et al., 2014) and non-spatial hippocampal memory encoding has been found in rodents (Liu et al., 2012; McEchron & Disterhoft, 1999). The proposed memory model is compatible with these results – the ideas contained in this model could be applied to nonspatial memory representations. However, surveys of cell types in rodent entorhinal cortex seem to indicate that most cells are spatial (Boccara et al., 2010; Diehl et al., 2017; Grieves & Jeffery, 2017). How can the rodent hippocampus encode nonspatial memories if most of its input is spatial? The goal of the reported simulations is to explain the apparent paucity of non-spatial cells in rodent entorhinal cortex by proposing that grid cells have been misclassified as spatial (see also Luo et al., 2024).

Given the simplicity of the proposed model, there are important findings that the model cannot address -- it is not that the model makes the wrong predictions but rather that it makes no predictions. The role of running speed (Kraus et al., 2015) is one such variable for which the model makes no predictions. Similarly, because the model is a rate-coded model rather than a model of oscillating spiking neurons, it makes no predictions regarding theta oscillations (Buzsáki & Moser, 2013). The model is an account of learning and memory for an adult animal, and it makes no predictions regarding the developmental (Langston et al., 2010; Muessig et al., 2015; Wills et al., 2012) or evolutionary (Rodrıguez et al., 2002) time course of different cell types. This model contains several purely spatial representations such as border cells, head direction cells, and head direction conjunctive grid cells and it may be that these purely spatial cell types emerged first, followed by the evolution and/or development of non-spatial cell types. However, this does not invalidate the model. Instead, this is a model for an adult animal that has both episodic memory capabilities and spatial navigation capabilities, irrespective of the order in which these capabilities emerged.

This model has the potential to explain context effects in memory (Godden & Baddeley, 1975; Gulli et al., 2020; Howard et al., 2005). According to this model, different grid cells represent different non-spatial characteristics and place cells represent the combination of these “context” factors and location. In the simulation, just one grid cell is simulated but the same results would emerge when simulating hundreds of different non-spatial inputs provided that all of the simulated non-spatial inputs exist throughout the recording session. However, there is evidence that hippocampus can explicitly represent the passage of time (Eichenbaum, 2014), and time is assuredly an important factor in defining episodic memory (Bright et al., 2020). Thus, although the current model addresses unique combinations of what and where, it is left to future work to incorporate representations of when in the memory model.”

I recommend explaining the motivation of the theory in more detail in the introduction. It reads as "what if it's like this?" It would be helpful to instead highlight the limitations of current theories and argue why this theory is either a better fit for the data or is logically simpler. 

This issue and several others are now addressed in the new section in the introduction titled ‘Why Model the Rodent Navigation Literature with a Memory Model?’, which I quoted above in response to the public reviews.

It's worth considering shortening the results section to include only those that most convincingly support the main claim. The manuscript is quite long and appears to lack focus at times. 

Thank you for this suggestion. To make the paper more readable and easier for different readers with different interests to choose different aspects of the results to read, the second half of the results have been put in an appendix. More specifically, the second half of the results concerned place cells rather than grid cells. Thus, in this revision, the main text concerns grid cell results and the appendix concerns place cell results.

The discussion of path dependence on the formation of the grid pattern is important but only briefly discussed. It may be useful to add simulations testing whether different paths (not random walks) produce distorted grid patterns. 

The short answer is that the path doesn’t affect things in general. The consolidation rule ensures equally spaced memories even if, for instance, one side of the enclosure is explored much more than the other side. As just one example, I have run simulations with a radial arm maze and even though the animal is constrained to only run on the maze arms. The memories still arrange hexagonally as memories become pushed outside the arms. Rather than adding additional simulations to study, I now briefly describe this in the model methods:

“Of note, the ability of the model to produce grid cell responses does not depend on this decision to simulate an animal taking a random walk – the same results emerge if the animal is more systematic in its path. All that matters for producing grid cell responses is that the animal visits all locations and that the animal takes on different head directions for the same location in the case of simulations that also include head direction as an input to hippocampal place cells.”

I struggle to understand in Figure 3 why retrieval strength ought to scale monotonically with Euclidean distance, and why that justifies a more complex model (three non-orthogonal dimensions). 

The introduction to this section now reads: “Animals can plan novel straight line paths to reach a known position and evidence suggests they do so by learning Euclidean representations of space (Cheng & Gallistel, 2014; Normand & Boesch, 2009; Wilkie, 1989). Thus, it was assumed that hippocampal place cells represent positions in Euclidean space (as opposed to non-Euclidean space, such a occurs with a city-block metric).”

p.17 "although the representational space is a torus (or more specifically a three-torus), it is assumed that the real-world two-dimensional surface is only a section of the torus (e.g., a square piece of tape stuck onto the surface of a donut)." I fail to understand how the realworld surface is only a part of the torus. In the existing theoretical and experimental work on toroidal topology of grid cell activity, the torus represents a very small fraction of the real world, and repeating activity on the toroidal manifold is a crucial feature of how it maps 2D space in a regular manner. Why then here do you want the torus to be larger than the realworld? 

This section has been rewritten to better explain these assumptions. The relevant paragraphs now read:

“The cosine tuning curves of the simulated border cells represent distance from the border on both sides of the border (i.e., firing rate increases as the animal approaches the border from either the inside or the outside of the enclosure). Experimental procedures do not allow the animal to experience locations immediately outside the enclosure, but these locations remain an important part of the hypothetic representation, particularly when considering the modification of memories through consolidation (i.e., a memory created inside the enclosure might be moved to a location outside the enclosure). This symmetry about the border cell’s preferred location is needed to maintain an unbiased representation, with a constant sum of squares for the border cell inputs (see methods section). Rather than using linear dimensions, all dimensions were assumed to be circular to keep the model relatively simple. This assumption was made because head direction is necessarily a circular dimension and by having all dimensions be circular, it is easy to combine dimensions in a consistent manner to produce multidimensional hippocampal place cell memories. Thus, the border cells define a torus (or more accurately a three-torus) of possible locations. This provides a hypothetical space of locations that could be represented.

In light of the assumption to represent border cells with a circular dimension, when a memory is pushed outside the East wall of the enclosure, it would necessarily be moved to the West wall of the enclosure if the period of the circular dimension was equal to the width of the enclosure. If this were true, then the partial grid field on one side of the enclosure would match up with its remainder on the other side. Such a situation would cause the animal to become completely confused regarding opposite sides of the enclosure (a location on the West wall would be indistinguishable from the corresponding location on the East wall). To reduce confusion between opposite sides of the enclosure, the width of the enclosure in which the animal navigated (Figure 5) was assumed to be half as wide as the full period of the border cells. In other words, although the space of possible representations was a three-torus, it was assumed that the real-world twodimensional enclosure encompassed a section of the torus (e.g., a square piece of tape stuck onto the surface of a donut). The torus is better thought of as “playing field” in which different sizes and shapes of enclosure can be represented (i.e., different sizes and shapes of tape placed on the donut). Furthermore, this assumption provides representational space that is outside the box without such locations wrapping around to the opposite side of the box.”

p.28 "More specifically, egocentric grid cells (e.g., head direction conjunctive grid cells) require stabilization of the place cell memories in the face of ongoing consolidation whereas allocentric grid cells reflect on-average place field positions." and p.32 "if place cells represent episodic memories, it seems natural that they should include head direction (an egocentric viewpoint)." But the head direction signal is not egocentric, it is allocentric. I'm unsure whether this is a typo or a potentially more serious conceptual misunderstanding. 

Any reference to egocentric has been removed in this revision. In the initial submission, when I used egocentric, I was referring to memories that depended on the head direction of the animal at the time of memory formation. I was using “egocentric” in relation to whether the memory was related to the animal’s personal bodily experience at the time of memory formation. But I concede that this is confusing since the ego/allo distinction is typically used to differentiate angular directions that are relative to the person (left/right) versus earth (East/West). Instead, throughout the manuscript I now refer to these as view-dependent memories since head direction would entail having a different view of the environment at the time of memory formation. I still refer to the stacking of multiple view-dependent memories on the same X/Y location as being the development of an allocentric representation however, since this can be thought of as one way to learn a cognitive map of the enclosure that is view independent.

p.37 "But if the border cells had changed their alignment with the new enclosure (e.g., if the E border dimension aligned with the North-South borders), then the place cells would have appeared to undergo global remapping as their positions rotated by 90 degrees and the grid pattern would have also rotated." But this would not be interpreted as global remapping by standard analyses of place and grid cell responses. A coherent rotation of firing patterns is not interpreted as remapping. 

This sentence now reads: “But if the border cells had changed their alignment with the new enclosure (e.g., if the E border dimension aligned with the North-South borders), then the place cells would remain in their same positions relative to the now-rotated borders (i.e., no remapping relative to the enclosure) and the corresponding grid cells would also retain their same alignment relative to the enclosure.”

p.37 "this is more accurately described as partial remapping (nearly all place fields were unaffected)." If nearly all place fields were unaffected, this should be interpreted as a stable map. Partial remapping is a mix of stability, rate remapping, and global remapping within a population of place cells. 

This sentence has been removed.

p.40 "The dependence of grid cell responses on memory may help explain why grid cells have been found for bats crawling on a two-dimensional surface (Yartsev et al., 2011), but three-dimensional grid cells have never been observed for flying bats." This is not true. Ginosar et al. (2021) observed 3D grid cells in flying bats.  

Thank you for highlighting this issue. In the initial submission I was using “grid cell” to mean a cell that produced a precise hexagonal grid, which is not the case for the 3D grid cells in bats. In this revision, I now discuss grid cell that produce irregular grid fields, writing:

“According to this model, hexagonally arranged grid cells should be the exception rather than the rule when considering more naturalistic environments. In a more ecologically valid situation, such as with landmarks, varied sounds, food sources, threats, and interactions with conspecifics, there may still be remembered locations were events occurred or remembered properties can be found, but because the non-spatial properties are non-uniform in the environment, the arrangement of memory feedback will be irregular, reflecting the varied nature of the environment. This may explain the finding that even in a situation where there are regular hexagonal grid cells, there are often irregular non-grid cells that have a reliable multi-location firing field, but the arrangement of the firing fields is irregular (Diehl et al., 2017). For instance, even when navigating in an enclosure that has uniform properties as dictated by experimental procedures, they may be other properties that were not well-controlled (e.g., a view of exterior lighting in some locations but not others), and these uncontrolled properties may produce an irregular grid (i.e., because the uncontrolled properties are reliably associated with some locations but not others, hippocampal memory feedback triggers retrieval of those properties in the associations locations).

In this memory model, there are other situations in which an irregular but reliable multi-location grid may occur, even when everything is well controlled. In the reported simulations, when the hippocampal place cells were based on variation in X/Y (as defined by Border cells), nothing else changed as a function of location, and the model rapidly produced a precise hexagonal arrangement of hippocampal place cell memories. When head direction was included (i.e., real-world variation in X, Y, and head direction), the model still produced a hexagonal arrangement as per face centered cubic packing of memories, but this precise arrangement was slower to emerge, with place cells continuing to shift their positions until the borders of the enclosure were sufficiently well learned from multiple viewpoints. If there is realworld variation in four or more dimensions, as is likely the case in a more ecologically valid situation, it will be even harder for place cell memories to settle on a precise regular lattice. Furthermore, in the case of four dimensions, mathematicians studying the “sphere packing problem” recently concluded that densest packing is irregular (Campos et al., 2023). This may explain why the multifield grid cells for freely flying bats have a systematic minimum distance between firing fields, but their arrangement is globally irregular (Ginosar et al., 2021). Assuming that the memories encoded by a bat include not just the three realworld dimensions of variation, but also head direction, the grid will likely be irregular even under optimal conditions of laboratory control.”

Multiple typos are found on page 25, end of paragraph 3: "More specifically, if there is one set of non-orthogonal dimensions for enclosure borders and the movement of one wall is too modest as to cause avoid global remapping, this would deform the grid modules based the enclosure border cells."

As detailed above in the response the public reviews, this paragraph has been rewritten.

According to all known laws of aviation,

there is no way a bee should be able to fly.

Its wings are too small to get its fat little body off the ground.

The bee, of course, flies anyway

because bees don't care what humans think is impossible.

Yellow, black. Yellow, black. Yellow, black. Yellow, black.

Ooh, black and yellow! Let's shake it up a little.

Barry! Breakfast is ready!

Ooming!

Hang on a second.

Hello?

Barry?

Adam?

Oan you believe this is happening?

I can't. I'll pick you up.

Looking sharp.

Use the stairs. Your father paid good money for those.

Sorry. I'm excited.

Here's the graduate. We're very proud of you, son.

A perfect report card, all B's.

Very proud.

Ma! I got a thing going here.

You got lint on your fuzz.

Ow! That's me!

Wave to us! We'll be in row 118,000.

Bye!

Barry, I told you, stop flying in the house!

Hey, Adam.

Hey, Barry.

Is that fuzz gel?

A little. Special day, graduation.

Never thought I'd make it.

Three days grade school, three days high school.

Those were awkward.

Three days college. I'm glad I took a day and hitchhiked around the hive.

You did come back different.

Hi, Barry.

Artie, growing a mustache? Looks good.

Hear about Frankie?

Yeah.

You going to the funeral?

No, I'm not going.

Everybody knows, sting someone, you die.

Don't waste it on a squirrel. Such a hothead.

I guess he could have just gotten out of the way.

I love this incorporating an amusement park into our day.

That's why we don't need vacations.

Boy, quite a bit of pomp… under the circumstances.

Well, Adam, today we are men.

We are!

Bee-men.

Amen!

Hallelujah!

Students, faculty, distinguished bees,

please welcome Dean Buzzwell.

Welcome, New Hive Oity graduating class of…

…9:15.

That concludes our ceremonies.

And begins your career at Honex Industries!

Will we pick ourjob today?

I heard it's just orientation.

Heads up! Here we go.

Keep your hands and antennas inside the tram at all times.

Wonder what it'll be like? A little scary. Welcome to Honex, a division of Honesco

and a part of the Hexagon Group.

This is it!

Wow.

Wow.

We know that you, as a bee, have worked your whole life

to get to the point where you can work for your whole life.

Honey begins when our valiant Pollen Jocks bring the nectar to the hive.

Our top-secret formula

is automatically color-corrected, scent-adjusted and bubble-contoured

into this soothing sweet syrup

with its distinctive golden glow you know as…

Honey!

That girl was hot.

She's my cousin!

She is?

Yes, we're all cousins.

Right. You're right.

At Honex, we constantly strive

to improve every aspect of bee existence.

These bees are stress-testing a new helmet technology.

What do you think he makes? Not enough. Here we have our latest advancement, the Krelman.

What does that do? Oatches that little strand of honey that hangs after you pour it. Saves us millions.

Oan anyone work on the Krelman?

Of course. Most bee jobs are small ones. But bees know

that every small job, if it's done well, means a lot.

But choose carefully

because you'll stay in the job you pick for the rest of your life.

The same job the rest of your life? I didn't know that.

What's the difference?

You'll be happy to know that bees, as a species, haven't had one day off

in 27 million years.

So you'll just work us to death?

We'll sure try.

Wow! That blew my mind!

"What's the difference?" How can you say that?

One job forever? That's an insane choice to have to make.

I'm relieved. Now we only have to make one decision in life.

But, Adam, how could they never have told us that?

Why would you question anything? We're bees.

We're the most perfectly functioning society on Earth.

You ever think maybe things work a little too well here?

Like what? Give me one example.

I don't know. But you know what I'm talking about.

Please clear the gate. Royal Nectar Force on approach.

Wait a second. Oheck it out.

Hey, those are Pollen Jocks! Wow. I've never seen them this close.

They know what it's like outside the hive.

Yeah, but some don't come back.

Hey, Jocks! Hi, Jocks! You guys did great!

You're monsters! You're sky freaks! I love it! I love it!

I wonder where they were. I don't know. Their day's not planned.

Outside the hive, flying who knows where, doing who knows what.

You can'tjust decide to be a Pollen Jock. You have to be bred for that.

Right.

Look. That's more pollen than you and I will see in a lifetime.

It's just a status symbol. Bees make too much of it.

Perhaps. Unless you're wearing it and the ladies see you wearing it.

Those ladies? Aren't they our cousins too?

Distant. Distant.

Look at these two.

Oouple of Hive Harrys. Let's have fun with them. It must be dangerous being a Pollen Jock.

Yeah. Once a bear pinned me against a mushroom!

He had a paw on my throat, and with the other, he was slapping me!

Oh, my! I never thought I'd knock him out. What were you doing during this?

Trying to alert the authorities.

I can autograph that.

A little gusty out there today, wasn't it, comrades?

Yeah. Gusty.

We're hitting a sunflower patch six miles from here tomorrow.

Six miles, huh? Barry! A puddle jump for us, but maybe you're not up for it.

Maybe I am. You are not! We're going 0900 at J-Gate.

What do you think, buzzy-boy? Are you bee enough?

I might be. It all depends on what 0900 means.

Hey, Honex!

Dad, you surprised me.

You decide what you're interested in?

Well, there's a lot of choices. But you only get one. Do you ever get bored doing the same job every day?

Son, let me tell you about stirring.

You grab that stick, and you just move it around, and you stir it around.

You get yourself into a rhythm. It's a beautiful thing.

You know, Dad, the more I think about it,

maybe the honey field just isn't right for me.

You were thinking of what, making balloon animals?

That's a bad job for a guy with a stinger.

Janet, your son's not sure he wants to go into honey!

Barry, you are so funny sometimes. I'm not trying to be funny. You're not funny! You're going into honey. Our son, the stirrer!

You're gonna be a stirrer? No one's listening to me! Wait till you see the sticks I have.

I could say anything right now. I'm gonna get an ant tattoo!

Let's open some honey and celebrate!

Maybe I'll pierce my thorax. Shave my antennae.

Shack up with a grasshopper. Get a gold tooth and call everybody "dawg"!

I'm so proud.

We're starting work today! Today's the day. Oome on! All the good jobs will be gone.

Yeah, right.

Pollen counting, stunt bee, pouring, stirrer, front desk, hair removal…

Is it still available? Hang on. Two left! One of them's yours! Oongratulations! Step to the side.

What'd you get? Picking crud out. Stellar! Wow!

Oouple of newbies?

Yes, sir! Our first day! We are ready!

Make your choice.

You want to go first? No, you go. Oh, my. What's available?

Restroom attendant's open, not for the reason you think.

Any chance of getting the Krelman? Sure, you're on. I'm sorry, the Krelman just closed out.

Wax monkey's always open.

The Krelman opened up again.

What happened?

A bee died. Makes an opening. See? He's dead. Another dead one.

Deady. Deadified. Two more dead.

Dead from the neck up. Dead from the neck down. That's life!

Oh, this is so hard!

Heating, cooling, stunt bee, pourer, stirrer,

humming, inspector number seven, lint coordinator, stripe supervisor,

mite wrangler. Barry, what do you think I should… Barry?

Barry!

All right, we've got the sunflower patch in quadrant nine…

What happened to you? Where are you?

I'm going out.

Out? Out where?

Out there.

Oh, no!

I have to, before I go to work for the rest of my life.

You're gonna die! You're crazy! Hello?

Another call coming in.

If anyone's feeling brave, there's a Korean deli on 83rd

that gets their roses today.

Hey, guys.

Look at that. Isn't that the kid we saw yesterday? Hold it, son, flight deck's restricted.

It's OK, Lou. We're gonna take him up.

Really? Feeling lucky, are you?

Sign here, here. Just initial that.

Thank you. OK. You got a rain advisory today,

and as you all know, bees cannot fly in rain.

So be careful. As always, watch your brooms,

hockey sticks, dogs, birds, bears and bats.

Also, I got a couple of reports of root beer being poured on us.

Murphy's in a home because of it, babbling like a cicada!

That's awful. And a reminder for you rookies, bee law number one, absolutely no talking to humans!

All right, launch positions!

Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz!

Black and yellow!

Hello!

You ready for this, hot shot?

Yeah. Yeah, bring it on.

Wind, check.

Antennae, check.

Nectar pack, check.

Wings, check.

Stinger, check.

Scared out of my shorts, check.

OK, ladies,

let's move it out!

Pound those petunias, you striped stem-suckers!

All of you, drain those flowers!

Wow! I'm out!

I can't believe I'm out!

So blue.

I feel so fast and free!

Box kite!

Wow!

Flowers!

This is Blue Leader. We have roses visual.

Bring it around 30 degrees and hold.

Roses!

30 degrees, roger. Bringing it around.

Stand to the side, kid. It's got a bit of a kick.

That is one nectar collector!

Ever see pollination up close? No, sir. I pick up some pollen here, sprinkle it over here. Maybe a dash over there,

a pinch on that one. See that? It's a little bit of magic.

That's amazing. Why do we do that?

That's pollen power. More pollen, more flowers, more nectar, more honey for us.