At what point in the process does one have a conceptualization for the overall outline of what they're writing?

In cases where it's earlier than others, then heavy linking and organization may not be as necessary.

She strips the focus away from the incident itself to focus on the degrading joke.

Author Response

Reviewer #1 (Public Review):

1) In family 2, the variant was detected by routine trio-based WES diagnostics. Sanger confirmation was not performed. IGV images can be added as supplementary material. Furthermore, median coverage was 75× which might not be sufficient for the identification of all heterozygous variants.

We thank reviewer for pointing it out for clarification. Obviously, at the time (2016) of the reporting of this variant this was our laboratory’s thoroughly validated protocol, which shows that median (!) coverage of 75x with the technology at the time is more than sufficient for robust variant calling. This particular variant was actually below 75X in coverage (at 65x), but Sanger confirmation was not necessary (based on thorough validation of the robustness of calling and GATK scores and other quality parameters for de novo calling). In addition, when coverage goes below 30-35X Sanger confirmation is warranted.

2) Proband 2 (P2) was born as the second child of non-consanguineous parents of Caucasian descent after an uneventful pregnancy and delivery. The boy was macrosomic at birth. Since there was macrosomia, how would the pregnancy be uneventful? At the last assessment at 10 years of age, obesity associated with hyperphagia was of concern; the weight of the patient should be clarified. P2 was diagnosed with autism spectrum disorder but a normal cognitive profile. The identified NM_001014809.2(CRMP1_v001):c.1280C>T variant is very rare and reported in GnomAD exomes with allele frequency 0.0000041.

Routine echographia during pregnancy did not result in any concerns. The pregnancy was indeed uneventful. BMI at last evaluation was 26.1. We included the details in the revised manuscript.

3) Proband 3 (P3) is the first of three children of a non-consanguineous family of European descent. There is a familial history of obesity on both parental sides, and the father is macrocephalic (head circumference: 60.5 cm). Macrocephaly can be isolated and benign, such as in benign familial macrocephaly. However, P3 presented with moderate intellectual disability and an autism spectrum disorder. Since P3 has a macrocephaly also, the PTEN gene should be further interrogated by detailed WGS data analysis as well as an additional orthogonal method(s) since it has pseudogenes.

We have not noted any pathogenic variant of the PTEN gene in the genetic analysis.

Reviewer #2 (Public Review):

Weaknesses of the article include:

1) Spelling errors and difficult-to-understand language. The use of "variant" is now preferred over mutation. According to current nomenclature, predicted but not experimentally confirmed protein alterations should be written as p.(Phe351Ser) rather than p.Phe351Ser.

We apologise for the spelling errors and the difficult-to-understand language in the manuscript. We considered the reviewers comments seriously and corrected the errors and rephrased the sentences wherever necessary.

2) Inconsistent use of in silico pathogenicity predictors and conservation metrics. These should be standardized for each case and should include at least phylop, CADD, and REVEL.

We have applied consistency in the description of in silico pathogenicity predictors and conservation metrics for each patient.

3) CRMP1 is under significant constraint against loss-of-function variation in gnomAD - pLI = 0.99, LOEUF 0.28. Genes in the top decile are highly enriched for haploinsufficiency as a disease mechanism. This should be considered in the interpretation of this data and incorporated into the manuscript.

We thank the reviewer for the comment. As per reviewer’s suggestion, we have included a statement in the revised manuscript under ‘Subjects and Methods’ section.

4) I am not convinced the data supports a dominant-negative interpretation. The variants do not oligomerize as well as wild-type CRMP1, and when co-expressed with wild-type CRMP1 there is an increase in monomeric wild-type CRMP1. While this could support a dominant-negative interpretation, an alternative explanation is these are loss-of-function alleles that cannot oligomerize, and at the stoichiometry of this artificial overexpression system, this leads to increased monomeric wild-type CRMP1. The axonal outgrowth studies are more compelling, but without a loss-of-function control allele, it is difficult to interpret.

The experiments in Figure 2 should be replicated, quantitated, and their statistical significance confirmed.

We thank reviewer for raising concern about the experiment and interpretation of the data. We performed size exclusion chromatography experiments and included the data in the revised Figure 2. Unfortunately, we could not reproduce the experiments for Figure 2B. From our current experimental results, we prove that the CRMP1 variants affect the homo-oligomerization process.

Reviewer #3 (Public Review):

1) The major weakness is Figure 2, as it is not performed up to high standards like the rest of the paper. Panel A does not show any loading control and does not confirm. Panel B at 720 kDa band is not convincing. Results should be repeated with size exclusion chromatography and/or another method to determine molecular weight and should be quantified from triplicate experiments. Panel C is also not convincing and should be repeated to more carefully show results, and quantified.

We thank reviewer for this important concern raised on our Figure 2 experimental data. We addressed the comments in the revised manuscript. We performed size exclusion chromatography and presented the results in the revised manuscript and discussed accordingly in page 23-24.

Fig. 2A: This panel shows the recombinant CRMP1 wildtype and the variants from E-coli expressing system. We repeated the expression several times and obtained similar partially cleaved proteins. Fig. 2A is Coomassie Brilliant Blue staining. Protein size marker and loading control (BSA) were applied on the same gel as shown in Fig.2A original.

Fig.2B: Due to limited protein expression of T313M and P475L mutants, we could not repeat the gel-filtration experiments.

Fig. 2C, 2D: It is difficult to adjust the expression level of each construct (CRMP1 wildtype, T313M, or P475L) in HEK293T cells (input). Therefore, we measured the signal intensity of myc-IP band and input ratio of V5 blot in each condition. Fig. 2D shows the ratio from four independent experiments.

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Reply to the reviewers

Manuscript number: RC-2022-01707

Corresponding author(s): Sarah Butcher, Richard Lundmark

1. General Statements [optional]

We thank the reviewers for their insightful comments. The inclusion of the points raised by the referees have strengthened the manuscript. However, some of the reviewer suggestions are beyond the scope of the work (see below), but will doubtlessly be touched upon in future studies by the authors. In addition to incorporating changes relevant to answering the reviewers’ comments, we have edited the manuscript for increased clarity and precision.

2. Description of the planned revisions

1. Liposome flotation assay Reviewer #1 suggested that we should perform a liposome floatation assay to separate possible C protein aggregation from membrane binding: "I would strongly recommend supplementing the current liposome sedimentation assay by liposome flotation assay. In contrast to liposome co-sedimentation, the flotation assay can discriminate protein aggregates from proteins bound to liposomes. Although the SDS PAGE shown in Fig. 1A looks pretty convincing, a faint protein band in the „P" lane of the middle panel for the (-) sample is evident. Therefore, C protein aggregation cannot be ruled out and it would be indistinguishable from liposome binding examined by mere co-sedimentation assay”

Response: We agree that this is a necessary control experiment to add, and we will perform it with liposomes containing 40 % POPS. As we detected complete C protein co-sedimentation with this lipid composition, performing the flotation experiment with the same composition will prove that the earlier result indicates lipid binding and not protein aggregation.

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

1. Reviewer #1
2. “In addition, it needs to be clarified which TBEV C protein construct, whether full-length or truncated, was used for co-sedimentation fragmentation.”

Response: We have clarified in this section of the manuscript that the full-length C protein construct was used for the liposome co-sedimentation assays by adding “full-length” prior to instances of “C protein” e.g. in the paragraph starting line 118.

1. “How to understand the finding that „the C protein forms a very rigid layer when adsorbed to the membrane". Can the aggregation of C-protein be ruled-out? Following the 1M NaCl wash of C-protein-bound to SLB, the authors stated: „This shows that initial membrane recruitment of C protein is strongly dependent on its interactions with the negatively-charged lipid headgroups. However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization": does it mean that there are several layers of C protein, the first held by electrostatic interactions, overlayed by non-electrostatically bound C protein? If yes, the illustration of single-layered C-protein adsorbed onto SLB in Fig. 2A, B is not correct. ”

Response: We understand the confusion regarding the term “rigid” which was used as a way to describe how we interpret the relatively minor change in the dissipation upon membrane binding. What we intended to describe was that this indicates that the protein is attached in a stable way that does not add viscoelastic properties to the system. These data indicate that the protein does not form large aggregates that non-specifically attach to the membrane in different protrusive orientations. We have clarified this in the manuscript and specified that the as there is no dissipation change, there is no aggregation. We added the following to line 168 “This, in turn, indicates that the C protein does not bind as non-specific aggregates as these would have changed the viscoelastic properties of the system.”

We do not mean that there are several layers of C protein. We consider, due to the highly charged nature of C, that the most likely explanation is that there are multiple modes of C binding but the result is only one layer, with multiple C-proteins interacting with each other within that layer. We have modified the text at line 184 to: “However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization within the bound layer.”

1. “The sentence: “To confirm that the C protein is biologically active, we investigated its ability to bind RNA" seems to be a little odd because it suggests the model membrane binding assays do not require biological active proteins. However, considering that the interactions leading to binding either negatively-charged lipid or negatively-charged RNA are electrostatic - this sentence must be rewritten.” Response: We thank the reviewer and have now rephrased this sentence to the following at line 249 “Since RNA binding is crucial for the NC assembly, we investigated the C protein’s ability to perform this function.”

2. “The authors´ statement in the Abstract: „....we investigate nucleocapsid assembly..." is too speculative because the assembly was not studied in their work. It needs to be reformulated.” Response: We agree, and the statement has been removed from the abstract.

3. “Despite this clear and valuable methodological contribution, the authors' contribution to our knowledge of the coordination of the nucleocapsid components to the sites of assembly and budding is not so obvious. Contrary to the earlier idea that the flavivirus is asymmetrically charged (that is, hydrophobic on one side (α2) and positively charged on the other side (α4), recent studies show that the entire surface of the protein is highly electropositive (Mebus-Antunnes et al., 2022). Therefore, a well-ordered neutralization of the flaviviral C proteins' highly positive surface seems critical for the proper organization and assembly of nucleocapsid. I am afraid that the authors do not shed much light on this issue.” Response: The recent structure of the TBEV C protein, published after we submitted the manuscript, shows that indeed the C protein is highly positively charged on all surfaces (updated Supplementary Figure 1 and Selinger et al., 2022). The recruitment of C protein to the membrane, that we demonstrate is dependent on negatively-charged head groups, provides a biologically relevant mechanism for charge neutralization on the C protein surface that interacts with the lipids. The remaining surface charge can be then neutralized by RNA recruitment. Mebus-Antunnes et al. made their observations with just RNA and C protein from Dengue virus in the context of artificial surfaces e.g. mica. However, our experiments utilize the TBEV C protein and specifically include a membrane, the third critical component of NC assembly. Thus, we build upon the work of Mebus-Antunnes et al. by adding a second biologically relevant charge-neutralising component and comparing with a distantly-related virus. We have changed the discussion section of the manuscript to reflect this new structure and to emphasize the advance here. Starting from line 371 we changed the text to: “Recently, it has been shown that the neutralization of the C protein surface positive charge is important for RNA binding in the distantly-related Dengue virus (DENV) (Mebus-Antunes et al, 2022). The recruitment of C protein to the membrane, that we demonstrate is dependent on negatively-charged head groups, provides a biologically relevant mechanism for charge neutralization on the C protein surface that interacts with the lipids. The remaining surface charge can be then neutralized by RNA recruitment.”

Reviewer #2 1. “What results demonstrate C protein inserts into membrane? The current results support the C protein interacts with membranes with positive charge, but do not seem to demonstrate membrane insertion. If the C protein inserts into the membrane, which regions (helices) play this role?”

Response: The Langmuir-Blodgett trough tensiometry experiments with monolayers directly measure the insertion of a protein into the monolayer. By determining the maximum insertion pressure of the C protein constructs, we also show that the membrane insertion can occur in bilayers. We show that the N-terminus is not inserting into the membrane, further work, beyond the scope of this manuscript, is needed to pinpoint the residues responsible for insertion, for instance by hydrogen-deuterium exchange or FRET measurements that would not affect folding. To clarify the use of the LB trough, we added the following at line 216: “To investigate if the C protein membrane binding includes insertion into the membrane after the initial electrostatic binding, we used Langmuir-Blodgett trough monolayer experiments. In this approach, the insertion of a protein into a lipid monolayer can be detected by following the pressure (π) of the monolayer after protein injection into the aqueous subphase, with increases in π corresponding to protein injection (Brockman, 1999; Liu et al, 2022).“

1. “The authors should discuss several previous papers reporting the effect of partial deletions of the C gene on the replication of TBEV, West Nile virus, and other flaviviruses.” Response: We agree that this is a necessary addition, and have now added a paragraph in the discussion section starting at line 333: “N-terminally truncated flaviviral C proteins have been shown to be assembly competent and in vitro, able to bind RNA, which is consistent with our results with N-terminally truncated TBEV C protein (Khromykh & Westaway, 1996; Kofler et al, 2002; Patkar et al, 2007; Schlick et al, 2009). One role of C is in the modulation of host responses to infection and the N-terminus maybe involved in that (Yang et al, 2002; Limjindaporn et al, 2007; Colpitts et al, 2011; Bhuvanakantham & Ng, 2013; Katoh et al, 2013; Urbanowski & Hobman, 2013; Samuel et al, 2016; Slomnicki et al, 2017; Fontaine et al, 2018). The membrane insertion directly detected in our experiments is central to C protein function. Other studies have found that deletions in the hydrophobic region of the α2 helix significantly impair particle assembly (Kofler et al, 2002; Patkar et al, 2007; Schlick et al, 2009). In the light of this evidence, we consider that the α2 helix could be responsible for membrane insertion (Markoff et al, 1997; Kofler et al, 2002; Nemésio et al, 2011, 2013).”

Reviewer #3 1. “In Figure 4, the band (256:1) that are supposedly in the wells (red arrow) is not clear- it is only slightly darker than the other wells.”

Response: This confusion was the result of unclear wording. We have now revised the figure legend at line 278 to : “The black arrow indicates the bands of freely-migrating RNA, and the red arrow the wells. On lanes 624:1 and 256:1, RNA has been immobilized in the wells.”

1. “Figure S1A, the N-terminal end (which is truncated in the mutant) should be colored on the cyan molecule.” Response: We have coloured the truncated part of the cyan molecule in the figure (now S1B) according to the reviewer’s comment.

Other 1. As the nuclear magnetic resonance structure of the truncated TBEV C protein has recently been released (Selinger et al, 2022), we have updated the manuscript and Figure S1 to include the information from this structure. We have also generated a new homology model of the full-length TBEV C protein using this structure as a template and included that in Figure S1.

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

1. Reviewer #1
2. “However, we do not know whether in the infected cells, the C protein is pre-bound to ER membrane or to viral RNA. Having such a unique assay in their hands, I wonder whether the authors could use the pre-bound C protein with genomic RNA (i.e. the experiment shown in Fig. 4A) ribonucleoprotein complex in the SLB binding assay. If doable, this experiment would be exciting and could bring some important information about NC assembly.”

Response: We agree that it would be very interesting to decipher if the C-protein first binds to RNA or to membranes using the QCM-D methodology. Yet, our data on pre-incubated C-protein and RNA suggests that large aggregates are formed which would hamper the interpretation of the QCM-D data. Furthermore, based on the suggested experiment, we will not be able to firmly conclude whether or not the C-protein first binds to RNA or to membranes since the time of the experiment will allow rearrangement of preformed complexes between C-protein and RNA. Additionally, the QCM-D measurement cannot differentiate if the preformed complexes bind on their own, or if excess unbound C protein binds the membrane and then recruits the complex. Therefore, addressing this question would require major adjustments to the RNA model system and methodology that we feel are beyond the scope of this study.

Reviewer 2 1. “The authors should use the lipids detected in the virions to confirm C protein binding experiments.”

Response: In the mass spectrometry characterization of the TBEV virions, we detected lipids from 9 classes (Car, PE, PS, PI, PG, PC, Cer, HexCer & TG). We have tested four of them (PE, PS, PI, PC) in the liposome sedimentation assay. Additionally, we tested GalCer, which, like HexCer, are cerebrosides. Our liposome binding experiments clearly demonstrate that the C protein does not bind to a specific lipid class, but instead to lipids with negatively-charged headgroups. Therefore, we would argue that doing additional sedimentation experiments with Car, PG, Cer, and TG would not add extra insight to the manuscript.

Additionally, while the population of lipid species in the TBEV envelope is diverse, the diversity mostly comes from differences in the lipid tails, which do not generally affect the head group-mediated binding of proteins. Therefore, performing additional lipid binding experiments with varying tail lengths would not likely lead to new observations.

Finally, to perform the authentic experiment of testing C protein binding to liposomes formed from lipids extracted from purified virions would require orders of magnitude more virus sample than our research laboratory is capable of producing. Therefore, we argue that this experiment is beyond the scope of this study.

1. “The study may be strengthened by performing virus mutagenesis experiments.” Response: While we agree that, ultimately, experiments on virus and cells would help to understand the role of the C protein in the biological context, we think these experiments are beyond the scope of this study. For virus mutagenesis, candidate residues should be first identified with biochemical and biophysical studies, which is already beyond the scope of this work. Additionally, the C protein has multiple functions in the host cell in addition to NC assembly, and interpreting the effect on the mutations on e.g. virus titer is difficult.

Reviewer #3 1. “In all figure legends, authors should write a conclusion line after the description of the experiments - what conclusion is drawn from each experiment.”

Response: While we agree that adding such a conclusion line would make it easier for the reader to understand each figure, the format of the figure legends is highly subject to journal policy. Therefore, we think that the addition of such lines will be an editorial decision and will depend on the journal. We have, however strived to make the figure titles as informative as possible in lieu of such concluding lines.

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Referee #1

Evidence, reproducibility and clarity

The authors characterized the interactions of recombinant, bacterially expressed full-length and N-terminally truncated C proteins of tick borne encephalitis virus with model membrane systems. They used a unique combination of biophysical methods, including protein liposome co-sedimentation, QCM-D measurement and Langmuir-Blodgett trough monolayer experiments. Their experiments showed that the binding of TBEV C to both liposomes and supported lipid bilayer (SLB) is strongly dependent on the presence of negatively charged lipids. They also showed that following the initial electrostatic binding to the model lipid membrane, both C protein variants absorb to the SLB and form rigid layers which are stabilized by non-electrostatic interactions. By Langmuir-Blodgett trough monolayer experiments they demonstrated that negatively charged lipids are needed for C protein membrane insertion. The SLB bound C proteins, either full-length or N-terminally truncated, were shown to bind in vitro transcribed TBEV genomic RNA. Finally, to prove their major finding that negatively charged lipid head groups are crucial for C protein interaction with the lipid membrane, the authors analyzed the lipid content of the purified virions.

This work deals with the central role of the C protein, namely with its binding to the lipid membrane and genomic RNA. In the infected cells, this process leads to nucleocapsid assembly, a step which is poorly understood. The authors demonstrate that the membrane affinity of the C protein is conditioned by the presence of negatively charged polar heads. The text and figures are clear and accurate. The results obtained from three independent methodological approaches are solid and confirm the importance of electrostatic interactions for a contact of C protein with the membrane. As highly interesting, I considered the observation that the C protein, while bound to the model membrane (SLB), still retains its ability to bind RNA. Although their data did not show anything about the orientation of the C protein in SLB, this methodology opens the way to how, using suitable mutants of TBEV C, this can be found. I am sure that the authors are aware of the possibilities of studying a series of the TBEV C mutants with impaired membrane or RNA binding. Therefore, I assume that the authors' primary focus here is to show new methodological approaches to the simultaneous measurement of C protein interactions with model membranes and RNA, and some data obtained on the abovementioned mutants will be published afterwards.

Major comments:

1. One of the fundamental challenges of the work with flaviviral capsid proteins is that they tend to form amorphous aggregates to neutralize their highly positive surface charge. As the authors state themselves, „ We cannot rule out that the C protein preparation is heterogeneous..." I would strongly recommend supplementing the current liposome sedimentation assay by liposome flotation assay. In contrast to liposome co-sedimentation, the flotation assay can discriminate protein aggregates from proteins bound to liposomes. Although the SDS PAGE shown in Fig. 1A looks pretty convincing, a faint protein band in the „P" lane of the middle panel for the (-) sample is evident. Therefore, C protein aggregation cannot be ruled out and it would be indistinguishable from liposome binding examined by mere co-sedimentation assay. In addition, it needs to be clarified which TBEV C protein construct, whether full-length or truncated, was used for co-sedimentation fragmentation.
2. In section: Initial C protein recruitment to the membrane is of an electrostatic nature How to understand the finding that „the C protein forms a very rigid layer when adsorbed to the membrane". Can the aggregation of C-protein be ruled-out?

Following the 1M NaCl wash of C-protein-bound to SLB, the authors stated: „This shows that initial membrane recruitment of C protein is strongly dependent on its interactions with the negatively-charged lipid headgroups. However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization": does it mean that there are several layers of C protein, the first held by electrostatic interactions, overlayed by non-electrostatically bound C protein? If yes, the illustration of single-layered C-protein adsorbed onto SLB in Fig. 2A, B is not correct. 3. C protein inserts into membranes It is beyond the frame of this work; however, it would be nice to show whether mutations of amino acid residues within the hydrophobic segment of TBEV C, which are in other flaviviral C proteins considered responsible for hydrophobic interaction, can abolish the membrane interaction. 4. Membrane-bound C protein can recruit TBEV genomic RNA. The sentence „ To confirm that the C protein is biologically active, we investigated its ability to bind RNA" seems to be a little odd because it suggests the model membrane binding assays do not require biological active proteins. However, considering that the interactions leading to binding either negatively-charged lipid or negatively-charged RNA are electrostatic - this sentence must be rewritten. 5. The authors state, "These data show that membrane-bound C protein is capable of recruiting TBEV genomic RNA at the membrane, suggesting that this also happens in the context of NC assembly". However, we do not know whether in the infected cells, the C protein is pre-bound to ER membrane or to viral RNA. Having such a unique assay in their hands, I wonder whether the authors could use the pre-bound C protein with genomic RNA (i.e. the experiment shown in Fig. 4A) ribonucleoprotein complex in the SLB binding assay. If doable, this experiment would be exciting and could bring some important information about NC assembly.

Minor comments:

The authors´ statement in the Abstract: „....we investigate nucleocapsid assembly..." is too speculative because the assembly was not studied in their work. It needs to be reformulated.

Referees cross-commenting

I agree with the Reviews by reviewers #2 and #3

Significance

This manuscript's major novelty and originality are in using a unique combination of biophysical methods, including quartz crystal microbalance with dissipation monitoring and Langmuir-Blodgett trough. Using quartz crystal microbalance with dissipation, the authors confirmed the necessity of negatively charged lipid components of the model lipid membrane for C-protein binding. Furthermore, this method also allows them to measure the formation of a rigid layer of C protein stabilized by non-electrostatic interactions. By Langmuir-Blodgett trough monolayer experiments, they demonstrated the insertion of TBEV C protein into the model membrane. However, I do not have sufficient expertise to evaluate the correctness of the experiments done by these two methodologies.

Despite this clear and valuable methodological contribution, the authors' contribution to our knowledge of the coordination of the nucleocapsid components to the sites of assembly and budding is not so obvious. Contrary to the earlier idea that the flavivirus is asymmetrically charged (that is, hydrophobic on one side (α2) and positively charged on the other side (α4), recent studies show that the entire surface of the protein is highly electropositive (Mebus-Antunnes et al., 2022). Therefore, a well-ordered neutralization of the flaviviral C proteins' highly positive surface seems critical for the proper organization and assembly of nucleocapsid. I am afraid that the authors do not shed much light on this issue.

In my opinion this statement is very true. Even though it is challenging at times , digital humanities can be fun. When collecting and improving the quality of data to represent reality it creates a thrill for digital humanists. The challenge allows humanists to think creatively and find different solutions to the problems presented to them

This is an example of fragmentation. The author has pieces of vastly different places and people fighting each other in his head and doesn't come out with a sense of wholeness

Reference to one of the "holy cities" and to the biblical story about the walls of the red sea parting and crashing.

Why does the author choose to use similes and metaphors, such as "I roll dizzily toward them like the bowling ball of very bad bowler" and "they meet in my head like the walls of the Red Sea crashing together"?

I think this statement is interesting. I believe that everyone was made in God's image and should be viewed as a child of God. While it can be hard for me to wrap my head around the idea of there being more than two genders, I cannot make a valid argument because it is not something I experience. Therefore, it is not my place to state if it is wright or wrong. I think people who identify as Catholic should just be classified as that and not a certain type of Catholic.

I too agree with Trump that gender is defined as either male or female, and can not still quite wrap my head around the fact of how this would not be true.

I can only slightly relate to this feeling as I to have cut down tree's and had to dig up the stump/roots! Of course I had better equipment and help but if this is the manual labor that the habitants had to do in order to live on the land it's no wonder the rent was low.

Dogs have been mans best friend for a very long time. My dog is always at the window watching, waiting for us to come home, such love!

This just makes me appreciate running water and water bottles/coolers.

I have thought long about the same issue and beyond. The triple (wiki, Hypothesis, donations) could be a working way to search for OER, form a social group processing them, and optionally support the creators.

I imagine that as follows: a person wants to learn about X. They can head to the wiki site about X and look into its Hypothesis annotations, where relevant OER with their preferred donation method can be linked. Also, study groups interested in the respective resource or topic can list virtual or live meetups there. The date of the meetups could be listed in a format that Hypothesis could search and display on a calendar.

Wiki is integral as it categorizes knowledge, is comprehensive, and strives to address biases. Hypothesis stitches websites together for the benefit of the site owners and the collective wisdom that emerges from the discussions. Donations support the creators so they can dedicate their time to creating high-quality resources.

Main inspirations:

Deschooling Society - Learning Webs

Building the Global Knowledge Graph

Schoolhouse calendar

The story you tell yourself creates reality.

To add some other intermediary services:

• ko-fi (site for contribution)
• GitHub sponsors (for GitPages)
• itch.io (for games)
• Gumroad (for sites and repositories)
• Patreon (for fan interaction)

To add a service for groups:

• Open Collective

To add a service that enables fans to support the creators directly and anonymously via microdonations or small donations by pre-charging their Coil account to spend on content streaming or tipping the creators' wallets via a layer containing JS script following the Interledger Protocol proposed to W3C:

• Web Monetization

If you want to know more, head to Web Monetization or Community or Explainer

Disclaimer: I am a recipient of a grant from the Interledger Foundation, so there would be a Conflict of Interest if I edited directly. Plus, sharing on Hypothesis allows other users to chime in.

this is so interesting! also the fact that these surrealist journals are not well-known

typo

Methods can also have the property of "idempotence" in that (aside from error or expiration issues) the side-effects of N > 0 identical requests is the same as for a single request. The methods GET, HEAD, PUT and DELETE share this property

Barbara adds a new element to the conditioning. It shows Peter that others do not find the fuzzy creature scary, which starts the process of reversing the fear. Social conditioning and learning is a powerful component and shows Peter that perhaps his response is not "normal" and that others do not find the rat as a problem. It is also interesting that he is possessive of the beads and does not want them violated by the scary creature. The fear is eventually forgotten with time and Peter is free to move about as normal as the conditioning wears off.

Detta är viktigt för liksom folkhälsan, men passar det in i hur mycket vi ska spendera/hur vi ska prioritera?

eLife assessment

This is an important study linking metabolic traits and head and neck cancer risk using Mendelian randomisation. The findings, well supported by the data, were inconclusive. This work will be of interest to researchers working in head and neck cancer.

Reviewer #2 (Public Review):

The authors used Mendelian randomisation to study the relationships between metabolic traits and oral/oropharyngeal/head and neck cancers. This study was conducted as the relationships between these traits and cancers are unclear based on observational data. Evidence for relationships between these traits and cancers is inconclusive, which is a relevant finding in the context of previous observational data.

Strengths include using large studies to develop the instrumental variables used in MR and examining multiple metabolic traits. Weaknesses include relatively low power to detect associations and a lack of discussion around any possible pleiotropy of SNPs associated with any of the metabolic traits. Based on these strengths and weaknesses, it is unclear whether the authors achieved their goal and whether the results support their conclusions.

This work is relevant to researchers interested in oral cancers and their etiology. Several issues would need to be addressed to make the evidence more reliable.

Reviewer #3 (Public Review):

This manuscript describes a Vegfc-independent mechanism of lymphatic vessel formation that is controlled by Svep1 and an orphan endothelial receptor tyrosine kinase Tie1. Based on similarities in the phenotype of svep1 and tie1 mutant zebrafish in the head and trunk vasculature, as well as genetic interaction between the two during parachordal lymphangioblast migration, the authors propose that svep1 is a component of the tie1 signaling pathway. Specifically, svep1 and tie1 mutants show a unique phenotype with the absence of facial collecting lymphatic vessel (that forms in Vegfc mutants) while other facial vessels (that are dependent on Vegfc/Vegfr3 signaling) were only partially affected. Svep1 and tie1 mutants also show similar defects in the formation of brain LECs, the number and migration of parachordal lymphangioblasts from the horizontal myoseptum, and DLAV formation. In contrast, tie2, which is the major angiopoietin receptor in mammals, was found to be dispensable for vascular development in zebrafish.

The presented experiments are performed well and the data are conclusive. The novel findings are the identification of a role of tie1 in zebrafish lymphatic development, and svep1 as a component of the tie1 signaling pathway. The latter raises the possibility that svep1 regulates the activity of Angiopoietin or even acts as a ligand for tie1. However, the conclusion on Svep1 and Tie1 being in the same pathway is based solely on the comparison of mutant phenotypes and genetic interaction studies. Any biochemical data on how svep1 regulates tie1 signaling would greatly strengthen this conclusion.

can not help the situation, negative self talk begins, blaming self + giving surface level solution for deeper mental issue

Relative to whole body, head cells were more affected by a high dose of RECS1. Cell death was also dependent on the presence of C-terminal segment of RECS1.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary

PIWI-interacting RNAs (piRNAs) are required for transposon repression and are transcribed from discrete genomic loci termed piRNA clusters. Torimochi was identified as a piRNA cluster in silkworm in 2012, but the incomplete genome assembly hindered its further characterisation. Here, Shoji and colleagues characterised torimochi using the current, recently improved, genome assembly, combined with long-read (MinION) and Sanger sequencing. This reveals that torimochi is a regular Gypsy LTR transposon. Comparison of copy number across strains reveals that torimochi has been particularly active in the BmN4 cell line, showing different insertions between strains. Moreover, piRNAs are produced from multiple torimochi copies across the genome. Lastly, the authors show that torimochi has an open chromatin conformation. The authors propose that torimochi may be a young and still growing piRNA cluster, capable of both trapping other transposable elements and transgenes and of producing piRNAs.

Major comments

How are the torimochi-derived piRNAs produced? Which part of the piRNA pathway are required for their production? Determining this would significantly strengthen the study and potentially support the idea that torimochi is a "young and still growing piRNA cluster". Currently, it is unclear what evidence there is for torimochi acting as a piRNA cluster rather than a regular LTR transposon.

We thank the Reviewer for raising this important point. We have now re-analyzed our piRNA sequencing data and confirmed that 1) production of torimochi-derived piRNAs requires Siwi, the core PIWI protein component in silkworms and 2) torimochi-derived piRNAs show the ping-pong signature, as observed for other typical piRNAs. These data strengthen the idea that torimochi is a cluster that produces canonical piRNAs.

As originally shown in Fig. 4, torimochi is the most actively translocated transposon in BmN4 cells with extremely high transcription and piRNA production levels and open chromatin structure, thereby representing those transposons that have gained the piRNA production activity in BmN4 cells. To further investigate if torimochi has any special features even among those piRNA-producing transposons in BmN4 cells, we have now performed a new analysis. It is known that well-established piRNA clusters in Drosophila (e.g., the 42AB cluster) have a specialized system for transcriptional activation. However, those specialized transcriptional activators such as Rhino (HP1 variant) and Moonshiner (TFIIA variant) are conserved only within the Drosophila genus, and thus the transcriptional activation systems of piRNA clusters are likely to be different in different organisms. Keeping this in mind, we asked if the transcription mechanism of torimochi is any different from other piRNA-producing transposons in BmN4 cells. Since specific transcriptional activators of piRNAs clusters remain unknown in silkworms (as in many other animals except for Drosophila), we decided to differentiate BmN4 cells into adipocytes so that they lose their “germline-ness” (Akiduki et al., 2007). As expected, the expression of the adipocyte marker BmFABP1 (Fatty Acid-Binding Protein 1) was markedly increased (Fig. 5a), while the expression levels of piRNA-related factors such as Vasa were decreased (Fig. 5b). Importantly, transcription of torimochi was drastically reduced by adipocyte differentiation (Fig. 5c), whereas most other transposons, including those piRNA-producing transposons in BmN4 cells, remained unrepressed or rather increased by differentiation (Fig. 5c and 5d). These findings suggest that, even among those piRNA-producing transposons in BmN4 cells, torimochi has started to gain a specialized, germline-specific transcriptional activation system and thus can be used as a good model as a “young and still growing piRNA cluster.” We will include these data and discussion in the revised manuscript.

In figure 1F, the positive control (P50T) is missing. Based on the description, this one should show a band, but doesn't or at least doesn't do very clearly. The authors need to repeat this assay.

We agree that the P50T band was quite faint, although it was clearly present at the expected molecular size. We will repeat this assay with more PCR cycles so that the band will appear more clearly.

The authors should perform a qPCR (or similar assay) on the different torimochi loci (and across different strains) to assess their individual transcriptional activity. Generally, showing that torimochi is an active transposable element is crucial to support the claim that it is still expanding.

We have now re-analyzed our RNA-seq data to assess the individual transcriptional activity of different torimochi loci. We found that, as expected, torimochi mRNAs are a mixture of transcripts from various loci, just like torimochi-derived piRNAs. We will include these data in the revised manuscript.

I would also recommend the authors to perform ping-pong analysis on all piRNAs mapping to torimochi. The hypothesis that torimochi acts as a piRNA cluster would be supported showing phased biogenesis, and a lack of a ping-pong signature (i.e., 10A). Please provide evidence that the piRNAs mapping to the different torimochi insertions are not produced via Post Transcriptional Gene Silencing.

We would like to note that silkworms have no homolog of Drosophila Piwi, the PIWI protein that is specialized for the phased piRNA biogenesis pathway. Instead, silkworm Siwi participates in both the ping-pong pathway and the phased piRNA pathway (Izumi and Shoji et al., 2020). As expected, torimochi-derived piRNAs show both the ping-pong signature and the head-to-tail phasing signature in Trimmer knockout BmN4 cells. We would also like to note that, even in Drosophila, dual-strand piRNA clusters (e.g., 42AB) are known to show the ping-pong signature, while uni-strand piRNA clusters (e.g., flamenco) lack it (refs).

Line 265; "Torimochi has the open chromatin structure and can trap foreign transgenes as well as endogenous transposons" - The evidence for "trapping" transposable elements is circumstantial. Transposons are known to insert into each other. One occasion of a transgene inserting in torimochi is not strong enough evidence to support the made claim.

We appreciate the Reviewer’s concern. We would like to note that, in the previous paper (Kawaoka et al., 2009), the GFP transgene was inserted into torimochi (not once but) at least three times independently; there were three out of eight independent lines that contained the GFP transgene inserted into torimochi for piRNA-mediated silencing. This observation highlights the especially efficient “trapping” ability of torimochi. We will revise the text to clarify this point.

Please provide a size distribution of all the piRNAs that are mapping on torimochi. In the methods section it is stated that small-RNAs of length 20-42 nt are mapped. This range is too generous as it also includes siRNA on the low end, and other ncRNAs on the long end. Please use the appropriate piRNA size range, i.e., 23-30 nt.

We will be happy to include the size distribution data of all small RNAs mapped to torimochi, which shows that only 6% of them are siRNAs (~21 nt) and the majority (82%) of them can be considered as piRNAs (23–32 nt).

Please include the sequences of the newly identified transposon families.

We will be happy to determine the exact sequences of the newly identified transposons in BmN4 cells by PCR and Sanger sequencing and deposit them in a public database.

Minor comments

Line 71-72; "However, it was recently shown that these large piRNA clusters are evolutionarily labile and mostly dispensable for transposon suppression", this is misleading in the context of flamenco since flamenco is essential for transposon suppression. Please rephrase.

We agree that flamenco is essential for transposon suppression in the somatic follicle cells in Drosophila, and we will rephase the sentence accordingly.

Line 100; "Therefore, torimochi may serve as a model for young piRNA clusters, which are still "alive" and active in transposition, can trap other transposons, and produce de novo piRNAs.". It is unclear how this is evidenced? Would not any transposon be able to "trap" external sequences (e.g., PMID: 33347429). It is unclear to me how torimochi is different from any active transposon that is silenced by the piRNA pathway.

As discussed above, our new data show that torimochi is not only a representative of transposons that have gained piRNA-producing activity in BmN4 cells but also a unique transposon that has started to gain a specialized transcriptional activation system as seen in well-established piRNA clusters in Drosophila. Therefore, we believe that torimochi will serve as a good model for young piRNA clusters.

Line 117; "Therefore, torimochi is not a unique sequence in the genome but should be now interpreted as a gypsy-type transposon" - Even if there is one copy in the genome, torimochi could still is a transposable element.

We agree that, even if there is one copy in the genome, it could still be a transposable element. We will change this part into "Therefore, torimochi is not a unique sequence in the genome as was thought in the past but should be now interpreted as a gypsy-type transposon with multiple copies in the genome".

Line 133; "a presumed ancestor of Bombyx mori" - both species are extant, so none of them can be an ancestor of the other.

Yes, Bombyx mandarina is also an extant species. We will change the wording to “a wild progenitor of Bombyx mori.”

Line 135 Change "species" into "strains"?

Yes, “strains” is appropriate and we will change it accordingly

Please provide the coverage for every SNP in figures 2D and 2E. Having an idea of the coverage (i.e., how many reads support this SNP) would strengthen the conclusions made.

We will add a Figure that shows the coverage of each SNPs at the top of the current Figure or as a Supplemental Figure.

Supplementary figure 2I/J; The insert depiction of the GFP cassette is incorrect, it currently is displayed as a small vertical strip, whereas it should be a large block.

We originally intended to show the situation around the GFP cassette for the sake of consistency with Supplemental Figure S2A–H. We will redraw this figure with including the GFP cassette.

Methods: More details are needed on the computational analysis. Please include parameters used for different tools as well as custom scripts. Where multi-mappers used to quantify piRNAs across the torimochi insertions?

We will include precise parameters used for different tools and upload our custom scripts on GitHub.

Display of Supplementary Table 2 and Supplementary Table 3 partially obscured.

We are sorry for the problems caused by the conversion. We will amend them.

Introduction/discussion: I would suggest that the authors also discuss how torimochi could be mis-identified as a piRNA cluster previously.

We will include the following statement to explain why torimochi was originally thought as a unique piRNA cluster in the genome. “The silkworm genome published in 2008 had many unassembled regions, which had masked two out of the three torimochi copies that we now found to exist in the p50T genome. In other words, the 2008 silkworm genome appeared as if there was only one region to which torimochi-derived piRNAs were mappable. Back then, the apparent difference in the chromosomal position of torimochi between BmN4 cells and silkworm ovaries was thought to be due to a large rearrangement of the corresponding genomic region.”

CROSS-CONSULTATION COMMENTS

Reviewer #2 raised three interesting points and the manuscript would be strengthened by addressing these.

We will also fully address the three points raised by Reviewer #2.

Reviewer #1 (Significance):

Significance

I find the topic both important and timely with the ongoing re-examination of whether piRNA clusters or dispersed euchromatic transposon insertions fuel the piRNA pathway. However, I feel that the current study on torimochi is relatively shallow and descriptive and does not take us much closer to resolving the issue. Re-examining the torimochi cluster is on its own of minor significance, since there are only five publications on torimochi since 2012. However, the current study has potential and torimochi could act as a model to study how piRNAs are produced.

We are grateful to the Reviewer for recognizing the potential importance of our current study. All the comments by the Reviewer were of great help in significantly improving our manuscript. In particular, new Fig. 5 (related to Major Point #1) is an important addition to support the idea that torimochi is a young and still growing piRNA cluster, and we thank the Reviewer again for his/her constructive comments.

Reviewer #2 (Evidence, reproducibility and clarity):

The author performed a straightforward of long read DNA sequencing data, which indicates that torimochi is not a single locus, but a gypsy-like LTR transposon that has massively expanded in BmN4 cells. The data are clear and convincing, and raise a number of interesting questions:

1. The authors present data on single nucleotide polymorphisms in torimochi insertions (Figure 2), but the element can capture transgenes and produce silencing piRNAs. Does the long read data reveal capture of transposon insertions by any of the torimochi elements? Do any appear to be expanding due to recurrent insertion?

In original Fig. S3A, we demonstrated that an endogenous transposon named mejiro is indeed inserted into the torimochi element . We plan to perform additional long read sequencing and further analyze the data to see if there are other examples of transposon capture events by any of the torimochi elements.

1. The data indicate that torimochi is active and transcribed, but also the source of piRNAs that can silence transgenes. Why isn't torimochi silenced by piRNAs derived from the dispersed insertions?

We believe that torimochi is indeed being silenced by piRNAs, but just not 100%. The GFP transgene trapped by torimochi was also not 100% silenced and some GFP signals were clearly detectable even in the silenced cell lines (Kawaoka et al., 2011). This must be also the case for any other transposons, although the silencing efficiency (the current result of the tug-of-war between transposons and the host’s piRNA system) should vary.

1. Comparisons with the silkworm genome indicates that torimochi has been very active since BmN4 were isolated, and the element appears to active now, based on transcription. However, activation could have occurred when the cell line was established. If transposition is ongoing, BmN4 cells maintained as independent stock should have different insertions. This could be tested by sequence analysis of stocks from different labs. This experiment isn't essential to publication, but could be informative.

We thank the Reviewer for raising this important point. Indeed, there exist BmN4 cells that have been independently maintained, and we have now obtained another stock of BmN4 cells from a different lab. We plan to perform long-read sequencing of genomic DNA using these cells to compare the insertion sites of torimochi. The results will allow us to determine whether activation of torimochi occurred when the cell line was established or its transposition is ongoing. Either result would be informative and helpful to further improve our manuscript.

Reviewer #2 (Significance):

piRNAs have a conserved role in transposon silencing. In many systems the most abundant piRNAs are derived from distinct chromosomal loci, termed clusters, that are composed of complex arrays of transposon fragments. Available data indicate that these loci can produce trans-silencing piRNAs, and the flam locus is required for fertility and silencing of Gyspsy transposons in flies. However, several major clusters, in flies and mice, are not required for fertility or transposon silencing, and dispersed mobile elements can produce piRNAs. The nature and function of piRNA source loci thus remains to be established. Shoji et al. address that nature of piRNA source loci through a reevaluation of the torimochi cluster In silkworm BmN4 cells. The authors show that torimochi is actually a gypsy-like LTR transposon that has massively expanded in BmN4 cells, and may represent an emerging piRNA clusters, falling between established clusters that look like “transposon graveyards”, and single euchromatic insertions that appear to have epigenetically converted to “mini-clusters”. The data raise a number of interesting questions, and should stimulate studies in other systems for similar elements.

We are grateful to the Reviewer for precisely understanding the significance of our current study.

Reviewer #1 (Public Review):

In this work, Li & Meister provide an extensive description of functional cell types within the posterior-medial part of the mouse superior colliculus, corresponding to the upper lateral visual field. Presenting a battery of visual stimuli to head-fixed wild-type mouse lines, they use calcium imaging and subsequent clustering of functional responses to identify 24 functional cell types. Besides the comprehensive sampling of SC cell types, a major strength of the manuscript in my view is the direct comparison with the previously published in vitro RGC data. Overall, the manuscript and the associated data promise to be a valuable resource to experimentalists and computational neuroscientists comparing visual processing across the major processing stages of the mouse visual system. However, in the current form the manuscript still comes with some limitations. In my view, these are related to some parts, where statistical justifications for the conclusions are still missing, where the findings should be more strongly embedded into the current and past literature, and where more efforts should be made to relate the findings in the different mouse lines to those for the overall population.

The use of language is very descriptive and puts an image in my head.

As an educator, you have a plan in your head that you want to execute. A plan that you think will benefit every student but the reality is nothing goes according to the way you picture. Teaching is about adapting to your classroom the different personalities and backgrounds of each student

Does it work for them, though? (maybe the answer is in the linked article)

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

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary<br /> The authors have set out to study the Drosophila immune response against the fungus Aspergillus fumigatus. They found that Aspergillus fumigatus kills Drosophila Toll pathway mutants. The fungus does this without invasion because its dissemination is blocked by melanization. They suggest that there is a role for Toll in host defense distinct from resistance. The findings are interesting, and looks like the mycotoxins play a role. It also seems that there is some role of the Bomanins here, but I find that in particular Figure4 experiments are not convincing enough to provide a mechanistic insight as to what is going on. I think the authors need to think through what their results mean, and also, explain better (especially regarding Fig 4) their ideas and how the data fits them.

We thank the reviewer for scrutinizing our manuscript as well as for suggestions to improve it.

The role of mycotoxins is demonstrated:

i) the fungus does not proliferate nor disseminate, also in Toll pathway mutant flies: thus, it must kill through diffusible substances, in as much as these immuno-deficient flies exhibit tremors toward the end of the infection;

ii) a fungal strain devoid of the capacity to produce secondary metabolites is no longer virulent, even in Toll pathway mutant flies.

The role of Bomanins is also demonstrated: the finding of a susceptibility of Bom__D__55C deletion flies to A. fumigatus and to mycotoxin challenges clearly shows that at least one or several Bomanin genes are required in the host defense against these challenges. The observation that this susceptibility can be rescued by the genetic overexpression of specific Bomanins indicates which ones are likely to mediate protection. The novel data we have included with the protection from mycotoxin action in neurons point clearly to BomS6 being the major mediator of protection against verruculogen action since it is the only one of two Bom genes to be induced in the head and with a proven potential for rescue of the Bom__D__55C phenotype.

As regards the concept of the article, it is simple: we show that the Toll pathway does not control A. fumigatus infection by directly attacking the fungus but does so by neutralizing the effects of secreted virulence factors such as restrictocin and verruculogen. We further identify some of the relevant effectors such as Bomanins by using a genetic complementation strategy. To make our point clearer, we have now included additional data in which we show that BomS6 and BomS4 are the only Bomanins induced in the head of flies upon the injection of these two toxins. We next determine that BomS6 and not BomS4 expression in the nervous system dominantly protects the flies from the deleterious effects of verruculogen injection, both in terms of recovery from tremors and survival. Mechanistically, the Toll pathway protects the host from the action of verruculogen by expressing and likely secreting BomS6 from neurons.

Major comments:<br /> Page 5: .."the fungal burden did not increase much in MyD88 flies challenged with 50 conidia (Fig. 1B)" - What do you mean did not increase much? There is a clear increase in Myd88 mutants compared to controls; would you expect a bigger increase (e.g. log scale induction)? Explain.

When the injected dose is higher than 50 injected colonies, the fungal burden remains very close to that of the injected inoculum (Fig. EV1_F, J_). As for other pathogens regulated by the Toll pathway, it has been published that the microbial burden increases by log factors for filamentous fungi (Huang et al.., in revision), pathogenic yeasts (e.g., work from our laboratory Quintin et al. Journal of Immunology, 2013), bacteria (e. g., Duneau et al., eLife 2017; Huang et al., in revision). The pathogens usually proliferate exponentially in immuno-deficient hosts, which is clearly not the case of A. fumigatus, the first example we know of.

Page 6: "the SPZ/Toll/MyD88 cassette is required for host defense against A. fumigatus infections, even though this pathogen only mildly stimulates the Toll pathway." - Should you rather say that A. fumigatus only mildly induces the Toll pathway target gene Drosomycin?

The answer is negative. Fig. EV1_C_ clearly shows that BomS1 is also modestly induced as compared to an infection with E. faecalis. The promoter of BomS1 contains a canonical Dif-response element (Busse et al., EMBO J., 2007_)_. For a more thorough discussion of this point, please, see reply to Reviewer 2, Major Comment 2.

Page 6: "...we tested Hayan mutant flies defective for this arm of innate immunity (Nam et al., 2012)." - elaborate this, which arm/which pathway?

The title of the paragraph is “Drosophila melanization curbs A. fumigatus invasion”. The full first sentence of the paragraph actually read: “As melanization is a host defense of insects effective against fungal infections, we tested Hayan mutant flies defective for this arm of innate immunity”.

This has not been introduced in the introduction. Explain.

We have now added a couple of lines (82-83) to introduce melanization for the nonspecialist reader.

Can you really draw this conclusion: "We conclude that melanization limits the proliferation and the dissemination of A. fumigatus injected into wild-type flies yet does not eradicate it at the injection site, where a melanization plug forms." Maybe you can based on the function/importance of the pathway to melanization, but you need to explain.

Melanization is mediated by the Hayan protease and three phenol oxidases (two in adults) that catalyze the enzymatic reactions leading to melanin production (for Drosophila, please see Nam et al. EMBO J. (2012), Bingelli et al., PLoS Pathogen (2014), Dudzic et al., BMC Biology (2015), Cell Reports, 2019). Thus, finding that there is an increased proliferation and dissemination in null Hayan mutants is a strong indication for a role of melanization. The identification of a similar phenotype for PPO2 and PO1-PPO2 mutants demonstrates that melanization is curbing A. fumigatus. Our sentence is therefore fully justified.

Page 10: "The cleavage of the 18S RNA was however much less pronounced in wild-type flies as compared to MyD88" - I am not sure what this means. Do you mean 28S?

We thank the reviewer for pointing out this mistake that has now been corrected.

And that the 28S peak is lower? Is this a quantitative method?

The technique is liquid electrophoresis on a microchip. It is both a qualitative and quantitative technique that replaces traditional agarose or polyacrylamide gels.

Fig. legend: "Arrows show the position of the 28S RNA sarcin fragment" - there are three arrows in both Fig 4E and F; specify which arrows point what.

The thick arrow is now indicated in the figure legend to correspond to the much smaller sarcin fragment whereas the thin arrows on the graph clearly specify the position of the 28S RNA peaks.

Based on the results, I am not convinced about the conclusion, that "restrictocin is able to inhibit translation to a detectable degree in vivo, likely through the cleavage of the ribosomal 28S a-sarcin/ricin loop as described in vitro." <- Do you draw this conclusion before doing the actual in vitro experiment, which is described next in the text (The rabbit reticulocute assay, S2 cells)?

The existing literature (line 259 for a few selected references) has largely proven that restrictocin cleaves 28S RNA in vitro. We are demonstrating that this also happens in vivo in flies based on the generation of the alpha-sarcin fragment as well as the decreased 28S peaks. Our transgenic approach also indicates that restrictocin blocks translation in vivo. The in vitro approach has been implemented so that we could test the effect of synthetic BomS1 and BomS3 in cell culture. As to our knowledge, no one had demonstrated that restrictocin blocks translation in Drosophila cultured cells. It was therefore important to demonstrate it in cell culture using well-characterized in vitro techniques mastered by AT and FM.

4H: Not sure what should be seen here, is it the darkest band at 0 uM that disappears?

We have improved the figure and added an arrow to point out to the relevant band on the gel.

HI & J need more explanation than what is now included in the text or Figure legend, is the conclusion that there is no difference? Write the stats above the Figs 4I & J (n.s.?).

We have added NS on the figures and made our conclusion clearer (lines 295-298).

Minor comments:

It would have helped commenting if the manuscript contained line numbers

We apologize for having initially provided a version in which lines were not numbered. At the prompting of Review Commons we immediately provided such a version, that was actually used by Reviewer 2.

Why do you have the title "Hayan" on top of Fig 1F; you don't have this marking system in the other survival curves

This point has now been addressed and the survival experiments checked for consistency.

Fig 2A: Can you speculate why MyD88 flies die rapidly at day 10 if you inject PBST (your control)? What would happen to uninjected controls in otherwise the same conditions? (you could include an uninjected control here?)

We suspect that this is linked to the trauma induced by the injection. Trauma has been shown to impact the homeostasis of the midgut epithelium (Lee & Miura, Current Topics Developmental Biology 2014, Chakrabarti et al., PLoS Genetics (2016)), and we suspect that it may lead to a leakiness of the gut allowing the passage of some bacteria from the gut microbiota that can proliferate in the hemocoel. Hence, we checked axenic and antibiotics-treated MyD88 flies to exclude that the limited sensitivity to trauma was not significantly contributing to the phenotypes we describe. It is also linked to the thickness of the needle and the problem is alleviated by using thinner needles.

The uninjected control is now shown in Fig. EV8_E_.

Please, see also the answer to Reviewer 2 Major comment 1.

Fig 2E: Not sure what would be the best way of presenting the curves - different colors, dotted lines or something? Now if there are too many lines, they are hard to tell apart. because the symbols are not that visible. Like in 2E if you want to compare the light red/orange colored lines.

We agree with the reviewer that the lines are hard to tell apart. This is however not a significant issue since the glip mutants display curves similar to that of the wt A. fumigatus control strain.

For consistency add the caption also to Fig 3D (I assume it is the same as 3C)

The caption was present in our version and is present in the revised version.

For consistency, should you add Verruculogen on top of Fig 3F?

Same reply as for the previous comment.

Chronologically, how it is explained in the text, Figs 4A and B are in the wrong order.

We fully agree with the reviewer. This problem has been addressed in the revised version.

The quality of Fig 4 is not great, the text is hard to read (too small) and becomes blurry upon magnification.

We fully agree with the reviewer. This problem has been addressed in the revised version.

Page 12; "These data then suggest that a process akin to the immune surveillance of core cellular processes first described in C. elegans may also exist in Drosophila" - I think this sentence belongs to the discussion, this is not directly drawn from the results.

We have followed the reviewer suggestion and have now developed our Discussion paragraph now entitled “Induction of the expression of specific Bomanin genes upon mycotoxin challenge”

Referees cross-commenting

I think we share many thoughts among all the reviewers.

The main problem is that the manuscript language is quite strong; from the results many times it is not ok to make such strong statements. Some experiments need further analysis and clarification.

I think in most cases, this could be achieved by softening the statements and adding more discussion, and not by making new experiments (some may be needed).

We respectfully disagree with the reviewer on this point. There were obviously some misunderstandings that might be traced to the short format of the initial version. We have now developed the Discussion to clarify our conclusions as suggested by the reviewer.

Minor things are that experiments are not advancing in a logical order between the text and the figures and there are problems with resolution in some figures.

Statistics in some figures needs to be added.

Please, see above.

Reviewer #1 (Significance):

The nature of the work is conceptual for the field, to understand the role of the Toll pathway and Bomanins in particular, in this fungal infection model. The work is interesting to a somewhat limited audience, mainly immunologists and in particular, people interested in the Drosophila model for immunity. The work may be interesting conceptually in understanding fungal infections.

We are not certain that immunologists represent a limited audience. We agree that work on fungal infections is insufficiently funded with respect to the medical importance of these infections, as highlighted in our introduction and Perspective section of the Discussion.

My expertise: I am a Drosophila immunity researcher with nearly 20 years of experience in working with fly immunity, in particular the Toll and the Imd pathways.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:

Xu et al. describe how A. fumigatus kills Toll-deficient fruit flies not by hyperproliferation, but more likely by virulence factors. Melanization is important for suppressing fungal spread. The Bomanin genes have an unknown function, and here the data suggest a reasonably convincing role for Toll in resilience. Overall the manuscript is thorough and presents a diversity of approaches that show Toll and the Bomanins in particular contribute to this resilience effect. The idea that Toll effectors are essential for resilience is interesting as other fly stress response pathways like JAK-STAT are better known for helping the fly cope with damages, while Toll is better known as an antifungal response.

I believe the study, with some careful considerations added, would add a valuable series of observations to understanding how the host immune system promotes survival after infection. Overall I am quite positive about the results, and the authors have made a significant effort.

We thank the reviewer for the positive evaluation of our work that actually spans many years of research on the Aspergillus fumigatus Drosophila infection model that is a major topic of our work at the Sino-French Hoffmann Institute of Guangzhou Medical University.

Any experiment suggestions I make are strictly to improve the confidence in the interpretations of the results, but the language could alternately be softened to address those concerns. My major critique is that the authors repeatedly extend beyond what is shown, and occasionally in defiance of what is shown (if I understand the results correctly).

We have chosen to perform additional experiments when needed. We have also clarified points where there were obvious misunderstandings by expanding our text that had been written under a very concise format.

It is not thoroughly clear what the reviewer has in mind when using the word defiance. We suppose it refers to the work of Scott Lindsay with whom we are in contact. He actually attempted to monitor the C. glabrata burden but did not pursue this line of investigations as he already saw a difference after one hour and he thought that the Toll pathway cannot be induced so rapidly. Actually, David Duneau mentions a time of two to three hours for the Toll pathway to control E. faecalis infections (eLife, 2017) and Sandrine Uttenweiler-Joseph already saw by MALDI-TOF MS an induction of Bomanins and other DIMs at the earliest point tested, six hours (PhD thesis). There is absolutely no critique of the work of the Wasserman laboratory who has greatly contributed to our understanding of Bomanin functions. Some of our unpublished data clearly point out to an AMP role for at least one Bomanin gene against E. faecalis and we certainly do not exclude an AMP role for BomS against C. glabrata. This however does not dismiss the possibility that Bomanins may also have other roles in dealing with microbial toxins. We have been studying Candida infections in Drosophila for many years and have documented the host defense against C. glabrata (Quintin et al., JI, 2013). We do suspect that C. glabrata likely secretes virulence factors that have not been identified so far. We mention this as a possibility and certainly not as a truth. One should remember that investigators were unaware for a long period of the role of Candidalysin, a pore-forming toxin, in C. albicans infections.

Finally, a dual role as AMP and protecting from secreted toxins has been clearly shown in the case of alpha-mammalian Defensins that we now are describing in our Revised Discussion (Kudryashova,Immunity, 2014).

Comments below.

Major comments:

1) The language is too strong. Specifically the use of the phrase "anti-toxin" is too generalist, especially as the authors show that their candidate Bomanin does not bind to the toxin directly.

We have checked all of the submitted documents: the term anti-toxin was never used (just found “anti” in antimicrobial, antifungal, antibiotics..), in this manuscript as well as in the companion article. and we have never excluded an indirect effect, quite to the contrary because of the in vitro experiment with restrictocin mentioned by the reviewer and other observations now included (see further below). We use the terms “protection” or “counteract”, which have not such a meaning. It is burdensome for the reader to read each time “counteract or protect from the actions of the toxins or the effects of the toxin.

Instead, Toll mutants seem susceptible to damage/stress caused by injury/toxins. MyD88 even show general susceptibility to vehicle controls in Fig3C-D.

The effects of stress related to the infection conditions and injury are clearly distinct from the much stronger ones exerted by the toxins themselves. As requested by the reviewer further below, we have submitted wild-type and immuno-deficient flies to several stresses such as heat or the injection of hydrogen peroxide or salt solution (Fig. EV8_B-E_). While the latter did not reveal any difference, MyD88 flies succumbed slightly faster to a strong 37°C stress; in contrast, they survived better to a 29°C exposure, the temperature at which we perform most experiments. However, the difference started to be visible only after some 15 days whereas the time frame in which flies succumb to A. fumigatus or toxin challenges is definitely much shorter by some 10 days. We also note that Bom__D__55C mutant flies behave like the isogenized wild-type controls in these assays, further excluding a potential role for general stress sensitivity as a contributor to the effect of toxins.

As regards DMSO, there is indeed a general mild sensitivity of flies to DMSO, but not specifically affecting MyD88 mutant (Rebuttal Fig. 1J). We find that this effect is lessened when using thinner needles. Thus, the problem has become minor as we became more experienced. We had checked axenics- and antibiotics-treated flies to exclude a contribution from the microbiota. Finally, to uncouple the effects of verruculogen from those of DMSO, we have also challenged flies directly by introducing the powder, using a technique similar to that of the septic injury. While it is quantitatively less accurate, it clearly proves that verruculogen produces the reported effects (Fig. 3C) and was useful to measure Bom and Drosomycin expression by digital PCR in the heads of challenged flies, e.g., Fig. EV6_J-K_ and Figs EV_11&12_.

Toll is important for development, so it may be expected that Toll flies could have development defects impacting resilience even if/when Toll flies can survive to adulthood. I don't say this to be too negative on the findings, which are quite convincing. But I am not sure that the phrase "anti-toxin" is right for what is shown.

We fully agree with the reviewer on this point. We have failed to find RNAi lines that are efficient enough to mimic the Toll pathway phenotype when expressed ubiquitously at the adult stage. However, Bom__D__55C mutants do not seem to display a developmental phenotype and display a phenotype similar to that of MyD88 flies. Furthermore, our rescue experiments of the Bom__D__55C sensitivity phenotype to mycotoxin challenge is achieved by the overexpression of specific Boms that are induced only at the adult stage, making it unlikely that this sensitivity phenotype reflects a developmental problem, as had been shown to be the case for 18-wheeler that had initially been proposed to encode the IMD pathway receptor.

A very interesting recent study shows Dif has a role in the synapse of neurons to protect from alcohol sensitivity. Could secreted Bomanins participate? This emphasizes a mechanism through which Toll mutants likely have defective neural development, which could make them stress response defective, especially to things like neurotoxins. See: https://pubmed.ncbi.nlm.nih.gov/35273084/

We are aware of this study first presented at the 2019 Fly Meeting in Dallas and this author did discuss with the authors of the study. However, we have found that Dif (and Dorsal) mutants are not sensitive to A. fumigatus infections nor to injected mycotoxins, as was the case already for C. glabrata (Quintin et al., JI, 2013).

Lin et al. (2019) also showed lack of Bomanin secretion from the fat body in Bombardier mutants causes loss of tolerance (resilience?). So does Bomanin disruption increase susceptibility to stresses more generally, rather than specifically fungal toxins? And is this a development role, rather than an immune response role?

The authors could try to use other stresses (NaCl, oxygen, heat, alcohol) to test the contribution of Bomanins to this resilience, which may reflect defective neural development rather than a role for secreted systemic immune-response peptides.

Please, see replies above.

2) The authors present a paradox. On the one hand, A. fumigatus hardly induces Drs/Bomanins (Fig. S1). Yet on the other, they propose that inducible Bomanins protect the fly from mycotoxins. Why do the authors say Toll is hardly induced by A. fumigatus at the start of the study (Fig S1), but later use the same data to argue that Bomanin induction underlies the resilience phenotype (Fig5).

The reviewer raises an interesting point. Of note, we have added new data in Fig. EV2_B_ that document that all 55C Bomanin genes, BomS4-_excepted, are induced by a systemic infection. There is indeed somewhat of a paradox. The _Bom__D__55C deletion phenotype clearly establishes that Bomanins play a major role in the protection against mycotoxins and A. fumigatus. The rescue experiments rely on ectopic expression and therefore establish that specific Bomanins can mediate the protective effect. Our data on verruculogen suggest that there might be local inductions, e. g., in the head of BomS6 and BomS4. The brain represents a compartment that is separated from the hemocoel by the blood-brain-barrier. We have not been able to generate BomS6 null mutants so far. In this case, the relevant response may not be systemic. We only detect a weak signal for BomS peptides in the hemolymph of unchallenged flies, making it unlikely that a basal expression is important, at least as regards a systemic infection. We cannot however exclude local inductions at the level of tissues. This would not rely on hemocytes as “hemoless” flies are not susceptible to A. fumigatus or toxin challenges. This topic definitely warrants further investigations.

In Fig 5, it looks like DMSO is nearly identical to A. fumigatus, so can the authors really suggest that equal induction to DMSO is relevant?

We had stated that an induction of the Bomanins by the injection of DMSO alone precluded us from analyzing the effects of verruculogen on Bom gene expression. We have now bypassed this difficulty through direct challenges by the undissolved powder (Fig. 6_J-K,_ Fig. EV11).

The authors' discussion of these points would benefit from considering Vaz et al. (2019; Cell Rep) to frame how much PAMP is injected given equal numbers of fungal cells vs. bacterial cells. To me the lower induction by injecting a few fungal cells with much lower surface area to volume ratio means equal microbe mass has exponentially less PAMP in fungal conidia cell walls (2-3um diameter) vs. equal mass of bacteria (0.5-1um diameter).

We fully agree with the reviewer and now mention that C. glabrata also led to a milder induction of the Toll-mediated humoral response (Quintin et al. JI, 2013). In addition, it has been shown previously that ß-(1-3)-glucans, which are sensed by GNBP3 in Drosophila (Gottar et al., Cell, 2006), are concealed by the cell wall (germinating conidia) or hydrophobins (Wheeler et al., PLoS Pathogens, 2006; Aimanianda et al., Nature, 2009) . In the case of yeasts, these glucans are accessible only at the budding scar (Gantner et al., EMBO J., 2003).

Fig S1O is not convincing that Boms alone are present. There is significant noise near Drs in FigS1 infected, which likely saturates the detector before Drs can fly to it. I say this because DIM4 (Daisho) indicates that Toll is strongly induced. The authors should show a larger mass range on the x-axis including peaks of other Toll-induced peptides like the BaramicinA DIM10, DIM12 and DIM13 peptides of their companion paper and DIM14 (Daisho), which are closer in mass to the Bomanins and less likely disrupted by the noise at 4300 m/z. The maldi-tof calibration to correct ranges is critical for arguments of quantification.

We provide the primary data in the Rebuttal figures at the end of this document. These are the results obtained from three single flies (Files A29683PBUG22, A29684PBUG23 and A29684PBUG24). The first three spectra correspond to the full scale based on the major peaks observed (DIM4/BomS5) in two out of three spectra. At this scale, no signal is visible for Drosomycin at 4891 and the “noise” at 4278 is modest. Next, the multi-spectra report allows to put all three samples on the same sheet, this time zooming on the peaks of interests in the region 4300 (“noise”) and 4891 (Drosomycin). Finally, the next two pages zoom in on the BomS peptide signals and the next page keeps the same scale to document the 4300-5000 region. On the last page, it is obvious that the signal around 4300 is very modest and too distant to influence the Drosomycin ion, thereby excluding any effect of suppression. Of note, in the systemic immune response, Drosomycin is the most induced AMP with a concentration estimated to be around 0.3µM, an order of magnitude higher than other AMPs. Finally, these experiments have been performed by PB who initially developed the technique (Uttenweiler-Joseph, PNAS, 1998) and has been using and developing it ever since.

Combined with comments in Major Concern 1, I am not convinced that the -inducible- Bomanin response mediates the resilience phenotype.

Besides our replies above, we do hope that the new data we have included in Fig. 6 that document an induction of only two BomS genes in the heads of Drosophila upon verruculogen and the finding that BomS6 expression in the nervous system protects the fly from the effects of verruculogen will convince this reviewer.

3) The author's language is very strong to disregard a possible antimicrobial activity.

As noted above, this is a misunderstanding that we hope is dispelled in the revised discussion (see also above and replies to Reviewer 1).

Previous studies showed increased Candida growth and decreased hemolymph killing activity in Bom55C flies (Lindsay et al. 2018 and Hanson et al. 2019).

Please, see reply above. Factually, Lindsay et al. did not study the C. glabrata titer in vivo but using collected hemolymph. The killing activity likely requires a cofactor regulated by the Toll pathway. Hanson investigated the burden of the dimorphic C. albicans pathogen that in flies is filamentous and not C. glabrata.

Also see minor concern (i).<br /> I grant that the data are consistent with a resilience role. However the authors found no binding of Bomanin to restrictocin, countering their idea of a -direct- anti-toxin effect.

We are surprised by this comment. We certainly did not favor this idea nor developed it in the original manuscript, even though we cannot formally exclude it at this stage. Future experiments will focus on BomS6 potential interactions with these two mycotoxins.

At present the authors cannot rule out a direct antimicrobial role, or even the possibility of two different roles for the same peptides (ex: one in resilience, one antimicrobial). For instance, it is difficult to explain the loss of killing activity of Bom-deficient hemolymph ex vivo from Lindsay et al. if Bomanins are strictly anti-toxins. Surely they must also do something generalist?

Please, see our replies above and the paragraph dedicated to this topic in the Discussion.

4) In most figures, the authors do not compare flies with shared genetic backgrounds.

The MyD88 allele we are using is a transposon insertion from the Exelixis collection and we are using the wA5001 strain that was used to generate the collection of insertion (Thibault et al., Nat. Genetics 2004). We thank the reviewer for this comment as we realized we had forgotten to mention the Bom__D__55C strain. Lines 603-604 state that the deficiency line has been isogenized in the wA5001 background.

The phenotypes are usually strong so I am not concerned.

However the rescue effect of Bom transgenes in Fig 5C-D is based on smaller differences. Were these genetic backgrounds controlled?

Yes, as much as we reasonably could. The fact that most BomS transgenes did not rescue gives further confidence in the data.

Were transgenes inserted at the same site?

We used the strategy for overexpression developed by the Basler laboratory (Bishof et al., Development 2013, Nat. Protocols 2014) that relies on insertions at the same site.

The authors seemingly used a heat shock to express transgenes.

Heat-shocks are usually a short exposure to higher temperatures, usually 37°C. Here, we have used the inducible Gal4-Gal80ts system developed by McGuire and Davis (Trends in Genetics, 2004). The Gal80 repressor inhibits Gal4 function at the permissive temperature (18°C) and becomes inactive at the restrictive temperature (29°C). Thus, we use a temperature shift and not a bona fide heat shock.

Given a resilience effect is being studied, this heat stress approach is sub-optimal. Earlier experiments showing effect/no effect of Bomanin on heat shock resilience would improve confidence here. I would recommend assaying temperatures that can kill wild-type in order to confirm that Bom do not succumb earlier (ex. up to 37'C).

The results have been discussed above and show that 29°C is not a concern for Bom__D__55C and not much of a significant problem as regards MyD88.

In Fig5C the time resolution is poor, and the effect inconsistent across Bomanins. What are the differences in the Bomanins that the authors suspect could cause this? And how consistent are the experiments?

We provide all the primary survival data in Rebuttal Fig.1 A-H. The partial protection effects of BomBc1, BomS3 and BomS6 against restrictocin are consistent in the three independent experiments (Fig. 5D and Rebuttal Fig. 1 A-B). As regards the seven independent experiments performed with verruculogen, we observed a strong protection conferred by BomS6 expression in six experiments whereas we detected a milder protection conferred by BomS1 in four out of seven experiments and no protection in the three other ones. The effects were always there after 24 hours, in keeping with our novel data showing that BomS6 expression allows a faster recovery, around 10 hours, from verruculogen-induced tremors (Fig. 6E-F).

Since the effect is finished by 24h, perhaps a boxplot of percent survival at this time would better show the consistency across experiments.

Given the argument presented just above and considering that this rebuttal letter will be published alongside the article, this may not be needed.

Minor concerns:

i) The authors say the fungal burden of Bom55C flies remains low in Fig 5B, but they never measure flies that are near death when fungal load is greatest, or FLUD like in other figures. Given low mortality at the following time points, it seems likely that A. fumigatus would grow beyond initial loads in those individuals and kill them. I grant that these loads are less than what is seen in Hayan mutants. I just might suggest a more careful consideration of the time points used and what can be said about the trends shown here.

This is certainly a relevant point. The FLUD data are now presented in Fig. EV8_A_ and do not reveal any additional growth.

ii) Could the authors comment somewhere about the levels of toxin they were required to inject to get a phenotype vs. the level of toxins the authors expect are found in the fly during infection? I appreciate that toxin injection likely requires much higher doses, but it would be good to know just how far the authors have pushed their experimental system beyond its natural range.

This a question that is difficult to answer accurately as we are not sure the techniques exist to measure toxin levels in these small flies. We have tested a range of concentrations. It is clear that we push the system and likely use concentrations that are higher than those actually secreted by A. fumigatus during infection. Indeed, the mutant strains defective for the production of verruculogen or restrictocin display only a mildly reduced virulence in MyD88 flies. This makes it even more remarkable that wild-type flies are able to withstand these high, unphysiological concentrations, an argument for an indirect effect independent of the dose as pointed out now in the Discussion. How fungal pathogens balance the expression of the hundreds of secreted virulence factors, proteins and secondary metabolites, is a major frontier for future investigations be them plant or animal pathogenic fungi/

Again regarding toxins vs. general stresses, one could manage to inject salt into the hemolymph and show a stress-sensitized fly would succumb at lower doses than wild-type, emphasizing the relevance of defining concentrations.

We feel that just monitoring the survival of flies after a challenge that produces an effect is sufficient (Fig. EV8_C_).

The authors could also write toxin concentrations clearly in the figure/legend per experiment.

Corrected.

iii) Throughout the manuscript, the order that figures/panels are cited is inconsistent. Perhaps the text could be re-written so the reader can follow the figures more intuitively while going through the text?

Corrected.

iv) There are a few points where run-on sentences, involving many commas, make it hard to follow the logic. I might suggest a careful reading to break up long sentences into two sentences to ensure clarity.

We hope to have addressed this concern.

v) Line 279-281: this is the first and only mention of the immune surveillance hypothesis in nematodes. This is strange, given the authors are effectively describing an analogous idea exists in flies? Perhaps this could be added somewhere in the introduction or discussion.

We have followed the advice of the reviewers and now discuss this point more fully in the Discussion under its own subheading.

Small points

• What timepoints are the gene expression data from? Could the authors indicate this in figures/legends?

Done

• Line 133-135: "We conclude that MyD88 flies succumb to a low A. fumigatus burden..." - could the authors cite a figure panel here to emphasize what evidence they're referring to.

Done

• Line 151-152: Dudzic et al. (2019- Cell Reports Figure 3) showed that PPO2 was regulated by Hayan, while PPO1 by Sp7. This relevant study should be cited here or in the introduction/discussion.

Excellent suggestion, this was indeed an important study. Done

• Line 179-180: could the authors define the gliotoxin mutant strain here in the text for clarity?

Done

• Line 196: Fig. 4A-B should be Fig. **S4 A-B?

Corrected.

• Fig4A: perhaps the authors could reduce the x-axis to focus on the early time points? If I understand correctly, aspf1 has slightly delayed killing compared to akuB (˜50% difference at 2 days), but both kill 100% by 3 days.

Done

• Fig4G: can the authors define the GFP transgene on pg10? Not clear what this is, or what this means. Brain? Fat body? The legend of Fig4G and the key in the top left... it's not easy to quickly understand what is shown in Fig4G.

Done

• Line 247: I would drop the "at the intracellular level" part. I'm not sure this is robustly shown given the use of an in vitro model where there is no closed extracellular environment. The data are convincing of the effect, this is just a semantic point.

We agree that there is no closed extracellular environment and that therefore any signal emitted by the cells might get too diluted. However, the addition of EGF will activate the Toll intracellular through the chimeric EGFR-Toll receptor. As restrictocin is known to act intracellularly, one might have though that there might be some intracellular effectors mediating the Toll-dependent protection against restrictocin. Our sentence excludes this possibility.

• Line 257-258: Cohen et al. (2020- Front Imm) never used Bomanin mutants. Did the authors mean to cite Hanson et al. (2019) here, which seems to fit their described citation re: Bom55C vs. Toll mutant flies (Fig. 2)? Given Hanson et al. infected Toll mutant and Bom55C flies with many bacteria/fungi including A. fumigatus, it's strange this study is not discussed currently.

The reviewer is correct. Cohen et al. did use A. fumigatus, but on Daisho mutants and MyD88 and not Bom__D__55C as a control. We are now citing Hanson et al., 2019 in lines 443-449 (Discussion).

• Fig5C-D: the labeling is difficult to follow.

This is difficult to address unless multiplying EV figures. We feel this is not needed: the important curves are in color and each such curve is seen on the graphs.

• Line 318: a -possible- AMP role of Bomanins was proposed because of the aforementioned killing activity of wt but not Bom mutant hemolymph, alongside rescue by single Bom genes. To say this was based only on survival experiments is incorrect.

The paragraph has been rewritten and expanded to dispel any misunderstanding.

• Line 324-328: could the authors cite appropriate references after "inhibition of calcium-activated K+ channels" ?

Done

• comment re-Line 334: Toll10b flies have melanotic tumors and are in general in a stressed state. Might their rescue be due to increased stress tolerance by pre-activated stress responses?

This is a developmental effect occurring during larval stages, also observed for Cactus mutants. Here, we use a UAS-Tl10B transgene that is induced only at the adult stage using the Gal4-Gal80ts system. Thus, any stress is minimized as much as possible. Furthermore, we can phenocopy this phenotype to a large extent using a UAS-BomS6 driver, even though the phenotypes are subtly different as regards the protection against verruculogen-induced tremors.

Referees cross-commenting

Yes I agree that the data themselves are not the issue, nor even the direction of the results. But there are many overly-strong statements that go so far as to refute ideas which are supported by other studies, and for which the authors here do not provide any contradictory evidence.

We hope that this revised, extended version has clarified any misunderstanding in the initial version.

As per my review, I would be happy with a re-write that softened the language overall. I genuinely wonder if these Bomanin mutants simply have poor development, and so they are susceptible to neurotoxins/stress because their nervous system/development leaves them less resilient in general. Experiments testing their resilience to different stresses would greatly elevate the ability to make confident insights in the present manuscript. Currently the authors have only investigated one type of phenotype and interpreted it as if that is evidence of the evolved purpose of the peptides. This approach does not account for many other possible (and reasonable) explanations.

We have performed the experiments suggested by the reviewer. While we see a modest effect of heat on MyD88, it is not found in Bom__D55C flies, which display essentially the same phenotype as MyD88 with regards to the sensitivity to A. fumigatus or some of its secreted mycotoxins_._

Reviewer #2 (Significance):

This paper should be of broad interest to the study of immunology, where roles for effectors are typically thought of as cytokines. In fruit flies and other invertebrates that lack adaptive immunity, immune effectors are more thought of as direct actors likely with antimicrobial properties. The finding that Toll might mediate resilience is interesting, and implicating well known Toll effectors provides an important step forward towards a mechanistic basis behind this resilience effect.

We thank the reviewer for his appraisal of the significance of our work.

My expertise is in insect and innate immunity.

Is there one or more voices? What are the voices saying? How loud are the voices? Are the voices telling negative things about you?

In addition to repeating your message, body movements can also regulate conversations. Nodding your head to indicate that you are listening may encourage the customer to continue asking questions. Holding your hand up, palm out, may signal them to stop and provide a pause where you can start to answer.

I'm struck by how often hair is described this way in Hurston's novels (straight/loose curls) partially or rarely covered. I'm not sure what to do with this fact.

The setup of this reminds me of the courtroom scene in Their Eyes Were Watching God, where Janie is tried for killing her husband

Brain is expressive query system that, given some constraints about some information, is able to retrieve lots of associated information from context. It's notably fairly difficult to demand precisely the information you want to receive.

Reference?

Importance of educators to implement the same tools of education that benefit white students. Thinking about the traditonal concept of education as white supremacy followed by damaging narratives from the mainstream media about black youth. An example of this off the top of my head is thinking about gun violence in chicago and how often the news is always reporting shots on the southside of chicago, when something happens vise versa it does not get as much coverage and I think this is to protect the "image" of the community that it is flawless and perfect which is a damaging concept.

Reviewer #1 (Public Review):

In this manuscript by Feng et al., the authors investigate the mechanism regulating the development of the levator veli palatini (LVP) in the posterior palate/pharyngeal region. While set up as a model to understand how myogenic progenitors migrate to discrete sites to form individual muscles, it is not clear how applicable the findings are to other subpopulations, though this is not a weakness. The mechanisms driving LVP development are of great interest to a broad group of developmental biologists, as LVP malformation is a common problem even in mild cases of cleft palate. The authors hypothesized that the perimysial population within palatal mesenchyme cells is a niche required for pharyngeal muscle development. Using exquisite analysis of scRNA-seq data from E13.5-E15.5 palatal cells, the authors illustrate that TGFb signaling is likely involved in perimysial cell development, using gene expression analysis in wild-type palatal sections to show that TGFb signaling precedes the arrival of myogenic cells. Inactivating ALk5 in palatal mesenchyme cells results in failure of LVP formation. The authors continue by identifying a number of transcription factors that presumably function downstream of TGFb signaling that drive LVP development. Among these are Fgf18, in which SMAD sites observed in the upstream region were validated to bind Smad2/3. The authors also identify Creb5 as a potential regulator of Fgf18. Overall, this is a remarkable use of scRNA-seq data, in which findings are supported by subsequent in vivo analysis of gene function using knockout mouse models. These findings will drive further analysis of LVP development and may shed light on the myogenesis of pharyngeal muscle in general.

Strengths

1) The treatment of scRNA-seq data using a variety of bioinformatic programs illustrates the utility of this type of data when using sufficient analysis software. The description of the approach is very clear and concise and the controls appear excellent. Further, the use of multiple time points further improves the analysis.

2) The focus of perimysial cell expression patterns supports the hypothesis of the authors, though as with this type of data, one probably can make a story out of several pathways. The use of RNAscope to carefully examine where TGFb signaling in the posterior pharynx occurs between E12.5 and E16.5 is critical to the setup of this manuscript and is well done. Further aiding the interpretation of these results are cartoons associated with the staining, which illustrate where the staining is occurring, though never over-stating the observed patterns.

3) Careful histological analysis illustrates the poor myogenic differentiation in the LVP of OSr1-Cre;Alk5fl/fl embryos.

4) Identifying that TGFb is more important for regulating late perimysial cell development is important in identifying the targets of TGFb signaling.

5) The use of CellChat to identify sending and receiving cells is well done and further supports the late function of TGFb signaling, in this context working through Fgf18 and Lama4.

6) The attempt to build a signaling network again using CellChat (Figure 6) is admirable, though there are a few caveats to that approach (see below).

7) While bead implant studies have been used for 40 years, the approach of culturing a piece of the pharynx and then performing a bead implant to prove that Fgf18 can positively influence myogenic development is admirable.

Weaknesses

1) In general, the authors are careful to not suggest that staining is significant unless showing quantification, though, at several points, this is not true.

2) The authors identify five putative Smad2 sites upstream of Fgf18, using one of them in a Cut and Run assay whose results suggest enhanced Smad2/3 binding. The problem is that this likely would have worked with the other Smad sites and probably would have worked for any other putative site that one might pick. Proving that a putative site can be bound by its cognate transcription factor is not the same as proving that this occurs in vivo and is sufficient to control the process of LVP development. One would need reporter assays using that TF binding site to better support the points being made by the authors.

3) In a similar manner, the authors try to define which factors might function with TGFb signaling to regulate myogenic development. Using SCENIC, the authors found a number of genes that might be involved in perimysial fibroblast development. Of these, they illustrate that Creb5 siRNA knockdown decreases Fgf18 expression in cultured palates. The focus on Creb5 was based on it showing, "the most specific expression patterning the late perimysial cells (Figure 6H)....". In fact, Creb5 appears the most broad, appearing to be expressed across the entire LVP, not just in the area where myogenic precursors are found. Thus, any statement or discussion about Creb5 being a direct regulator of Fgf18 should be removed probably needs to be reworded. However, the second problem is that Creb5 knockdown reducing Fgf18 expression does not prove any direct regulation. Both of these are rather circuitous arguments.

4) While the disorganization of myogenic fibers in the posterior LVP is somewhat obvious, it is not as clear as the authors suggest. This change (which I believe) needs to be better quantified (length, width, area, etc.).

We thank the reviewer for these “Public Review” comments. For point 1, we have added more quantification for clarification and rephased the wording when quantification was not performed. For point 4, we added measurement to quantify the changes of volume and cross-section area of the LVP in Osr2Cre;Fgf18fl/fl mice (Figure 7M-V).

Reviewer #2 (Public Review):

In this study, the authors take advantage of unbiased scRNA-seq datasets of the developing mouse soft palate that they previously reported and performed a new bioinformatic analysis to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. They found a strong association of TGF-beta signaling pathway activity with the perimysial cells and validated through immunofluorescent detection of pSmad2, which led to their hypothesis that TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. They generated and analyzed Osr2-Cre;Alk5fl/fl mice and showed those mice have cleft soft palate and disruption of the levator veli palataini (LVP) muscle. They then performed a comparative scRNA-seq analysis of the soft palate tissues from E14.5 Osr2-Cre;Alk5fl/fl and control embryos and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited defects in the perimysial cells, in particular reduction in Tbx15+ perimysial fibroblasts that directly associate with the LVP muscle progenitors. The FGF18 is one of the most highly enriched signaling molecules in the perimysial cells and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited reduced Fgf18 expression together with loss of MyoD+ myoblasts in the prospective LVP region. Further data showed that pSmad2 bound in the Fgf18 promoter region in the developing soft palate tissues. In addition, bioinformatic gene regulatory network analysis of the scRNA-seq data identified Creb5 as a potential tissue-specific transcription factor in the perimysial cells and RNAi knockdown assays in palatal mesenchyme culture suggested that Creb5 is required for Fgf18 expression. Further studies identified a subtle deficiency in LVP in Osr2-Cre;Fgf18fl/fl mice and showed that exogenous Fgf18 bead implantation in explants of E14 Osr2-Cre;Alk5fl/fl embryonic head increased the MyoD+ myoblast population in the prospective LVP region. The authors concluded that TGF-beta signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development. While the study used multiple complementary approaches and the data presented are solid, important questions need to be addressed to resolve reasonable alternative explanations of the data to the authors' main conclusion.

We thank the reviewer for the evaluation and suggestions. Responses to each of the suggested revisions are detailed below.

Major points:

1) TGF-beta signaling is known to be crucial for neural crest-derived palatal mesenchyme cell proliferation from E13.5 to E14.5. The Osr2-Cre;Alk5fl/fl mutant embryos exhibited obvious disruption of LVP myogenesis and reduced soft palatal shelf size at E14.5 (Fig3-Sup2A-D and Fig 4H-K). The cellular and molecular defects likely started prior to E14.5. Thus, it is important to examine at earlier stages (E13.5/E14.0) whether the palatal mesenchyme was already defective in cell proliferation/survival and/or perimysial cell marker expression, including Creb5 and Tbx15, to resolve whether the primary defect in the Osr2-Cre;Alk5fl/fl palatal mesenchyme could be a reduction in perimysial progenitor cell proliferation and/or differentiation of the myoblast-associated subset, for which Tbx15 and Fgf18hi act as marker genes rather than direct molecular targets. Furthermore, the apparent loss of Tbx15+ cells coincided with a specific reduction of Fgf18 expression in the myoblast-associated perimysial cells (Fig 4J/K versus Fig 5H-K), which raises the possibility that TGF-beta signaling regulates the differentiation of the Tbx5+ population from the mesenchymal progenitors while the reduction in Fgf18 expression might be a secondary consequence of the cellular defect. The data in Fig 6O showing a lack of significant induction of Fgf18 expression in the palatal mesenchyme culture in both control and Creb5-RNAi cells is also consistent with this alternative explanation.

We thank the reviewer for the valuable suggestion to identify the primary defects of the perimysial cells. We compared the expression of Creb5, Tbx15 and Fgf18 as well as Smoc2 in E13.5-E14.5 palatal mesenchyme from control and Osr2-Cre;Alk5fl/fl mice (Osr2Cre;Tgfbr1fl/fl mice). We found that expression of Creb5 is prominent from E13.5 to E14.5 and is not affected in Osr2Cre;Tgfbr1fl/fl mice, suggesting that Creb5 may not be a downstream factor but just a “partner” for TGF-β signaling. At E13.5, Tbx15 is not expressed, while Smoc2 is expressed extensively in the palatal mesenchyme but is not affected in the Osr2Cre;Tgfbr1fl/fl mice. In contrast, Fgf18 is expressed as early as E13.5 and this expression was already reduced in the palatal of Osr2Cre;Tgfbr1fl/fl mice relative to controls at this stage, suggesting the changes of Fgf18 expression are primary and precede changes in the perimysial populations. While the proliferation and apoptosis at E13.5 remain unchanged in Osr2Cre;Tgfbr1fl/fl mice, Smoc2 expression in the palate starts to be reduced at E14.0 in Osr2Cre;Tgfbr1fl/fl mice. This suggests that TGF-β signaling is required for the activation of Smoc2 during E13.5-E14.0. In parallel, Tbx15 expression is just starting to be activated in a few cells at E14.0 and this expression increased between E14.0-E14.5 in the control but failed to increase in Osr2Cre;Tgfbr1fl/fl mice. This suggests that TGF-β signaling is also required for the activation of Tbx15 during E14.0-E14.5. Thus, loss of TGF-β signaling leads to differentiation defects of both Smoc2+ and Tbx15+ perimysial cells. For Figure 6O, we performed a time-course experiment of TGF-β induction and found a significant increase of Fgf18 expression after 4 to 18 hours of treatment (instead of 24 hours used in previous experiments), with more obvious changes at 4 hours, confirming the early response of Fgf18 expression to TGF-β induction. These results have been added to Figure 4-figure supplement 2, Figure 5I-L, 5U, Figure 6-figure supplement 2, and Figure 6C.

2) Since the Osr2-Cre;Fgf18fl/fl mice exhibited much subtler palatal and LVP defects than the Osr2-Cre;Alk5fl/fl mice even though the latter still had a lot of Fgf18-expressing perimysial cells at E14.5, Fgf18 is likely a minor player in the TGF-beta mediated gene regulatory network regulating LVP formation. The major players acting downstream of TGF-beta signaling in the palatal mesenchyme, that control initial LVP progenitor migration to and/or proliferation in the soft palate region, remain to be identified and functionally validated. Whether and how Fgf18 directly regulates the perimysial-myoblast interaction is also not known.

We agree with the reviewer that the phenotype of Osr2-Cre;Fgf18fl/fl mice is much milder than that of Osr2-Cre;Alk5fl/fl mice, as we postulate that Fgf18 is just one of several perimysial-derived signals that may be affected. It will be of great interest to explore the function of other players in future studies. However, we are more inclined toward the possibility that there may be no single “major” player but rather a combination of many signals associated with different aspects of the muscle development. For example, loss of Fgf18 seems to mainly affect the Myf5+ cell proliferation in Osr2-Cre;Fgf18fl/fl mice (Osr2Cre;Fgf18fl/fl mice), as we do not observe any differentiation defect except the reduced muscle size. It is likely that other factors may also play specific functions in specific subpopulations as well. To clarify whether Fgf18 can directly affect the myogenic cell fate, we treated C2C12 mouse myogenic cells with exogenous FGF18 and found that this treatment could indeed significantly increase the proliferation of these cells. We have added these results to Figure 7—figure supplement 2.

3) While the title and the main conclusion of this manuscript imply a crucial role of Creb5 in the regulation of pharyngeal muscle development, there is no data supporting such a crucial role. Do Creb5-/- mice have specific defects in pharyngeal muscle development?

We thank the reviewer for this insight. We agree that it is very likely that Creb5 itself may have many roles in the regulation of palatal development or pharyngeal muscle development, given the prominent expression of Creb5 throughout soft palate development and in other myogenic sites of the pharyngeal muscles. Creb5-/- mice (reported as Cre-bpa-/- mice) die immediately after birth; however, the detailed phenotype of this mice was merely described as “data not shown” in a previous publication and defects of craniofacial development in these mice remain unclear (Maekawa et al., 2010). In this study, we focused on the role of Creb5 as a partner of TGF-β signaling, but we plan to generate a Creb5fl/fl mouse model to thoroughly evaluate Creb5’s functions in craniofacial development as an independent study following this work.

4) Data in Fig 6 are not sufficient to conclude that TGF-beta signaling and Creb5 cooperatively regulate Fgf18. The TGFb1 treatment did not significantly induce Fgf18 expression in either the control or Creb5-RNAi palate mesenchyme cells (Fig 6O). No data regarding how they act cooperatively to regulate Fgf18 expression.

We appreciate the reviewer for carefully reviewing our data. We re-evaluated the temporal response of Fgf18 expression following TGF- induction and found a significant increase of Fgf18 expression 4 hours post-treatment (instead of 24 hours post-treatment as used in previous experiments). We repeated the Creb5-siRNA treatment experiment using the new experimental condition and replaced the previous Figure 6O with new results showing a significant increase of Fgf18 after TGF-β induction, which was attenuated by Creb5-RNAi treatment, suggesting a requirement of Creb5 for TGF-β-mediated Fgf18 expression. The new result is now included in Figure 6Q.

Reviewer #3 (Public Review):

In this study, the authors investigated cell-cell communication between perimysial cells and skeletal muscle progenitors during soft palate development in the mouse. The authors have previously reported on the development of this structure and here they propose that a TGF-β signaling and Creb5 act to regulate Fgf18, and this pathway regulates pharyngeal muscle development through the indicated cell populations. The study is of high quality, very nicely illustrated, and uses multiple approaches including inferences from single cell transcriptomics, validations on sections, and lineage-specific gene activations. In addition, the authors successfully optimized an organ culture system from thick sections to test locally the role of FGF signaling (bead implantation). The results largely confer with the conclusions and provide a valuable example of how subjacent cell populations cooperate to establish an embryonic structure.

We thank the reviewer for the evaluation and suggestions.

This shows the impact that it had in shooting the animal, and how it stuggling to stay up right or just laying down resulted in it being shot again.

The two visualizations below both share the general overview of the landscape in contrast to the university however the second map goes more in detail of the campus. I can see how the second map is more appealing to someone who may be new to the area and is need of more detailed visualization but the first is useful in its own right as it allows for a user to quickly identify key areas such as the Kailash Mital Theatre that you wouldn't find to quickly on the second map.

I will also agree with bhanhart that the second map may be more appealing to people with anxiety as it solves a lot of issues that they may face such as wanting to know where parking is or the general walking paths that others may take, etc...

As with my personal experience at UNBC , I would prefer the second map if I were a new resident in the area but as I would get more antiquated with my area I would slowly transition to my memory as a mental image of the mapping would begin to integrate into my head. (Which is something a lot people take for granted!)

喪失動力時的自我診斷：

• 腦：這項任務對我真的重要，且與我的目標相關嗎？
• 心：我喜歡這項任務嗎？
• 手：我擅長這項任務嗎？

發現自己缺失的元素，然後便可依其制定策略與行動

Their punishment is rather twisted... I have picked up on they are long and drawn out. They want the most pain to be felt before death. Nothing quick and easy.

why?

I found this really interesting because I've seen roses used as symbolism before with vampires however i've not actually seen it used as a deterrent in modern vampire media

Bruh you could've put that lightly

"Is Teddy gonna talk about the vampire scare again?" Yes yes they are. This was a real thing that was done to suspected vampires, even when there was a degree of uncertainty as to whether they were vampires or not, so that they would not come back and make havoc on a town. They would exhume bodies postmortem from their graves and cut their heads off and hearts out among other gory procedures, not always all of the possible procedures as there was a regionality to these practices.

Using the similar function from NLTK, "throat" in Dracula is similar to the following words:

eyes, face, heart, room, hand, place, head, neck, lips, life, forehead, soul, and, time, sleep, door, diary, husband, work, love

See About the Project page for more info.

Using the similar function from NLTK, "lips" in Dracula is similar to the following words:

eyes, face, horses, hand, head, arms, cheeks, heart, throat, others, room, house, blood, feet, ship, left, way, work, count, bed

See About the Project page for more info.

Using the similar function from NLTK, "garlic" in Dracula is similar to the following words:

time, head, hand, place, way, door, room, eyes, fear, heart, tomb, anxiety, morning, east, books, light, work, castle, south, world

See About the Project page for more info.

Although aspects of Van Helsing's physical being are mentioned in earlier parts of the novel i.e. bushy eyebrows, this is the first time we get a fully detailed description on Van Helsing's appearance. It is an interesting choice to have a female character be the one to account for these details. Mina's glamorized recounting of Van Helsing perhaps leans into the plausibility of Van Helsing being a hero one can romanticize while reading. His physical being is in stark contrast to that of Dracula's who Jonathan Harker described with words like "peculiar" , "cruel looking" and "extraordinary pallor". The image of Dracula has definitely been sexed up in later adaptions but Stoker made it a point to distinguish the Count as a monster among men. The Vampire Almanac states that the film and stage versions of Dracula drastically altered the image of the vampire so as to be more openly accepted in to middle-class British society.

Dracula was so stupid angry that he couldn't drink Lucy that he * tried to summon wolves * but no wolves appeared * so he stomped all over London * finally found a zoo * summoned one (1) wolf * lured it all the way back to Lucy's house * and rocket launched it through her window

original post

original

First almost bite is played out in a seductive manner. Almost as if describing a passion filled kiss. We see Jonathan Harker repulse in fear but also feel stimulated for the act that is about to occur. The lustful nature of a vampire bite is one that is often emphasized in other popular vampire works.

The fact they molested, mutated and killed a goof-off black boy with no bodily strength or power to fight back, just in an effort to "how blacks where they belong" is discusting and horrifying.

The singer is shown to have this echo of Weary Blues playing in his head as he sleeps, suggesting that the song has a larger meaning for him. By comparing the sleeping man like a dead one, it calls back to the song and the line, "'I ain't happy no mo'/ And I wish that I had died.'". Again, this shows the way that the singer's song is much more personal to them and shows more of his soul.

Improve economic efficiency, but also surveillance

I really like the spacing between your sections even though you don't need to. It definitely makes it easier to read!

the code is very clean and there is also mention in comments like which part starts from where and it looks very good so that anyone can know which coding is for which thing

I had no idea that what I do has a term to it but I do tend to find myself king of traumatizing myself when I get reeled in to these kind of things. However, other times, when I scroll into bad news off guard, I then try to forget it but the thought will still linger in my head.

missing head and closing head before the body tag

eLife assessment

The paper will be of interest to cognitive neuroscientists in the field of spatial navigation as well as to systems neuroscientists interested in neural representations. Using simultaneous electrophysiological recordings in the anterior thalamus and the retrosplenial cortex, the study investigates the coordination of neurons coding for the head direction in this thalamocortical network. Environmental manipulations led to a near-synchronous update of the head direction signal encoded by the two populations. Further data analysis is needed to support the main claim of the study.

Reviewer #1 (Public Review):

The authors convincingly show directionally tuned signals in AD and RSC. RSC is found to have a lower proportion of HD cells than AD, and RSC HD cells are more sensitive to angular velocity than AD HD cells. Importantly, HD responses are shown to be tightly correlated between the two areas. Population decoding of head direction, performed on AD neuron ensembles or RSC ensembles, revealed similar shifts following visual cue rotation and also similar HD drift in darkness, indicating that the HD representation across both areas is coordinated. The study further finds that AD-to-RSC connections are relatively frequent, while RSC-to-AD connectivity is very sparse. This asymmetry in functional connectivity is matched by viral tracing results. Together, the results lead the authors to the conclusion that this corticothalamic connection is likely not driving visual landmark updating of the global head direction system.

This is a welcome piece of work, providing the first assessment of the high degree of coherence between AD and RSC HD representations, using pairwise and population-level analysis methods, which had not been accessible before. It will be a valuable reference for researchers interested in inter-area interactions in the head direction system, leaving the question of how and where visual reference updates are fed into the HD circuit open for further investigations.

Reviewer #3 (Public Review):

The work provides direct evidence for the coherent activity of head-direction (HD) cells in the anterior thalamus and retrosplenial cortex (RSC). RSC is one of two major direct cortical recipients of the subcortical HD signal, the other being the postsubiculum (POS). While it is established that POS inherits its HD tuning from ADN (Peyrache et al, 2015), it is not known whether HD cells in RSC show similar coordination with ADN. The manuscript employs technically challenging dual electrophysiological recordings from ADN and RSC to establish that the local internal representations of HD encoded in ADN and RSC are coherent during free exploration but also show coordinated realignment after cue rotation as well as coordinated drift in darkness. The work thus provides evidence that HD and RSC assemblies represent the same internal heading direction, at least in the behavioural paradigms tested and at the investigated temporal resolution. The manuscript also makes a claim that the RSC is unlikely to mediate the realignment of the HD signal following cue rotation because the HD signal realigns itself synchronously across the two brain regions. This claim is additionally supported by the sparse anatomical projection and the paucity of putative direct synaptic connections from RSC to ADN.

The manuscript convincingly demonstrates overall ADN-RSC coordination in two different paradigms. While such coordination is expected in instances when HD representations in both areas are precisely aligned with the current HD, it may not be the case in instances of sensory conflict or limited sensory information. The fact that internal HD in both ADN and RSC drifts coherently in darkness provides strong evidence of the tight functional coupling between the two areas. Additionally, while the cue rotation paradigm used in the study often failed to elicit the full realignment of the HD signal, this variability was certainly utilized to the manuscript's advantage as it makes the coupling evident even when the HD signal realigns only partially. The overall conclusions of the manuscript are largely supported by the presented data but the strength of the argument, especially with regard to the zero-lag coupling between ADN and RSC, is somewhat affected by the technical limitations.

1) The manuscript relies heavily on supervised decoding of the internal HD from population activity in RSC and ADN and in turn suffers from relatively low numbers of simultaneously recorded neurons, which is especially evident in the representative images in Figure 2C. The reported average decoding errors are much higher than those reported elsewhere (Peyrache et al, 2015; Viejo et al, 2018; Xu et al, 2019), which may occlude the effects of RSC activity on ADN that are more subtle and/or occur at shorter timescales than the bin size used in the decoding algorithm. The manuscript includes no discussion of how much these factors could contribute to the observed variability in the data.

2) RSC-HD cells recorded in the study are relatively poorly tuned to HD, which is contrary to the reports of HD cells recorded in RSC (Lozano et al, 2017; Javob et al, 2017; Keshavarzi et al, 2021). In fact, the median directional information score for RSC-HD cells is the same as that for non-HD cells in ADN (Supplementary Figure 2B). In fact, due to their relatively low HD modulation, it may be more appropriate to refer to them as 'HD-modulated' cells. While the electrode positions indicate that RSC was sampled across layers and sub-regions so missing the HD cell 'hot spots' like granular RSCb is unlikely, the apparent poor directional tuning of RSC cells could possibly be due to the nature of the recording environment (e.g. low light condition with the LED landmark being the only light source). Importantly, the manuscript lacks a control 'baseline' condition in which HD cells are recorded in a standard, well-lit open field, as well as a discussion of the discrepancy between the observed HD tuning and that reported in the literature.

3) Analysis of decoding error, which features prominently in the manuscript, is critically dependent on the quality of behavioural tracking - errors in tracking could lead to the accumulation of decoding errors and this could dominate decoding error analyses. Indeed, Figure 2A shows many gaps in the tracked HD of the mouse, which may point to the sub-optimal quality of the behavioural tracking. This is especially important for analyses like the one in Figure 2D which shows that internal HD representations in ADN and RSC are coordinated at zero lag (+/- 20ms). The observed zero-lag peak could be instead explained by errors in behavioural tracking dominating the analysis, which would affect both representations simultaneously and show spurious zero-lag positive correlations. As such, the analysis that is missing is the difference between internal HD decoded from ADN and RSC at different time lags, without reference to the HD tracked behaviourally.

4) The work often uses a number of trials as their 'n' sample size for statistical analyses and the methods state that tetrodes were regularly advanced, but there is no indication of whether multiple trials at the same tetrode position were included in the same statistical comparison (except for recordings '4 days apart' for the HD tuning and synaptic connectivity analyses). Multiple trials with a high likelihood of recording the same cell population should not be counted as separate samples when calculating statistical significance.

https://www.youtube.com/watch?v=32W3J7XaNH8

This passage reminds me of the control and expectations of the workers seen in this poem and the soldiers mentioned in the song. The lyrics,"Head in the dust, feet in the fire," not only symbolize the smoldering heat of a place like a wasteland, but also are akin to the men walking up the hill while looking at their feet. They are unmovable and pressured to keep working. This song continues with, "Soldier keep on marchin' on / Head down 'til the work is done," which is also what is expected of these men in T.S. Eliot's work. There is a deadened hope in both figures from the song and poem that further the feeling of restraint and suffocation of life and hope. This is T.S. Eliot's fear as he sees the 'wasteland' as devoid of growth and prosperity.

The poem sees this life as a death to society and happiness while the poem sees this stage as a stepping stone to rising up. This is clear in the line, "Careful son, you got dreamer's plans."

The picture is well described and seems simple enough but it certainly can hold a lot of symbolism for the family unit being described. Details such as the hand holding and head cocked likely indicate the connection and reliance to her maternal figure yet hear head position indicates some degree of oddity.

Such a well written description, there is sometimes something so peaceful about looking back at old family pictures and remembering that period of time and also being able to see how far you’ve come since then

Nice. The details of the photograph are described so as to mirror the thesis outlined in the next paragraph. Well, not exactly a thesis so much as a central story of self-individuation.

Beautifully described. I can picture this in my head easily.

5th empiree

Mike Masnick https://knightcolumbia.org/content/protocols-not-platforms-a-technological-approach-to-free-speech Aug 2019. n:: "Build protocols, not platforms" (reminds me of the convo's around Jabber/XMPP I listened into in the early '00s) Also, IndieWeb / ActivityPub

1. open an investigation 展开调查(carry out the investigation) · investigation /ɪnˌvestəˈɡeɪʃ(ə)n/ n. 调查 · sth. is under investigation 某事正在调查之中 · The cause of the fire is under investigation.

火灾原因正在调查之中。

1. crush /krʌʃ/ n. 拥挤的人群
2. crowd control 人群管控
3. could have done 本可以…… · She could have avoided the accident. 她本来可以避免这场事故的。
4. surge /sɜːrdʒ/ n.(数量上的)激增，猛增；(人群)蜂拥
5. head into 朝着某个方向走

Do not forget the U of R or instructor names, due date, page number, NO RUNNING HEAD

not looking forward to watching this, sounds terrifying

I find it very interesting that these headrests have similar makes and use but very different beliefs. Egyptians saw sleep as a deathlike state and cast protective spells on their "pillows," whereas the Africans mad the "pillow" seem more like a piece of artwork to help facilitate sleep.

This is a metaphor that entails avoiding some sort of trouble/ controversy.

Another metaphor was in the headline, 'kicking everybody's ass'. This entails being successful or even outstandingly successful amongst others.

Author Response

Reviewer #1 (Public Review):

In order to study odor response dynamics in the olfactory peripheral organ, Kim et al. employs extracellular sensillum recording from the locust antenna to a set of 4 odors at different concentrations. Using spike sorting to assign odor responses to single olfactory sensory neurons (OSNs), the authors demonstrate that OSNs exhibit four distinct response motifs comprising two types of excitation, namely fast and delayed excitatory responses, as well as inhibitory responses in form of offset responses and inhibition. Notably, OSNs can switch between these four motifs depending on the odor applied. This finding is highly interesting and facilitates odor classification as demonstrated by computational modeling in this study. Furthermore, the authors demonstrate that each response motifs follows different adaptation profiles which further results in an increased coding space. The authors conclude and provide evidence with their model that the experimentally observed response dynamics also facilitate determining the distance to the odor source. The obtained results are novel and demonstrate a new dimension of odor response properties at the peripheral level. However, given that the authors used a very limited set of chemically similar odors and considering that the broad tuning and wiring of OSNs in the locust is special and follows different rules compared to the olfactory circuitry of OSNs in other insects (i.e. locust OSNs do not converge onto a single glomerulus but target multiple glomeruli), I wonder whether the observed distinct response motifs are a general phenomenon or a rather special case. I therefore recommend that the authors discuss their findings in the light of these key issues before general conclusions with regard to odor coding rules is being drawn. Do these response motifs also occur for highly ecologically relevant odors, such as PAN, where a rather specialized olfactory circuit would be assumed? Hence, the MS would benefit if those questions would be addressed as well. In addition, the computational modeling approach is written in specialized terms and is therefore difficult to grasp for readers lacking modeling expertise.

We thank the reviewer for this very positive and helpful assessment of our work. We agree with suggestions to expand our discussion of (1) olfactory circuitry following OSNs and of (2) responses to highly ecologically relevant odors. We have also extensively revised the description of our computational modeling approach to make it understandable to non-specialists.

In brief:

(1) The results we present here address only peripheral activity – we do not record or model responses of follower neurons. Because our conclusions do not depend to any extent upon the architecture of the locust's olfactory system, we would prefer to limit necessarily speculative discussion or analyses of these factors. We agree these factors provide interesting context for our work, so we have now expanded our discussion to include: “In other species, how exactly ORN response patterns are utilized downstream may depend on species-specific variations in connectivity between ORNs and the antennal lobe and its glomeruli” (lines 490-492). More investigation is needed to address this important question. Nevertheless, our study shows ORN response motifs provide useful information, and conveying this information to downstream circuits augments coding space.

(2) We share the reviewers’ concern that our odor set should include ecologically particularly relevant odors. Indeed, it was for this reason that our odor set includes components of the locust diet, wheat grass: 1-Octanol, 1-Hexanol, and Cyclohexanol. As above, though, we are reluctant to speculate on the responses of downstream circuits. But to acknowledge the reviewer’s important point, we have added the following text to our discussion in lines 401-405: “For these studies we used odorants known to be ecologically relevant to locusts, including several found in the head space of wheat grass. Future experiments with larger sets of odorants, including blends or locust pheromones like 4-vinylanisole (4VA) and phenylacetonitrile (PAN), may help clarify the logic of motif switching.”

Reviewer #2 (Public Review):

This manuscript provides additional data about how smell is encoded by insects. The study includes both new experimental measurements and simulations. At present, there are questions about whether simulations are appropriately performed to support experimental measurements.

The main experimental finding reported here is that the same olfactory receptor neurons (ORN) can respond with different temporal dynamics to different odorants. This finding is of interest. However, it is very important to discuss whether the differences in temporal dynamics can be explained by differences in how this odorant is carried by air, as has been described here: https://pubmed.ncbi.nlm.nih.gov/23575828/.

We agree this phenomenon is of great interest, and we have now expanded our discussion section to address it.

In the cited paper (see also Su et al, 2011), PID response characteristics were indeed quite different for different odors, reflecting “fast” and “slow” intrinsic odor dynamics. We are aware of these studies and shared the reviewer’s concern, and for this reason we also made PID recordings during odor presentations. These recordings show our odor set included only “fast” odorants (please see the figure below). We also note that, across our extensive dataset, all odors could elicit all four response motifs. These observations rule out the possibility that differences in how odorants are carried by air underlie the different temporal dynamics we observed in OSN responses.

We now discuss this important point in the text, as follows: “Earlier work established that the intrinsic dynamic properties of odorants, described as “fast” or “slow,” can contribute to variations in the timing of ORN responses (Su et al., 2011; Martelli et al., 2013). However, our experiments ruled out the possibility that intrinsic odorant dynamics underly the response motifs we describe here. First, across our extensive dataset, all odors could elicit all four response motifs; second, photoionization detector recordings of our odor presentations all revealed “fast” dynamics (not shown). It seems likely that “slow” odors would elicit concentration-dependent elaborations in the response motifs we observed. In future work it will be interesting to investigate ways intrinsic odor dynamics interact with ORN response motifs. We predict such interactions would further increase ORN response dimensionality” (lines 370-380).

There are several questions that need to be addressed regarding the simulations part of the manuscript.

1) There is a mismatch between the number of ORNs used in the model and in the insect system studied.

The exact number of ORNs in the locust is not known, but estimates range from 45,000 to 113,000 per antenna (Leitch & Laurent 1996; Perez-Orive et al 2002; Galizia & Sachse 2010). We chose to model a smaller but still large set of ORNs (10,000) which we believe is a reasonable compromise between the ideal size (which would be true number of ORNs in locust), and limitations needed to achieve practical computational efficiency. Indeed, almost all computational models are unavoidably scaled-down versions of the biological organisms.

2) The demonstration in Figure 5 that motif switching improves odor classification includes motif switching for a given odorant, which is not observed experimentally.

We regret that our description of the experiment presented in Figure 5 was confusing, and we have revised extensively to clarify this in our revision. In brief, the simulation shown in Figure 5 was not, as the reviewer understood, an attempt to model motif switching that occurs when a given odorant is presented repeatedly; rather, it shows how responses to two different, similar odors (Odor 1 and Odor 2) become increasingly different from each other when the probability of motif switching increases.

We have now revised the text to clarify this point as follows: “With our model we could independently vary odor-elicited response motifs and response magnitudes (Figure 4E), allowing us to evaluate the extent to which motif switching benefitted odor classification in a way that cannot be tested in vivo. Thus, we simulated a realistically large number of ORNs (10,000) and compared the relative success of classifying two different odors (Odor 1 and Odor 2) with three different versions of our model in which we systematically varied the probability of motif switching. Model Version 1: the probability of switching response motif when switching from Odor 1 to Odor 2 was 0%; Version 2: 10%; Version 3: 50%. We found that the model versions that simulated higher motif switching probability made it easier to distinguish these two similar odors.” (lines 191-195, 206-209).

We have also revised the figure caption as follows: “Computational model shows response motif switching substantially improves odor classification. A) Simulated ORN spiking illustrates different motif switching probabilities. Odors 1 and 2 are similar (see Methods). Each ORN response is sorted by motifs elicited by Odor 1. Raster plots show the responses to Odor 2 become increasingly different from responses to Odor 1 as motif switching probability increases. B) ORN odor-elicited response trajectories in reduced PCA space show motif switching increases the separation between responses to similar Odors 1 and 2; response to Odor 1 (blue) is the same in each panel; response to Odor 2 (red) changes with switching probability. C) Odor classification success as a function of odor similarity and motif switching probability for 1s (top) and 4s (bottom) stimulus pulses; even low switching probabilities improve classification performance; darker shading indicates lower classification accuracy. Odor similarity is quantified by angles (degrees) between odor vectors (see Methods)” (lines 231-239).

3) The methodology for estimating neural temporal dynamics needs to be corrected to apply to the natural stimuli used here.

We agree and thank the reviewer for raising this important point. To appropriately account for natural correlations present in the stimuli we used in experiments, we have now completely redone our analysis, substantially revised Figure 6, and rewritten the Methods section titled “Temporal filters using linear non-linear models.” Using methods appropriate for strongly correlated and natural odorant stimuli delivered experimentally, we obtained results consistent with those in the previous version of our manuscript.

Reviewer #3 (Public Review):

In this contribution, the authors align an extensive analysis of in vivo recordings of olfactory receptor neuron (ORN) responses to odors in the locust with a data-driven mathematical model of ORN population coding. Their results provide novel insights into the temporal dynamics of peripheral encoding of time-varying and naturalistic olfactory input.

The manuscript presents three central experimental results: 1) ORNs odor responses can be grouped into 4 distinct response motifs (response profiles). This has partly been known with respect to the typical excitatory phasic-tonic motif and odor offset responses. The exhaustive data set here is however unprecedented. 2) Individual ORNs can switch their response motif, e.g. from excitatory to inhibitory responses. To my knowledge, this is entirely new, highly interesting, and has strong implications. For one it implies an increased coding space and odor separability, which is supported by the authors' model study. It also bears implications for our understanding of processing in the antennal lobe where projection neurons were shown to exhibit property but this has largely been attributed to network processing within the AL. The authors discuss ephaptic interactions as a possible underlying mechanism. 3) ORNs not only show classical within and across pulse adaptation where the response amplitude reduces, but also the novel result that the offset response can increase across repeated pulses with short inter-stimulus intervals. The data-driven model reproduces the experimental observations and a population model that confirms the assumed increase in coding space. In the temporal domain, the authors then perform simulations that mimic realistic stimulus statistics with stochastic arrival of odor packets of variably short duration. The model with a trained linear filter and a non-linear transfer function faithfully predicts the experimental firing rates.

These results, based on an exhaustive set of experimental data, provide a novel view of peripheral odor coding in insects and they will have a particularly strong impact on biologically realistic computational (spiking) circuit models of sensory processing and sensory-to-motor transformations during odor source navigation in naturalistic simulated odor environments where conclusive data and analysis of ORN signaling has thus far been lacking.

We thank the reviewer for this very thoughtful and positive assessment of our work.

Author Response

Reviewer #1 (Public Review):

Authors introduced new strategy of genetic manipulation in mice to reveal functional development of the retrotrapezoid nucleus (RTN) neurons that is known as an important brainstem region for central chemoreception and the dysfunction is relate to congenital central hypoventilation syndrome (CCHS) neuropathology. They used a conditional mutation of Phox2b within Atoh1derived cells (Atoh1Cre/Phox2bΔ8 mice) and examined a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. They found that 1) mice with mutant Phox2b expression showed a suppressed breath activity to hypoxia and hypercapnia in neonates; 2) adult mutant mice presented irregular breathing pattern, partial recovery of the ventilatory response to hypoxia and complete recovery of response to hypercapnia; 3) anatomical data showed reduced number of activated neurons by hypercapnia and Phox2b immunoreactivity in the RTN. They concluded that conditionally expression of Phox2b mutation by Atoh1 affected development of the RTN neurons and suggested that Atoh1/Phox2b system in the RTN was essential for the activation of breathing under hypoxic and hypercapnia condition. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology. The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with results of various previous studies performed by different genetic strategies for the RTN development.

We would like to thank the reviewer for the comments on our manuscript. In the present version, we made several corrections as suggested by the reviewers to facilitate interpretation and strength the manuscript.

Reviewer #2 (Public Review):

Mutations in the Phox2B gene can lead to congenital central hypoventilation syndrome with variable presentations. Two distinct classes of causative mutations have been found in the human population. The first group consists of mutations that result in trinucleotide, polyalanine repeat expansions, referred to as PARM. The second group are non- polyalanine repeat expansion mutations (NPARM) that includes missense, nonsense, and frameshift mutations. Each group (and even specific mutations) present with differing clinical phenotype severity, with NPARM mutations typically being more severe. As Phox2B is expressed across a multitude of cell types across the life an individual, there remains much to be understood as to the cell specific effects of various Phox2B mutations on phenotype. To add to our understanding, the authors utilized a conditional Phox2bΔ8 allele that, upon recombination, replaces Exon 3 and UTR with a mutated exon and IRES GFP reporter. This approach allows for an inducible NPARM mutation and reporter expression in a targeted cell type. The authors focused on Atoh1 expressing cells using an Atoh1 expressing Cre recombinase line (Atoh1_Cre). Atoh1 has been shown to also be coexpressed in the RTN and in the para and inter-trigeminal regions of the Pons. After inducing the Phox2B mutations in one allele, the authors examined respiratory features in both adults and neonate mice under room air, hypercapnia (7%) and Hypoxia (8%). The Atoh1_Cre; Phox2bΔ8 adult mice showed a significant body weight difference. Under their plethysmography approach neonate mice breathing room air showed few differences with a potential difference in tidal volume. Notably adult mice show irregularity in their breathing. Both adult and neonate mice may show compromised chemosensory deficits. A potential hypercapnic deficit likely resolves in the adult but there may remain a compromised hypoxic reflex in the adult. Notably, Atoh1_Cre; Phox2bΔ8 mice showed reduced cfos expression in the RTN after hypercapnic stimulation and reduced Phox2B immuno-reactivity.

The premise of the paper is to examine how a distinct mutation in a specific cellular context may contribute to clinical outcomes. The potential phenotypes are interesting and illuminate how differing mutations may drive different phenotypes or phenotype severity. While the RTN is likely a key mediator of the reported phenotypes, the conclusions drawn by the authors cannot be fully supported with the data presented.

We would like to thank the reviewer for the comments. In the present version, we have made all changes suggested and we performed new sets of additional experiments to strengthen the work. We are very enthusiastic about the new version of the manuscript, and we believe it opened new questions that could be addressed in the future.

The authors assign all phenotypes to RTN function. However, there are other documented and potential undocumented areas of Atoh1 and Phox2b overlap that could either impact breathing directly or indirectly through metabolism and stress responses (PMID 8184995). As noted above, para trigeminal neurons including those in the ITR also co-express Atoh1 and Phox2B and are captured in the Atoh1_Cre; Phox2bΔ8 mouse model. The inter-trigeminal region is associated with apneic reflexes and jaw opening (PMID: 19914183). Thus, perturbations to this center may underlie the increased irregularity seen in adult life. A potential role in chemosensory function cannot be entirely ruled out either. While Rose et al. assert that the RTN and para- and inter- trigeminal neurons are the only ones co-expressing Atoh1 and Phox2B (using antibodies), the persistent cumulative GFP labeled fate map offered by the Atoh1_Cre; Phox2bΔ8 model would allow the authors to rule in or rule out any other uncharacterized overlapping populations. Such a fate map may also help to inform as to why the adult mice are significantly underweight. The weight phenotype may stem from metabolic dysregulation, changes in behavior, or feeding. Changes in metabolism may drive secondary changes in breathing and chemosensory reflexes that play a role in the reported phenotypes. Ultimately, the relative roles of para-trigeminal and RTN neurons in these phenotypes should be dissected out.

Yes, we ran a new series of experiments and noticed that Phox2b+ neurons in the pons as well as the number of TH cells in the A1, A2, A6, and C1 were not affected by the mutation. Unfortunately, we were unable to quantify the number of Phox2b-expressing neurons in the paratrigeminal region.

Both the adult and neonate plethysmography was not collected in line with current best practices. Adult whole body plethysmography is best carried out in a temperature controlled chamber held at thermo-neutrality. This minimizes any thermo-regulatory and metabolic effects on respiratory drive. Concurrent measurement of one or more metabolic parameters such as VO2 or VCO2 is required to determine if baseline breathing, and chemosensory reflex phenotypes may be affected by changes metabolism or persistent metabolic imbalances (acidosis or alkalosis). Whole body measurements in neonates are do not allow for accurate assessment of tidal volume. Rather head out or facemark pneumotachography are more accurate, (PMID: 25017785).

We totally agree with the reviewer. In fact, some information and misconception were noticed in the previous version. We added the correct way in which the respiratory parameters were measured in both neonate and adult mice. Additionally, we performed head-out plethysmograph in a subset of neonates (control and mutant) and added it in the result section. We also measure VO2 and VE/VO2 in neonates and adults.

Reviewer #3 (Public Review):

The work by Ferreira and colleagues set to define the functional consequences of a PHOX2B (Phox2bdelta8) mutation, belonging to the group of non-polyalanine repeat expansions, when restricted to Atoh1 expressing cells. In doing so, the authors generated a new mouse model (Atoh1Cre,Phox2bdelta8 mice) for the study of the central respiratory chemoreceptor circuit. Ferreira et al., found that these conditional mutants present with largely unaffected breathing parameters in postnatal life. However, neonatal breathing irregularities, normally observable in control neonates, are not corrected with the maturation of the conditional mutants. Furthermore, the authors found that conditional Atoh1Cre,Phox2bdelta8 neonates fail to display ventilatory responses to hypoxic (low O2 content in air) and hypercapnic (high CO2 content in air) challenges. The authors show that Atoh1Cre,Phox2bdelta8 adult mice appear to "recover" the capacity to response to hypercapnic, but not hypoxic, challenges. Lastly, the authors found reduced numbers of Phox2b+ cells in an "area" where the retrotrapezoid nucleus, a key center in the respiratory chemoreceptor circuit, normally locates.

Strengths:

The most exciting aspect of this work is the modelling of the Phox2bdelta8 mutation in one element of the central neuronal circuit mediating respiratory reflexes, that is in the retrotrapezoid nucleus. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with central congenital hypoventilation syndrome (CCHS), a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. Two distinct types of PHOX2B mutations have been identified in CCHS patients: i) polyalanine repeat expansions, and ii) non-polyalanine repeat expansions. Non-polyalanine repeat expansions tend to be more prevalent in severe cases of CCHS. Thus, the characterization of the Phox2bdelta8 mutation could allow for a better understanding of the etiology behind CCHS.

Weaknesses:

Whereas the most exciting part of this work is the modelling of the Phox2bdelta8 mutation in retrotrapezoid neurons using conditional mutagenesis driven by Atoh1 (i.e. Atoh1Cre,Phox2bdelta8 mice), the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere, for instance the use of Atoh1Cre,Phox2bflox/flox and P2b::CreBAC1;Atoh1lox/lox mice (Ruffault et al., 2015, DOI: 10.7554/eLife.07051), Egr2cre;P2b27Alacki (Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011), Atoh1Phox2bCKO mice (Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027) and Egr2cre;Lbx1FS (Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115).

Several conclusions presented in this work are not directly supported by the provided data. For instance, the reduction in the number of retrotrapezoid neurons in Atoh1Cre,Phox2bdelta8 mice or the reduction of fos+ activated retrotrapezoid neurons after CO2 exposure, as the identity of retrotrapezoid neurons was not thoroughly determined. Furthermore, the authors conclude from their plethysmograph (respiratory recordings) data that Atoh1Cre,Phox2bdelta8 neonatal mice display an impaired ventilatory responses to hypoxia (low O2 in air) and hypercapnia (high CO2 in air), but that these mutant animals recover the capacity to respond to hypercapnia, but not to hypoxia, in the adult life. This is a bit of an overstatement, as their plethysmograph recordings show that adult Atoh1Cre,Phox2bdelta8 mice do respond to low O2 in air, as these mice accelerate respiration, increase tidal volumes and minute ventilation in the same fashion as control mice. However, what the presented data show is that adult Atoh1Cre,Phox2bdelta8 mice do not sustain the ventilatory response as efficient as control mice.

We would like to thank the reviewer for the comments, strengths, and weakness of our study. In the present version, we have made a significant change throughout the manuscript as suggested by the editor and reviewers. In addition, we performed new sets of experiments to strengthen our work. We are very enthusiastic about the current version, and we believe it will open new questions that need to be addressed in future studies

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Reply to the reviewers

Author response (Tane at al: RC-2022-01646)

Reviewer #1 (Evidence, reproducibility and clarity (Required)): * Comments The work described in this manuscript starts with an in-silico analysis of the primary amino-acid sequence of CAP-H proteins that reveals the presence in vertebrate orthologs of an N-terminal extension of ~80 amino acids in length which contains 19 serine or threonine residues and also, in its centre, a stretch of conserved basic amino acids predicted to form a helix. These features suggest a regulatory module. Using xenopus egg extracts depleted of endogenous condensins and supplemented with recombinant condensin I holocomplexes, either wildtype or mutants, the authors show that deleting the N-terminal tail of CAP-H, or just the central helix (CH), increases the association condensin I with chromatin in mitotic egg extracts and accelerates the formation of mitotic chromosomes. Interestingly, they also show that deleting the N-tail enables a substantial amount of condensin I to associate with chromatin in interphase extracts and to form chromosome-like structures, while WT condensin I cannot. Using in vitro assays and naked DNA as substrate, the authors further show that removing the N-terminal tail of CAP-H improves both the topological (salt-resistant) association of condensin I with DNA and it loop extrusion activity. These experiments appear to me as are clear and robust. They convincingly reveal that N-tail of human CAP-H hinders the binding of condensin I to DNA and both its loop-extrusion and chromosome-shaping activities. However, the mechanism through which such hindrance is achieved remains elusive (see major comments 1-3). A complementary part of the work tackles the important question of the cell cycle control of such counteracting effect. Using newly-designed antibodies against two phospho-serine residues within the tail, the authors provide evidence that the tail is phosphorylated in mitosis-specific manner. This points towards phosphorylation as a biological mean to modulate the effect of the tail on condensin's binding during the cell cycle. In support to this idea, using non-phosphorylatable or phosphomimic substitutions of all the serine and threonine residues within the tail (n =19), including one substitution within the CH domain (Ser 70), the authors show that non-phosphorylatable mutations (H-N19A) or phosphomimic mutations (H-N19D) respectively reduce or improve condensin I binding to chromatin in mitotic egg extracts. This suggests that the phosphorylation of the N-terminal tail in mitosis might relieve its negative effect on condensin I binding to chromatin. The weaknesses I see in this part of the study concern (1) the validation of the phospho-antibodies, which appears to me as insufficiently described (major comment 4), (2) the possibility the bulk changes in amino acids (n=19 out of 80) could impact the folding of the central helix (minor comment X) and (3) that some substitutions could impact the binding of condensin I by different mechanisms (minor comment X).

Major comments:

1. On the model. The authors propose that the N-tail could stabilise an interaction between the N-terminal part of CAP-H and SMC2's neck, which would restrain the transient opening of a DNA entry gate within the ring, necessary for the topological engagement of DNA and loop formation. Although the model is sound, I see no direct data that support it in the manuscript. Such model predicts that a CAP-H protein containing or not the N-terminal tail (or the central helix) should exhibit different binding strengths to SMC2 in vitro. It seems to me that the authors could easily test this prediction using the recombinant proteins they produced in the context of this study. *

Response

We thank the reviewer for pointing out this important issue. To test whether the CAP-H N-tail indeed contributes to the stabilization of the SMC2-kleisin gate, we set up a highly sophisticated functional assay described by Hassler et al (2019). The authors used this assay to demonstrate that an N-terminal fragment of kleisin (engineered to be cleaved by TEV protease) is released from the rest of the condensin complex in an ATP-dependent (i.e., head-head engagement-dependent) manner. We reasoned that this assay is most powerful to prove our hypothesis in a mechanistically relevant context. We envisioned that the CAP-H fragment lacking its N-tail can readily be released whereas the CAP-H fragment retaining its N-tail is more difficult to be released (because of the postulated stabilization of the SMC2-CAP-H interaction). Despite substantial efforts in making TEV-cleavable constructs and in testing various releasing conditions, we have not been able to recapitulate the ATP-dependent release even with the holo(H-dN) construct. Thus, unfortunately, this trial enabled us to neither prove nor disprove our hypothesis.

We are fully aware that the full reconstitution of ATP-dependent and phosphorylation-stimulated gate-opening reaction in vitro is a very important direction in the future. It is beyond the scope of the current study, however.

2. On ATP-hydrolysis. Given the importance of ATP hydrolysis for the engagement of condensin into a topological mode of association with DNA and for its loop extrusion activity, I suggest that the authors measure the impact of the N-tail and of the CH domain on the rate of ATP hydrolysis by condensin I holocomplexes. I suppose that it can be relatively easily done (PMID: 9288743) using the recombinant WT and mutant versions they purified in the course of this study.

Response

We appreciate this constructive comment. In fact, we did a preliminary experiment and found that ATPase activities (either in the absence or presence of DNA) were not significantly different between holo(WT) and holo(H-dN). We were not surprised with this result because our previous study on condensin II indicated that enhanced ATP hydrolysis by a class of mutant complexes is not directly coupled to their enhanced association with chromosomes (Yoshida et al., 2022, eLife). We consider that other functional assays, such as the topological loading assay and the loop extrusion assay shown in the current manuscript, are more informative assays to address ATP-dependent activities of the condensin complexes.

3. A conundrum with previous work? In Kimura et al. Science 1998 (PMID: 9774278), the lab of Tatsuya Hirano has shown that xenopus condensin I purified from mitotic egg extracts induces the supercoiling of plasmid DNA in vitro, but fails to do so when it is purified from interphase egg extracts. This echoes the inhibitory effect of the N-tail of the topological binding of condensin I described in the current manuscript. However, using a gel shift assay, Kimura et al. 1998 also provide evidence that interphase and mitotic condensin I bind plasmid DNA in vitro with similar efficiencies. At first sight, this prior observation seems to contradict the idea that the N-tail of CAP-H restrains DNA binding unless it is phosphorylated in mitosis. Is it possible that the in vitro binding assays used in Kimura et al. 1998 and in this work might assess different modes of binding? I suggest that this apparent conundrum should to be discussed.

Response

We thank the reviewer for following our early studies. As discussed below, we are confident that our conclusion reported in the current study by no means contradicts our previous observations.

We reason that the confusion expressed by the reviewer stems from intrinsic, technical limitations of the gel-shift assay. Such limitations become apparent especially when it is applied to the functional analyses of complicated protein machines such as condensins. For instance, the DNA-binding activity of condensin I detected by the gel-shift assay is neither ATP-dependent nor phosphorylation-dependent (Kimura and Hirano, 1997; Kimura et al., 1998). It is fundamentally different from the ATP-dependent activities detected by the topological loading and loop extrusion assays reported in the current study (It remains unknown whether the two activities are stimulated by mitotic phosphorylation). Thus, the DNA-binding activity detected by the gel-shift assay does not reflect “productive” DNA interactions that depend on ATP hydrolysis in vitro. We therefore consider that gel-shift analyses of holo(WT) and holo(H-dN) would not produce any useful information.

*Related to that, could it be possible for the authors to assess the impact of the N-tail on the salt-sensitive binding of condensin to DNA, i.e. by reproducing the topological binding assay but omitting the high salt washes? I guess this information could be useful to fully apprehend the impact of the N-tail on the binding of condensin. *

Response

When we set up the topological loading assay, we actually tested a low-salt wash condition that the reviewer suggests here. Although a much higher level of DNA recovery was observed with the low-salt condition than with the high-salt wash condition, no nucleotide dependency was detectable with the low-salt condition. Moreover, no difference in DNA recovery between holo(WT) and holo(H-dN) was observed. Thus, the low-condition condition allowed us to detect the “bulk” DNA-binding activity that is equivalent to that detected by the gel-shift assay. These results were fully consistent with the discussion above.

4. Validation of phospho-antibodies and by extension showing the phosphorylation of the tail. The newly-designed phospho-serine antibodies used in this study to show that the N-tail is phosphorylated at serine 17 and serine 76 in mitosis (Fig. EV3) are, in my view, not characterized enough. This piece of data is key to substantiate the idea that the tail is phosphorylated in mitosis. Yet, showing that these antibodies detect epitopes on WT condensin I from mitotic egg extracts but not on the H-N19A counterpart, nor on WT condensin I from interphase extracts, does not demonstrate the phospho-specificity of such antibodies. I suggest that a PPase treatment should be conducted to assess the phospho-specificity of these antibodies. Moreover, since the lab of Tatsuya Hirano has the know-how to deplete Cdc2/CDK1 from xenopus egg extract, such strategy could/should be used to further validate the antibodies and assess to which extent the N-tail is phosphorylated in a Cdc2-dependent manner.

Response

We have performed two sets of experiments to confirm the specificity of the phosphoepitopes recognized by anti-hHP1 and anti-hHP2. In the first set, we performed a phosphatase treatment assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated using an antibody against hCAP-G, treated with l protein phosphatase in the presence or absence of phosphatase inhibitors, and analyzed by immunoblotting using anti-hHP1 and anti-hHP2. The results (now shown in Supplementary Fig 3C) demonstrated that the epitopes recognized by anti-hHP1 and anti-hHP2 are sensitive to phosphatase treatment. In the second set, we performed a phosphopeptide competition assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated and subjected to immunoblotting. The membranes were triplicated and probed with anti-hHP1 in the presence of no competing peptide, hHP1 or hHP2. Similarly, another set of triplicated membranes was probed with anti-hHP2 in the presence of no competing peptide, hHP1 or hHP2. We found that the signal recognized by anti-hHP1 competed with hHP1, but not with hHP2, and that the signal recognized by anti-hHP2 competed with hHP2, but not with hHP1. The results (now shown in Supplementary Fig 3D) demonstrated the sequence specificity of the phosphoepitopes recognized by the two antibodies. The procedures for these experiments have been described in Materials and Methods.

Because Cdk1 depletion from M-HSS creates an HSS equivalent to I-HSS, we do not consider that the suggested experiment will provide additional information.

*Minor comments:

1. The impact of the 19 mutations, A or D, introduced within the tail on the folding of the central helix? The idea that the negative effect of the N-tail is relieved by phosphorylation is based on the chromatin binding phenotypes exhibited by the H-N19D or H-N19A mutant holocomplexes, in which 19 amino-acids out of 80 have been modified, include one in the central helix. The authors also provide evidence that the central helix (CH) located within the tail plays a key role in the negative regulation of condensin I binding. Thus, I wonder to which extent the folding of the central helix could be impacted by the mutations introduced in the tail and notably the one within the central helix itself. Could the author assess the structure of mutated tails using Alpho-fold and/or discuss this point? *

Response

According to the reviewer’s suggestion, we performed structure predictions using Alphafold2, and found that neither the N19A nor N19D mutations alter the original prediction of helix formation that was made for the wild-type CH sequence. A conventional secondary structure prediction using Jpred4 reached the same conclusion.

2. Phosphorylation of serine 70 in the central helix by Aurora-B kinase? A prior study by Tada et al. (PMID: 21633354) has shown (1) that serine 70 of the N-tail of hCAP-H is phosphorylated by Aurora-B kinase, (2) that the mutation S70A reduces the binding of condensin I to chromatin in HeLa cells and (3) that hCAP-H interacts with histone H2A in an Aurora-B dependent manner. This draws a picture in which the phosphorylation of Ser70 by Aurora-B would improve condensin I binding to chromatin by promoting an interaction between hCAP-H and histone H2A/nucleosomes. Intriguingly, Ser 70 in Tada et al. correspond to the serine residue located within the conserved central helix analysed in this study, and this Ser70 residue is mutated in the H-N19D or H-N19A holocomplexes that show reduced chromatin binding in this study. This raises the question as what could be the contribution of the S70A or S70D substitution to the chromatin binding phenotypes shown by the H-N19D or H-N19A holocomplexes. This is not discussed in the manuscript, and the authors do not cite this earlier work (PMID: 21633354) in their manuscript. Is there any reason for that? I suggest it should be cited and discussed.

Response

We thank the reviewer for bringing up this issue. In many respects, we do not trust the data reported by Tada et al (2011) and the resultant model they proposed. Previous and emerging lines of evidence reported from our own and other laboratories indicate that histones compete with condensins for DNA binding, strongly arguing against the possibility that histone H2A acts as a “chromatin receptor” for condensins. We formally discussed and criticized the Tada 2011 model in our previous publications, which included Shintomi et al (2015) NCB, Shintomi et al (2017) Science, Hirano (2016) Cell and Kinoshita/Hirano (2017) COCB. We thought that those were enough. That said, we also consider that the reviewer is right. The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional line of evidence that argues against the Tada model. A critical comparison between the Tada model and our current model would benefit the readers. In the revised manuscript, we have added the following discussion:

In terms of the regulatory role of the CAP-H N-tail, it would be worthy to discuss the model previously proposed by Tada et al (2011). According to their model, aurora B-mediated phosphorylation of the CAP-H N-tail allows its direct interaction with the histone H2A N-tail, which in turn triggers condensin I loading onto chromatin. Accumulating lines of evidence, however, strongly argue against this model: (i) aurora B is not essential for single chromatid assembly in Xenopus egg extracts (MacCallum et al., 2002) or in a reconstitution assay (Shintomi et al., 2015); (ii) the H2A N-tail is dispensable for condensin I-dependent chromatid assembly in the reconstitution assay (Shintomi et al., 2015); (iii) even whole nucleosomes are not essential for condensin I-mediated assembly of chromatid-like structures (Shintomi et al., 2017). The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional piece of evidence against the model proposed by Tada et al (2011).

3. Other minor comments - Please provide a microscope image of DNA loop in Fig. 4D.

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.

*- The authors do not compare the kleisin of condensin I with the one of condensin II with respect to the features tackled in this work. Given the different behaviours condensin I and II, such comparison could be informative for the readers. *

Response

We thank the reviewer for this constructive comment. In the revised manuscript, we have added the following statement:

It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II.

*- The authors do not reference the work of Robellet et al. Genes & Dev (2015) (PMID: 25691469) on the regulation of condensin binding in budding yeast by an SMC4 phospho-tail. I suggest that the analogy should be discussed. *

Response

According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.

Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015; Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.

- In the introduction section, lane 5, the sentence "Many if not all eukaryotic species have two different condensin complexes" appears inappropriate since budding and fission yeast cells possess a single condensin complexes, similar to condensin I in term of primary amino-acid sequence.

Response

We thought that the original wording “Many if not all” was good enough to imply that some species, which include budding yeast and fission yeast, have only a single condensin complex. To make it clear, however, we have modified the sentence in the revised manuscript as follows:

Many eukaryotic species have two different condensin complexes although some species including fungi have only condensin I.

*- page 4; typo: motif I and V bind to the SMC neck and the SMC4 cap regions, respectively. Should read SMC2 neck. *

Response

The reviewer is right. It should read the SMC2 neck. Corrected.

*- Are the data and the methods presented in such a way that they can be reproduced? YES - Are the experiments adequately replicated and statistical analysis adequate? YES - Are prior studies referenced appropriately? Not all of them (see above) - Are the text and figures clear and accurate? YES

CROSS-CONSULTATION COMMENTS I consider the comments from all reviewers as helpful for the authors.

Reviewer #1 (Significance (Required)):

Summary Condensins are genome organisers of the family of SMC ATPase complexes and are best characterized as the drivers of mitotic chromosome assembly (condensation). It is acknowledged that condensins shape mitotic chromosomes by massively associating with DNA upon mitotic entry (loading step) and by folding chromatin fibres into arrays of loops, most likely through an ATP-dependent extrusion of DNA into loops, as seen in vitro. What remains unclear, however, are the mechanisms by which condensins load onto DNA and fold it into arrays of loops in vivo, and how these reactions are coupled with the cell cycle, i.e. restricted mostly to mitosis. Condensins are ring shaped pentamers that change conformation upon ATP-hydrolysis. In vitro studies suggest that condensins bind DNA via ATP-hydrolysis-independent, direct electrostatic contacts between condensin subunits and DNA. Such electrostatic contacts are salt-sensitive in in-vitro assays. Upon ATP-hydrolysis, condensins engage into an additional mode of binding that is resistant to high salt concentration and likely to be topological in nature. Both modes of association are necessary to form DNA loops. Vertebrates possess two types of condensin complexes, condensin I and II, each composed of a same SMC2-SMC4 ATPase core but associated with two different sets of three non-SMC subunits; a kleisin and two HEAT-repeat proteins. Condensin II is nuclear during interphase and stably binds chromatin upon mitotic entry, while condensin I is located in the cytoplasm during interphase and binds chromatin in a dynamic manner upon nuclear envelope breakdown. How the spatiotemporal control of condensin I and II is achieved remains poorly understood. Previous studies have shown that the phosphorylation of condensin I by mitotic kinases, such as CDK1, Aurora-B and Polo, play a positive role in its binding to chromatin and/or its functioning, but the underlying mechanisms remain to be characterised. In this manuscript, Shoji Tane and colleagues provide good evidence that the N-terminal tail of the human kleisin subunit of condensin I, hCAP-H, serves as a regulatory module for the cell-cycle control of condensin I binding to chromatin and chromosome shaping activity. The authors clearly show that the N-tail of CAP-H hinders the binding of condensin I to chromatin in xenopus egg extracts and, using in vitro assays, that the N-tail also hinders the topological association of condensin I with DNA and its loop extrusion activity. The authors provide additional data suggesting that the phosphorylation of the N-tail of CAP-H, in mitosis, relieves its inhibitory effect on condensin I binding. Based on their findings, Tane et al. propose a model suggesting that the N-terminal tail of CAP-H constitutes a gate keeper that maintains condensin-rings in a closed conformation that is unfavourable for topological binding to DNA, and whose locking effect is relieved in mitosis by phosphorylation.

Taken as a whole, this work has the potential to reveal a molecular basis for the cell cycle regulation of condensin I in vertebrate cells and as such to significantly improve our understanding of the integrated functioning condensin I. The characterisation of the inhibitory effect of the N-tail on condensin binding to chromatin and to naked DNA in vitro is well described, the data are clear and robust and the results convincing. On the other hand, some of the data on the phospho-regulation appear to me as are more debatable and I think that some of the results described here deserve to be discussed in the context of previous works. Finally, I see no data in the manuscript that directly supports the mechanistic model proposed by the authors, while it seems to me that such model could have been easily tested exprimentally. Thus, I suggest that Tane and colleagues should perform a couple of relatively easy experiments to strengthen their claims and that a few omitted prior studies on the topic should be referenced and discussed. *

Reviewer #2 (Evidence, reproducibility and clarity (Required)): * The manuscript reveals that the N-terminal region of CAPH could play a role in regulating condensin I activity, using a range of in vitro methods. They propose that the N-terminal region of CAPH inhibits complex activity, and this is turned off upon deletion or phosphorylation, by using truncations, phospho-mimics or phospho-deficient mutations. While the results are interesting to the field, and helps to address the question as to how condensin complexes are controlled in a cell cycle dependent manner, some key data and controls are necessary to ensure the conclusion is robust.

Main comments

• What is meant by "unperturbed I-HSS" on page 7, ie membrane containing versus membrane free or condensin depleted? *

Response

We apologize for having created unnecessary confusion. We meant that the “unperturbed I-HSS” is the “undepleted I-HSS”. As far as the issue of membrane-containing vs membrane-free is concerned, we explicitly mentioned that “we used membrane-free I-HSS in the following experiments” several lines above. In the revised manuscript, we have revised the statement accordingly.

In many of the protein gels eg figure 4B, the bands for SMC2 and 4 are more intense that the non-SMC components. The method for protein purification also does not include a size exclusion step to ensure sample homogeneity. Authors should perform some sort of quality control checks on samples such as analytical gel filtration or mass photometry to ensure stoichiometry/homogeneity. This is particularly important for samples eg the N19A, where activity is reduced compared to wild-type as poor protein behaviour could create false negative results.

Response

As the reviewer is fully aware, the reconstitution and purification of multiprotein complexes, such as condensins, is by no means an easy task. We notice that many groups in the field share common concerns about sample homogeneity and subunit stoichiometry, and that these concerns cannot completely be eliminated even after size exclusion chromatography. Because the current study handles a large number of mutant complexes, we consider that the purification by two-step column chromatography is the most practical approach. We do not notice any abnormal behaviors of holo(H-N19A) in the processes of expression and purification. It is also important to emphasize that the H-N19D mutations cause the completely opposite phenotype. Taken all together, we are confident of our current conclusions.

That said, in the revised manuscript, we have added the following statement in Results:

Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….

• Loop extrusion assays in figure 4D-G shows no example data i.e. no pictures or videos of loops being formed. These should also be included.*

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.

• Given the mutant holo(H-dN) has higher activity than wild-type, a negative control such as holo(H-dN) without ATP or holo(H-dN) ATPase deficient mutant should also be measured in loop extrusion assays, to ensure the activity is derived from the ATPase activity.*

Response

In the revised manuscript, we have added loop formation data for both holo(WT) and holo(H-dN) in the absence or presence of ATP (Supplementary Fig 5). We are confident that both complexes support loop extrusion strictly in an ATP-dependent manner.

• According to the methods, this work performs the same loop extrusion assay as described in Kinoshita et al, 2022, however, in Kinoshita et al, wild type condensin I makes loops in 30-50% of DNA molecules, where in this study the percentage is less than half that. Can the author please explain the discrepancy given the same method was used?*

Response

First of all, we wish to remind the reviewer that the holo(WT) constructs used in the two studies are not identical: CAP-H was N-terminally HaloTagged in all constructs used in Kinoshita et al (2022), whereas the same subunit was C-terminally HaloTagged in the pair of constructs used in the current study. Because we wanted to compare the activities between the full-length CAP-H and N-terminally deleted version of CAP-H (H-dN), we reasoned that it would be inappropriate to put the HaloTag to the N-terminus of CAP-H. The difference in the constructs could explain the observed discrepancy, even if it might not be the sole reason.

The design of the constructs was accurately described in each manuscript, but the statements were not very explicit about the positions of the HaloTag. To clarify this issue, we have added the following sentences in the revised manuscript:

Note that the HaloTag was fused to the C-terminus of CAP-H in the current study because we wanted to investigate the effect of the N-terminal deletion of CAP-H. We used N-terminally HaloTagged CAP-H constructs in our previous study (Kinoshita et al., 2022).

• In the concluding statement the author suggests "Upon mitotic entry, multisite phosphorylation of the N-tail relieves the stabilization, allowing the opening of the DNA entry gate, hence, the loading of condensin I onto chromosomes." This seems unlikely as fusion the N-terminus of the of the kleisin to the C-terminus of SMC2 is able to function for yeast (Shaltiel et al 2022) and condensin II (Houlard et al 2021), and equivalently in cohesin (Davidson et al 2019).*

Response

We appreciate the reviewer’s concern. In our view, however, the issue of the “DNA-entry gate” remains under debate in the SMC field (e.g., Higashi et al [2020] Mol Cell; Taschner and Gruber [2022] bioRxiv). For instance, Shaltiel et al (2022) demonstrated that neck-gate fusion constructs can support in vitro activities including topological loading under certain conditions, but also showed that such constructs greatly reduce the cell viability, leaving the possibility that the gate opening is required for some physiological functions.

That said, it is true that the data reported in the current manuscript do not exclude the possibility that the SMC2 neck-kleisin interface is not used as a DNA entry gate for condensin I loading. In the revised manuscript, we have added the following statement in Discussion:

Although our model predicts that the SMC2 neck-kleisin interface is used as a DNA entry gate, we are aware that several studies reported evidence arguing against this possibility (e.g., Houlard et al [2021]; Shaltiel et al [2022]). Our current data do not exclude other models.

*Reviewer #2 (Significance (Required)):

This is an interesting story that reveals new insights about condensin regulation.

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

This paper reveals a role of an N-terminal extension of CAP-H in the regulation of condensin-I activity in Xenopus extracts using biochemical reconstitution experiments. The authors demonstrate that a motif in the N-terminal tail that is conserved in vertebrates acts as an inhibitor of condensin I activity. Using several mutant constructs, it is shown that the inhibition by this motif is in turn counteracted by the phosphorylation of neighbouring serine and threonine residues in mitosis, presumably at least in part by CdK. Mutants that have lost this inhibition are able to condense chromatin into chromatid-like structures more efficiently and to some degree even in interphase extracts. Moreover, one such mutant is characterized in detail by biochemical and biophysical experiments and shown to have increased capacity in salt-stable DNA loading and in DNA loop extrusion.

Major comments: This is a beautiful and thorough study that is presented in a clear and concise manner. The main conclusions are well justified. No additional experiments are needed to support them. Replication and statistical analysis appear adequate. The final model is however largely speculative. Recent work has indicated that loading of yeast condensin does not require gate opening. The authors may thus want to include alternative scenarios or remain more vague. *

Response

This comment is related to the last comment of Reviewer#2. See above for our response.

*The H-N19A mutant has a loss of function phenotype (possibly due to folding problem caused by 19 point mutations rather than lack of phosphorylation), the authors could consider to rescue the phenotype by also including the CH motif mutations in this construct (or make an explanatory statement in the text). *

Response

We understand the reviewer’s logic here, but overlaying additional mutations into the H-N19A mutations could cause an unforeseeable effect, potentially making the interpretation of the outcome complicated.

We also wish to point out that it may be inappropriate to regard the phenotype exhibited by holo(H-N19A) as a simple loss-of-function phenotype. This is because the opposite, accelerated loading phenotype exhibited by holo(H-dN) can be regarded as a consequence of loss of negative regulation. Like holo(H-dN), the phosphomimetic mutant complex holo(H-N19D) displayed an accelerated loading phenotype, fully supporting our conclusion. In the revised manuscript, we have added the following statement in Results:

Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….

*Albeit not necessary for the main conclusions, the authors could possibly significantly strengthen their study by testing for binding partners of the N-tail and the CH motif by running AlphaFold predictions against the condensin I subunits. *

Response

We appreciate this constructive comment. We attempted to predict possible interactions between SMC2 and a CAP-H fragment containing its N-tail and motif I using

ColabFold (Mirdita et al., 2022, Nat. Methods). The algorism excellently predicted the proper folding of the CAP-H motif I and its interaction with the SMC2 neck. Under this condition of predictions, however, the N-tail remained largely disordered (except for the CH), and no interaction with any part of SMC2 was predicted. The same was true when the N19D mutations were introduced in the N-tail sequence. Thus, this trial did not provide much information about the potential interaction target(s) of the CAP-H N-tail.

*The efficiency of depletion of condensin subunits from I-HSS extracts is not documented (in contrast to M-HSS extracts - figure EV1C). While any condensin remaining in these extracts might not be active (or interfering), the authors may want to at least comment on this in the text. *

Response

We check the efficiency of immunodepletion every time by immunoblotting and confirm that a high level of depletion is achieved from both M-HSS and I-HSS. According to the reviewer’s comment, the following statement was placed in Materials and Methods:

The efficiency of immunodepletion was checked every time by immunoblotting. An example of immunodepletion from M-HSS was shown in Supplemental figure 1C. We also confirmed that a similar efficiency of immunodepletion was achieved from I-HSS.

*The authors should include information on the quantification of chromatid morphology. Is the analysis based on chromatids taken from the same images/imaging session, from technical replicates, biological replicates? *

Response

In the revised manuscript, we have added statements on image presentation and experimental repeats in the appropriate figure legends and methods section. During the revision process, we repeated the experiments shown in Supplementary Fig 2, and obtained the same results. In the revised manuscript, the original set of data has been replaced with the new set of data along with panel C (Quantification of the intensity of mSMC4 per DNA area).

Minor comment: The colour scheme in Figure 5A is confusing. Use less colour? The orange and red colours are moreover quite similar.

Response

According to the reviewer’s comment, we have modified Figure 5A.

*Reviewer #3 (Significance (Required)):

The findings provide new insights into how condensin-I activity is restricted outside of mitosis. It was previously assumed that this regulation was (largely) due to the exclusion of condensin I from the nucleus prior to nuclear envelope breakdown. This study shows that another pathway is contributing to the regulation and implies that controlling condensin I activity is more important than previously appreciated. Whether all residual nuclear condensin I is inactivated, remains to be determined. The physiological impact of loss of autoinhibition on chromosome segregation and cell cycle progression also remains to be uncovered. The observed effects are robust and appear significant. Future research on condensin I using reconstitution will likely benefit from being able to control or eliminate the self-inhibition.

This reviewer has expertise on the biochemistry and structural biology of SMC protein complexes.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Mitotic chromosome formation is a cell cycle-regulated process that is crucial for eukaryotic genome stability. The chromosomal condensin complex promotes chromosome condensation, but the temporal control over condensin function is only scantly understood. In this impressive manuscript, "Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit", Tane and colleagues provide important new insight into the cell cycle-regulation of condensin. The authors identify a kleisin N-tail that acts as a negative regulator of condensin's DNA interactions. Removal of this N-tail, or its cell cycle-dependent phosphorylation, relieves inhibition and activates condensin. This is a simple, yet very important story, that advances our molecular understanding of chromosome formation. The experiments are performed to a very high technical standard and support the authors conclusions. This manuscript is highly suitable for publication in any molecular biology journal, once the authors have considered the following points.

1. Introduction. a) The authors could better explain their own prior work (Kimura et al. 1998), which has identified the condensin XCAP-D2 and XCAP-H as the targets of phosphoregulation. The current manuscript explains the role of XCAP-H phosphorylation. *

Response

According to the reviewer’s comment, we have added the following sentence in Introduction:

The major targets of mitotic phosphorylation identified in these studies included the CAP-D2 and CAP-H subunits.

1. b) Given the limited knowledge about condensin cell cycle regulation, it seems prudent to provide a brief summary of what is known. Fission yeast Smc4 phosphorylation regulates condensin nuclear import (Sutani et al. 1999), while budding yeast Smc4 phosphorylation slows down the dynamic turnover of the condensin complex on chromosomes (Robellet et al. 2015 and Thadani et al. 2018).

Response

We appreciate this constructive comment. According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.

Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015 and Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.

2. Extracts were mixed with mouse sperm nuclei. If there is a reason why mouse rather than Xenopus sperm nuclei were used, this would be interesting to know.

Response

The original motivation for introducing mouse sperm nuclei into Xenopus egg extracts was to test the functional contribution of nucleosomes to mitotic chromosome assembly. When mouse sperm nuclei are incubated with an extract depleted of the histone chaperone Asf1, the assembly of octasomes can be suppressed almost completely. Remarkably, we found that even under this “nucleosome-depleted” condition, mitotic chromosome-like structures can be assembled in a manner dependent on condensins (Shintomi et al., 2017, Science). Xenopus sperm nuclei cannot be used in this type of experiment because they endogenously retain histones H3 and H4 and are therefore competent in assembling octasomes even in the Asf1-depleted extract. During this study, we realized that the use of mouse sperm nuclei in Xenopus egg extracts provides additional and deep insights into the basic mechanisms of mitotic chromosome assembly. For instance, the functional contribution of condensin II to chromosome assembly could be observed more prominently when mouse sperm nuclei are used as a substrate than when Xenopus sperm nuclei are used (Shintomi et al., 2017, Science). We suspected that the slow kinetics of nucleosome assembly on the mouse sperm substrate creates an environment in favor of condensin II’s action. For these reasons, our laboratory now extensively uses mouse sperm nuclei for the functional analyses of condensin II (Yoshida et al., 2022. eLife) and other purposes (Kinoshita et al., 2022, JCB). Yoshida et al (2022) used experimental approaches analogous to the current study, and found that the deletion of the CAP-D3 C-tail, causes accelerated loading of condensin II. One of the long-term goals in our laboratory is to critically compare and contrast the actions of condensin I and condensin II in mitotic chromosome assembly. Thus, the use of the same substrate in the two complementary studies can be fully justified.

During the preparation of this response, we realized that the readers would benefit from a brief statement about the comparison between condensin I and condensin II. In the revised manuscript, we have added the following statement in Discussion:

It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II. Interestingly, however, a recent study from our laboratory has shown that the deletion of the CAP-D3 C-tail causes accelerated loading of condensin II onto chromatin (Yoshida et al., 2022). It is therefore possible that condensins I and II utilize similar IDR-mediated regulatory mechanisms, but they do so in different ways.

3. Page 5. "we next focused on the conserved helix (CH) [...], that is enriched with basic amino acids." Based on the provided sequence alignment, the helix contains an equal number of both basic and acidic residues. Is it correct to characterize this helix as positively charged?

Response

The reviewer is right. In the revised manuscript, we have used a more neutral expression as follows:

we next focused on the conserved helix (CH) [...], that contains conserved basic amino acids.

4. To prevent N-tail phosphorylation, the authors create a (H-N19A) allele, referring to Cdk promiscuity. Cdk cooperation with other mitotic kinases can also be expected. Nevertheless, in case the authors created a variant with only the 4 Cdk consensus sites mutated, it would be interesting to know its consequences.

Response

We consider that this is a reasonable question. In our early experiments, we noticed that introduction of multiple SP/TP sites in the non-SMC subunits of condensin I including CAP-H caused a relatively mild phenotype in mitotic chromosome assembly in Xenopus egg extracts. Then we found that the deletion of the CAP-H N-tail caused a very clear, accelerated loading phenotype, prompting us to focus on the regulatory function of the CAP-H N-tail. As the reviewer correctly points out, the current study does not pinpoint the number and position of target sites involved in the proposed phosphoregulation by the CAP-H N-tail. We wish to address this important issue in the near future, along with reconstitution of the phosphoregulation using purified components.

5. Fig EV3A, a second region of mitotic condensin phosphorylation is XCAP-D2. The authors state that XCAP-D2 phosphorylation does not impact on condensin function in their assays. This is very relevant to the current paper, so it would be good to see the Yoshida et al. 2022 Elife publication (in press) as an accompanying manuscript.

Response

We thank the reviewer for pointing out this issue, but it is not necessarily clear to us what the reviewer requests. In the original manuscript, we cited Yoshida et al (2022) in Discussion as follows:

Recent studies from our laboratory showed that the deletion of the CAP-D2 C-tail, which also contains multiple SP/TP sites (Supplementary Figure 3A), has little impact on condensin I function as judged by the same and related add-back assays using Xenopus egg extracts (Kinoshita et al, 2022; Yoshida et al, 2022).

We believe that the current statement is good enough.

6. One of the authors' most striking results is chromosome formation in interphase egg extracts using condensin (H-dN). At the same time, condensin (H-dN) is unable to support DNA supercoiling or chromosome reconstitution with recombinant components. More emphasis might be placed on this important piece of information, and possible reasons should be discussed. Can Cdk-treatment restore condensin (H-dN) biochemical activity? If not, then condensin (H-dN) might have lost more than just an inhibitory N-tail. The cohesin N-tail is thought to fulfil a positive role during DNA loading (Higashi et al. 2020). Could it be that the condensin N-tail encompasses both positive and negative roles?

Response

We were also surprised to find that holo(H-dN) gains the ability to assemble mitotic chromosome-like structures in interphase extracts. It should be stressed, however, that the formation of mitotic chromosome-like structures in I-HSS requires a much higher concentration (150 nM) than the standard concentration used in M-HSS (35 nM). Thus, the deletion of the CAP-H N-tail alone cannot make the condensin I complex fully active in I-HSS. We think that the negative regulation by the CAP-H N-tail is not the sole mechanism responsible for the very tight cell cycle regulation of condensin I function. We emphasize this important point by mentioning that “our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes” in Summary.

At the end of Discussion, we describe the limitations of the current study: “we have so far been unsuccessful in using these recombinant complexes to recapitulate positive DNA supercoiling or chromatid reconstitution, both of which require proper Cdk1 phosphorylation in vitro”. We are fully aware that full reconstitution of phosphorylation-dependent activation of condensin I in vitro is one of the most important directions in the future.

Although we currently do not have any evidence to suggest that the H N-tail has a positive role, we do not exclude such a possibility.

7. Here comes my main question for the authors (though I should stress that I do not expect an answer for publication in a Review Commons journal). The authors now have a unique opportunity to gain key mechanistic insight into condensin by answering the question, 'how does the kleisin N-tail inhibit condensin'? The authors allude to a model in which the N-tail interacts with Smc2 to close/obstruct the kleisin N-gate, through which the DNA likely enters the condensin ring. Can the authors biochemically recapitulate an interaction between an isolated N-tail (or N-terminal section of XCAP-H) and Smc2? Does Cdk phosphorylation alter this interaction?

Response

This comment is related to Comment #1 of Reviewer#1. See above for our response.

*Minor points. 8. The condensin loop extrusion results would benefit from a supplementary movie or time-series, to illustrate the comparison. Details of how loop rate, duration and sizes were assessed should be added to the methods section. *

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E. We have also added the following explanations for how the parameters of loop extrusion reactions were assessed in Materials and Methods:

To determine the loop size, the fluorescence intensity of the looped DNA was divided by that of the entire DNA molecule for each image, and multiplied by the length of the entire DNA molecule (48.5 kb). The loop rate was obtained by averaging the increase in looped DNA size per second. The loop duration was calculated by measuring the time from the start of DNA loop formation until the DNA loop became unidentifiable.

9. Figure EV3A legend, "hHP4" should probably read "hHP2".

Response

The reviewer is right. It should read hHP2. Corrected.

*Reviewer #4 (Significance (Required)):

see above *

This is an interesting way of evaluating a discipline: considering how many items must be remembered and recalled in active thought during the performance of a task. Machines can only get so far; holding the whole program in your head is the best way to go.

This is interesting for itself.

(reference: Sicker in the Head)

It's also interesting because it's an example of regular rehearsal that actors, comedians, storytellers, performers and even salespeople often do to slowly hone and improve their performance or pitch. Each retelling and the response it gives provides subtle hints and clues as to how to improve the story or performance on the next go round, or at least until the thing is both perfected and comes out the same way every time.

In Miracle and Wonder: Conversations with Paul Simon, Malcolm Gladwell comments on the prodigious memories of both Paul Simon with respect to sounds and Lebron James with respect to basketball game play.

Where these sorts of situational memories built and exercised over time or were they natural gifts? Or perhaps natural gifts that were also finely tuned over time?

Code is proof of itself; the ability to write code to compute some solution is proof by construction of a solution, and its behavior can be observed by demonstrating the code.

From what I've read about the process of Feynman and others, this isn't entirely true. Though there is power in being able to hold all of math in your head, the work is also on paper, or in the theorem prover, a symbiotic relationship that can track much more than the conscious mind.

I understand why Diller wanted to create another network to compete although at first glance it seems like a bad investment. If he was able to resuscitate the film division of Fox, then there would be no reason to believe he cannot do the same thing with network television.

she is making a metaphor about how it felt to get attacked by an owl, they said its like getting punched by a fist with rings.

This shows how hard these people had to fight without actually fighting. Holding back is harder than fighting something head on especially when people are pushing you to your limits. Going to jail is a serious thing that they had to face and having to do that when they did not deserve it tells us how much they went through just to try to get equal treatment.

<br> Source Excerpt: “Article 141

The President of the Republic performs the duties of the Executive Branch of Government and is the Head of State and Government and is in charge of public administration.

The Executive Branch is comprised of the Office of the President and the Office of the Vice-President of the Republic, the Ministries of State and the other organizations and institutions needed to fulfill, in the framework of their competence, the attributions of Leadership, planning, implementation and evaluation of national public policies and plans that are created to implement them.”

Data Source: https://doi.org/10.5064/F6WZUAYU/I9ME03

Full Citation: Constitution of Ecuador. Article 141. 2008. Page 60.

<br> Source Excerpt: “Article 141

The President of the Republic performs the duties of the Executive Branch of Government and is the Head of State and Government and is in charge of public administration.

The Executive Branch is comprised of the Office of the President and the Office of the Vice-President of the Republic, the Ministries of State and the other organizations and institutions needed to fulfill, in the framework of their competence, the attributions of Leadership, planning, implementation and evaluation of national public policies and plans that are created to implement them.”

Data Source: https://doi.org/10.5064/F6WZUAYU/I9ME03

Full Citation: Constitution of Ecuador. Article 141. 2008. Page 60.

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

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Reply to the reviewers

We really appreciate the reviewers’ insightful comments, which help improve the quality of this work. We have responded to the reviewers’ questions/comments point by point in the following text and made the corresponding changes in the revised manuscript. Lastly, we added one more figure (Fig. 7) with lineage tracing experiments demonstrating the conversion of id2a+ liver ductal cells to hepatocytes in extreme hepatocyte loss condition.

Reviewer #1 (Evidence, reproducibility and clarity):

Mi and Andersson describe a method for creating efficient 3' knock-ins in zebrafish using a combination of end-modified dsDNA and Cas9/gRNA RNPs. They tested their method on four genetic loci where they introduced Cre recombinase endogenously, and obtained high F0 mosaicism and germline transmission. The authors included fluorescent proteins with self-cleaving peptides to determine that endogenous expression patterns are observed. By crossing their knock-in Cre lines with lineage tracing reporter lines, the authors temporally traced lineage divergences in zebrafish liver and pancreas.

The authors should clarify the following points before I can recommend publication:

Overall, I suggest that the authors consider paring down their figures. Throughout the paper, multiple figure panels convey the same point but for different genes. Furthermore, many construct configurations are shown that are not used in the subsequent panels. For example, the mNeonGreen only (no Cre) constructs and the EGFP constructs are largely not used in downstream experiments. The authors could pick the important constructs and show the relevant data, and summarize all their other constructs in one supplementary figure. The authors also jump around in different parts of the paper with regards to using iCre or CreERT2 and ubi:Switch or ubi:CSHm. It's not clear to me why they're doing that? It makes the paper hard to follow. For example, why use iCre - it's not temporal if I understand correctly (and I'm not sure what improved Cre is - could they reference a paper and include a small explanation) so CreERT2 seems suitable especially for their temporal lineage tracing experiments. Why not limit the description to CreERT2 in the main text/figures? Also, isn't ubi:Switch and ubi:CSHm pretty similar except the latter is nuclear mCherry due to H2B? Why not only focus on ubi:CSHm experiments? I found the paper to be unnecessarily long and think it would benefit from editing to describe the most important concepts and experiments.

Response: Thank you for your constructive and helpful comments. We do agree that sometimes the schematic constructs seem redundant. This is because the krt4, nkx6.1, and id2a genes have similar gRNA targeting sites (all spanning over the stop codon). However, we prefer to keep these schematic constructs as we have all the statistical results showing the knock-in efficiency in the subsequent figure panels. Such layout can allow readers to make comparisons and better understand the efficacy of this method. However, combined with the comments from the second reviewer, we indeed need to add more detailed information, including the sequence and the length of the short left and right homologous arms in the schematics, to enable the readers to follow this strategy more easily. Meanwhile, we added a new supplementary figure with the sequences of the long left and right homologous arms, as well as the genetic cassettes/point mutations for krt92 knock-in (Figure EV1).

As for the color switch lines we used, we appreciate your comments and replaced Fig. 5E-G with new fluorescent images using zebrafish larvae carrying the ubb:CSHm transgene. For most of the lineage tracing experiments in this study, we used Tg(ubb:CSHm) as the H2BmCherry is more stable, located in the nucleus, and the fluorescence intensity is stronger than in Tg(ubb:Switch). However, for the lineage tracing experiments in the liver injury model, we believe that Tg(ubb:switch) is a better option than Tg(ubb:CSHm). In the absence of a hepatocyte specific far-red reporter line, we can distinguish the hepatocytes derived from the id2a+ origin using the Tg(ubb:Switch) line, as the cells with Cre recombination express mCherry in the cytoplasm; i.e. we can tell the cell types based on the cell morphology in combination with the ductal anti-vasnb staining. This strategy was previously used by Dr. Donghun Shin’s group in their 2014 Gastroenterology paper (Figure 4B, DOI: 10.1053/j.gastro.2013.10.019). Therefore, we still kept the ubb:switch in the Fig. 1F schematic, and we have elaborated on why we chose Tg(ubb:switch) line for the id2a+ cell conversion experiments in Fig. 7 and Figure EV14.

The iCre we used is a codon-improved Cre (iCre). The original cDNA sequence was from pDIRE (Addgene plasmid #26745; provided by Dr. Rolf Zeller, University of Basel) (Osterwalder et al., 2010).

At the beginning of this project, we actually didn’t know whether there were any differences between iCre and CreERT2 in labelling of the cells of interest. Here, using both the iCre and CreERT2 lines, we for the first time, formally show the developmental lineage path of nkx6.1-expressing cells in the zebrafish pancreas. Our data suggested that the early nkx6.1-expressing cells are multipotent pancreatic progenitors giving rise to all three major cell types in the pancreas (endocrine, ductal and acinar cells, shown by nkx6.1 knock-in iCre) and gradually the nkx6.1-expressing cells become restricted in the ductal/endocrine lineages (shown by the nkx6.1 knock-in CreERT2 treated with 4-OHT at different timepoints). In addition, we also aim to use these knock-in lines for multiple studies in which we need to perform many quantitative experiments. As expected, we are unable to reach 100% labeling using the knock-in CreERT2 lines, even if we treated the larvae with very high concentration of 4-OHT over a long period of time. This means that the CreERT2 induced recombination will introduce more variation for quantitative experiments (for instance, the number of regenerated beta-cells from the ductal origin). As we were quite confident with the efficiency of this knock-in strategy, we decided to make both iCre and CreERT2 lines in krt4, nkx6.1, and id2a locus and just observe how they performed. We often use iCre knock-in lines for lineage tracing experiments, because the iCre lines reach near 100% labeling efficiency. Such iCre lines are particularly useful if they only label terminally differentiated cell types. Thus, the near 100% labeling efficiency in iCre lines can be of great help for initial experiments, which later can be confirmed by temporal labeling using CreERT2 lines.

1. Could the authors describe the purpose of the 5'AmC6 modification earlier in the paper? I didn't see much text about it until the discussion. It seems that the speculation is that it provides end protection and prevents degradation (based on in vitro studies in human). This should be inserted into the introduction as a reader might be wondering about this and won't find an answer until near the end. Also, is this the first in vivo use of this modification for knock-ins? If so, that should be highlighted in the text.

Response: This is a helpful comment. In the revised manuscript, we elaborate more on why we chose 5'AmC6 modification in our donors. To our knowledge, this is the first time this 5’ modification is used in vivo, however, bulky 5’modification (5'Biotin - 5x phosphorothioate bonds) has been used in medaka (DOI: https://doi.org/10.7554/eLife.39468.001, 2018 Elife, as we previously referenced). The cell division rate is much faster in zebrafish embryos compared with medaka embryos during early development, so we speculate that such modification might be of more importance in zebrafish to achieve early integration. Another advantage is that the 5'AmC6 modification is commercially available, allowing researchers to prepare the donor dsDNA in a handy fashion. We have now expanded on these details and advantages in the introduction.

1. The authors do not show any sequencing data confirming that their insert was knocked-in as designed with no disruption to the immediate upstream and downstream endogenous sequences. Can they sequence the loci to confirm?

Response: This is indeed a question we frequently get – thank you for making us relay this information more clearly! We have put the raw Sanger sequencing data in a public repository (mentioned in the Data Availability section), and included the sequencing primers in the method paragraph. Now we also refer to this data in the discussion section in conjunction to highlighting that the integrations were correctly placed in the loci. If you think there are better ways to show the sequencing results, please let us know.

1. I found the descriptions of the long and short HA to be confusing when describing the results, especially since the first tested gene krt92 only has long and all subsequent ones are short. The discussion made it more clear that short HA is more efficient and applicable when gRNAs span the stop codon. Perhaps that wasn't possible with krt92, but the authors could prevent the confusion by clearly stating the design requirements of long and short HA and that they wanted to test which is more efficient before starting to describe the data. I also didn't see a description of what the length difference between long and short HA is? How short is short HA?

Response: This is a great question that is well worth discussing. In the revised manuscript, we changed the order in which the parts are described, with nkx6.1 knock-in in front of krt4 knock-in. Here we explain why we would like to do that:

At the beginning of this project, we did not know if the 5’ modified dsDNA could be an effective donor. To test our hypothesis, we chose the krt92 gene as our first target, as this is a keratin protein and expressed in the epithelial cells. We can easily detect the fluorescence in the epithelial cells (most notably in the skin), which allow us to sort the F0 mosaic embryos with high percentage of integration. Notably, from our experience, the most difficult part of the knock-in method is the sorting step (usually performed during 1-3 dpf). This is because the fluorescence signal is highly dependent on the endogenous gene expression level and is usually dimmer with an overall integration efficiency that is lower compared to canonical transgenesis. Therefore, we thought that targeting an epithelial cell marker would be informative and help us to evaluate the validity and reproducibility of the method. If it worked, then we could move on targeting genes expressing in more restricted tissues or cell types. For krt92 gene, the gRNA targets the region upstream of the stop codon. To prevent the cleavage of the donor template, we had to introduce several point mutations and at the same time keep the amino acid sequence intact. However, such mutations can restrict the knock-in and lower the integration efficiency when using shorter arms (due to the sequence mismatch).

After we managed to make the krt92 knock-in, our next question was, what about using a gRNA spanning over the stop codon region? In this way, we don’t need to introduce point mutations on neither the left nor the right homologous arm. Also, for the purpose of our biological study, the nkx6.1 were on top of our gene list for lineage tracing experiments and we luckily identified that there is very good gRNA targeting this locus. After we successfully made the nkx6.1 knock-in, we were thinking that we could simplify the protocol even further, i.e. switching to short homologous arms so that we can prepare the donor by a one-step PCR instead of making complicated constructs. We tested that hypothesis in nkx6.1, krt4, and id2a sites and obtained very promising efficiency. Also, we did some further testing with dsDNA without the 5’ modifications and showed that the 5’ modifications indeed greatly increased integration efficiency. Therefore, although the short homologous arm method is a highlight here, we also point out that it was not planned from the beginning. In the revised manuscripts, we want to convey our method in a logical way and show how we modify the method in a step-by-step fashion.

Moreover, with regards to the comments from the second reviewer, we now added the length of the homologous arms as well as the mutation site on the schematics. We chose short homologous arm because in previous literature it was suggested that short homologous arms (36-48 bp, which we now write out in both the results and the methods) can promote microhomology-mediated end joining (doi: 10.1096/fj.201800077RR). We also noticed that the recent Geneweld method (DOI: 10.7554/eLife.53968) also adheres to a similar length for homology mediated integration. In this study, HAs even shorter than 36 bp also perform well.

1. The authors state that they could not use in situs to confirm krt92 endogenous and knock-in expression overlap, but rather say that they match based on data from an intestine scRNA-seq dataset. Can they elaborate on this? Which clusters/cell types show overlap? Furthermore, is there any krt92:GFP transgenic line that can be used as a reference for expression as well? This point is also applicable for krt4 described in Fig.2

Response: We appreciated this point. In the beginning, we contacted Molecular Instruments to synthesize krt92 HCR3.0 in situ hybridization probes. However, the technical staff there told us that they are unable to make specific probes due to high sequence similarity to other keratin protein families. We can see that the sequence similarity mostly occurs in the middle of krt92 genes, and the HCR3.0 probes rely on a probe set (preferably 20-30 probes with different sequences) to target the mRNA.

The scRNA-seq data that we referenced are from 10X platform, which is based on a 3’enrichment methodology. The reads mapping to krt92 genes are mostly located on the 3’ end. This is good as there is much less similarity to other cytoskeleton genes in the 3’ end of the gene. Unfortunately, there is no krt92 transgenic lines available, so we relied on the single-cell data to correlate expression patterns in this case.

There are two zebrafish intestine single-cell data sets available, with the following links:

(1): https://singlecell.broadinstitute.org/single_cell/study/SCP1675/zebrafish-intestinal-epithelial-cells-wt-and-fxr?genes=krt92#study-visualize

(2): https://singlecell.broadinstitute.org/single_cell/study/SCP1623/zebrafish-intestine-conventional-and-germ-free-conditions?genes=krt92#study-visualize

We can see that krt92 is widely expressed in different types of intestinal epithelial cells (absorptive enterocytes, secretory enteroendocrine/goblet cells and ionocyte).

For the krt4 gene, we now added the HCR3.0 in situ hybridization and immunofluorescence for both krt4 knock-in EGFP-t2a-CreERT2 lines and the Tg(krt4:EGFP-rpl10a) transgenic line (a construct from Anna Huttenlocher, https://www.addgene.org/128839/, which has been widely used to label skin cells). The results are shown in Figure EV9. We show that krt4 has very high expression in the intestinal bulb and hindgut based on the HCR3.0 in situ. The Immunofluorescence of the krt4 knock-in fully recapitulate the krt4 expression pattern in the intestine, while there is almost no fluorescence signal in Tg(krt4:EGFP-Mmu.Rpl10a). We believe this is another advantage of using the knock-in method, over transgenics, for cellular labeling and lineage tracing. Classical transgenics often rely on short promoters of the proximal/enhancer region upstream of ATG with various length (arbitrarily or based on clues from motif analysis/DNA methylation sites). However, different tissues/cell types tend to use different cis-_regulatory elements and the chromatin structure/enhancer-promoter loops might differ dramatically among different cell types. It is hard to predict the exact region of the regulatory sequences that is sufficient for driving the gene expression in a certain cell type. Thus, such reasoning consolidates with that our knock-in lines recapitulate the endogenous _krt4 gene expression. Therefore, we believe that the knock-in based genetic lineage tracing will become the standard in the zebrafish field, as theoretically it avoids both the lack of relevant expression and leakage problems of transgenics.

1. I think Figure 2A needs the dotted lines on the last construct to be fixed (points to p2A)

Response: Thank you for noticing! This was due to a bug in the IBS software, and we changed it manually using Adobe Illustrator in the revised manuscript.

1. There are a few instances where the authors describe performing 4-OHT treatment for long period (e.g. over a 20 hour or 24 hour period). Is fresh 4-OHT added after a certain amount of time or is it a one-time addition? Is such long periods of 4-OHT required or has maximal recombination already occurred within a few hours after addition of 4-OHT?

Response: For 4-OHT treatment, we referred to the method described by Dr. Christian Mosimann (DOI: 10.1371/journal.pone.0152989). We actually tried different conditions (dosage, duration, refresh or not). This is particularly important for the knock-in CreERT lines because the level of CreERT2 is highly dependent upon the endogenous gene expression level. In our case, the nkx6.1 and id2a are transcriptional regulators and relatively lowly expressed compared with structural proteins. We maximized the labeling efficiency by using the highest concentration and longest duration suggested for 4-OHT treatment. The 4-OHT was stored in -20 ℃ and it would become less effective after 30 days of storage. Therefore, we first incubated the 4-OHT in 65 ℃ for 10 min (as recommended by Dr. Christian Mosimann) in order to convert it to a bioactive form. Next, we treated the zebrafish embryos with 4-OHT using a final concentration of 20 μM for 24 hours. We didn’t refresh the 4-OHT since there was no significant difference compared with a one-time addition. Moreover, using higher dosage or longer treatment time can lead to less survival and increased deformity rate. 20 μM 4-OHT treatment for shorter time periods (6 or 12 hours) can cause high labeling variability (some larvae have good labeling while others not). In the end, after several rounds of experiments, we settled on 20 μM 4-OHT treatment for 24 hours as it can reach the highest labeling efficiency, lower variability, and good survival.

1. For Figures 4-6 where confocal images of lineage tracing experiments are shown, there is no indication of how many times the experiments were repeated, how many sections were images, how many animals used, how many cells counted. All of this information should be included in the figure legends and plots should be added showing quantification and statistical analysis (where appropriate).

Response: The reviewer makes a good point and we have now added the number of larvae used and statistical results for the quantitative experiments. The quantification of experiments in Figure 3E-H (originally Figure 4E-H) are shown in Figure EV6D using box/dotplot. We randomly selected 3 secondary islets of different sizes (large, middle, and small) from each juvenile fish (n=5) and pooled the number of mCherry/ins double positive cells and ins positive cells together. The quantification of the lineage-tracing efficiency in the experiments in Figure 6 are shown in Figure EV13.

1. Figure 4 C, C' - I'm not sure what to look for. Is the message that there is no Cherry positive cells that are vasnb negative when labelling is done at 8 somite? But the vasnb positive cells that are also Cherry positive remain? The vasnb staining seems much weaker/harder to see in C C' compared to B, B'. As mentioned above, these data should be quantified and statistical significance indicated.

Response: Thank you for pointing this out; the second reviewer made a similar point. We redid the experiments using zebrafish larvae carrying the ptf1α:EGFP transgene to indicate the acinar cells (Figure 3B-D, Figure EV4G). We also quantified the results and performed statistical testing.

1. I recommend the authors include a short section in the discussion comparing the efficiency of their method to other knock-in strategies used in zebrafish. This is an important claim of the paper yet it is not clear how much better it is (if at all) in terms of frequency of F0 mosaicism and identification of founders relative to other methods. I do appreciate the relative simplicity of the molecular steps of construct design/generation.

Response: This is indeed important. It is also tricky since we are unable to make head-to-head comparisons between different methods as we are targeting different genetic loci and do not have the other methods up and running in our lab. However, the general comparison is based on the statistics shown in the hallmark papers describing these other methods, regardless of which genes were selected for targeting. In the discussion, we added a list of points that are novel/improved with our method versus previous ones, including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product. We believe this is critical for the field of developmental biology, regenerative medicine, and disease modeling in zebrafish – and perhaps a similar 3’ knock-in based lineage-tracing method can become commonly used to delineate the cell differentiation and plasticity during homeostatic and diseased conditions in additional organisms.

Reviewer #1 (Significance):

Overall, the study contributes a new knock-in strategy in zebrafish that appears to be more user-friendly and results in high germline transmission. The authors also identify nkx6.1+ ductal cells as progenitors of endocrine cells in the pancreas highlighting the biological applications of their method. I think this study represents an important advancement in zebrafish genetics and will have future impact in lineage tracing during development, regeneration, and disease.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:

Here, the authors present a strategy where they performed knock-in at the level of the STOP codon, taking care of not perturbing the coding region. They integrate cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides.

The cassettes are done by PCR with primers with 5' AmC6 modifications and they test short (36 to 46 bp) or long homologous arms (~950bp). For nkx6.1 gene, they observed a dramatic increase of recombination efficiency when injecting the donors with short Homology arms compared to long arms suggesting that short arms could be used. Indeed, short arms used with krt4 and id2a allow them to obtain K.I lines.

The techniques described here look promising. Indeed, even if the proportion of F0 showing adequate reporter expression is low (usually about 2%), the percentages of founders among these mosaic F0 were quite high (between 50% and 100%). And this is the most important aspect as it is usually the most time-consuming aspect of the work.

Major comment:

The authors claim that the knock-in lines can precisely reflect the endogenous gene expression, as visualized by optional fluorescent proteins. But are the authors sure that the integration of the cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides, will not affect the level of expression of the targeted genes . Indeed, it has been shown that sometimes self-cleavable peptides could affect the expression of the genes of the cassette like for example in this reference ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034980]. Therefore it is important that the authors check whether the cassette affect the level of expression of the targeted gene if they want to claim that the knock-in lines precisely reflect the endogenous gene expression.

Response: Thank you for your insightful comments. With regards to the endogenous gene expression, we now use qPCR for further validation. We added the qPCR results to the supplement material (Figure EV15) in the revised manuscript. In brief, we pooled 4 larvae in one tube per biological replicate and have 4 biological replicates for each knock-in line. We didn’t see a significant change in the endogenous expression for any gene. In addition, we have grown up homozygous knock-in lines to adulthood and they are fertile without any overt phenotype.

The highlighted reference is dealing with a cardiomyocyte specific transgenic line, and we assume figure 3-Supplementary figure 1 is what the reviewer is referring to. The altered level of erbb2 expression might be due to the experimental conditions (no treatment or 3 days post treatment). Also, it is possible multiple transgenic insertions occur, as well as gene silencing at some insertion sites. However, such issues would not present, or very limited, with knock-in methods.

Minor comments:

General points:

I believed that the authors should improve the presentation of their data. Indeed, based on what they present, it would be impossible for me to reproduce their technique. Indeed, it is not clear at all how they design the short and long arm, where they are exactly located, which mutations they have done (for fig1), where is located the guide RNA compared to the STOP codon and the HA arms. Graphics that exactly place all these sequences are absolutely required to understand the strategy used and should be placed in figure 1, 2, 3 and 4.

Response: Thank you for these comments. In the revised version, we added the sequence information of the short homologous arms in each of the schematics. As for the krt92 gene, we added the sequence information in the first supplement results (Figure EV1) with the genetic cassettes and point mutation information. We list all the primer information in the methods. Also, we have uploaded our vector templates in the public repository (as listed in the Data availability section). Lastly, we added a key resource table in the supplement file with all the detailed information of reagents for the ease of reproducibility (including all the primers sequences used). We are also willing to share our constructs with the scientific community upon request.

Specific points:

Introduction:

"In zebrafish, the NHEJ-mediated methods have been intensively investigated in 5'knock-in upstream of ATG using donor plasmid containing in vivo linearization site flanking the insertion sequences (11,12,17-20). The 3' knock-in method has also been examined using circular plasmid as the donor with either long or short homologous arms (HAs) flanked by in vivo linearization sites (14, 21-23). Recently, intron-based and exon-based knock-in approaches have remarkably expanded the knock-in toolbox by targeting genetic loci beyond the 5' or 3' end (8-10,13,24-26)."<br /> This part should be explained better in order that the readers could really understand the differences between these old studies and this new one. And really insist on what is the novelty of their technique.

Response: Good points. In the revised version, we elaborated more on the previous discoveries, the major challenges, the knowledge gap in zebrafish knock-in methodology, and what is novel and improved with our new technique. Please, see clarifications and the expanded text in both the introduction and discussion.

Results:

Page 4: To my opinion, the first paragraph should be removed and the technique directly explained based on krt92 strategy as this paragraph does not allow to understand the technique. As indicated above, figure 1 should indicate more clearly the location of the long arms and which mutations they have done and where is located the guide RNA.

Figure 1G: The expression in the skin is far from obvious and the image should be improved (for example with some inset).

Response: Thank you for the comments. We added a new supplementary figure (Figure EV1) and show the sequences of left and right homologous arms, the genetic cassettes, as well as the point mutations with different background color highlight. We added the insets to show the magnified regions of interest. Also, we added the images from the fluorescent microscope used for sorting, to show the EGFP signals in live zebrafish embryos (Figure EV2D and Figure EV8D).

Figure 3E: The authors say that "cells expressing nkx6.1 (displayed by the green fluorescence) were located on the ventral side of the spinal cord whereas H2BmCherry positive cells, which include all the progenies of nkx6.1+ cells after the iCre recombination, resided in both the ventral and dorsal parts of spinal cord". This differential expression in the spinal cord is not obvious and a more closer view should be provided.

Response: Thank you for the comment. First, we changed the order and now describe all nkx6.1 content in Figure 2 and 3 and the krt4 content in Figure 4. We added insets to show the magnified regions and better display the expression pattern of the two fluorescence proteins in Figure 2E-G. One can now clearly see from the magnified insets that the green signals driven by the endogenous nkx6.1 gene are present in the ventral part of the spinal cord, while the red signals are present in both the ventral and the dorsal side of the spinal cord.

Fig S4H: The authors say that" using lineage tracing, we could trace back all three major cell types in the pancreas (acinar, ductal and endocrine cells) to nkx6.1 lineage (Figure 3H-H',Supplementary Figure S4G, H)". While this is obvious for endocrine, the colocalisation with ela3l:GFP is not obvious and the figure should be improved.

Response: This is a very good point, and the first reviewer gave similar suggestions. In the revised version (shown in Figure EV4H and I), we added the insets to show the magnified regions to better display the expression pattern of two fluorescence proteins. The ela3l reporter line is using a short promoter to drive the expression of H2B-EGFP (doi: 10.1242/dmm.026633). However, this short promoter cannot reach 100% labeling of acinar cells, so we also use the ptf1α:EGFP transgene for further validation (new Figure EV4G). Both transgenic reporter lines showed many EGFP and mCherry double-positive cells, indicating that these acinar cells are derived from a nkx6.1-expressing origin. Here we did not use the anti-GFP antibody, as our color switch lines contains CFP and anti-GFP antibody can also recognize CFP. However, the GFP signal is strong enough to show the expression. We hope the additional experiments and insets clarifies this point.

Page 8: the authors say that "The immunostaining at 6 dpf showed that both intrapancreatic ductal cells and a portion of acinar cells can be lineage traced when the 4-OHT treatment started at the 6 somite stage (Figure 4B and B'). The identification of the acinar cells has been done based on the absence of the ductal marker vasnb. To trace efficiently the acinar cells, this should be done with an acinar marker.

Response: Another good point also mentioned by reviewer one. We redid the analyses using zebrafish larvae containing the ptf1α:EGFP transgene to indicate the acinar cells and the co-expression pattern with the lineage-tracing (the data is shown in new Figure 3B-D).

Reviewer #2 (Significance):

I do not have enough expertise in the KI field to evaluate whether this strategy is really novel and as mentioned above, the authors should better explain what is really the novelty of their strategy.

Response: In our answers to the comments of the first reviewer, we elaborated more on the points that are novel/improved with our method vs previous methods, as reiterated here:

“…including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product.”

Moreover, the first reviewer asked about the difference between the krt4 knock-in and krt4 transgenics, and based on the in situ data, we showed that our krt4 knock-in can fully recapitulate the endogenous gene expression, while the krt4 transgenics can hardly label the intestinal bulb and hindgut. This might be due to that different tissues/cell types may depend on different _cis-_regulatory elements to drive the gene expression. The chromatin structure and the enhancer/promoter loop might also differ dramatically among different tissues. Therefore, the transgenics might be useful for one type of cells, while they might be not useful at all for other cell types. In the future, we believe that, similar to the mouse field, the 3’ knock-in based lineage tracing methods might become the standard method in the zebrafish field, to delineate cellular differentiation and plasticity during homeostatic and diseased conditions.

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Manuscript number: RC-2022-01490

Corresponding author(s): Cariboni, Anna; Howard, Sasha R

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

The current manuscript in question is well written and of general interest to the reproductive neuroendocrinology field. Overall it is a well written and substantiated.

Reply: We thank the reviewer for his/her positive and supportive comments on our manuscript.

The primary problem with the paper is the data derived from the microarray. While the experimental design included replicates (n = 3), although weak, the actual microarray data was based on a single data point. A major weakness. This experiment should be repeated using more up-to-date approaches such as RNA-seq or left out of the manuscript, because this data set is compromised due to the data collection procedure.

Reply: We thank the Reviewer for raising these points, which we wish to clarify. We respectfully disagree that the microarray data generated in this study is not valuable. The transcriptomic analysis of immortalized cells was performed on 3 biological replicates (specifically, RNA was extracted from n=3 samples, obtained from each cell line at 3 different passages) and run as 3 independent samples (for a total of 6, 3 for GN11 cells and 3 for GT1-7 cells). For the primary embryonic GFP-GnRH neurons, given the difficulty of isolating with FACS a sufficient number of GFP+ cells from each embryo due their very small number (around 1000 GnRH neurons/head), we had to pool sorted cells from 2-3 embryos for each time-point. Thus, although the primary cell microarrays were run on one sample for each time point, the RNA was not derived from one embryo only, but from at least 2/3 embryos.

Nevertheless, to overcome the issue of low number of replicates for the primary embryonic cells, we revised our manuscript by re-running our analyses, using as the starting dataset the analyses obtained from immortalized cells, which were based on a ‘true’ n=3 of biological replicates. In this context, we filtered DEGs from this microarray using logFC>2 and adj. p-value1) found in primary GFP-GnRH neurons. We believe that this revised analysis is statistically more powerful, as the core bioinformatic analyses were performed on triplicate samples, with a second filtering step to take advantage of biologically relevant data obtained from n=1 primary GFP-GnRH neurons to confirm in vivo the expression of selected genes. Whilst RNAseq offers wider coverage of the genome and has advantages over microarray, we do not believe that this renders unimportant the data generated from these unique experiments and the novel genomic discoveries it facilitated.

In line with this, our work may be considered as a proof-of-principle that transcriptomic profiles from rodent GnRH neurons can be exploited at different levels, including the possibility to identify novel GD candidate genes. Overall, our work also highlights the existence of similarities between two immortalized GnRH neuron cell lines with primary GnRH neurons, which was so far demonstrated by several functional studies, but not at molecular level.

The manuscript has been now edited as per the above amendments (see first and second paragraph of Results section, lines 86-135).

__CROSS-CONSULTATION COMMENTS __Notwithstanding the importance of neuroligin 3 during glutaminergic synaptogenesis, I agree with the reviewers on both points. Further screenings of the patient's family members should be done and the microarray data should be removed or potentially moved to a supplementary status.

Reply: we thank the reviewer for their comments and, accordingly with their suggestion, we revised the filtering strategy starting from immortalized cells microarray and therefore moved a substantial part of the microarray data from primary GFP+ neurons as supplementary data. We also unsuccessfully tried to collect information of the brother from case 2 and investigated datasets from both the DECIPHER and 100,000 genome projects, but have been limited to two cases for which we have familial consent to publish.

Reviewer #1 (Significance (Required)): The paper is of significance based on the neuroligin 3 data, which is indicative of abnormal synaptogenesis. However, these defects seem to only have a limited effect on the functionality of GnRH neuron system and do not seem to cause elimination of GnRH neurons themselves. Nevertheless these data do open end a new direction that may help explain some dysfunctions in reproductive health.

Reply: we thank the reviewer for their comments and agree that our findings have the potential to facilitate new avenues for the investigation of reproductive disorders.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Oleari et al performed comparative transcriptome analysis on the different developmental stages of GnRH neurons, as well as two immortalized GnRH neuronal cells GT1-7 and GN11 which represent mature and immature GnRH neurons. As a results, they identified a panel of differentially expressed genes (DEG). They further used top DEGs as candidate disease-related genes for GnRH-deficiency (GD), a disorder characterized with absent of delayed puberty and infertility. To this end, they found two loss-of-function mutations in NLGN3 in patients with GD combined with autism. This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. I only have some minor concerns.

1. According to the pedigree, both probands (case 1 and 2) inherited their NLGN3 mutations from their unaffected mother, consistent with an X-linked recessive inheritance. However, only "parent" was used in the manuscript, therefore, it is not clear if this "parent" is the probands' mother or father. __Reply: __Thank you for this comment. We were limited to the use of non-gendered terminology due to medRxiv policies. We have now amended the text and changed ‘parent’ to ‘mother’, lines 161, 173, 179, 185 and 730. We also integrated this sentence highlighting the X-linked pattern of inheritance: “Sanger sequencing of the probands’ mothers confirmed them to be the heterozygous carrier in each family, consistent with an X-linked recessive inheritance pattern.”, lines 185-186.

It is suggested to integrate Figure 2 as a panel in Figure 1.

__Reply: __We thank the reviewer for this suggestion. Due to our revision of first two Results paragraphs, we have now edited the Figures and the filtering flowchart has been added in Figure 2.

What is the meaning of Peak LH and Peak FSH, and how are they measured in Table 2?

Reply: This refers to peak value obtained after standard protocol GnRH stimulation testing with 100mcg GnRH (Gonadorelin) as an IV bolus and measurement of serum LH and FSH at 0, 20 and 60 minutes intervals. (e.g. Harrington et al., 2012, doi:10.1210/jc.2012-1598). This clarification has been added to the text in Table 2 legend (lines 681-683).

A genotyping for the elder brother of Case 2 will be a strong evidence to support NLGN3 as a GD-associated gene.

__Reply: __We thank the reviewer for this important point. In view of this issue, we have strived to collect DNA from this individual. Unfortunately, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.

We also identified a third case via a public database with central hypogonadism who carried a stop-gain variant in NLGN3, but unfortunately the family did not release their consent for publishing this case.

The authors claimed neither probands carried deleterious variants in known GD genes. It is suggested to indicate the exclusion criteria (which genes? How do they define a variant is deleterious?)

Reply: We thank this reviewer for raising this important point of clarification. Inclusion criteria for variants in known GD genes (updated gene list available in Supplemental Table 3) were as per Saengkaew et al., 2021 (doi: 10.1530/EJE-21-0387): “Only variants that met the ACMG criteria for pathogenicity, likely pathogenicity, or variants of uncertain significance (VUS) were retained in the analysis”. We have added this sentence in the manuscript, lines 150-151.

Please also include a sequence chromatogram for proband 2.

Reply: We thank the reviewer for their comment. We added the chromatograms for proband 2 and his heterozygous mother in revised Figure 3.

CROSS-CONSULTATION COMMENTS I agree with Reviewer 3, the genetics is not very strong, as NLGN3 mutations were only found in one GD case from their cohort and one pre-pubertal case from the literature. It will be nice to analyze the genotype and phenotype of Case 2's older brother. Further, it is important to screen NLGN3 rare sequencing variants in larger GD cohorts.

Reply: We thank the reviewer for their comment, but respectfully disagree with this assertion. The second case is not from the literature, but is a second case found thanks to GeneMatcher, an international tool that allows researchers to collaborate on novel gene discovery. We have also explored other cohorts that were available to us, including the DECIPHER and 100,000 genome project, but have been limited to two cases for which we have familial consent to publish. We anticipate that further international patient cohorts will be screened following the publication of this manuscript (added in Discussion section, lines 306-308). As described above, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.

Reviewer #2 (Significance (Required)): This study provides a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. It may welcome by researchers and clinicians in the filed of neurodevelopment.

Reply: We thank the reviewer for their positive and supportive comments.

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

__Summary: Oleari et al used murine GnRH1, and immortalized GnRH cell lines (GT1-7, Gn11) to define genes of interest in GnRH development and used this list to filter exome sequencing data from patients with some evidence for GnRH Deficiency.

Title: I am concerned that the title of the paper overstates the results and conclusions.

Intro: use of "candidate causative genes" overstates the evidence presented.

__Reply: __We thank the reviewer for their comment and have revised the title to reflect the findings of the study. We have also edited the sentence in the abstract reporting "candidate causative genes" as follows: “Here, we combined bioinformatic analyses of primary embryonic and immortalized GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in GD pathogenesis”, lines 40-43.

Results: The transcriptomic profile of the developing human GnRH neuron has been published via in vitro differentiation protocols twice (Lund et al 2020, and Keen et al 2021). Gene set data is publicly available. This should be explicitly compared in results not relegated to discussion -- two or three examples it not enough to say mouse can be used instead of human.

__Reply: __We thank the reviewer for this comment. We apologize if our sentence in the Discussion was misleading, as we did not intend to make a conclusion on the similarities of the two datasets/cell types, neither to suggest the use of rodent instead of human.

Although we are aware that differences among species might exist, mouse/rodent models including immortalized cells have been instrumental to understand the molecular mechanisms of GnRH neuron development and to predict candidate genes. Indeed, our aim was to demonstrate that transcriptomic profiles of rodent GnRH neurons could be integrated with exome sequencing data from human patients to reveal novel candidate genes.

Therefore, the aim of our study was different to that of the Lund and Keen publications. Further, caution should be exercised in any deeper comparative analyses with our transcriptomes, for following reasons: first, the GnRH neurons generated from human iPSC and cultured for 20 and 27 days cannot be objectively defined for their ‘age’ in order to be then compared to immortalized or primary embryonic GnRH neurons; second, in these datasets a different and more extensive transcriptomic technique has been used (RNAseq vs microarrays).

There was no intention to relegate to the discussion the possible similarities with other transcriptomic datasets, but we felt that these comparative analyses were beyond the scope of our work.

However, following the Reviewer’s suggestion, we have tried to make comparative analyses with the publicly available datasets from Lund et al 2020 and Keen et al 2021, and with a paper just published (Wang et al 2022), as follows.

In Lund et al. paper, GnRH-like neurons were obtained from human iPSCs by dual SMAD inhibition and FGF8 treatment. We selected data obtained from cells treated with FGF8 and cultured for 20 days and 27 days for comparison with our early and late genes, respectively.

Because the authors of this paper did not publish the full list of differentially expressed genes (DEGs) from this specific comparison (20 vs 27days) and we were not able to retrieve it upon request, we used the normalized counts of these samples (available at ArrayExpress repository) to compare the two experimental groups with DESeq (Bioconductor release 3.15). To increase stringency of our analysis, we considered as differentially expressed those genes which displayed both an adjusted p-value of less than 0.05 and an absolute fold change of >2. The number of DEGs obtained was different and greater (5981) than from the published data, and this large number of genes may, by chance alone, contain a large fraction of any gene dataset (including the genes that we found with our analysis). For this reason, this particular comparison in this dataset cannot be informative or useful.

Next, we considered the dataset from Keen et al. In this paper, the authors have tested different differentiation protocols to obtain GnRH-like neurons from human wild-type or mCherry embryonic stem cells (hESC). They transcriptomically profiled hESC-mCherry-derived GnRH neurons at 8,15 and 25 days of culture.

Again, although we cannot precisely define the matching embryonic stage of cells cultured for 8, 15 or 25 days, we compared the lists of DEGs from immortalized GnRH neurons (GN11vsGT1-7) with the transcriptomic profiles of mCh-hESC at day 15 vs day 8 and mCh-hESC at day 25 vs day 15, respectively. We considered as differentially expressed the genes that displayed both an adjusted p-value of less than 0.05 and logFC>2. We found that the majority of the genes that were differentially expressed in one dataset were not in the other. However, the few genes that were differentially expressed in both datasets demonstrated a good correlation, i.e. the same expression trend. Although this latter approach was more fruitful, by suggesting a partial similarity between primary GFP-GnRH neurons and hESCs-derived GnRH neurons at day 25 vs day 15 time-point, we do not feel that we could draw significant and reliable conclusions.

Further, if we compare these two datasets obtained by RNAseq from hiPSC and hESC, even by taking into account the large amount of DEGs found in our re-analysis of Lund et al., 2021 raw data, a relatively small number of common DEGs were found. These data also suggest that there is transcriptomic heterogeneity even among human-derived GnRH neurons.

In addition to these two datasets, while our manuscript was under revision, a new paper was published, in which the authors dissected iPSC-derived GnRH neuron transcriptome with RNA-seq at single cell level (Wang et al., 2022, doi:10.1093/stmcls/sxac069). Again, although the same concerns may apply in comparing this dataset with ours and raw data of DEGs were not publicly available in this case, we compared the expression trends of our 29 candidates with gene expression trajectories identified in this work. As a result, 24/29 candidate genes, including NLGN3, were found to have an expression trend consistent with our dataset. The few remaining genes exhibited an opposite trend (2/29) or were not found in available data from this work (3/29). As this is a purely qualitative analysis, we do not feel it would be appropriate to include it in the Results section, but have included commentary on these comparative dataset analyses in the Discussion section (lines 247-257). A future study could be designed to mine the raw data from all the available transcriptomic profiles of developing GnRH neurons, but this is beyond the scope of our current manuscript.

The authors need to comment on other GnRH1 expression in the brain of developing rodent and if they think the GnRH1 sorted neurons are just "GnRH Neurons" associated with reproduction (Parhar et al 2005) due to microdissection.

__Reply: __We thanks the reviewer for raising this point of clarification. We have carefully selected by microdissection nasal areas from E14, nasal and basal forebrain areas from E17 and basal forebrain from E20 rat embryos (see revised Methods, lines 325-327). We are therefore confident that what we have obtained is RNA from ‘reproductive’ GnRH neurons only.

Questions about Cases/Missing Phenotypic Information: 1) Case 1: the patient underwent increased testicular volume on testosterone therapy -- testosterone therapy does not increase testicular volume. Has this patient undergone or been assessed for reversal of his hypogonadism?

__Reply: __We thank the reviewer for their comment. The patient had minimal testicular development on testosterone (from 10ml to 12ml) but did not increase testes volume beyond 12mls, consistent with a partial HH phenotype. He has had two trial periods of 3-4 months off testosterone treatment and during these periods had both low serum testosterone concentrations and symptoms of hypogonadism (tiredness, low energy and reduced muscle strength).

2) Case 2: Is too young to be classified as having a pubertal defect. Microphallus is mentioned but what size, was this diagnosed at birth and treated? I think the case for GD is overstated in the results and discussion (especially with the discussion of small testes).

Reply: We thank the reviewer for requesting these clarifications. The patient has not received any treatment for his microphallus (2.5 cm length in mid-childhood). We agree that this case is too young to be classified as having a pubertal defect, but the presence of microphallus and small testes volume in infancy and early childhood, in association with low gonadotrophins and absent erections, are well recognized as red flag signs for hypogonadotropic hypogonadism (Swee & Quinton, 2019, doi:10.3389/fendo.2019.00097). We added this information to the Results section, lines 175-177.

Genetic Information: Since this was a candidate gene search -- what other candidate genes were uncovered in these probands?

Reply: The revised list of 29 candidate genes were screened in the two probands from our study using the whole exome sequencing datasets for these individuals, and only the variants of interest in NLGN3 described in the manuscript were found.

By searching for mutations of the revised list of candidate genes in our GD cohort, we identified nonsense variants only in NLGN3 and no splice variants. We also found few rare and predicted damaging missense variants in this gene list identified. Indeed, two rare (MAF 25) missense variants were identified in the genes PLXNC1 and CLSTN2 in two further probands (now summarized in Supplemental table 4). We have not identified further probands with PLXNC1 or CLSTN2 variants of interest from additional cohorts and thus at present we have not yet taken these gene variants further for molecular characterization, but we will examine the relevance of this gene variant in future work.

Do the probands have a clear explanation for their developmental disability other than the gene noted?

__Reply: __We thank the reviewer for raising this point. Proband exomes were also screened for genes related to developmental delay and no other causal gene variant were identified. We added this information in the text, lines 183-185.

I would encourage the authors to update Table 3: they are missing IHH/KS genes such as GLI3, SEMA7A, SOX2, STUB1, TCF12. I suggest they update the Table and analyses.

Reply: we thank the reviewer for highlighting this point. Since we performed a new analysis, we also performed a new candidate gene prioritization using a more up-to-date gene list to instruct ToppGene (please see revised Supplemental table 3).

CROSS-CONSULTATION COMMENTS Dear Reviewer #2, I am concerned that the paper presents only a single case of GD to support the scientific work. What do you think?

__Reply: __We would like to highlight that, as we describe above, GD can be diagnosed prior to pubertal age in individuals with red flag phenotypic signs and biochemical evidence of hypogonadism.

Dear Reviewer #1: In addition to the weakness in the microarray data, what do you think about the authors using publicly available data from human GnRH neuron transcriptomics for analysis?

__Reply: __please see the above discussion on the comparison with publicly available datasets.

Reviewer #3 (Significance (Required)):

There is not high significance to this paper: This is not the first article with GnRH transcriptomes. I would argue the human data is more relevant. Developmental disability has been previously linked the GnRH deficiency (as even cited in this paper) The article presents one case of GnRH deficiency, and one pre-pubertal case -- providing some modest evidence for a candidate gene, NLGN3.

__Reply: __We would like to rebuff this assessment of the paper’s significance. To our knowledge, this is the first report of transcriptomes from primary GnRH neurons isolated at key embryonic developmental time points. Other published reports refer to iPSC-derived or adult GnRH neurons (Keen et al., 2021; Lund et al., 2020; Wang et al., 2022; Vastagh et al., 2016 and 2020).

Similarly, the association of central hypogonadism with developmental disabilities have been reported in registry-based studies, but few causative genes have been identified, nor patient variants functionally validated in order to investigate the molecular biology underpinning this association. In the Discussion, in the light of a recent paper (Manfredi-Lozano et al., 2022, doi: 10.1126/science.abq4515), we also postulate that NLGN3 might be required for neuritogenesis of extra-hypothalamic projections of GnRH neurons thus contributing to the pathogenesis of NDD (lines 294-300).

Regarding to human data, we would like to acknowledge that we had a third case that we were not able to publish due to family consent. NLGN3 deficiency is likely to be a rare disorder, but that should not obviate the impact of investigating the molecular etiology – indeed, many insights into human biology have come from private mutations in rare disease.

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La réparation homme/femme des têtes s’avère problématique, ou à moins qu’elle relève d’une interprétation d’A Benoit ?<br/><br/> N^o 167 = Male Clay Statue Head ♂<br/> N^o 168 = Male Clay Mask ♂<br/> N^o 169 = Female Clay Statue Head ♀<br/>

E. O. Negahban, Excavations at Haft Tepe, Iran, Philadelphie, 1991, p. 37 :

Two large portrait heads and a mask were found in the artist’s workshop on the eastern side of Terrace Complex I (Plan 5) in association with many other beautifully made and crafted objects. <span style="background-color: #ffff00; padding: 0px 2px 0px 2px;">Included are the portrait head of a man, possibly King Tepti Ahar (167), the portrait head of a woman who may be his queen (169), and a mask of a man (168), possibly also Tepti Ahar since the facial features of the mask are similar to those of the male head.</span>

What does this mean.

This is a solid report.

None of this is solid, factual information.

This is interesting in two ways! The will to question everything and the perception based on some observations that are fairly clear.

This reminds me of a game called Pathologic about curing a plague, it is also a kind of depressing game in that lots of odds are stacked against you and sometimes you can do everything right and still not get a good result (I haven't actually played just heard a lot about it). I sometimes wonder what the point of these games are because usually people wants stories for the escapism right? I think as this creator talks about, there is a sort of power being able to face horrible things head on and video games can help you do that emotionally while still remaining physically safe.

This is a literacy problem akin to a nation-wide fire hazard.

Future proofing by asking "what if we're wrong?"

It is rumored to be a dice game and Takeoff was shot in the head.

1. backlash /ˈbæklæʃ/ n. 强烈抵制或反对
2. alter ego 另一个自我

3. fatphobic /fætˈfoʊbɪk/ adj. 恐胖的、恐脂的 · fatphobia /fætˈfoʊbiə/ n. 恐脂症 · phobia /ˈfoʊbiə/ n. 恐惧症 · phobic /ˈfoʊbɪk/ adj. 恐惧的

4. reinforce /ˌriːɪnˈfɔːrs/ v. 加强，增强

5. negative connotations 负面含义 · connotation /ˌkɑːnəˈteɪʃ(ə)n/ n. 隐含意义，联想意义

When we think philosophically (that is, by non-scientific methods) about problems like "What is thinking? Who am I? Do we have free will?", these words immediately cue us into using heuristic cognition, rather than causal cognition. This cueing is so immediate, that we are often unaware that there is a cueing, or that we could have tried using causal cognition.

When we finally become aware of this, there is another problem: trying to apply causal cognition to these problems destroys heuristic cognition.

For example, some old TV can be fixed by hitting on it. A person ignorant of causal information would use heuristic cognition, and see that, after being hit becomes "afraid" and would "start doing its job in earnest". With enough causal information about TV, the person would use causal cognition, and see that hitting on the TV simply shakes some loosened electric contacts into tighter contact.

Crucially, causal cognition doesn't "save the phenomena". In causal cognition, there's no "fear of being punished" in the TV. The fear is purely in the mind of a person using heuristic cognition. It is a convenient approximation used in human minds, not a real phenomenon.

Now, try applying it to humans. Causal cognition doesn't have to save all the phenomena. Perhaps humans really are not conscious, and the only thing left to explain is why humans model humans as conscious. Why is it convenient to model humans as conscious? That's a well-defined question. What really is conscious? That is not a well-defined question.

"But we can really see how we feel! Seeing the fear in a TV is an unconscious inference, but seeing that I am feeling is a direct perception, not an inference!"

"You can also see emotions in strange places -- pareidolia. You simply see emotions in the shapes of human head-meat, directly... until you meet someone with face-blindness, then you realize that seeing emotions is not a direct thing. You simply see what you are feeling, directly... until you meet someone with Cotard's delusion, then you realize that seeing your own feelings is not a direct thing."

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This is perhaps similar to the Yoruba notion of an ori (head/destiny) combining with ẹmi (breath/spirit) in a body (ara) to form a person, and the body itself being a composite of several other important organs such as the heart (ọkan) and brain (ọpọlọ), but there is no Cartesian duality between the physical and mental/spiritual aspects of these organs/elements.

As a result, however, there is a celestial pre-existence of at least some of these elements of the self, and they have something akin to a sense of agency, indicating a level of consciousness, which would suggest that it is not the unification of these organs that produces consciousness, but perhaps a particular type of consciousness.

This opening paints a vivid picture of a child looking confused at something.

Needs the citation that I'm not gonna go find right now – but TikTok is notably less divided in this way than e.g. YouTube. A much larger portion of their users create videos.

Bradstreet begins the poem with dramatic hyperboles and what is hers is ultimately his. There are no doubts that they are one.

Another interesting line and the way she uses color to get her point across about her own personal feelings.

This is poem is off to a quick start especially talking about emotion of the bat and the joy.

Line spacing is too much in most areas.

a woman who is the head of a family or tribe.