Joint Public Review:

In this manuscript, Kipfer et al describe a method for a fast and accurate SARS-CoV2 rescue and mutagenesis. This work is based on a published method termed ISA (infectious subgenomic amplicons), in which partially overlapping DNA fragments covering the entire viral genome and additional 5' and 3' sequences are transfected into mammalian cell lines. These DNA fragments recombine in the cells, express the full length viral genomic RNA and launch replication and rescue of infectious virus.

CLEVER, the method described here significantly improves on the ISA method to generate infectious SARS-CoV2, making it widely useful to the virology community.

Specifically, the strengths of this method are:<br /> 1) The successful use of various cell lines and transfection methods.<br /> 2) Generation of a four-fragment system, which significantly improves the method efficiency due to lower number of required recombination events.<br /> 3) Flexibility in choice of overlapping sequences, making this system more versatile.<br /> 4) The authors demonstrated how this system can be used to introduce point mutations as well as insertion of a tag and deletion of a viral gene.<br /> 5) Fast-tracking generation of infectious virus directly from RNA of clinical isolates by RT-PCR, without the need for cloning the fragments or using synthetic sequences.<br /> 6) The authors further expanded this method to work on additional plus-strand RNA viruses beyond SARS-CoV-2 (CHIKV, DENV)

The manuscript clearly presents the findings, and the proof-of-concept experiments are well designed.

Overall, this is a very useful method for SARS-CoV2 research. Importantly, it can be applicable to many other viruses, speeding up the response to newly emerging viruses than threaten the public health.

eLife assessment

This manuscript provides fundamental findings on the association between sleep regularity and mortality in the UK Biobank, which is a popular topic in recent sleep and circadian research in population-based studies. The study is based on a large accelerometer study with validated follow-up of incident diseases and deaths, and the data quality and large sample size are convincing and strengthen the credibility of the conclusion. This will be of wide interest to researchers in the sleep study field, epidemiologists, practicing clinicians and the general public.

Reviewer #1 (Public Review):

This manuscript provides important evidence on the association between sleep regularity and mortality in the UK Biobank, which is a popular topic in recent sleep and circadian research in population-based studies. The analysis reported robust associations between sleep irregularity and increased total, CVD and cancer mortality, and provided evidence to support the role of sleep and circadian health in disease progression and longevity in human populations. The Sleep Regularity Index (SRI) used in this study is a novel metric that quantifies the consistency in rest-activity rhythms over consecutive 24 hour periods, thus providing objective assessment of potential circadian disruption. The study is based on a large accelerometer study with validated follow-up of incident diseases and deaths. The data quality and large sample size strengthen the credibility of the conclusion. Overall, the analyses are appropriately done and the manuscript is clearly written.

Reviewer #2 (Public Review):

This interesting research commendably revealed irregular sleep-wake patterns are associated with higher mortality risk. However, as authors acknowledged, the analysis can not to accurately identify the cause and effect. In my opinion, the causality is important for this topic, cuz, sleep regularity and health (e.g. chronic disease) were long-term simultaneous status. especially given the participants are older. I suggest that the author could utilize MR analysis to find out for more evidence.

eLife assessment

This small-sized clinical trial comparing nebulized dornase-alfa to the best available care in patients hospitalized with COVID-19 pneumonia is valuable, but in its present form the paper is incomplete.

Joint Public Review:

In this study by Porter et al reports on outcomes from a small, open-label, pilot randomized clinical trial comparing dornase-alfa to the best available care in patients hospitalized with COVID-19 pneumonia. As the number of randomized participants is small, investigators describe also a contemporary cohort of controls and the study concludes about decrease of inflammation (reflected by CRP levels) after 7 days of treatment but no other statistically significant clinical benefit.

Suggestions to the authors:

• Please re-analyze findings by omitting from all Tables and Figures all data of comparators who were not randomized (BAC). I understand the difficulties of running this trial but the results of excess reduction of mortality do not allow the publication of a trial where comparators do not come from the randomized patient population.<br /> • The presentation remains confusing and the manuscript should be critically revised for clarity. There is a repetition of methods (e.g. lines 176-187 repeat 160-175) and redundant results (e.g. Figure S2, Table 3). At Table 4: the authors should select one method of illustration for lab results, either Table or figure, without repetitions<br /> • Regarding inclusion criteria, it is unclear whether high radiological suspicion is sufficient for inclusion or whether PCR based confirmation is required in all instances (differences in wording between lines 153 and 191), and under which oxygen requirements (lines 155 and 192)<br /> • Table 1 should be merged with Table S2 and a better description of cohort baseline severity (P/F, SOFA, APACHE, organ support, number of patients in each point of the WHO severity score) and treatments should be made available

eLife assessment

This important study unravels the interaction between effort cost, pupil-indexed brain state, and movement (saccadic) vigor during foraging decisions in marmoset monkeys. Based on a normative computational model, the authors derive the prediction that anticipated effort should affect both decisions and movement vigor during foraging; and then provide solid behavioural and pupillometric evidence for this prediction in a foraging task. This paper will be of interest to decision and motor neuroscience as well as to all researchers studying animal behavior.

Reviewer #1 (Public Review):

The manuscript by Hage et al. presents interesting results from a foraging behavior in Marmosets that explores the interactions of saccade and lick vigor with pupil dilation and performance as well as a marginal value theory and foraging theory-inspired value-based decision-making model thereof. The results are generally robust and carefully presented and analyses, particularly of vigor, are carefully executed.

Reviewer #2 (Public Review):

Hage et al examine how the foraging behavior of marmoset monkeys in a laboratory setting systematically takes into account the reward value and anticipated effort cost associated with the acquisition and consumption of food. In an interesting comprehensive framework, the authors study how experimental and natural variation of these factors affect both the decisions and actions necessary to gather and accumulate food, as well as the actions necessary to consume the food.

The manuscript proposes a computational model of how the monkeys may guide all these aspects of behavior, by maximizing a food capture rate that trades off the food that can be gathered with the effort and duration of the underlying actions. They use this model to derive qualitative predictions for how monkeys should react to an increase in the effort associated with food consumption: Monkeys should work longer before deciding to consume the accumulated food, but should move more slowly. The model also predicts that monkeys should show a different reaction to an increase in reward value of the food, also working longer but moving faster. The authors test these predictions in an interesting experimental setup that requires monkeys to collect small increments of food rewards for successful eye movements to targets. The monkeys can decide freely when to interrupt work and consume the accumulated food, and the authors measure the speed of the eye movements involved in the food acquisition as well as the tongue movements involved in the food consumption.

By and large, the behavioral findings fall in line with the qualitative model predictions: When the effort involved in food consumption increases, monkeys collect more food before deciding to consume it, and they move slower both during food acquisition and food consumption. In a second test, the authors approximate the effects of reward value of the food at stake, by comparing monkey behavior during different days with natural variations in body weight. These quasi-experimental increases in the reward value of food also lead to longer work times before consumption, but to faster movements during food consumption. Finally, the authors show that these effects correlate with pupil size, with pupils dilating more for low-effort foraging actions with increased saccade speed and decreased work duration. The authors conclude that the effort associated with anticipated actions can lead to changes in global brain state that simultaneously affect decisions and action vigor.

The paper proposes an interesting model for how one unified action policy may simultaneously affect multiple types of decisions and movements involved in foraging. The methods employed to measure behavior and test these predictions are generally sound, and the paper is well written.

eLife assessment

This study presents valuable observations indicating that the electrophysiological excitability of cultured sympathetic motor neurons progressively increase during aging, and are inversely correlated with the magnitude of KCNQ currents. The alterations in membrane excitability are broadly relevant for those interested in understanding how the nervous system changes during aging. While the data as a whole are solid in showing that the excitability of sympathetic neurons increases in neurons cultured from older mice, the mechanism of the underlying changes and in vivo relevance is incomplete.

Reviewer #1 (Public Review):

Summary:<br /> The authors study age-related changes in the excitability and firing properties of sympathetic neurons, which they ascribe to age-related changes in the expression of KCNQ (Kv7, "M-type") K+ currents in rodent sympathetic neurons, whose regulation by GPCRs has been most thoroughly studied for over 40 years. The authors suggest the ingestion of rapamycin may partially reverse the age-related decrease in M-channel expression. With the rapamycin part included, it is unclear how this work will impact the field of age-related neuronal dysfunction, as the mechanistic information is not strong.

Strengths:<br /> The strengths include the rigor of the current-clamp and voltage-clamp experiments, the lovely, crisp presentation of the data, and the expert statistics. The separation of neurons into tonic, phasic, and adapting classes is also interesting, and informative. The writing is also elegant, and crisp. The above is especially true of the manuscript up until the part dealing with the effects of rapamycin, which becomes less compelling.

Weaknesses:<br /> Where the manuscript becomes less compelling is in the rapamycin section, which does not provide much in the way of mechanistic insights. As such, the effect is more of an epi-phenomenon of unclear insight, and the authors cannot ascribe a signaling mechanism to it that is supported by data. Thus, this latter part rather undermines the overall impact and central advance of the manuscript. The problem is exacerbated by the controversial and anecdotal nature of the entire mTor/aging field, some of whose findings have very unfortunately had to be recently retracted.

I would strongly recommend to the authors that they end the manuscript with their analysis of the role of M current/KCNQ channels in the numerous age-related changes in sympathetic neuron function that they elegantly report, and save the rapamycin, and possible mTor action, for a separate line of inquiry that the authors could develop in a more thorough and scholarly way.

Reviewer #2 (Public Review):

Summary:<br /> This research shows compelling and detailed evidence showing that aging influences intrinsic membrane properties of peripheral sympathetic motor neurons such that they become more excitable. Furthermore, the authors present convincing evidence that the oral administration of the anti-aging drug Rapamycin partially reversed hyperexcitability in aged neurons. This study also investigates the molecular mechanisms underlying age-associated hyperexcitability in mouse sympathetic motor neurons. In that regard, the authors found an age-associated reduction of an outward current having properties similar to KCNQ2/Q3 potassium current. They suggested a reduction of KCNQ2/Q3 current density in aged neurons as a potential mechanism behind their overactivity.

Strengths:<br /> Detailed and rigorous analysis of electrical responses of peripheral sympathetic motor neurons using electrophysiology (perforated patch and whole-cell recordings). Most of the conclusions of this paper are well supported by the data.

Weaknesses:<br /> 1) The identity of the age-associated reduced current as KCNQ2/Q3 is not corroborated by pharmacology (blocking the current with the specific blocker XE-991).<br /> 2) The manuscript does not include a direct test of the reduction of KCNQ current as the mechanism behind age-induced hyperexcitability.

eLife assessment

In Drosophila melanogaster, the Store-operated Ca2+ entry (SOCE) channel, Orai, is required for the development of flight-promoting dopaminergic neurons. Here, Mitra et al. determine that expression of a loss-of-function Orai1 mutant during the 72-96 hour window of pupal development impairs gene expression in dopaminergic flight neurons in part through the expression of Set2, a histone methyltransferase. The authors identify a large number of genes that are controlled by Set2, and show that Set2 is controlled by the Trl/GAF transcription factor. Although the findings reported here are important, the evidence supporting some of the claims is incomplete.

Author Response

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

We appreciate the critical review of our manuscript. We believe that we have addressed the questions and concerns raised by the reviewers to the best of our ability. As part of the revision, we conducted two new experiments to enhance the rigor of the conclusions and to provide more insights into the mechanism of STEAP proteins, and we reorganized the Results section, as suggested by the reviewers, following to a clearer logical thread. The new data are briefly summarized below.

1) Reduction of L230G STEAP1 by reduced FAD. We made Leu230Gly STEAP1 mutant and measured the rate of heme reduction by reduced FAD. We found that the rate of heme reduction in L230G STEAP1 is slower than that in the wild type STEAP1. Since Leu230 is solvent accessible only from the intracellular side, this result supports the conclusion that reduced FAD binds to STEAP1 on the intracellular side and reduces the heme. This result also indicates that leucine, which is found at the equivalent position in STEAP1, 2 and 3, and Phe359 in STEAP4, has a significant role in mediating electron transfer from FAD to the bound heme.

2) Reduction of STEAP2 by reduced FAD. We showed that STEAP2 can be reduced when supplied with reduced FAD, and that the rate of heme reduction is significantly slower than that of reduction of STEAP1 by reduced FAD. This result is consistent with presence of the oxidoreductase domain (OxRD)† in STEAP2, which hampers direct entrance of the isoalloxazine ring of FAD to its binding pocket in the transmembrane domain (TMD). On the other hand, the rate of heme reduction by reduced FAD is much faster than that of heme reduction in the presence of NADPH and FAD, indicating that reduction of FAD by NADPH is rate-limiting in the electron transfer chain in STEAP2.

†: To be consistent with literature, we adopted the nomenclature “oxidoreductase domain (OxRD)” for the N-terminal soluble domain in STEAP proteins. We used the term “reductase domain (RED)” in the previous version of our manuscript.

Reviewer #1 (Public Review):

This important study reveals the structure of human STEAP2 for the first time and suggests the electron transport pathway, but some questions remain regarding the interpretation of the in vitro electron transport experiments, the lack of available redox couples, and potential alternative hypotheses that would if addressed, strengthen the claims in the manuscript.

Strengths

One of the clear strengths of the manuscript that stands out is the determination of the structure of human STEAP2. The structures of some other homologs are known, but STEAP2's structure was not, and STEAP2 seems to have an unusually low activity towards certain metal chelates. The approach of producing the human STEAP2 in insect cells with the supplementation of cofactor biogenesis components nicely results in cofactor-replete protein. The structure of STEAP2 reveals a domain-swapped trimer, with the NADPH-binding domain of the neighboring protomer within electron-transport distance of the FAD-heme axis. The FAD has an interesting and somewhat unusual extended conformation and abuts a Leu residue that may regulate electron transport. Another strength of the manuscript is the demonstration that STEAP1, which does not have the internal NADPH binding domain, can interact modestly and shuttle electrons to the heme in STEAP1 through FAD. These experiments nicely expand information on the function of STEAP1 and provide a structural basis for electron transport in STEAP2.

Weaknesses

A major weakness in the manuscript lies with the kinetics data and how the data, as presented, are unclear to the reader regarding their impact and their connection to the purported electron transport scheme. While multiple sets of data are reported, the analysis in all cases is simply the reduction of a hexacoordinate heme and its related spectra and kinetic parameters. In most cases, it's unclear to the reader which part of the electron pathway is being tested in which experiment. Simple diagrams would be helpful in each case. However, it's also unclear if all of the potential order of addition experiments were actually performed; i.e., flavin but no NADPH; NADPH but no flavin; flavin before NADPH; flavin after NADPH, etc. As there are multiple permutations that should be tested to demonstrate the electron transport pathway, presenting the data in a way that makes it clear to the reader is challenging. Particularly missing are the determined redox potentials of the hemes in both STEAP1 and STEAP2. Could differences in these heme redox potentials be drivers of the difference in metal reduction rates?

We re-structured the manuscript to follow a clearer logical thread. We provided explanations for which electron transfer steps are being examined in each experiment.

We cannot reliably determine EM due to insufficient amount of purified proteins. We are inclined to think that the bound heme on STEAP1 and STEAP2 have similar EM, based on their similar coordination geometry and nearly identical UV-Vis and MCD spectra. Thus, different rates of Fe3+-NTA reduction by STEAP1 and STEAP2 are likely due to differences in substrate binding site rather than different EM.

Also, the text indicates that STEAP2 does not show a reduction rate dependence on the [Fe3+NTA], but Figure 1A shows a difference in rates dependent on [Fe3+-NTA], and the shape of the reduction curve also changes based on [Fe3+-NTA]. This discrepancy should be rectified.

We fixed this error. The reduction of Fe3+-NTA by ferrous STEAP2 shows multiple phases and the reaction rates within the initial 2 seconds are weakly dependent on [Fe3+-NTA].

A second major weakness is the lack of any verification of the relevance of the STEAP2 oligomerization to its in vivo function. Is the same domain-swapped trimer known to exist in vivo? If the protein were prepared in other detergents, is the oligomerization preserved? It is alluded to in the text that another STEAP protein is also a trimer. Was this oligomerization verified in vivo?

The domain-swapped assembly is an interesting phenomenon, and it seems to provide a solution for bringing the FAD closer to heme. The same domain swapped trimeric assembly is also observed in the structure of STEAP4, which was purified in a different detergent (Nat Commun (2018), 9, page 4337). It is likely that this feature is shared by STEAP2, 3, and 4, and preserved during the purification process.

Could this oligomerization be disrupted to impede or abrogate electron transport to underscore the oligomerization relevance? This point is germane, as it would further suggest that the domain-swapped trimer observed in the STEAP2 cryo-EM structure is physiologically relevant, especially given how far the distance between the NADPH and the FAD would otherwise be to support electron transport.

We agree with the reviewer’s reasoning that the oligomeric assembly is required for proper function of STEAPs and thus could potentially be utilized for functional regulation. However, we are not aware of any physiologically relevant stimuli or treatment that would allow regulation of STEAP functions by inducing or forming different oligomeric states. Our experience with STEAP proteins is that the trimeric assembly is stable and well-preserved during the purification process and can only be disrupted under denaturing conditions such as SDS-PAGE.

Beyond these two areas in which the manuscript could be improved there are a couple of minor considerations. First, the modest resolution of the STEAP2 structure prevents assigning the states of NADP+/NADPH and FAD/FADH2 with confidence. An orthogonal measure would be useful for modeling the accurate states in the structure.

We agree. We clarified the ambiguity and stated in the main text that the cryo-EM structure of STEAP2 was determined in the presence of NADP+ and FAD.

Finally, the BLI b5R/STEAP1 binding/unbinding fits seem somewhat poor, especially at high concentrations of b5R in the dissociation regime, which likely influences the derived value of Kd. A different fitting equilibrium might yield better agreement between the experimental and theoretical results. Moreover, whether this binding strength is influenced by the reduction state of the NADPH would be helpful in understanding and contextualizing the weak binding affinity.

We think that non-specific binding likely causes deviations from the simple binding model at higher b5R concentrations. We made a comment on this in the main text. We agree with the reviewer that the interactions between b5R and STEAP1 could be redox dependent, for example, a reduced FAD on b5R may enhance the affinity. We could implement this by performing the binding experiments in an anaerobic chamber, but this is beyond the scope of the current study.

Reviewer #2 (Public Review):

The manuscript provides new insight into a family of human enzymes. It demonstrates that STEAP2 can reduce iron and STEAP1 can be promiscuous regarding the source of electron donors that it can use. The quality of the kinetics experiment and the structural analysis is excellent. I am less enthusiastic about the interpretation of data and the take-home message that the manuscript intends to deliver. Above all, the work combines data on STEAP2 and STEAP1 and it remains unclear which questions are ultimately addressed. A second critical point is about the interpretation of the experiment demonstrating that STEAP1 can be reduced by cytochrome b5 reductase. The abstract states that "We show that STEAP1 can form an electron transfer chain with cytochrome b5 reductase" whereas the main text discusses the data by suggesting that "we speculate that FAD on b5R may partially dissociate to straddle between the two proteins". The two statements seem to be partly contradictory. Cytochrome b5 reductases do not easily release FAD but rather directly donate electrons to heme-protein acceptors (PMID: 36441026). According to the methods section, no FAD was added to the reaction mix used for the cytochrome b5 reductase experiment. Overall, the data seem to indicate that the reductase might reduce the heme of STEAP1 directly. Would it be possible to check whether FAD addition affects the kinetics of the process?

We agree with the reviewer on this point. We do not have evidence indicating that FAD fully or partially dissociates from b5R to interact with STEAP1. We removed the statement in the revision.

We have not tried to add free reduced FAD to the mixture of STEAP1/b5R/NADH, because we feel that this would increase the complexity of the system and complicate data interpretation. We are working on determining the structure of b5R in complex with STEAP1 to visualize the electron transfer pathway, and we hope that such a structure would provide a framework for understanding electron transfer between the two proteins.

And to perform a control experiment to check that NAD(P)H does not directly reduce the heme of STEAP1 (though unlikely)?

We did the control experiment and included data in Fig. S3A. No reduction of heme by NADH alone.

A final point concerns the "slow Fe3+-NTA reduction by STEAP2". The reaction is not that slow as the initial phase is 2 per second. The reaction does not show dependence on the substrate concentration suggesting "poor substrate binding". I am not convinced by this interpretation. Poor substrate binding would give rise to substrate dependency as saturation would not be achieved. A possible interpretation could be that substrate binding is instead tight and the enzyme is saturated by the substrate. Can it be that the reaction is limited by non-productive substrate-binding and/or by interconversions between active and non-active conformations? We re-analyzed the data and revised the Results and Discussion.

We agree with the reviewer on this point. We re-analyzed the data and found that the reaction rates within the first 2 seconds are weakly dependent on [Fe3+-NTA] while the rates beyond 2 seconds do not show dependence on [Fe3+-NTA]. More studies are required to unravel the mechanism that leads to the complicated kinetic data.

Reviewer #3 (Public Review):

The six-transmembrane epithelial antigen of the prostate (STEAP) family comprises four members in metazoans. STEAP1 was identified as integral membrane protein highly upregulated on the plasma membrane of prostate cancer cells (PMID: 10588738), and it later became evident that other STEAP proteins are also over expressed in cancers, making STEAPs potential therapeutic targets (PMID: 22804687). Functionally, STEAP2-4 are ferric and cupric reductases that are important for maintaining cellular metal uptake (PMIDs: 16227996, 16609065). The cellular function of STEAP1 remains unknown, as it cannot function as an independent metalloreductase. In the last years, structural and functional data have revealed that STEAPs form trimeric assemblies and that they transport electrons from intracellular NADPH, through membrane bound FAD and heme cofactors, to extracellular metal ions (PMIDs: 23733181, 26205815, 30337524). In addition, numerous studies (including a previous study from the senior authors) have provided strong implications for a potential metalloreductase function of STEAP1 (PMIDs: 27792302, 32409586).

This new study by Chen et al. aims to further characterize the previously established electron transport chain in STEAPs in high molecular detail through a variety of assays. This is a wellperformed, highly specialized study that provides some useful extra insights into the established mechanism of electron transport in STEAP proteins. The authors first perform a detailed spectroscopic analysis of Fe3+-NTA reduction by STEAP2 and STEAP1, confirming that both purified proteins are capable of reducing metal ions. A cryo-EM structure of STEAP2 is also presented. It is then established that STEAP1 can receive electrons from cytochrome b5 reductase, and the authors provide experimental evidence that the flavin in STEAP proteins becomes diffusible.

The specific aims of the study are clear, but it is not always obvious why certain experiments are performed only on STEAP2, on STEAP1, or on both isoforms. A better justification of the performed experiments through connecting paragraphs and proper referencing of the literature would improve readability of the manuscript. Experimentally, the conclusions are appropriate and mostly consistent with the experimental data, although one important finding can benefit from further clarification. Namely, the observation that STEAP1 can form an electron transfer chain with cytochrome b5 reductase in vitro is an exciting finding, but its physiological relevance remains to be validated. The metalloreductase activity of STEAP1 in vitro has been described previously by the authors and by others (PMIDs: 27792302, 32409586). However, when over expressed in HEK cells, STEAP1 by itself does not display metal ion reductase activity (PMID: 16609065), and it was also found that STEAP1 over expression does not impact iron uptake and reduction in Ewing's sarcoma (cancer) cells (PMID: 22080479). Therefore, the physiological relevance of metal ion reduction by STEAP1 remains controversial. The current work establishes an electron transfer chain between STEAP1 and cytochrome b5 reductase in vitro with purified proteins. However, the conformation of this metalloreductase activity of the STEAP1-cytochrome b5 complex will be required in a cell line to prove that the two proteins indeed form a physiological relevant complex and that the results are not just an in vitro artefact from using high concentrations of purified proteins.

The work will be interesting for scientists working within the STEAP field. However, some of the presented data are redundant with previous findings, moderating the study's impact. For instance, the new structural insights into STEAP2 are limited because the structure is virtually identical to the published structures of STEAP4 and STEAP1 (PMIDs: 30337524, 32409586), which is not surprising because of the high sequence similarity between the STEAP isoforms. Moreover, the authors provide experimental evidence to prove the previous hypothesis (PMID: 30337524) that the flavin in STEAP proteins becomes diffusible, but the molecular arrangement of a STEAP protein, in which the flavin interacts with NADPH, remains unknown. Based on the manuscript title, I would also expect the in-depth characterization of STEAP1/STEAP2 hetero trimers (first identified by the authors), but this is only briefly mentioned. When taken together, this study by Chen et al. strengthens and supports previously published biochemical and structural data on STEAP proteins, without revealing many prominent conceptual advances.

We thank the reviewer for information and the broader context. We have revised the manuscript to have a clearer logical thread.

Reviewer #1 (Recommendations For The Authors):

Please see the "Public Review" for recommendations.

Reviewer #2 (Recommendations For The Authors):

Specific suggestions

-The introduction should more clearly state which questions are being addressed and why STEAP1 and STEAP2 are investigated.

We have revised the Introduction to make that clearer.

-The manuscript should discuss more extensively and provide possible explanations for the substrate-independent kinetics of iron-reduction by STEAP2.

We re-analyzed the data and found the rate constants of the reactions before 2 s are weakly [Fe3+NTA]-dependent. We ascribe the weak [Fe3+-NTA]-dependence to the partial rate-limiting by substrate binding. However, we do not have a good interpretation for the reaction kinetics after 2 s which does not show [Fe3+-NTA]-dependence.

-"The rate of STEAP1(Fe(II)) oxidation by Fe3+-NTA is similar to those by Fe3+-EDTA or Fe3+-citrate, but the rates are significantly faster than STEAP2(Fe(II)) re-oxidation by Fe3+NTA (Fig. 1B)." The rates for STEAP1 should be given to substantiate this statement.

We added Table S1 in the supplementary materials that includes the 2nd order association (kon) and the 1st order dissociation rate constants (koff) of iron substrates in STEAP1 and STEAP2. Data on Fe3+-EDTA or Fe3+-citrate by STEAP1 are from our previous study (Biochemistry, 2016). We also calculated the KDs of different iron substrates for STEAP1 and STEAP2.

• "Our results indicate that STEAP2 can supply reduce FAD to initiate electron transfer in STEAP1." As discussed above, this statement should be discussed and analyzed

We mixed 0.9 μM STEAP1, 1.1 μM STEAP2, and 2.2 μM FAD and added 60 μM NADPH to the system and found that the heme on both STEAP1 and STEAP2 are reduced. Since adding NADPH to STEAP1 plus FAD alone does not reduce the heme (Fig. S3B), we reasoned that reduction of the heme on STEAP1 is achieved by the reduced FAD produced on STEAP2. The reduced FAD likely dissociates from STEAP2 and then bind to STEAP1.

-Experiments on "STEAP1 reduction by STEAP2" The experiments show that "STEAP2 can supply reduce FAD to initiate electron transfer in STEAP1." Could these results be explained by heterotrimer formation in agreement with the previous data published by the authors?

In our experience, STEAP1 and STEAP2 homotrimers are stable and do not form heterotrimers when mixed. STEAP1/2 heterotrimers form only when the two proteins are co-expressed in cells (Biochemistry (2016) 55, 6673-6684).

Reviewer #3 (Recommendations For The Authors):

Major points:

1) As a very general point: the order in which the results are presented could be greatly improved to increase the readability for non-experts. To elaborate: The manuscript starts with the spectroscopic characterization of STEAP2, then suddenly the reductase activities of STEAP1 and STEAP2 are compared; subsequently, experiments are described involving STEAP1 and cytochrome b5 reductase; then the cryo-EM structure of STEAP2 is presented etc. As a non-expert reader, this presentation of the results is confusing, especially because the paragraphs are not always connected well, and there is a lot of switching between STEAP1 and STEAP2 data. A more logical order would be to first present the STEAP2 spectroscopy and structural data, then write a connecting paragraph on why it is important to also study the electron transfer chain in STEAP1, followed by the comparison of the STEAP isoforms and the data on STEAP1 alone. The authors should include sentences on why they performed each experiment. For example, why did they determine the structure of STEAP2. What were they after that they could not retrieve from the homologous STEAP4 and STEAP1 structures? Justification of the performed experiments will make it easier for the reader, and will establish a better connection between the paragraphs.

We reorganized the data presentation in Results per the reviewer’s suggestions.

2) The physiological relevance of metal ion reduction by STEAP1 remains controversial. Because the current work establishes an electron transfer chain between STEAP1 and cytochrome b5 reductase, could the authors perform an easy experiment where they over express both STEAP1 and cytochrome b5 reductase in a cell line? If such an experiment would reveal STEAP1-dependent metal-ion reduction, it would greatly improve this part of the manuscript. If no activity is observed, the established electron transfer chain could just represent an in vitro artifact from using high concentrations of purified proteins.

This is an excellent point. We are not set up to perform the proposed experiment but will do so in the future.

3) The manuscript states that metal ion reduction of purified STEAP2 is slow, and the authors explain this by the absence of density for the extracellular region between helices 3 and 4 that are present in the structures of STEAP4 and STEAP1, resulting in a less-well defined substratebinding site. Can the authors exclude that the less-well defined substrate-binding site is a result of the detergent extraction of STEAP2? Would it be possible to measure the reductase activity of STEAP2 in purified membranes?

Detergent mostly interacts with the transmembrane domains and since the TMD region of STEAP2 aligns well with those of STEAP1 and STEAP4, we do not think that the disordered substrate binding region in STEAP2 is a consequence of detergent solubilization. It is difficult to conduct pre-steady state kinetic experiments using STEAP2 in membrane fractions.

4) The manuscript would greatly benefit from citing the literature more comprehensively to acknowledge insightful findings from authors in the field; for example, the important work by the Lawrence lab from 2015 (PMID: 26205815), which biochemically proved that STEAPs bind a single heme and that FAD bridges the TMD and RED, is not cited. The authors also mention that STEAP proteins belong to the same family as NOX proteins and cite some NOX structure papers. However, they fail to cite the first NOX structure paper (PMID: 28607049), as well the manuscript that structurally compares NOXs and STEAPs (PMID: 32815713). Similarly, the authors use SerialEM for their cryo-EM data collection but cite an old paper instead of the more recent (and relevant) SerialEM publication (PMID: 31086343).

We agree and added the references.

5) Generally, the data presented in the manuscript appear of good technical quality. However, a 'Table 1' with all relevant cryo-EM data collection and refinement statistics is completely missing as far as I can see. The authors should definitely add this to allow for the judgement of structural data quality. Without it, the manuscript is not suitable for publication.

We added Table S2 that includes relevant cryo-EM statistics.

Minor points:

6) The authors write in the abstract: 'STEAP2 - 4, but not STEAP1, have an intracellular domain that binds to NADPH and FAD'. This is not correct, because it has clearly been established that FAD also majorly binds to the transmembrane domain (this is even shown by the authors in the current manuscript as well).

Agree, we corrected that in the revision.

7) Sentence from the abstract and introduction state: 'It is also unclear whether STEAP1 has metal ion reductase activity' and 'it is unclear whether STEAP1 can form a competent electron transfer chain from NADPH'. The authors should definitely add "physiologically relevant" to these sentences. Namely, the senior authors themselves concluded in their 2016 Biochemistry paper (PMID: 27792302) that STEAP1 is capable of reducing metal ion complexes. Further indications that the transmembrane domain of STEAP1 displays metalloreductase activity was published by the Gros lab (PMID: 32409586), and it was also shown that fusing the RED of STEAP4 to the TMD of STEAP1 yields a functional protein in cells that reduces metal ions.

Good point and we revised the text and included the references.

8) Why is scheme 1 not just a summarizing figure?

We could change Scheme 1 to a Figure if required by the copy editor.

9) What is the purpose of Fig. 6? A larger depiction of Fig. 5e would likely be more relevant and should be considered as a replacement. Alternatively, the structure of STEAP1 (pdb 6y9b) could be shown in combination with Fig. 7, as the mutation is performed in STEAP1.

We agree and made changes to the structural figures to enhance clarity.

10) The manuscript now contains many, single panel figures. Certain main figures could easily be combined, for example, Fig. 1 and 2 and/or Fig. 3 and 4.

We agree and made changes to reduce single panel figures.

11) In Fig. 2, 3 and 4, the spectra show changes in peak heights as a result of the ferric to ferrous heme transition. However, a time component is missing in the legend. How long do these transitions take?

We added the reaction times to the figure legends.

12) The last part of the discussion states: 'The assembly of an intracellular RED with a membrane-embedded TMD observed in NOX, DUOX, and STEAPs naturally led to the notion that NADPH, FAD, and heme form an uninterrupted rigid electron-transfer chain that shuttles electron from the intracellular cellular NADPH to the extracellular substrates. While this may be true for NOX and DUOX, in which rapid supply of electrons to their extracellular substrates are essential to their biological functions, it may not apply similarly to STEAPs since it has only one heme in the TMD, and their electron transfer relies on shuttling of FAD.' The authors should mention here that the activity of NOX and DUOX is tightly regulated by accessory proteins, Ca2+ etc. Similarly, do the authors expect that the large distance between NADPH and FAD in the structures could represent a way to regulate/dampen the metal ion reduction rates of STEAPs in vivo?

We agree. We mentioned the regulation of NOX and DUOX in Discussion. We remain puzzled by the large distance between NADPH and FAD in STEAPs and are in pursuit of a structure in which the two cofactors are “in touch” for electron transfer.

eLife assessment

This study provides useful insights into the mechanisms of electron transport in STEAP proteins, consistent with current models. The work strengthens and supports previously published biochemical and structural data, and the experimental results are of solid technical quality. The manuscript will be of interest to colleagues who work on STEAP proteins and related electron transfer systems.

Reviewer #1 (Public Review):

In the revised manuscript presented by Chen, Wang, and coworkers, the authors examine two proteins, STEAP1 and STEAP2, which are transmembrane hemoproteins that are involved in Fe and Cu homeostasis and are implicated in certain cancer states. The authors produce recombinant forms of STEAP1 and STEAP2 and attempt to reconstruct the electron-transport chains of both; under certain conditions, the electron transport chain of STEAP2 consists of an internal reductase domain that binds NADPH and transfers electrons to an internal FAD molecule prior to the heme b, while STEAP1 can use an independent/external b5 reductase instead of an intrinsic reductase domain to accomplish the same electron transport pathway. A strong feature of this manuscript is the determination of the cryo-EM structure of the human STEAP2 protein resolved to 3.2 Å globally and bound to heme, FAD (in an extended conformation), and NADP+/NADPH.

This revised study aims to address the previous weaknesses that were noted, such as the unclear presentation of the kinetics data, the lack of determined redox couples, the lack of in vivo oligomerization verification, and some minor weaknesses such as the fit of the BLI data and the exact redox states of the bound coenzymes. In general, the authors have sought to rectify these weaknesses chiefly through textual edits. Through these revisions, the kinetics data are now better presented and may be more easily interpreted by the reader, how the samples for cryo-EM were prepared with the respective coenzymes is clearer, and a comparison between the oligomerization of STEAP2 and STEAP4 suggests conservation of oligomerization. The determination of the redox potentials of the hemes in both STEAP1 and STEAP2 would still be a strong addition to the data presented, but it is recognized that the limitations in the ability to prepare sufficient quantities of recombinant enzyme limits the ability to determine the measurements and may represent another publication outside of the scope of this publication.

Reviewer #2 (Public Review):

Human STEAPs form a family of transmembrane heme-bound proteins. They are implicated in cancer given their high expression levels in tumor cells. Previous work has revealed that STEAPs 1-4 are iron and copper reductases. The recent structure determination of STEAP1 and STEAP4 unveiled their trimeric arrangement. STEAP1 is an outlier because it lacks the cytosolic reductase domain present in STEAPs 2-5. The present work adds to our knowledge of the family. It reports on the cryoEM structure of STEAP2 that is similar to the known structures of STEAP4 and STEAP1. The structural analysis provides additional support to a FAD-dependent heme-reduction mechanism whereby FAD oscillates between two conformations. The excellent kinetics experiments show that STEAP1 can be promiscuous regarding the source of electron donors that it can use. Indeed, cytochrome b5 can directly reduce the heme prosthetic group of STEAP1 thereby establishing an electron transfer chain that conveys electrons from NADP(P)H to the extracellular iron. Remarkably, STEAP1 can also accept electrons from free reduced FAD. Most interestingly, the manuscript demonstrates that STEAP2 can be a source of reduced FAD so that STEAP2 can create the reducing power needed for its own activity and the activity of STEAP1. This work further convincingly shows that STEAP1 can reduce iron whereas STEAP2 is less effective in iron reduction. The manuscript indicates that STEAP2 might accept other substrates providing a hint about the distinct biochemical and physiological roles of the STEAP paralogs. The manuscript does not address this point that remains open for further investigations. Aside from this minor weakness, the manuscript will advance the fields of STEAP and iron biochemistry. It has benefited from the advice given by the Reviewers leading to a high-quality presentation and data analysis.

Reviewer #3 (Public Review):

The six-transmembrane epithelial antigen of the prostate (STEAP) family comprises four members in metazoans. STEAP1 was identified as integral membrane protein highly upregulated on the plasma membrane of prostate cancer cells (PMID: 10588738), and it later became evident that other STEAP proteins are also over expressed in cancers, making STEAPs potential therapeutic targets (PMID: 22804687). Functionally, STEAP2-4 are ferric and cupric reductases that are important for maintaining cellular metal uptake (PMIDs: 16227996, 16609065). The cellular function of STEAP1 remains unknown, as it cannot function as an independent metalloreductase. In the last years, structural and functional data have revealed that STEAPs form trimeric assemblies and that they transport electrons from intracellular NADPH, through membrane bound FAD and heme cofactors, to extracellular metal ions (PMIDs: 23733181, 26205815, 30337524). In addition, numerous studies (including a previous study from the senior authors) have provided strong implications for a potential metalloreductase function of STEAP1 (PMIDs: 27792302, 32409586).

This new study by Chen et al. aims to further characterize the previously established electron transport chain in STEAPs in high molecular detail through a variety of assays. This is a well-performed study that provides new insights into the established mechanism of electron transport in STEAP proteins. The authors first perform a detailed spectroscopic analysis of STEAP1, and present the interesting observation that STEAP1 can receive electrons from cytochrome b5 reductase. Then, a similar spectroscopic analysis is performed on another STEAP family member, STEAP2, followed by experiments that show how reduced FAD can diffuse from STEAP2 to STEAP1 to reduce the heme of STEAP1. Finally, the cryo-EM structure of STEAP2 is presented.

Experimentally, the conclusions are appropriate and consistent with the experimental data. The observation that STEAP1 can form an electron transfer chain with cytochrome b5 reductase in vitro is an exciting finding, but its physiological relevance remains to be validated. The metalloreductase activity of STEAP1 in vitro has been described previously by the authors and by others (PMIDs: 27792302, 32409586). However, when over expressed in HEK cells, STEAP1 by itself does not display metal ion reductase activity (PMID: 16609065), and it was also found that STEAP1 over expression does not impact iron uptake and reduction in Ewing's sarcoma (cancer) cells (PMID: 22080479). Therefore, the physiological relevance of metal ion reduction by STEAP1 remains controversial. Future studies will have to elucidate if the established interaction between STEAP1 and cytochrome b5 reductase is relevant in cells.

The work will be interesting for scientists working within the STEAP field and for those working on other oxidoreductases. The spectroscopic data is robust and However, the new structural insights into STEAP2 are limited because the structure is virtually identical to the published structures of STEAP4 and STEAP1 (PMIDs: 30337524, 32409586), which is not surprising because of the high sequence similarity between the STEAP isoforms. When taken together, this study by Chen et al. strengthens and supports previously published biochemical and structural data on STEAP proteins, making an important contribution to the STEAP field.

Author Response

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

Reviewer #1 (Recommendations For The Authors):

The conclusions of this paper are mostly well supported by data, but some aspects need to be corrected.

1) Line 99. The title is not suitable for summarizing this part of the results. In this paragraph, the results mainly describe SRSF1 expression pattern and binding of spermatogonia-associated gene's transcripts in testes. There is no functional assay to conclude SRSF1 has an essential role in mouse testes. The data only indicate that SRSF1 may have a vital role in posttranscriptional regulation in the testes.

Thank you for the professional suggestions. Following this advice, we have corrected the text in this revised version (Page 4, Line 98 and 112).

2) Line 141. In the mating scheme, Vasa-Cre Srsf1Fl/del mice should be obtained instead of Vasa-Cre Srsf1Fl/Fl mice.

Thank you for the professional suggestions. Following this advice, we have corrected the text in this revised version (Page 4, Line 118).

3) Fig 2 C, "PZLF" should be corrected to "PLZF".

Thank you very much for the helpful comments. We have corrected this in Figure 2C.

4) Fig 5 B, "VASA" and "Merge" should be interchanged.

Thank you very much for the helpful comments. We have interchanged "VASA" and "Merge" in Figure 5B.

5) Fig 5 D, "Ctrl" should be added in the up panel.

Thank you very much for the helpful suggestions. We have added "Ctrl" in Figure 5C.

6) The legend for Figure 6 D should be revised.

Thank you very much for the helpful suggestions. We have revised the legend for Figure 7D

7) The legend for Figure 7 G should be revised.

Thank you very much for the helpful suggestions. We have revised the legend for Figure 8D

8) Immunoprecipitation mass spectrometry (IP-MS) data showed that t SRSF1 interacts with other RNA splicing-related proteins (e.g., SRSF10, SART1, RBM15, SRRM2, SF3B6, and SF3A2). The authors should verify the interactions in testis or cells.

We thank the reviewer for the professional comments and suggestions. Following this advice, we performed co-transfection and co-IP to verify the protein-protein interactions in 293T cells, the results showed that the RRM1 domain of SRSF1 interacted with SART1, RBM15 and SRSF10 in 293T cells. In addition, the fluorescence results showed complete co-localization of mCherry-SRSF1 with eGFP-SART1, eGFP-RBM15 and eGFP-SRSF10 in 293T cells. Therefore, we have incorporated the data into the Figure 9G-J. Meanwhile, these have been incorporated into the text, given descriptions, and highlighted (Page 17, Lines 338-347).

9) To avoid overstatement, the authors should pay attention to the use of adjectives and adverbs in the article, especially when drawing conclusions about the role of Tail1.

We thank the reviewer for the professional comments and suggestions. To avoid overstatement, we have revised the entire text (Page 4, Lines 98, and 112; Page 16, Lines 308; Page 17, Lines 346-347; Page 20, Lines 413-414; Page 21, Lines 432-433).

Reviewer #2 (Recommendations For The Authors):

Major

1) I find the use of "SSC homing" misleading/confusing because this "homing" or relocation of postnatal gonocytes/nascent spermatogonia to the basement membrane precedes the maturation of the nascent spermatogonia into SSCs. In addition, "SSC homing" is commonly used in the SSC transplantation field to describe a transplanted SSC's ability to find and colonize its niche within the seminiferous tubules. I appreciate that "postnatal gonocytes/nascent spermatogonia homing" is not easily grasped by a broader audience. Perhaps "homing of precursor SSCs" is more appropriate.

Thank you very much for the helpful comments and suggestions. Following this advice, we have corrected the text in this revised version (Line 1-2, 39, 44, 49, 54-55, 68, 70, 72-73, 77, 84, 93-95, 191, 201, 240, 384-387, 397, 417-422, and 433)

2) If I am misunderstanding the description of the Srsf1 cKO phenotype, and the authors truly believe SSCs have formed in the Srsf1 cKO testis, I strongly recommend immunostaining to show that the cKO germ cells robustly express SSC markers, not just markers of undifferentiated spermatogonia.

We thank the reviewer for the professional suggestions. We fully agree with the reviewer. Immunohistochemical staining for FOXO1 and statistical results indicated a reduced number of prospermatogonia (Figure 6C-E). So, we have corrected the text in this revised version (Line 1-2, 39, 44, 49, 54-55, 68, 70, 72-73, 77, 84, 93-95, 191, 201, 240, 384-387, 397, 417-422, and 433).

3) If the authors have the available resources, the significance of this report would be enhanced by additional characterization of the cKO phenotype at the transition from gonocyte to nascent spermatogonia. Do any cKO germ cells exhibit defects in maturing from gonocytes to nascent spermatogonia at the molecular level? I.e., by P5-7, do all cKO germ cells express PLZF and localize FOXO1 to cytoplasm, as expected of nascent spermatogonia? If the cKO germ cells are actually a heterogenous population of gonocytes and nascent spermatogonia, what is the distribution of each subpopulation in the lumen vs basement membrane?

Thank you for the professional suggestions. Following this advice, immunohistochemical staining for FOXO1 was performed on 5 dpp mouse testis sections (Figure 6C). Further, germ cell statistics of FOXO1 expression in the nucleus showed a reduced number of prospermatogonia in cKO mice (Figure 6D). And germ cells in which FOXO1 is expressed in the nucleus similarly undergo abnormal homing (Figure 6E). Thus, all the above data indicated that SRSF1 has an essential role in the homing of precursor SSCs. we have incorporated the data into the Figure 6C-E. Meanwhile, these have been incorporated into the text, given descriptions, and highlighted (Page 9, Lines 191-201; Page 20, Lines 389-391).

Minor

1) Could the authors clarify why Tial1 exon exclusion in the cKO results in reduced protein expression? Is it creating a transcript isoform that undergoes nonsense-mediated decay?

Thank you for the professional suggestions. Following this advice, we analyzed Tial1 transcripts again, and we found that Tial1 exon exclusion resulted in reduced expression of protein isoform X2 (Figure 8J). Since this region is not in the CDS, no clear evidence of nonsense-mediated decay was found in the analysis.

2) Could the authors confirm that the TIAL1 antibody is not detecting the portion of the protein encoded by the alternatively spliced exon?

Thank you for the helpful comments. The TIAL1 monoclonal antibody is produced by Proteintech Group under the product number 66907-1-Ig. Immunogen is TIAL1 fusion protein Ag11981. The sequence is as follows. MDARVVKDMATGKSKGYGFVSFYNKLDAENAIVHMGGQWLGGRQIRTNWATRKPPAPKSTQENNTKQLRFEDVVNQSSPKNCTVYCGGIASGLTDQLMRQTFSPFGQIMEIRVFPEKGYSFVRFSTHESAAHAIVSVNGTTIEGHVVKCYWGKESPDMTKNFQQVDYSQWGQWSQVYGNPQQYGQYMANGWQVPPYGVYGQPWNQQGFGVDQSPSAAWMGGFGAQPPQGQAPPPVIPPPNQAGYGMASYQTQ The homology was 99% in mice and all TIAL1 isoforms were detected. So, TIAL1 antibody is detecting the portion of the protein encoded by the alternatively spliced exon.

3) Lines 143: should "cKO" actually be "control"?

Thank you for the helpful suggestions. There is a real problem in the text description. we have corrected the text in this revised version (Page 6, Line 138-139).

4) Lines 272-3 "visual analysis using IGV showed the peak of Tial1/Tiar was stabilized in 5 dpp cKO mouse testes (Figure 7H)": "peak stabilization" is not evident to me from the figure nor do I see Tial1 listed as differentially expressed in the supplemental. I would refrain from using IGV visualization as the basis for the differential abundance of a transcript.

Thank you very much for the helpful comments and suggestions. Tial1/Tiar is one of 39 stabilizing genes that are bound by SRSF1 and undergo abnormal AS. Following this advice, we have substituted Tial1/Tiar's FPKM for his peaks (Figure 8H). Meanwhile, we have corrected the text in this revised version (Page 15, Line 296-300; Page 16, Line 303-304).

5) Lines 468-473: please clarify the background list used for GO enrichment analyses. By default, the genes expressed in the testis are enriched for spermatogenesis-related genes. To control for this and test whether a gene list is enriched for spermatogenesis-related genes beyond what is already seen in the testis, I recommend using a list of all expressed genes (for example, defined by TPM>=1) as the background list.

We thank the reviewer for the professional comments and suggestions. Following this advice, all expressed genes (TPM sum of all samples >=1) are listed background for GO enrichment analyses. The results of GO enrichment analysis of the AS gene turned out to be the same. The results of GO enrichment analysis of the SRSF1 peak-containing genes, differential genes, and IP proteins-associated genes have corrected in the figure (Figure 2A, 7E, and 9E)

6) Figure 2B: Could the authors mark where the statistically significant peaks appear on the tracks? There are many small peaks and it's unclear if they are significant or not.

Thank you for the helpful suggestions. Following this advice, we have marked the areas of higher peaks in the figure (Figure 2B). We generally believe that any region above the peaks of IgG is likely to be a binding region, and of course, the higher the peak value, the more pre-mRNA is bound by SRSF1 in that region.

7) Figure 7A: I assume the SRSF1 CLIP-seq genes are all the genes from the adult testis experiments. I would suggest limiting the CLIP-seq gene set to only those expressed in the P5 RNA-seq data, as if the target is not expressed at P5, there's no way it will be differentially expressed or differentially spliced in at P5.

Thank you very much for the helpful comments and suggestions. Following this advice, we found that 3543 of the 4824 genes bound by SRSF1 were expressed in testes at 5 dpp. we have corrected in the figure (Figure 8A). these have been incorporated into the text, given descriptions, and highlighted (Page 14, Lines 274-277).

8) Figure 7F: Could the authors clarify where the alternatively spliced exon is relative to the total transcript, shown in 7H?

Thank you for the helpful suggestions. Following this advice, we have labeled the number of exons where variable splicing occurs. (Figure 8F).

9) Please include where the sequencing and mass spec data will be publicly available.

Thank you very much for the helpful comments and suggestions. Following this advice, these have been incorporated into the text, given descriptions, and highlighted (Page 25, Lines 560-565).

Reviewer #3 (Recommendations For The Authors):

Suggestions for improving the data and analysis

1) The claim that TIAL1 mediates SRSF1 effects is not well supported; this claim should be adjusted or additional supporting data should be provided. To support a claim that alternative splicing of Tial1 mediates the effects of SRSF1, at least two additional pieces of data are needed: first, a demonstration that the two alternative protein isoforms have different molecular functions, either in vitro or in vivo; and second, a better quantitation of the levels and ratios of expression of the two different isoforms in vivo.

Thank you for the helpful comments and suggestions. Following this advice, we quantified the expression levels and ratios of two different isoforms in vivo, and we found that Tial1 exon exclusion resulted in reduced expression of protein isoform X2 (Figure 8J). However, it is not possible to prove that the two alternative protein isoforms have different molecular functions. So, this claim has been adjusted in the text. these have been incorporated into the text, given descriptions, and highlighted (Lines 1-2, 43-45, 95, 306, 323-325, 408, 413-414).

2) Likewise, the claim that "SRSF1 is required for "homing and self-renewal" of SSCs should be adjusted or better supported. As of now, the data supports a claim that SRSF1 is required for the establishment of the SSC population in the testis after birth. This could be due to defects in homing, self-renewal, or survival. To support claims about homing and self-renewal, these phenotypes should be tested more directly, for example by quantitating numbers of spermatogonia at the basal membrane in juvenile testes (homing) and expression of SSC markers in addition to the pan-germ cell marker VASA across early postnatal time points.

Thank you very much for the helpful comments and suggestions. Immunohistochemical staining for FOXO1 was performed on 5 dpp mouse testis sections (Figure 6C). Further, germ cell statistics of FOXO1 expression in the nucleus showed a reduced number of prospermatogonia in cKO mice (Figure 6D). And germ cells in which FOXO1 is expressed in the nucleus similarly undergo abnormal homing (Figure 6E). Thus, all the above data indicated that SRSF1 has an essential role in the homing of precursor SSCs. we have incorporated the data into the Figure 6C-E. These have been incorporated into the text, given descriptions, and highlighted (Page 9, Lines 191-201; Page 20, Lines 387-389). Meanwhile, "homing and self-renewal" of SSCs have corrected the text in this revised version (Line 1-2, 39, 44, 49, 54-55, 68, 70, 72-73, 77, 84, 93-95, 191, 201, 240, 384-387, 397, 417-422, and 433).

3) Additional, more detailed analyses of CLIP-seq and RNA-seq data at least showing that the libraries are of good quality should be provided.

Thank you very much for suggestions. Following this advice, detailed analyses of RNA-seq data have been incorporated the data into the figures (Figure S2). But detailed analyses of CLIP-seq have already been used in another paper (Sun et al., 2023), and we have not provided it in order to avoid multiple uses of one figure. Meanwhile, we made a citation in the article (Page 4, Lines 105; Page 25, Lines 564-565).

4) Gene Ontology analyses should be redone with a more appropriate background gene set.

Thank you for the helpful suggestions. All expressed genes (TPM sum of all samples >=1) are listed background for GO enrichment analyses. The results of GO enrichment analysis of the AS gene turned out to be the same. The results of GO enrichment analysis of the SRSF1 peak-containing genes, differential genes, and IP proteins-associated genes have been corrected in the figure (Figure 2A, 7E, and 9E)

Minor points about the text and figures

5) The species (mouse) should be stated earlier in the Introduction.

Thank you for the professional suggestions. Following this advice, the mouse has been stated earlier in the Introduction (Page 3, Line 65).

6) In Fig. 1C (Western blot), the results would be more convincing if quantitation of band intensities normalized to the loading control was added.

Thank you very much for comments and suggestions. Following this advice, ACTB served as a loading control. The value in 16.5 dpc testes were set as 1.0, and the relative values of testes in other developmental periods are indicated. Therefore, we have incorporated the data into the figures (Figure 1C).

7) In Fig 5D, TUNEL signal in the single-channel image is difficult to see; please adjust the contrast.

Thank you for the professional suggestions. Following this advice, the images of the channels have been replaced by enlarged images for better visibility (Figure 5C).

Major comments

1) In Fig 1D, it appears that SRSF1 is expressed most strongly in spermatogonia by immunofluorescence, but this is inconsistent with the sharp rise in expression detected by RT-qPCR at 20 days post partum (dpp) (Fig. 1B), which is when round spermatids are first added; this discrepancy should be explained or addressed.

We appreciate the important comments from the reviewer. In another of our studies, we showed that SRSF1 expression is higher in pachytene spermatocytes and round spermatids (Sun et al., 2023). So, it is normal for the sharp rise in expression detected by RT-qPCR at 20 days post partum (dpp).

Author response image 1.

Dynamic localization of SRSF1 in male mouse germ cells. (Sun et al., 2023)

2) It is important to provide a more comprehensive basic description of the CLIP-seq datasets beyond what is shown in the tracks shown in Fig. 2B. This would allow a better assessment of the data quality and would also provide information about the transcriptome-wide patterns of SRSF1 binding. No information or quality metrics are provided about the libraries, and it is not stated how replicates are handled to maximize the robustness of the analysis. The distribution of peaks across exons, introns, and other genomic elements should also be shown.

Thank you very much for the helpful comments and suggestions. In fact, detailed analyses of CLIP-seq have already been presented in another paper (Sun et al., 2023), and we have not provided it in order to avoid multiple uses of one figure. Meanwhile, we made a citation in the article (Page 4, Lines 105; Page 25, Lines 564-565). In addition, the distribution of peaks in exons, introns, and other genomic elements is shown in Figure 2B.

3) The claim that SRSF1 is required for "homing and self-renewal" of SSCs is made in multiple places in the manuscript. However, neither homing nor self-renewal is ever directly tested. A single image is shown in Fig. 5E of a spermatogonium at 5dpp that does not appropriately sit on the basal membrane, potentially indicating a homing defect, but this is not quantified or followed up. There is good evidence for depletion of spermatogonia starting at 7 dpp, but no further explanation of how homing and/or self-renewal fit into the phenotype.

Thank you very much for the helpful comments and suggestions. Following this advice, immunohistochemical staining for FOXO1 was performed on 5 dpp mouse testis sections (Figure 6C). Further, germ cell statistics of FOXO1 expression in the nucleus showed a reduced number of prospermatogonia in cKO mice (Figure 6D). And germ cells in which FOXO1 is expressed in the nucleus similarly undergo abnormal homing (Figure 6E). Thus, all the above data indicated that SRSF1 has an essential role in the homing of precursor SSCs. we have incorporated the data into the Figure 6C-E. These have been incorporated into the text, given descriptions, and highlighted (Page 9, Lines 191-201; Page 20, Lines 387-389). Meanwhile, "homing and self-renewal" of SSCs have corrected the text in this revised version (Line 1-2, 39, 44, 49, 54-55, 68, 70, 72-73, 77, 84, 93-95, 191, 201, 240, 384-387, 397, 417-422, and 433).

4) In Fig. 6A (lines 258-260) very few genes downregulated in the cKO are bound by SRSF1 and undergo abnormal splicing. The small handful that falls into this overlap could simply be noise. A much larger fraction of differentially spliced genes are CLIP-seq targets (~33%), which is potentially interesting, but this set of genes is not explored.

Thank you for the helpful comments. Following this advice, this was specifically indicated by the fact that 39 stabilizing genes were bound by SRSF1 and underwent abnormal AS. In our study, Tial1/Tiar is one of 39 stabilizing genes that are bound by SRSF1 and undergo abnormal AS. Therefore, we fully agree with the reviewers' comments. These have been added in this revised version (Page 14, Lines 279-280; Page 15, Lines 296-300).

5) The background gene set for Gene Ontology analyses is not specified. If these were done with the whole transcriptome as background, one would expect enrichment of spermatogenesis genes simply because they are expressed in testes. The more appropriate set of genes to use as background in these analyses is the total set of genes that are expressed in testis.

We thank the reviewer for the professional comments and suggestions. All expressed genes (TPM sum of all samples >=1) are listed background for GO enrichment analyses. The results of GO enrichment analysis of the AS gene turned out to be the same. The results of GO enrichment analysis of the SRSF1 peak-containing genes, differential genes, and IP proteins-associated genes have been corrected in the figure (Figure 2A, 7E, and 9E)

6) In general, the model is over-claimed: aside from interactions by IP-MS, little is demonstrated in this study about how SRSF1 affects alternative splicing in spermatogenesis, or how alternative splicing of TIAL1 specifically would result in the phenotype shown. It is not clear why Tial1/Tiar is selected as a candidate mediator of SRSF1 function from among the nine genes that are downregulated in the cKO, are bound by SRSF1, and undergo abnormal splicing. Although TIAL1 levels are reduced in cKO testes by Western blot (Fig. 7J), this could be due just be due to a depletion of germ cells from whole testis. The reported splicing difference for Tial1 seems very subtle and the ratio of isoforms does not look different in the Western blot image.

Thank you very much for the helpful comments and suggestions. In our study, Tial1/Tiar is one of 39 stabilizing genes that are bound by SRSF1 and undergo abnormal AS. However, Western blotting showed that expression levels of TIAL1/TIAR isoform X2 were significantly suppressed (Figure 8J). So, the data indicate that SRSF1 is required for TIAL1/TIAR expression and splicing.

Sun, L., Chen, J., Ye, R., Lv, Z., Chen, X., Xie, X., Li, Y., Wang, C., Lv, P., Yan, L., et al. (2023). SRSF1 is crucial for male meiosis through alternative splicing during homologous pairing and synapsis in mice. Sci Bull 68, 1100-1104. 10.1016/j.scib.2023.04.030.

Joint Public Review:

Summary:<br /> In this study, the authors seek to characterize the role of splicing factor SRSF1. Using a conditional deletion of Srsf1 in germ cells, they find that SRSF1 is required for male fertility. Via immunostaining and RNA-seq analysis of the Srsf1 conditional knockout (cKO) testes, combined with SRSF1 CLIP-seq and IP-MS data from the testis, they conclude that Srsf1 is required for homing of precursor spermatogonial stem cells (SCCs) due to alternative splicing of Tial1. They further show that spermatogonia-related genes (Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes by CLIP-seq. They show that SRSF1 coordinates with other RNA splicing-related proteins to directly bind and regulate the expression of several spermatogonia-related genes, including Tial1/Tiar, via alternative splicing Ultimately, the study shows that SRSF1's effects on alternative splicing are required to establish spermatogenesis. In the absence of Srsf1, the postnatal gonocytes do not properly mature into spermatogonia and consequently never initiate spermatogenesis.

Strengths:<br /> This study shows a role of SRSF1-mediated alternative splicing in establishment and survival of precursor SSCs, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying the formation of SSC pools. The histological analysis of the Srsf1 cKO traces the origins of the fertility defect to the postnatal testis, and the authors have generated interesting CLIP-seq, IP-MS, and RNA-seq datasets characterizing SRSF1's RNA targets and interacting proteins specifically in the testis. Together, this study provides detailed phenotyping of the Srsf1 cKO, which convincingly supports the Sertoli Cell Only phenotype, establishes the timing of the first appearance of the spermatogonial defect, and provides new insight into the role of splicing factors and SRSF1 specifically in spermatogenesis. The experiments are well-designed and conducted, the overall methods and results are robust and convincing.

Weaknesses:<br /> This study does not provide a full mechanistic explanation connecting altered splicing with defects in SSC precursors. The claim that altered splicing of the Tial1 transcript mediates the effect of SRSF1 loss is not convincingly supported. In addition, some regions of the text suggest that misregulated splicing of Tial1 disrupts spermatogonial survival; while Tial1 is required for primordial germ cell survival in embryonic gonads (E11.5-13.5; Beck et al 1998), it is unclear if Tial1 is required for germline development beyond this embryonic stage.

Reviewer #1 (Public Review):

Summary:<br /> In this research article, the authors utilized the zebrafish embryo to explore the idea that two different cell types emerge with different morphodynamics from the floor of the dorsal aorta based on their apicobasal polarity establishment. The hypothesis that the apical-luminal polarity of the membrane could be maintained after EHT and confer different functionality to the cell is exciting, however, this could not be established. There is a general lack of data supporting several of the main statements and conclusions. In addition, the manuscript is difficult to follow and needs refinement. We present below some questions and suggestions with the goal of guiding the authors to improve the manuscript and solidify their findings.

Strengths:<br /> New transgenic zebrafish lines developed. Challenging imaging.

Weaknesses:<br /> 1. The authors conclude that the truncated version of Podxl2 fused to a fluorophore is enriched within the apical site of the cell. However, based on the images provided, an alternative interpretation is that the portion of the membrane within the apical side is less stretched than in the luminal side, and therefore the fluorophore is more concentrated and easier to identify by confocal. This alternative interpretation is also supported by data presented later in the paper where the authors demonstrate that the early HE is not polarized (membranes are not under tension and stretched yet). Could the authors confirm their interpretation with a different technique/marker like TEM?

2. Could the authors confirm that the engulfed membranes are vacuoles as they claimed, using, for example, TEM? Why is it concluded that "these vacuoles appear to emanate from the abluminal membrane (facing the sub-aortic space) and not from the lumen?" This is not clear from the data presented.

3. It is unclear why the authors conclude that "their dynamics appears to depend on the activity of aquaporins and it is very possible that aquaporins are active in zebrafish too, although rather in EHT cells late in their emergence and/or in post-EHT cells, for water chase and vacuolar regression as proposed in our model (Figure 1 - figure supplement 1B)." In our opinion, these figures do not confirm this statement.

4. Could the authors prove and show data for their conclusions "We observed that both EHT pol+ and EHT pol- cells divide during the emergence"; "both EHT pol+ and EHT pol- cells express reporters driven by the hematopoietic marker CD41 (data not shown), which indicates that they are both endowed with hematopoietic potential"; and "the full recovery of their respective morphodynamic characteristics (not shown)?".

5. The authors do not demonstrate the conclusion traced from Fig. 2B. Is there a fusion of the vacuoles to the apical side in the EHT pol+ cells? Do the cells inheriting less vacuoles result in pol- EHT? It looks like the legend for Fig. 2-fig supp is missing.

6. The title of the paper "Tuning apico-basal polarity and junctional recycling in the hemogenic endothelium orchestrates pre-hematopoietic stem cell emergence complexity" could be interpreted as functional heterogeneity within the HSCs, which is not demonstrated in this work. A more conservative title denoting that there are two types of EHT from the DA could avoid misinterpretations and be more appropriate.

7. There are several conclusions not supported by data: "Finally, we have estimated that the ratio between EHT pol+ and EHT pol- cells is of approximately 2/1". "We observed that both EHT pol+ and EHT pol- cells divide during the emergence and remain with their respective morphological characteristics". "We also observed that both EHT pol+ and EHT pol- cells express reporters driven by the hematopoietic marker CD41 (data not shown), which indicates that they are both endowed with hematopoietic potential." These conclusions are key in the paper, and therefore they should be supported by data.

Reviewer #2 (Public Review):

In this study, Torcq and colleagues make careful observations of the cellular morphology of haemogenic endothelium undergoing endothelial to haematopoietic transition (EHT) to become stem cells, using the zebrafish model. To achieve this, they used an extensive array of transgenic lines driving fluorescent markers, markers of apico-basal polarity (podocalixin-FP fusions), or tight junction markers (jamb-FP fusions). The use of the runx truncation to block native Runx1 only in endothelial cells is an elegant tool to achieve something akin to tissue-specific deletion of Runx1. Overall, the imaging data is of excellent quality. They demonstrate that differences in apico-basal polarity are strongly associated with different cellular morphologies of cells undergoing EHT from HE (EHT pol- and EHT pol+) which raises the exciting possibility that these morphological differences reflect the heterogeneity of HE (and therefore HSCs) at a very early stage. They then overexpress a truncated form of Runx1 (just the runt domain) to block Runx1 function and show that more HE cells abort EHT and remain associated with the embryonic dorsal aorta. They identify pard3aa and pard3ab as potential regulators of cell polarity. However, despite showing that loss of runx1 function leads to (late) decreases in the expression of these genes, no evidence for their role in EHT is presented. The FRAP experiments and the 2d-cartography, albeit very elegant, are difficult to interpret and not very clearly described throughout the text, making interpretation difficult for someone less familiar with the techniques. Finally, while it is clear that ArhGEF11 is playing an important role in defining cell shapes and junctions between cells during EHT, there is very little statistical evidence to support the limited data presented in the (very beautiful) images.

There is a sense that this work is both overwhelming in terms of the sheer amount of imaging data, and the work behind it to generate all the lines they required, and at the same time that there is very little evidence supporting the assertion that pard3 (and even ArhGEF11) are important mediators of cell morphology and cell fate in the context of EHT. For instance, the pard3 expression data, and levels after blocking runx1 (part of Figure 3 and Figure 4) don't particularly add to the manuscript beyond indicating that the pard3 genes are regulated by Runx1.

Weaknesses<br /> The writing style is quite convoluted and could be simplified for clarity. For example, there is plenty of discussion and speculation throughout the presentation of the results. A clearer separation of the results from this speculation/discussion would help with understanding. Figures are frequently presented out of order in the text; modifying the figures to accommodate the flow of the text (or the other way around) - would make it much easier to follow the narrative. While the evidence for the different cellular morphologies of cells undergoing EHT is strong, the main claim (or at least the title of the manuscript) that tuning apico-basal polarity and junctional recycling orchestrate stem cell emergence complexity is not well supported by the data.

Reviewer #2 (Public Review):

Summary<br /> This work investigates how multiple regulatory elements combine to regulate gene expression. The authors use an episomal reporter assay which measures the transcriptional output of the reporter under the regulation of an enhancer-enhancer-promoter triple. The authors test all combinations of 8 promoters and 59 enhancers in this assay. The main finding is that enhancer pairs generally combine additively on reporter output. The authors also find that the extent to which enhancers increase reporter output is inversely related to the intrinsic strength of the promoter.

This manuscript presents a compact experiment that investigates an important open question in gene regulation. The results and data will be of interest to researchers studying enhancers. Given that my expertise is in modeling and computation, I will take the experimental results at face value and focus my review on the interpretation of the results and the computational methodology. I find the result of additivity between enhancers to be well supported. The findings on differential responsiveness between promoters are very interesting but the interpretation of such responses as 'non-linear' or 'following a power-law' may be misleading. More broadly, I think a more rigorous description of the mathematical methodology would increase the clarity and accessibility of this manuscript. A major unanswered question is whether the findings in this study apply to enhancers in their native genomic context. Regardless, investigating such questions in an episomal reporter assay is valuable.

Main comments<br /> Applicability to native genomic context: The applicability of the results in this paper to enhancers in their native genomic context is unclear. As the authors state in the discussion section, the reporter gene is not integrated into the genome, the spacing between enhancers does not match their native context etc. It is thus unclear whether this experimental design is able to detect the non-additivity between enhancers which is known to be present in the genome. This could be investigated by testing the enhancer-enhancer-promoter tuples for which non-additivity has been observed in the genome (references are given in the introduction) in this assay.

Interpretation of promoter responses as non-linear and following a power-law: In Fig 5, the authors demonstrate that enhancer-enhancer pairs boost reporter output more for weak promoters as opposed to strong promoters. I agree the data supports this finding, but I find the interpretation of such data as promoters scaling enhancers according to a power-law (as stated in the abstract) to be misleading. As mentioned on line 297, it is not possible to define an intrinsic measure of enhancer strength, thus the authors assign the base of the power-law to be the average boost index of the enhancer-enhancer pair across the 8 promoters. But this measure incorporates some aspect of a promoter and is not solely a property of enhancers. It would also be useful to know whether the results in Fig 5 apply to only enhancer-enhancer-promoter triples or also to enhancer-promoter pairs.

Enhancer-promoter selectivity: As a follow-up to a previous study (Martinez-Ara et al, Molecular Cell 2022) the authors mention that the data in this study also shows that enhancers show selectivity for certain promoters. The authors mention that both studies use the same statistical methodology and the data in this study is consistent with the data from the 2022 paper. However, I think the statistical methodology in both studies needs further exposition. This section of the review is thus meant to ensure that I understand the author's methodology, to guide the reader in understanding how the authors define 'selectivity', and to probe certain assumptions underlying the methodology.

My understanding of the approach is as follows: The authors consider an enhancer to be not selective if its 'boost index' is the same across a set of promoters. 'Boost index' is defined to be the ratio of the reporter output with the enhancer and promoter divided by the reporter output with just the promoter. Conceptually, I think that considering the boost index is a reasonable way to quantify selectivity.

The authors use a frequentist approach to classify each enhancer as selective or not selective. The null hypothesis is that the boost index of the enhancer is equal across a set of promoters. This can be visualized in Fig. 2C where the null hypothesis is that the mean of each vertical distribution is equal. Note that in Figure S4 of this paper (and in Figure 4B of their 2022 paper) the within-group variance is not plotted. Statistical significance is assessed using a Welch F-test. This is a parametric test that assumes that the observations within each vertical distribution in Fig 2C are normally distributed (this test does allow for heteroskedasticity - which means that the variance may differ within each vertical distribution). Does the normality assumption hold? This analysis should be reported. If this assumption does not hold, is the Welch test well calibrated?

eLife assessment

Understanding how genomic regulatory elements interact to control spatiotemporal gene expression is essential to explaining cell type diversification, function, and delineating genetic variation and disease. In this important study, the authors provide solid evidence showing that, in general, enhancers influence gene expression in an additive way. The findings contribute to ongoing discussions about the selectivity and combination of regulatory elements. Improved clarity regarding the statistical analysis, computational methods, and definitions used would strengthen the conclusions.

Reviewer #1 (Public Review):

This manuscript by Martinez-Ara et al investigates how combinations of cis-regulatory elements combine to influence gene expression. Using a clever iteration on massively parallel reporter assays (MPRAs), the authors measure the combinatorial effects of pairs of enhancers on specific promoters. Specifically, they assayed the activity of 59x59 different enhancer-enhancer (E-E) combinations on 8 different promoters in mouse embryonic stem cells. The main claims of the paper are that E-E pairs combine nearly additively, and that supra-additive E-E pairs are rare and often promoter-dependent. The data in this study generally support these claims.

This paper makes a good contribution to the ongoing discussions about the selectivity of gene regulatory elements. Recent works, such as those by Martinez-Ara et al. and Burgman et al., have indicated limited selectivity between E-P pairs on plasmid-based assays; this paper adds another layer to that by suggesting a similar lack of selectivity between E-E pairs.

An interesting result in this manuscript is the observation that weak promoters allow more supra-additive E-E interactions than strong promoters (Figure 4b). This nonlinear promoter response to enhancers aligns with the model previously proposed in Hong et al. (from my own group), which posited that core promoter activities are nonlinearly scaled by the genomic environment, and that (similar to the trend observed in Figure 5b) the steepness of the scaling is negatively correlated with promoter strength.

My only suggestion for the authors is that they include more plots showing how much the intrinsic strengths of the promoters and enhancers they are working with explain the trends in their data.

Specific Suggestions<br /> Supplementary Figure 4 is presented as evidence for selectivity between single enhancers and promoters. Could the authors inspect the relationship between enhancer/promoter strength and this selectivity? Generating plots similar to Figure 4B and Figure 5B, but for single enhancers, should show if the ability of an enhancer to boost a promoter is inversely correlated to that promoter's intrinsic strength. Also, in Supplementary Figure 4, coloring each point by promoter type would clarify if certain promoters (the weak ones) consistently show higher boost indices across all enhancers. If they do not, the authors may want to speculate how single enhancers can show selectivity for promoters while the effect of adding a second enhancer to an existing E-P has little selectivity. An alternate explanation, based solely on the strength of the elements, would be that when the expression of a gene is low the addition of enhancer(s) has large effects, but when the expression of a gene is high (closer to saturation) the addition of enhancer(s) have small effects.

Can anything more be said about the enhancers in E-E-P combinations that exhibit supra-additivity? Specifically, it would be interesting to know if certain enhancers, e.g. strong enhancers or enhancers with certain motifs, are more likely to show supra-additivity with a given promoter.

Reviewer #2 (Public Review):

Summary:<br /> Deng et al. investigate, for the first time to my knowledge, the role that hippocampal dentate gyrus mossy cells play in Fragile X Syndrome. They provide strong evidence that, in slice preparations from Fmr1 knockout mice, mossy cells are hypoactive due to increased Kv7 function whereas granule cells are hyperactive compared to slices from wild-type mice. They provide indirect evidence that the weakness of mossy cell-interneuron connections contributes to granule cell hyperexcitability, despite converse adaptations to mossy cell inputs. The authors show that application of the Kv7 inhibitor XE991 is able to rescue granule cell hyperexcitability back to wild-type baseline, supporting the overall conclusion that inhibition of Kv7 in the dentate may be a potential therapeutic approach for Fragile X Syndrome. However, any claims regarding specific circuit-based intervention or analysis are limited by the exclusively pharmacological approach of the manipulations.

Strengths:<br /> Thorough electrophysiological characterization of mossy cells in Fmr1 knockout mice, a novel finding.

Their electrophysiological approach is quite rigorous: patched different neuron types (GC, MC, INs) one at a time within the dentate gyrus in FMR1 KO and WT, with and without 'circuit blockade' by pharmacologically inhibiting neurotransmission. This allows the most detailed characterization possible of passive membrane/intrinsic cell differences in the dentate gyrus of Fmr1 knockout mice.

Provide several examples showing the use of Kv7 inhibitor XE991 is able to rescue excitability of granule cell circuit in Fmr1 knockout mice (AP firing in the intact circuit, postsynaptic current recordings, theta-gamma coupling stimulation).

Weaknesses:<br /> The implications for these findings and the applicability of the potential treatment for the disorder in a whole animal are limited due to the fact that all experiments were done in slices.

The authors' interpretation of the word 'circuit-based' is problematic - there are no truly circuit-specific manipulations in this study due to the reliance on pharmacology for their manipulations. While the application of the Kv7 inhibitor may have a predominant effect on the circuit through changes to mossy cell excitability, this manipulation would affect many other cells within the dentate and adjacent brain regions that connect to the dentate that express Kv7 as well.

eLife assessment

This is a fundamental work that significantly advances our understanding of the role of mossy cells in the dentate gyrus in Fragile X Syndrome. Carefully designed experiments provide evidence that changes in their excitability occur due to up-regulation of Kv7 currents. While the evidence supporting the authors' conclusions is solid, some of their claims did not consider other potential factors and explanations. The work will be of interest to neuroscientists working on unveiling the mechanisms of Fragile X pathology.

Reviewer #1 (Public Review):

Summary:<br /> In this work, the authors provide evidence to show that an increase in Kv7 channels in hilar mossy cells of Fmr1 knock out mice results in a marked decrease in their excitability. The reduction in excitatory drive onto local hilar interneurons produces an increased excitation/inhibition ratio in granule cells. Inhibiting Kv7 channels can help normalize the excitatory drive in this circuit, suggesting that they may represent a viable target for targeted therapeutics for fragile-x syndrome.

Strengths:<br /> The work is supported by a compelling and thorough set of electrophysiological studies. The authors do an excellent job of analysing their data and present a very complete data set.

Weaknesses:<br /> There are no significant weaknesses in the experimental work, however the complexity of the data presentation and the lack of a schematic showing the organizational framework of this circuit make the data less accessible to non-experts in the field. I highly encourage a graphical abstract and network diagram to help individuals understand the implications of this work.

The work is important as it identifies a unique regional and cell-specific abnormality in Fmr1 KO mice, showing how the loss of one gene can result in region-specific changes in brain circuits.

Reviewer #3 (Public Review):

The paper by Deng, Kumar, Cavalli, Klyachko describes that, unlike in other cell types, loss of Fmr1 decreases the excitability of hippocampal mossy cells due to up-regulation of Kv7 currents. They also show evidence that while muting mossy cells appears to be a compensatory mechanism, it contributes to the higher activity of the dentate gyrus, because the removal of mossy cell output alleviates the inhibition of dentate principal cells. This may be important for the patho-mechanism in Fragile X syndrome caused by the loss of Fmr1.

These experiments were carefully designed, and the results are presented ‎in a very logical, insightful, and self-explanatory way. Therefore, this paper represents strong evidence for the claims of the authors. In the current state of the manuscript, there are only a few points that need additional explanation.

One of the results, which is shown in the supplementary dataset, does not fit the main conclusions. Changes in the mEPSC frequency suggest that in addition to the proposed network effects, there are additional changes in the synaptic machinery or synapse number that are independent of the actual activity of the neurons. Since the differences of the mEPSC and sEPSC frequencies are similar and because only the latter can signal network effects, while the former is typically interpreted as a presynaptic change, it cannot be claimed that sEPSC frequency changes are due to the hypo-excitability of mossy cells.

An apparent technical issue may imply a second weak point in the interpretation of the results. Because the IPSCs in the PP stimulation experiments (Fig 8) start within a few milliseconds, it is unlikely that its first ‎components originate from the PP-GC-MC-IN feedforward inhibitory circuit. The involvement of this circuit and MCs in the Kv7-dependent excitability changes is the main implication of the results of this paper. But this feedforward inhibition requires three consecutive synaptic steps and EPSP-AP couplings, each of them lasting for at least 1ms + 2-5ms. Therefore, the inhibition via the PP-GC-MC-IN circuit can be only seen from 10-20ms after PP stimulation. The earlier components of the cPSCs should originate from other circuit elements that are not related to the rest of the paper. Therefore, more isolated measurements on the cPSC recordings are needed ‎which consider only the later phase of the IPSCs. This can be either a measurement of the decay phase or a pharmacological manipulation that selectively enhances/inhibits a specific component of the proposed circuit.

I suggest refraining from the conclusions saying "‎MCs provide at least ~51% of the excitatory drive onto interneurons in WT and ~41% in KO mice", because too many factors (eg. IN cell types, slice condition, synaptic reliability) are not accounted for in these actual numbers, and these values are not necessary for the general observation of the paper.

There are additional minor issues about the presentation of the results.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors investigated the relationship between menopause (including status, type, and age of onset) with measures of brain health, including cognition, Alzheimer's disease (including age of onset), and structural brain imaging.

Strengths:<br /> A key strength is the use of propensity matching to address the confound of age. However, further clarification and justification regarding the study design, methodology, reporting, and discussion of the results is required.

Weaknesses:<br /> Overall, the strength of evidence is uncertain/incomplete, given the methodological limitations present in the design, analyses, and reporting of results. The findings are useful, however, much of the relevant literature in this area is missing and the findings have therefore not been appropriately contextualised nor compared with previous results, including those using the same dataset.

eLife assessment

This study presents useful findings from a large sample of participants from the UK Biobank on the relationship between menopause (including status, type, and age of onset), cognition, neuroanatomical measures derived from magnetic resonance imaging, and Alzheimer's disease. The strength of evidence is incomplete, and the study would benefit from clearer methodological descriptions, more careful consideration of potential confounds, and better theoretical integration with prior work in the field. This paper will be of interest to people working in the fields of cognitive neuroscience, endocrinology, and dementia.

Reviewer #1 (Public Review):

Summary:<br /> Costantino et al report on data from thousands of participants from the UK Biobank whereby they assessed relationships between menopausal status, menopause type (surgical or natural), and age at menopause with cognition, neuroanatomical measures derived from magnetic resonance imaging and Alzheimer's disease (AD) risk.

Strengths:<br /> This is a really important field of research. Alzheimer's disease is a leading cause of death in women and better understanding whether hormonal and brain changes associated with the menopause transition are contributing to this risk is a crucial research question. Access to such a large database, with cognitive assessment alongside structural MRI data, is a strength of this study. The authors report a positive association between earlier age of menopause as well as surgical menopause and a higher risk of developing AD. The authors also report associations between age at natural menopause and performances on various cognitive tests. Positive associations were found between the age of menopause and fluid intelligence, numeric memory, and pair matching.

Weaknesses:<br /> The manuscript would benefit from further clarification about the sample and descriptions of analyses. At the moment, it is difficult to determine whether the conclusions align with the results. In terms of the method, this is a cross-sectional analysis, with different subgroups selected depending on the research question and model. Some further clarification on the full sample and the participants selected for each analysis would be helpful. Some clarification on how menopause status and AD diagnosis were determined would be helpful. The results and discussion refer to menopause having an impact on specific cognitive tasks - the domains that these tasks assess would be worthy of some discussion.

Author Response

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

Reviewer #1 (Public Review):

This manuscript represents an elegant bioinformatics approach to addressing causal pathways in vascular and liver tissue related to atherosclerosis/coronary artery disease, including those shared by humans and mice and those that are specific to only one of these species. The authors constructed co-expression networks using bulk transcriptome data from human (aorta, coronary) and mouse (aorta) vascular and liver tissue. They mapped human CAD GWAS data onto these modules, mapped GWAS SNPs to putatively causal genes, identified pathways and modules enriched in CAD GWAS hits, assessed those shared between vascular and liver tissues and between humans and mice, determined key driver genes in CAD-associated supersets, and used mouse single-cell transcriptome data to infer the roles of specific vascular and liver cell types. The overall approach used by the authors is rigorous and provides new insights into potentially causal pathways in vascular tissue and liver involved in atherosclerosis/CAD that are shared between humans and mice as well as those that are species-specific. This approach could be applied to a variety of other common complex conditions.

The conclusions are largely supported by the analyses. Some specific comments:

1) It appears that GWAS SNPs were mapped to genes solely through the use of eQTLs. Current methods involve a number of other complementary approaches to map GWAS SNPs to effector genes/transcripts and there is the thought that eQTLs may not necessarily be the best way to map causal genes.

We agree with the reviewer that multiple approaches can be used to map GWAS SNPs to genes, and eQTLs is only one way to do so. We focused on eQTLs mainly because we aim to address tissue-specificity of eQTLs and the relative higher abundance of eQTLs compared to other tissue-specific functional genomics data, such as pQTLs and epiQTLs. We now acknowledge this limitation in the discussion section in our revised manuscript and point to future studies utilizing other approaches to map GWAS signals to downstream effectors.

2) Given the critical causal role of circulating apoB lipoproteins in atherosclerosis in both mice and humans and the central role of the liver in regulating their levels, perhaps the authors could use the 'metabolism of lipids and lipoproteins' network in Fig 3B as a kind of 'positive control' to illustrate the overlap between mice and humans and the driver genes for this network.

We appreciate the reviewer’s excellent suggestion and now elaborate the findings in Fig 3B as a positive control in the results section.

3) Is it possible to infer the directionality of effect of key driver genes and pathways from these analyses? For example, ACADM was found to be a KD gene for a human-specific liver CAD superset pathway involving BCAA degradation. Are the authors able to determine or predict the effect of genetically increased expression of ACADM on BCAA metabolism and on CAD? Or the directionality of the effect of the hepatic KD gene OIT3 on hepatic and plasma lipids and atherosclerosis.

The Bayesian networks only have information on which genes likely regulate the others but not the up or down-regulation direction, and the network key driver analysis only considers the enrichment of GWAS candidate genes in the neighborhood of each key driver. Therefore, it is not possible to directly infer whether increasing or decreasing a key driver will lead to up or downregulation of the downstream pathways based on our current analysis. We could, however, examine correlations of key driver genes with downstream genes, or disease traits in relevant datasets. For instance, we checked the mouse atherosclerosis HMDP datasets for the correlations between select key drivers and clinical traits and found various key drivers shared and species-specific in aorta and liver significantly correlate with aortic lesion area and other traits of interest such as LDL levels, and inflammatory cytokines. We have added these new findings into the results section and supplemental tables.

4) While likely beyond the scope of this manuscript, the substantial amount of publicly available plasma proteomic and metabolomic data could be incorporated into this multiomic bioinformatic analysis. Many of the pathways involve secreted proteins or metabolites that would further inform the biology and the understanding of how these pathways relate to atherosclerosis.

We appreciate the reviewer’s valuable suggestion. Here we focused on liver and aorta gene regulatory networks to understand the tissue-specific mechanisms at the gene level. Indeed, plasma proteomic and metabolomic data could be further incorporated in future studies to understand the pathways captured in the circulation that can capture cross-tissue interactions mediated by secreted proteins and metabolites from different tissues. We have addressed this as a future direction in the discussion section.

The findings here will motivate the community of atherosclerosis investigators to pursue additional potential causal genes and pathways through computational and experimental approaches. It will also influence the approach around the use of the mouse model to test specific pathways and therapeutic approaches in atherosclerosis. In addition, the computational approach is robust and could (and likely will) be applied to a variety of other common complex conditions.

Reviewer #2 (Public Review):

Summary:

Mouse models are widely used to determine key molecular mechanisms of atherosclerosis, the underlying pathology that leads to coronary artery disease. The authors use various systems biology approaches, namely co-expression and Bayesian Network analysis, as well as key driver analysis, to identify co-regulated genes and pathways involved in human and mouse atherosclerosis in artery and liver tissues. They identify species-specific and tissue-specific pathways enriched for the genetic association signals obtained in genome-wide association studies of human and mouse cohorts.

Strengths:

The manuscript is well executed with appropriate analysis methods. It also provides a compelling series of results regarding mouse and human atherosclerosis.

Weaknesses:

The manuscript has several weaknesses that should be acknowledged in the discussion. First, there are numerous models of mouse atherosclerosis; however, the HMDP atherosclerosis study uses only one model of mouse atherosclerosis, namely hyperlipidemic mice, due to the transgenic expression of human apolipoprotein ELeiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). Therefore, when drawing general conclusions about mouse pathways not being identified in humans, caution is warranted. Other models of mouse atherosclerosis may be able to capture different aspects of human atherosclerosis.

We appreciate the reviewer’s valuable insight! Indeed, the specific HMDP atherosclerosis model may miss important mouse pathways that could have overlapped with the human pathways. We have added this important point to the limitations section under the discussion to caution the interpretation of the human-specific pathways, as they could be present in mice but are missed by the specific HMDP atherosclerosis dataset used.

Second, the mouse aorta tissue is atherosclerotic, whereas the atherosclerosis status of the GTEX aorta tissues is not known. Therefore, it is possible that some of the human or mouse-specific gene modules/pathways may be due to the difference in the disease status of the tissues from which the gene expression is obtained.

We agree with the reviewer that GTEx vascular tissues have unclear atherosclerosis status. However, in addition to GTEx, we also included the human STARNET dataset which contains vascular tissues from human patients with CAD. Therefore, we believe the comparability of the human and mouse vascular tissue datasets is reasonable.

Third, it is unclear how the sex of the mice (all female in the HMDP atherosclerosis study and all male in the baseline HMDP study) and the sex of the human donors affected the results. Did the authors regress out the influence of sex on gene expression in the human data before performing the co-expression and preservation studies? If not, this should be acknowledged.

We acknowledge that the effect of sex in the mouse and human datasets were not regressed out in our analysis. We have added this under the limitations section.

Fourth, some of the results are unexpected, and these should be discussed. For example, the authors identify that the leukocyte transendothelial migration pathway and PDGF signaling pathway are human-specific in their vascular tissue analysis. These pathways have been extensively described in mouse studies. Why do the authors think they identified these pathways as human-specific? Similarly, the authors identified gluconeogenesis and branched-chain amino acid catabolism as human and mouseshared modules in the vascular tissue. Is there evidence of the involvement of these pathways in atherosclerosis in vascular cells?

We agree with the reviewer that these unexpected findings warrant further discussion. As pointed out by this reviewer, it is possible that the mouse HMDP atherosclerosis dataset cannot fully represent all mouse atherosclerosis biology and therefore missed the leukocyte migration and PDGF pathways that were identified in the human datasets. Regarding the surprising findings of pathways such as BCAA catabolism in vascular tissues, we acknowledge that future studies will need to further investigate such pathway predictions but also highlight that these pathway terms have many shared genes with more commonly known pathways such as the TCA cycle, which may indicate the involvement of energy metabolism in vascular tissues in CAD development. We have added these points to the discussion section under limitations and concluding remarks.

Overall, acknowledging these drawbacks and adding points to the discussion will strengthen the manuscript.

Reviewer #1 (Recommendations For The Authors):

1) Could the authors comment on why MEGENA produces so many more co-expression modules per tissue than WCGNA?

As described in the methods section, MEGENA uses a multi-scale clustering structure to generate network modules at different scales, with each scale representing a different compactness level of the modules. At lower compactness scales larger modules are generated; at higher compactness scales, smaller modules are generated. By using all modules obtained from different scales, the total number of modules is much larger than WGCNA which only generates a network at one scale.

2) Much of the results section involves repeating in the text lists of pathways, modules, and genes that are also listed in Figures 2 and 3. The text in this part of the results could be used more productively to focus on illustrative examples or potential new biology.

We have revised the results section to reduce repeating long lists of pathways, modules, and genes as suggested.

Reviewer #2 (Recommendations For The Authors):

In addition to the weaknesses I mentioned in the public review comments, there are a few minor issues that I outline below:

1) The authors should introduce atherosclerosis as the underlying cause of CAD in the Introduction. In fact, I believe there are many places in the manuscript where the authors mean atherosclerosis instead of coronary artery disease, especially when presenting and discussing mouse results since the HMDP study did not examine the coronary arteries of mice. I believe the authors should make the appropriate changes throughout the manuscript.

We have made the changes as suggested.

2) The authors state in the introduction, "For example, mice tend to develop atherosclerotic lesions in the aorta and carotids, while humans often develop lesions in coronary arteries (Ma et al., 2012)." This is not entirely correct, so this sentence should be revised. Several models of mice show coronary artery atherosclerosis development, but most researchers study lesions in larger aortas. Further, humans develop lesions throughout the arterial tree, but perhaps what the authors meant was the most consequential plaque development is in the coronary arteries. Please rephrase.

We have rephrased the statement as suggested.

3) Last line of page 5 should read "...which will drive modules and pathways that are more likely..." not "derive"

Typo corrected.

eLife assessment

In this important study, the authors integrated genetic and genomic datasets from humans and mice to unveil shared networks and pathways associated with coronary artery disease. Their compelling analysis led to the identification of new regulatory genes and pathways in vascular tissues and in the liver, allowing for a more in-depth understanding of the pathogenesis of coronary artery disease.

Reviewer #1 (Public Review):

This manuscript represents an elegant bioinformatics approach to addressing causal pathways in vascular and liver tissue related to atherosclerosis/coronary artery disease, including those shared by humans and mice and those that are specific to only one of these species. The authors constructed co-expression networks using bulk transcriptome data from human (aorta, coronary) and mouse (aorta) vascular and liver tissue. They mapped human CAD GWAS data onto these modules, mapped GWAS SNPs to putatively causal genes, identified pathways and modules enriched in CAD GWAS hits, assessed those shared between vascular and liver tissues and between humans and mice, determined key driver genes in CAD-associated supersets, and used mouse single-cell transcriptome data to infer the roles of specific vascular and liver cell types. The overall approach used by the authors is rigorous and provides new insights into potentially causal pathways in vascular tissue and liver involved in atherosclerosis/CAD that are shared between humans and mice as well as those that are species-specific. This approach could be applied to a variety of other common complex conditions.

Reviewer #2 (Public Review):

Summary:<br /> Mouse models are widely used to determine key molecular mechanisms of atherosclerosis, the underlying pathology that leads to coronary artery disease. The authors use various systems biology approaches, namely co-expression and Bayesian Network analysis, as well as key driver analysis, to identify co-regulated genes and pathways involved in human and mouse atherosclerosis in artery and liver tissues. They identify species-specific and tissue-specific pathways enriched for the genetic association signals obtained in genome-wide association studies of human and mouse cohorts.

Strengths:<br /> The manuscript is well executed with appropriate analysis methods. It also provides a compelling series of results regarding mouse and human atherosclerosis.

Author Response

We appreciate the editor's and reviewers' time to review our manuscript. We will work on the suggestions and have provided an initial assessment of what we can do for our revised submission.

Reviewer #1 (Public Review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

We thank the reviewer for their positive comments.

Weaknesses:

1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla.

We considered two approaches initially. The first approach was to look at specific projections to the motor regions, focusing on the MLR. The second approach was to utilize a whole-brain analysis that is presented here. Given what we know about the zona incerta, especially its integrative role, we felt that a reasonable starting point was to examine the full connectome. The value of the whole-brain approach is that it provides a high-level overview of the afferents and efferents to the region. The changes in the brain that occur following Parkinson-like lesions, such as those in the nigrostriatal pathway, are known to be complex and can affect neighbouring regions such as the A13. Therefore, we wished to highlight the A13, which we considered a therapeutic target, and examine changes in connectivity that could occur following acute lesions affecting the SNc. We acknowledge that this study does not provide a causal link, but it presents the fundamental background information for subsequent hypothesis-driven, focused, region-specific analysis.

The terms provided were from the Allen Brain Atlas terminology and were presented as abbreviations. We have looked at other ways to present it, including a greater emphasis on raw numbers and highlighting motor-related subareas. We will rewrite the connectomics section to make it more accessible, reflecting the change in the figures.

Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

This is a great point and could help simplify the whole-brain results. We can present the motor-related inputs and outputs as part of a new figure in the main paper and add accompanying text in the results section. This will help highlight possible therapeutic pathways. We can also enhance our discussion of these motor-related pathways. We will retain the entire dataset and present it in a supplementary table for those who are interested.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease and a new perspective on the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

Thank you for the advice, and as pointed out, Kleinfeld’s group had a nice, focused presentation of their data. For the connectomic piece, we can certainly adopt their reporting style, which, as you point out, may highlight key motor-related regions. There are a few ideas here that we can explore further, as mentioned above.

2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

Thank you for these suggestions. We will revise as suggested. We will provide additional and/or updated data in revised figures and text. We will also move Supplementary Figures S1 and S2, which present additional locomotor data, into the main text to partly address the reviewers' points.

Reviewer #2 (Public Review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection. The study suggests that if the remodeling of the A13 region connectome does not promote recovery following chronic dopaminergic depletion, photostimulation of the A13 region restores locomotor functions.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients.

Weaknesses:

Electrical stimulation of the medial Zona Incerta, in which the A13 region is located, has been previously reported to promote locomotion (Grossman et al., 1958). Recent mouse studies have shown that if optogenetic or chemogenetic stimulation of GABAergic neurons of the Zona Incerta promotes and restores locomotor functions after 6-OHDA injection (Chen et al., 2023), stimulation of glutamatergic ZI neurons worsens motor symptoms after 6-OHDA (Lie et al., 2022).

Thank you - we will add this reference. It is useful as Grossman did stimulate the zona incerta in the cat and elicit locomotion, suggesting that stimulation of the area in normal mice has external validity. The area targeted by Chen et al. (2023) is in the lateral aspect of central/medial zona incerta, formed by dorsal and ventral zona incerta, which may account for the differing results. Our data were robust for stimulation of the medial aspect of the rostromedial zona incerta. The thigmotactic behaviour that we observed in our work that focused on CamKII neurons has not been observed with chemogenetic, optogenetic activation or with photoinhibition of GABAergic central/medial ZI (Chen et al. 2023).

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, behavioral results of this study raise questions about the neuronal population targeted in the vicinity of the A13 region. Moreover, if YFP and CHR2-YFP neurons express dopamine (TH) within the A13 region (Fig. 2), there is also a large population of transduced neurons within and outside of the A13 region that do not, thus suggesting the recruitment of other neuronal cell types that could be GABAergic or glutamatergic.

We found that CamKII transfection of the A13 region was extremely effective in promoting locomotor activity, which was critical for our work in exploring its possible therapeutic potential. We acknowledge that specific viral approaches that target the GABAergic, glutamatergic, and dopaminergic circuits would be very useful. The range of tools to target A13 dopaminergic circuits is more limited than the SNc, for example, because the A13 region lacks DAT, and TH-IRES-Cre approaches, while useful, are less specific than DAT-Cre mouse models. Intersectional approaches targeting multiple transmitters (glutamate & dopamine, for example) may be one solution as we do not expect that a single transmitter-specific pathway would work, as well as broad targeting of the A13 region. Recent work suggests that GABAergic neuron activation may have more general effects on behaviour rather than control of ongoing locomotor parameters. However, this is in contrast to recent work showing a positive valence effect of dopamine A13 activation on motivated food-seeking behavior, which differs from consummatory behavior observed with GABAergic modulation (Ye, Nunez, and Zhang 2023). Chemogenetic inactivation and ablation of dopaminergic A13 revealed that they contribute to grip strength and prehensile movements, uncoupling food-seeking grasping behavior from motivational factors (Garau et al. 2023). Overall, this suggests differing effects of GABA compared to DA and/or glutamatergic cell types, consistent with our effects of stimulating CamKII.

Regarding the analysis of interregional connectivity of the A13 region, there is a lack of specificity (the viral approach did not specifically target the A13 region), the number of mice is low for such correlation analyses (2 sham and 3 6-OHDA mice), and there are no statistics comparing 6-OHDA versus sham (Fig. 4) or contra- versus ipsilesional sides (Fig. 5). Moreover, the data are too processed, and the color matrices (Fig. 4) are too packed in the current format to enable proper visualization of the data. The A13 afferents/efferents analysis is based on normalized relative values; absolute values should also be presented to support the claim about their upregulation or downregulation.

Generally, papers using tissue-clearing imaging approaches have low sample sizes due to technical complexity and challenges. The technical challenges of obtaining these data were substantial in both collection and analysis. There are multiple technical complexities arising from dual injections (A13 and MFB coordinates) and targeting the area correctly. The A13 region is difficult to target as it spans only around 300 µm in the anterior-posterior axis. While clearing the brain takes weeks, and light-sheet imaging also takes time, the time necessary to analyze the tissue using whole-brain quantification is labor intensive, especially with a lack of a standardized analysis pipeline from atlas registrations, signal segmentations, and quantifications. The field is still relatively new, requiring additional time to refine pipelines.

Correlation matrices are often used in analyzing connectivity patterns on a brain-wide scale, as they can identify any observable patterns within a large amount of data. We used correlation matrices to display estimated correlation coefficients between the afferent and efferent proportions from one brain subregion to another across 251 brain regions in total in a pairwise manner (not for hypothesis testing). We provided descriptive statistics (mean and error bars) in Figure 5C and G. As mentioned in comments for Reviewer 1, we will also present data in a revised Figure 5 and/or a new figure that focuses specifically on motor-related pathways to provide information on possible therapeutic pathways. As suggested, absolute values will be shared in a supplemental table.

In the absence of changes in the number of dopaminergic A13 neurons after 6-OHDA injection, results from this correlation analysis are difficult to interpret as they might reflect changes from various impaired brain regions independently of the A13 region.

We acknowledge that models of Parkinson’s disease, particularly those using 6-OHDA, induce plasticity in various regions, which may subsequently affect A13 connectivity. Our aim is to emphasize the residual, intact A13 pathways that could serve as therapeutic targets in future investigations. This emphasis is pertinent in the context of potential clinical applications, as the overall input and output to the region fundamentally dictate the significance of the A13 region in lesioned nigrostriatal models. We agree with the reviewer that the changes certainly can be independent of A13; however, the fact that there was a significant change in the connectome post-6-OHDA injection and striatonigral degeneration is in and of itself important and important to document.

There is no causal link between anatomical and behavioral data, which raises questions about the relevance of the anatomical data.

This point was also addressed earlier in response to a comment from Reviewer 1. Focusing on specific motor pathways is one avenue to explore. However, given that the zona incerta acts as an integrative hub, we believed it is prudent to initially examine both afferent and efferent pathways using a brain-wide approach. For instance, without employing this methodology, the potential significance of cortical interconnectivity to the A13 region might not have been fully appreciated. As mentioned previously, we will place additional emphasis on motor-related regions in our revised paper, thereby enhancing the relevance of the anatomical data presented. With these modifications, we anticipate that our data will underscore specific motor-related targets for future exploration, employing optogenetic targeting to assess necessity and sufficiency.

Overall, the study does not take advantage of genetic tools accessible in the mouse to address the direct or indirect behavioral and anatomical contributions of the A13 region to motor control and recovery after 6-OHDA injection.

We acknowledge that our study has not specifically targeted neurons that express dopaminergic, glutamatergic, or GABAergic properties (refer to earlier comment for more detail). However, like others, we find that targeting one neuronal population often does not result in a pure transmitter phenotype. For instance, evidence suggests co-localization of dopamine neurons with a subpopulation of GABA neurons in the A13/medial zona incerta (Negishi et al. 2020). In the hypothalamus, research by Deisseroth and colleagues (Romanov et al. 2017) indicates the presence of multiple classes of dopamine cells, each containing different ratios of co-localized peptides and/or fast neurotransmitters. Consequently, we believe our work lays the foundation for the investigations suggested by the reviewer. Furthermore, if one considers this work in the context of a preclinical study to determine whether the A13 might be a target in human Parkinson's disease, the existing technology that could be utilized is deep brain stimulation (DBS) or electrical modulation, which would also affect different neuronal populations in a non-specific manner. While optogenetic stimulation therapy is longer term, using CamKII combined with the DJ hybrid AAV could be a translatable strategy for targeting A13 neuronal populations in non-human primates (Watakabe et al. 2015; Watanabe et al. 2020).

Reviewer #3 (Public Review):

Kim, Lognon et al. present an important finding on pro-locomotor effects of optogenetic activation of the A13 region, which they identify as a dopamine-containing area of the medial zona incerta that undergoes profound remodeling in terms of afferent and efferent connectivity after administration of 6-OHDA to the MFB. The authors claim to address a model of PD-related gait dysfunction, a contentious problem that can be difficult to treat with dopaminergic medication or DBS in conventional targets. They make use of an impressive array of technologies to gain insight into the role of A13 remodeling in the 6-OHDA model of PD. The evidence provided is solid and the paper is well written, but there are several general issues that reduce the value of the paper in its current form, and a number of specific, more minor ones. Also, some suggestions, that may improve the paper compared to its recent form, come to mind.

Thank you for the suggestions and careful consideration of our work - it is appreciated.

The most fundamental issue that needs to be addressed is the relation of the structural to the behavioral findings. It would be very interesting to see whether the structural heterogeneity in afferent/effects projections induced by 6-OHDA is related to the degree of symptom severity and motor improvement during A13 stimulation.

As mentioned in comments for Reviewer 1, we will be highlighting motor-related A13 pathways in a revised Figure 5 and/or a new figure. We hope that our work will provide a roadmap for future studies to disentangle divergent or convergent A13 pathways that are involved in different or all PD-related motor symptoms. Because we could not measure behavioural change in the same animals studied with the anatomic study (essentially because the optrode would have significantly disrupted the connectome we are measuring), we cannot directly compare behaviour to structure.

The authors provide extensive interrogation of large-scale changes in the organization of the A13 region afferent and efferent distributions. It remains unclear how many animals were included to produce Fig 4 and 5. Fig S5 suggests that only 3 animals were used, is that correct? Please provide details about the heterogeneity between animals. Please provide a table detailing how many animals were used for which experiment. Were the same animals used for several experiments?

The behavioral set and the anatomical set were necessarily distinct. In the anatomical experiments, we employed both anterograde and retrograde viral approaches to target the afferent and efferent A13 populations with fluorescent proteins. For the behavioral approach, a single ChR2 opsin was utilized to photostimulate the A13 region; hence combining the two populations was not feasible. We were also concerned that the optrode itself would interfere with connectomics. A lower number of animals were used for the whole-brain work due to technical limitations described earlier. We will provide more details regarding numbers we can identify as a table and text.

While the authors provide evidence that photoactivation of the A13 is sufficient in driving locomotion in the OFT, this pro-locomotor effect seems to be independent of 6-OHDA-induced pathophysiology. Only in the pole test do they find that there seems to be a difference between Sham vs 6-OHDA concerning the effects of photoactivation of the A13. Because of these behavioral findings, optogenic activation of A13 may represent a gain of function rather than disease-specific rescue. This needs to be highlighted more explicitly in the title, abstract, and conclusion.

We agree with the reviewer that this aspect needs to be highlighted more. Optogenetic activation of A13 may represent a gain of function in both healthy and 6-OHDA mice, highlighting a parallel descending motor pathway that remains intact. 6-OHDA lesions have multiple effects on motor and cognitive function. This makes a single pathway unlikely to rescue all deficits observed in 6-OHDA models. We can say that the lack of locomotion observed in 6-OHDA models can be reversed by A13 region stimulation. We have discussed some aspects of the gain of function possibility but will augment this in other areas of the paper as well, as suggested.

The authors claim that A13 may be a possible target for DBS to treat gait dysfunction. However, the experimental evidence provided (in particular the lack of disease-specific changes in the OFT) seems insufficient to draw such conclusions. It needs to be highlighted that optogenetic activation does not necessarily have the same effects as DBS (see the recent review from Neumann et al. in Brain: https://pubmed.ncbi.nlm.nih.gov/37450573/). This is important because ZI-DBS so far had very mixed clinical effects. The authors should provide plausible reasons for these discrepancies. Is cell-specificity, which only optogenetic interventions can achieve, necessary? Can new forms of cyclic burst DBS achieve similar specificity (Spix et al, Science 2021)? Please comment.

Thank you for the useful comments - we will update our discussion accordingly.

Our study highlights a parallel motor pathway provided by the A13 region that remains intact in 6-OHDA mice and can be sufficiently driven to rescue the hypolocomotor pathology observed in the OFT and overcome bradykinesia and akinesia. The photoactivation of ipsilesional A13 also has an overall additive effect on ipsiversive circling, representing a gain of function on the intact side that contributes to the magnitude of overall motor asymmetry against the lesioned side. The effects of DBS are rather complex, ranging from micro-, meso-, to macro-scales, involving activation, inhibition, and informational lesioning, and network interactions. This could contribute to the mixed clinical effects observed with ZI-DBS, in addition to differences in targeting and DBS programming among the studies (see review (Ossowska 2019)). Also the DBS studies targeting ZI have never targeted the rostromedial ZI which extends towards the hypothalamus and contains the A13. Furthermore, DBS and electrical stimulation of neural tissue, in general, are always limited by current spread and lower thresholds of activation of axons (e.g., axons of passage), both of which can reduce the specificity of the true therapeutic target. Optogenetic studies have provided mechanistic insights that could be leveraged in overcoming some of the limitations in targeting with conventional DBS approaches. Spix et al. (2021) provided an interesting approach highlighting these advancements. They devised burst stimulation to facilitate population-specific neuromodulation within the external globus pallidus. Moreover, they found a complementary role for optogenetics in exploring the pathway-specific activation of neurons activated by DBS. To ascertain whether A13 DBS may be a viable therapy for PD gait, it will be necessary to perform many more preclinical experiments, and tuning of DBS parameters could be facilitated by optogenetic stimulation in these murine models.

In a recent study, Jeon et al (Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus, 2022, Cell Reports) provided evidence on the topographically graded organization of STN afferents and McElvain et al. (Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon, 2021, Neuron) have shown similar topographical resolution for SNr efferents. Can a similar topographical organization of efferents and afferents be derived for the A13/ ZI in total?

The ZI can be subdivided into four subregions in the antero-posterior axis: rostral (ZIr), dorsal (ZId), ventral (ZIv), and caudal (ZIc) regions. The dorsal and ventral ZI is also referred together as central/medial/intermediate ZI. There are topographical gradients in different cell types and connectivity across these subregions (see reviews: (Mitrofanis 2005; Monosov et al. 2022; Ossowska 2019). Recent work by Yang and colleagues (2022) demonstrated a topographical organization among the inputs and outputs of GABAergic (VGAT) populations across four ZI subregions. Given that A13 region encompasses a smaller portion (the medial aspect) of both rostral and medial/central ZI (three of four ZI subregions) and coexpress VGAT, A13 region likely falls under rostral and intermediate medial ZI dataset found in Yang et al. (2022). With our data, we would not be able to capture the breadth of topographical organization shown in Yang et al (2022).

In conclusion, this is an interesting study that can be improved by taking into consideration the points mentioned above.

Reviewer #1 (Recommendations For The Authors):

1) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in the healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.

Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

Agreed - we will add quantification and create graphs to present the data in Figure 2.

2) Refer to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Fgure 2A-E but it should be replaced with 3A-E. Please do that.

Will be done

Reviewer #2 (Recommendations For The Authors):

Abstract

Page 1: Inhibitory or lesion studies will be necessary to support the claim that the global remodeling of afferent and efferent projections of the A13 region highlights the Zona Incerta's role as a crucial hub for the rapid selection of motor function.

We believe that overall, there is quite a bit of evidence that the zona incerta is a hub for afferent/efferents. Mitrofanis (2005) and, more recently, Wang et al. (2020) summarize some of the evidence. Yang (2022) illustrates that the zona incerta shows multiple inputs to GABAergic neurons and outputs to diverse regions. Recent work suggests that the zona incerta contributes to various motor functions such as hunting, exploratory locomotion, and integrating multiple modalities (Zhao et al. 2019; Wang et al. 2019; Monosov et al. 2022; Chometton et al. 2017). We will update our paper to reflect these references.

Introduction

Page 2, paragraph 2: "However, little attention has been placed on the medial zona incerta (mZI), particularly the A13, the only dopamine-containing region of the rostral ZI" Is the A13 region located in the rostral or medial ZI or both?

It should have been written “rostromedial” ZI. The A13 is located in the medial aspect of rostromedial ZI. We will update the introduction.

Page 2, para 3: Li et al (2021) used a mini-endoscope to record the GCaMP6 signal. Masini and Kiehn, 2022 transiently blocked the dopaminergic transmission; they never used 6-OHDA. Please correct through the text.

We will correct this.

Page 2, para 4: the A13 connectome encompasses the cerebral cortex,... MLR. The MLR is a functional region, correct this for the CNF and PPN.

Thank you, we will correct this.

Page 3, the last paragraph of the introduction could be clarified by presenting the behavioral data first, followed by the anatomy.

We will correct this.

Figure 1 is nice and clear, and well summarizes the experimental design.

Thank you.

Figure 2 shows an example of the extent of the ChR2-YFP expression and the position of an optical fiber tip above the dopaminergic A13 region from a mouse. Without any quantification, these images could be included in Figure 1. Despite a very small volume (36.8nL) of AAV, the extent of ChR2-YFP expression is quite large and includes dopaminergic and unidentified neurons within the A13 region but also a large population of unidentified neurons outside of it, thus raising questions about the volume and the types of neurons recruited.

This is an important consideration. As mentioned previously, we will provide more information on viral spread and optrode location. The issue of viral spread is complex and depends on factors including tissue type, serotype, and promotor of the virus. Li et al. (2021), for example, used different virus serotypes and promotors, injecting 150 nL, whereas we used AAV DJ, injecting 36.8nL. AAV-DJ is a hybrid viral type consisting of multiple serotypes. It has a high transduction efficiency, which leads to greater gene delivery than single-serotype AAV viral constructs (Mao et al. 2016). A secondary consideration regarding translation was that AAV-DJ could effectively transduce non-primate neurons (Watanabe et al. 2020). We have addressed the issue of neurons recruited earlier and will provide c-Fos quantification to illustrate the extent of co-localization with TH.

Anatomical reconstruction of the extent of the ChR2-YFP expression and the location of the tip of the optical fiber will be necessary to confirm that ChR2-YFP expression was restricted to the A13 region.

We will provide additional information regarding viral spread, ferrule tip placement, and c-fos cell counts.

Page 5, 1st para: Double-check the references, as not all of them are 6-OHDA injections in the MLF.

Will correct.

Page 5, 1st para, 4th line: Replace ferrule with optical canula or fiber.

Will correct.

Page 5, 1st para, 9th line: Replace Figure 2 with Figure 3.

Will correct.

Page 5, 2nd para: About the refractory decrease in traveled distance by sham-ChR2 mice: is this significant?

It was not significant (Figure S1, 1-way RM ANOVA: F5,25 = 0.486, P = 0.783)). We will update this.

Figure 3 showing behavioral assessments is nice, but the stats are not always clear. In Fig 3A, are each of the off and on boxes 1 minute long? The figure legend states the test lasts 1 min, but isn't it 4 minutes? In Figure 3B-E and 3J-M, what are the differences? Do the stats identify a significant difference only during the stimulation phase? Fig. 3F-I are nice and could have been presented as primary examples prior to data analysis in Fig. 3B-E. Group labels above the graph would help.

Yes, the off-on boxes are 1 minute long. We will correct the error in the legend. Great suggestion for F-I - we will move them ahead of the summary figures.

Fig. 3L-M, what do PreSur, Post, and Ferrule mean? I assume that Ferrule refers to mice tested with the optical fiber without stimulation, whereas Stim. refers to the stimulation. It would be helpful to standardize the format of stats in Fig. 3B-E and 3-J-M. What are time points a, b, and c referring to?

We will do this.

Figure S2A: the higher variability in 6-OHDA-YFP mice in comparison to 6-OHDA-ChR2 mice prior to stimulation suggests that 6-OHDA-YFP mice were less impaired. Why use boxplots only for these data? Would a pairwise comparison be more appropriate?

Data did not follow a normal distribution and thus, were plotted as box and whiskers with the horizontal line through the box indicating the group median, interquartile range indicated by the limits of the box, and group minimum and maximum indicated by the whiskers. And indeed, a non-parametric equivalent of paired t-test (Wilcoxon signed-rank test) was used.

Fig. S2B: add the statistical marker.

Will do

Page 7, para 1, line 8: to add "in comparison to 6-OHDA-YFP and YFP mice" to during photostimulation... (Figure 3E).

Will do

Page 7, para 3, line 5: about larger improvement, replace "sham ChR2" with "6-OHDA."

Will do

Page 8, para 1, line 4: Perier et al., 2000 reported that 6-OHDA injection increased the firing frequency of the ZI over a month.

We will add that time frame. Agreed, it is shorter than the behavioral work, which was started 3 weeks after 6-OHDA injection.

Page 8, para 2, line 1: Since the results were expected, add some references.

Will do

Page 8, para 3, line 4. Double-check the reference.

Will correct and update

Page 8: About large-scale changes in the A13 region, the relevance of correlation matrices is difficult to grasp. Analysis of local connectivity would have been more informative in the context of GABAergic and glutamatergic neurons of the ZI in the vicinity of the A13 region.

We will explore alternative methods to present the data.

Page 8, para 3, line: given Fig. 2, there is concern about the claim that only the A13 region was targeted. The time of the analysis after 6-OHDA should be mentioned. Some sections of the paragraph could be moved to methods.

As mentioned earlier, we will provide additional information regarding viral spread, ferrule tip placement, and c-fos cell counts. We will mention analysis time after 6-OHDA and update Figure 1a to include this.

Fig. 4: The color code helps the reader visualize distribution differences. However, statistical analyses comparing 6-OHDA versus sham should be included. Quantification per region would greatly help readers visualize the data and support the conclusion. The relationship between the type of correlation (positive or negative) and absolute change (increase or decrease) is unknown in the current format, which limits the interpretation of the data. Moreover, examples of raw images of axons and cells should be presented for several brain regions. The experimental design with a timeline, as in Fig. 1, would be helpful. The legend for Fig. 4 is a bit long. Some sections are very descriptive, whereas others are more interpretive.

We will explore alternative methods of presenting the data, as suggested in a previous comment. Should we retain the correlation matrix, we will incorporate the reviewer’s suggestions.

Page 10, para 1, line 1: add "afferent" to "changes in -afferent and- projection patterns."

Will do

Page 10, para 1, line 9: remove the 2nd "compared to sham" in the sentence.

Will do

10, para 1, line 10: remove "coordinated" in "several regions showed a coordinated reduction in afferent density." We cannot say anything about the timing of events, as there is only info at 1 month.

Will do

Page 10, para 2: the section should be written in the past tense.

Will do

Page 13, para 2, the last sentence is overstated. Please remove "cells" and refer to the A13 region instead.

Will do

About differential remodelling of the A13 region connectome: Figure 5C and 5G: The proportion of total afferents ipsi- and contralateral to 6-OHDA injection argues that the A13 region primarily receives inputs from the cortical plate and the striatum. Unfortunately, there are no statistics.

Due to the small sample size, we provided descriptive statistics (mean and error bars) in Figure 5C and G. As mentioned in comments for Reviewers 1 and 2, we will revise Figure 5 to present data focusing on motor-related pathways to provide clarity. In addition, absolute values will be shared in a supplemental table.

Figure 5 D and 5H: Changes in the proportion of total afferents/projections are relatively modest (less than 10% of the whole population for the highest changes). There is no standard deviation for these data and no statistics. Do they reflect real changes or variability from the injection site?

The changes are relatively modest (less than 10%) since a small brain region usually provides a very small proportion of total input (McElvain et al. 2021; Yang et al. 2022). The changes in the proportions reflect real differences between average proportions observed in sham and 6-OHDA mice. The variability in the total labeling of neurons and fibers was minimized by normalizing individual regional counts against total counts found in each individual animal.

Fig 5F and H: The example in F shows a huge decrease in the striatum, but H indicates only a 2% change, which makes the example not very representative. Absolute values would be helpful.

While a 2% change may seem small, it represents a relatively large change in the A13 efferent connectome. To provide further clarity, we will provide absolute values as suggested in our new supplemental table.

Figure 6 is inaccurate and unnecessary.

Agree - it is too simplistic. We will remove it and replace it with one outlined in comments to Reviewer 1.

Discussion

Although interesting, the discussion is too long.

We will make it more concise in the revised paper.

Page 12: para 2. If the A13 region has a pro-locomotor effect and has therapeutical potential; the claim about its plasticity relies on Fig. 4 and 5, which have a limited scope in the current analysis and presentation (see comments above).

We will revise the paper per the comments above and then update this accordingly.

Methods

Page 17, para 1: include the stereotaxic coordinates of the optical cannula above the A13 region.

We will include this information.

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Reviewer #2 (Public Review):

Summary:<br /> The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection. The study suggests that if the remodeling of the A13 region connectome does not promote recovery following chronic dopaminergic depletion, photostimulation of the A13 region restores locomotor functions.

Strengths:<br /> Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients.

Weaknesses:<br /> Electrical stimulation of the medial Zona Incerta, in which the A13 region is located, has been previously reported to promote locomotion (Grossman et al., 1958). Recent mouse studies have shown that if optogenetic or chemogenetic stimulation of GABAergic neurons of the Zona Incerta promotes and restores locomotor functions after 6-OHDA injection (Chen et al., 2023), stimulation of glutamatergic ZI neurons worsens motor symptoms after 6-OHDA (Lie et al., 2022).

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, behavioral results of this study raise questions about the neuronal population targeted in the vicinity of the A13 region. Moreover, if YFP and CHR2-YFP neurons express dopamine (TH) within the A13 region (Fig. 2), there is also a large population of transduced neurons within and outside of the A13 region that do not, thus suggesting the recruitment of other neuronal cell types that could be GABAergic or glutamatergic.

Regarding the analysis of interregional connectivity of the A13 region, there is a lack of specificity (the viral approach did not specifically target the A13 region), the number of mice is low for such correlation analyses (2 sham and 3 6-OHDA mice), and there are no statistics comparing 6-OHDA versus sham (Fig. 4) or contra- versus ipsilesional sides (Fig. 5). Moreover, the data are too processed, and the color matrices (Fig. 4) are too packed in the current format to enable proper visualization of the data. The A13 afferents/efferents analysis is based on normalized relative values; absolute values should also be presented to support the claim about their upregulation or downregulation.

In the absence of changes in the number of dopaminergic A13 neurons after 6-OHDA injection, results from this correlation analysis are difficult to interpret as they might reflect changes from various impaired brain regions independently of the A13 region. There is no causal link between anatomical and behavioral data, which raises questions about the relevance of the anatomical data.

Overall, the study does not take advantage of genetic tools accessible in the mouse to address the direct or indirect behavioral and anatomical contributions of the A13 region to motor control and recovery after 6-OHDA injection.

eLife assessment

This useful study summarises the effect of optical stimulation of the A13 region on locomotion in healthy mice and experimental Parkinsonism and could potentially be of interest to basic and clinical neuroscientists. Behavioural analyses and evidence for pro-locomotor effects of stimulation are solid. However, anatomical analyses are incomplete and do not yield mechanistic insights due to various issues with specificity, sample size, statistical analysis, and data presentation in the present form of the study.

Reviewer #1 (Public Review):

Summary:<br /> This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:<br /> These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion - the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

Weaknesses:<br /> 1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla. Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease and a new perspective on the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

Reviewer #3 (Public Review):

Kim, Lognon et al. present an important finding on pro-locomotor effects of optogenetic activation of the A13 region, which they identify as a dopamine-containing area of the medial zona incerta that undergoes profound remodeling in terms of afferent and efferent connectivity after administration of 6-OHDA to the MFB. The authors claim to address a model of PD-related gait dysfunction, a contentious problem that can be difficult to treat with dopaminergic medication or DBS in conventional targets. They make use of an impressive array of technologies to gain insight into the role of A13 remodeling in the 6-OHDA model of PD. The evidence provided is solid and the paper is well written, but there are several general issues that reduce the value of the paper in its current form, and a number of specific, more minor ones. Also, some suggestions, that may improve the paper compared to its recent form, come to mind.

The most fundamental issue that needs to be addressed is the relation of the structural to the behavioral findings. It would be very interesting to see whether the structural heterogeneity in afferent/effects projections induced by 6-OHDA is related to the degree of symptom severity and motor improvement during A13 stimulation.

The authors provide extensive interrogation of large-scale changes in the organization of the A13 region afferent and efferent distributions. It remains unclear how many animals were included to produce Fig 4 and 5. Fig S5 suggests that only 3 animals were used, is that correct? Please provide details about the heterogeneity between animals. Please provide a table detailing how many animals were used for which experiment. Were the same animals used for several experiments?

While the authors provide evidence that photoactivation of the A13 is sufficient in driving locomotion in the OFT, this pro-locomotor effect seems to be independent of 6-OHDA-induced pathophysiology. Only in the pole test do they find that there seems to be a difference between Sham vs 6-OHDA concerning the effects of photoactivation of the A13. Because of these behavioral findings, optogenic activation of A13 may represent a gain of function rather than disease-specific rescue. This needs to be highlighted more explicitly in the title, abstract, and conclusion.

The authors claim that A13 may be a possible target for DBS to treat gait dysfunction. However, the experimental evidence provided (in particular the lack of disease-specific changes in the OFT) seems insufficient to draw such conclusions. It needs to be highlighted that optogenetic activation does not necessarily have the same effects as DBS (see the recent review from Neumann et al. in Brain: https://pubmed.ncbi.nlm.nih.gov/37450573/). This is important because ZI-DBS so far had very mixed clinical effects. The authors should provide plausible reasons for these discrepancies. Is cell-specificity, which only optogenetic interventions can achieve, necessary? Can new forms of cyclic burst DBS achieve similar specificity (Spix et al, Science 2021)? Please comment.

In a recent study, Jeon et al (Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus, 2022, Cell Reports) provided evidence on the topographically graded organization of STN afferents and McElvain et al. (Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon, 2021, Neuron) have shown similar topographical resolution for SNr efferents. Can a similar topographical organization of efferents and afferents be derived for the A13/ ZI in total?

In conclusion, this is an interesting study that can be improved by taking into consideration the points mentioned above.

eLife assessment

This study presents useful insights into core genome mutations that could have contributed to the emergence of the Staphylococcus aureus lineage USA300, a frequent cause of community-acquired infections. The solid approach used is innovative in combining genome-wide association studies and RNA-expression analyses, both applied to extensive publicly available datasets. This strategy reduces the rate of false positives attributed to high genome-wide linkage disequilibrium. It is noted that this method cannot be used for most phenotype-genotype studies, especially those requiring essential population structure correction, and it can therefore not be readily replicated in different datasets.

Reviewer #1 (Public Review):

Summary:<br /> This is large-scale genomics and transcriptomics study of the epidemic community-acquired methicillin-resistant S. aureus clone USA300, designed to identify core genome mutations that drove the emergence of the clone. It used publicly available datasets and a combination of genome-wide association studies (GWAS) and independent principal-component analysis (ICA) of RNA-seq profiles to compare USA300 versus non-USA300 within clonal complex 8. By overlapping the analyses the authors identified a 38bp deletion upstream of the iron-scavenging surface-protein gene isdH that was both significantly associated with the USA300 lineage and with a decreased transcription of the gene.

Strengths:<br /> Several genomic studies have investigated genomic factors driving the emergence of successful S. aureus clones, in particular USA300. These studies have often focussed on acquisition of key accessory genes or have focussed on a small number of strains. This study makes a smart use of publicly available repositories to leverage the sample size of the analysis and identify new genomics markers of USA300 success.<br /> The approach of combining large-scale genomics and transcriptomics analysis is powerful, as it allows to make some inferences on the impact of the mutations. This is particularly important for mutations in intergenic regions, whose functional impact is often uncertain.<br /> The statistical genomics approaches are elegant and state-of-the-art and can be easily applied to other contexts or pathogens.

Weaknesses:<br /> The main weakness of this work is that these data don't allow a casual inference on the role of isdH in driving the emergence of USA300. It is of course impossible to prove which mutation or gene drove the success of the clone, however, experimental data would have strengthened the conclusions of the authors in my opinion.<br /> Another limitation of this approach is that the approach taken here doesn't allow to make any conclusions on the adaptive role of the isdH mutation. In other words, it is still possible that the mutation is just a marker of USA300 success, due to other factors such as PVL, ACMI or the SCCmecIVa. This is because by its nature this analysis is heavily influenced by population structure. Usually, GWAS is applied to find genetic loci that are associated with a phenotype and are independent of the underlying population structure. Here, authors are using GWAS to find loci that are associated with a lineage. In other words, they are simply running a univariate analysis (likely a logistic regression) between genetic loci and the lineage without any correction for population structure, since population structure is the outcome. Therefore, this approach can't be applied to most phenotype-genotype studies where correction for population structure is critical.<br /> Finally, the approach used is complex and not easily reproduced in another dataset. Although I like DBGWAS and find the network analysis elegant, I would be interested in seeing how a simpler GWAS tool like Pyseer would perform.

Reviewer #2 (Public Review):

Summary:

The work of Poudel et al. identified potential causal mutations related to the successful emergence of the virulent USA300 community-associated MRSA clone within clonal complex 8. To achieve this, the authors employed a methodology that combines the genome-wide association studies (GWAS) with the inference of a transcriptional regulatory network (TRN) through the independent component analysis (ICA) method from publicly available transcriptomic data. Thus, they identified genes with altered expression in the iModulons calculated by ICA and enriched mutations obtained from the De Bruijn graph genome-wide association study (DBGWAS) in the USA300 strains versus non-USA300 strains. The results revealed a deletion of 38 base pairs, containing a binding site for the Fur repressor, and an A→T mutation, both occurring in the upstream region of the isdH gene, whose expression level in USA300 strains exhibited a general increase compared to the other group. IsdH encodes the iron-regulated surface determinant protein H, which plays a crucial role in iron acquisition from heme and immune system evasion - two essential processes for the pathogenicity of S. aureus.

Strengths:

The clonal complex 8 (CC8), one of the most prevalent among S. aureus, encompasses strains responsible for both community-associated MRSA infections (CA-MRSA) and healthcare-associated (HA) infections (HA-MRSA and HA-MSSA). Within the CC8, one of the most prominent lineages is USA300, which emerged in the early 2000s and has since become a leading cause of CA-MRSA infections in the United States. The key genetic traits that characterize USA300 strains include the presence of the Panton-Valentine leukocidin (PVL) encoded by the genes lukF-PV and lukS-PV, the staphylococcal chromosomal cassette mec IVa (SCCmecIVa), and the arginine catabolic mobile element (ACME). Investigating the phenotypic impact of individual mutations on the success of epidemic strains through GWAS poses a challenge due to two main confounding factors: genome-wide linkage disequilibrium (LD) and population structure. The genome-wide LD is associated with false positives, where linked non-causal mutations are mistakenly identified as causal due to the same genomic backgrounds. Therefore, the strength of this work lies in the use of publicly available transcriptomic data to construct a TRN based on ICA. This approach validates the mutations enriched by GWAS and reduces the occurrence of false positives attributed to high genome-wide LD. By integrating various 'omics' data sources, this method enhances the reliability of the results and has successfully identified new potential genetic markers specific to USA300 strains. Furthermore, it revealed mutations within core genes and intergenic regulatory regions, findings that can be validated through experimental data.

Weaknesses:

GWAS aims to identify statistically significant associations that suggest a causal link between genotype and the specific phenotype of interest while simultaneously filtering out spurious associations caused by confounding factors. While the method described in this study minimizes the impact of genome-wide linkage disequilibrium (LD), it does not extend to addressing population structure. This is because the objective was precisely to identify mutations associated with the emergence of the USA300 clone. In this context, the confounding element arising from shared ancestry becomes the subject of analysis rather than an issue to be corrected. Therefore, it is essential to highlight that the method proposed in this work can not be applied to genome-wide association studies, where correction for population structure is critical for distinguishing genuine causal associations from spurious ones. This correction is crucial and necessary to most of the studied phenotypes of interest.

Another limitation is that, although the authors emphasize the mutation in the isdH gene, the analyses conducted in this study do not provide insight into any potential adaptive function associated with it. Similarly, like the other genes exhibiting distinct expression patterns associated with enriched mutations from DBGWAS in USA300 strains, isdH is among the potential markers related to the success of the clone. This group includes well-established markers, such as ACME, which carries relevant genes like the arc operon and the speG gene that contribute to virulence and survival at infection sites.

Finally, despite the availability of the codes on GitHub, the analysis itself is not easily reproducible or adaptable to other datasets.

Author Response

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

eLife assessment

This important study provides a framework bearing on the role of Eph-Ephrin signaling mechanisms in the clinically condition of amyotrophic lateral sclerosis. It provides compelling evidence for the roles of glial cells in this condition. This novel astrocyte-mediated mechanism may help identify future therapeutic targets.

Drs. Huang and Zaidi: Thank you for considering this revision of our manuscript for potential publication in eLife. We have addressed the excellent comments of the two reviewers, including the addition of new data. We have included detailed response-to-reviewer comments below to address each specific point, and we have highlighted all the changes in the manuscript text (using a red font color) made in response to these comments. Based on the reviewers’ critiques, we feel our re-working of the manuscript has made for a greatly improved study.

Reviewer #1 (Public Review):

In the manuscript by Urban et al., the authors attempt to further delineate the role which non-neuronal CNS cells play in the development of ALS. Toward this goal, the transmembrane signaling molecule ephrinB2 was studied. It was found that there is an increased expression of ephrinB2 in astrocytes within the cervical ventral horn of the spinal cord in a rodent model of ALS. Moreover, the reduction of ephrinB2 reduced motoneuron loss and prevented respiratory dysfunction at the NMJ. Further driving the importance of ephrinB2 is an increased expression in the spinal cords of human ALS individuals. Collectively, these findings present compelling evidence implicating ephrinB2 as a contributing factor towards the development of ALS.

We thank Reviewer #1 for the very helpful critique. We address each of the specific comments below (in the “Recommendations for the Authors” section of this Response to Reviewer Comments document), and have made changes to the manuscript based on these excellent points.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediator of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from postmortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the presymptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied by the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function.

We thank Reviewer #2 for the very helpful critique. We address each of the specific comments below, and have made changes to the manuscript based on all of these excellent points.

The major limitation of the manuscript as currently written is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many others that comprise the ventral gray matter, have not been investigated in this study. Thus, the primary conclusion that astrocytes drive ephrinB2-mediated pathogenesis in ALS mice is largely correlative.

This is an excellent point. While the majority of transduced cells were astrocytes, we did not identify the lineage of a portion of the transduced cells, which could consist of cell types such as microglia, endothelial cells and others, some of which have been linked to ALS pathogenesis. Nevertheless, we find that the cells expressing high levels of ephrinB2 in ventral horn of SOD1G93A mice are all astrocytes (as seen in Figure 1O-Q), strongly suggesting – though not definitively demonstrating – that astrocyte ephrinB2 is the pathogenic source in this model (even if our viral transduction did not solely target astrocytes).

In the revised version of the manuscript, we now include an extensive paragraph in the Discussion section dedicated to this point.

Importantly, we have toned down our conclusion by modifying the title by removing “…in spinal cord astrocytes…”. We changed the title from “EphrinB2 knockdown in spinal cord astrocytes preserves diaphragm innervation in a mutant SOD1 mouse model of ALS" to “EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS”.

Further, it is interesting to note that no other functional outcomes were improved in this study. The C3-C5 region of the spinal cord consists of many motor pools that innervate forelimb muscles. CMAP recordings conducted at the diaphragm are a reflection of intact motor pools. This type of assessment of neuromuscular health is hard to re-capitulate in the kind of forelimb task that is being employed to test motor function (grip strength). Thus, it would be interesting to see if CMAP recordings of forelimb muscles would capture the kind of motor function preservation observed in the diaphragm muscle.

We did perform forelimb grip strength analysis on these animals and found no effect of focal ephrinB2 knockdown. However, this functional assay is impacted more by distal forelimb muscle groups controlled by motor neuron pools located at more caudal locations of the spinal cord (i.e. low cervical and high thoracic), likely explaining the lack of effect on grip strength.

Unfortunately, we did not perform this CMAP recording on forelimb muscle, and these mice have all already been sacrificed. We have added discussion of this point to the revised manuscript.

On a similar note, the functional impact of increased CMAP amplitude has not been presented. An increase in CMAP amplitude does not necessarily translate to improved breathing function or overall ventilation. Thus, the impact of this improvement in motor output should be clearly presented to the reader.

This is a very important point. While CMAP recording is a powerful assay of functional innervation of diaphragm muscle by phrenic motor neurons, it does not directly measure respiratory function. There are assays to test outcomes such as ventilatory behavior and gas exchange (e.g. whole-body plethysmography; blood gas measurements, etc.). We did not however perform these analyses. Respiratory function involves contribution of a number of other muscle groups, and these muscles are innervated by various motor neuron pools located across a relatively-large expanse of the CNS neuraxis. As we focally targeted ephrinB2 knockdown to only a small area, we would not expect effects on these other functional assays, which is why we restricted our testing to CMAP recording since this can be used to specifically study the phrenic motor neuron pool (and can be combined with detailed histological analyses in the cervical enlargement and at the diaphragm NMJ).

Importantly, this is why we chose to use “preserves diaphragm innervation” in the manuscript title, as opposed to wording such as “preserves diaphragm function” in the title. In addition, have added this point to the Discussion section in the revised manuscript.

Further, to the best of my knowledge, expression of Eph (or EphB) receptors has not been explicitly shown at the phrenic motor pool. It is thus speculative at best that the mechanism that the authors suggest in preserving diaphragm function is in fact mediated through Eph-EphrinB2 signaling at the phrenic motor pool. This aspect of the study would warrant a deeper discussion.

We address this important comment with multiple pieces of data showing that Eph receptors are expressed in the phrenic motor neuron pool. EphrinB2 binds and activates EphBs, as well as EphAs such as EphA4. Importantly, previous work has linked expression of EphA4 in motor neurons to the rate of ALS progression (Van Hoecke, et al. Nature Medicine. 2012). Consistent with these studies, single-nucleus RNAseq on mouse cervical spinal cord shows that alpha motor neurons of cervical spinal cord express various EphA and EphB receptors (http://spinalcordatlas.org/; Blum et al., Nature Neuroscience, 2021; Alkaslasi et al., Nature Communications, 2021). In addition, this dataset identifies a phrenic motor neuron-specific marker (ErbB4); when we specifically look at the expression profile of only the ErbB4-expressing alpha motor neurons, the data reveal that phrenic motor neurons express a number of EphA and EphB receptors, including EphA4.

To validate expression specifically of EphA4, we performed IHC for phosphorylated EphA4 (a marker of activated EphA4) on C3-C5 spinal cord sections from SOD1G93A mice injected with shRNAephrinB2 or control vector. We find that large ventral horn neurons are positive for phosphorylated EphA4. The ventral horn at these cervical spinal cord levels includes motor neuron pools in addition to just phrenic motor neurons; therefore, this result by itself does not conclusively show that phrenic motor neurons express EphA4, though they likely do since we find EphA4 expression in most ventral horn neuron cell bodies in C3-C5. A representative image is included in Supplemental Figure 1.

In the revised manuscript, we added a paragraph to the Discussion section to address this important comment from the reviewer, including describing these data on Eph receptor expression.

Lastly, although authors include both male and female animals in this investigation, they do not have sufficient power to evaluate sex differences. Thus, this presents another exciting future of investigation, given that ALS has a slightly higher preponderance in males as compared to females.

As the reviewer notes, our studies are under-powered with respect to examining possible sex-specific effects. We now include a brief discussion of this issue in the revised manuscript.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Recommendations for the authors: please note that you control which revisions to undertake from the public reviews and recommendations for the authors.

Both reviewers were enthusiastic about your paper. Reviewer (1) had some technical queries (see his/her items 2 and 4). Reviewer (2) had some questions about principles (items 1 and 2) with the remaining points being technical queries.

We have addressed all comments of both reviewers. We detail our responses in this Response to Reviewer Comments document and have made the associated modifications to the revised manuscript.

Reviewer #1 (Recommendations For The Authors):

Questions and/or Recommendations:

There is convincing evidence that there is increased expression of ephrinB2 over time in the mouse model of ALS. Is there a corresponding increase in astrocytes in this animal model?

We previously published data showing quantification of astrocyte numbers within the spinal cord of this same SOD1G93A mouse model. Specifically, we performed this quantification in the ventral horn of the lumbar spinal cord following disease onset. We found that there was a modest increase in the number of GFAP+ astrocytes at this location and disease time point.

[ Lepore et al. Selective ablation of proliferating astrocytes does not affect disease outcome in either acute or chronic models of motor neuron degeneration. Experimental Neurology. 211 (2): 423-32, 2008. ]

One could speculate that the increase in ephrinB2 expression we observe across the ventral horn in the mutant SOD1 mice was solely due to this modest increase in astrocyte number. However, this is highly unlikely to be the case, as in wild-type mice and in mutant SOD1 mice prior to disease onset astrocytes (and all other cell types) express very low levels of ephrinB2. Throughout disease course in these mutant SOD1 mice, the ephrinB2 expression level in individual astrocytes dramatically increases (including across most or all astrocytes), suggesting that the total increase in ephrinB2 expression across the ventral horn was not due to just this modest increase in astrocyte numbers but was instead due to the dramatically elevated eprhinB2 expression in most/all astrocytes. We have added this point to the Discussion section in the revised manuscript.

It would help the reviewer and readers to show a lower magnification image of Figure 2, panels O and P to demonstrate the reduction of ephrin B2 in the ventral horns.

We have added the lower magnification images to Figure 2.

It is commended that not all data was "positive". Figure 4 especially shows some of the limitations of eprhinB2 knockdown. This provides a realistic image - strengths and limitations - of this approach. Very well done!

Thank you! In future work, we could employ alternative vector-based strategies to restrict transduction/knockdown to only astrocytes. With such experiments, it’s possible that the impact of ephrinB2 knockdown would not be the same, if ephrinB2 actions in non-astrocytes also plays a role in disease pathogenesis. We have added discussion of this same point to the revised manuscript in response to a similar comment above from Reviewer #2.

Reviewer comment 4: Fig 6 - if possible can you please add demographic (age/sex) with each band?

We have added this information to the Legend. For aesthetic reasons, we chose not to add this information directly to the figure itself and instead included all of this information for each sample/band in the Legend.

Reviewer #2 (Recommendations For The Authors):

Overall, the manuscript addresses a novel aspect of the role of astrocytes in mediating ALS pathogenesis. I commend the authors for a well thought-out and clearly presented study. However, a few concerns limit the enthusiasm and deserve attention to improve the clarity of the report.

The biggest limitation of this study is that microglia or other cell types (endothelial cells) have not been explored in this study. They constitute a big proportion of cell types in the spinal cord and to conclude that only astrocytes mediate ephrinB2 signaling in the ALS model would be a stretch without the proper stains.

Please see our comments above to address this same point from Reviewer #2.

A clear premise for the investigation of EphrinB2 ligands has not been presented in the introduction. The authors provide a good background on the emerging role of EphEphrin interactions in neurodegenerative diseases. But it is unclear how the authors landed on this sub-class of ephrins.

We have added this premise to the Introduction section of the revised manuscript. In published work, ephrinB2 has been shown to be upregulated in reactive astrocytes and to be involved in disease pathogenesis in other neurological disease models (e.g. traumatic spinal cord injury).

There are several acronyms that have not been defined in the manuscript, e.g. GPI.

We now define “GPI” and all other abbreviations in the revised manuscript.

While the authors state that males and females had been included in the study, their individual n's for various outcomes have not been presented in the results section. Further, n's are missing from the figure legends, which will aid the clarity of the presentation. Further, please clarify the ages of the mice in the methods section.

(1) We now provide the n’s for males versus females for all analyses in the figure legends. (2) We also now include the total n for each experimental condition in all of the figure legends. (3) We also now state the ages of the mice for the various analyses in the Methods section.

It appears that several statistical interactions have been summarized in the results section but inconsistently reported on figures.

We now provide the exact n’s for each analysis in all figure legends. We include all of the details of the statistical analysis in the text of the Results section and do not include this text in the Legends; we do this for all figures to maintain consistency.

I presume that when the authors write "the number of neurons with somal diameter greater than 200 μm and with an identifiable nucleolus was determined", the 200 was a typo. Mouse motor neurons do not have a diameter of 200 μm and perhaps the authors meant an area of 200μm2?

We have corrected this: 200 μm2

Authors should consider adding a quantification for the human tissue immunoblots.

We have added the quantification of these human tissue data for ephrinB2.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediators of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied with the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function. This is an expected result, given the fairly focal approach of ephrinB2 knockdown in C3-C5 spinal segments.

The major limitation of the study is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many other that comprise the ventral gray matter, have not been investigated in this study. Nonetheless, there is convincing evidence to suggest astrocytes play a significant role, as compared to oligodendrocytes in promoting ALS pathogenesis.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

eLife assessment

This is a valuable study of Eph-Ephrin signaling mechanisms generating pathological changes in amyotropic lateral sclerosis. There are exciting findings bearing on the role of glial cells in this pathology. The study emerges with solid evidence for a novel astrocyte-mediated mechanism for disease propagation. It may help identify potential therapeutic targets.

Reviewer #1 (Public Review):

In the manuscript by Urban et al., the authors attempt to further delineate the role with which non-neuronal CNS cells play in the development of ALS. Towards this goal, the transmembrane signaling molecule ephrinB2 was studied. It was found that there is an increased expression of ephrinB2 in astrocytes within the cervical ventral horn of the spinal cord in a rodent model of ALS. Moreover, reduction of ephrinB2 reduced motoneuron loss and prevented respiratory dysfunction at the NMJ. Further driving the importance of ephrinB2 is an increased expression in the spinal cords of human ALS individuals. Collectively, these findings present compelling evidence implicating ephrinB2 as a contributing factor towards the development of ALS.

eLife assessment

This manuscript describes an important NMR investigation of allosteric interactions within Abl kinase. The authors identify helix I as a major element that couples the Abl active site with the myristate-binding pocket. The convincing findings have implications for understanding Abl kinase activation and how to target Abl kinase in diseases.

Reviewer #1 (Public Review):

Summary:<br /> The authors identify a mechanical model of activation of Abelson kinase involving the modification of stability of an alpha helix by mutations and different classes of inhibitors. They use NMR chemical shifts of mutant sequences of the alpha helix in a model of Abelson kinase including the regulatory and kinase domains.

Strengths:<br /> The mechanism of inhibition of this important drug target is highly complex involving multiple domains' interactions, While crystal structures can establish end states well, the details of more dynamic interactions among the components can be assessed by NMR studies, The authors previously established {Sonti, 2018, PMID29319304} that different inhibitors and assembled states result from changes of stabilisation of the assembly involving the kinase and the SH3 domain. This is extended here to<br /> illuminate the role of the kinase C terminal alpha helic I' to the domains' interface, expanding the previous identification of this area of the protein as key to agonist/antagonist action at the allosteric myristlylation binding site.

Weaknesses:<br /> The conclusions are based on the relationship with prior observations of classes of chemical shift perturbation, with a set of deletion mutants limited by expression issues. The origin of the force involving the straight or bent helix is not readily apparent. The deletion mutants are treated as solely limiting the helix length irrespective of residue type, and their interactions may be more subtle, beyond the helix stabilization, in other interactions, and in the indirect nature of NMR chemical shift perturbations.

Reviewer #2 (Public Review):

In this paper, Paladini and colleagues investigate the concerted motions within the Abl kinase that control its conformational transition between the active (disassembled) and inactive (assembled state). This work follows their previously published findings that binding of the type II inhibitor, imatinib to the active site of Abl, leads to kinase core disassembly via the force imposed by the P-loop and other regions of the N-lobe on the SH3 domain. Interestingly, imatinib-induced disassembly is prevented when an allosteric inhibitor, asciminib, binds to the myristate-binding pocket. Key to asciminib and myristate binding are motions of helix I, located in the C-lobe, and thus, helix I is hypothesized to be the sensor of the imatinib-induced changes. Specifically, bending of helix I upon engagement of myristate or asciminib was postulated to be important for re-assembly of the autoinhibited Abl core, and thus, reducing the "force" with which kinase N-lobe pushes against the SH2 domain upon binding imatinib.

The authors use NMR to measure conformational transitions in the several 15N-labeled Abl kinase constructs that display different degrees of helix I truncations. This analysis is slightly limited by the instability of the constructs that carry truncations beyond the helix I "bend". Nevertheless, it is sufficient to establish that truncation of helix I that removes its fragment, which is in contact with myristate or asciminib ligands, results in loss of the ability of helix I to impose "force" on the SH2 domain that results in kinase core disassembly, even in the presence of imatinib binding. In the absence of this force, the allosteric coupling between the helix I/SH2 and KD/SH3 interfaces is compromised. Principle component analysis is used to analyze the NMR data, and it is very clear and convincing.

A compelling evidence in support of the proposed allosteric mechanism comes from the analysis of the E528K disease mutation, identified in the Abl1 malformation syndrome. The authors show that this mutant, poised to break a salt bridge formed between E528 in the C-terminal portion of helix I and R479 on the kinase domain, increases helix I outward motions resulting in core disassembly and higher Abl kinase activity. Together, these results reinforce that helix I motions are central to the mechanism of kinase activation via core disassembly. I find that all authors' claims are supported by the experimental data. A couple of suggestions on how to expand and improve the discussion of the data are listed in specific feedback to the authors.

eLife assessment

This study provides a valuable resource that documents the protein-protein interactions (PPI) network for alpha-arrestins in both human and Drosophila based on affinity purification/mass spectrometry and the SAINTexpress method followed by a series of bioinformatic and functional assessments. Through these, the authors confirmed the roles of known and novel interactions, including proteins involved in RNA splicing and helicase, GTPase-activating proteins, and ATP synthase. This study represents a convincing example of how to adopt comparative molecular interactions and how to interpret the functional implications.

Reviewer #1 (Public Review):

The study provides a complete comparative interactome analysis of α-arrestin in both humans and drosophila. The authors have presented interactomes of six humans and twelve Drosophila α-arrestins using affinity purification/mass spectrometry (AP/MS). The constructed interactomes helped to find α-arrestins binding partners through common protein motifs. The authors have used bioinformatic tools and experimental data in human cells to identify the roles of TXNIP and ARRDC5: TXNIP-HADC2 interaction and ARRDC5-V-type ATPase interaction. The study reveals the PPI network for α-arrestins and examines the functions of α-arrestins in both humans and Drosophila. The authors have carried out the necessary changes that were suggested, and the manuscript can now be accepted.

Comments: I would like to congratulate the authors and the corresponding authors of this manuscript for bringing together such an elaborate study on α-arrestin and conducting a comparative study in drosophila and humans.

Introduction: The introduction provides a rationale behind why the comparison between humans and Drosophila is performed.

Results: The results cover all the necessary points concluded from the experiments and computational analysis. The images are elaborate and well-made. The authors have a rigorous amount of work added together for the success of this manuscript. The authors have provided a database of network of α-arrestins in both humans and Drosophila which can be used by other researchers working in the same subject to study the interacting genes.

Discussion: the authors have utilized and discussed the conclusion they draw from their study. But could highlight more on ARRDCs and why it was selected out of the other arrestins.

References: the authors have considered the suggestion and added the necessary references.

The authors have provided future work directions associated with their work.

Reviewer #2 (Public Review):

In this manuscript, the authors present a novel interactome focused on human and fly alpha-arrestin family proteins and demonstrate its application in understanding the functions of these proteins. Initially, the authors employed AP/MS analysis, a popular method for mapping protein-protein interactions (PPIs) by isolating protein complexes. Through rigorous statistical and manual quality control procedures, they established two robust interactomes, consisting of 6 baits and 307 prey proteins for humans, and 12 baits and 467 prey proteins for flies. To gain insights into the gene function, the authors investigated the interactors of alpha-arrestin proteins through various functional analyses, such as gene set enrichment. Furthermore, by comparing the interactors between humans and flies, the authors described both conserved and species-specific functions of the alpha-arrestin proteins. To validate their findings, the authors performed several experimental validations for TXNIP and ARRDC5 using ATAC-seq, siRNA knockdown, and tissue staining assays. The experimental results strongly support the predicted functions of the alpha-arrestin proteins and underscore their importance.

Reviewer #3 (Public Review):

Lee, Kyungtae and colleagues have discovered and mapped out alpha-arrestin interactomes in both human and Drosophila through the affinity purification/mass spectrometry and the SAINTexpress method. Their work revealed highly confident interactomes, consisting of 390 protein-protein interactions (PPIs) between six human alpha-arrestins and 307 preproteins, as well as 740 PPIs between twelve Drosophila alpha-arrestins and 467 prey proteins.

To define and characterize these identified alpha-arrestin interactomes, the team employed a variety of widely recognized bioinformatics tools. These analyses included protein domain enrichment analysis, PANTHER for protein class enrichment, DAVID for subcellular localization analysis, COMPLEAT for the identification of functional complexes, and DIOPT to identify evolutionary conserved interactomes. Through these assessments, they not only confirmed the roles and associated functions of known alpha-arrestin interactors, such as ubiquitin ligase and protease, but also unearthed unexpected biological functions in the newly discovered interactomes. These included involvement in RNA splicing and helicase, GTPase-activating proteins, and ATP synthase.

The authors carried out further study into the role of human TXNIP in transcription and epigenetic regulation, as well as the role of ARRDC5 in osteoclast differentiation. It is particularly commendable that the authors conducted comprehensive testing of TXNIP's role in HDAC2 in gene expression and provided a compelling model while revised the manuscript. Additionally, the quantification of the immunocytochemistry data presented in Figure 6 convincingly supports the authors' hypothesis.

Overall, this study holds important value, as the newly identified alpha-arrestin interactomes are likely aiding functional studies of this protein group and advance alpha-arrestin research.

eLife assessment

In this study, the authors (1) build a detailed model of the process whereby visual information is stored in neuronal activity from the moment that a stimulus is presented until some time later when such information needs to be recalled, and (2) test numerous mechanistic assumptions by fitting the model to rich psychophysical experiments performed by human participants. The evidence supporting the claims is convincing, utilizing detailed model comparison to evaluate potential mechanisms. Overall, the results represent a valuable advance in our understanding of how sensory representations are encoded in and then recalled from, working memory.

Reviewer #1 (Public Review):

Summary:<br /> This study investigates how the neural representation of a stimulus transitions from that evoked by the presence of the stimulus (sensory) to one that exists only as a memory trace once the stimulus disappears (mnemonic). In simple terms, it explores the transition from so-called "iconic memory" (akin to residual sensory-driven neural activity) to working memory proper (self-sustained activity). The authors build a computational model for this transition and test it against data from two new psychophysical experiments plus two datasets from prior experiments.

Strengths and weaknesses:<br /> I really liked this work. It considers a fairly complex process but builds a mechanistically comprehensive scheme that is intuitive and testable. This is a hefty paper; the full model built by the authors has a lot of moving parts. But these are all carefully justified, and in fact, many of them are specifically tested by fitting customized variants of the model to the experimental data (which are rich enough to distinguish all of these variants, not only quantitatively but also qualitatively). Said differently, both the assumptions used to build the model and the conclusions drawn after comparison with the experimental data are well justified. In the end, although it takes some effort to put the whole scheme together, I think the reader learns a lot about memory mechanisms. The Discussion is rich, as beyond working memory per se, the work relates to numerous issues (e.g., perception, attention, neural dynamics, population coding). Importantly, although part of the value of the study lies in the way it integrates many prior results into a cohesive framework, it also makes an important novel point: that iconic and working memory are not qualitatively different things, but rather just different extreme manifestations of the one, continuous process whereby perceptual information is stored (as a pattern of neural activation) and made accessible to other cognitive functions. In this conceptualization, working memory corresponds to a readout of activity a significant time (typically > 1 s) after stimulus offset, whereas iconic memory is consistent with a readout from the same neural population but immediately or very shortly after stimulus offset. This account not only is parsimonious but also provides a specific hypothesis (or a set of hypotheses) that can be tested further.

I did not find any major weaknesses. The paper does require some time and effort in order to appreciate all that it contains, but this is inevitable, as it aims to (1) build a compact but mechanistically detailed account of a process that is somewhat complex, and (2) test key predictions through psychophysical experiments that must be sufficiently rich. In the end, I found the effort quite rewarding.

Reviewer #2 (Public Review):

Summary:<br /> Previous work has shown subjects can use a form of short-term sensory memory, known as 'iconic memory', to accurately remember stimuli over short periods of time (several hundred milliseconds). Working memory maintains representations for longer periods of time but is strictly limited in its capacity. While it has long been assumed that sensory information acts as the input to working memory, a process model of this transfer has been missing. To address this, Tomic and Bays present the Dynamic Neural Resource (DyNR) model. The DyNR model captures the dynamics of processing sensory stimuli, transferring that representation into working memory, the diffusion of representations within working memory, and the selection of memory for report.

The DyNR model captures many of the effects observed in behavior. Most importantly, psychophysical experiments found the greater the delay between stimulus presentation and the selection of an item from working memory, the greater the error. This effect also depended on working memory load. In the model, these effects are captured by the exponential decay of sensory representations (i.e., iconic memory) following the offset of the stimulus. Once the selection cue is presented, residual information in iconic memory can be integrated into working memory, improving the strength of the representation and reducing error. This selection process takes time, and is slower for larger memory loads, explaining the observation that memory seems to decay instantly. The authors compare the DyNR model to several variants, demonstrating the importance of the persistence of sensory information in iconic memory, normalization of representations with increasing memory load, and diffusion of memories over time.

Strengths:<br /> The manuscript provides a convincing argument for the interaction of iconic memory and working memory, as captured by the DyNR model. The analyses are cutting-edge and the results are well captured by the DyNR model. In particular, a strength of the manuscript is the comparison of the DyNR model to several alternative variants.

The results provide a process model for interactions between iconic memory and working memory. This will be of interest to neuroscientists and psychologists studying working memory. I see this work as providing a foundation for understanding behavior in continuous working memory tasks, from either a mechanistic, neuroscience, perspective or as a high-water mark for comparison to other psychological process models.

Finally, the manuscript is well written. The DyNR model is nicely described and an intuition for the dynamics of the model is clearly shown in Figures 2 and 4.

Weaknesses:<br /> Despite its strengths, the paper does have some (relatively minor) weaknesses. In particular, the authors could consider the role of sensory processing, and its limitations, and variability in selecting an item from working memory as other factors affecting memory accuracy.

Reviewer #3 (Public Review):

Summary:<br /> The authors set out to formally contrast several theoretical models of working memory, being particularly interested in comparing the models regarding their ability to explain cueing effects at short cue durations. These benefits are traditionally attributed to the existence of a high capacity, rapidly decaying sensory storage which can be directly read out following short latency retro-cues. Based on the model fits, the authors alternatively suggest that cue-benefits arise from a freeing of working memory resources, which at short cue latencies can be utilized to encode additional sensory information into VWM.

A dynamic neural population model consisting of separate sensory and VWM populations was used to explain temporal VWM fidelity of human behavioral data collected during several working memory tasks. VWM fidelity was probed at several timepoints during encoding, while sensory information was available, and maintenance when sensory information was no longer available. Furthermore, set size and exposure durations were manipulated to disentangle contributions of sensory and visual working memory.

Overall, the model explained human memory fidelity well, accounting for set size, exposure time, retention time, error distributions, and swap errors. Crucially the model suggests that recall at short delays is due to post-cue integration of sensory information into VWM as opposed to direct readout from sensory memory. The authors formally address several alternative theories, demonstrating that models with reduced sensory persistence, direct readout from sensory memory, no set-size dependent delays in cue processing, and constant accumulation rate provide significantly worse fits to the data.

I congratulate the authors for this rigorous scientific work. I have only very few remarks that I hope the authors can clarify.

Author Response

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

We would like to thank the editors and reviewers for their overall positive assessment of this work. We have carefully revised the manuscript and implemented near all reviewers’ public and confidential recommendations. We believe these modifications have strengthened the manuscript and hope it will further convince the editors and reviewers.

We below provide a point-by-point response to the reviewers’ comments.

Reviewer #1 (Public Review):

To further understand the plasticity of vestibular compensation, Schenberg et al. sought to characterize the response of the vestibular system to short-term and partial impairment using gaze stabilization behaviors. A transient ototoxic protocol affected type I hair cells and produced gain changes in the vestibulo-ocular reflex and optokinetic response. Interestingly, decreases in vestibular function occurred in coordination with an increase in ocular reflex gain at frequencies where vestibular information is more highly weighted over visual. Moreover, computational approaches revealed unexpected detriment from low reproducibility on combined gaze responses. These results inform the current understanding of visual-vestibular integration especially in the face of dysfunction.

Strengths

The manuscript takes advantage of VOR measurements which can be activated by targeted organs, are used in many species including clinically, and indicate additional adverse effects of vestibular dysfunction. The authors use a variety of experimental procedures and analysis methods to verify results and consider individual performance effects on the population data. The conclusions are well-justified by current data and supported by previous research and theories of visuo-vestibular function and plasticity.

The authors thank reviewer 1 for emphasizing these positive aspects of the work.

Weaknesses

The manuscript describes the methodology as inducing reversible changes (lines 44, 67,) but the data shows a reversible effect only in hair cell histology (Fig 3A-B) not in function as demonstrated by the persistent aVOR gain reduction in week 12 (Fig 1C) and increase of OKR gain in weeks 6-12 (Fig 4C/D).

Rodents exposed to IDPN in the drinking water show from complete to null reversibility of the function loss depending on the IDPN concentration and duration of exposure, and the relationship between exposure and effect varies as a function of species, strain and sex of the exposed animals (Llorens and Rodríguez-Farré, Neurotoxicol. Teratol., 1997; Seoane et al., J. Comp. Neurol. 2001; Sedó-Cabezón et al., Dis. Model. Mech., 2015; Greguske et al., Arch. Toxicol., 2019). In addition, there is individual variability. The concentration of IDPN and time of exposure used in this study were selected to result in a loss followed by complete reversion but, as noted by the referee, the reversion was complete on Hair cells, while the gaze stabilizing reflexes show differential degrees of recovery depending on the functional tests (complete recovery on OCR; partial on aVOR and OKR). These make the IDPN subchronic protocol an interesting methodology to study the long term consequences of partial/reversible inner ear impairment. To be more accurate in the description of the reversibility, we have now introduced the following changes:

Lines 43: Subchronic exposure to IDPN in drinking water at low doses allowed for progressive ototoxicity, leading to a partial and largely reversible loss of function.

Lines 67-68: We demonstrate that despite the significant recovery in their vestibulo-ocular reflexes, the visuo-vestibular integration remains notably impaired in some IDPN-treated mice

Lines 578: Previous experiments (Greguske et al., 2021) had demonstrated that at these concentrations, ototoxic lesions produced by IDPN are largely reversible.

Reviewer 1: The manuscript begins with the mention of fluctuating vestibular function clinically, but does not connect this to any specific pathologies nor does it relate its conclusions back to this motivation.

Thank you. We have now added a conclusion (lines 525-552) to discuss the results in a clinical perspective.

Reviewer 1: The conclusions of frequency-specific changes in OKR would be stronger if frequency-specific aVOR effects were demonstrated similar to Figure 4D.

We have presented the frequency-selective effects in Figure 1 supplement and related text; changes observed in aVOR are mostly (see below) comparable for all frequencies >0.2Hz. However, we have edited the text to better highlight when the IDPN differentially affect aVOR tested at different frequencies (see lines 97-99).

Reviewer #2 (Public Review):

This is a very nice study showing how partial loss of vestibular function leads to long term alterations in behavioural responses of mice. Specifically, the authors show that VOR involving both canal and otolith afferents are strongly attenuated following treatment and partially recover. The main result is that loss of VOR is partially "compensated" by increased OKR in treated animals. Finally, the authors show that treatment primarily affects type I hair cells as opposed to type II. Overall, these results have potentially important implications for our understanding of how the VOR Is generated using input from both type I and type II hair cells. As detailed below however, more controls as well as analyses are needed.

The authors thank reviewer 2 for positive evaluation regarding the potential implication of the work.

Major points:

Reviewer 2: The authors analyze both canal and otolith contributions to the VOR which is great. There is however an asymmetry in the way that the results are presented in Figure 1. Please correct this and show time series of fixations for control and at W6 and W12. Moreover, the authors are plotting table and eye position traces in Fig. 1B but, based on the methods, gains are computed based on velocity. So please show eye velocity traces instead. Also, what was the goodness of fit of the model to the trace at W6? If lower than 0.5 then I think that it is misleading to show such a trace since there does not seem to be a significant VOR.

Figure 1 was modified as suggested. Panel B now shows velocity traces, and goodness of fit is reported in figure legend. Panel E now shows raw OCR traces at W0, W6, W12.

Reviewer 2: This is important to show that the loss is partial as opposed to total. It seems to me that the treatment was not effective at all for aVOR for at least some animals. What happens if these are not included in the analysis?

The reviewer is correct, there is indeed variability in the alteration observed during the treatment, as previously described and expected from previous experiments (Llorens and Rodríguez-Farré, Neurotoxicol. Teratol., 1997; Seoane et al., J. Comp. Neurol. 2001; SedóCabezón et al., Dis. Model. Mech., 2015; Greguske et al., Arch. Toxicol., 2019). It was actually one of the goal of the study to compare hair cell loss and VOR responses in individuals. The individual aVOR gain and phase responses during the IDPN treatment are all presented in Figure 1 supplement. aVOR was reduced in all animals, although 2/21 only showed a decrease of less than 10% of their initial gain at W6. If these were excluded from the analysis, the statistical differences between the 2 groups would be reinforced.

Reviewer 2: Figure 2A shows a parallel time course for gains of aVOR and OCR at the population level. Is this also seen at the individual level?

Yes, this is seen in individuals. This result is presented in Figure 2C and 2D which illustrate the similar effect of IDPN on aVOR and OCR responses at week 6 and week 12 at the individual level (each symbol represents an individual mouse). The plotted delta gain of both aVOR and OCR represents the relative loss of vestibular function for each individual mouse at W6 and W12, respectively.

Reviewer 2: Figure 3: please show individual datapoints in all conditions.

Figure 3 was modified to show individual datapoints in all conditions (see Figure 3 A2, A3, C2 and C3).

Reviewer 2: Figure 4: The authors show both gain and phase for OKR. Why not show gain and phase for aVOR and OCR in Figure 1. I realize that phase is shown in sup Figures but it is important to show in main figures. The authors show a significant increase in phase lead for aVOR but no further mention is made of this in the discussion. Moreover, how are the authors dealing with the fact that, as gain gets smaller, the error on the phase will increase. Specifically, what happens when the grey datapoints are not included?

As pointed by the reviewer, we have included all aVOR phase results in Figure 1 supplement and also stated it in the main text (lines 100-102). There is however no phase calculated for the OCR which is a static test, as better illustrated in new Figure 1E. Error in phase calculations increases as gain gets smaller. To take this into account, the phase corresponding to the grey points (VAF<0,5; corresponding to Gains<0.10) were not included in the statistical analysis of the aVOR phase. This point is now made clearer in methods lines 639-640.

Reviewer 2: Discussion: As mentioned above, the authors should discuss the mechanisms and implications of the observed phase lead following treatment. Moreover, recent literature showing that VN neurons that make the primary contribution to the VOR (i.e., PVP neurons) tend to show more regular resting discharges than other classes (i.e., EH cells), and that such regularity is needed for the VOR should be discussed (Mackrous et al. 2020 eLife). Specifically, how are type I and type II hair cells related to discharge regularity by central neurons in VN?

We have now added discussion regarding mechanisms and implications of the phase changes in lines 363-371. The authors thank reviewer 1 for pointing at the Mackrous et al. 2020 eLife paper which is now included in the updated discussion. The relations between type I and type II and discharge regularity in afferents and central VN is further discussed 442-449.

Below we provide answers to specific recommendations for the authors.

Reviewer #1 (Recommendations For The Authors):

Reviewer 1: Were hair cells counted for the whole organ? what was the control for epithelial size differences?

The effect of the treatment on hair cells was estimated by counting numbers of cells in square area of the central and peripheral parts of the sensory epithelia. The text has been modified to better describe the method, lines 748-751.

Reviewer 1: The title of the article leads readers to expect more emphasis on hair cell changes, while the content of the manuscript is more functional and encompassing the visual and vestibular systems.

We have retained the original tittle.

Reviewer 1: Please provide acronym definitions before they are used. Examples: HC (line 63), W6 etc (line 82-83)

Done as suggested on lines 63, 82 and 107.

Reviewer 1: Please describe the ages of animals used in the study.

The animals used in the study were 6 weeks old at the beginning of the protocol and 20 weeks old at the end. The text has been modified accordingly, line 564.

Reviewer 1: Consider changing "until" to "through" when describing time ranges (initially line 88), as the following time mentioned is included in the statement. E.g., line 216-217 sounds as if gain was insignificantly different at W12.

Done as suggested, lines 88 and 219.

Reviewer 1: Line 162: lower case for "immunostaining".

Done, line 164.

Reviewer 1: Consider regrouping or renumbering panels of Figure 3 for more clarity.

Panels in Figure 3 were regrouped as suggested, with first the canal-related data in panels A-B followed by the utricule-related data in panels C-D.

Reviewer 1: Lines 222-223: reword as gain increased not frequency.

Thank you, the text has been reworded, line 224-225.

Reviewer 1: It is unclear if the two subgroups revealed in CGR analysis (line 288) are relevant to the two groups described in VOR responses (line 137-138). Please clarify if these are the same mice or distinct clusters.

The two subgroups found in the CGR analysis differ from the clusters revealed by the decrease of the aVOR gain; the text has been modified lines 300-301.

Reviewer 1: Consider adding that irregular afferents + calyces are relevant specifically to type I HCs (lines 411-426).

The text has been modified to clarify the contacts between the two types of vestibular afferents and hair cells, lines 431-435.

Reviewer 1: Line 434: clarify which "scheme" given context before and after this phrase

In order to clarify this part of the discussion, the text has been modified and this term no longer appears.

Reviewer 1: Please indicate the time gap from surgery to treatment.

The time gap from the surgery to treatment, at least 72h, has been updated in the methods, lines 575.

Reviewer 1: Line 619-620: It is unclear if VOR and OKR sessions were randomized in order or if the authors have considered training or adaptive effects from the initial test session.

VOR and OKR sessions were performed on different days to limit cross effects, lines 639-640.

Reviewer 1: Line 688: typo-change ifG to IgG.

modified, line 744.

Reviewer 1: Line 692-693: were hair cells counted for the whole organ? what was the control for epithelial size differences?

The effect of the treatment on hair cells was estimated by counting numbers of cells in square area of the central and peripheral parts of the sensory epithelia. The text has been modified to better explain the method, lines 748-751.

Reviewer 1: Change decimal indicator to be consistent (commas used in lines 732, 759, 776, Figure 6C),

Thank you; modified as suggested.

Reviewer 1: Line 763: "stimulation at 0.5Hz &10{degree sign}/s" is unclear.

The text has been modified, line 817.

Reviewer 1: Line 765: bold (E)

The police format has been updated, line 820.

Reviewer 1: Line 770-771: A) insert OKR to be "mean delta aVOR and delta OKR gain", B) plot is OKR as a function of VOR.

Thank you, done as suggested. The text has been modified, line 824. Reviewer 1: Describe Figure 6 delta at initial mention (line 784 instead of 786) Authors: thank you, done as suggested, line 839.

Reviewer 1: It is unclear why the tables are included if never mentioned in the text.

The tables are now mentioned, lines 90 and 218.

Reviewer 1: Figure 1: is the observed gain for Sham group expected value rather than closer to 1?

Yes, as the value reported on Figure 1 is a mean of the values obtained during aVOR test in the dark at frequencies in range 0.2-1Hz (see also Figure 1 Supplement).

Reviewer 1: Figure 2: A) give enough space to see error bars at W2. Consider making test data more easily distinguishable. B) is OCR mean or specific stimulation? C/D) move 1Hz label from title to x-axis label as it does not describe OCR test. Figure 5: C) consider making color specific to frequency for better distinction on C+D as symbols previously indicated individual data. D) 1Hz specific to OKR? move to axis label instead of title

The Figures 2 and 5 have been modified according to reviewer 1 suggestions.

Reviewer 1: Figure 6: A/B) what time point are these, W12?

Those points correspond to W6 and W12, the text has been updated to specify the time points, lines 834 and 835.

Reviewer #2 (Recommendations For The Authors):

The authors should perform additional analyses that will help strengthen their results.

We are unsure about the exact implementation of this recommendation. However, we have strengthened our results by following all reviewers’ specific recommendations.

eLife assessment

This paper provides a fundamental expansion of vestibular compensation into transient and partial dysfunction, as well as insights into the adaptation of visual reflexes in this process. The conclusions are convincingly supported with paired histological and behavioral measurements, which are additionally modeled for further interpretation. This work would be of interest to neuroscientists working in multisensory integration and recovery mechanisms.

Reviewer #1 (Public Review):

To further understand the plasticity of vestibular compensation, Schenberg et al. sought to characterize the response of the vestibular system to short-term and partial impairment using gaze stabilization behaviors. A transient ototoxic protocol affected type I hair cells and produced gain changes in the vestibulo-ocular reflex and optokinetic response. Interestingly, decreases in vestibular function occurred in coordination with an increase in ocular reflex gain at frequencies where vestibular information is more highly weighted over visual. Moreover, computational approaches revealed unexpected detriment from low reproducibility on combined gaze responses. These results inform the current understanding of visual-vestibular integration especially in the face of dysfunction.

Strengths<br /> The manuscript takes advantage of VOR measurements that can be activated by targeted organs, are used in many species including clinically, and indicate additional adverse effects of vestibular dysfunction.

The authors use a variety of experimental procedures and analysis methods to verify results and consider individual performance effects on the population data.

The conclusions are well-justified by current data and supported by previous research and theories of visuo-vestibular function and plasticity.

Reviewer #2 (Public Review):

This is a very nice study showing how partial loss of vestibular function leads to long term alterations in behavioural responses of mice. Specifically, the authors show that VOR involving both canal and otolith afferents are strongly attenuated following treatment and partially recover. The main result is that loss of VOR is partially "compensated" by increased OKR in treated animals. Finally, the authors show that treatment primarily affects type I hair cells as opposed to type II hair cells. Overall, these results have important implications for our understanding of how the VOR Is generated using input from both type I and type II hair cells.

The major strength of the study lies in the use of partial inactivation of hair cells to look at the effects on behaviors such as VOR and OKR. Some weaknesses stem from the fact that the effects of inactivation are highly variable across specimens and that there is no recovery of behavioral function.

Author Response

Reviewer #1 (Public Review):

Assessment:

The manuscript titled 'Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal 1 homeostasis' by Gaur et al discusses the role of Rab7 in the development of ulcerative colitis by regulating the lysosomal degradation of Clca1, a mucin protease. The manuscript presents interesting data and provides a potential molecular mechanism for the pathological alterations observed in ulcerative colitis. Gaur et al demonstrate that Rab7 levels are lowered in UC and CD. However, a similar analysis of Rab7 levels in ulcerative colitis (UC) and Crohn's disease (CD) patient samples was conducted recently (Du et al, Dev Cell, 2020) which showed that Rab7 levels are found to be elevated under these conditions. While Gaur et al have briefly mentioned Du et al's paper in passing in the discussion, they need to discuss these contradictory results in their paper and clarify these differences. Additionally, Du et al are not included in the list of references.

Strengths:

The manuscript used a multi-pronged approach and compares patient samples, mouse models of DSS, and protocols that allow differentiation of goblet cells. They also use a nanogel-based delivery system for siRNAs, which is ideal for the knockdown of specific genes in the gut.

Weaknesses:

Du et al, Dev Cell 2020 (https://doi.org/10.1016/j.devcel.2020.03.002) have previously shown that Rab7 levels are elevated in a similar set of colonic samples (age group, number etc) from UC and CD patients. Gaur et al have not discussed this paper or its findings in detail, which directly contradicts their results. Clarification regarding this should be provided.

We thank and appreciate the reviewer for bringing this point.

The results shown by Du et al, Dev Cell, 2020 depict elevated expression of Rab7 in UC and CD patients compared to controls. In first occurrence, these results appear contradictory, but there may be a few possible explanations for this.

Firstly, Rab7 expression levels may fluctuate in the tissue depending on the degree of the gut inflammation. This can be concluded from our observations in DSS-mice dynamics model and the human patient samples with mild and moderate UC. Furthermore, Du et al provide no information of the severity of the condition among the patients employed in the study. Our motive, in the current work, was to emphasise this aspect. This point was mentioned in the discussion section of the manuscript. However, in view of the reviewer’s concern, we now intend to add a detailed comment on this in the main text of the revised version of the manuscript.

Secondly, the control biopsies in our investigation were acquired from non-IBD patients, and not what was done by Du et al., wherein biopsies from the normal para-carcinoma region of the colorectal cancer patients was used. One can not overlook the fact that physiological and molecular changes are apparent even in non-inflamed regions in the gut of an IBD or CRC patient. It is possible that the observed discrepancy arises due to the differences in the sample type used for comparing the Rab7 expression.

Finally, the main sub-tissue region showing a decrease in Rab7 expression in UC samples, appeared to be the Goblet cells which was not covered by Du et al.

Keeping these points in mind we do not think that there is a contradiction in our findings with that of Du et al., 2020. In the revised submission some of these explanations will be incorporated. Include Du et al in the reference list and add the comment in main text.

This was an oversight from our side. We have actually mentioned Du et al., 2020 in the discussion (line number 338) but somehow the reference was missing in the main list. We will ensure that the reference is included in the revised version and that their findings are included both in main text and in the discussion.

Reviewer #2 (Public Review):

Summary:

In this work, the authors report a role for the well-studied GTPase Rab7 in gut homeostasis. The study combines cell culture experiments with mouse models and human ulcerative colitis patient tissues to propose a model where, Rab7 by delivering a key mucous component CLCA1 to lysosomes, regulates its secretion in the goblet cells. This is important for the maintenance of mucous permeability and gut microbiota composition. In the absence of Rab7, CLCA1 protein levels are higher in tissues as well as the mucus layer, corroborating with the anticorrelation of Rab7 (reduced) and CLCA1 (increased) from ulcerative colitis patients. The authors conclude that Rab7 maintains CLCA1 level by controlling its lysosomal degradation, thereby playing a vital role in mucous composition, colon integrity, and gut homeostasis.

Strengths:

The biggest strength of this manuscript is the combination of cell culture, mouse model, and human tissues. The experiments are largely well done and in most cases, the results support their conclusions. The authors go to substantial lengths to find a link, such as alteration in microbiota, or mucus proteomics.

Weaknesses:

There are also some weaknesses that need to be addressed. The association of Rab7 with UC in both mice and humans is clear, however, claims on the underlying mechanisms are less clear. Does Rab7 regulate specifically CLCA1 delivery to lysosomes, or is it an outcome of a generic trafficking defect? CLCA1 is a secretory protein, how does it get routed to lysosomes, i.e. through Golgi-derived vesicles, or by endocytosis of mucous components? Mechanistic details on how CLCA1 is routed to lysosomes will add substantial value.

We thank the reviewer for the insightful comment. We would like to bring forth the following explanation for each these concerns:

(a) Our immunofluorescence imaging experiments revealed co-localization of Rab7 protein with CLCA1 and the lysosomes (Fig 7I). In addition, the absence of Rab7 affects the transport of CLCA1 to lysosomes (Fig 7J). This demonstrates that Rab7 may be involved in regulation of CLCA1 transport (presumably along with other cargo), to lysosomes selectively. However, we do recognise that the point raised by the reviewer about possible effect of a generic trafficking defect is valid. (b) As mentioned in the manuscript, the trafficking of CLCA1 protein or CLCA1-containing vesicles within the goblet cell is unknown, with no information on the proteins involved in its mobility. The switching of CLCA1 containing vesicles from the secretory route to lysosomes needs extensive investigation involving overall trafficking of the protein. Taken together, the complete answer to both these important questions will need a series of experiments and those may be interesting avenues for future research.

(a) Why does the level of Rab7 fluctuate during DSS treatment (Fig 1B)? (b) Does the reduction seen in Rab7 levels (by WB) also reflect in reduced Rab7 endosome numbers?

This is a very thoughtful point from the reviewer. We detected a distinct pattern of Rab7 expression fluctuation in intestinal epithelial cells after DSS-dynamics treatment in mice. Perhaps, these changes are the result of complex cellular signalling in response to the DSS treatment. Rab7, being a fundamental protein involved in protein sorting pathway, is expected to undergo alteration based on cells requirement. Presently there are no reports suggesting the regulatory mechanisms that govern Rab7 levels in the gut. (b) We observed reduction in Rab7 expression both at RNA and protein levels. To confirm whether this alteration will lead to reduced Rab7 positive endosome numbers may require detailed investigations.

Are other late endosomal (and lysosomal) populations also reduced upon DSS treatment and UC? Is there a general defect in lysosomal function?

There are no direct evidences showing reduction in the late endosomal and lysosomal population during gut inflammation, but few studies link lysosomal dysfunction with risk for colitis (doi: 10.1016/j.immuni.2016.05.007).

The evidence for lysosomal delivery of CLCA1 (Fig 7 I, J) is weak. Although used sometimes in combination with antibodies, lysotracker red is not well compatible with permeabilization and immunofluorescence staining. The authors can substantiate this result further using lysosomal antibodies such as Lamp1 and Lamp2. For Fig 7J, it will be good to see a reduction in Rab7 levels upon KD in the same cell.

We used Lysotracker red in live cells followed by fixation. So, permeabilization issues were resolved. Lamp1, as suggested by the reviewer, is definitely a better marker for lysosomes in immunofluorescence studies, but is also shown to mark late endosomes (doi: 10.1083/jcb.132.4.565). As Rab7 protein also marks the late endosomes, using Lamp1 may leave the ambiguity of CLCA1 in Rab7 positive late endosomes versus lysosomes. Nevertheless, we will be carrying out this experiment and the data will be shared in revised version of the work.

In this connection, Fig S3D is somewhat confusing. While it is clear that the pattern of Muc2 in WT and Rab7-/- cells are different, how this corroborates with the in vivo data on alterations in mucus layer permeability -- as claimed -- is not clear.

The data in Fig. S3D suggest the involvement of Rab7 in packaging of Muc2. The whole idea for doing this experiment was to support our observation in the Rab7KD-mice model where mucus layer was seen to be loose and more permeable in Rab7 deficient mice.

Overall, the work shows a role for a well-studied GTPase, Rab7, in gut homeostasis. This is an important finding and could provide scope and testable hypotheses for future studies aimed at understanding in detail the mechanisms involved.

We thank the reviewer for this comment.

eLife assessment

The study provides important insights into the mechanisms underlying ulcerative colitis, a chronic and debilitating gastrointestinal condition. The article provides solid evidence of the role of the vesicular trafficking protein Rab7 in regulating the colonic mucus system and its implications in ulcerative colitis.

Reviewer #1 (Public Review):

Assessment:

The manuscript titled 'Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal 1 homeostasis' by Gaur et al discusses the role of Rab7 in the development of ulcerative colitis by regulating the lysosomal degradation of Clca1, a mucin protease. The manuscript presents interesting data and provides a potential molecular mechanism for the pathological alterations observed in ulcerative colitis. Gaur et al demonstrate that Rab7 levels are lowered in UC and CD. However, a similar analysis of Rab7 levels in ulcerative colitis (UC) and Crohn's disease (CD) patient samples was conducted recently (Du et al, Dev Cell, 2020) which showed that Rab7 levels are found to be elevated under these conditions. While Gaur et al have briefly mentioned Du et al's paper in passing in the discussion, they need to discuss these contradictory results in their paper and clarify these differences. Additionally, Du et al are not included in the list of references.

Strengths:

The manuscript used a multi-pronged approach and compares patient samples, mouse models of DSS, and protocols that allow differentiation of goblet cells. They also use a nanogel-based delivery system for siRNAs, which is ideal for the knockdown of specific genes in the gut.

Weaknesses:

Du et al, Dev Cell 2020 (https://doi.org/10.1016/j.devcel.2020.03.002) have previously shown that Rab7 levels are elevated in a similar set of colonic samples (age group, number etc) from UC and CD patients. Gaur et al have not discussed this paper or its findings in detail, which directly contradicts their results. Clarification regarding this should be provided.

Reviewer #2 (Public Review):

Summary:

In this work, the authors report a role for the well-studied GTPase Rab7 in gut homeostasis. The study combines cell culture experiments with mouse models and human ulcerative colitis patient tissues to propose a model where, Rab7 by delivering a key mucous component CLCA1 to lysosomes, regulates its secretion in the goblet cells. This is important for the maintenance of mucous permeability and gut microbiota composition. In the absence of Rab7, CLCA1 protein levels are higher in tissues as well as the mucus layer, corroborating with the anti-correlation of Rab7 (reduced) and CLCA1 (increased) from ulcerative colitis patients. The authors conclude that Rab7 maintains CLCA1 level by controlling its lysosomal degradation, thereby playing a vital role in mucous composition, colon integrity, and gut homeostasis.

Strengths:

The biggest strength of this manuscript is the combination of cell culture, mouse model, and human tissues. The experiments are largely well done and in most cases, the results support their conclusions. The authors go to substantial lengths to find a link, such as alteration in microbiota, or mucus proteomics.

Weaknesses:

There are also some weaknesses that need to be addressed. The association of Rab7 with UC in both mice and humans is clear, however, claims on the underlying mechanisms are less clear. Does Rab7 regulate specifically CLCA1 delivery to lysosomes, or is it an outcome of a generic trafficking defect? CLCA1 is a secretory protein, how does it get routed to lysosomes, i.e. through Golgi-derived vesicles, or by endocytosis of mucous components? Mechanistic details on how CLCA1 is routed to lysosomes will add substantial value.

Why does the level of Rab7 fluctuate during DSS treatment (Fig 1B)? Does the reduction seen in Rab7 levels (by WB) also reflect in reduced Rab7 endosome numbers? Are other late endosomal (and lysosomal) populations also reduced upon DSS treatment and UC? Is there a general defect in lysosomal function?

The evidence for lysosomal delivery of CLCA1 (Fig 7 I, J) is weak. Although used sometimes in combination with antibodies, lysotracker red is not well compatible with permeabilization and immunofluorescence staining. The authors can substantiate this result further using lysosomal antibodies such as Lamp1 and Lamp2. For Fig 7J, it will be good to see a reduction in Rab7 levels upon KD in the same cell. In this connection, Fig S3D is somewhat confusing. While it is clear that the pattern of Muc2 in WT and Rab7-/- cells are different, how this corroborates with the in vivo data on alterations in mucus layer permeability -- as claimed -- is not clear.

Overall, the work shows a role for a well-studied GTPase, Rab7, in gut homeostasis. This is an important finding and could provide scope and testable hypotheses for future studies aimed at understanding in detail the mechanisms involved.

Reviewer #2 (Public Review):

Summary:

Using in vitro and in vivo approaches, the authors first demonstrate that BEST4 inhibits intestinal tumor cell growth and reduces their metastatic potential, possibly via downstream regulation of TWIST1.

They further show that HES4 positively upregulates BEST4 expression, with direct interaction with BEST4 promoter region and protein. The authors further expand on this with results showing that negative regulation of TWIST1 by HES4 requires BEST4 protein, with BEST4 required for TWIST1 association with HES4. Reduction of BEST1 expression was shown in CRC tumor samples, with correlation of BEST4 mRNA levels with different clinicopathological factors such as sex, tumor stage, and lymph node metastasis, suggesting a tumor-suppressive role of BEST4 for intestinal cancer.

Strengths:

• Good quality western blot data.<br /> • Multiple approaches were used to validate the findings.<br /> • Logical experimental progression for readability.<br /> • Human patient data / In vivo murine model / Multiple cell lines were used, which supports translatability / reproducibility of findings.

Weaknesses:

• Interpretation of figures and data (unsubstantiated conclusions).<br /> • Figure quality.<br /> • Figure legends lack information.<br /> • Lacking/shallow discussion.<br /> • Requires more information for reproducibility regarding materials and methods.

eLife assessment

The findings of this valuable manuscript advance our understanding of the significance of Bestrophin isoform 4 (BEST4) in suppressing colorectal cancer (CRC) progression. The authors used appropriate and validated methodology, such as the knockout of BEST4 using CRISPR/Cas9 in CRC cells, to provide a solid foundation for elucidating the potential link between BEST4 and CRC progression.

Reviewer #1 (Public Review):

Summary:

In this study, the authors describe the participation of the Hes4-BEST4-Twist axis in controlling the process of epithelial-mesenchymal transition (EMT) and the advancement of colorectal cancers (CRC). They assert that this axis diminishes the EMT capabilities of CRC cells through a variety of molecular mechanisms. Additionally, they propose that reduced BEST4 expression within tumor cells might serve as an indicator of an adverse prognosis for individuals with CRC.

Strengths:

• Exploring the correlation between the Hes4-BEST4-Twist axis, EMT, and the advancement of CRC is a novel perspective and gives readers a fresh standpoint.<br /> • The whole transcriptome sequence analysis (Figure 5) showing low expression of BEST4 in CRC samples will be of broad interest to cancer specialists as well as cell biologists although further corroborative data is essential to strengthen these findings (See Weaknesses).

Weaknesses:

• The authors employed three kinds of CRC cell lines, but not untransformed cells such as intestinal epithelial organoids which are commonly used in recent research.<br /> • The authors use three different human CRC cell lines with a lack of consistency in the selection of them. Please clarify 1) how these lines are different from each other, 2) why they pick up one or two of them for each experiment. To be more convincing, at least two lines should be employed for each in vitro experiment.<br /> • The authors demonstrated associations between BEST4 and cell proliferation/viability as well as migration/invasion, utilizing CRC cell lines, but it should be noted that these findings do not indicate a tumor-suppressive role of BEST4 as mentioned in line 120. Furthermore, while the authors propose that "BEST4 functions as a tumor suppressor in CRC" in line 50, there seems no supporting data to suggest BEST4 as a tumor suppressor gene.<br /> • The HES4-BEST4-Twist1 axis likely plays a significant role in CRC progression via EMT but not CRC initiation. Some sentences could lead to a misunderstanding that the axis is important for CRC initiation.<br /> • The authors mostly focus on the relationship of the HES4-BEST4-Twist1 axis with EMT, but their claims sometimes appear to deviate from this focus.<br /> • Some experiments do not appear to have a direct relevance to their claims. For example, the analysis using the xenograft model in Figure 2E-J is not optimal for analyzing EMT. The authors should analyze metastatic or invasive properties of the transplanted tumors if they intend to provide some supporting evidence for their claims.<br /> • In Figure 4H, ZO-1 and E-cad expression looks unchanged in the BEST4 KD.<br /> • The in vivo and in vitro data supporting the whole transcriptome sequence analysis (Figure 5) is mostly insufficient. Including the following experiments will substantiate their claims: 1) BEST4 and HES4 immunostaining of human surgical tissue samples, 2) qPCR data of HES4, Twist1, Vimentin, etc. as shown in Figure 5C, 5D.<br /> • Some statements are inconsistent probably due to grammatical errors. (For example, some High/low may be reversed in lines 234-244.)

Author Response

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

eLife assessment

This study and associated data is compelling, novel, important, and well-carried out. The study demonstrates a novel finding that different chemotherapeutic agents can induce nucleolar stress, which manifests with varying cellular and molecular characteristics. The study also proposes a mechanism for how a novel type of nucleolar stress driven by CDK inhibitors may be regulated. The study sheds light on the importance of nucleolar stress in defining the on-target and offtarget effects of chemotherapy in normal and cancer cells.

We are thankful to the reviewers and the editor for their feedback and thorough assessment of our work. Our responses to the comments and suggestions are below.

Reviewer #1 (Public Review):

The study titled "Distinct states of nucleolar stress induced by anti-cancer drugs" by Potapova and colleagues demonstrates that different chemotherapeutic agents can induce nucleolar stress, which manifests with varying cellular and molecular characteristics. The study also proposes a mechanism for how a novel type of nucleolar stress driven by CDK inhibitors may be regulated. As a reviewer, I appreciate the unbiased screening approach and I am enthusiastic about the novel insights into cell biology and the implications for cancer research and treatment. The study has several significant strengths: i) it highlights the understudied role of nucleolar stress in the on- and off-target effects of chemotherapy; ii) it defines novel molecular and cellular characteristics of the different types of nucleolar stress phenotypes; iii) it proposes novel modes of action for well-known drugs. However, there are several important points that should be addressed:

• The rationale behind choosing RPE cells for the screen is unclear. It might be more informative to use cancer cells to study the effects of chemotherapeutic agents. Alternatively, were RPE cells selected to evaluate the side effects of these agents on normal cells? Clarifying these points in the introduction and discussion would guide the reader.

RPE1, a non-cancer-derived cell line, was chosen for this study to evaluate the effects of anticancer drugs on normal nucleolar function, with the underlying premise that nucleolar stress in normal cells can contribute to non-specific toxicity. This clarification is added to the introduction. Another factor that played in selecting a normal cell line for the drug screen and subsequent experiments was the spectrum of known and unknown genetic and metabolic alterations present in various cancer cell lines. These variables are often unique to a particular cancer cell line and may or may not impact nucleolar proteome and function. Therefore, the nucleolar stress response can be influenced by the spectrum of alterations inherent to each cancer. Our primary focus was to determine the impact of these drugs under normal conditions.

That said, the selected hits of main drug classes were validated in a panel of cell lines that included two other hTERT lines (BJ5TA and CHON-002) and two cancer lines (DLD1 and HCT116). In cancer cells starting nucleolar normality scores were lower than in hTERT cells, suggesting that genetic and metabolic changes in these cells may indeed affect nucleolar morphology. Nonetheless, all drugs from a panel of selected hits from different target classes validated in both cancer cell lines (Fig. 2F).

• Figure 2F indicates that DLD1 and HCT116 cells are less sensitive to nucleolar changes induced by several inhibitors, including CDK inhibitors. It would be crucial to correlate these differences with cell viability. Are these differences due to cell-type sensitivity or variations in intracellular drug levels? Assessing cell viability and intracellular drug concentration for the same drugs and cells would provide valuable insights.

One of the reasons for the reduced magnitude of the effects of selected drugs in DLD1 and HCT116 cells is their lower baseline normality scores compared to hTERT cells (now shown in Sup. Fig. 1B-C). Other potential factors include proteomic and metabolic shifts and alterations in signaling pathways that control ribosome production. The less-likely possibility of variations in intracellular drug levels cannot be excluded, but measuring this for every compound in every cell line was not feasible in this study. These limitations are now noted in the results section.

Regarding the point about viability - our initial screen output, in addition to normality scores, included cell count (cumulative count of cells in all imaged fields), which serves as a proxy for viability. By this measure, all hit compounds in our screen were cytostatic or cytotoxic in RPE1 cells (Fig. 2C). The impact of these drugs on the viability of cancer cells that can have various degrees of addiction to ribosome biogenesis merits a separate study of a large cancer cell line panel.

• Have the authors interpreted nucleolar stress as the primary cause of cell death induced by these drugs? When cells treated with CDK inhibitors exhibit the dissociated nucleoli phenotype, is this effect reversible? Is this phenotype indicative of cell death commitment? Conducting a washout experiment to measure the recovery of nucleolar function and cell viability would address these questions.

Whether nucleolar toxicity is the primary cause of cytotoxicity for a given chemotherapy drug is an incisive and thought-provoking question. Our screen did not discern whether the cytotoxic effects of our hits were due to inhibition of their intended targets, their impact on the nucleolus, or a combined effect. This point is now mentioned in the results section. Regarding the reversibility of the nucleolar disassembly phenotype seen in CDK inhibitors –in the case of flavopiridol, which is a reversible CDK inhibitor, we demonstrated that nucleoli re-assembled within 4-6 hours after the drug was washed out. An example of this is shown in Sup. Figure 3 and in Video 5. For these experiments, cells were pretreated with the drug for 5 hours, not long enough to cause cell death.

• The correlation between the loss of Treacle phosphorylation and nucleolar stress upon CDK inhibition is intriguing. However, it remains unclear how these two events are related. Would Treacle knockdown yield the same nucleolar phenotype as CDK inhibition? Moreover, would point mutations that abolish Treacle phosphorylation prevent its interaction with Pol-I? Experiments addressing these questions would enhance our understanding of the correlation/causation between Treacle phosphorylation and the effects of CDK inhibition on nucleolar stress.

We agree that the Treacle finding is interesting and warrants further investigation. In our attempts to knock down Treacle with siRNA, its protein levels were reduced by no more than 50%, which was not sufficient to cause a strong nucleolar stress response. Therefore, these data were not incorporated into the manuscript. However, in our view, Treacle is unlikely to be the only nucleolar CDK substrate whose dephosphorylation is causing the “bare scaffold” phenotype caused by the transcriptional CDK inhibitors. Our phospho-proteomics studies identified multiple nucleolar CDK substrates with established roles in the formation of the nucleolus. For instance, the granular component protein Ki-67 was also dephosphorylated on multiple sites and dispersed throughout the nucleus (shown in Sup. Fig 4). Given that CDKs typically phosphorylate many substrates that can have multiple phosphorylation sites, identifying a sole protein or phosphorylation site responsible for nucleolar disassembly may be an unattainable target.

Overall, this study is significant and novel as it sheds light on the importance of nucleolar stress in defining the on-target and off-target effects of chemotherapy in normal and cancer cells.

Thank you, we appreciate the positive and constructive assessment of our study.

Reviewer #2 (Public Review):

This is an interesting study with high-quality imaging and quantitative data. The authors devise a robust quantitative parameter that is easily applicable to any experimental system. The drug screen data can potentially be helpful to the wider community studying nucleolar architecture and the effects of chemotherapy drugs. Additionally, the authors find Treacle phosphorylation as a potential link between CDK9 inhibition, rDNA transcription, and nucleolar stress. Therefore I think this would be of broad interest to researchers studying transcription, CDKs, nucleolus, and chemotherapy drug mechanisms. However, the study has several weaknesses in its current form as outlined below.

1) Overall the study seems to suffer from a lack of focus. At first, it feels like a descriptive study aimed at characterizing the effect of chemotherapy drugs on the nucleolar state. But then the authors dive into the mechanism of CDK inhibition and then suddenly switch to studying biophysical properties of nucleolus using NPM1. Figure 6 does not enhance the story in any way; on the contrary, the findings from Fig. 6 are inconclusive and therefore could lead to some confusion.

This study was specifically designed to examine a broad range of chemotherapy drugs. The newly created nucleolar normality score enabled us to measure nucleolar stress precisely and in high throughput. Our primary objective was to find drugs that disrupt the normal nucleolar morphology and then study in-depth the most interesting and novel hits. We have made revisions to emphasize that these are the primary focal points of the manuscript.

As context, we were motivated to explore the biophysical properties of the nucleolus because they are thought to underlie its formation and function, which also suggested a potential predictive value for modeling nucleolar responses to drug treatments. For this, we edited the RPE1 cell line by endogenously tagging NPM1, a granular component protein that behaves in line with the phase-separation paradigm in vitro and when over-expressed. We fully expected to confirm that its behavior in vivo would be consistent with LLPS, but instead found that even in an untreated scenario, the dynamics of endogenous NPM1 could not be fully explained by the phase separation theory (Fig. 6 A-C). Our message is that accurately predicting drug responses using the nucleolar normality score as a readout, based on our current understanding of the biophysical forces governing nucleolar assembly, is unworkable. For instance, normality scores decrease and NPM1 dynamics increase radically when CDKs are inhibited, without changes in NPM1 concentration or concentrations of other protein components (Fig.6 E-H). These observations are important because they highlight our gaps in understanding the relative contribution of phase separation versus active assembly in nucleolar formation. We believe that these observations are worth sharing with the scientific community.

2) The justification for pursuing CDK inhibitors is not clear. Some of the top hits in the screen were mTOR, PI3K, HSP90, Topoisomerases, but the authors fail to properly justify why they chose CDKi over other inhibitors.

We decided to focus on CDK inhibitors for several reasons. First, their effects were completely new and unexpected, suggesting the existence of an unknown mechanism regulating nucleolar structure and function. In addition, CDK inhibitors caused a very strong and distinct nucleolar stress phenotype with the lowest normality scores that merited its own term, the “bare scaffold” phenotype. One more reason for pursuing CDK-inhibiting drugs was their high rate of failure in clinics because of the intense and hard-to-explain toxicity. We suspect that this toxicity may be due at least in part to their profound effect on nucleolar organization and ribosome production throughout the body. We stated this rationale more explicitly in the manuscript.

3) In addition to poor justification, it seems like a very superficial attempt at deciphering the mechanism of CDK9imediated nucleolar stress. I think the most interesting part of the study is the link between CDK9, Pol I transcription, and nucleolar stress. But the data presented is not entirely convincing. There are several important controls missing as detailed below.

We agree with the reviewer that follow-up studies of CDK9, Pol I, and nucleolar stress connection are important long-term goals. However, the primary objective of this study was to ascertain the scope of anticancer agents that can cause nucleolar stress and the establishment of nucleolar stress categories. This is an important advance and could serve as the foundation for a standalone in-depth study or multiple studies. We have included the complete screen, proteomics, and phospho-proteomics results (Sup. Tables 1, 2, and 3), which will enable other investigators to mine the screen information based on their specific interests. Furthermore, we have made multiple text revisions to clarify rationale and interpretation, and incorporated additional data that strengthen the manuscript.

4) The authors did not test if inhibition of CDK7 and/or CDK12 also induces nucleolar stress. CDK7 and CDK12 are also major kinases of RNAPII CTD, just like CDK9. Importantly, there are well-established inhibitors against both these kinases. It is not clear from the text whether these inhibitors were included in the screen library.

Our anticancer compound library contained CDK7 inhibitor THZ1⦁2HCL, and it was a hit at both 1 and 10 uM concentrations (Sup. Table 1). However, its nucleolar stress phenotype was morphologically distinct from CDK9 inhibitors, resembling the stress caps phenotype instead of the bare scaffold phenotype. We did not pursue CDK7 because of its two hard-to-separate functions: in addition to its role as an RNAPII CTD kinase, it also acts as a CDK-activating kinase (CAK) by promoting the associations of multiple CDKs with their cyclin partners. This dual role of CDK7 makes the interpretation of THZ1-induced nucleolar stress phenotype difficult because it could be attributed to either or both of these functions. Moreover, it was reported to cause DNA damage, which may explain why it causes stress caps. An image depicting nucleolar stress phenotype caused by THZ1⦁2HCL is provided in Author response image 1.

Author response image 1.

Control and THZ1 - treated RPE1 cells, images from screen plates.

We are not aware of specific inhibitors of CDK12, as they also reportedly inhibit CDK13. None of the CDK12/CDK13 inhibitors were present in our library, therefore we can neither confirm nor exclude the possible involvement of these kinases in regulating nucleolar structure. Many other existing CDK inhibitors were absent from our library. Our work highlights the importance of assessing their potential to induce nucleolar stress and offers an approach for this assessment.

5) In Figure 4E, the authors show that Pol I is reduced in nucleolus/on rDNA. The authors should include an orthogonal method like chromatin fractionation and/or ChIP

We acknowledge the reviewer’s request for additional validation of reduced occupancy of rDNA by Pol I.<br /> Nucleolar chromatin fractionation in cells treated with CDK inhibitors is unlikely to work due to nearly complete nucleolar disassembly. Chromatin immunoprecipitation would require finding and validating a suitable ChIP-grade antibody. Moreover, the evaluation of repetitive regions by ChIP is non-trivial and error-prone. To help address this request and further confirm the POLR1A immunofluorescence results in 4E, we included additional immunofluorescence data obtained with a different POLR1A antibody (Sup. Fig. 3D), and the results were similar.

6) In Fig. 5D, in vitro kinase lacks important controls. The authors should include S to A mutants of Treacle S1299A/S1301A to demonstrate that CDK9 phosphorylates these two residues specifically.

7) To support their model, the authors should test if overexpression of Treacle mutants S1299A/S1301A can partially phenocopy the nucleolar stress seen upon CDK9 inhibition. This would considerably strengthen the author's claim that reduced Treacle phosphorylation leads to Pol I disassociation from rDNA and consequently leads to nucleolar stress.

8) Additionally, it would be interesting if S1299D/S1301D mutants could partially rescue CDK9 inhibition.

Points (6-8):

We reiterate that transcriptional CDKs target multiple nucleolar proteins, and the observed phenotype might be due to the combined effects of de-phosphorylation of multiple substrates. We concur that deconstructing the role of Treacle phosphorylation sites is very interesting and warrants further in-depth studies. The phospho-proteomics enrichment method, while an effective first-pass strategy, might not capture 100% of the phosphorylated sites. Treacle is a phospho-protein with an abundance of serine and threonine residues. It could potentially have been selectively dephosphorylated on more sites than were detected by this method. Therefore, the suggested mutations may not be the exclusive contributors responsible for the functional phenotype. Additionally, overexpressing Treacle impairs the viability of RPE1 cells, complicating the interpretation of experiments involving overexpression of both wild-type and mutant proteins. A conceivable strategy would involve generating phosphomimetic and non-phosphorylatable mutants by gene editing, studying their interactions by biochemical approaches, and determining their impact on nucleolar function, but this may take years of additional work. We hope that our work will inspire further studies that explore Treacle phosphorylation and other functions of transcriptional CDKs in nucleolar formation.

Thank you for the thoughtful review and suggestions.

Reviewer #2 (Recommendations For The Authors):

1) The manuscript could be re-organized to focus on 'CDK9-Treacle-Pol I-nucleolar stress' as the central part of the story.

While we acknowledge this suggestion, it's important to emphasize that the primary focus of this manuscript is on the identification of anticancer drugs that induce nucleolar stress and the establishment of nucleolar stress categories.

2) Include a "no ATP" control in the in vitro kinase assay and indicate molecular sizes.

We provided an additional kinase assay (Sup. Fig. 4B) that includes no ATP control lanes and a fragment of a Coomassie blue stained gel showing molecular weight markers. No ATP control assays (lanes 4 and 5) were blank as expected. Molecular weight markers were added to all other kinase assays based on the known sizes of isolated Pol II holoenzyme subunits Rbp1 (191 kDa) and Rbp2 (138 kDa).

3) For in vitro phosphorylation, please provide an explanation for using CDK9/cyclin K instead of Cyclin T1 which is the predominant cyclin for CDK9

Recombinant CDK9/cyclin K complex was used for in vitro kinase assays for a technical reason: CDK9/cyclin T obtained from the same vendor appeared to be low quality, as it showed only minimal activity toward our positive control, the isolated Pol II complex. The kinase assays using recombinant CDK9/cyclin T in parallel with CDK9/cyclin K are now presented it Sup. Fig. 4B. The first two assays in this experiment contained Pol II as a substrate, and it is evident that Pol II was phosphorylated much stronger by CDK9/cyclin K than CDK9/cyclin T (comparing lane 1 vs lane 2). Therefore, the lack of detectable Treacle phosphorylation by CDK9/Cyclin T (lane 7), in contrast to strong phosphorylation by CDK9/cyclin K (lane 6), was likely attributable to poor reagent quality rather than physiological differences. We can conclude that CDK9/cyclin K reliably phosphorylates Treacle in vitro, but CDK9/cyclin T kinase assays were inconclusive.

eLife assessment

This study and associated data is compelling, novel, important, and well-carried out. The study demonstrates a novel finding that different chemotherapeutic agents can induce nucleolar stress, which manifests with varying cellular and molecular characteristics. The study also proposes a mechanism for how a novel type of nucleolar stress driven by CDK inhibitors may be regulated. The study sheds light on the importance of nucleolar stress in defining the on-target and off-target effects of chemotherapy in normal and cancer cells.

Joint Public Review:

Summary:

This is an interesting study with high quality imaging and quantitative data. The authors devise a robust quantitative parameter that is easy applicable to any experimental system. The drug screen data can potentially be helpful to the wider community studying nucleolar architecture and effects of chemotherapy drugs. Additionally, the authors find Treacle phosphorylation as a potential link between CDK9 inhibition, rDNA transcription and nucleolar stress. Therefore I think this would be of broad interest to researchers studying transcription, CDKs, nucleolus and chemotherapy drug mechanisms.

Revised manuscript:

While most of my concerns related were addressed, a PolI ChIP on rDNA would be an important experiment to establish the relevance of some of the conclusions of the paper using well established protocols with validated antibodies for PolI ChIP. Furthermore, additional S to A mutants of Treacle S1299A/S1301A is an important control which could have provided evidence if indeed S1299/S1301 were the only sites being phosphorylated by CDK9. To support their model, the authors should test if overexpression of Treacle mutants S1299A/S1301A can partially phenocopy the nucleolar stress seen upon CDK9 inhibition. This would considerably strengthen the author's claim that reduced Treacle phosphorylation leads to Pol I disassociation from rDNA and consequently leads to nucleolar stress. If not, it would have strengthened the authors' argument that Treacle could have multiple sites targeted by CDK9 and that mutating any one or two may not be sufficient to cause disassociation from PolI.

Overall, I believe the primary conclusions regarding the impact of various chemotherapy drugs on nucleolar state are solid and valuable to the broader scientific community. However, the mechanistic exploration of CDK9i is not sufficiently developed, and the authors have not adequately addressed the feedback provided in the original manuscript.

eLife assessment

This manuscript describes valuable findings on the expression pattern of orexin receptors in the midbrain and how manipulating this system influences several behaviors, such as context-induced locomotor activity and exploration. The overall strength of evidence - which includes anatomical, viral manipulation studies, and brain imaging - is solid and broadly supports claims in the paper, however, there are several areas in which the conclusions are only partially supported by the data provided. These results have implications for understanding the neural underpinnings of reward and will be of interest to neuroscientists and cognitive scientists with an interest in the neurobiology of reward.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, the role of orexin receptors in dopamine neurons is studied. Considering the importance of both orexin and dopamine signalling in the brain, with critical roles in arousal and drug seeking, this study is important to understand the anatomical and functional interaction between these two neuromodulators. This work suggests that such interaction is direct and occurs at the level of SN and VTA, via the expression of OX1R-type orexin receptors by dopaminergic neurons.

Strengths:<br /> The use of a transgenic line that lacks OX1R in dopamine-transporter-expressing neurons is a strong approach to dissecting the direct role of orexin in modulating dopamine signalling in the brain. The battery of behavioural assays to study this line provides a valuable source of information for researchers interested in the role of orexin-A in animal physiology.

Weaknesses:<br /> The choice of methods to demonstrate the role of orexin in the activation of dopamine neurons is not justified and the quantification methods are not described with enough detail. The representation of results can be dramatically improved and the data can be statistically analysed with more appropriate methods.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript examines the expression of orexin receptors in the midbrain - with a focus on dopamine neurons - and uses several fairly sophisticated manipulation techniques to explore the role of this peptide neurotransmitter in reward-related behaviors. Specifically, in situ hybridization is used to show that dopamine neurons predominantly express the orexin receptor 1 subtype and then go on to delete this receptor in dopamine neurons using a transgenic strategy. Ex vivo calcium imaging of midbrain neurons is used to show that in the absence of this receptor orexin is no longer able to excite dopamine neurons of the substantia nigra.

The authors proceed to use this same model to study the effect of orexin receptor 1 deletion on a series of behavioral tests, namely, novelty-induced locomotion and exploration, anxiety-related behavior, preference for sweet solutions, cocaine-induced conditioned place preference, and energy metabolism. Of these, the most consistent effects are seen in the tests of novelty-induced locomotion and exploration in which the mice with orexin 1 receptor deletion are observed to show greater levels of exploration, relative to wild-type, when placed in a novel environment, an effect that is augmented after icv administration of orexin.

In the final part of the paper, the authors use PET imaging to compare brain-wide activity patterns in the mutant mice compared to wildtype. They find differences in several areas both under control conditions (i.e., after injection of saline) as well as after injection of orexin. They focus on changes in the dorsal bed nucleus of stria terminalis (dBNST) and the lateral paragigantocellular nucleus (LPGi) and perform analysis of the dopaminergic projections to these areas. They provide anatomical evidence that these regions are innervated by dopamine fibers from the midbrain, are activated by orexin in control, but not mutant mice, and that dopamine receptors are present. Thus, they argue these anatomical data support the hypothesis that behavioral effects of orexin receptor 1 deletion in dopamine neurons are due to changes in dopamine signaling in these areas.

Strengths:<br /> Understanding how orexin interacts with the dopamine system is an important question and this paper contains several novel findings along these lines. Specifically:<br /> (1) The distribution of orexin receptor subtypes in VTA and SN is explored thoroughly.<br /> (2) Use of the genetic model that knocks out a specific orexin receptor subtype from only dopamine neurons is a useful model and helps to narrow down the behavioral significance of this interaction.<br /> (3) PET studies showing how central administration of orexin evokes dopamine release across the brain is intriguing, especially since two key areas are pursued - BNST and LPGi - where the dopamine projection is not as well described/understood.

Weaknesses:<br /> The role of the orexin-dopamine interaction is not explored in enough detail. The manuscript presents several related findings, but the combination of anatomy and manipulation studies does not quite tell a cogent story. Ideally, one would like to see the authors focus on a specific behavioral parameter and show that one of their final target areas (dBNST or LPGi) was responsible or at least correlated with this behavioral readout. In addition, some more discussion on what the results tell us about orexin signaling to dopamine neurons under normal physiological conditions would be very useful. For example, what is the relevance of the orexin-dopamine interaction blunting novelty-induced locomotion under wildtype conditions?

In some places in the Results, insufficient explanation and reporting is provided. For example, when reporting the behavioral effects of the Ox1 deletion in two bottle preference, it is stated that "[mutant] mice showed significant changes..." without stating the direction in which preference was affected.

The cocaine CPP results are difficult to interpret because it is unclear whether any of the control mice developed a CPP preference. Therefore, it is difficult to conclude that the knockout animals were unaffected by drug reward learning. Similarly, the sucrose/sucralose preference scores are also difficult to interpret because no test of preference vs. water is performed (although the data appear to show that there is a preference at least at higher concentrations, it has not been tested).

Author Response

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

Reviewer #1 (Public Review):

The manuscript by Muthana et al. describes the effect of injection of an antibody specific for human CTLA4 conjugated to a cytotoxic molecule (Ipi-DM1) in knock-in mice expressing human CTLA4. The authors show that Ipi-DM1 administration causes a partial decrease (about 50% in absolute number) of mature B cells in blood and bone marrow 9-14 days after the beginning of treatment. Ipi-DM1 also results in a partial decrease in Foxp3+ Tregs (about 40% in absolute number) and a slight increase in activation of conventional T cells (Tconvs) in the blood at D9. Tconv depletion, CTLA4-Ig or anti-TNF mAb partially prevents the effect of ipi-DM1 on B cells. This work is interesting but has the following major limitations:

1) This work could have been of more interest if the Ipi-DM1 molecule would be used in the clinic. As this is not the case, the intimate mechanism of the effect of this molecule in mice is of reduced interest.

The goal of the current study is to use Ipi-DM1 ADC as probe to study mechanism of B cell loss observed in Treg-deficient host.

2) The fact that a partial deletion of Tregs is associated with activation of Tconvs and a decrease in B cells has been published several times and is therefore not new. According to the authors, their work would be the first to show that activation of Tconvs would lead to B cell depletion. However, this is shown in an indirect way and the mechanisms are not really elucidated. Indeed, this work shows a correlation between an increase in Tconv activation and a decrease in the number of B cells in the blood. The experiments to try to show a causal link are of 2 types: deletion of T cells (Fig 4) and blocking T cell activation with CTLA4-Ig (Fig 5) (neutralization of TNF addresses another question). Neither of these 2 experiments is totally convincing. Indeed, the absence of B cell depletion when T cells are deleted can be explained by other mechanisms than the preservation of B cell destruction by activated T cells. The phenomenon could be explained by B cell recirculation to lymphoid tissues or an effect of massive T cell death for example. The experiment shown in Fig. 5 with Belatacept is more convincing because this time the effect is targeted to activated T cells only. However, the prevention of B cell ablation is only partial. Again, since only blood is analyzed, other mechanisms could explain the B cell loss, such as their recirculation in lymphoid tissues.

While the concept of treg depletion leads to activation of Tconv cells and reduced B cells has been previously published, B cell loss was explained on basis of defective B cell lymphopoiesis due to low production of stroma cell-derived IL-7 or destruction of stromal cells by effector T cells. Our new data established that loss of B cells in the context of Treg depletion was not due to defects in the number of pre-/pro-B cells. Rather it is the death of mature B cells in the bone marrow.

To address the reviewer’s concern that the B cell loss was merely caused by a change in circulating pattern, we performed a new study on the effect of the ADC on B cells in bone marrow. Our new data reveal loss of mature bone marrow B cells, and that such loss is associated with increased apoptosis of mature B cells. Therefore, the loss of B cells in the peripheral blood is not due to a changed circulation. Furthermore, our data show that B cell progenitor, Pre-B, cells are not changed. Therefore, B cell lymphopoiesis is not the reason for B cell loss in our model system.

3) It is disappointing that only the blood (and sometimes the bone marrow) was studied in this work. The interest of doing experiments in mice is to have access to many tissues such as the spleen, lymph nodes, colon, lung, and liver. To conclude that there is B cell deletion without showing lymphoid organs (where the majority of B cells reside) is insufficient. As discussed above, the drop in B cells in the blood could be due to their recirculation in lymphoid organs. In addition, there is no measurement of functional B cells activity. Do mice treated with Ipi-DM1 have a decreased ability to develop an antibody response following immunization?

We have analyzed lymph nodes and spleen at the same time points. Unfortunately, Treg depletion was no longer observed at these time points. As expected, we did not see a clear depletion of B cells (Figure 1-figure supplement 6). In regards to functional B cell activity, we observed an increase of plasma immunoglobulins especially IgE which are now shown in Figure 3-figure supplement 1.

4) Although it is difficult to study in vivo, there is not a single evidence of increased B cell death after injection of Ipi-DM1.

Figure 2 & Figure 2-supplement 1 provides B cell death comparisons between IpiDM1 and hIgGFc group for bone marrow, blood, spleen, and lymph nodes. Statistically significant increase in B cell death is observed in mature B cells in bone marrow.

5) In most of the experiments, B cells are quantified with the B220 marker alone, but this marker, in some cases, can be expressed by other cells. It would have been preferable to use a marker more specific to B cells such as CD19 for example.

We have added data to support the death of mature B cells using other markers.

Minor points.

1) It should be indicated whether human CTLA4 binds normally to mouse CD80 CD86. We do not know if knock-in mice with human CTLA4 have a fully functional immune system.

We have indicated this point as suggested and cited our previous work line 226-227 (ref 23 & 24)

2) The manuscript is too long. Some of the data in the figures should be moved to supplemental figures. This is the case, for example, for some trivial stainings (Fig 1F, Fig 4B, 4F, Fig 5A, D, F, G). The figure legends and the Materials and Methods section are far too long. On the other hand, Fig 5-Fig Sup 1 could go into the main figures.

The figure legends, materials, and methods may be too long, but our intention is to provide as much info as possible for others who may be interested in our model system.

3) The anti-CTLA4 ADC reagent should be better explained and defined in the text.

The anti-CTLA-4 ADC reagent synthesis described in materials/methods under “Antibody-drug conjugate preparation.”

Reviewer #2 (Public Review):

Despite the fact that CTLA-4 is a critical molecule for inhibiting the immune response, surprisingly individuals with heterozygous CTLA-4 mutations exhibit immunodeficiency, presenting with antibody deficiency secondary to B cell loss. Why the loss of a molecule that regulates T cell activation should lead to B cell loss has remained unclear. In this study, Muthana and colleagues use an anti-CTLA-4 antibody drug conjugate (aCTLA-4 ADC) to delete cells expressing high levels of CTLA-4, and show that this leads to a reduction in B cells. The aCTLA-4 ADC is found to delete a subset of Tregs, leading to hyperactivation of T cells that is associated with B cell depletion. Using blocking antibodies, the authors implicate TNFa in the observed B cell loss.

The reciprocal regulation of T and B cell homeostasis is an important research area. While it has been shown that Treg defects are associated with B cell loss, the mechanisms at play are incompletely understood. CTLA-4 is not normally expressed in B cells so an indirect mechanism of action is assumed. The authors show that the decrease in Treg following aCTLA-4 ADC treatment is associated with activation of T cells, and that B cell loss is blunted if T cells are depleted. A role for both CD4 and CD8 T cells is identified by selective CD4/CD8 depletion. T cells appear to require CD28 costimulation in order to mediate B cell loss, since the response is partially inhibited in the presence of the costimulation blockade drug belatacept (CTLA-4-Ig). Finally, experiments using the anti-TNFa antibody adalimumab suggest a potential role for TNFa in the depletion of B cells.

While the manuscript makes a useful contribution, a number of questions remain. Perhaps most important is the extent to which this model mimics the natural situation in individuals with CTLA-4 mutations (or following CTLA-4-based clinical interventions). aCTLA-4 ADC treatment permits acute deletion of Treg expressing high levels of CTLA-4, whereas in patients the Treg population remains but is specifically impaired in CTLA-4 function. Secondly, although the requirement for T cells to mediate B cell loss is convincingly demonstrated, the incomplete reversal by TNFa blockade suggests additional unidentified factors contribute to this effect. Finally, although the manuscript favours peripheral killing of mature B cells over alterations to B cell lymphopoiesis, one concern is that this may simply reflect the model employed: the shortterm (6 day) treatment used here may be too acute to alter B cell development, but this may nevertheless be a feature of prolonged immune dysregulation in humans.

We appreciate reviewer’s comments and the difference between short-term depletion and permanent inactivation of Treg by genetic mutation is discussed. We would note that apart from mutation, dynamic Treg perturbation does occur under autoimmune conditions. Therefore, our data have significant implications for T-B cell interactions.

TNF-alpha is implicated in B cell loss as evidenced by the partial rescue with Anti-TNF treatment. We did not try to exclude the possibility that other mechanisms are involved.

Our data shows loss of circulating B cell in peripheral blood and mature bone marrow B cells. B cell progenitor, Pre-B, cells are not changed due Ipi-DM1 induced treg impairment, therefore B cell lymphopoiesis is not the reason for B cell loss in our model system. Evidence of increased cell death is only observed in mature B cells (Figure 2).

1) Following aCTLA-4 ADC treatment, it is surprising how subtle the deletion of Treg is (from ~8% to ~7%, Fig 1G), compared to the marked deletion of CTLA-4-expressing CHO cells. Is this a feature of in vivo versus in vitro treatment? If Treg are treated in vitro is deletion more efficient? How does the expression level of CTLA-4 in the CHO cells compare with the Treg in these assays?

We appreciate reviewer’s comments. The anti-CTLA-4 ADC targets CTLA-4 on cell surface. On average about 5% of Tregs express surface CTLA-4 at given moment while human CTLA-4 expressing CHO cell line stains > 90%. Nevertheless, Treg cell number in peripheral blood is reduced by >40%. Additionally, we have included bone marrow data, which shows a greater percentage of Treg depletion (Figure 1J).

2) The decrease in CTLA-4 seen after ipi-DM1 is complicated by the fact that the control DM1 conjugate (IgG1-DM1) appears to significantly increase CTLA-4 expression (Fig 1 supplement 2). It would be useful to clarify when hIgGFc is used versus hIgGFc-DM1 given the additional complexity introduced here (comparisons lacking a payload differ in more than one variable, while the hIgGFc-DM1 is clearly not inert).

We appreciate reviewer’s comments. We agree that the hIgGFc-DM1 control slightly increased CTLA-4 level; nevertheless, it did not alter B cells, T cells or their proliferation capacity when compared to hIgGFc. Our point here is that B cell depletion is not mediated by DM1 payload off target release (new-version Figure 1-Figure supplement 4, old version Figure 1-figure supplement 2). As for the clarification comment when hIgGFc is used versus hIgGFcDM1 is used, the information is clarified in the figure legend. Comparisons are made between (hIgGFc VS Ipi-DM1) or (hIgGFc VS hIgGFc-DM1).

3) T cell-derived IFNg is another potential contender for influencing B cell homeostasis - have you considered testing whether this also contributes in your model?

We appreciate reviewer’s suggestion. IFN was reported to induce apoptosis and cell arrest in Pre- B cells, however these studies are invitro studies Garvey et.al Immunology. 1994 Mar; 81(3): 381–388; Grawunder et.al Eur. J. Immunol. 23, 544–551. Since we did not observe any effect on Pre-B cells, we have not followed the literature to investigate the role of IFNy in B cell loss in our model.

Reviewer #3 (Public Review):

The co-suppressive molecule CTLA-4 has a critical role in the maintenance of peripheral tolerance, primarily by Treg mediated control of the co-stimulatory molecules CD80 and CD86. As stated by the authors, previous studies have found a variety of effects of anti-CTLA-4 antibody treatment or genetic loss of CTLA-4 on B-cells. These include increased B-cell activation and antibody production, autoantibody production, impairment of B-cell production in the bone marrow and loss of peripheral B-cells. In this article Muthana et al use a CTLA-4 humanized mouse model and examine the effects of drug conjugated CTLA-4 on the immune system. They observe a transient loss of B-cells in the blood of the treated mice. They then use a range of immune interventions such as T-cell depletion and blocking antibodies to demonstrate that this effect is dependent on T-cell activation.

Since anti-CTLA-4 immunotherapy is in active clinical use exploration of its effects are welcome, this is helped by the use of a humanized CTLA-4 system which should be considered a strength of the paper. However, currently, the central premise of this paper, that B-cells are depleted, seems underexplored. Direct evidence of T-cell killing of B-cells is never presented, rather it is inferred from the reduced numbers of B-cells in the blood. The status of B-cells in sites that contain a large proportion of B-cells such as the spleen and lymph nodes is not examined. Additionally, no examination of B-cell antibody production is performed.

We appreciate reviewer’s comments. To address the reviewer’s concerns we performed additional experiments to evaluate the impact on B cells in other organs, as detailed in our responses to specific questions.

1) Examination of B-cell apoptosis/cell death and T-cell mediated cytotoxicity is needed. The authors repeatedly refer to auto destructive T-cells without ever demonstrating their presence or any direct evidence that B-cells are dying. This is particularly important in the context of the blood since an alternative hypothesis would be a change in B cell trafficking and infiltration into tissues.

We appreciate reviewer’s comments. To address the reviewer’s concern that B cell loss in blood might be caused by a change in B cell trafficking pattern. We performed new study on the effect of the ADC on B cells in bone marrow. Our new data reveal loss of mature bone marrow B cells, and that such loss is associated with increased apoptosis of mature B cells (Figure 2). Therefore, the loss of B cells in the peripheral blood is not due to B cell trafficking and infiltration into tissues.

2) The authors demonstrate that B-cells are mostly reduced in blood at around days 10 to 15, I believe it is critical to determine if this is also reflected in the lymphoid organs such as the spleen and lymph nodes.

We appreciate reviewer’s comments. We have analyzed lymph node and spleen at the same time points. Unfortunately, Treg depletion was no longer observed at these time points. As expected, we did not see a clear depletion of B cells (Figure 1-figure supplement 6).

3) Related to the above point do the authors see evidence of Splenomegaly or lymphadenopathy?

We appreciate reviewer’s comment. Evidence of splenomegaly and lymphadenopathy is presented in Figure 3-figure supplement 2.

4) Minimal examination of the status of the B-cells or antibody production is performed. Previous reports would suggest that plasma cell induction and antibody responses may be expected. Do serum antibody levels change in this system?

We appreciate reviewer’s comment. Increases of plasma immunoglobulins especially IgE are now shown in Figure 3-figure supplement 1.

5) Its unclear how the authors interpret their experiment with anti-TNFa (figure 6). Are they suggesting that TNFa itself depletes B-cells or that it is part of the inflammatory milieu that contributes to wider T-cell activation and, in turn, B-cell depletion?

We have discussed these possibilities in the revised manuscript.

Reviewer #1 (Public Review):

The manuscript by Muthana et al. describes the effect of injection of an antibody specific for human CTLA4 conjugated to a cytotoxic molecule (Ipi-DM1) in knock-in mice expressing human CTLA4. The authors show that Ipi-DM1 administration causes a partial decrease (about 50% in absolute number) of mature B cells in blood and bone marrow 9-14 days after the beginning of treatment. B cell progenitors and pre-B cells in the BM are not affected. Ipi-DM1 also results in a partial decrease in Foxp3+ Tregs (about 40% in absolute number) and a slight increase in activation of conventional T cells (Tconvs) in the blood, spleen, BM and LNs at D9 as well as increased plasma immunoglobulins especially IgE. Tconv depletion, CTLA4-Ig or anti-TNF mAb partially prevents the effect of ipi-DM1 on B cells. This effect of Ipi-DM1 on the reduction B cells and Tregs at D9 is not observed in the spleen and lymph nodes (maybe not the good timing to see it), and there is even an increase in the number of Treg and the frequency and number of B cells in lymph nodes. This work is interesting but has the following major limitations:

1- This work could have been of more interest if the Ipi-DM1 molecule would be used in the clinic. As this is not the case, the intimate mechanism of the effect of this molecule in mice is of reduced interest.

2- The fact that a partial deletion of Tregs is associated with activation of Tconvs and a decrease in B cells is not new. According to the authors, their work would be the first to show that activation of Tconvs would lead to B cell death. However, this is shown in an indirect way and the mechanisms are not really elucidated. The experiments to try to show a causal link are of 2 types: deletion of T cells (Fig 5) and blocking T cell activation with CTLA4-Ig (Fig 6). These 2 experiments are not fully convincing. The absence of B cell depletion in the blood when T cells are deleted can be explained by other mechanisms, such as B cell recirculation to lymphoid tissues or an effect of massive T cell death for example. The experiment with CTLA4-Ig is more convincing because the effect is targeted to activated T cells only. However, the prevention of B cell ablation is only partial. Since only blood is analyzed, other mechanisms could explain the B cell loss, such as their recirculation in lymphoid tissues.

3- The authors propose that the drop in B cell numbers in the blood in mice treated with Ipi-DM1 results from reduced mature B cells in the bone marrow. However, B cells are continuously recirculating between the blood and secondary lymphoid tissues. The drop of blood B cells could be well explained by an increased recirculation to lymphoid organs. The increased numbers of B cells in lymph nodes support this latter hypothesis.

4- The new Figure 2 suggests direct evidence of apoptosis of mature B cells in the BM of treated mice using a PI/annexin V staining assay. This is an important point to support the point of the manuscript. However, using the same assay, the level of B cell apoptosis is of 80% in lymph nodes and 50% in the spleen in control mice (see new Figure 2-figure supplement 1), which is way too high and questions the reliability of this assay. It is likely that B cells enter apoptosis only in vitro due to some artefactual stress.

Reviewer #2 (Public Review):

Despite the fact that CTLA-4 is a critical molecule for inhibiting the immune response, surprisingly individuals with heterozygous CTLA-4 mutations exhibit immunodeficiency, presenting with antibody deficiency secondary to B cell loss. Why the loss of a molecule that regulates T cell activation should lead to B cell loss has remained unclear. In this study, Muthana and colleagues use an anti-CTLA-4 antibody drug conjugate (aCTLA-4 ADC) to delete cells expressing high levels of CTLA-4, and show that this leads to a reduction in B cells. The aCTLA-4 ADC is found to delete a subset of Tregs, leading to hyperactivation of T cells that is associated with B cell depletion. Using blocking antibodies, the authors implicate TNFa in the observed B cell loss.

The reciprocal regulation of T and B cell homeostasis is an important research area. While it has been shown that Treg defects are associated with B cell loss, the mechanisms at play are incompletely understood. CTLA-4 is not normally expressed in B cells so an indirect mechanism of action is assumed. The authors show that the decrease in Treg following aCTLA-4 ADC treatment is associated with activation of T cells, and that B cell loss is blunted if T cells are depleted. A role for both CD4 and CD8 T cells is identified by selective CD4/CD8 depletion. T cells appear to require CD28 costimulation in order to mediate B cell loss, since the response is partially inhibited in the presence of the costimulation blockade drug belatacept (CTLA-4-Ig). Finally, experiments using the anti-TNFa antibody adalimumab suggest a potential role for TNFa in the depletion of B cells.

While the manuscript makes a useful contribution, a number of limitations remain. Perhaps most important is the extent to which this model mimics the natural situation in individuals with CTLA-4 mutations (or following CTLA-4-based clinical interventions). aCTLA-4 ADC treatment permits acute deletion of Treg expressing high levels of CTLA-4, whereas in patients the Treg population remains but is specifically impaired in CTLA-4 function. Secondly, although the requirement for T cells to mediate B cell loss is convincingly demonstrated, the incomplete reversal by TNFa blockade suggests additional unidentified factors contribute to this effect. Finally, although the manuscript favours peripheral killing of mature B cells over alterations to B cell lymphopoiesis, one concern is that this may simply reflect the model employed: the short-term (6 day) treatment used here may be too acute to alter B cell development, but this may nevertheless be a feature of prolonged immune dysregulation in humans.

Reviewer #3 (Public Review):

The co-suppressive molecule CTLA-4 has a critical role in the maintenance of peripheral tolerance, primarily by Treg mediated control of the co-stimulatory molecules CD80 and CD86. As stated by the authors, previous studies have found a variety of effects of anti-CTLA-4 antibody treatment or genetic loss of CTLA-4 on B-cells. These include increased B-cell activation and antibody production, autoantibody production, impairment of B-cell production in the bone marrow and loss of peripheral B-cells. In this article Muthana et al use a CTLA-4 humanized mouse model and examine the effects of drug conjugated CTLA-4 on the immune system. They observe a transient loss of B-cells in the blood of the treated mice. They then use a range of immune interventions such as T-cell depletion and blocking antibodies to demonstrate that this effect is dependent on T-cell activation.

Since anti-CTLA-4 immunotherapy is in active clinical use exploration of its effects are welcome, this is helped by the use of a humanized CTLA-4 system which should be considered a strength of the paper. However, currently the central premise of this paper, that B-cells are depleted seems underexplored. Direct evidence of T-cell killing of B-cells is never presented, rather it is inferred from the reduced numbers of B-cells in the blood and increased apoptosis in the bone marrow. It is not made clear if B-cell numbers in the bone marrow are reduced.

Upon examining lymphoid organs it seems that the spleen is relatively unchanged while the lymph nodes have a large increase in B-cells alongside increased serum antibody levels. The paper does underline the importance of looking at the differences of multiple immune compartments and interesting phenomenon are described in each compartment. Simultaneous inhibition of B-cell lymphopoiesis and blood trafficking with strong activation and antibody production of lymphoid resident (presumably germinal center) B-cells appears to be occurring. However the current overall interpretation that B-cells are broadly depleted is perhaps too simplistic and largely ignores the lymphoid organs and serum antibodies.

eLife assessment

This study reports that neural activity in the auditory cortex (field L) of singing male songbirds can be modulated by social context. These potentially important findings indicate that the presence of a female conspecific alters the response of auditory cortical neurons to the male bird's own song and to perturbations of auditory feedback that the bird has been trained to expect. While they extend recent work showing that the activity of dopaminergic neurons in songbirds is also affected by an audience, the evidence presented is incomplete since it is unclear how much of the apparent modulation of cortical neurons may be due to other factors, such as changes in the recorded neurons or their properties over time, which will require additional analyses to work out.

Reviewer #1 (Public Review):

Summary:<br /> This study examines the context-dependent modulation of auditory cortical neurons in response to expected sensory input, either self-generated sounds or expected perturbations of self-generated sounds. Specifically, using songbirds, the authors ask whether social context (the presence of a female conspecific) affects 1) the response of auditory cortical neurons to the bird's own song when he is singing; and 2) the response of neurons to perturbations of auditory feedback that the bird has been trained to expect.

Strengths:<br /> First, the authors report that across the population, the responses of the neurons does not differ when a male bird sings alone or if he sings to a female. A fraction of auditory cortical neurons, however, do show significant differences in the firing rate, precision, and/or degree of burst firing when males sing alone vs. when they sing to females. This finding is broadly consistent with the literature showing that sensory neurons (visual, auditory, somatosensory, etc.) can be rapidly reconfigured into different "information processing modes" depending on behavioral state (e.g, quiescence vs vigilance).

For the perturbation experiments, the authors trained birds to expect distorted auditory feedback during a particular syllable. They found that some neurons showed greater responses during perturbation when a female was present (compared to when males were alone) while other neurons had smaller responses during perturbation when a female was present. In addition, the response of a small number of auditory cortical neurons were not affected by behavioral state. These results contrast with their prior report that the responses of midbrain dopaminergic neurons that project to the basal ganglia are "uniformly reduced" in the presence of a female, raising a question of how an evaluation signal is transformed in the circuit from the primary sensory region to the midbrain.

Weaknesses:<br /> While the experiments and analysis are solid, the finding that social context can alter responses of auditory cortical neurons in a multitude of ways (increase, decrease or no change) raises several questions that can be examined with additional analysis. For example, do context-dependent differences in auditory responses derive from context-dependent differences in the songs? Are context-dependent differences present in all classes of neurons and throughout the auditory system?

The observed heterogeneity in the firing properties of auditory cortical neurons, both in response to self-generated sounds and during perturbations of auditory feedback, raises the question of which neurons are sensitive to social context (which likely can be addressed by the authors in a revision). The authors should provide additional details about the recordings:

a) What are the locations of the recording sites?<br /> Prior work has shown that there is an organized map of spectrotemporal features of sounds in the auditory cortex of songbirds; spectral tuning widths change along the medial-lateral axis and temporal tuning widths differ between the input and output layers of Field L. Were the recordings primarily in Field L2 (thalamo-recipient region), L1 or L3? Were some recordings lateral to Field L in secondary auditory regions? Were the neurons that showed context-dependent changes in firing properties localized or distributed throughout Field L (i.e., were the context-dependent differences in neural responses truly brain-wide)? At a minimum, the authors should include a schematic showing the different regions of Field L and a summary of the location of the recording sites. Images of the processed tissue with electrolytic lesions would also be helpful.

b) Was the context-dependent modulation limited to a particular class of neurons (distinguished by spike waveform shape, spontaneous firing rate, or other feature)?

While the authors attribute differences in the responses of single auditory cortical neurons to the presence of a female, other potential explanations for the observed differences should be examined (and potentially ruled out):

a) Prior work has shown that songs of zebra finches differ slightly when males sing alone compared to when they sing to females: songs are faster; pitch is less variable; and the number of introductory elements is greater when males sing to females. Do some of the observed social context-dependent differences in the responses of auditory neurons reflect differences in the songs in the two conditions? This idea is supported in part by a prior study in juvenile zebra finches (Keller & Hahnloser, 2009) showing that ~20% of the neurons they recorded in Field L and a secondary auditory region (CLM) showed anticipatory activity even before the onset of a song bout, suggesting a source of premotor (or at least non-auditory drive) to neurons in the auditory cortex. Did the authors of this study also find premotor activity in Field L, and if so, did it differ between the two social contexts? Might differences in Field L responses reflect motor/song differences?

b) For the perturbation experiments, the authors report heterogeneous responses to playback, with some neurons firing more and other firing less when a female is present compared to when the male is alone. Keller and Hahnloser (2009) found that in juvenile birds, responses of Field L to perturbations of auditory feedback were sensitive to sound amplitude; perturbation responses increased with relative perturbation amplitude. This raises a question of whether perturbation amplitude is different when a male is alone and when a female is present (i.e., the male may move towards the female when she is present and if the speaker is close to the female, the perturbation may be louder than when the male is alone; alternatively, the male be more active when he is alone so the loudness of the perturbation may be more variable across song bouts). It would be useful to know if (and how much) perturbation amplitude varied depending on the location inside the cage as well as whether the sound pressure level of the underlying song was higher (e.g., Lombard effect). Addition of details of the experimental setup/procedure would help to allay concerns that the amplitude of the white noise varied significantly depending on behavioral context.

Finally, I am still trying to make sense of the differences in the context-dependent modulation of responses of auditory cortical neurons vs. midbrain dopaminergic neurons. Given the heterogeneity of responses in Field L, both to self-generated sounds and to expected perturbations during singing, how are the signals decoded downstream of Field L? At the population level, neither the mean firing rate nor the timing of firing of Field L neurons changed with courtship. Similarly, across the population, the responses to perturbations of auditory feedback were not affected by courtship state (error signal attenuated in 11 neurons, increased in 22 neurons and not affected in 10 neurons). Yet, the courtship state "uniformly" reduces the response of midbrain dopaminergic neurons to auditory perturbation. It would be helpful if the authors could include a model and/or more discussion of how this change may arise.

Reviewer #2 (Public Review):

Summary:

In the manuscript 'Auditory cortical error signals retune during songbird courtship', Jones and Goldberg study auditory cortex in male zebra finches. They explore song-related responses in two different contexts, when the male is either alone or in the presence of a female. Social-context related responses are hypothesized based on previous results on downstream VTA neurons where such modulation is found. They play jamming stimuli through a loudspeaker to probe sensitivity of song-related neural responses to these external stimuli. They find a heterogeneity of responses, in line with auditory cortical neurons computing the social modulation of responses found in VTA.

Strengths:

In general, the work is interesting and sheds light onto auditory processing and self-perception mechanisms in songbirds.

Weaknesses:

Stability of responses has not been studied: some neurons seem to have responses that slowly drift in time, which could lead to observed differences between alone and with-female conditions. Also, possible motor confounds and sound-of-audience confounds should be addressed. The language is often imprecise.

Stability and Reversal: It is a bit unfortunate that stability of effects seemingly has not been studied by reversing experimental conditions. The work would be much stronger if authors could show that audience-dependent tuning is robust in individual cells. Did they record from some neurons during reversal back to the alone condition? Ideally, the responses should be identical before and after recording with an audience. This would control for possible non-stationarities in their neuron recordings/spike-sorting/circadian trends. If authors do not have such data, it would be worth wile to even just try to divide the dataset for each neuron and condition (either the audience or isolate condition) into two parts to verify that the response is the same in either part (provided sufficient song renditions are recorded). See also my comment below about Fig. 2A.

Motor responses: Does DAF playback change song? If so, especially if it applies only in one of the two conditions (audience/no audience), then the observed response differences could be motor-related rather than auditory responses. Analyses of song spectrograms right after DAF would presumably provide the answer.

Similarly, motif-aligned spiking activity was time warped to the median duration of undirected or directed motifs. Could the shorter motifs during directed song (as has been reported in other studies) lead to alignment differences that would account for the different error responses in alone/wfemale conditions? In other words, could increased error responses be due to the fixed 100 ms analysis window of the audience condition that extends into a song region beyond the 100 ms region of the no-audience condition where there is increased firing? And vice versa for observed decreases in error responses, i.e. is there a firing pause just after the offset of the 100 ms window in the no-audience condition that causes audience dependence of responses? A simple compensation of song tempo differences by shortening/stretching the analysis window in one of the two conditions would allow to test for this.

Audience versus sound of audience: In the first sentence of the discussion authors write: we discovered that auditory representations of an animal's own vocalizations change with an audience. Is it truly the audience that causes the difference in error responses or is it the sounds the audience makes? To control for that would be to play back stimuli that simulate a non-silent audience through a loudspeaker to see whether error responses depend on the soundscape created by a typical audience (either present or absent). Authors probably do not have such data and to record it would go beyond the scope of this study, but it would be important to discuss this possibility or perform some analysis in that vein.

Reviewer #3 (Public Review):

Summary:

In this study, Jones et al. examine how neural activity in a primary auditory area (field L) of singing male songbirds is modulated by the presence or absence of an audience (a female conspecific). Prior work has demonstrated that the presence of an audience attenuates the responses of dopaminergic neurons to distortions of auditory feedback (DAF). Here the authors report that even in a region that is primarily considered sensory, responses to DAF are also modulated by the audience, although in a heterogeneous manner that does not readily explain previously observed attenuation. These findings address an interesting question and will potentially be important in adding to an understanding of how non-sensory factors can alter response properties of neurons even in primary sensory regions in a context dependent fashion. However, to be fully persuasive, additional analyses will be required to address how much of the apparent modulation by audience may be explained by other factors such as changes in recorded neurons or their properties over time.

Full Public Review:

In this study, Jones et al. examine how neural activity in a primary auditory area (field L) of singing male songbirds is modulated by the presence or absence of an audience (a female conspecific). They test whether activity in Field L differs between conditions in which the male is singing to a female (directed song) or alone (undirected song) and whether response to distortions of auditory feedback (DAF) differ between these conditions. Previous work has shown that in other parts of the songbird brain, sensory-motor activity can differ between directed and undirected song, and that responses to DAF are attenuated when males sing directed song versus undirected song. These prior results raise the interesting question of the extent to which such modulations of activity by the presence of an audience are already present in primary sensory areas such as Field L. This possibility is also motivated by prior work that has shown that Field L activity is not exclusively explained by auditory input, but can also be modulated by the bird's state - whether it is singing or not.

Against this background, the questions asked here are of interest for two inter-related reasons:

1) the authors address whether the presence of an audience (a female conspecific) alters activity in a primary auditory area during singing. Primary auditory areas such as Field L, and analogous mammalian thalamo-recipient cortical regions such as A1, are often thought of as responding very specifically to the features of sensory stimuli, but are also understood to be modulated by a variety of factors including the attentional and behavioral state of the animal. For audition, such modulation includes whether or not animals are vocalizing and listening to themselves or listening to playback of their own vocalizations. Cited works from Keller (2009) as well as Eliades and Wang (2008) have indicated that the act of vocalizing can modulate auditory responses to self-generated feedback in primary auditory areas relative to those arising from playback of the same sounds. Here, the question is whether responses to self-generated feedback differ between conditions of singing alone versus singing to a female audience. A demonstration that the presence of an audience matters to responses in Field L would add to a general understanding of how it is that non-auditory factors can modulate sensory responses.

2) the authors address the possible source of an audience-dependent modulation of responses to feedback perturbation in the VTA previously reported by Goldberg and colleagues (2023). In the VTA, responses to perturbations during singing are consistently attenuated when males are singing to females versus when they are singing alone, but the underlying mechanisms of this modulation are unknown. Here, the authors test the possibility that such modulation by an audience is already present at the level of Field L. The previously reported attenuation in VTA is quite striking and reflects a nice example of how neural processing can differ with varying behavioral priorities. Understanding whether this modulation of responses to DAF arises already in primary auditory areas would further a mechanistic understanding of an intriguing example of state-dependent modulation of sensory processing and behavior, and lend broad insight into related phenomena.

The authors report 1) that activity in Field L differs between directed and undirected singing at many individual recording sites, but that these changes are heterogeneous, with both increases and decreases in activity, so that there is no consistent change across the population and 2) that the responses to DAF differ between directed and undirected song, but that there is no consistent attenuation of response (as observed in the VTA) and instead heterogeneous increases and decreases in response to DAF so that there is no net change at the population level.

These findings, if firmly established, are important and of general interest. While they do not readily explain the source of the audience-dependent attenuation of auditory responses to DAF in the VTA, the demonstration of audience-dependent modulation of self-generated feedback and its disruption in a primary auditory area is an exciting result that would provide an opportunity for further investigation of how changes in social context influence brain and behavior. The manuscript is generally well written, although the presentation is terse. My main reservations about the current manuscript relate to aspects of experimental design and analysis that need to be clarified and addressed before these conclusions will be fully persuasive. There are also some places where further discussion of the findings and their relationship to prior studies would be helpful.

1. A central concern relates to whether the main reported effects associated with differences in singing directed versus undirected song reflect only those changes in conditions, versus contributions from changes in unit isolation or response properties over time. The authors record undirected song in a block in the morning and only after collecting at least 40 renditions do they later record responses during directed song over a series of repeated exposures to a female. Therefore, differences between data collected during undirected song and directed song also reflect differences between data collected initially during the morning versus later. It is unclear from methods whether any of these recordings during undirected and directed conditions are interleaved, but if this is not the case, then it is crucial to ask how stable were neural recordings with respect to unit isolation, and potential changes to response properties, over the duration of the experiments. This would be less of a concern if the results mirrored those observed in the VTA, where attenuation of responses was observed across the entire population during directed versus undirected conditions - it is hard to explain a phenomenon that is consistently observed across the population as arising from a change in which neurons and spikes are contributing to responses, or other forms of non-stationarity. However, because there are no significant differences reported at the population level in the current study, it is important to address the possibility that observed differences between conditions reflect some form of noise or drift in recorded units, rather than being entirely due to directed versus undirected singing. I have elaborated in more detail below on this concern, including places where the data seems to suggest some non-stationarity of responses, and have some suggestions for ways in which this concern might be addressed.

2. A second concern, related to this first one, has to do with the categorical definition of 'error neurons'. The authors note in their text that it could be problematic to apply categorical definitions to continuous distributions, and yet that seems to be what they then do. The authors have a metric of error sensitivity that they apply to each neuron's response to DAF in both undirected and directed conditions (the error score). They show that there is a continuous distribution of error scores (Figure 2 - figure supplement 1) across the population, with no bimodality that would be suggestive of distinct error sensitive and error-insensitive neurons. One nice feature of their analysis is that they also show the distribution of error scores computed in an analogous fashion for a period of neural activity in the song prior to DAF. This control data set makes it persuasive that there is a significant response to DAF, but also shows that there can be a broad range of error scores even when no DAF has been played, and that this range of 'noise' responses to DAF overlaps substantially with the actual responses to DAF. Despite the continuum of error scores, the authors define a subset of neurons as error responsive only if their responses to DAF exceed a specific threshold (2.5 standard deviations). One of the main conclusions of the paper is based on finding a subset of 22 neurons that exhibited error responses (by this definition) only during singing to a female and 11 neurons that exhibited error responses only when singing alone. These neurons are described as 'retuned' because they have error responses in only one condition.

The problem here is that for some, if not many, of the neurons that are categorically defined as being responsive to DAF in only one condition (directed versus undirected) there is almost certainly not a significant difference in the actual responses to DAF between conditions. This is apparent in the relevant data figure (figure 2 - figure supplement 1) and is a consequence of using a threshold to split a continuous distribution into groups defined as error responsive or not. For example, several neurons in this plot that have almost identical scores in the directed and undirected condition are counted as examples of retuning because the error scores are just a bit over 2.5 in the directed condition and just a bit under 2.5 in the undirected condition.

That this kind of categorical approach may be problematic is apparent in the control data in the plot. Despite the absence of any perturbation, there are error responsive neurons present in these data that are considered selective for directed versus undirected singing - this is an expected consequence of using a threshold on dispersed or noisy biological data. Shifting to a more stringent threshold of three standard deviations, as the authors do, does not help with this problem, as that still treats as categorically different responses that fall on either side of a line, even if only by a tiny amount. I suggest that the authors devise a measure for each neuron to test whether the responses to DAF are significantly different under the two conditions (directed versus undirected). As noted above, this measure should take into account some assessment of the stationarity of responses, as well as the distribution of responses (which, in some of the examples does not seem to be Gaussian around a mean response level, but rather highly variable across trials).

3. There are several places where further discussion of the previous literature and how the current results relate to that literature would be helpful. This includes:

3a. Some discussion of what is already known about the auditory tuning of field L, and the extent to which responses associated with distortion of feedback may reflect the frequency tuning of field L neurons versus something that might be construed as more specifically as detecting an error in perceived feedback. For example, Field L neurons have previously been characterized as having relatively simple spectro-temporal receptive fields, often with a single frequency band that is excitatory and nearby frequency bands that are inhibitory. It would be beyond the scope of this paper to directly assess the extent to which both song responses and responses to DAF are well predicted by simple STRFs that might be measured for the recorded neurons, or computed from activity during a range of vocalizations, but perhaps worth discussing whether a neuron with such frequency tuning would potentially exhibit 'error responses' of the sort described here, simply because the DAF stimulus happens to fall into the excitatory or inhibitory regions of the neuron's receptive field. While it is OK to use the term 'error responsive' in the current study, it would be good to make clear that changes in firing associated with playing DAF should be expected even for neurons that have simple auditory receptive fields (i.e. with center surround tuning to specific frequencies in a tonotopic map, as has been described for Field L) without necessarily indicating that these neurons are specifically registering any deviation or 'error' between expected feedback and experienced feedback. In this respect, there are multiple subdivisions of Field L with different tuning properties. Please specify further what criteria were used to determine recording locations and how these correspond with previously defined subdivisions.

3b. It would also be useful to discuss further previous work on differences in auditory tuning or responses between conditions when subjects are vocalizing, versus when vocalizations are played back (as in Keller, Eliades) and whether the results in the current study are similar or different. For example, this prior work has indicated that efference copy or other signals that precede vocalizations can reach and influence activity in auditory areas - with the most compelling evidence for this being the modulation of activity prior to the onset of vocalizations. Was this also observed in the current study, and to what extent might this kind of mechanism contribute to the processing of feedback distortions? With respect to this kind of efference signal, or other possibilities, can the authors provide some discussion or speculation about possible mechanisms that might be differentially engaged between conditions of singing directed versus undirected song?

3c. The previous study on DAF responses in VTA indicates enhanced responses to female calls during directed song. To what extent did the current study control for any vocalizations or other sounds produced by females during the directed singing, and could this have contributed to differences in Field L activity between conditions? This question is motivated partly by the highly variable responses in raster plots even within one condition - might some of this reflect motifs during which transient noises are produced from female calling or other movements by the male or female?

More regarding stability of recordings:

The data presented in Figure 1D illustrate some of my concerns about the stationarity of recordings. In the directed condition there are no spikes at all following the first handful of motif renditions. Were the directed and undirected recordings interleaved here? If not, could the recorded neuron simply have been lost, changed in amplitude of recorded spikes so that it was no longer counted, or reduced its responsiveness over the course of the recordings? Because the recordings of undirected and directed singing are described as occurring sequentially, it seems likely that this type of change in recorded signal could contribute to changes in measured responses over time, independently of effects due to directed versus undirected singing.

A minor issue of this example is that the raw example trace with male alone does not seem to have a corresponding set of points in the roster plot. For panel E, I also cannot find rasters that correspond to the example recordings shown at top.

Figure 2A also shows a neuron that looks like it has non-stationarity; for the alone condition without altered feedback, the main peak has no spikes for the bottom half of the rasters. For the directed condition, much of the difference between control and distorted feedback conditions seems to come from a few trials towards the bottom of the raster plot that show more and earlier firing than most other rasters.

Other more subtle examples are suggested in the figures, such as Figure 1F where responses in the alone condition seem to increase over the course of recordings. A related issue apparent in some of the raster plots is that the firing rate distributions within a given condition sometimes appear to be very non-gaussian, with some motifs during which there is a lot of activity, or apparent bursting, and others in which there is little activity. In addition to the examples above, this includes<br /> responses in Fig 1E and Fig 2F. Does anything distinguish these cases or trails? Where differences between conditions are driven by firing differences that are present on only a subset of trials, such as in Fig 2A, there is some deviation from the normal criteria for use of T-tests/Z-scores. Please consider this point and discuss any caveats and/or apply other tests (Monte Carlo? Non-parametric?) as appropriate.

These potential issues of non-stationarily, and non-Gaussian firing rate distributions in each condition, make it complicated to think about what differences in activity reflect changes from undirected to directed conditions versus these other factors.

Approaches to addressing this issue could include more specifically indicating examples in which recordings from the alone condition and directed condition are interleaved and exhibit reversible (between conditions) changes in the pattern of responses (both without DAF in comparing alone versus directed, and with DAF demonstrating differences in DAF influences between conditions). Some good interleaved examples of this sort would be very helpful to illustrate the robustness of differences between conditions. More generally, the methods and or raster plots should include some further explanation of the time periods over which recordings were made in the alone versus directed conditions, and the extent to which they are interleaved or not.

Another approach that could be used if there are not many instances of inter-leaved recordings is to try to document the stationarily or stability of unit isolation and/or responses over time. It would be most helpful when applied to recordings from a given singing condition (i.e. alone or directed) that are interleaved, but even in cases where this is not possible perhaps one could assess the stability of waveforms and unit isolation across time. For example in Figure 2 - Supplementary figure 2, the left-hand and middle examples appear to have quite good unit isolation, and might be the sorts of cases where measures of unit isolation and waveform stability could be used to argue that a gain or loss of spikes due to drift in recordings or changes to SNR and spike detection are not contributing to changes in firing patterns over time (and across conditions).

It potentially would also be informative to present the prevalence of the main effects reported in the study as a function of some measures of unit isolation, SNR, and recording stability. It would be reassuring to see that significant differences between conditions are equally or more prevalent under the conditions of greatest unit isolation and recording stability than in cases with worse SNR or stability.

One other way that the authors might be able to address my main concern would be to look at the stability of firing patterns within conditions, where differences across trials most directly indicate the potential contributions of technical or biological changes in neural activity over time that are not related to the experimental conditions.

To further address some of these issues, it would be helpful to have additional explanations in this paper (rather than by reference to Goldberg and Fee, 2010) of the criteria that were used for counting spikes, and assessing stability of recordings. All I found about this in the Goldberg and Fee, 2010 reference was that "Spikes were sorted off-line using custom Matlab software" Does this require human inspection and judgment? Is there a simple threshold, or waveform measurement used for detecting spikes from single units? Are some sort of signal to noise measures, or ISI violations used to score how well units are isolated?

For the specific examples shown in figures, it would be useful to indicate by small tick marks or otherwise which spikes were counted as single units. For example in figure 2 column B, for the condition with female, did only the 1-3 largest spikes get counted, or also the spikes of medium height?

Page 11: "Many channels on the probes recorded multi-unit activity, which were taken note of but not analyzed in this study."

What were the criteria for this? For several of the examples in the figures there are spikes of varying amplitudes and as mentioned above it would be helpful to clarify how the spikes were sorted into single units in such cases.

Categorical scores:

Page 13: "Neurons with error responses greater than 2.5 in only one condition (undirected versus directed) were considered to have retuned; neurons with error scores greater than 2.5 in both conditions were considered not to have retuned."

This definition results in cases where responses of 2.45 vs 2.55 are described as 'retuned', even if these responses are not significantly different. The figure (Figure 2 - figure supplement 1) indicates that multiple neurons that were scored as retuning had responses that fall very near the threshold in this way.

Page 13, "Our results did not fundamentally change with ... a more stringent threshold of 3..."

The stringency is not issue here, rather the categorical threshold. Retuning would be more persuasively demonstrated if the authors could provide a test of whether or not the responses for individual neurons differ significantly between conditions appropriately taking into account multiple comparisons, stability of recordings, non-Gaussian firing rate distributions across motif renditions, etc. and use this metric to report effects, rather than setting a categorical threshold.

eLife assessment

This important study discovered DBT as a novel gene implicated in the resistance to MG132-mediated cytotoxicity and potentially also in the pathogenesis of ALS and FTD, two fatal neurodegenerative diseases. The authors provided solid evidence to support a mechanism by which loss of DBT suppresses MG132-mediated toxicity. While activation of autophagy is shown to be associated with DBT knockdown, it remains unclear if this is the underlying mechanism driving improved survival.

Reviewer #1 (Public Review):

Summary:<br /> Through an unbiased genomewide KO screen, the authors identified loss of DBT to suppress MG132-mediated death of cultured RPE cells. Further analyses suggested that DBT reduces ubiquitinated proteins by promoting autophagy. Mechanistic studies indicated that DBT loss promotes autophagy via AMPK and its downstream ULK and mTOR signaling. Furthermore, loss of DBT suppresses polyglutamine- or TDP-43-mediated cytotoxicity and/or neurodegeneration in fly models. Finally, the authors showed that DBT proteins are increased in ALS patient tissues, compared to non-neurological controls.

Strengths:<br /> The idea is novel, the evidence is mostly convincing, and the data are clean. The findings have implications for human diseases.

Weaknesses:<br /> More experiments are needed to establish the connections between DBT and autophagy. The mechanistic studies are somewhat biased, and it's unclear whether the same mechanism (i.e., AMPK-->mTOR) can be applied to TDP-43-mediated neurodegeneration. Also, some data interpretation has to be more accurate.

Reviewer #2 (Public Review):

Summary:<br /> Hwang, Ran-Der et al utilized a CRISPR-Cas9 knockout in human retinal pigment epithelium (RPE1) cells to evaluate for suppressors of toxicity by the proteasome inhibitor MG132 and identified that knockout of dihydrolipoamide branched chain transacylase E2 (DBT) suppressed cell death. They show that DBT knockout in RPE1 cells does not alter proteasome or autophagy function at baseline. However, with MG132 treatment, they show a reduction in ubiquitinated proteins but with no change in proteasome function. Instead, they show that DBT knockout cells treated with MG132 have improved autophagy flux compared to wildtype cells treated with MG132. They show that MG132 treatment decreases ATP/ADP ratios to a greater extent in DBT knockout cells, and in accordance causes activation of AMPK. They then show downstream altered autophagy signaling in DBT knockout cells treated with MG132 compared to wild-type cells treated with MG132. Then they express the ALS mutant TDP43 M337 or expanded polyglutamine repeats to model Huntington's disease and show that knockdown of DBT improves cell survival in RPE1 cells with improved autophagic flux. They also utilize a Drosophila model and show that utilizing either a RNAi or CRISPR-Cas9 knockout of DBT improves eye pigment in TDP43M337V and polyglutamine repeat-expressing transgenic flies. Finally, they show evidence for increased DBT in postmortem spinal cord tissue from patients with ALS via both immunoblotting and immunofluorescence.

Strengths:<br /> This is a mechanistic and well-designed paper that identifies DBT as a novel regulator of proteotoxicity via activating autophagy in the setting of proteasome inhibition. Major strengths include careful delineation of a mechanistic pathway to define how DBT is protective. These conclusions are largely justified, but additional experiments and information would be useful to clarify and extend these conclusions.

Weaknesses:<br /> The large majority of the experiments are evaluating suppression of drug (MG132) toxicity in an in vitro epithelial cell line, so the generalizability to disease is unclear. Indeed, MG132 itself has been shown to modulate autophagy, and off-target effects of MG132 are not addressed. While this paper is strengthened by the inclusion of mouse-induced motor neurons, Drosophila models, and postmortem tissue, the putative mechanisms are minimally evaluated in these models.

Also, this effect is only seen with MG132 treatment, at a dose that causes markedly impaired cell survival. In this setting, it is certainly plausible that changes in autophagy could be the result of differences in cell survival, as opposed to an underlying mechanism for cell survival. Additional controls would be useful to increase confidence that DBT knockdown is protective via modulation of autophagy.

While the authors report increased DBT in postmortem ALS tissue as suggestive that DBT may modulate proteotoxicity in neurodegeneration, this point would be better supported with the evaluation of overexpression of DBT in their model.

eLife assessment

This fundamental study provides an unprecedented understanding of the roles of different combinations of NaV channel isoforms in nociceptors' excitability, with relevance for the design of better strategies targeting NaV channels to treat pain. Although the experimental combination of electrophysiological, modeling, imaging, molecular biology, and behavioral data is convincing and supports the major claims of the work, some conclusions need to be strengthened by further evidence or discussion. The work may be of broad interest to scientists working on pain, drug development, neuronal excitability, and ion channels.

Reviewer #1 (Public Review):

Summary:<br /> In this work, Xie, Prescott, and colleagues have reevaluated the role of Nav1.7 in nociceptive sensory neuron excitability. They find that nociceptors can make use of different sodium channel subtypes to reach equivalent excitability. The existence of this degeneracy is critical to understanding neuronal physiology under normal and pathological conditions and could explain why Nav subtype-selective drugs have failed in clinical trials. More concretely, nociceptor repetitive spiking relies on Nav1.8 at DIV0 (and probably under normal conditions in vivo), but on Nav1.7 and Nav1.3 at DIV4-7 (and after inflammation in vivo).

The conclusions of this paper are mostly well supported by data, and these findings should be of broad interest to scientists working on pain, drug development, neuronal excitability, and ion channels.

Strengths:<br /> The authors have employed elegant electrophysiology experiments (including specific pharmacology and dynamic clamp) and computational simulations to study the excitability of a subpopulation of DRGs that would very likely match with nociceptors (they take advantage of using transgenic mice to detect Nav1.8-expressing neurons). They make a strong point showing the degeneracy that occurs at the ion channel expression level in nociceptors, adding this new data to previous observations in other neuronal types. They also demonstrate that the different Nav subtypes functionally overlap and are able to interchange their "typical" roles in action potential generation. As Xie, Prescott, and colleagues argue, the functional implications of the degenerate character of nociceptive sensory neuron excitability need to be seriously taken into account regarding drug development and clinical trials with Nav subtype-selective inhibitors.

Weaknesses:<br /> The next comments are minor criticisms, as the major conclusions of the paper are well substantiated. Most of the results presented in the article have been obtained from experiments with DRG neuron cultures, and surely there is a greater degree of complexity and heterogeneity about the degeneracy of nociceptors excitability in the "in vivo" condition. Indeed, the authors show in Figures 7 and 8 data that support their hypothesis and an increased Nav1.7's influence on nociceptor excitability after inflammation, but also a higher variability in the nociceptors spiking responses. On the other hand, DRG neurons targeted in this study (YFP (+) after crossing with Nav1.8-Cre mice) are >90% nociceptors, but not all nociceptors express Nav1.8 in vivo. As shown by Li et al., 2016 ("Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity"), there is a high heterogeneity of neuron subtypes within sensory neurons. Therefore, some caution should be taken when translating the results obtained with the DRG neuron cultures to the more complex "in vivo" panorama.

Although the authors have focused their attention on Nav channels, it should be noted that degeneracy concerning other ion channels (such as potassium ion channels) could also impact the nociceptor excitability. The action potential AHP in Figure 1, panel A is very different comparing the DIV0 (blue) and DIV4-7 examples. Indeed, the conductance density values for the AHP current are higher at DIV0 than at DIV7 in the computational model (supplementary table 5). The role of other ion channels in order to obtain equivalent excitability should not be underestimated.

Reviewer #2 (Public Review):

Summary:<br /> The authors have noted in preliminary work that tetrodotoxin (TTX), which inhibits NaV1.7 and several other TTX-sensitive sodium channels, has differential effects on nociceptors, dramatically reducing their excitability under certain conditions but not under others. Partly because of this coincidental observation, the aim of the present work was to re-examine or characterize the role of NaV1.7 in nociceptor excitability and its effects on drug efficacy. The manuscript demonstrates that a NaV1.7-selective inhibitor produces analgesia only when nociceptor excitability is based on NaV1.7. More generally and comprehensively, the results show that nociceptors can achieve equivalent excitability through changes in differential NaV inactivation and NaV expression of different NaV subtypes (NaV 1.3/1.7 and 1.8). This can cause widespread changes in the role of a particular subtype over time. The degenerate nature of nociceptor excitability shows functional implications that make the assignment of pathological changes to a particular NaV subtype difficult or even impossible.

Thus, the analgesic efficacy of NaV1.7- or NaV1.8-selective agents depends essentially on which NaV subtype controls excitability at a given time point. These results explain, at least in part, the poor clinical outcomes with the use of subtype-selective NaV inhibitors and therefore have major implications for the future development of Nav-selective analgesics.

Strengths:<br /> The above results are clearly and impressively supported by the experiments and data shown. All methods are described in detail, presumably allow good reproducibility, and were suitable to address the corresponding question. The only exception is the description of the computer model, which should be described in more detail.

The results showing that nociceptors can achieve equivalent excitability through changes in differential NaV inactivation and expression of different NaV subtypes are of great importance in the fields of basic and clinical pain research and sodium channel physiology and pharmacology, but also for a broad readership and community. The degenerate nature of nociceptor excitability, which is clearly shown and well supported by data has large functional implications. The results are of great importance because they may explain, at least in part, the poor clinical outcomes with the use of subtype-selective NaV inhibitors and therefore have major implications for the future development of Nav-selective analgesics.

In summary, the authors achieved their overall aim to enlighten the role of NaV1.7 in nociceptor excitability and the effects on drug efficacy. The data support the conclusions, although the clinical implications could be highlighted in a more detailed manner.

Weaknesses:<br /> As mentioned before, the results that nociceptors can achieve equivalent excitability through changes in differential NaV inactivation and NaV expression of different NaV subtypes are impressive. However, there is some "gap" between the DRG culture experiments and acutely dissociated DRGs from mice after CFA injection. In the extensive experiments with cultured DRG neurons, different time points after dissociation were compared. Although it would have been difficult for functional testing to examine additional time points (besides DIV0 and DIV4-7), at least mRNA and protein levels should have been determined at additional time points (DIV) to examine the time course or whether gene expression (mRNA) or membrane expression (protein) changes slowly and gradually or rapidly and more abruptly. It would also be interesting to clarify whether the changes that occur in culture (DIV0 vs. DIV4-7) are accompanied by (pro-)inflammatory changes in gene and protein expression, such as those known for nociceptors after CFA injection. This would better link the following data demonstrating that in acutely dissociated nociceptors after CFA injection, the inflammation-induced increase in NaV1.7 membrane expression enhances the effect of (or more neurons respond to) the NaV1.7 inhibitor PF-71, whereas fewer CFA neurons respond to the NaV1.8 inhibitor PF-24.

The results shown explain, at least in part, the poor clinical outcomes with the use of subtype-selective NaV inhibitors and therefore have important implications for the future development of Nav-selective analgesics. However, this point, which is also evident from the title of the manuscript, is discussed only superficially with respect to clinical outcomes. In particular, the promising role of NaV1.7, which plays a role in nociceptor hyperexcitability but not in "normal" neurons, should be discussed in light of clinical results and not just covered with a citation of a review. Which clinical results of NaV1.7-selective drugs can now be better explained and how?

Another point directly related to the previous one, which should at least be discussed, is that all the data are from rodents, or in this case from mice, and this should explain the clinical data in humans. Even if "impediment to translation" is briefly mentioned in a slightly different context, one could (as mentioned above) discuss in more detail which human clinical data support the existence of "equivalent excitability through different sodium channels" also in humans.

Although speculative, it would be interesting for readers to know whether a treatment regimen based on "time since injury" with NaV1.7 and NaV1.8 inhibitors might offer benefits. Based on the data, could one hypothesize that NaV1.7 inhibitors are more likely to benefit (albeit in the short term) in patients with neuropathic pain with better patient selection (e.g., defined interval between injury and treatment)?

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors used patch-clamp to characterize the implication of various voltage-gated Na+ channels in the firing properties of mouse nociceptive sensory neurons. They report that depending on the culture conditions NaV1.3, NaV1.7, and NaV1.8 have distinct contributions to action potential firing and that similar firing patterns can result from distinct relative roles of these channels. The findings may be relevant for the design of better strategies targeting NaV channels to treat pain.

Strengths:<br /> The paper addresses the important issue of understanding, from an interesting perspective, the lack of success of therapeutic strategies targeting NaV channels in the context of pain. Specifically, the authors test the hypothesis that different NaV channels contribute in a plastic manner to action potential firing, which may be the reason why it is difficult to target pain by inhibiting these channels. The experiments seem to have been properly performed and most conclusions are justified. The paper is concisely written and easy to follow.

Weaknesses:<br /> 1) The most critical issue I find in the manuscript is the claim that different combinations of NaV channels result in equivalent excitability. For example, in the Abstract it is stated that: "...we show that nociceptors can achieve equivalent excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8". The gating properties of these channels are not identical, and therefore their contributions to excitability should not be the same. I think that the culprit of this issue is that the authors reach their conclusion from the comparison of the (average) firing rate determined over 1 s current stimulation in distinct conditions. However, this is not the only parameter that determines how sensory neurons convey information. For instance, the time dependence of the instantaneous frequency, the actual firing pattern, may be important too. Moreover, the use of 1 s of current stimulation might not be sufficient to characterize the firing pattern if one wants to obtain conclusions that could translate to clinical settings (i.e., sustained pain). A neuron in which NaV1.7 is the main contributor is expected to have a damping firing pattern due to cumulative channel inactivation, whereas another depending mainly on NaV1.8 is expected to display more sustained firing. This is actually seen in the results of the modelling.

2) In Fig. 1, is 100 nM TTX sufficient to inhibit all TTX-sensitive NaV currents? More common in literature values to fully inhibit these currents are between 300 to 500 nM. The currents shown as TTX-sensitive in Fig. 1D look very strange (not like the ones at Baseline DIV4-7). It seems that 100 nM TTX was not enough, leading to an underestimation of the amplitude of the TTX-sensitive currents.

3) Page 8, the authors conclude that "Inflammation caused nociceptors to become much more variable in their reliance of specific NaV subtypes". However, how did the authors ensure that all neurons tested were affected by the CFA model? It could be that the heterogeneity in neuron properties results from distinct levels of effects of CFA.

eLife assessment

This study provides valuable insights into how researchers can use perceptual metamers to formally explore the limits of visual representations at different processing stages. While the study is overall convincing in terms of approach and results, issues were identified with respect to novelty, sample size, incomplete psychophysical methodology, and better motivation of the models tested.

Reviewer #1 (Public Review):

This is an interesting study of the nature of representations across the visual field. The question of how peripheral vision differs from foveal vision is a fascinating and important one. The majority of our visual field is extra-foveal yet our sensory and perceptual capabilities decline in pronounced and well-documented ways away from the fovea. Part of the decline is thought to be due to spatial averaging ('pooling') of features. Here, the authors contrast two models of such feature pooling with human judgments of image content. They use much larger visual stimuli than in most previous studies, and some sophisticated image synthesis methods to tease apart the prediction of the distinct models.

More importantly, in so doing, the researchers thoroughly explore the general approach of probing visual representations through metamers-stimuli that are physically distinct but perceptually indistinguishable. The work is embedded within a rigorous and general mathematical framework for expressing equivalence classes of images and how visual representations influence these. They describe how image-computable models can be used to make predictions about metamers, which can then be compared to make inferences about the underlying sensory representations. The main merit of the work lies in providing a formal framework for reasoning about metamers and their implications, for comparing models of sensory processing in terms of the metamers that they predict, and for mapping such models onto physiology. Importantly, they also consider the limits of what can be inferred about sensory processing from metamers derived from different models.

Overall, the work is of a very high standard and represents a significant advance over our current understanding of perceptual representations of image structure at different locations across the visual field. The authors do a good job of capturing the limits of their approach and I particularly appreciated the detailed and thoughtful Discussion section and the suggestion to extend the metamer-based approach described in the MS with observer models. The work will have an impact on researchers studying many different aspects of visual function including texture perception, crowding, natural image statistics, and the physiology of low- and mid-level vision.

The main weaknesses of the original submission relate to the writing. A clearer motivation could have been provided for the specific models that they consider, and the text could have been written in a more didactic and easy-to-follow manner. The authors could also have been more explicit about the assumptions that they make.

Reviewer #2 (Public Review):

Summary<br /> This paper expands on the literature on spatial metamers, evaluating different aspects of spatial metamers including the effect of different models and initialization conditions, as well as the relationship between metamers of the human visual system and metamers for a model. The authors conduct psychophysics experiments testing variations of metamer synthesis parameters including type of target image, scaling factor, and initialization parameters, and also compare two different metamer models (luminance vs energy). An additional contribution is doing this for a field of view larger than has been explored previously.

General Comments<br /> Overall, this paper addresses some important outstanding questions regarding comparing original to synthesized images in metamer experiments and begins to explore the effect of noise vs image seed on the resulting syntheses. While the paper tests some model classes that could be better motivated, and the results are not particularly groundbreaking, the contributions are convincing and undoubtedly important to the field. The paper includes an interesting Voronoi-like schematic of how to think about perceptual metamers, which I found helpful, but for which I do have some questions and suggestions. I also have some major concerns regarding incomplete psychophysical methodology including lack of eye-tracking, results inferred from a single subject, and a huge number of trials. I have only minor typographical criticisms and suggestions to improve clarity. The authors also use very good data reproducibility practices.

Specific Comments

Experimental Setup<br /> Firstly, the experiments do not appear to utilize an eye tracker to monitor fixation. Without eye tracking or another manipulation to ensure fixation, we cannot ensure the subjects were fixating the center of the image, and viewing the metamer as intended. While the short stimulus time (200ms) can help minimize eye movements, this does not guarantee that subjects began the trial with correct fixation, especially in such a long experiment. While Covid-19 did at one point limit in-person eye-tracked experiments, the paper reports no such restrictions that would have made the addition of eye-tracking impossible. While such a large-scale experiment may be difficult to repeat with the addition of eye tracking, the paper would be greatly improved with, at a minimum, an explanation as to why eye tracking was not included.

Secondly, many of the comparisons later in the paper (Figures 9,10) are made from a single subject. N=1 is not typically accepted as sufficient to draw conclusions in such a psychophysics experiment. Again, if there were restrictions limiting this it should be discussed. Also (P11) Is subject sub-00 is this an author? Other expert? A naive subject? The subject's expertise in viewing metamers will likely affect their performance.

Finally, the number of trials per subject is quite large. 13,000 over 9 sessions is much larger than most human experiments in this area. The reason for this should be justified.

Model<br /> For the main experiment, the authors compare the results of two models: a 'luminance model' that spatially pools mean luminance values, and an 'energy model' that spatially pools energy calculated from a multi-scale pyramid decomposition. They show that these models create metamers that result in different thresholds for human performance, and therefore different critical scaling parameters, with the basic luminance pooling model producing a scaling factor 1/4 that of the energy model. While this is certain to be true, due to the luminance model being so much simpler, the motivation for the simple luminance-based model as a comparison is unclear.

The authors claim that this luminance model captures the response of retinal ganglion cells, often modeled as a center-surround operation (Rodieck, 1964). I am unclear in what aspect(s) the authors claim these center-surround neurons mimic a simple mean luminance, especially in the context of evidence supporting a much more complex role of RGCs in vision (Atick & Redlich, 1992). Why do the authors not compare the energy model to a model that captures center-surround responses instead? Do the authors mean to claim that the luminance model captures only the pooling aspects of an RGC model? This is particularly confusing as Figures 6 and 9 show the luminance and energy models for original vs synth aligning with the scaling of Midget and Parasol RGCs, respectively. These claims should be more clearly stated, and citations included to motivate this. Similarly, with the energy model, the physiological evidence is very loosely connected to the model discussed.

Prior Work:<br /> While the explorations in this paper clearly have value, it does not present any particularly groundbreaking results, and those reported are consistent with previous literature. The explorations around critical eccentricity measurement have been done for texture models (Figure 11) in multiple papers (Freeman 2011, Wallis, 2019, Balas 2009). In particular, Freeman 20111 demonstrated that simpler models, representing measurements presumed to occur earlier in visual processing need smaller pooling regions to achieve metamerism. This work's measurements for the simpler models tested here are consistent with those results, though the model details are different. In addition, Brown, 2023 (which is miscited) also used an extended field of view (though not as large as in this work). Both Brown 2023, and Wallis 2019 performed an exploration of the effect of the target image. Also, much of the more recent previous work uses color images, while the author's exploration is only done for greyscale.

Discussion of Prior Work:<br /> The prior work on testing metamerism between original vs. synthesized and synthesized vs. synthesized images is presented in a misleading way. Wallis et al.'s prior work on this should not be a minor remark in the post-experiment discussion. Rather, it was surely a motivation for the experiment. The text should make this clear; a discussion of Wallis et al. should appear at the start of that section. The authors similarly cite much of the most relevant literature in this area as a minor remark at the end of the introduction (P3L72).

White Noise:<br /> The authors make an analogy to the inability of humans to distinguish samples of white noise. It is unclear however that human difficulty distinguishing samples of white noise is a perceptual issue- It could instead perhaps be due to cognitive/memory limitations. If one concentrates on an individual patch one can usually tell apart two samples. Support for these difficulties emerging from perceptual limitations, or a discussion of the possibility of these limitations being more cognitive should be discussed, or a different analogy employed.

Relatedly, in Figure 14, the authors do not explain why the white noise seeds would be more likely to produce syntheses that end up in different human equivalence classes.

It would be nice to see the effect of pink noise seeds, which mirror the power spectrum of natural images, but do not contain the same structure as natural images - this may address the artifacts noted in Figure 9b.

Finally, the authors note high-frequency artifacts in Figure 4 & P5L135, that remain after syntheses from the luminance model. They hypothesize that this is due to a lack of constraints on frequencies above that defined by the pooling region size. Could these be addressed with a white noise image seed that is pre-blurred with a low pass filter removing the frequencies above the spatial frequency constrained at the given eccentricity?

Schematic of metamerism:<br /> Figures 1,2,12, and 13 show a visual schematic of the state space of images, and their relationship to both model and human metamers. This is depicted as a Voronoi diagram, with individual images near the center of each shape, and other images that fall at different locations within the same cell producing the same human visual system response. I felt this conceptualization was helpful. However, implicitly it seems to make a distinction between metamerism and JND (just noticeable difference). I felt this would be better made explicit. In the case of JND, neighboring points, despite having different visual system responses, might not be distinguishable to a human observer.

In these diagrams and throughout the paper, the phrase 'visual stimulus' rather than 'image' would improve clarity, because the location of the stimulus in relation to the fovea matters whereas the image can be interpreted as the pixels displayed on the computer.

Other<br /> The authors show good reproducibility practices with links to relevant code, datasets, and figures.

eLife assessment

This study provides valuable findings about pre-saccadic foveal prediction and the extent to which it is influenced by the visibility of the saccade target relative to its background. The results and research methodology are technically solid, but there are questions about the interpretation of data which if addressed, would benefit our understanding of this phenomenon. This work should be of broad interest to visual neuroscientists, as well as those interested in understanding perception in the context of eye movements and in modeling visually guided actions.

Reviewer #1 Public Review:

Summary:<br /> This study examines to what extent this phenomenon varies based on the visibility of the saccade target. Visibility is defined as the contrast level of the target with respect to the noise background, and it is related to the signal-to-noise ratio of the target. A more visible target facilitates the oculomotor behavior planning and execution, however, as speculated by the authors, it can also benefit foveal prediction even if the foveal stimulus visibility is maintained constant. Remarkably, the authors show that presenting a highly visible saccade target is beneficial for foveal vision as the detection of stimuli with an orientation similar to that of the saccade target is improved, the lower the saccade target visibility, the less prominent the effect.

Strengths:<br /> The results are convincing and the research methodology is technically sound.

Weaknesses:<br /> Discussion on how this phenomenon may unfold in natural viewing conditions when the foveal and saccade target stimuli are complex and are constituted by different visual properties is lacking. Some speculations regarding feedforward vs feedback neural processing involved in the phenomenon and the speed of the feedforward signal in relation to the visibility of the target, are not well justified and not clearly supported by the data.

Reviewer #2 Public Review:

Summary:<br /> In this manuscript, the authors ran a dual task. Subjects monitored a peripheral location for a target onset (to generate a saccade to), and they also monitored a foveal location for a foveal probe. The foveal probe could be congruent or incongruent with the orientation of the peripheral target. In this study, the authors manipulated the conspicuity of the peripheral target, and they saw changes in performance in the foveal task. However, the changes were somewhat counterintuitive.

Strengths:<br /> The authors use solid analysis methods and careful experimental design.

Weaknesses:<br /> I have some issues with the interpretation of the results, as explained below. In general, I feel that a lot of effects are being explained by attention and target-probe onset asynchrony etc, but this seems to be against the idea put forth by the authors of "foveal prediction for visual continuity across saccades". Why would foveal prediction be so dependent on such other processes? This needs to be better clarified and justified.

Specifics:<br /> The explanation of decreased hit rates with increased peripheral target opacity is not convincing. The authors suggest that higher contrast stimuli in the periphery attract attention. But, then, why are the foveal results occurring earlier (as per the later descriptions in the manuscript)? And, more importantly, why would foveal prediction need to be weaker with stronger pre-saccadic attention to the periphery? What is the function of foveal prediction? What of the other interpretation that could be invoked in general for this type of task used by the authors: that the dual task is challenging and that subjects somehow misattribute what they saw in the peripheral task when planning the saccade. i.e. foveal hit rates are misperceptions of the peripheral target. When the peripheral target is easier to see, then the foveal hit rate drops.

The analyses of Fig. 3C appear to be overly convoluted. They also imply an acknowledgment by the authors that target-probe temporal difference matters. Doesn't this already negate the idea that the foveal effects are associated with the saccade generation process itself? If the effect is related to target onset, how is it interpreted as related to a foveal prediction that is associated with the saccade itself? Also, the oscillatory nature of the effect in Fig. 3C for 59% and 90% opacity is quite confusing and not addressed. The authors simply state that enhancement occurs earlier before the saccade for higher contrasts. But, this is not entirely true. The enhancement emerges then disappears and then emerges again leading up to the saccade. Why would foveal prediction do that?

The interpretation of Fig. 4 is also confusing. Doesn't the longer latency already account for the lapse in attention, such that visual continuity can proceed normally now that the saccade is actually eventually made? In all results, it seems that the effects are all related to the dual nature of the task and/or attention, rather than to the act of making the saccade itself. Why should visual continuity (when a saccade is actually made, whether with short or long latency) have different "fidelity"? And, isn't this disruptive to the whole idea of visual continuity in the first place?

Small question: is it just me or does the data in general seem to be too excessively smoothed?

eLife assessment

In this study, the authors develop a useful strategy for fluorophore-tagging endogenous proteins in human induced pluripotent stem cells (iPSCs) using a split mNeonGreen approach. Experimentally, the methods are solid, and the data presented support the author's conclusions. Overall, these methodologies should be useful to a wide audience of cell biologists who want to study protein localization and dynamics at endogenous levels in iPSCs.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, the authors have applied an asymmetric split mNeonGreen2 (mNG2) system to human iPSCs. Integrating a constitutively expressed long fragment of mNG2 at the AAVS1 locus, allows other proteins to be tagged through the use of available ssODN donors. This removes the need to generate long AAV donors for tagging, thus greatly facilitating high-throughput tagging efforts. The authors then demonstrate the feasibility of the method by successfully tagging 9 markers expressed in iPSC at various, and one expressed upon endoderm differentiation. Several additional differentiation markers were also successfully tagged but not subsequently tested for expression/visibility. As one might expect for high-throughput tagging, a few proteins, while successfully tagged at the genomic level, failed to be visible. Finally, to demonstrate the utility of the tagged cells, the authors isolated clones with genes relevant to cytokinesis tagged, and together with an AI to enhance signal-to-noise ratios, monitored their localization over cell division.

Strengths:<br /> Characterization of the mNG2 tagged parental iPSC line was well and carefully done including validation of a single integration, the presence of markers for continued pluripotency, selected off-target analysis, and G-banding-based structural rearrangement detection.

The ability to tag proteins with simple ssODNs in iPSC capable of multi-lineage differentiation will undoubtedly be useful for localization tracking and reporter line generation.

Validation of clone genotypes was carefully performed and highlights the continued need for caution with regard to editing outcomes.

Weaknesses:<br /> IF and flow cytometry figures lack quantification and information on replication. How consistent is the brightness and localization of the markers? How representative are the specific images? Stability is mentioned in the text but data on the stability of expression/brightness is not shown.

The localization of markers, while consistent with expectations, is not validated by a second technique such as antibody staining, and in many cases not even with Hoechst to show nuclear vs cytoplasmic.

For the multi-germ layer differentiation validation, NCAM is also expressed by ectoderm, so isn't a good solo marker for mesoderm as it was used. Indeed, the kit used for the differentiation suggests Brachyury combined with either NCAM or CXCR4, not NCAM alone.

Only a single female parental line has been generated and characterized. It would have been useful to have several lines and both male and female to allow sex differences to be explored.

The AI-based signal-to-noise enhancement needs more details and testing. Such models can introduce strong assumptions and thus artefacts into the resolved data. Was the model trained on all markers or were multiple models trained on a single marker each? For example, if trained to enhance a single marker (or co-localized group of markers), it could introduce artefacts where it forces signal localization to those areas even for others. What happens if you feed in images with scrambled pixel locations, does it still say the structures are where the training data says they should be? What about markers with different localization from the training set? If you feed those in, does it force them to the location expected by the training data or does it retain their differential true localization and simply enhance the signal?

Reviewer #2 (Public Review):

Summary:<br /> The authors have generated human iPSC cells constitutively expressing the mNG21-10 and tested them by endogenous tagging multiple genes with mNG211 (several tagged iPS cell lines clones were isolated). With this tool, they have explored several weakly expressed cytokinesis genes and gained insights into how cytokinesis occurs.

Strengths:<br /> Human iPSC cells are used.

Weaknesses:<br /> i) The manuscript is extremely incremental, no improvements are present in the split-fluorescent (split-FP) protein variant used nor in the approach for endogenous tagging with split-FPs (both of them are already very well established and used in literature as well as in different cell types).

ii) The fluorescence intensity of the split mNeonGreen appears rather low, for example in Figure 2C the H2BC11, ANLN, SOX2, and TUBB3 signals are very noisy (differences between the structures observed are almost absent). For low-expression targets, this is an important limitation. This is also stated by the authors but image restoration could not be the best solution since a lot of biologically relevant information will be lost anyway.

iii) There is no comparison with other existing split-FP variants, methods, or imaging and it is unclear what the advantages of the system are.

Reviewer #3 (Public Review):

The authors report on the engineering of an induced Pluripotent Stem Cell (iPSC) line that harbours a single copy of a split mNeonGreen, mNG2(1-10). This cell line is subsequently used to take endogenous protein with a smaller part of mNeonGreen, mNG2(11), enabling the complementation of mNG into a fluorescent protein that is then used to visualize the protein. The parental cell is validated and used to construct several iPSC lines with endogenously tagged proteins. These are used to visualize and quantify endogenous protein localisation during mitosis.

I see the advantage of tagging endogenous loci with small fragments, but the complementation strategy has disadvantages that deserve some attention. One potential issue is the level of the mNG2(1-10). Is it clear that the current level is saturating? Based on the data in Figure S3, the expression levels and fluorescence intensity levels show a similar dose-dependency which is reassuring, but not definitive proof that all the mNG2(11)-tagged protein is detected.

Do the authors see a difference in fluorescence intensity for homo- and heterozygous cell lines that have the same protein tagged with mNG2(11)? One would expect two-fold differences, or not?

Related to this, would it be favourable to have a homozygous line for expressing mNG2(1-10)?

The complementation seems to work well for the proteins that are tested. Would this also work for secreted (or other organelle-resident) proteins, for which the mNG2(11) tag is localised in a membrane-enclosed compartment?

The authors present a technological advance and it would be great if others could benefit from this as well by having access to the cell lines.

eLife assessment

This study describes a method to track MHC class II binding peptides on dendritic cell (DC) surfaces using a tetracystein tag and a thiol-reactive dye, which can then be investigated in vitro and in vivo. This is a valuable study for the impact on immunology and potentially other areas where the detection of cell-associated peptides is required. The methods are convincing based on the use of MHC class I/II deficient mice that have significantly reduced signal, but the non-zero background is detected, and it is not clear that this is lower than if the peptides were directly labelled with fluorophores.

Reviewer #1 (Public Review):

Summary:<br /> The authors develop a method to fluorescently tag peptides loaded onto dendritic cells using a two-step method with a tetracystein motif modified peptide and labelling step done on the surface of live DC using a dye with high affinity for the added motif. The results are convincing in demonstrating in vitro and in vivo T cell activation and efficient label transfer to specific T cells in vivo. The label transfer technique will be useful to identify T cells that have recognised a DC presenting a specific peptide antigen to allow the isolation of the T cell and cloning of its TCR subunits, for example. It may also be useful as a general assay for in vitro or in vivo T-DC communication that can allow the detection of genetic or chemical modulators.

Strengths:<br /> The study includes both in vitro and in vivo analysis including flow cytometry and two-photon laser scanning microscopy. The results are convincing and the level of T cell labelling with the fluorescent pMHC is surprisingly robust and suggests that the approach is potentially revealing something about fundamental mechanisms beyond the state of the art.

Weaknesses:<br /> The method is demonstrated only at high pMHC density and it is not clear if it can operate at at lower peptide doses where T cells normally operate. However, this doesn't limit the utility of the method for applications where the peptide of interest is known. It's not clear to me how it could be used to de-orphan known TCR and this should be explained if they want to claim this as an application. Previous methods based on biotin-streptavidin and phycoerythrin had single pMHC sensitivity, but there were limitations to the PE-based probe so the use of organic dyes could offer advantages.

Reviewer #2 (Public Review):

Summary:<br /> The authors here develop a novel Ovalbumin model peptide that can be labeled with a site-specific FlAsH dye to track agonist peptides both in vitro and in vivo. The utility of this tool could allow better tracking of activated polyclonal T cells particularly in novel systems. The authors have provided solid evidence that peptides are functional, capable of activating OTII T cells, and that these peptides can undergo trogocytosis by cognate T cells only.

Strengths:<br /> -An array of in vitro and in vivo studies are used to assess peptide functionality.<br /> -Nice use of cutting-edge intravital imaging.<br /> -Internal controls such as non-cogate T cells to improve the robustness of the results (such as Fig 5A-D).<br /> -One of the strengths is the direct labeling of the peptide and the potential utility in other systems.

Weaknesses:<br /> 1. What is the background signal from FlAsH?<br /> The baselines for Figure 1 flow plots are all quite different. Hard to follow. What does the background signal look like without FLASH (how much fluorescence shift is unlabeled cells to No antigen+FLASH?). How much of the FlAsH in cells is actually conjugated to the peptide? In Figure 2E, it doesn't look like it's very specific to pMHC complexes. Maybe you could double-stain with Ab for MHCII. Figure 4e suggests there is no background without MHCII but I'm not fully convinced. Potentially some MassSpec for FLASH-containing peptides.

2. On the flip side, how much of the variant peptides are getting conjugated in cells? I'd like to see some quantification (HPLC or MassSpec). If it's ~10% of peptides that get labeled, this could explain the low shifts in fluorescence and the similar T cell activation to native peptides if FlasH has any deleterious effects on TCR recognition. But if it's a high rate of labeling, then it adds confidence to this system.

3. Conceptually, what is the value of labeling peptides after loading with DCs? Why not preconjugate peptides with dye, before loading, so you have a cleaner, potentially higher fluorescence signal? If there is a potential utility, I do not see it being well exploited in this paper. There are some hints in the discussion of additional use cases, but it was not clear exactly how they would work. One mention was that the dye could be added in real-time in vivo to label complexes, but I believe this was not done here. Is that feasible to show?

4. Figure 5D-F the imaging data isn't fully convincing. For example, in 5F and 2G, the speeds for T cells with no Ag should be much higher (10-15micron/min or 0.16-0.25micron/sec). The fact that yours are much lower speeds suggests technical or biological issues, that might need to be acknowledged or use other readouts like the flow cytometry.

eLife assessment

This valuable work by Rivera et al. probes to understand how the regulation of oligodendrocyte progenitor cell (OPC) remyelination and function contributes to the treatment of multiple sclerosis. The authors provide incomplete evidence for the platelets to mediate OPC differentiation and remyelination. Both reviewers have raised important questions. This work will be of broad interest to biologists in general.

Reviewer #1 (Public Review):

Summary:<br /> The authors have studied the effects of platelets in OPC biology and remyelination. For this, they used mutant mice with lower levels of platelets as a demyelinating/remyelinating scenario, as well as in a model with large numbers of circulating platelets.

Strengths:<br /> -The work is very focused, with defined objectives.<br /> -The work is properly done.

Weaknesses:<br /> -There is no clear effect on a single cell type and/or mechanism involved.

Reviewer #2 (Public Review):

Summary:<br /> This paper examined whether circulating platelets regulate oligodendrocyte progenitor cell (OPC) differentiation for the link with multiple sclerosis (MS). They identified that the interaction with platelets enhances OPC differentiation although persistent contact inhibits the process in the long-term. The mouse model with increased platelet levels in the blood reduced mature oligodendrocytes, while how platelets might regulate OPC differentiation is not clear yet.

Strengths:<br /> The use of both partial platelet depletion and thrombocytosis mouse models gives in vivo evidence. The presentation of platelet accumulation in a time-course manner is rigorous. The in vitro co-culture model tested the role of platelets in OPC differentiation, which was supportive of in vivo observations.

Weaknesses:<br /> How platelets regulate OPC differentiation is not clear. What the significance of platelets is in MS progression is not clear.

eLife assessment

This study presents a valuable finding on the mechanism of glucocorticoid-induced osteonecrosis of the femoral head. The data were collected and analyzed using solid and validated methodology and can be used as a starting point for functional studies of the development of glucocorticoid-induced osteonecrosis. This paper would be of interest to cell biologists and biophysicists working on the potential pharmacological treatments for glucocorticoid-induced osteonecrosis.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript by Xia et al. investigated the mechanisms underlying Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). The authors observed that abnormal osteogenesis and adipogenesis are associated with decreased β-catenin in the necrotic femoral head of GONFH patients, and that the inhibition of β-catenin signalling leads to abnormal osteogenesis and adipogenesis in GONFH rats. Of interest, the deletion of β-catenin in Col2-expressing cells rather than in osx-expressing cells leads to a GONFH-like phenotype in the femoral head of mice.

Strengths:<br /> A strength of the study is that it sets up a Col2-expressing cell-specific β-catenin knockout mouse model that mimics the full spectrum of osteonecrosis phenotype of GONFH. This is interesting and provides new insights into the understanding of GONFH. Overall, the data are solid and support their conclusions.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors reported a study to uncover that β-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). In this study, they first observed abnormal osteogenesis and adipogenesis associated with decreased β-catenin in the necrotic femoral head of GONFH patients, but the exact pathological mechanisms of GONFH remain unknown. They then performed in vivo and in vitro studies to further reveal that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation bone marrow stromal cells (BMSCs) by inhibiting β-catenin signaling in glucocorticoid-induced GONFH rats, and specific deletion of β-catenin in Col2+ cells shifted BMSCs commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice.

Strengths:<br /> This innovative study provides strong evidence supporting that β-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation that contributes to the development of GONFH. This study also identifies an ideal genetically modified mouse model of GONFH. Overall, the experiment is logically designed, the figures are clear, and the data generated from humans and animals is abundant supporting their conclusions.

Weaknesses:<br /> There is a lack of discussion to explain how the Wnt agonist 1 works. There are several types of Wnt ligands. It is not clear if this agonist only targets Wnt1 or other Wnts as well. Also, why Wnt agonist 1 couldn't rescue the GONFH-like phenotype in β-cateninCol2ER mice needs to be discussed.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors are trying to delineate the mechanism underlying the osteonecrosis of the femoral head.

Strengths:<br /> The authors provided compelling in vivo and in vitro data to demonstrate Col2+ cells and Osx+ cells were differentially expressed in the femoral head. Moreover, inducible knockout of β-catenin in Col2+ cells but not Osx+ cells lead to a GONFH-like phenotype including fat accumulation, subchondral bone destruction, and femoral head collapse, indicating that imbalance of osteogenic/adipogenic differentiation of Col2+ cells plays an important role in GONFH pathogenesis. Therefore, this manuscript provided mechanistic insights into osteonecrosis as well as potential therapeutic targets for disease treatment.

Weaknesses:<br /> However, additional in-depth discussion regarding the phenotype observed in mice is highly encouraged.

eLife assessment

The authors have shown a valuable tumor suppressive function of the non-core regions of RAG1/2 recombinases, by using a set of animal models. The work is solid and the conclusions are supported by their data. Some areas of mechanistic work can be improved.

Reviewer #1 (Public Review):

Summary:<br /> In this report, Yu et al ascribe potential tumor suppressive functions to the non-core regions of RAG1/2 recombinases. Using a well-established BCR-ABL oncogene-driven system, the authors model the development of B cell acute lymphoblastic leukemia in mice and found that RAG mutants lacking non-core regions show accelerated leukemogenesis. They further report that the loss of non-core regions of RAG1/2 increases genomic instability, possibly caused by increased off-target recombination of aberrant RAG-induced breaks. The authors conclude that the non-core regions of RAG1 in particular not only increase the fidelity of VDJ recombination, but may also influence the recombination "range" of off-target joints, and that in the absence of the non-core regions, mutant RAG1/2 (termed cRAGs) catalyze high levels of off-target recombination leading to the development of aggressive leukemia.

Strengths:<br /> The authors used a genetically defined oncogene-driven model to study the effect of RAG non-core regions on leukemogenesis. The animal studies were well performed and generally included a good number of mice. Therefore, the finding that cRAG expression led to the development of more aggressive BCR-ABL+ leukemia compared to fRAG is solid.

Weaknesses:<br /> In general, I find the mechanistic explanation offered by the authors to explain how the non-core regions of RAG1/2 suppress leukemogenesis to be less convincing. My main concern is that cRAG1 and cRAG2 are overexpressed relative to fRAG1/2. This raises the possibility that the observed increased aggressiveness of cRAG tumors compared to fRAG tumors could be solely due to cRAG1/2 overexpression, rather than any intrinsic differences in the activity of cRAG1/2 vs fRAG1/2; and indeed, the authors allude to this possibility in Fig S8, where it was shown that elevated expression of RAG (i.e. fRAG) correlated with decreased survival in pediatric ALL. Although it doesn't mean the authors' assertions are incorrect, this potential caveat should nevertheless be discussed.

Some of the conclusions drawn were not supported by the data.<br /> 1. I'm not sure that the authors can conclude based on μHC expression that there is a loss of pre-BCR checkpoint in cRAG tumors. In fact, Fig. 2B showed that the differences are not statistically significant overall, and more importantly, μHC expression should be detectable in small pre-B cells (CD43-). This is also corroborated by the authors' analysis of VDJ rearrangements, showing that it has occurred at the H chain locus in cRAG cells.

2. The authors found a high degree of polyclonal VDJ rearrangements in fRAG tumor cells but a much more limited oligoclonal VDJ repertoire in cRAG tumors. They concluded that this explains why cRAG tumors are more aggressive because BCR-ABL induced leukemia requires secondary oncogenic hits, resulting in the outgrowth of a few dominant clones (Page 19, lines 381-398). I'm not sure this is necessarily a causal relationship since we don't know if the oligoclonality of cRAG tumors is due to selection based on oncogenic potential or if it may actually reflect a more restricted usage of different VDJ gene segments during rearrangement.

3. What constitutes a cancer gene can be highly context- and tissue-dependent. Given that there is no additional information on how any putative cancer gene was disrupted (e.g., truncation of regulatory or coding regions), it is not possible to infer whether increased off-target cRAG activity really directly contributed to the increased aggressiveness of leukemia.

4. Fig. 6A, it seems that it is really the first four nucleotide (CACA) that determines fRAG binding and the first three (CAC) that determine cRAG binding, as opposed to five for fRAG and four for cRAG, as the author wrote (page 24, lines 493-497).

5. Fig S3B, I don't really see why "significant variations in NHEJ" would necessarily equate "aberrant expression of DNA repair pathways in cRAG leukemic cells". This is purely speculative. Since it has been reported previously that alt-EJ/MMEJ can join off target RAG breaks, do the authors detect high levels of microhomology usage at break points in cRAG tumors?

6. Fig. S7, CDKN2B inhibits CDK4/6 activation by cyclin D, but I don't think it has been shown to regulate CDK6 mRNA expression. The increase in CDK6 mRNA likely just reflects a more proliferative tumor but may have nothing to do with CDKN2B deletion in cRAG1 tumors.

Insufficient details in some figures. For instance, Fig. 1A, please include statistics in the plot showing a comparison of fRAG vs cRAG1, fRAG vs cRAG2, cRAG1 vs cRAG2. As of now, there's a single p-value (0.0425) stated in the main text and the legend but why is there only one p-value when fRAG is compared to cRAG1 or cRAG2? Similarly, the authors wrote "median survival days 11-26, 10-16, 11-21 days, P < 0.0023-0.0299, Fig. S2B." However, it is difficult for me to figure out what are the numbers referring to. For instance, is 11-26 referring to median survival of fRAG inoculated with three different concentrations of GFP+ leukemic cells or is 11-26 referring to median survival of fRAG, cRAG1, cRAG2 inoculated with 10^5 cells? It would be much clearer if the authors can provide the numbers for each pair-wise comparison, if not in the main text, then at least in the figure legend. In Fig. 5A-B, do the plots depict SVs in cRAG tumors or both cRAG and fRAG cells? Also in Fig. 5, why did 24 SVs give rise to 42 breakpoints, and not 48? Doesn't it take 2 breaks to accomplish rearrangement? In Fig. 6B-C, it is not clear how the recombination sizes were calculated. In the examples shown in Fig. 4, only cRAG1 tumors show intra-chromosomal joins (chr 12), while fRAG and cRAG2 tumors show exclusively inter-chromosomal joins.

Insufficient details on certain reagents/methods. For instance, are the cRAG1/2 mice of the same genetic background as fRAG mice (C57BL/6 WT)? On Page 23, line 481, what is a cancer gene? How are they defined? In Fig. 3C, are the FACS plots gated on intact cells? Since apoptotic cells show high levels of gH2AX, I'm surprised that the fraction of gH2AX+ cells is so much lower in fRAG tumors compared to cRAG tumors. The in vitro VDJ assay shown in Fig 3B is not described in the Method section (although it is described in Fig S5b). Fig. 5A-B, do the plots depict SVs in cRAG tumors or both cRAG and fRAG cells?

Reviewer #2 (Public Review):

Summary: In the manuscript, the authors summarized and introduced the correlation between the non-core regions of RAG1 and RAG2 in BCR-ABL1+acute B lymphoblastic leukemia and off-target recombination which has certain innovative and clinical significance.

eLife assessment

The authors studied key characteristics of MYC-driven cancers: dysregulated pre-mRNA splicing and altered metabolism. The reviewers agree that this is an interesting study and that the findings are important. Overall the data was considered solid although the paper would benefit from revisions. The manuscript has the potential to be of broad interest to cancer biologists due to its therapeutic implications.

Reviewer #1 (Public Review):

Summary of Author's Objectives:

The authors aimed to explore JMJD6's role in MYC-driven neuroblastoma, particularly in the interplay between pre-mRNA splicing and cancer metabolism, and to investigate the potential for targeting this pathway.

Strengths:

1. The study employs a diverse range of experimental techniques, including molecular biology assays, next-generation sequencing, interactome profiling, and metabolic analysis. Moreover, the authors specifically focused on gained chromosome 17q in neuroblastoma, in combination with analyzing cancer dependency genes screened with Crispr/Cas9 library, analyzing the association of gene expression with prognosis of neuroblastoma patients with large clinical cohort. This comprehensive approach strengthens the credibility of the findings. The identification of the link between JMJD6-mediated pre-mRNA splicing and metabolic reprogramming in MYC-driven cancer cells is innovative.

2. The authors effectively integrate data from multiple sources, such as gene expression analysis, RNA splicing analysis, JMJD6 interactome assay, and metabolic profiling. This holistic approach provides a more complete understanding of JMJD6's role.

3. The identification of JMJD6 as a potential therapeutic target and its correlation with the response to indisulam have significant clinical implications, addressing an unmet need in cancer treatment.

Weaknesses:

1. The manuscript contains complex technical details and terminology that may pose challenges for readers without a deep background in molecular biology and cancer research. Providing simplified explanations or additional context would enhance accessibility.

2. It would be beneficial to explore whether treatment with JMJD6 inhibitors, both in vitro and in vivo, can effectively target the enhanced pre-mRNA splicing of metabolic genes in MYC-driven cancer cells.

Appraisal of Achievement and Conclusion Support:

The authors have effectively met their objectives by offering valuable insights into JMJD6's role in MYC-driven neuroblastoma. The results robustly underpin their conclusions about JMJD6's contribution to metabolic reprogramming through alternative splicing and its connection to the therapeutic response to indisulam.

Likely Impact on the Field and Utility of Methods/Data:

The study's findings have the potential to significantly impact the field of cancer research by identifying JMJD6 as a promising therapeutic target for MYC-driven cancers. The methods and data presented in the manuscript offer valuable resources to the research community for further investigations into cancer metabolism and splicing regulation.

Additional Context for Interpretation:

Understanding the complex interplay between cancer metabolism and splicing regulation is crucial for developing effective cancer treatments. This study sheds light on a previously poorly understood aspect of MYC-driven cancers and opens new avenues for targeted therapies. However, the transition from preclinical findings to clinical applications may face challenges, which should be considered in future research and clinical trials.

Reviewer #2 (Public Review):

Summary:

Jablonowski and colleagues studied key characteristics of MYC-driven cancers: dysregulated pre-mRNA splicing and altered metabolism. This is an important field of study as it remains largely unclear as to how these processes are coordinated in response to malignant transformation and how they are exploitable for future treatments. In the present study, the authors attempt to show that Jumonji Domain Containing 6, Arginine Demethylase And Lysine Hydroxylase (JMJD6) plays a central role in connecting pre-mRNA splicing and metabolism in MYC-driven neuroblastoma. JMJD6 collaborates with the MYC protein in driving cellular transformation by physically interacting with RNA-binding proteins involved in pre-mRNA splicing and protein regulation. In cell line experiments, JMJD6 affected the alternative splicing of two forms of glutaminase (GLS), an essential enzyme in the glutaminolysis process within the central carbon metabolism of neuroblastoma cells. Additionally, the study provides in vitro (and in silico) evidence for JMJD6 being associated with the anti-proliferation effects of a compound called indisulam, which degrades the splicing factor RBM39, known to interact with JMJD6.

Overall, the findings presented by Jabolonowski et al. begin to illuminate a cancer-promoting metabolic, and potentially, a protein synthesis suppression program that may be linked to alternative pre-mRNA splicing through the action of JMJD6 - downstream of MYC. This discovery can provide further evidence for considering JMJD6 as a potential therapeutic target for the treatment of MYC-driven cancers.

Strengths:

Alternative Splicing Induced by JMJD6 Knockdown: the study presents evidence for the role of JMJD6 in alternative splicing in neuroblastoma cells. Specifically, the RNA immunoprecipitation experiments demonstrated a significant shift from the GAC to the KGA GLS isoform upon JMJD6 knockdown. Moreover, a significant correlation between JMJD6 levels and GAC/KGA isoform expression was identified in two distinct neuroblastoma cohorts. This suggests a causative link between JMJD6 activity and isoform prevalence.

Physical Interaction of JMJD6 in Neuroblastoma Cells: The paper provides preliminary insight into the physical interactome of JMJD6 in neuroblastoma cells. This offers a potential mechanistic avenue for the observed effects on metabolism and protein synthesis and could be exploited for a deeper investigation into the exact nature, and implications of neuroblastoma-specific JMJD6 protein-protein interactions.

Weaknesses:

There are several areas that would benefit from improvements with regard to the current data supporting the claims of the paper (i.e., the conclusion presented in Figure 8).

Neuroblastoma Modelling Strategy: The study heavily relies on cell lines without incorporating patient-derived cells/biomaterials. Using databases to fill gaps in the experimental design can only fortify the observations to a certain extent. A critical oversight is the absence of non-cancerous control cells in many figures, and the rationale for selecting specific cell lines for assays/approaches remains somewhat unclear. A foundational control for such experiments should involve the non-transformed neural crest cell line, which the authors have readily available. Are the observed splicing and metabolic effects of JMJD6 specific to neuroblastoma? Is there a neuroblastoma-specific JMJD6 interactome? Is MYC function essential?

In Vivo Modelling: The inclusion of a genetic mouse model combined with an inducible JMJD6 knockdown, would enhance the study by allowing examination of JMJD6's role during both tumor initiation and growth in vivo. For instance, the TH-MYCN mice overexpressing MYCN in neural crest cells, could be a promising choice.

Dependence on Colony Formation Assay: The study leans on 2D and semi-quantitative colony formation assays to assess malignant growth. To validate the link between the mechanistic insights discussed (e.g., reduced protein synthesis) and JMJD6-mediated malignant growth as a potential therapeutic target, evidence from in vivo or representative 3D models would be crucial.

Data Presentation and Rigor: The presented data is predominantly qualitative and necessitates quantification. For instance, Western blots should be quantified. The RNAseq, metabolism, and pull-down data should be transparently and numerically presented. The figure legends seem elusive and their lack of transparency (often with regards to biological repeats, error bars, cell line used etc.) is concerning. Adequate citation and identification of all data sources, including online resources, are imperative. The manuscript would also benefit from a more rigorous depiction and quantification of RNA interference of both stable and transient knockdowns with quantitative validation at mRNA and protein levels.

Novelty Concerns: The emphasis on JMJD6 as a novel neuroblastoma target is contingent on the new mechanistic revelations about the JMJD6-centered link between splicing, metabolism, and protein synthesis. Given that JMJD6 has been previously linked to neuroblastoma biology, the rationale (particularly in Figure 1) for concentrating on JMJD6 may stem more from bias rather than data-driven reasoning.

Depth of Mechanistic Investigation: Current evidence lacks depth in key areas such as JMJD6-RNA binding. A more thorough approach would involve pinpointing specific JMJD6 binding sites on endogenous RNAs using techniques such as cross-linking and immunoprecipitation, paired with complementary proximity-based methodologies. Regarding the presented metabolism data, diving deeper into metabolic flux via isotope labeling experiments could shed light on dynamic processes like TCA and glutaminolysis. As it stands, the 'pathway cartoon' in Figure 6d appears overly qualitative.

eLife assessment

This paper proposes a valuable new method for the assessment of the mean kurtosis for diffusional kurtosis imaging by utilizing a recently introduced sub-diffusion model. The evidence supporting the claims that this technique is robust and accurate in brain imaging is incomplete. The work could be of interest in the research and clinical arena.

Reviewer #1 (Public Review):

Summary:<br /> This study introduces an innovative method for assessing the mean kurtosis, utilizing the mathematical foundation of the sub-diffusion framework. In particular, a new fitting technique that incorporates two different diffusion times is proposed to estimate the parameters of the sub-diffusion model. The evaluation of this technique, which generates kurtosis maps based on the sub-diffusion framework, is conducted through simulations and the examination of data obtained from human subjects.

Strengths:<br /> The utilization of the sub-diffusion model for tissue characterization is a significant conceptual advancement for the field of diffusion MRI. This study adeptly harnesses this approach for an accurate estimation of the parameters of the widely employed diffusion model, DKI, leveraging their established analytical interconnection as evidenced in prior research. Notably, this approach not only proposes a robust, fast, and accurate technique for DKI parameter estimation but also underscores the viability of deploying the sub-diffusion model for tissue characterization, substantiated by both simulated and human subject analyses. The paper is very-well written; well-organized; and coherent. The simulation study included different aspects of water diffusion as captured by diffusion-weighted MRI such as varying diffusion times and different b-value subpopulations, resulting in a comprehensive and thorough discussion.

Weaknesses:<br /> The primary objective of this study is to demonstrate a robust approach for estimating DKI parameters by directly calculating them using the parameters of the sub-diffusion model. This premise, however, relies on the assumption that the sub-diffusion model effectively characterizes the diffusion MRI signal and that its parameters are both robust and accurate. Throughout the manuscript, the term "ground truth kurtosis K" is frequently used to denote the "true K" value in the context of the simulation study. Nonetheless, given that the data is simulated using the new sub-diffusion model - an approximation of the DKI-based signal expression- this value cannot truly be considered the "ground truth K". The simulation study highlights the robustness and accuracy of D* and K*, but it inherently operates under the assumption that the observed data is in the form of the sub-diffusion model.

Reviewer #2 (Public Review):

Summary: The authors present a technique for fitting diffusion magnetic resonance images (dMRI) to a sub-diffusion model of the diffusion process within brain imaging. The authors suggest that their technique provides robust and accurate calculation of diffusional kurtosis imaging parameters from which high quality images can be calculated from short dMRI data acquisitions at two diffusion times.

Strengths: If the authors can show that the dMRI signal in brain tissue follows a sub-diffusion model decay curve then their technique for accurately and robustly calculating diffusional kurtosis parameters from multiple diffusion times would be of benefit for tissue microstructural imaging in research and clinical arenas.

Weaknesses: The applied sub-diffusion model has two parameters that are invariant to diffusion time, D_β and β which are used to calculate the diffusional kurtosis measures of a diffusion time dependent D* and a diffusion time invariant K*. However, the authors do not demonstrate that the D_β, β and K* parameters are invariant to diffusion time in brain tissue. The authors' results visually show that there is time dependence of the K* measure (in Figure 6) that is more apparent in white matter with K* values being higher for diffusion times of ∆=49 ms than ∆ = 19 ms. The diffusion time dependence of K* indicates there is also diffusion time dependence of β. Furthermore, Figure 7 shows that there is a tissue specific root mean squared error in model fitting over the two diffusion times which indicates greater deviation from the model fit in white matter than grey matter. To show that the sub-diffusion model is robust and accurate (and consequently that K* is robust and accurate) the authors would have to demonstrate that there is no diffusion time-dependence in both D_β and β in application to brain imaging data for each diffusion time separately. Simulated data should not be used to demonstrate the robustness and accuracy of the sub-diffusion model or to determine optimization of dMRI acquisition parameters without first demonstrating that D_β and β are invariant to diffusion time. This is because simulated signals calculated by using the sub-diffusion charateristic equation of dMRI signal decay will necessarily have diffusion time invariant D_β and β parameters.

Without further information demonstrating diffusion time invariance of D_β, β and K* it is not possible to determine whether the authors have achieved their aims or that their results support their conclusions.

Author Response

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

Thank you for reviewing and assessing our paper. Reviewer2 had only posive comments. Reviewer 1 also had posive comments but included a list of suggesons. The revised version includes text edits to address the suggesons.

Reviewer 1:

… First, it is unclear whether the experiments and analyses were set up to be able to rule out more specific candidate funcons of the ZI.

The list of possible funcons performed by the ZI is broad. Nevertheless, our study considers a rather long list of neural processes related to the behaviors listed below.

Second, many important details of the experiments and their results are hard to decipher given the current descripons and presentaons of the data.

The procedures used in the present study have all been used and described in our previous studies (cited). We used the same descripons and presentaons as in the prior studies. We have gone over the Methods and figures to ensure that all details required to understand the experiments are provided, but we also added further details following the suggesons noted below.

The paper could be significantly strengthened by including more details from each experiment, stronger jusficaons for the limited behaviors and experimental analyses performed, and, finally, a broader analysis of how the recorded acvity in the ZI relates to behavioral parameters.

The paper studied several behaviors including: 1) spontaneous movement of head-fixed mice on a spherical treadmill, 2) tacle (whisker, and body parts) and auditory (tones and white noise) smuli applied to head fixed mice, 3) spontaneous movement iniaon, change, and turns in freely moving mice, 4) auditory tone (frequency and SPL) mapping in freely behaving mice, 5) auditory-evoked orienng head movements (responses) in the context of several behavioral tasks, 6) signaled acve avoidance responses and escapes (AA1), 7) unsignaled/signaled passive avoidance responses (AA2ITI/AA3-CS2), 8) sensory discriminaon (AA3), 9) CS-US interval ming discriminaon (AA4), and 10) USevoked unsignaled escape responses.

In freely moving experiments, the behavior is connuously tracked and decomposed into translaonal and rotaonal movement components. Discrete responses are also evaluated (e.g., acve avoids, escapes, passive avoids, errors, intertrial crossings, latencies, etc.). These behavioral procedures evaluate many neural processes, including decision making (Go/NoGo in AA1-3), response control/inhibion (unsignaled and signaled passive avoidance in AA2/3), and smulus discriminaon (AA3). The applied smuli, discrete responses, and tracked movement are always related to the recorded ZI acvity using a variety of techniques (e.g., cross-correlaons, PSTHs, event-triggered me extracons, etc.), which relate the discrete and me-series parameters to the neural acvity. We do not think all this qualifies as, “limited behaviors”.

(1) Anatomical specificaon: The ZI contains many disnct subdivisions--each with its own topographically organized inputs/outputs and putave funcons. The current manuscript doesn't reference these known divisions or their behavioral disncons, and one cannot tell exactly which poron(s) of the ZI was included in the current study. Moreover, the elongated structure of the ZI makes it very difficult to specifically or completely infect virally. The data could be beter interpreted if the paper included basic informaon on the locaons of recordings, the extent of the AAV spread in the ZI in each viral experiment, and what fracon of infected neurons were inside versus outside ZI.

Our experiments employed Vgat-Cre mice to target ZI neurons. In this line, GABAergic neurons from the enre ZI express Cre, including the dorsal and ventral subdivisions (see (Vong et al., 2011; Hormigo et al., 2020)). Consequently, AAV injecons in Vgat-Cre mice produce restricted expression in the ZI that can fully delineate the nucleus as shown in the papers referenced above (including ours). There is nil expression in structures above or below ZI because they do not express Cre in these mice (e.g., thalamus and subthalamic nucleus), which allows for selecve targeng of ZI. Our optogenec manipulaons and photometry recordings were not aimed at specific ZI subdivisions. We targeted the area of ZI indicated by the stereotaxic coordinates (see Methods), which are aimed at the center of the structure to maximize success in recording/manipulang neurons within ZI. While all the animals included in the study expressed opsins and GCaMP within ZI that in many animals fully delineated the nucleus, there was normal variability in the locaon of opcal fibers, but we did not detect any differences in the results related to these variaons.

Fiber photometry and optogenecs experiments are performed with rather large diameter opcal probes, which record/manipulate relavely large areas of the targeted structure. This is useful because our goal was to idenfy funconal roles of the enre ZI, which could then be parsed. In the present study, we did not perform experiments to target specific ZI populaons (e.g., retrograde Cre expression from target areas), which may have revealed differences atributed to their projecon sites. However, in the last experiment, we selecvely excited ZI fibers targeng three different areas (midbrain tegmentum, superior colliculus, and posterior thalamus), which revealed clear differences on movement. Thus, future experiments should explore these different populaons (e.g., using retrograde/anterograde expression systems), which may be in different subdivisions.

We have enhanced the Methods secon to clarify these points, including the addion of these references.

(2) Electrophysiological recording on the treadmill: The authors are commended for this technically very difficult experiment. The authors do not specify, however, how they knew when they were recording in ZI rather than surrounding structures, parcularly given that recording site lesions were only performed during the last recording session. A map of the locaons of the different classes of units would be valuable data to relate to the literature.

We have added details about this procedure in the Methods secon. These recordings are performed based on coordinates, and categorizing neurons as belonging to ZI is obviously an esmate based on the final histological verificaon. Nevertheless, the marking lesions revealed that the electrodes were on target, which likely resulted from the care taken during the surgical procedure to define reference points used later during the recording sessions (see Methods). Regarding a map of the unit locaons, we performed several analyses that did not reveal clear differences based on site. For example, we compared depth vs cell class, “There was no difference in recording depth between the four classes of neurons (ANOVA F(3,337)= 1.06 p=0.3676)”. Future experiments that employ addional methods (labelling, opto-tagging, etc.) would be more appropriate to address mapping quesons. Finally, as we state in the paper, “However, these recordings do not target GABAergic neurons and may sample some neurons in the tissue surrounding the zona incerta. Therefore, we used calcium imaging fiber photometry to target GABAergic neurons in the zona incerta”.

(3) The raonale of the analysis of acvity with respect to “movement peak”: It is unclear why the authors did not assess how ZI acvity correlates with a broad set of movement parameters, but rather grouped heterogeneous behavioral epochs to analyze firing with respect to “movement peaks”.

The reviewer is referring to movement peaks on the spherical treadmill. On the treadmill, we used the forward locomotor movement of the animal because this is the main acvity of the mice on the treadmill. We considered “all peaks” (or movements) and “>4 sec peaks”, which select for movement onsets. Compared to the treadmill, in freely movement condions during various behavioral tasks, there is a richer behavioral repertoire, which was analyzed in more detail (i.e., translaonal, and rotaonal components during spontaneous ongoing movement and movement onsets, movement related to various behaviors such as orienng, acve and passive avoidance, escape, sensory smulaon, discriminaon, etc.). Thus, we focused on a broader set of movement parameters in the Cre-defined ZI cells of freely behaving mice.

(4) The display of mean categorical data in various figures is interesng, however, the reader cannot gather a very detailed view of ZI firing responses or potenal heterogeneity with so litle informaon about their distribuons.

The PCA performs the heterogeneity classificaon in an unbiased manner, which we feel is a thoughul approach. The firing rates and correlaons with movement for each category of neurons are detailed in the results. Furthermore, the sensory responses for these neurons are also detailed. Together, we think this provides a detailed view of the units we recorded in awake/head-fixed mice. As already stated, further study would benefit from an addional level of cell site verificaon.

(5) Somatosensory firing responses in ZI: It is unclear why the authors chose the specific smuli used in the study. How oen did they evoke reflexive motor responses? What was the latency of sensory-evoked responses in ZI acvity and the latency of the reflexive movement?

These are broad quesons, and we assume that the reviewer is asking about somatosensory evoked responses on the spherical treadmill. We used air-puffs applied to the whiskers and on the back (le vs right) because the whiskers represent an important sensory representaon for mice, and the back is a part of the body (trunk), which we oen use to movate the animals to move forward on the treadmill. Regarding the latency of the somatosensory evoked responses, in this case, we did not correct them based on the me it takes the air-puff to travel to the whiskers or body part, and therefore we did not provide latencies. Moreover, air-puffs are not a very good method to quanfy whisker-evoked latencies, which are beter measured using other methods (whisker deflecons of single/mulple whiskers using piezo-devices or other mechanical devices, as we and others have done in many studies). We are not sure what the reviewer means by “reflexive behavior”; we did not measure any reflexive behavior under these condions. We have gone over the Methods and Results to ensure that sufficient details are provided about these experiments.

(6) It would be valuable to see example traces in Figure 3 to get a beter sense of the me course and contexts under which Ca signals in ZI tracks movement. What is the typical latency? What is the typical range of magnitudes of responses? Does the Ca signal track both fast and slow movements? How are the authors sure that there are no movement arfacts contribung to the calcium imaging? It seems there is more informaon in the dataset that could be valuable.

As is well known, fiber photometry calcium imaging is a slow populaon signal. We do not think it would be valuable to get into ming issues beyond what is already detailed in the study (i.e., magnitudes measured as areas or peaks, and ming as me-to-peaks). Regarding “movement arfacts”, these signals are absent (flat) in animals that do not express GCAMP. We agree that there must be addional valuable informaon in our datasets (as in most me-series). However, the current paper is already rather extensive. We will connue to peruse our datasets and report addional findings in new papers.

(7) Figure 4: The raonale for quanfying the F/Fo responses over a 6-second window, rather than with respect to discrete movement parameters, is not well explained. What types of movement are binned in this approach and might this broad binning hinder the ability to detect more specific relaonships between acvity and movement?

Figure 4 is focused on characterizing the relaonship between turns (ipsiversive and contraversive) during movement and ZI acvity. We tested different binning windows to find differences, including the 6 sec window in figure 4 for populaon measures (-3 to 3 sec around the turns). This binning approach is effecve at revealing differences where they exist (e.g., superior colliculus) as shown in our previous studies (e.g. (Zhou et al., 2023)). Moreover, the turns in the different direcons can be considered discrete responses at their peak, and the ming of the related acvaons (e.g., me to peaks), which we evaluated, are rather sensive and would have revealed differences, but we did not find them.

(8) Separaon of sensory and motor responses in Figure 5: The current data do not adequately differenate whether the responses are sensory or motor given the high correlaon of the sensory inputs driving motor responses. Because isoflurane can diminish auditory responses early in the auditory pathway, this reviewer is not convinced the isoflurane experiments are interpretable.

The reviewer is referring to Fig. 5C,D. Indeed, the point of this experiment was to show that it is difficult to differenate whether neural responses are sensory or motor in awake and freely moving condions. As we stated in the Results secon, “Although arousal and movement were not dissected in the present experiment (this would likely require paralyzing and ventilating the animal), the results indicate that activation of zona incerta neurons by sensory stimulation is primarily associated with states when sensory-evoked movement is also present”. This is followed in the Discussion by, “…as already noted, the suppression of sensory responses may be due to changes in arousal (Castro-Alamancos, 2004; Lee and Dan, 2012) and not caused by the abolishment of the movements per se”.

(9) Given the broad duraon of the mean avoidance response (Fig. 6 C, botom), it would be useful to know to what extent this plot reflects a prolonged behavior or is the result of averaging different animals/trials with different latencies. Given that the shapes of the F/Fo responses in ZI appear similar across avoids and escapes (Fig. 6D), despite their apparent different speeds and movement duraons (Fig 6C), it would be valuable to know how the ming of the F/Fo relates to movement on a trial-by-trial basis.

The duraon of the avoidance response cannot be ascertained from CS onset (panel 6C botom) and avoids are not wide but rather sharp. We have now made this clearer when Fig. 6C is first menoned (“note that since avoids occur at different latencies after CS onset they are best measured from their occurrence as in Fig. 6D”). Like other related condioned and uncondioned responses, avoids and escapes are similar, varying in the noted parameters. Regarding ming, as already menoned above, we think that the characteriscs of the populaon calcium signal make it unsuitable for further ming consideraons than what we included, parcularly for movements occurring at the fast speeds of avoids and escapes.

(10) Lesion quanficaon: One cannot tell what rostral-caudal extent of ZI was lesioned and quanfied in this experiment. It would be easier to interpret if also ploted for each animal, so the reader can tell how reliable the method is. The mean ablaon would be beter shown as a normalized fracon of cells. Although the authors claim the lesions have litle impact on behavior, it appears the incompleteness of the lesions could warrant a more conservave interpretaon.

The lesion experiment was a complement to the optogenecs inacvaon experiments we performed in our preceding ZI paper and in the present paper. Thus, the finding that the lesions had litle impact on behavior is supporve of the optogenecs findings. Regarding cell counts, we did not select any parts of the ZI to quanfy the number of neurons in either control or lesion mice. We considered the full rostrocaudal extent in our measurements. We are not sure what “fracon” the reviewer is suggesng, considering that these counts are from two different groups of mice (control vs lesion). Note that the red-marked neurons, as shown in Fig. 8A, reveal healthy non-Vgat-Cre neurons outside ZI that mark the extent of the AAV diffusion, which as shown spanned the full extent of the ZI in the coronal plane (and in other planes as the AAV spreads in all direcons).

(11) Optogenecs: the locaon of infected neurons is poorly described, including the rostral-caudal extent and the fracon of neurons inside and outside of ZI. Moreover, it is unclear how strongly the optogenec manipulaons in this study are expected to affect neuronal acvity in ZI.

We discussed the first point in (1) above. Regarding, how optogenec manipulaons are expected to affect neuronal acvity in ZI and its targets, we have conducted extensive electrophysiological recordings in slices and in vivo to detail the effects of our manipulaons on GABAergic neurons (e.g. (Hormigo et al., 2016; Hormigo et al., 2019; Hormigo et al., 2021a; Hormigo et al., 2021b), including ZI neurons (Hormigo et al., 2020). In fact, we never use an opsin we have not validated ourselves using electrophysiology. Moreover, our experiments employ a spectrum of optogenec light paterns (including trains/cont at different powers) that trate the optogenec effects within each session/animal. As shown in fig. 11 and 12, these paterns produce different behavioral effects related to the different levels of neural firing they induce. For ChR2-expressing neurons in ZI, firing is frequency dependent and maximal during Cont blue light (at the same power). For Arch-expressing neurons only Cont is used, and inhibion is a funcon of the green light power. When blue light is applied in ZI fibers targeng different areas, this relaonship changes. Blue light trains (1-ms pulses) at 40-66 Hz become the most effecve means of inducing sustained postsynapc inhibion compared to Cont or low frequencies.

References

Castro-Alamancos MA (2004) Dynamics of sensory thalamocorcal synapc networks during informaon processing states. Progress in Neurobiology 74:213-247.

Hormigo S, Vega-Flores G, Castro-Alamancos MA (2016) Basal Ganglia Output Controls Acve Avoidance Behavior. J Neurosci 36:10274-10284.

Hormigo S, Zhou J, Castro-Alamancos MA (2020) Zona Incerta GABAergic Output Controls a Signaled Locomotor Acon in the Midbrain Tegmentum. eNeuro 7.

Hormigo S, Zhou J, Castro-Alamancos MA (2021a) Bidireconal control of orienng behavior by the substana nigra pars reculata: disnct significance of head and whisker movements. eNeuro. Hormigo S, Vega-Flores G, Rovira V, Castro-Alamancos MA (2019) Circuits That Mediate Expression of Signaled Acve Avoidance Converge in the Pedunculoponne Tegmentum. J Neurosci 39:45764594.

Hormigo S, Zhou J, Chabbert D, Shanmugasundaram B, Castro-Alamancos MA (2021b) Basal Ganglia Output Has a Permissive Non-Driving Role in a Signaled Locomotor Acon Mediated by the Midbrain. J Neurosci 41:1529-1552.

Lee SH, Dan Y (2012) Neuromodulaon of brain states. Neuron 76:209-222.

Vong L, Ye C, Yang Z, Choi B, Chua S, Jr., Lowell BB (2011) Lepn acon on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71:142-154.

Zhou J, Hormigo S, Busel N, Castro-Alamancos MA (2023) The Orienng Reflex Reveals Behavioral States Set by Demanding Contexts: Role of the Superior Colliculus. J Neurosci 43:1778-1796.

eLife assessment

This important study uses a range of technical approaches to investigate the responses of zona incerta neurons to movement and sensory stimuli. The majority of neurons exhibited movement related activity but only a small proportion were modulated by whisker deflections. The major conclusion of the study is that the zona incerta distributes a general motor signal. The evidence supporting this claim is solid, although the study would be improved by greater transparency and discussion of experimental methods and histological verification of recording sites, viral spread, and which territories of the zona incerta were investigated. The work will be of interest to behavioral and physiological neuroscientists.

Joint Public Review:

The manuscript presents compelling evidence for the role of the zona incerta area of the brain in regulating movement and sensory stimuli in mice. The study uses appropriate and validated methodology in line with the current state-of-the-art, including optogenetic manipulation and recording of single-unit activity. The authors' claims and conclusions are well-supported by their data, which includes a comprehensive review of previous research on the zona incerta. Overall, the manuscript provides solid evidence for the role of the zona incerta in regulating movement and sensory processing.

Major strengths and weaknesses of the methods and results.<br /> The zona incerta have many integrative functions that link sensory stimuli with motor responses to guide behavior.<br /> The study explored the activation of zona incerta GABAergic neurons during cued avoidance tasks and found that these neurons activate during goal-directed avoidance movement. Optogenetic manipulation of these neurons affected movement speed and performance during active avoidance tasks.<br /> The findings suggest that the zona incerta area of the brain plays a significant role in regulating movement and responding to salient auditory tones in association with movement in mice. The evidence presented is fundamental and provides a comprehensive review of previous research on the zona incerta and its involvement in various behaviors and sensory processing.

The article is very well written, with a correct hypothesis and a cutting-edge methodology to achieve the expected objectives. Moreover, they use statistical rigorous approaches in the analysis of the results. Also, analyzes are performed using scripts that automate all aspects of data analysis, ensuring their objectivity. The results are very novel, and provides solid evidence for the role of the zona incerta in regulating movement and sensory processing.

Author Response

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

We thank the editor and the reviewers for their very useful and constructive comments. We went through the list and gladly received all their suggestions. The reviewers mostly pointed to minor revisions in the text, and we acted on all of those. The one suggestion that required major work was the one raised in point 13, about the processing pipeline being unconvincingly scattered between different tools (R → Python → Matlab). I agree that this was a major annoyance, and I am happy to say we have solved it integrating everything in a recent version of the ethoscopy software (available on biorxiv with DOI https://www.biorxiv.org/content/10.1101/2022.11.28.517675v2 and in press with Bioinformatics Advances). End users will now be able to perform coccinella analysis using ethoscopy only, thus relying on nothing else but Python as their data analysis tool. This revised version of the manuscript now includes two Jupyter Notebooks as supplementary material with a “pre-cooked” sample recipe of how to do that. This should really simplify adoption and provides more details on the pipeline used for phenotyping.

Please find below a point-by-point description of how we incorporated all the reviewers’ excellent suggestions.

Recommendations for the authors: please note that you control which, if any, revisions, to undertake

1) Line 38: "collecting data simultaneously from a large number of individuals with no or limited human intervention" is a bit misleading, as the entire condition the individuals are put in are highly modified by humans and most times "unnatural". I understand the point that once the animals are placed in these environments, then recording takes place without intervention, but it would be nice to rephrase this so that it reflects more accurately what is happening.

We have now rephrased this into the following (L39):

Collecting data simultaneously from a large number of individuals, which can remain undisturbed throughout recording.

2) Line 63: please add a reference to the Ethoscopes so that readers can easily find it.

Done.

2b) And also add how much they cost and the time needed to build them, as this will allow readers to better compare the proposed system against other commercially available ones.

This information is available on the ethoscope manual website (http://lab.gilest.ro/ethoscope). The price of one ethoscope, provided all necessary tools are available, is around ~£75 and the building time very much depends on the skillset of the builder and whether they are building their first ethoscope or subsequent ones. In our experience, building and adopting ethoscopes for the first time is not any more time-expensive than building a (e.g.) deeplabcut setup for the first time. We have added this information to L81

Ethoscopes are open source and can be manufactured by a skilled end-user at a cost of about £75 per machine, mostly building on two off-the-shelf component: a Raspberry Pi microcomputer and a Raspberry Pi NoIR camera overlooking a bespoke 3D printed arena hosting freely moving flies.

3) Line 88: The authors describe that in the current setting, their system is capable of an acquisition rate of 2.2 frames per second (FPS). Would reducing the resolution of the PiCamera allow for higher FPS? I raise this point because the authors state that max velocity over a ten second window is a good feature for classifying behaviors. However, if animals move much faster than the current acquisition rate, they could, for instance, be in position X, move about and be close to the initial position when the next data point is acquired, leading to a measured low max velocity, when in fact the opposite happened. I think it would be good to add a statement addressing this (either data from the literature showing that the low FPS does not compromise data acquisition, or a test where increasing greatly FPS leads to the same results).

We have previously performed a comparison of data analysed using videos captured at different FPSs, which is published in Quentin Geissman’s doctoral Thesis (2018, DOI: https://doi.org/10.25560/69514 ) in chapter 2, section 2.8.3, figure 2.9 ). We have now added this work as one of the references at L95 (reference 19).

4) Still on the low FPS, would a Raspberry Pi 4 help with the sampling rate? Given that they are more powerful than the RPi3 used in the paper?

It would, but it would be a minor increase, leading from 2.2 to probably 3-5 FPS. A significantly higher number of FPSs would be best achieved by lowering the camera’s resolution, as the reviewer’s suggested, or by operating offline. I think the interesting point being implied by the reviewers is that, for Drosophila, the current limits of resolution are more than sufficient. For other animals, perhaps moving more abruptly, they may not. The reviewer is right that we should add a line of caveat about this. We now do so in the discussion, lines 215-224.

Coccinella is a reductionist tool, not meant to replace the behavioural categorization that other tools can offer but to complement it. It relies on raspberry PIs as main acquisition devices, with associated advantages and limitations. Ethoscopes are inexpensive and versatile but have limitations in terms of computing power and acquisition rates. Their online acquisition speed is fast enough to successfully capture the motor activity of different species of Drosophilae28, but may not be sufficient for other animals moving more swiftly, such as zebrafish larvae. Moreover, coccinella cannot apply labels to behaviour (“courting”, “lounging”, “sipping”, “jumping” etc.) but it can successfully identify large behavioural phenotypes and generate unbiased hypothesis on how behaviour – and a nervous system at large – can be influenced by chemicals, genetics, artificial manipulations in general.

5) Along the same line of thought, would using a simple webcam (with similar specs to the PiCamera - ELP has cameras that operate on infrared and are quite affordable too) connected to a more powerful computer lead to higher FPS? - The reason for the question about using a simple webcam is that this would make your system more flexible (especially useful in the current shortage of RPi boards on the market) lowering the barrier for others to use it, increasing the chances for adoption.

Completely bypassing ethoscopes would require the users to setup their own tracking solution, with a final result that may or may not match what we describe here. If a greater temporal resolution is necessary, the easiest way to achieve more FPSs would be to either decrease camera resolution or use the Pis to take videos offline and then process those videos at a later stage. The combination of these two would give FPS acquisition of 60 fps at 720p, which is the maximum the camera can achieve. We now made this clear at lines 83-92.

The temporal and spatial resolution of the collected images depends on the working modality the user chooses. When operating in offline mode, ethoscopes are capable to acquire 720p videos at 60 fps, which is a convenient option with fast moving animals. In this study, we instead opted for the default ethoscope working settings, providing online tracking and realtime parametric extraction, meaning that images are analysed by each raspberry Pi at the very moment they were acquired (Figure 1b). This latter modality limits the temporal resolution of information being processed (one frame every 444 ms ± 127 ms, equivalent to 2.2 fps on a Raspberry Pi3 at a resolution of 1280x960 pixels with each animal being constricted in an ellipse measuring 25.8 ± 1.4 x 9.85 ±1.4 pixels - Figure 1a) but provides the most affordable and high-throughput solution, dispensing the researcher from organising video storage or asynchronous video processing for animals tracking.

6) One last point about decreasing use barrier and increasing adoption: Would it be possible to use DeepLabCut (DLC) to simply annotate each animal (instead of each body part) and feed the extracted data into your current analysis with coccinella? This way different labs that already have pipelines in place that use DLC would have a much easier time in testing and eventually switching to coccinella? I understand that extracting simple maximal velocity this way would be an overkill, but the trade-off would again be a lowering of the adoption barrier.

It would certainly be possible to calculate velocity from the whole animal pose measurement and then use this with HCTSA or Catch22, thus mimicking the coccinella pipeline, but it would be definitely overkilled, as the reviewers correctly points out. Given that we are trying to make an argument about high-throughput data acquisition I would rather not suggest this option in the manuscript.

7) Line 96: The authors state that once data is collected, it is put through a computational frameworkthat uses 7700 tests described in the literature so that meaningful discriminative features are found. I think it would be interesting to expand a bit on the explanation of how this framework deals multiple comparison/multiple testing issues.

We always use the full set of features on aggregate to train a classifier (e.g., TS_Classify in HCTSA) and that means no correction is necessary because the trained classifier only ever makes a single prediction (only one test is performed), so as long as it is done correctly (e.g., proper separation of training and test sets, etc.) then multiple hypothesis correction is not appropriate. This has been confirmed with the HCTSA/Catch22 author (Dr Ben Fulcher, personal communication). We have added a clarifying sentence about this to the methods (L315-318)

8) It would be nice to have a couple of lines explaining the choice of compounds used for testing and also why in some tests, 17 compounds were used, while in others 40, and then 12? I understand how much work it must be in terms of experiment preparation and data collection for these many flies and compounds, but these changes in the compounds used for testing without a more detailed explanation is suboptimal.

This is another good point. We have now added this information to the methods, in a section renamed “choice, handling and preparation of drugs” L280-285, which now reads like this:

The initial preliminary analysis was conducted using a group of 12 compounds “proof of principle” compounds and a solvent control. These compounds were initially used to compare both the video method and ethoscope method. After testing these initial compounds, it was found that the ethoscope methodology was more successful, and then the compound list was expanded to 17 (including the control) only using the ethoscope method. As a final test, we included additional compounds for a single concentration, bringing up the total to 40 (including control), also for the ethoscope method.

9) Line 119 states: "A similar drop in accuracy was observed using a smaller panel of 12 treatments (Supplementary Figure 2a)". It is actually Supplementary Figure 1c.

Thank you for noticing that! Now corrected. The Supplementary figures have also been renamed to obey eLife’s expected nomenclature (both Figure 1 – Figure supplements)

10) In some places the language seems a little outlandish and should either be removed or appropriately qualified. a- Lines 56-59 pose three questions that are either rhetorical or ill-posed. For example, "...minimal amount of information...behavior" implies there is a singular response but the response depends on many details such as to what degree do the authors want to "classify behavior".

Yes, those were meant as rhetorical questions indeed, but we prefer to keep them in, because we are hoping to generate this type of thoughts with the readers. These are concepts that may not be so obvious to someone who is just looking to apply an existing tool and may spring some reflection about what kind of data do they really want/need to acquire.

b) Some of the criticisms leveled at the state-of-the-art methods are probably unwarranted because the goals of the different approaches are different. The current method does not yield the type of rich information that DeepLabCut yields. So, depending on the application DeepLabCut may be the method of choice. The authors of the current manuscript should more clearly state that.

In the introduction and discussion we do try to stress that coccinella is not meant to replace tools like DLC. We have now added more emphasis to this concept, for instance to L212:

[tools like deeplabcut] are ideal – and irreplaceable – to identify behavioural patterns and study fine motor control but may be undue for many other uses.

And L215:

Coccinella is a reductionist tool not meant to replace the behavioural categorization that other tools can offer but to complement it

11) The application to sleep data appears suddenly in the manuscript. The authors should attempt to make with text change a smoother transition from drug screen to investigation into sleep.

I agree with this observation. We have now tried to add a couple of sentences to contextualise this experiment and hopefully make the connection appear more natural. Ultimately, this is a proof-ofprinciple example anyway so hopefully the reader will take it for what it is (L169).

Finally, to push the system to its limit, we asked coccinella to find qualitative differences not in pharmacologically induced changes in activity, but in a type of spontaneous behaviour mostly characterised by lack of movement: sleep. In particular, we wondered whether coccinella could provide biological insights comparing conditions of sleep rebound observed after different regimes of sleep deprivation. Drosophila melanogaster is known to show a strong, conserved homeostatic regulation of sleep that forces flies to recover at least in part lost sleep, for instance after a night of forceful sleep deprivation.

11b) Additionally, the beginning section of sleep experiments talks about sleep depth yet the conclusion drawn from sleep rebound says more about the validity of the current 5 min definition of sleep than about sleep depth. If this conclusion was misunderstood, it should be clarified. If it was not, the beginning text of the sleep section should be tailored to better fit the conclusion.

I am afraid we did not a good job at explaining a critical aspect here: the data fed to coccinella are the “raw” activity data, in which we are not making any assumption on the state of the animal. In other words, we do not use the 5-minutes at this or any other point to classify sleep and wakening. Nevertheless, coccinella picks the 300 seconds threshold as the critical one for discerning the two groups. This is interesting because it provides a full agnostic confirmation of the five minutes rule in D. melanogaster. We recognise this was not necessarily obvious from the text and now added a clarification at L189-201:

However, analysis of those same animals during rebound after sleep deprivation showed a clear clustering, segregating the samples in two subsets with separation around the 300 seconds inactivity trigger (Figure 3d). This result is important for two reasons: on one hand, it provides, for the third time, strong evidence that the system is not simply overfitting data of nought biological significance, given that it could not perform any better than a random classifier on the baseline control. On the other hand, coccinella could find biologically relevant differences on rebound data after different regimes of sleep deprivation. Interestingly enough, the 300 seconds threshold that coccinella independently identified has a deep intrinsic significance for the field, for it is considered to be the threshold beyond which flies lose arousal response to external stimuli, defining a “sleep quantum” (i.e.: the minimum amount of time required for transforming inactivity bouts into sleep bouts23,24,28). Coccinella’s analysis ran agnostic of the arbitrary 5-minutes threshold and yet identified the same value as the one able to segregate the two clusters, thus providing an independent confirmation of the fiveminutes rule in D. melanogaster.

12) Line 227: (standard food) - please add a link to a protocol or a detailed description on what is "standard food". This way others can precisely replicate what you are using. This is not my field, but I have the impression that food content/composition for these animals makes big changes in behaviour?

Yes, good point. We have now added the actual recipe to the methods L240:

Fly lines were maintained on a 12-hour light: 12-hour dark (LD) cycle and raised on polenta and yeast-based fly media (agar 96 g, polenta 240 g, fructose 960 g and Brewer’s yeast 1,200 g in 12 litres of water).

13) Data acquisition and processing: please add links to the code used.

Both the code and the raw data used to generate all the figures have been uploaded on Zenodo and available through their repository. Zenodo has a limit of 50GB per uploaded dataset so we had to split everything into two files, with two DOIs, given in the methods (L356, section “code and availability” - DOIs: 10.5281/zenodo.7335575 and 10.5281/zenodo.7393689). We have now also created a landing page for the entire project at http://lab.gilest.ro/coccinella and linked that landing page in the introduction (L64).

13b) Also your pipeline seems to use three different programming languages/environments... Any chance this could be reduced? Maybe there are R packages that can convert csv to matlab compatible formats, so you can avoid the Python step? (nothing against using the current pipeline per se, I am just thinking that for usability and adoption by other labs, the smaller amount of languages, the better?

This is a very important suggestion that highlights a clear limitation of the pipeline. I am happy to say that we worked on this and solved the problem integrating the Python version of Catch22 into the ethoscopy software. This means the two now integrate, and the entire analysis can be run within the Python ecosystem. HCTSA does not have a Python package unfortunately but we still streamlined the process so that one only has to go from Python to Matlab without passing through R. To be honest, Catch22 is the evolution of HCTSA and performs really well so I think that is what most users will want to use. We provide two supplementary notebooks to guide the reader through the process. One explains how to go from ethoscope data to an HCTSA compatible mat file. The other explains how ethoscope data integrate with Catch22 and provides many more examples than the ones found in the paper figures.

14) There are two sections named "References" (which are different from each other) on the manuscript I received and also on BioRxiv. Should one of them be a supplementary reference? Please correct it. I spent a bit of time trying to figure out why cited references in the paper had nothing to do with what was being described...

The second list of references actually applied only to the list of compounds in the supplementary table 1. When generating a collated PDF this appeared at the end of the document and created confusion. We have now amended the heading of that list in the following way, to read more appropriately:

eLife assessment

This study presents an important open-source resource for high-throughput behavioral screening. The protocols employ inexpensive, off the shelf hardware, and allow real-time analysis of hundreds of behaving flies. Although these protocols were developed using Drosophila melanogaster, they could easily be applied to other models. The evidence in support of the conclusions is solid and the revisions carried out by the authors go a long way towards providing the user with an integrated system that is also more user-friendly.

Joint Public Review:

In the current paper, Jones et al. describe a new framework, named "coccinella", for real-time high-throughput behavioral analysis aimed at reducing the cost of analyzing behavior. In the setup used here each fly is confined to a small circular arena and able to walk around on an agar bed spiked with nutrients or pharmacological agents. The new framework, built on the researchers' previously developed platform Ethoscope, relies on relatively low-cost Raspberry Pi video cameras to acquire images at ~0.5 Hz and pull out, in real time, the maximal velocity (parameter extraction) during 10 second windows from each video. Thus, the program produces a text file, and not voluminous videos requiring storage facilities for large amounts of video data, a prohibitive step in many behavioral analyses. The maximal velocity time-series is then fed to an algorithm called Highly Comparative Time-Series Classification (HCTSA)(which itself is based on a large number of feature extraction algorithms) developed by other researchers. HCTSA identifies statistically salient features in the time-series which are then passed on to a type of linear classifier algorithm called support vector machines (SVM). In cases where such analyses are sufficient for characterizing the behaviors of interest this system performs as well as other state-of-the-art systems used in behavioral analysis (e.g., DeepLabCut)

In a pharmacobehavior paradigm testing different chemicals, the authors show that coccinella can identify specific compounds as effectively as other more time-consuming and resource-consuming systems.

The new paradigm should be of interest to researchers involved in drug screens, and more generally, in high-throughput analysis focused on gross locomotor defects in fruit flies such as identification of sleep phenotypes. By extracting/saving only the maximal velocity from video clips, the method is fast. However, the rapidity of the platform comes at a cost--loss of information on subtle but important behavioral alterations. When seeking subtle modifications in animal behavior, solutions like DeepLabCut, which are admittedly slower but far superior in terms of the level of details they yield, would be more appropriate.

The manuscript reads well, and it is scientifically solid. The comments listed below were directed to the original submission and were satisfactorily addressed in the revised version.

1- The fact that Coccinella runs on Ethoscopes, an open source hardware platform described by the same group, is very useful because the relevant publication describes Ethoscope in detail. However, the current version of the paper does not offer details or alternatives for users that would like to test the framework, but do not have an Ethoscope. Would it be possible to overcome this barrier and have coccinella run with any video data (and, thus, potentially be used to analyze data obtained from other animal models)?

2- Readers who want background on the analytical approaches that the platform relies on following maximal velocity extraction, will have to consult the original publications. In particular, the current manuscript does not provide much explanation on Highly Comparative Time-Series Classification (HCTSA) or SVM; this may be reasonable because the methods were developed earlier by others. While some readers may find that the lack of details increases the manuscript's readability, others may be left wanting to see more discussion on these not-so-trivial approaches. In addition, it is worth noting that the same authors that published the HCTSA method, also described a shorter version named catch22, that runs faster with a similar output. Thus, explaining in more detail how HCTSA operates, considering is a relatively new method, will make the method more convincing.

eLife assessment

This paper presents an important contribution to the field of hippocampal registration by introducing a novel surface-based approach that utilizes the topological and morphological features of the hippocampus for anatomical registration across individuals, rather than volumetric-based methods commonly used in the literature. The study provides compelling evidence for the efficacy of this approach using histological samples from three different datasets and offers validation of the method through comparison with traditional volumetric registration. This is significant work given the large number of studies that examine hippocampal shape, thickness, and function in large cohorts, providing strong support for the use of hippocampal unfolding methods in future studies.

Author Response

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

Thank you for reviewing our manuscript. We do find that the reviews are constructive and meaningful. Accordingly, we incorporated most suggestions into our revision. We provided a point-by-point responses to the reviews below.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sexbiased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of nonsynonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

Thank you for your positive comments. Greatly appreciated.

There are, however, parts of the manuscript that are not clearly described or could be otherwise improved.

• The number of denovo-assembled unigenes seems large and I would like to know how it compares to the number of genes in other Cucurbitaceae species. The presence of alternatively assembled isoforms or assembly artifacts may be still high in the final assembly and inflate the numbers of identified sex-biased genes.

The majority of unigenes were annotated by homologs in species of Cucurbitaceae (63%), including Momordica charantia (16.3%), Cucumis melo (11.9%), Cucurbita pepo (11.9%), Cucurbita moschata (11.5%), Cucurbita maxima (10.1%) and other species of Cucurbitaceae (Fig. S1C). We admit that in the final assembly, transcripts may be still overestimated due to the unavoidable presence of isoforms, although we have tried our best to filter it by several strategies of clustering methods. Additionally, we assessed the transcripts using BUSCOv5.4.5 and embryophyta_odb10 database with 1,614 plant orthologs assessment. Some 95.0% of these orthologs were covered by the unigenes, in which 1447 (89.7%) BUSCO genes were “Complete BUSCOs”, 85 (5.3%) were “Fragmented BUSCOs”, and only 82 (5.0%) were “Missing BUSCOs” (Table S2). Overall, our assessment suggested that we have generated high-quality reference transcriptomes in the absence of a reference genome. Subsequently, we revised the manuscript (lines 175-181).

• It is interesting that the majority of sex-biased genes are present in the floral buds but not in the mature flowers. I think this pattern could be explored in more detail, by investigating the expression of male and female sex-biased genes throughout the flower development in the opposite sex. It is also not clear how the expression of the sex-biased genes found in the buds changes when buds and mature flowers are compared within each sex.

Thank you for your advice for further understanding of this interesting pattern. In the near future, we would like to study these issues through more development stages of flowers in each sex, probably with the aid of single-cell techniques and a reference genome. We have revised the manuscript to reflect these in Results, in the section "Tissue-biased/stage-biased gene expression" (lines 202216).

• The statistical analysis of evolutionary rates between male-biased, female-biased, and unbiased genes is performed on samples with very different numbers of observations, therefore, a permutation test seems more appropriate here.

Thank you for your suggestion. However, all comparisons between sex-biased and unbiased genes were tested using Wilcoxon rank sum test in R software, which is more commonly used. Additionally, we tested some datasets, which were consistent with Wilcoxon rank sum test.

• The impact of pleiotropy on the evolutionary rates of male-biased genes is speculative since only two tissue samples (buds and mature flowers) are used. More tissue types need to be included to draw any meaningful conclusions here.

Thank you for your advice for further understanding of the impact of pleitropy. In the near future, we would like make further investigations through more development stages of flowers and new technologies in each sex to consolidate the conclusion.

Reviewer #2 (Public Review):

Summary:

This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

Strengths:

The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

Weaknesses:

The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

Thank you for your meanful suggestions. We agree that the identification of chromosome origins for transcripts would greatly improve the insights of selection, and we will investigate these issues, probably with a reference genome in the near future.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

The main limitation of the study is the very low number of samples analyzed, with only three replicate individuals per sex (i.e. the whole study is built on six individuals only). This provides low power to detect differential expression. Along the same line, only three species were used to evaluate the rates of non-synonymous to synonymous substitutions, which also represents a very limited dataset, in particular when trying to fit parameter-rich models such as those implemented here.

A third limitation relates to the absence of a reference genome for the species, making the use of a de novo transcriptome assembly necessary, which is likely to lead to a large number of incorrectly assembled transcripts. Of course, the production of a reference transcriptome in this non-model species is already a useful resource, but this point should at least be acknowledged somewhere in the manuscript.

Each of these shortcomings is relatively important, and together they strongly limit the scope of the conclusions that can be made, and they should at least be acknowledged more prominently. The study is valuable in spite of these limitations and the topic remains grossly understudied, so I think the study will be of interest to researchers in the field, and hopefully inspire further, more comprehensive analyses.

We acknowledged that our sample size was relatively small. We will investigate these issues at the population level, probably with a reference genome in the near future. We acknowledged in the revised manuscript that there may be some incorrectly assembled transcripts. We assessed the transcripts using BUSCOv5.4.5 and the latest embryophyta_odb10 database with 1,614 plant orthologs assessment. As mentioned, 95.0% of these orthologs were covered by the unigenes, which of 1447 (89.7%) BUSCO genes were “Complete BUSCOs”, 85 (5.3%) were “Fragmented BUSCOs”, and only 82 (5.0%) were “Missing BUSCOs” (Table S2). In short, the quality of transcriptome was high in the absence of a reference genome.

Reviewer #1 (Recommendations For The Authors):

My main criticism of this manuscript is that it refers to gene names and orthogroups throughout the text, however, the assembled transcripts are not accessible. The reference trascriptome, orthology data, and alignments used for evolutionary analysis should be made available through a public repository to support reproducibility and efficient use of produced resources in this study.

We have uploaded these datasets in Researchgate (https://www.researchgate.net/publication/373194650_Trichosanthes_pilosa_datasets Positive_selection_and_relaxed_purifying_selection_contribute_to_rapid_evolution of_male-biased_genes_in_a_dioecious_flowering_plant).

Comments to the authors:

1) I have an issue with the tissue-biased gene expression analysis. Looking at Fig.3, it seems to me there are 3,204 male-biased genes that are expressed at the same level in male buds and mature flowers (same for 5,011 female-biased genes in female buds and flowers), however, only a handful of genes show sex bias between mature male and female flowers. Taking the male-biased genes as an example, if the 3,204 M1BGs experience the same expression levels in mature male flowers and are no longer male-biased when mature male vs female flowers are compared, why there are not found as female tissue biased (F2TGs)? I may be wrong, but one scenario would be that the M1BGs increase their expression in female flowers and become unbiased. However, that increase in expression (low expression in the female buds → higher expression in the female flowers) should classify them as female tissue-biased genes (F2TGs). Can you please clarify how are the M1BGs and F1BGs expressed in the flowers of the opposite sex?

As to Fig. 3A, 3,204 male-biased genes expressed in male floral buds are part of all male-biased genes (3204+286+724=4214), as shown in Fig.2A. However, only 233 male-biased genes (88+1+144=233, Fig.2B and Fig.3B) expressed in male mature flowers. So, they are not expressed at the same level between male floral buds and mature flowers. Only 288 genes are sex-biased (M1BGs), as well as tissue/stage-biased (M1TGs) in male floral buds. M1BGs (4,214 male-biased genes) and F1BGs (5,096 female-biased genes) are 0 overlaps, except for 44,326 unbiasedgenes shown in Fig.2A. That is, F1BGs (5,096 female-biased genes) are low expression or no expression in M1BGs (4,214 male-biased genes). The expression levels of some genes have been shown in Table S14.

2) Paragraph (407-416) describes the analysis of duplicated genes under relaxed selection but there is no mention of this in the results.

In fact, these results have been shown in Table S13. It is not necessary for us to describe them in detail in the results.

3) How did the authors conclude that the identified functions in male flowers make them more adapted to biotic and abiotic environments (line 347-350)? In the paragraph above (line 338-342) the authors describe that female buds are better equipped against herbivores, which are a biotic factor?

Following your concerns, we have revised the manuscript as follows: For line 338-342, we revised the text as “Indeed, functional enrichment analysis in chemical pathways such as terpenoid backbone and diterpenoid biosynthesis indicated that relative to male floral buds, female floral buds had more expressed genes that were equipped to defend against herbivorous insects and pathogens, except for growth and development (Vaughan et al., 2013; Ren et al., 2022) (Fig. S7A and Table S11).” For line 347-350, we revised text as “We also found that male-biased genes with high evolutionary rates in male buds were associated with functions to abiotic stresses and immune responses (Tables S12 and S13), which suggest that male floral buds through rapidly evolving genes are adapted to mountain climate and the environment in Southwest China compared to female floral buds through high gene expression.”

4) Line 417-418: decreasing codon usage bias is linked to decreasing synonymous substitution rates, should this be the opposite?

No. Codon usage bias was positively related to synonymous substitution rates. That is, stronger codon usage bias may be related to higher synonymous substitution rates (Parvathy et al., 2022).

5) Figures and Tables are not standalone and are missing details in the legends. - Fig.2C, which genes are plotted on the heatmap and what is the color scale corresponding to?

• All Supplementary figures are missing the descriptions of individual panels (A, B, C,etc.) in the legends. In addition, please add the numbers of observations under boxplots.

• Supplementary Fig.5 and 6: Panel B is not a Venn diagram, I suggest removing it from the figures.

• Supplementary Fig.7: Should be 'sex-biased genes'. What is the x-axis on the plot?

• Supplementary Fig.8: Please add the description of the abbreviations in the legend. - Supplementary Tables S4, S5, S6: Please add information about the foreground and background branches.

• Supplementary Table S6, S7, S8, S9, S10: Please add more details about the column headers (what is Model-A, background ω 2a, Unconstrained_1.p, K, which was the foreground branch etc.).

• Supplementary Table S11: Please add gene IDs for each KEGG category.

We have revised/fixed these issues following your concerns and suggetions.

Minor comments:

Line 28: 'algae' in place of 'algas'

Line 53-56: Please provide more recent references.

Line65: 'most' instead of 'almost'

Line 86-87: It is not clear from the sentence if the sex-biased expression was detected in flowers compared to leaves, or were the sex-biased genes detected between male and female leaves? Please clarify.

Line 107-108: positive selection is referred to as adaptive evolution, please choose one or the other.

Line 109: 'force' instead of 'forces'

Line 110: 'algae' instead of 'alga'

Line 132: '..mainly distributed from Southwest,' the country is missing.

Line 202: 'protein sequence evolution'?

Line 232: what does the 'number of evolutionary rates' refers to?

Line 253: please provide a reference for the RELAX model.

Line 274: 'relaxed selective male-biased genes' should be 'male-biased genes under relaxed purifying selection'?

Line 318: Please add a sentence explaining why the Cucurbitaceae family is a great model to study the evolution of sexual systems.

Line 321: 'genes' instead of 'gene'.

Line 366: male-biased genes experience 'higher' or 'more rapid' evolutionary rates. line 377: in the present study and in the case of Ectocarpus alga, positive selection plays an important role in male-biased genes evolution, but does not account for the majority of evolutionary change. Therefore, I would not call it a 'primary' force.

Line 477: missing reference for DESeq2 package.

Line 480: 'used'.

Line 498: 'coding sequences'.

Line516: 'to' instead of 'by'.

Line 553: 'the' is repeated twice.

Sorry for the typos and grammatical issues. We have revised them accordingly.

Reviewer #2 (Recommendations For The Authors):

There are two areas for improvement, one empirical and one theoretical.

Empirically, the analyses could be expanded by an attempt to distinguish between genes on the autosomes and the sex chromosomes. Genotypic patterns can be used to provisionally assign transcripts to XY or XX-like behavior when all males are heterozygous and all females are homozygous (fixed X-Y SNPs) and when all females are heterozygous and males are homozygous (lost or silenced Y genes). Comparing such genes to autosomal genes with sex-biased expression would sharpen the results because there are different expectations for the efficacy of selection on sex chromosomes. See this paper (Hough et al. 2014; https://www.pnas.org/doi/abs/10.1073/pnas.1319227111), which should be cited and does in fact identify faster substitution rates in Y-linked genes (and note that pollenexpressed genes, at least, are concentrated on the sex chromosome in this system: https://academic.oup.com/evlett/article/2/4/368/6697528, https://royalsocietypublishing.org/doi/10.1098/rstb.2021.0226).

We have cited Hough et al. 2014 and noticed that several species have been observed to exhibit rapid evolutionary rates of sequences on sex chromosomes compared to autosomes, which has been related to the evolutionary theories of fast-X or fast-Z (lines 482-484).

On the theoretical side, this study is making a very specific intervention, namely identifying more rapid evolutionary rates in genes with male-biased than femalebiased expression in a dioecious plant. The writing in the introduction and the discussion needs to be improved to differentiate between this comparison and similar comparisons, e.g. sex-biased expression in other dioecious plants (76-81), between Xlinked and Y-linked genes (Hough et al. 2014), sex chromosomes and autosome (several studies already cited), gametophytic and sporophytic tissue, and male and female reproductive tissue in hermaphroditic plants. Setting out this distinction early in the introduction will make the specific goals and novelty of this work clearer.

Thank you for your constructive suggestions. We have revised the relevant part of the Introduction accordingly (lines 74-107).

Specific comments by line:

Sorry for the typos or wording issues. We have revised them.

26 - driven not driving

28 - check house style (algae vs algas)

28-29 - consider clarifying the antecedent of "them" (evolutionary forces, not algas) 35 - maybe, but don't the signalling genes involved in stress responses function in many capacities, not just stress? Also, there's evidence that reproductive recognition machinery in plants may ultimately derive from immune function (e.g. https://doi.org/10.1111/j.1469-8137.2008.02403.x), so the GO category "biotic stress" may be too vague

39 - maybe clarify that "for the first time" refers to male rather than female, since there have been other studies in dioecious plants

66-68 - asserting that something is "essential" after describing how rare it is doesn't quite follow, since diecious plants - especially with sex chromosomes - are basically an exception. I agree that understanding the evolution of dioecious plants is important, but this isn't the most compelling way to make that case - perhaps try something else.

137ff - this sentence can be consolidated and streamlined

142 - "floral tissue" rather than "flowers tissue," here and elsewhere

144 - divergence (singular)

235 - "evidence for the contributions of" = "evidences" is unidiomatic 250 - efficiency or efficacy?

300 - why is "inositol" capitalized here and elsewhere?

300ff - are these typical patterns in male tissue in other species?

308 - is that interesting? It seems like exactly what I'd expect. Perhaps start with the unsurprising but reassuring observation (anther and pollen development genes are indeed expressed in male buds) before moving on to the more surprising findings.

319 - remove "the"

321 - genes (plural)

330 - replace "these differences" with "the differences" 336 - perhaps recap proportions / percents here?

340 - unnecessary comma after diterpenoid

341 - this seems like a big leap from the evidence, especially in the absence of supporting information about the chemical defenses of these species and how they differ by sex. Don't terpenoids have a diverse array of functions, not just defense? Here's a review: https://link.springer.com/chapter/10.1007/10_2014_295

We have revised the text as “Indeed, functional enrichment analysis in chemical pathways such as terpenoid backbone and diterpenoid biosynthesis indicated that relative to male floral buds, female floral buds had more expressed genes that were equipped to defend against herbivorous insects and pathogens, except for growth and development (Vaughan et al., 2013; Ren et al., 2022) (Fig. S7A and Table S11)” (lines 373-378).

349 - as mentioned in line 35, this is a big speculative leap. The discussion is the place for speculation, but consider other explanations too. How does the development of flowers work? Are male flowers suppressing or resorbing female primordial organs? Do male flowers in fact senesce faster? perhaps spell out the logic in more detail.

We have revised the text as “In addition, the enrichment in regulation of autophagy pathways could be associated with gamete development and the senescence of male floral buds (Table S14) (Liu and Bassham, 2012; Li et al., 2020; Zhou et al., 2021). In fact, it was observed that male flowers senesced faster (Wu et al., 2011). We also found that homologous genes of two male-biased genes in floral buds (Table S14) that control the raceme inflorescence development (Teo et al., 2014) were highly expressed compared to female floral buds. Taken together, these results indicate that expression changes in sex-biased genes, rather than sex-specific genes play different roles in sexual dimorphic traits in physiology and morphology (Dawson and Geber, 1999).” (lines 390-402).

351 - senescence of, not senescence for

363 - but Hough et al. 2014 did show rapid evolution of Y-linked genes, and those are by definition sex biased ...

391 - perhaps reiterate here that while some sex-BIASED genes did, sex-SPECIFIC genes did not, to avoid confusion

We also revised them accordingly.

Reviewer #3 (Recommendations For The Authors):

1- lines 56-57 : « have facilitated » : this wording confounds correlation with causation. Consider rephrasing as « is associated with »

2- lines 58-60 : vague wording : what are these variations ? e.g. which tissues and stages are generally enriched?

3- line 63 : this sentence is a bit misleading: consider changing it to « Most dioecious plants possess homomorphic sex-chromosomes » [and explain what homomorphic means in this context].

4- line 68 : a reference is missing here. Also perhaps, allude to the fact that sexual selection in plants has long been considered a contentious issue (e.g. https://doi.org/10.1016/j.cub.2010.12.035)

5- lines 72-76 : beyond simply describing the pattern, say what evolutionary processes are revealed by these observations.

6- line 92 : remind the reader what these 5 studies are.

7- line 94-95 : explain why the comparison of vegetative vs vegetative and vegetative vs reproductive tissues is a problem.

The published studies only compared gene expression in vegetative versus vegetative tissues and vegetative versus reproductive tissues. Because it limited our understanding of sexual selection at different floral development stages. Revised accordingly (lines 103-104). We are very interested in flower development stage for sex-biased genes. The datasets could investigate sexual selection using two developmental stage (buds + mature flowers).

8- line 100 « Evolutionary dynamic analyses » : this wording is vague

9- line 110 : brown algae are NOT plants

10- line 137-140 or in M&M : needs to describe somewhere how the male flowers differ from the female flowers and vice-versa: are the whole morphological structures related to female (male) reproduction entirely missing, or is their development arrested later on and they are still present but simply not producing gametes? This has consequences for the interpretation of the genes they express.

We have revised the typos or wording issues accordingly. However, because the sampled floral buds were equal or less than 3 mm in size, we did not observe much morphological structural difference. Indeed, the male and female flowers at antheses were markedly different in this dioecious plant as shown in Fig. 1. Additionally, we found that dioecy is the ancestral state of Trichosanthes, and transitions to monoecy (Guo et al., 2020) based on our analysis (not shown in this study), which suggest that in the early stages of flower development, female floral buds may tend to masculinize, and vice versa (Fig. 2C).

11- line 152 : it is important to be very transparent on the sample sizes here: « from three females and three males of the dioecious ... »

12- line 153 : along the same line, explain here why a de novo transcriptome had to be generated here: « In the absence of an assembled reference genome for this nonmodel species, we de novo assembled ... »

13- line 164-165 : « we have generated high-quality reference trancriptomes » : I am not entirely convinced of the quality of the transcriptome obtained without a reference genome, so I suggest simply removing this subjective sentence.

Our assessment suggested that we have generated high-quality reference transcriptomes in the absence of a reference genome, which will be the next step of our work.

14- line 169 : briefly explain the criteria used to call differentially expressed genes. Given the threshold (log-fold change >=1.3 if I read the figure correctly, but the M&M says >=1), explain how it was chosen.

Sorry, you may have misunderstood the X, Y coordinates. The value of y coordinate represents -log10(FDR), and the value of x coordinate represents log2 (Fold Change).

15- line 174 : Not clear to me how Fig2C is « revealing strong sexual dimorphism », since genes cluster neither by sex nor by tissue. This should be explained more clearly.

16- line 174-177 : The fact that more ex-biased genes were identified in early buds than in mature flowers is an interesting observation that could be given more prominence in the manuscript, but it is not really explained. Could it reflect the fact that more genes are expressed in early buds because meiotic processes happen early in flower development? Also, the genes involved in male and female organ cell fate determination might also be expected to be expressed early, with mostly organ growth genes being expressed in the mature flower.

17- line 181 : a wrap-up sentence might be useful here to drive the point home that sex-bias is more prevalent in buds than mature flowers.

18- line 184 : « tissue-biased » : a more appropriate wording here would be « stagebiased », no ? These are indeed the same tissues but at different developmental stages.

19- line 183-195 : this section could benefit from setting clear expectations in a hypothesis testing framework laying out the reasons to expect a different bias between stages and between sexes. How does that fit with the level of morphological divergence between sexes (relates to my point 10 above).

20- line 197-204. A number of essential pieces of information are missing here: how many species, how divergent, say that one other is dioecious, and precise their relative phylogenetic placement (which is important to understand the models used below). Maybe adding a phylogeny of these species in Figure 4 could be useful. Also, briefly explain the « two-ratio » and « free-ratio » models here.

21- line 196 and following: In these analyses, I could not understand the rationale for keeping buds vs mature flowers as separate analyses throughout. Why not combine both and use the full set of genes showing sex-bias in any tissue? This would increase the power and make the presentation of the results a lot more straightforward.

As you pointed earlier (in the public review, paragraphy 2), “the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers)”, we totally agree with your points and were very interested in floral development stages for sex-biased genes.

22- line 216 : say explicitly that the reason for not detecting a significant difference in spite of a relatively large effect size is probably related to the low number of genes, conferring low statistical power to detect a difference. An important feature also not highlighted here is that the trend (though not significant) is in the opposite direction than in the buds, and that both the 2-ratio and the free-ratio models agree on these inverted trends. This could be the basis for an interesting comparison.

Thank you for your suggestions.

23- line 220 : needs to explain more clearly how this « free-ratio » differs from the « two-ratio » model.

24- line 232-234 : I don't see why this is necessary. Why not combine both? See also my comment 21 above.

25- line 237 : the «A-model » was not defined before.

26- line 237 : « male-biased » is missing after 343.

27- line 253-258 : briefly explain what these other models are based on and how they are not redundant and instead complement the previous analyses and each other. 28- line 266-268 : the use of a more precise set of codons for male-biased genes than the others (if I understood correctly) could also be interpreted as another sign of stronger selective constraint, no?

Codon usage bias is influenced by many factors, such as levels of gene expression. Highly expressed genes have a stronger codon usage bias and could be encoded by optimal codons for more efficient translation (Frumkin et al., 2018; Parvathy et al., 2022).

29- line 269-291 : removing genes on a post-hoc basis seems statistically suspicious to me. I don't think your analysis has enough power to hand-pick specific categories of genes, and it is not clear what this brings here. I suggest simply removing these analyses and paragraphs.

30- line 325 : say whether this patterns parallels / or not those in animals.

31- line 335 : yes, these biological pieces of information are important and should be given in the introduction already.

32- the discussion should focus at some point on the observation that more femalebiased genes are found in general, but that male-biased genes seem to be under greater selection. How do you reconcile these two apparently contradictory observations?

We found that male-biased genes with high evolutionary rates in male floral buds were associated with functions to abiotic stresses and immune responses (Tables S12 and S13), which suggests that male floral buds through rapidly evolving genes are adapted to mountain climate and the environment in Southwest China compared to female floral buds through high gene expression (lines 387-390).

33- line 355 : not clear how this follows from the previous sentences.

34- line 356-358 : vagiue. not clear what the message of this sentence is.

35- line 378-383 : say that these conclusions rely on the quality of gene annotation in this non-model species, which is probably pretty low (just like any other non-model species).

36- line 403 : this conclusion seems far-fetched. Explain how exactly you reached this conclusion.

37- line 406-416: these speculations on the role of paralogs seem unnecessary, in particular since the de novo transcriptome onto which all analyses are based cannot distinguish orthologs from paralogs.

38- line 417-424. The discussion should not contain new results.

39- line 510 : why were genes with dN/dS >2 discarded here? This might strongly bias the comparison, no? This needs to be properly justified.

40- lines 516-523 : references to the models are missing.

41- line 527: « omega = 1.5 » : why/how was this arbitrary threshold chosen?

42- Fig 2 : write out « buds » and « mature flowers » on top of the graphs

43- Fig 4 : add a phylogeny of the species showing the branch being compared. Also, add the number of genes in each category on each plot.

Thanks, we revised/fixed these issues accordingly.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

eLife assessment

This valuable paper examines gene expression differences between male and female individuals over the course of flower development in the dioecious angiosperm Trichosantes pilosa. The authors show that male-biased genes evolve faster than female-biased and unbiased genes. This is frequently observed in animals, but this is the first report of such a pattern in plants. In spite of the limited sample size, the evidence is mostly solid and the methods appropriate for a non-model organism. The resources produced will be used by researchers working in the Cucurbitaceae, and the results obtained advance our understanding of the mechanisms of plant sexual reproduction and its evolutionary implications: as such they will broadly appeal to evolutionary biologists and plant biologists.

Reviewer #2 (Public Review):

Summary:

This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

Strengths:

The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

Weaknesses:

The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

Some aspects of the presentation have been improved in this new version of the manuscript. Specifically:

- the link between sex-biased and tissue-biased genes is now slightly clearer,<br /> - the limitation related to the de novo assembled transcriptome is now formally acknowledged,<br /> - the interpretation of functional categories of the genes identified is more precise,<br /> - the legends of supplementary figures have been improved<br /> - a large number of typos have been fixed.

However, overall the analyses are largely unchanged and the manuscript did not mature much in response to this first round of reviews. As I detail below, many of the relevant and constructive suggestions by the previous reviewers were not taken into account in this revision. For instance:

- Reviewer 2 made precise suggestions for trying to take into account the potential confonding factor of sex-chromosomes. This suggestion was not followed.<br /> - Reviewer 1 & 3 indicated that results were mentioned in the discussion section without having been described before. This was not fixed in this new version.<br /> - Reviewer 1 asked for a comparison between the number of de novo assembled unigenes in this transcriptome and the number of genes in other Cucurbitaceae species. I could not see this comparison reported.<br /> - Reviewer 1 pointed out that permutation tests were more appropriate, but no change was made to the manuscript.<br /> - Reviewer 3 pointed out the small sample size (both for the RNA-seq and the phylogenetic analysis), but again this limitation is not acknowledged very clearly.<br /> - Reviewer 1 & 3 pointed out that Fig 3 was hard to understand and asked for clarifications that I did not see in the text and the figure in unchanged.<br /> - Reviewer 3 suggested to combine all genes with sex-bias expression when evaluating the evolutionary rate, in addition to the analyses already done. This suggestion was not followed.<br /> - Reviewer 3 pointed out that hand-picking specific categories of genes was not statistically valid, and in fact not necessary in the present context. This was not changed.<br /> - Reviewer 1 asked for all data to be public, but I could not find in the manuscript where the link to the data on ResearchGate was provided.<br /> - Reviewers 1 & 3 pointed out that since only two tissues were compared, the claims on pleiotropy should have been toned down, but no change was made to the text.<br /> - Reviewer 1 asked for a clarification on which genes are plotted on the heatmap of Fig3C and an explanation of the color scale. No change was made.<br /> - Reviewer 1 asked for panel B in Fig 5 and 6 to be removed. They are still there. They asked for abbreviations to be explained in the legend of Fig S8. This was not done. They asked for details about coluln headers. Such detailed were not added. They asked for more recent references on line 53-56 : this was not done.

eLife assessment

This manuscript aims to identify the pacemaker cells in the lymphatic collecting vessels - the cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the exemplary use of existing approaches (genetic deletions and cytosolic calcium detection in multiple cell types), the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction. This fundamental discovery establishes a new standard for the field of lymphatic physiology.

Reviewer #1 (Public Review):

Summary:

This manuscript explores the multiple cell types present in the wall of murine-collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.

Strengths:

The experiments are rigorously performed, the data justify the conclusions, and the limitations of the study are appropriately discussed.

There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.

Weaknesses:

My only major comment would be that the manuscript provides a lot of rich information describing the cellular components of the muscular lymphatic vessel wall and that these data are not well represented by the title. The title (while currently accurate) could be tweaked to better represent all that is in this manuscript. Maybe something like "Characterization/Interrogation of the cellular components of murine collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions" or "Discovery/Confirmation of lymphatic muscle cells as innate pacemaker cells of lymphatic contraction through characterization of the cellular components of murine collecting lymphatic vessels". Potentially a cartoon summary figure of the components that make up the collecting lymphatic vessel wall could also be included. In my opinion, these changes will make this manuscript of more interest to a broader group of scientists. I have a few additional comments for consideration to improve the clarity and enhance the discussion of this work.

Reviewer #2 (Public Review):

Summary:

This is a well-written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine-collecting lymphatics. Using state-of-the-art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.

Strengths:

The use of a targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.

Weaknesses:

The only significant weakness was the lack of quantitative analysis of most of the imaging data shown in Figures 1-11. In particular, the colonization analysis should be extended to show cells not expected to demonstrate colocalization as a negative control for the colocalization analysis that the authors present.

Reviewer #3 (Public Review):

Summary:

Zawieja et al. aimed to identify the pacemaker cells in the lymphatic collecting vessels. Authors have used various Cre-based expression systems and optogentic tools to identify these cells. Their findings suggest these cells are lymphatic muscle cells that drive the pacemaker activity in the lymphatic collecting vessels.

Strengths:

The authors have used multiple approaches to test their hypothesis. Some findings are presented as qualitative images, while some quantitative measurements are provided.

Weaknesses:

- More quantitative measurements.<br /> - Possible mechanisms associated with the pacemaker activity.<br /> - Membrane potential measurements.

Author Response

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

We thank the reviewers and editors for their thoughtful assessment and critiques. As detailed below in the point-by-point replies, we have modified the text and figures to clarify points of ambiguity and to document statistical significance in places where we had inadvertently neglected to do so. The manuscript is clearer and more rigorous as a result of the review process.

Reviewer #1 (Public Review):

This study addresses the fundamental question of how the nucleotide, associated with the beta-subunit of the tubulin dimer, dictates the tubulin-tubulin interaction strength in the microtubule polymer. This problem has been a topic of debate in the field for over a decade, and it is essential for understanding microtubule dynamics.

McCormick and colleagues focus their attention on two hypotheses, which they call the "self-acting" model and the "interface-acting" model. Both models have been previously discussed in the literature and they are related to the specific way, in which the GTP hydrolysis in the beta-tubulin subunit exerts an effect on the microtubule lattice. The authors argue that the two considered models can be discriminated based on a quantitative analysis of the sensitivity of the growth rates at the plus- and minus-ends of microtubules to the concentration of GDP-tubulins in mixed nucleotide (GDP/GMPCPP) experiments. By combing computational simulations and in vitro observations, they conclude that the tubulin-tubulin interaction strength is determined by the interfacial nucleotide.

The major strength of the paper is a systematic and thorough consideration of GDP as a modulator of microtubule dynamics, which brings novel insights about the structure of the stabilizing cap on the growing microtubule end.

I think that the study is interesting and valuable for the field, but it could be improved by addressing the following critical points and suggestions. They concern (1) the statistical significance of the main experimental finding about the distinct sensitivity of the plus- and minus-ends of microtubules to the GTP-tubulin concentration in solution, and (2) the validity of the formulation of the "self-acting" model with an emphasis solely on the longitudinal bonds.

We thank the reviewer for the comment about statistical significance, and we regret our oversight to have not included that analysis in the original manuscript. We have now included an analysis of statistical significance for the main experimental results supporting the interface-acting model (Fig. 2C and the replotting of those data against a different abscissa in Fig. 3C,D), and more broadly we have ensured that all figure legends contain information about the number of measurements and whether error bars indicate SD or SEM.

The reviewers comment about the sole emphasis on longitudinal bonds helped us realize that a change to Fig. 1 (where we illustrate the two models) would improve clarity. We had originally chosen to illustrate Figure 1 using ‘pure’ longitudinal interactions (with no lateral contacts), and this may be what triggered the reviewer’s comment. We have now revised the figure to show ‘corner’ (longitudinal + lateral) interactions. There are two main reasons for this decision. First, the corner interactions are more long-lived and therefore more important for the phenomena under study. Second, because illustrating corner interactions provides a better basis for us to discuss what is a subtle aspect of our model – that the ‘GDP penalty’ affecting longitudinal or lateral interactions in a corner site is completely equivalent. Thus, our model is not quite as narrow/exclusive as the reviewer suggested. We appreciate having had the chance to clarify this.

Reviewer #2 (Public Review):

McCormick, Cleary et al., explore the question of how the nucleotide state of the tubulin heterodimer affects the interaction between adjacent tubulins.

(1) The setup of the authors' model, which attributes the dynamic properties of the growing microtubule only to the differences in interface binding affinities, is unrealistic. They excluded the influence of the nucleotide-dependent global conformational changes even in the 'Self-Acting Nucleodide' model (Fig. 1A). As the authors have found earlier, tubulin in its unassembled state may be curved irrespective of the species of the bound nucleotide (Rice et al., 2008, doi: 10.1073/pnas.0801155105), but at the growing end of microtubules, the situation could be different. Considering the recently published papers from other laboratories, it may be more appropriate to include the nucleotide-dependent change in the tubulin conformation in the Self-Acting Nucleotide model.

We understand the reviewer’s perspective, which may be summarized as: “We know conformational changes are happening and that they affect tubulin:tubulin interactions, so why isn’t your model trying to account for that?” In text added to the revised manuscript, we address this critique in the following ways. First, there is not a consensus in the field about how to parameterize the different conformations of tubulin and how they influence tubulin:tubulin interactions. Second, any attempt to explicitly account for different conformations of tubulin would substantially increase the number of adjustable model parameters, which in turn makes the fitting to growth rates more complicated. Third, compared to traditional ‘dynamics’ assays that use GTP, using mixtures of GMPCPP and GDP simplifies the biochemistry by eliminating GTPase. This results in a more uniform composition of nucleotide state in the body of the microtubule polymer, which diminishes the importance of explicitly modeling nucleotide-influenced changes in conformation. Fourth, it seems likely that different conformations of tubulin will modulate both longitudinal interactions (as tubulin becomes straighter the longitudinal contact area grows larger) and lateral interactions (as tubulin becomes straighter, the lateral contact areas on α- and β-tubulin come into better alignment). Our model treats longitudinal and corner (defined as longitudinal + lateral) interactions as independent, so in principle it could be implicitly capturing some of these conformational effects. By refining the strengths of the longitudinal and corner interactions independently, the model effectively allows the strength of longitudinal contacts to be different for pure longitudinal and corner interactions, which might implicitly capture some variations in longitudinal contacts for different tubulin conformations. Our model treats ‘bucket’-type sites (one longitudinal and two lateral interactions) as simply having an additional lateral interaction of equal strength as the first, but because bucket sites have such a high affinity, they rarely dissociate and this small oversimplification is unlikely to have a substantial effect. We have introduced text in several places (bottom of p. 7 and elsewhere) to cover these points.

(2) The result that the minus end is insensitive to GDP (Fig. 2) was previously published in a paper by Tanaka-Takiguchi et al. (doi: 10.1006/jmbi.1998.1877). The exact experimental condition was different from the one used in Fig. 2, but the essential point of the finding is the same. The authors should cite the preceding work, and discuss the similarities and differences, as compared to their own results.

Thank you for reminding us of this paper! We agree that it is an ‘on target’ citation, and have cited and discussed it in the revised manuscript (last paragraph of Introduction, third paragraph of Discussion).

Reviewer #1 (Recommendations For The Authors):

1) In my opinion, the way in which the authors have depicted their "self-acting" model in Fig. 1 and in Supplementary Figure 1, makes the model look intuitively implausible. The drawings seem to imply that at the plus-end the GTP hydrolysis in the beta-tubulin subunit somehow allosterically affects the alpha-tubulin subunit of the same dimer to weaken its longitudinal bond with adjacent tubulin dimer. Conversely, at the minus end, the same reaction now affects the very same beta-tubulin subunit, and modulates its longitudinal interaction with the next dimer.

However, a more realistic formulation of the "self-acting" model would be that the exchangeable nucleotide affects the lateral bonds, formed by the same beta-tubulin with its lateral neighbors. Although the experimental data in this regard are controversial, at least some supporting evidence for this idea comes from structural arguments, e.g. [Manka, S.W., Moores, C.A. Nat Struct Mol Biol 25, 607-615 (2018).] This "lateral selfacting", but not the "longitudinal self-acting" hypothesis, seems more natural, and it was the one previously implemented in the seminal paper by [Vanburen et al, 2002 Proceedings of the National Academy of Sciences 99.9 (2002): 6035-6040.] and later by other some other models as well.

This point has been addressed above, in part by modifying the cartoon in Fig. 1.

2) To better clarify, which exact models are considered in this manuscript, it would be helpful if the authors provided a detailed table with all simulation parameters, including, k_off_loner, k_off_bucket and k_off_corner, for both nucleotide states, in both the selfacting and the interface-acting models.

Thank you for the suggestion. We have added tables that show all simulation parameters, as well as the corresponding calculated on- and off-rates for each interaction.

3) I am not sure that using some 'arbitrarily chosen' parameters is very helpful in Chapter 1 of Results. In fact, the results, obtained with an unconstrained set of parameters may be misleading or provide ambiguous answers. In other words, how reliable are the conclusions, based on the arbitrary parameter set? For example, could the dependences of the microtubule growth rate on the GDP-tubulin content be more or less pronounced with a different set of arbitrarily chosen parameters, compared to the graphs in Fig. 1BC?

This is a fair criticism. In response, we have added three new sets of simulations that each test different choices of the biochemical parameters used in Figure 1. With respect to the original parameters, we tested a weaker and stronger choice for the longitudinal interaction (KDlong, a 100-fold range), the corner interaction (KDcorner, a 25-fold range), and the GDP weakening factor (a 100-fold range). The predicted supersensitivity of plus-end growth rates to GDP in the self-acting vs interface-acting mechanisms is robust across the range of different choices for the above parameters (Figure 1 Supplements 1 and 2). Parameters for these new simulations are shown in Tables 3 and 4.

4) It took me some time to comprehend why the minus-end growth rate is assumed to be dependent only on the concentration of the GMPCPP-tubulin (in section 2 of Results). It would be great if the authors simply plotted the simulated dependence of the growth rate on the GMPCPP-tubulin concentration in the case when no GDP-tubulin was added. As I understand, that curve should almost exactly match the dependence observed in Fig 1B, correct? Otherwise, it does not seem obvious, why GDP-tubulin does not impede the minus-end growth. Again, is this conclusion model- and parameterdependent? This question is related to point 3 above.

The minus-end growth rates decrease in proportion to the concentration of GMPCPPtubulin. We have added a note on minus-end growth rates in the Figure 1 legend.

5) I was not quite convinced by the evidence for distinct sensitivities of the plus- and minus-end growth rates to GDP-tubulin concentration (Figure 2C and Fig 3C, D). These are the key experimental measurements in the paper. Therefore, I suggest that the authors try to strengthen this point by additional measurements to increase statistics. Or at least, please, explain the data points, the error bars, and provide some information on the number of independent measurements and the statistical significance between the curves. Maybe, they could be directly compared after normalizing by the "all GMPCPP growth rate"? How was the "1.5-fold" ratio obtained in Fig 2C? Does that number refer only to a certain GDP-tubulin concentration or does that value somehow characterize the whole range of the concentrations measured?

This has been addressed above.

Reviewer #2 (Recommendations For The Authors):

These look identical to above and were addressed there.

(1) The setup of the authors' model, which attributes the dynamic properties of the growing microtubule only to the differences in interface binding affinities, is unrealistic. They excluded the influence of the nucleotide-dependent global conformational changes even in the 'Self-Acting Nucleodide' model (Fig. 1A). As the authors have found earlier, tubulin in its unassembled state may be curved irrespective of the species of the bound nucleotide (Rice et al., 2008, doi: 10.1073/pnas.0801155105), but at the growing end of microtubules, the situation could be different. Considering the recently published papers from other laboratories, it may be more appropriate to include the nucleotide-dependent change in the tubulin conformation in the Self-Acting Nucleotide model.

(2) The result that the minus end is insensitive to GDP (Fig. 2) was previously published in a paper by Tanaka-Takiguchi et al. (doi: 10.1006/jmbi.1998.1877). The exact experimental condition was different from the one used in Fig. 2, but the essential point of the finding is the same. The authors should cite the preceding work, and discuss the similarities and differences, as compared to their own results.

Reviewer #1 (Public Review):

This study addresses the fundamental question of how the nucleotide, associated with the beta-subunit of the tubulin dimer, dictates the tubulin-tubulin interaction strength in the microtubule polymer. This problem has been a topic of debate in the field for over a decade, and it is essential for understanding microtubule dynamics.

McCormick and colleagues focus their attention on two hypotheses, which they call the "self-acting" model and the "interface-acting" model. Both models have been previously discussed in the literature and they are related to the specific way, in which the GTP hydrolysis in the beta-tubulin subunit exerts an effect on the microtubule lattice. The authors argue that the two considered models can be discriminated based on a quantitative analysis of the sensitivity of the growth rates at the plus- and minus-ends of microtubules to the concentration of GDP-tubulins in mixed nucleotide (GDP/GMPCPP) experiments. By combing computational simulations and in vitro observations, they conclude that the tubulin-tubulin interaction strength is determined by the interfacial nucleotide.

The major strength of the paper is a systematic and thorough consideration of GDP as a modulator of microtubule dynamics, which brings novel insights about the structure of the stabilizing cap on the growing microtubule end.

eLife assessment

This important study combines in vitro experiments with simulations to identify the mechanisms governing modulation of microtubule dynamics by GTP hydrolysis. The authors introduce a convincing new approach by using a mixed GDP/GMPCPP lattice and varying GDP concentration to reveal that the nucleotide at the interface of two tubulin dimers determines the strength of the interaction between two dimers. Overall, the findings will be of interest to biophysicists and cell biologists, especially in the field of microtubule biology.

Reviewer #2 (Public Review):

In their manuscript, McCormick, Cleary et al., explore the question of how the nucleotide state of the tubulin heterodimer affects the interaction between adjacent tubulins. They use a solid combination of biochemical reconstitution assays and modeling to reveal that the nucleotide at the interface of two tubulin dimers determines the strength of the interaction between two dimers. Overall, the findings will be valuable to the field of microtubule biology.

Author Response

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

Response to Public Reviews

Reviewer #1:

We thank this reviewer for their comments on our paper. We have adjusted the methods secon to ensure it is clear, including an updated descripon of the stascs and in some cases updated stascal methods to ensure uniformity in analyses across datasets. The discussion has been modified so that the message regarding our results is set appropriately in the literature.

Reviewer #2:

We are grateful to this reviewer for their insight. We have modified the text of the discussion to address the points of this reviewer, including providing a greater focus on the significance of our results without overgeneralizing. We have addionally reframed our argument regarding the detecon of pescides by Bombus terrestris to more carefully consider conflicng results from other papers.

Response to Recommendaons For The Authors

Response to Reviewer #1

We thank this reviewer for their thoughul comments and ideas. We have made several changes to the text of the manuscript to improve the clarity of our wring, and we are grateful to the reviewer for raising several important points that we had not sufficiently discussed in the paper previously. We feel the paper has been improved with the inclusion of a more thorough discussion and clarified methods. Please see below our responses to the points they raised.

A few general thoughts that I had when reading your manuscript: I assume you have only tested the acve pescide ingredients, but not the formula generally applied in the field. Given that these formulas contain addional compounds but the acve ingredients, might it not be possible that they could be perceived by bees?

For this study, we were interested specifically with the taste of acve pescide compounds, although we agree it could be interesng to explore the taste of other formula compounds, it was not within the scope of this paper to test these.

Is there an alternave to quinine as a negave control? As you state, quinine is generally used in studies and likely oen in concentraons which are beyond what can be seen in e.g. floral nectar, which might explain its aversive effect. I would like to see it tested in natural concentraons and ideally in combinaon with other potenally toxic plant secondary metabolites (PSMs).

The purpose of including quinine in our study was to provide an in-depth characterizaon of “biter” taste responses using the sensilla on bumblebee labial palps and galea (i.e., through the atenuaon of GRN firing). This was not to show how bumblebees may interact with plants containing quinine in the field, or other PSMs. It would indeed be interesng to explore other plant secondary metabolites, however this was beyond the scope of our paper.

L177-187 AND 233-238 Could you, please, provide a photo or schemac drawing to illustrate your assay? I have a very hard me picturing the actual set-up.

We have provided a labeled diagram of the bumblebee mouthparts and sensillum types (Fig 1A), as well as an image of the bumblebee feeding from a capillary in the behavioural assay (Fig 1G). Further details about the feeding assay (including a JoVe video) can be found with the Ma 2016 paper that we cite throughout our methods secon.

L219 Why did you choose 5 sec here?

This feeding bout duraon was selected based on the criteria defined in Ma et al 2016. We have added a citaon to that sentence.

L221-224 How precisely was the behavior scored? Just length of bouts or also repeated short contacts? Please, specify.

We used the first bout duraon and the cumulave bout duraon in our analyses. A sentence has been added to specify this.

L231/233 Please, provide some brief details here, as many readers may find it annoying to find and read another study for methods' details.

We have added three sentences in the methods to further explain the electrophysiological method.

L238-245 See also my general methods comment: concentraons used for pescides and quinine differ quite substanally, which may explain their different effects on the bees' percepon. Are the concentraons used for quinine realisc? If not that is totally fine for a negave control, but it would be interesng to see a comparison of effects conducted for similar concentraons.

The concentraons used of quinine were selected so that they would elicit a known “biter response” – these concentraons are not meant to be field-realisc, and our data (and others, e.g., Tiedeken et al 2014) show that lower concentraons of quinine are not detected by bumblebees.

L277-301 I assume that this is a standard stascal approach to analyze electrophysiological data. However, I am really struggling with fully understanding what you did here. It might be good to add some addional explanaon/detail, e.g. on why you constructed firing rate histograms or how you derived slopes (aren't smulus and bin categorical variables?).

Firing rate histograms are indeed very commonly used for visualizing neuron spikes over me. We have changed the text somewhat in an effort to make it more clear. Likewise, the “slopes” were derived from the LMEs, and in this case “bin” is a connuous me variable – any me variable will involve some binning depending on the resoluon used but should not be considered categorical.

L291-295 As you were averaging and normalizing your data, could you, please, provide some informaon on variaon within animals?

We have now included informaon on the coefficient of variaon for spike rates across sensilla for a given animal/smulus (CV range, median, and IQR).

L295 I assume t-SNE represent a mulvariate approach for ordinaon, correct? Can you explain why you chose to use this approach? Did you use Euclidean Distance?

Yes, t-SNE is a mulvariate technique for dimensionality reducon. It is parcularly well-suited for the visualizaon of high-dimensional datasets, as it can reveal the underlying structure of the data by embedding it in a lower-dimensional space (e.g., 2D) while preserving the local structure of the data as much as possible. We used t-SNE because it has been shown to be effecve in visualizing clusters of similar data points in high-dimensional data. Euclidean distance was used as the distance metric for the t-SNE embedding. Euclidean distance is the default distance metric for most implementaons of t-SNE and is appropriate for connuous data like the spike counts in this study. We have adjusted the methods to clarify this.

L304 Why did you not always use LMEs?

We have adjusted the text to show that we used LME for all comparisons, and the stascs have been updated accordingly in the results secon. None of the outcomes changed with the implementaon of LME for all tests.

L306 Would it not make sense to also include the interacon between smulus and concentraon in your models?

We have now included a sentence to explain that the interacon term was removed due to it being nonsignificant, and the models without the interacon term having beter model fit (determined by having lower AIC and BIC values).

Results:<br /> L337, 339 and more: I would prefer to see actual p-values, not just "p > 0.05".

This has been adjusted on L337 and 339. As far as we are aware, there are no other instances where exact p-values were not given (except when p < 0.0001).

Discussion:<br /> L470 This is true, but the bees' behavior changed significantly, indicang that they may respond to this small change in firing paterns already?

It is true that the bees’ behaviour changed significantly with 0.1mM QUI, but this was not the case with the pescides. Bees drank less overall of 0.1mM QUI than OSR because of the rapid posngesve effects of this compound. It’s important that the duraon of the first bout was not affected (i.e., they didn’t directly avoid it by taste upon first encountering it, as they do with 1mM QUI), but just that they drank less of the 0.1mM QUI over 2 minutes. Post-ingesve effects may occur as quickly as 30s aer inial consumpon. For the pescides, the small changes in GRN firing were not associated with any effects on consumpon or our other measures of feeding behaviour, and we suggest this results from a lack of rapid negave posngesve consequences. We now include further discussion of these important points.

L481 But they consumed significantly less of the 0.1 mM QUI!?

This is true, but they did not reject it (i.e., not drink it at all), and there were no changes in the amount of me the bees spent in contact with the 0.1mM QUI soluon compared to OSR. We have adjusted the text for clarificaon.

L504/505 AND 520/521 AND 533-536 I see your point, but I am wondering whether the bees may need some me but are generally able to learn the taste of pescides, which may explain why e.g. Arce et al. only saw an effect over me. For example, learning to 'focus' on the pescide taste may require some internal feedback (bees not feeling well) or larvae feedback. If I understood right, you tested workers only, which might be less sensive than queens or larvae. I think these aspects should be discussed.

In our study, we invesgated the mechanism of taste detecon of pescides. We agree that bees likely use posngesve mechanisms to learn to associate the locaon (or another cue) of a food source with posive or negave posngesve cues. ‘Focus’ is a higher-order process that involves increased atenon to sensory smuli but does not affect sensaon at the level of the receptor. We show that bees are unable to taste pescides using the gustatory receptors on their mouthparts, so post-ingesve learning would not be able to associate the pescides with any taste cue. Indeed, there may be caste-specific differences with foraging queens, however a discussion of this would be beyond the scope of our paper.

I also recommend broadening the scope of your discussion. For example, you only focus on nectar, while the story might be different for pollen, which is also contaminated with pescides but represents a different chemical matrix with potenally different taste properes. Also, unlike nectar, pollen is collected with tarsae and hence on contact with other bee body parts.<br /> I would also like to see a discussion of your study's implicaons for other bee species and other potenally toxic compounds (e.g. PSMs).

We do not include any data in this paper regarding tarsal or antennal taste or other potenally toxic compounds. In this paper we present one mechanism of biter taste percepon (i.e., of quinine) and specifically show that the buff-tailed bumblebee is unable to taste certain pescides using their mouthparts. To avoid overgeneralizing, we have not included discussions about other species or compounds, which may or may not share similaries with our study.

Response to Reviewer #2

We thank this reviewer for their comments. We have adjusted the text to avoid overgeneralizaons with our conclusions, and atempted to soen language in the discussion that may have been construed as combave towards the Arce et al (2018) paper. We hope this reviewer finds these adjustments to be in line with their expectaons.

1) In two parts of the manuscript, the authors made broad predicons and conclusions beyond what the evidence in the paper can support and wrote "Future studies will be necessary to confirm this." (Lines 508-509) and " Future studies that survey a greater variety of compounds will be necessary to confirm this." (563-564). Instead of making conclusions based on what experimental data in future studies may support, I would ask the authors instead to make conclusions that their current study can support based on experimental evidence in this paper.

We have removed these predicons that extend beyond the scope of the paper.

2) Line 315 "GRNs encode differences in sugar soluon composion". The logic of the tle is wrong.

This has been fixed.

3) Since this study is only performed in one bumblebee species, then I would suggest that the tle be more specific e.g., "Mouthparts of the bumblebee Bombus terrestris exhibit poor acuity for the detecon of pescides in nectar".

We have made this change.

Reviewer #1 (Public Review):

Summary:<br /> Parkinson and colleagues address an interesting and important question, i.e. whether the bumblebee Bombus terrestris can receive field-realistic concentrations of different pesticides in a sugar solution mimicking nectar. The study directly follows up on a previous study conducted by the same team (Kessler et al. 2015, Nature), which was partly questioned by another more recent study (Arce et al. 2018, Proc R. Soc. B). The authors apply a combination of electrophysiological measurements and behavioral feeding tests to answer this question. Their results strongly suggest that B. terrestris workers are not able to perceive field-realistic doses of pesticides in a sugar solution. They additionally show that B. terrestris can physiologically differentiate between solutions varying in sugar composition.

Strengths:<br /> Sophisticated methodology, combination of approaches, clear and precise language. The stats questions have been addressed to my satisfaction. In terms of interpretation, however, several suggestions and comments were provided from an ecological perspective, which was deemed important, while the authors have expressed their intent to concentrate on the electrophysiological mechanism. Given that this study was motivated by conflicting results from earlier research, which were frequently employed to discuss the authors' findings, I still find that the discussion needs to be expanded in order to encompass a wider context.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript is part of the Wright lab's ongoing studies that investigate whether the bumblebee B. terrestris can detect the presence of pesticides when feeding. Previously, they showed that B. terrestris cannot detect neonicotinoids and would prefer food containing neonicotinoids (Kessler et al. 2015). However, in that paper, they showed that B. terrestris cannot taste neonicotinoids but did not provide evidence on why B. terrestris prefer food containing neonicotinoids. In the current paper, the authors continue to suggest that B. terrestris cannot taste neonicotinoids as well as another insecticide, sulfoxaflor, based on additional behavioral experiments and electrophysiological experiments focusing on specific GRNs. While the data from these experiments continue to suggest that B. terrestris cannot taste these insecticides using their mouthparts, whether B. terrestris can actually perceive these insecticides, and why this species prefers food containing these compounds remains unknown.

Strengths:<br /> The authors provided additional evidence that B. terrestris cannot taste neonicotinoids with their mouthparts. The authors have addressed my concerns regarding overgeneralization and that parts of the manuscript were written in a way that sounded combative with studies from other groups that had come to slightly different conclusions from their previous paper.

Author Response

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

We thank the reviewers for recognizing the importance of our work and for their insightful suggestions. A point-by-point response to their comments is listed underneath each reviewer’s section.

Reviewer #1 (Recommendations For The Authors):

Major comments

1) Have the authors optimized the expression level of dCas9? I cannot find this information in this paper or in their 2021 paper. It is important to avoid the toxicity phenomenon that occurs when using guide RNAs that share specific five base seed sequences (referred to as 'bad seeds').

Cui L., Vigouroux A., Rousset F., Varet H., Khanna V., Bikard D. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat. Commun. 2018; 9:1912.

Rostain W., Grebert T., Vyhovskyi D., Thiel Pizarro P., Tshinsele-Van Bellingen G., Cui1 L., Bikard D. Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Research, 2023, gkad170.

2) One guide per gene is highly unusual given that different guides block the RNA polymerase with different efficiency. This was even shown by the Machner lab in the Legionella context in Figure 1c of Ellis et al. 2021 for sidM and vipD. Typically, genes need three guides minimum to ensure that the gene of interest is knocked down fully unless it is not possible as the gene is too small and/or it is difficult to find an NGG sequence. The authors have used one guide per effector, how can they be sure that each gene is knocked down? The Machner lab themselves in Figure 3c of Ellis et al. 2021 shows not all genes targeted using multiplex CRISPRi are equally efficiently knocked down. Please justify why only one guide per gene was chosen and add controls to validate the results. The authors themselves state that the strategy of one guide may be problematic. Lines 315-316 it reads... A possible explanation was the incomplete knockdown of a seemingly important process.

3) Given what the Machner lab observed about spacer location in Ellis et al. 2021 would it not make more sense to take one set of redundant effectors and make multiplex randomized CRISPRi with them in different locations? For Figure 1 at least.

4) Following infection, it seems that the bacteria were not plated onto antibiotic media, so it is not known how well the plasmid harboring guides is kept through infection.

Specific comments

A) The first results paragraph describes the set-up of 10-plex synthesized CRISPR arrays, where 10 effector encoding genes of specific gene families are selected. The rationale of the choice of these genes is not given. Please explain.

B) Please also add some biological data on what these genes code for, and what is their known or predicted function. It is not very informative and exciting to have tables of lpg numbers without any knowledge of what these genes code for and why they were selected, at least some.

C) Figure 1 A Why are only some of the MC arrays shown? Please, at least include in supplementary the others. Again one array in detail would be more informative, showing true knockdown of all genes by qPCR and ideally by western blot.

D) I am not convinced that the gene silencing efficiency qPCR comparison is done in the correct way. In my opinion, each of the genes to be knocked down should be tested against the expression of a control gene e.g. rpoS and then these results should be compared and not the results of empty plasmid or CRISPR array containing plasmid directly. L. pneumophila are very sensitive to growth conditions and inoculum, thus the two strains might not be completely at the same growth stage when being compared which can impact the results.

E) Figure 1 B As stated in general comment number 4, the authors do not appear to plate onto antibiotic so we don't know how well the plasmid harboring the guides is kept through infection. The sustained presence of the guide is particularly important for CRISPRi.

F) The authors found only a few growth phenotypes and mainly this was due to single genes and not combinations of genes. This might again be due to the fact that only one guide per gene was used. How do the authors know that all genes targeted were indeed knocked down?

G) Line 119 Alternatively, the genes were not 100% all knocked down, escaping the knockdown effect expected. Could authors take three genes with three guides each and look at impact instead of only one?

H) The authors then develop the randomized multiplexed arrays and chose to test 44 TME encoding genes. Line 141 Justify why these effectors were chosen in the text.

I) Unfortunately, the method is not clearly described, and many parts are complicated and the text needs to be re-read several times to be understood (lines 150 - 166). Please re-write to better explain to the reader. In line 156 the authors point to a supplementary note 1. This information should be in the main text.

J) What is the copy number of the CRISPR plasmid? Please add in the Material and Method section also the origin of this plasmid.

Figure 2

K) In the paper (line 154-160) and the extra notes, it states that authors attempt to size select CRISPR arrays. However, this is not apparent in Figure 2 schematic. Or are the authors stating that plasmids only containing one guide were selected out? However, line 312 would suggest not. Please clarify

L) A limiting factor in making multiplex guide CRISPR, as the authors are trying to establish in this study, is cloning of multiple guides. In the pre-determined CRISPR arrays in this study, the guides were synthesized. For the randomized multiplex CRISPR in this study, the authors adapt a Golden Gate cloning method to generate multiple sgRNAs in the Cas9 vector. A similar protocol was established in the below paper. Please add this reference.

Zuckermann, M.; Hlevnjak, M.; Yazdanparast, H.; Zapatka, M.; Jones, D.T.W.; Lichter, P.; Gronych, J. A novel cloning strategy for one-step assembly of multiplex CRISPR vectors. Sci. Rep. 2018

M) As the authors note, Zuckermann et al. similarly note that plex of 3 or 4 is most common and above 5 is rare. This thus appears to still be the limiting step of multiplex CRISPR technology. Please discuss

Figure 4

N) The idea of multiplexed CRISPRi seq to address the biological phenomenon of redundancy is an interesting one, however, I am missing the in-depth functional characterization and discussion of at least one of the redundant functions discovered. Please add.

Figure5/6

O) As noted above, the strength of the experiments is undermined by how CRISPRi is set up. Having an average multiplex of 2 or three and again only using one guide per gene weakens the study and the results obtained. Furthermore, as stated in general comment number 4, the authors do not appear to plate onto antibiotic so again, we don't know how well the plasmid harboring the guides is kept through infection. The sustained presence of the guide is particularly important for CRISPRi. Please add a validation that the guides are all present.

Response to Reviewer #1

We are grateful to the reviewers for their insightful comments and suggestions on how to further improve the manuscript.

Regarding the issue of ‘bad seed sequences’ (comment #1), we had previously evaluated the expression level of dcas9 (plotted in Figure 1b of the 2021 Communications Biol paper) and tuned our induction conditions accordingly (40 ng/mL as described in the Methods). Since all strains used in this study express dcas9 from the chromosome, not a plasmid, this eliminates the possibility of fluctuations in expression levels due to variabilities in plasmid copy numbers.

In the rare event that toxicity by any given guide occurs, we would expect that guide to already be underrepresented or missing in the input pool following 24+ hours of CRISPRi induction during axenic growth. Our data, now discussed in the manuscript (Lines 211-216 and Figure S2), revealed that this was not the case and that all guide-encoding spacers were present in roughly equal amounts (median of >5000 occurrences). As with any knockdown study, the creation of true chromosome deletions was performed throughout as to alleviate the issue of false positives.

Regarding comments #2, #3, and specific comments made under point F, G, and O, on the topic of using single vs. multiple guides, we agree that there are circumstances under which using more than one guide per target may be advantageous, for example when attempting to delete a gene from mammalian cells using conventional CRISPR engineering. In the study described here, this is not the case. In fact, we did create a second array library with alternative guides targeting the same group of genes at locations other than the “optimal location” identified in our 2021 paper and found that these “sub-optimal” guides were inefficient for identifying critical effectors as described in Supplemental Note S1 under the heading “Sub-optimal annealing sites” (Lines 919+). These data suggest that adding sub-optimal guides to the arrays of optimal guides might ‘poison’ the arrays and limit rather than enhance their ability to identify gene combinations.

Regarding comment #2, #3, and specific comments made under point C, F, and G, on the topic of confirming efficient gene knockdown for the identification of critical genes, we remind Reviewer 1 that we did confirm knockdown of 60 of the target genes of the 10-plex screen to be at least 2-fold, with an average fold repression of one order of magnitude or more (Figure 1A). While knockdown of every gene in every 10-plex construct would be an unprecedented ask of any published CRISPR screen, we believe that these 60 genes provide a large enough sampling of all guides to elucidate the range of knockdown to be expected by our CRISPRi platform. As with other knockdown technologies, such as RNAi, there is no expectation of accomplishing complete knockdown for any given target. Hence, the data in Figure 1A suggest that the lack of identifying critical genes using pre-determined 10-plex arrays was not due to a lack of knockdown efficiency, but rather the difficulty to accurately predict redundancy within a cohort of uncharacterized genes, accentuating the need for array randomization with MuRCiS.

On the topic of antibiotic use for plasmid selection (comments #4, E and O), we would like to clarify that the CRISPR plasmids were selected by thymidine prototrophy, not antibiotic resistance, and we apologize for not making this clearer. The laboratory strain Lp02 is a thymidine auxotroph (thyA-) L. pneumophila variant, and plasmid retention is routinely achieved by including the thymidine biosynthesis gene (thyA) on the plasmid backbone. Only with a plasmid bearing the thyA gene can L. pneumophila grow on CYE (thymidine-) plates. Our use of vectors bearing thyA and plating on CYE plates is described in the Methods section. Further, in Figure 7 of our 2021 paper, we show that CRISPR plasmids are efficiently retained by Lp02 for the duration of a 48-hour infection, resulting in efficient multi-gene knockdown even at the end of the intracellular growth experiment.

Regarding comments A and B, on publishing the biological data used to classify genes in gene families for 10-plex silencing, we do not consider it critical to provide additional information beyond the broad classification (e.g. kinases, phosphatases, etc) described in Table S1. Structural predictions constantly change due to continuously evolving databases. Our initial analyses were made in 2015 using HHPRED Hidden-Markov models and, in all likelihood, those predictions have been refined since then. Moreover, with the recent advent of Alphafold, anyone interested in learning more about select effectors from our list is advised to simply access the most recent functional predictions directly on the Alphafold webpage (https://alphafold.ebi.ac.uk/). We clarify how predictions were made on Lines 97-101.

Regarding specific comment D, on our method for qPCR normalization and comparison, we point Reviewer 1 to the Methods section (Lines 460+) where we describe that data obtained from each CRISPRi strain were in fact normalized to the levels of rpsL prior to comparing them to the normalized data from the strain with the empty control plasmid. This normalization to rpsL, a gene encoding a ribosomal protein, also corrects for growth differences between samples.

Regarding specific comment H, the justification for studying 44 transmembrane effector-encoding genes was driven by the fact that activities mediated by transmembrane proteins are difficult (though not impossible) to be replaced by cytosolic proteins, for example the transport of metabolites across the LCV membrane. And since transmembrane regions can be predicted with high confidence, we decided to probe this group of TMEs for synthetic lethality with the randomized CRISPRi approach as proof-of-concept. To make this clearer, we have added more detail to the text (Lines 151-155).

Regarding specific comment I, we have further simplified the description of the cloning technique to increase clarity (Lines 156+). The information listed under Supplemental Note S1, though informative, is not critical for the overall understanding of this highly technical section, and since the reviewer already considered this section to be difficult to follow, we would prefer to not further complicate the text by including these non-essential details.

Regarding the origin of the CRISPRi plasmid (specific comment J), we point Reviewer 1 to the reference (Hammer BK and Swanson MS (Mol Microbiol 1999)) listed in Table S10: Strains and Plasmids Used in this Study.

Regarding specific comment K and O, on the clarity of depicting the CRISPR array size selection process, we have updated the Figure 2 schematic. Reviewer 1 is correct in that despite our best efforts to exclude short CRISPR arrays, inevitably some 1-plex arrays remained in our input vector pool. Still, the average length of all arrays used in our pilot study exceeded three crRNA-encoding spacers. Further, having a population of 1- or 2-plex arrays in our libraries did allow us to pin-point the most critical effectors of a larger arrays within the same MuRCiS experiment (Table S5 and Table S7), a strength of MuRCiS as described in the discussion (Lines 378+).

Regarding specific comment L, we appreciate Reviewer 1’s suggestion of an additional reference and we have added it to the manuscript as reference #23 (Line 71). While this reference does use a Golden Gate strategy to build a multiplex array, that array was not randomized but had a predefined order. Hence, our assembly method is unique due to its randomization.

Regarding specific comment M, on array length cloning limitations, we agree with the conclusion of Zuckermann in Figure 1d of their article that longer inserts are generally harder to get into vector backbones. The challenge of cloning longer inserts is a common phenomenon of general biology and is not unique to cloning CRISPR arrays. We have altered the wording in our manuscript to better describe the intrinsic competition between short and long inserts during cloning (Lines 162-164).

Regarding specific comment N, we second Reviewer 1’s desire to learn more about the critical effector pairs discovered here. With that said, the goal of this manuscript is to report the development of a novel MuRCiS pipeline to identify these critical pairs. Biochemical and molecular investigations of the encoded protein pairs are on-going and will be the topic of a future manuscript.

Reviewer #2 (Recommendations For The Authors):

Specific points

1) The effector repertoire of L. pneumophila seems to have evolved in response to the plethora of potential protozoan hosts (PMID: 31988381). To further assess evolutionary aspects of the vast L. pneumophila effector arsenal, it would be interesting to test the single and double effector mutant strains (Fig. 5FG, Fig. 6EF) for growth in protozoa other than A. castellanii.

2) Most CRISPR arrays comprising genes encoding functionally similar proteins or encoding evolutionarily conserved proteins did not substantially affect intracellular growth of L. pneumophila (Fig. 1B). This rather surprising result should be further discussed.

3) l. 118/119: "Similar results ..., where none of the MC arrays ..." This statement should be phrased more precisely, since some CRISPR arrays did indeed have an effect on intracellular growth of L. pneumophila in U937 macrophages, while none affected intracellular growth in A. castellanii (Fig. 1B).

4) Typos:

• l. 852: ... (arbitrarily set to -100).

• l. 862: ... Legionella-containing vacuole (LCV).

• l. 895: ... and so we would recommend ...

Regarding point 1, we thank Reviewer 2 for the suggestion of testing effector mutants in different hosts. While the primary purpose of the current manuscript was to optimize the MuRCiS platform, future studies using this technology to investigate specific biological questions related to Legionella infection would certainly benefit from including more than one amoebaean species.

Regarding point 2, we agree that the lack of substantial growth defects seems surprising. Yet only two of the seven core effectors (found in all Legionella sp.), lpg2300 and mavN, individually attenuated Legionella intracellular growth when deleted (Burstein 2016 Nat Genetics; Isaac et al., 2015 PNAS). Thus, we hypothesize that the functions many effectors fulfil are of such importance for intracellular survival that that redundancy reaches beyond the boundary of conservation or like-function. We have added a statement emphasizing this at the end of the Figure 1 results section (Line 122-125).

Regarding points 3 and 4, we thank Reviewer 2 for catching these errors and have corrected where needed in the text.

-l. 852 (now Line 874): … (arbitrarily set to -100,000) is correct for Figure 6E.

eLife assessment

This important study uses CRISPRi to silence multiple effectors in the pathogen, Legionella pneumophila. It provides a technique that will allow researchers to address functional redundancy amongst effectors, a problem that has persisted even after decades of study. The methodology used is convincing, and further improvement (such as using multiple guides per gene) can lead to the identification of novel virulence factors.

Reviewer #1 (Public Review):

The article "A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes" describes the development of a multiplex randomized CRISPRi screening method that they named MurCiS and applied it to study redundancy of L. pneumophila virulence factors. The authors used a L. pneumophila strain carrying dCas9 on the chromosome that they had constructed for a CRISPRi screen they had published recently and here combined it with self-assembly randomized multiplex CRISPR arrays that they developed. The strains carrying the dCas9 and the different CRISPRi arrays were used to infect U937 or Acanthamoeba castellanii cells and the intracellular growth phenotypes were recorded as readout. This allowed the authors to identify certain gene combinations that when knocked down induced a growth defect in either or both cells tested but not when they were knocked down alone. A particular gene combination caught their attention, as the genes lpg2888 and lpg3000 were inducing a growth defect only when both were knocked down in U937 cells but in A. castellanii cells lpg3000 alone was sufficient to cause a growth defect.

The concept of using CRISPRi to look at functional redundancy in effectors is a very useful one to the Legionella field and where biological redundancy limits studies. It has the potential to uncover virulence effectors of importance that have not been described before.

Comments on revised version: In this revised version the authors have answered our concerns satisfactorily except the point related to the use of only one guide per gene.

Reviewer #2 (Public Review):

The study by Ellis et al. documents the development of a CRISPR interference (CRISPRi) screen aiming at identifying virulence-critical genes of Legionella pneumophila, the facultative intracellular bacterium causing Legionnaires' disease. L. pneumophila employs the Dot/Icm type IV secretion system to translocate more than 300 different "effector proteins" into host cells. Many effector proteins appear to have redundant functions, and therefore, depleting several of them is required to observe a strong intracellular replication phenotype. In the current study, Ellis et al. develop a "multiplex, randomized CRISPRi sequencing" (MuRCiS) approach to silence several effector genes simultaneously and randomly, thereby possibly causing synthetic lethality for L. pneumophila upon infection of host cells.

The MuRCiS approach comprises the ligation of different CRISPR spacers flanked by repeats in presence of "dead end" oligonucleotide pairs capping a random array of building blocks to be inserted into a library vector. Thus, spacer arrays with an average of 3.3 spacers per array were obtained. As a proof-of-concept, spacer arrays targeting 44 transmembrane effector-encoding L. pneumophila genes were employed to screen for intracellular growth defects in macrophages and amoeba. Consequently, novel pairs of synergistically functioning effector genes were identified by comparative next-generation sequencing of the input and output pools of spacer arrays.

A major strength of this well-written and straightforward study is the construction and use of random and multiplexed CRISPRi arrays, allowing an unbiased and comprehensive screen for multiple genes affecting the intracellular growth of L. pneumophila. The ingenious approach established by Ellis et al. will be useful for further genetic analysis of L. pneumophila infection and might also be adopted for other pathogens employing a large set of (functionally redundant) virulence factors.<br /> The reviewer's suggestion to test the single and double L. pneumophila effector mutant strains for growth in protozoa other than A. castellanii was considered beyond the scope of the current manuscript describing the optimization of the MuRCiS platform. The authors have satisfactorily addressed the minor points raised previously.

Author Response

The following is the authors’ response to the previous reviews

Comments from reviewer 1:

Comment 1. Regarding SBSMMA, the authors may complement their discussion by mentioning recent work (PMID: 35738428) where SBSMMA was used to exemplify a potential fragment-based design approach for developing allosteric effectors for kinases.

Thank you for the suggestion, we have added a short summary of the work where SBSMMA is used as a basis for developing small molecules to target kinases using fragment-based design approach

eLife assessment

One of the most promising strategies in development of drugs targeting kinases is provided by using allosteric control that allows specific regulation and study of kinase function without directly targeting the active site. This important paper reviews convincingly the current repertoire of tools for regulating the activity of protein kinases with the ultimate goal of developing novel approaches in treating diseases associated with signal dysregulation.

Joint Public Review:

This concise review provides a clear and instructive picture of the state-of-the-art understanding of protein kinases' activity and sets of approaches and tools to analyse and regulate it.

Three major parts of the work include: methods to map allosteric communications, tools to control allostery, and allosteric regulation of protein kinases. The work provides an important and timely view of the current status of our understanding of the function of protein kinases and state-of-the-art methods to study its allosteric regulation and to develop allosteric approaches to control it.

Authorr Response

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

Reviewer #1 (Public Review):

The objective of this investigation was to determine whether experimental pain could induce alterations in cortical inhibitory/facilitatory activity observed in TMS-evoked potentials (TEPs). Previous TMS investigations of pain perception had focused on motor evoked potentials (MEPs), which reflect a combination of cortical, spinal, and peripheral activity, as well as restricting the focus to M1. The main strength of this investigation is the combined use of TMS and EEG in the context of experimental pain. More specifically, Experiment 1 investigated whether acute pain altered cortical excitability, reflected in the modulation of TEPs. The main outcome of this study is that relative to non-painful warm stimuli, painful thermal stimuli led to an increase on the amplitude of the TEP N45, with a larger increase associated with higher pain ratings. Because it has been argued that a significant portion of TEPs could reflect auditory potentials elicited by the sound (click) of the TMS, Experiment 2 constituted a control study that aimed to disentangle the cortical response related to TMS and auditory activity. Finally, Experiment 3 aimed to disentangle the cortical response to TMS and reafferent feedback from muscular activity elicited by suprathreshold TMS applied over M1. The fact that the authors accompanied their main experiment with two control experiments strengthens the conclusion that the N45 TEP peak could be implicated in the perception of painful stimuli.

Perhaps, the addition of a highly salient but non-painful stimulus (i.e. from another modality) would have further ruled out that the effects on the N45 are not predominantly related to intensity/saliency of the stimulus rather than to pain per se.

We thank the reviewer for their comment on the possibility of whether stimulus intensity influences the N45 as opposed to pain per se. We agree that the ideal experiment would have included multiple levels of stimulation. We would argue, however, that that in Experiment 1, despite the same level of stimulus intensity for all participants (46 degrees), individual differences in pain ratings were associated with the change in the N45 amplitude, suggesting that the results cannot be explained by stimulus intensity, but rather by pain intensity.

Reviewer #2 (Public Review):

The authors have used transcranial magnetic stimulation (TMS) and motor evoked potentials (MEPs) and TMS-electroencephalography (EEG) evoked potentials (TEPs) to determine how experimental heat pain could induce alterations in these metrics.
In Experiment 1 (n = 29), multiple sustained thermal stimuli were administered over the forearm, with the first, second, and third block of stimuli consisting of warm but non-painful (pre-pain block), painful heat (pain block) and warm but non-painful (post-pain block) temperatures respectively. Painful stimuli led to an increase in the amplitude of the fronto-central N45, with a larger increase associated with higher pain ratings. Experiments 2 and 3 studied the correlation between the increase in the N45 in pain and the effects of a sham stimulation protocol/higher stimulation intensity. They found that the centro-frontal N45 TEP was decreased in acute pain. The study comes from a very strong group in the pain fields with long experience in psychophysics, experimental pain, neuromodulation, and EEG in pain. They are among the first to report on changes in cortical excitability as measured by TMS-EEG over M1. While their results are in line with reductions seen in motor-evoked responses during pain and effort was made to address possible confounding factors (study 2 and 3), there are some points that need attention. In my view the most important are:

1) The method used to calculate the rest motor threshold, which is likely to have overestimated its true value : calculating highly abnormal RMT may lead to suprathreshold stimulations in all instances (Experiment 3) and may lead to somatosensory "contamination" due to re-afferent loops in both "supra" and "infra" (aka. less supra) conditions.

The method used to assess motor threshold was the TMS motor threshold Assessment Tool (MTAT) which estimates motor threshold using maximum likelihood parametric estimation by sequential testing (Awiszus et al., 2003; Awiszus and Borckardt, 2011). This was developed as a quicker alternative for calculating motor threshold compared to the traditional Rossini-Rothwell method which involves determining the lowest intensity that evokes at least 5/10 MEPs of at least 50 microvolts. The method has been shown to achieve the same accuracy of determining motor threshold as the traditional Rossini-Rothwell method, but with fewer pulses (Qi et al., 2011; Silbert et al., 2013).

We have now made this clearer in the manuscript:

“The RMT was determined using the TMS motor thresholding assessment tool, which estimates the TMS intensity required to induce an MEP of 50 microvolts with a 50% probability using maximum likelihood parametric estimation by sequential testing (Awiszus, 2003; Awiszus & Borckardt, 2011). This method has been shown to achieve the accuracy of methods such as the Rossini-Rothwell method (Rossini et al., 1994; Rothwell et al., 1999) but with fewer pulses (Qi, Wu, & Schweighofer, 2011; Silbert, Patterson, Pevcic, Windnagel, & Thickbroom, 2013). The test stimulus intensity was set at 110% RMT to concurrently measure MEPs and TEPs during pre-pain, pain and post-pain blocks.”

Therefore, the high RMTs in our study cannot be explained by the threshold assessment method. Instead, they are likely explained by aspects of the experimental setup that increased the distance between the TMS coil and the scalp, including the layer of foam placed over the coil, the EEG cap and the fact that the electrodes we used had a relatively thick profile. This has been explained in the paper:

“We note that the relatively high RMTs are likely due to aspects of the experimental setup that increased the distance between the TMS coil and the scalp, including the layer of foam placed over the coil, the EEG cap and relatively thick electrodes (6mm)”

Awiszus, F. (2003). TMS and threshold hunting. In Supplements to Clinical neurophysiology (Vol. 56, pp. 13-23). Elsevier.

Qi, F., Wu, A. D., & Schweighofer, N. (2011). Fast estimation of transcranial magnetic stimulation motor threshold. Brain stimulation, 4(1), 50-57.

Silbert, B. I., Patterson, H. I., Pevcic, D. D., Windnagel, K. A., & Thickbroom, G. W. (2013). A comparison of relative-frequency and threshold-hunting methods to determine stimulus intensity in transcranial magnetic stimulation. Clinical Neurophysiology, 124(4), 708-712.

2) The low number of pulses used for TEPs (close to ⅓ of the usual and recommended)

We agree that increasing the number of pulses can increase the signal to noise ratio. During piloting, participants were unable to tolerate the painful stimulus for long periods of time and we were required to minimize the number of pulses per condition.

We note that there is no set advised number of trials in TMS-EEG research. According to the recommendations paper, the number of trials should be based on the outcome measure e.g., TEP peaks vs. frequency domain measures vs. other measures and based on previous studies investigating test-retest reliability (Hernandez-Pavon et al., 2023). The choice of 66 pulses per condition was based on the study by Kerwin et al., (2018) showing that optimal concordance between TEP peaks can be found with 60-100 TMS pulses delivered in the same run (as in the present study). The concordance was particularly higher for the N40 peak at prefrontal electrodes, which was the key peak and electrode cluster in our study. We have made this clearer:

“Current recommendations (Hernandez-Pavon et al., 2023) suggest basing the number of TMS trials per condition on the key outcome measure (e.g., TEP peaks vs. frequency measures) and based on previous test-retest reliability studies. In our study the number of trials was based on a test-retest reliability study by (Kerwin, Keller, Wu, Narayan, & Etkin, 2018) which showed that 60 TMS pulses (delivered in the same run) was sufficient to obtain reliable TEP peaks (i.e., sufficient within-individual concordance between the resultant TEP peaks of each trial).”

Further supporting the reliability of the TEP data in our experiment, we note that the scalp topographies of the TEPs for active TMS at various timepoints (Figures 5, 7 and 9) were similar across all three experiments, especially at 45 ms post-TMS (frontal negative activity, parietal-occipital positive activity).

In addition to this, the interclass correlation coefficient (Two-way fixed, single measure) for the N45 to active suprathreshold TMS across timepoints for each experiment was 0.90 for Experiment 1 (across pre-pain, pain, post-pain time points), 0.74 for Experiment 2 (across pre-pain and pain conditions), and 0.95 for Experiment 3 (across pre-pain conditions). This suggests that even with the fluctuations in the N45 induced by pain, the N45 for each participant was stable across time, further supporting the reliability of our data. These ICCs are now reported in the supplementary material (subheading: Test-retest reliability of N45 Peaks).

Hernandez-Pavon, J. C., Veniero, D., Bergmann, T. O., Belardinelli, P., Bortoletto, M., Casarotto, S., ... & Ilmoniemi, R. J. (2023). TMS combined with EEG: Recommendations and open issues for data collection and analysis. Brain Stimulatio, 16(3), 567-593

Kerwin, L. J., Keller, C. J., Wu, W., Narayan, M., & Etkin, A. (2018). Test-retest reliability of transcranial magnetic stimulation EEG evoked potentials. Brain stimulation, 11(3), 536-544.

Lack of measures to mask auditory noise.

In TMS-EEG research, various masking methods have been proposed to suppress the somatosensory and auditory artefacts resulting from TMS pulses, such as white noise played through headphones to mask the click sound (Ilmoniemi and Kičić, 2010), and a thin layer of foam placed between the TMS coil and EEG cap to minimize the scalp sensation (Massimini et al., 2005). However, recent studies have shown that even when these methods are used, sensory contamination of TEPs is still present, as shown by studies that show commonalities in the signal between active and sensory sham conditions that mimic the auditory/somatosensory aspects of real TMS (Biabani et al., 2019; Conde et al., 2019; Rocchi et al., 2021). This has led many authors (Biabani et al., 2019; Conde et al., 2019) to recommend the use of sham conditions to control for sensory contamination. To separate the direct cortical response to TMS from sensory evoked activity, Experiment 2 included a sham TMS condition that mimicked the auditory/somatosensory aspects of active TMS to determine whether any alterations in the TEP peaks in response to pain were due to changes in sensory evoked activity associated with TMS, as opposed to changes in cortical excitability. Therefore, the lack of auditory masking does not impact the main conclusions of the paper.

We have made this clearer:

“… masking methods have been used to suppress these sensory inputs, (Ilmoniemi and Kičić, 2010; Massimini et al., 2005). However recent studies have shown that even when these methods are used, sensory contamination of TEPs is still present, as shown by commonalities in the signal between active and sensory sham conditions that mimic the auditory/somatosensory aspects of real TMS (Biabani et al., 2019; Conde et al., 2019; Rocchi et al., 2021). This has led many leading authors (Biabani et al., 2019; Conde et al., 2019) to recommend the use of sham conditions to control for sensory contamination.”

Ilmoniemi, R. J., & Kičić, D. (2010). Methodology for combined TMS and EEG. Brain topography, 22, 233-248.

Massimini, M., Ferrarelli, F., Huber, R., Esser, S. K., Singh, H., & Tononi, G. (2005). Breakdown of cortical effective connectivity during sleep. Science, 309(5744), 2228-2232.

Biabani, M., Fornito, A., Mutanen, T. P., Morrow, J., & Rogasch, N. C. (2019). Characterizing and minimizing the contribution of sensory inputs to TMS-evoked potentials. Brain stimulation, 12(6), 1537-1552.

Conde, V., Tomasevic, L., Akopian, I., Stanek, K., Saturnino, G. B., Thielscher, A., ... & Siebner, H. R. (2019). The non-transcranial TMS-evoked potential is an inherent source of ambiguity in TMS-EEG studies. Neuroimage, 185, 300-312.

Rocchi, L., Di Santo, A., Brown, K., Ibáñez, J., Casula, E., Rawji, V., ... & Rothwell, J. (2021). Disentangling EEG responses to TMS due to cortical and peripheral activations. Brain stimulation, 14(1), 4-18.

3) A supra-stimulus heat stimulus not based on individual HPT, that oscillates during the experiment and that lead to large variations in pain intensity across participants is unfortunate.

The choice of whether to calibrate or fix stimulus intensity is a contentious question in experimental pain research. A recent discussion by Adamczyk et al., (2022) explores the pros and cons of each approach and recommends situations where one method may be preferred over the other. That paper suggests that the choice of the methodology is related to the research question – when the main outcome of the research is objective (neurophysiological measures) and researchers are interested in the variability in pain ratings, the fixed approach is preferrable. Given we explored the relationship between MEP/N45 modulation by pain and pain intensity, this question is better explored by using the same stimulus intensity for all participants, as opposed to calibrating the intensity to achieve a similar level of pain across participants.

We have made this clearer:

“Given we were interested in the individual relationship between pain and excitability changes, the fixed temperature of 46ºC ensured larger variability in pain ratings as opposed to calibrating the temperature of the thermode for each participant (Adamczyk et al., 2022).”.

Adamczyk, W. M., Szikszay, T. M., Nahman-Averbuch, H., Skalski, J., Nastaj, J., Gouverneur, P., & Luedtke, K. (2022). To calibrate or not to calibrate? A methodological dilemma in experimental pain research. The Journal of Pain, 23(11), 1823-1832.

So is the lack of report on measures taken to correct for a fortuitous significance (multiple comparison correction) in such a huge number of serial paired tests.

Note that we used a Bayesian approach for all analyses as opposed to the traditional frequentist approach. In contrast to the frequentist approach, the Bayesian approach does not require corrections for multiple comparisons (Gelman et al., 2000) given that they provide a ratio representing the strength of evidence for the null vs. alternative hypotheses as opposed to accepting or rejecting the null hypothesis based on p-values. As such, throughout the paper, we frame our interpretations and conclusions based on the strength of evidence (e.g. anecdotal/weak, moderate, strong, very strong) as opposed to referring to the significance of the effects.

Gelman A, Tuerlinckx F. (2000). Type S error rates for classical and Bayesian single and multiple comparison procedures. Computational statistics, 15(3):373-90.

Reviewer #3 (Public Review):

The present study aims to investigate whether pain influences cortical excitability. To this end, heat pain stimuli are applied to healthy human participants. Simultaneously, TMS pulses are applied to M1 and TMS-evoked potentials (TEPs) and pain ratings are assessed after each TMS pulse. TEPs are used as measures of cortical excitability. The results show that TEP amplitudes at 45 msec (N45) after TMS pulses are higher during painful stimulation than during non-painful warm stimulation. Control experiments indicate that auditory, somatosensory, or proprioceptive effects cannot explain this effect. Considering that the N45 might reflect GABAergic activity, the results suggest that pain changes GABAergic activity. The authors conclude that TEP indices of GABAergic transmission might be useful as biomarkers of pain sensitivity.

Pain-induced cortical excitability changes is an interesting, timely, and potentially clinically relevant topic. The paradigm and the analysis are sound, the results are mostly convincing, and the interpretation is adequate. The following clarifications and revisions might help to improve the manuscript further.

1) Non-painful control condition. In this condition, stimuli are applied at warmth detection threshold. At this intensity, by definition, some stimuli are not perceived as different from the baseline. Thus, this condition might not be perfectly suited to control for the effects of painful vs. non-painful stimulation. This potential confound should be critically discussed.

In Experiment 3, we also collected warmth ratings to confirm whether the pre-pain stimuli were perceived as different from baseline. This detail has been added to them methods:

“In addition to the pain rating in between TMS pulses, we collected a second rating for warmth of the thermal stimulus (0 = neutral, 10 = very warm) to confirm that the participants felt some difference in sensation relative to baseline during the pre-pain block. This data is presented in the supplementary material”.

We did not include these data in the initial submission but have now included it in the supplemental material. These data showed warmth ratings were close to 2/10 on average. This confirms that the non-painful control condition produced some level of non-painful sensation.

2) MEP differences between conditions. The results do not show differences in MEP amplitudes between conditions (BF 1.015). The analysis nevertheless relates MEP differences between conditions to pain ratings. It would be more appropriate to state that in this study, pain did not affect MEP and to remove the correlation analysis and its interpretation from the manuscript.

The interindividual relationship between changes in MEP amplitude and individual pain rating is statistically independent from the overall group level effect of pain on MEP amplitude. Therefore, conclusions for the individual and group level effects can be made independently.

It is also important to note that in the pain literature, there is now increasing emphasis placed on investigating the individual level relationship between changes in cortical excitability and pain as opposed to the group level effect (Seminowicz et al., 2019; Summers et al., 2019). As such, it is important to make these results readily available for the scientific community.

We have made this clearer:

‘As there is now increasing emphasis placed on investigating the individual level relationship between changes in cortical excitability and pain and not only the group level effect, (Chowdhury et al., 2022; Seminowicz et al., 2018; Seminowicz, Thapa, & Schabrun, 2019; Summers et al., 2019) we also investigated the correlations between pain ratings and changes in MEP (and TEP) amplitude”

Chowdhury, N. S., Chang, W. J., Millard, S. K., Skippen, P., Bilska, K., Seminowicz, D. A., & Schabrun, S. M. (2022). The Effect of Acute and Sustained Pain on Corticomotor Excitability: A Systematic Review and Meta-Analysis of Group and Individual Level Data. The Journal of Pain, 23(10), 1680-1696.

Summers, S. J., Chipchase, L. S., Hirata, R., Graven-Nielsen, T., Cavaleri, R., & Schabrun, S. M. (2019). Motor adaptation varies between individuals in the transition to sustained pain. Pain, 160(9), 2115-2125.

Seminowicz, D. A., Thapa, T., & Schabrun, S. M. (2019). Corticomotor depression is associated with higher pain severity in the transition to sustained pain: a longitudinal exploratory study of individual differences. The Journal of Pain, 20(12), 1498-1506.

3) Confounds by pain ratings. The ISI between TMS pulses is 4 sec and includes verbal pain ratings. Considering this relatively short ISI, would it be possible that verbal pain ratings confound the TEP? Moreover, could the pain ratings confound TEP differences between conditions, e.g., by providing earlier ratings when the stimulus is painful? This should be carefully considered, and the authors might perform control analyses.

It is unlikely that the verbal ratings contaminated the TEP response as the subsequent TMS pulse was not delivered until the verbal rating was complete and given that each participant was cued by the experimenter to provide the pain rating after each pulse (rather than the participant giving the rating at any time). As such, it would not be possible for participants to provide earlier ratings to more painful stimuli.

We have made this clearer:

"To avoid contamination of TEPs by verbal ratings, the subsequent TMS pulse was not delivered until the verbal rating was complete, and the participant was cued by the experimenter to provide the pain rating after each pulse.”

4) Confounds by time effects. Non-painful and painful conditions were performed in a fixed order. Potential confounds by time effects should be carefully considered.

Previous research suggests that pain alters neural excitability even after pain has subsided. In a recent meta-analysis (Chowdhury et al., 2022) we found effect sizes of 0.55-0.9 for MEP reductions 0-30 minutes after pain had resolved. As such, we avoided intermixing pain and warm blocks given subsequent warm blocks would not serve as a valid baseline, as each subsequent warm block would have residual effects from the previous pain blocks.

Chowdhury, N. S., Chang, W. J., Millard, S. K., Skippen, P., Bilska, K., Seminowicz, D. A., & Schabrun, S. M. (2022). The Effect of Acute and Sustained Pain on Corticomotor Excitability: A Systematic Review and Meta-Analysis of Group and Individual Level Data. The Journal of Pain, 23(10), 1680-1696.

At the same time, given there was no conclusive evidence for a difference in N45 amplitude between pre-pain and post-pain conditions of Experiment 1 (Supplementary Figure 1), it is unlikely that the effect of pain was an artefact of time i.e., the explanation that successive thermal stimuli applied to the skin results an increase in the N45, regardless of whether the stimuli are painful or not. We will make this point in our next revision.

We have discussed this issue:

“Lastly, future research should consider replicating our experiment using intermixed pain and no pain blocks, as opposed to fixed pre-pain and pain blocks, to control for order effects i.e., the explanation that successive thermal stimuli applied to the skin results an increase in the N45 peak, regardless of whether the stimuli are painful or not. However, we note that there was no conclusive evidence for a difference in N45 peak amplitude between pre-pain and post-pain conditions of Experiment 1 (Supplementary Figure 1), suggesting it is unlikely that the observed effects were an artefact of time.”

5) Data availability. The authors should state how they make the data openly available.

We have uploaded the MEP, TEP and pain data on the Open science framework https://osf.io/k3psu/

Reviewer #1 (Recommendations For The Authors):

I think the study is quite solid and I only have very minor recommendations for the authors:

• Introduction, p. 3: "Functional magnetic resonance imaging has helped us understand where in the brain pain is processed". This is an overstatement. fMRI provides us with potential biomarkers (e.g. "the pain signature"), but the specificity of these responses for pain is debated and we still do not know where in the brain pain is processed.

We have amended to:

“functional magnetic resonance imaging has assisted in the localization of brain structures implicated in pain processing”

• Introduction, p. 5: "neural baseline" should be "neutral baseline"?

We thank the reviewer for identifying this – this has now been amended.

Reviewer #2 (Recommendations For The Authors):

INTRODUCTION

The introduction mentions how important extra-motor areas can be explored by TMS-EEG, then the effects of DLPFC rTMS on TEPs ... but you do not explore the DLPFC... Perhaps the introduction should be reframed.

The current work explores cortical excitability throughout the brain (as shown in our cluster-based permutation and source localization analyses), so our investigations are in line with the introductions statement about the importance of studying non-motor areas.

The reference to DLPFC rTMS was to highlight current existing research that has applied TMS-EEG to understand pain. It was not used as a methodological rationale to investigate the DLPFC in the present study. To make the research gap clearer, we state:

“While these studies assist us in understanding whether TEPs might mediate rTMS-induced pain reductions, no study has investigated whether TEPs are altered in direct response to pain”

Lignes 63-65 the term "TMS" is used to refer to motor corticospinal excitability measures, in contrast to TMS-EEG measures of TEPs. Then the authors come back to TMS-EEG and then again back to MEPs. This is rather confusing: TMS means TMS... the concept of MEP/ motor corticospinal excitability measures is not intuitive when using the term "TMS". I suggest using motor corticospinal excitability measures when referring to MEP/MEP-based measures of cortical excitability...) and M1TMS-EEG-evoked potentials (usually abbreviated to TEPs) to refer to TMS-EEG responses as measured here.

Throughout the manuscript, we now use the term TEPs when referring to TMS-EEG measures, and MEPs when referring to TMS-EMG measure. The use of TEPs vs. MEPs will make it easier for readers to follow which measures we are referring to.

Line 83: "As such, the precise origin of the pain mechanism cannot be localized." Please rephrase, the sentence conveys the idea that it is indeed possible to localize the origin of a pain mechanism with a different approach, and we know this is not currently possible, irrespective of the methodological setup.

We have replaced this with:

“This makes it unclear as to whether pain processes occur at the cortical, spinal or peripheral level.”

How can one predetermine the temperature that will be perceived as painful by someone else, and not base it on individual HPT? This is against principles of psychophysics. Please comment. Attesting all participants had HPT below 46 is important, but then being stimulated at 46C when our HPT is 45C is different from when our HPT is 39C. Please explain why the pain intensity was not standardised based on individual HPT.

Please refer to our response to the public review related to the issue

Line 38: "if we had used an alternative design with blocks of warm stimuli intermixed with blocks of painful stimuli, the warm stimuli blocks would not serve as a valid non-painful baseline". I do not understand why it is not possible to have a pain-free baseline, followed by a pain/warm sequence.

In our study, we had the choice of either intermixing blocks or to use a fixed sequence. Previous research suggests that pain alters neural excitability even after pain has subsided. In a recent meta-analysis (Chowdhury et al., 2022) we found effect sizes of 0.55-0.9 for MEP reductions 0-30 minutes after pain had resolved. As such, we avoided intermixing pain and warm blocks given subsequent warm blocks would not serve as a valid baseline, as each subsequent warm block would have residual effects from the previous pain blocks.

We have updated the manuscript to be clearer about why we used a fixed sequence:

“The pre-pain/pain/post-pain design has been commonly used in the TMS-MEP pain literature, as many studies have demonstrated strong changes in corticomotor excitability that persist beyond the painful period. Indeed, in a systematic review, we showed effect sizes of 0.55-0.9 for MEP reductions 0-30 minutes after pain had resolved (Chowdhury et al., 2022). As such, if we had used an alternative design with blocks of warm stimuli intermixed with blocks of painful stimuli, the warm stimuli blocks would not serve as a valid non-painful baseline”

Chowdhury, N. S., Chang, W. J., Millard, S. K., Skippen, P., Bilska, K., Seminowicz, D. A., & Schabrun, S. M. (2022). The Effect of Acute and Sustained Pain on Corticomotor Excitability: A Systematic Review and Meta-Analysis of Group and Individual Level Data. The Journal of Pain, 23(10), 1680-1696.

Please explain, and provide evidence that stimulation of people with predetermined temperatures is able to create warm/pain/warm sensations, without entraining pain in the last warm stimulation.

A previous study by Dube et al. (2011) used sequences of warm (36°C), painful and neutral (32° C) and found that participants did not experience pain at any time when the temperature was at a warm temperature of 36°C. We have now cited this study:

“Based on a previous study (Dubé & Mercier, 2011) which also used sequences of painful (50ºC) and warm (36°C) thermal stimuli, we did not anticipate that the stimulus in the pain block would entrain pain in the post-pain block”

Dubé, J. A., & Mercier, C. (2011). Effect of pain and pain expectation on primary motor cortex excitability. Clinical neurophysiology, 122(11), 2318-2323.

METHODS

It is not clear if participants with chronic pain, present in 20% of the general population, were excluded. If they were, please provide "how" in methods.

We excluded participants with a history or presence of acute/chronic pain. This has now been clarified:

“Participants were excluded if they had a history of chronic pain condition or any current acute pain”

Line 489: the definition of warm detection threshold is unusual, please provide a reference.

We used an identical method to Furman et al., (2020). We have made the reference to this clearer: “Warmth, cold and pain thresholds were assessed in line with a previous study (Furman et al., 2020)”

Furman, A. J., Prokhorenko, M., Keaser, M. L., Zhang, J., Chen, S., Mazaheri, A., & Seminowicz, D. A. (2020). Sensorimotor peak alpha frequency is a reliable biomarker of prolonged pain sensitivity. Cerebral Cortex, 30(12), 6069-6082.

In Experiment 2, please explain how the lack of randomisation between "pre-pain" and "pain" may have influenced results.

Given we tried to replicate Experiment 1’s methodology as close as possible (to isolate the source of the effect from Experiment 1) we chose to repeat the same sequence of blocks as Experiment 1: pre-pain followed by pain.

Given there was no conclusive evidence for a difference in N45 amplitude between pre-pain and post-pain conditions of Experiment 1 (Supplementary Figure 1), it is unlikely that the effect of pain was an order effect i.e., the explanation that successive thermal stimuli applied to the skin results an increase in the N45, regardless of whether the stimuli are painful or not.

We now discuss the issue of randomization:

“Lastly, future research should consider replicating our experiment using intermixed pain and no pain blocks, as opposed to fixed pre-pain and pain blocks, to control for order effects i.e. the explanation that successive thermal stimuli applied to the skin results an increase in the N45 peak, regardless of whether the stimuli are painful or not. However, we note that there was no conclusive evidence for a difference in N45 peak amplitude between pre-pain and post-pain conditions of Experiment 1 (Supplementary Figure 1), suggesting it is unlikely that the observed effects were an artefact of time”

Also, in Methods in general, disclose how pain intensity was assessed, and how.

Pain intensity was assessed using a verbal rating scale (0 = no pain, and 10 = most pain imaginable). We have provided more detail:

“During each 40 second thermal stimulus, TMS pulses were manually delivered, with a verbal pain rating score (0 = no pain, and 10 = worst pain imaginable) obtained between pulses. To avoid contamination of TEPs by verbal ratings, the subsequent TMS pulse was not delivered until the verbal rating was complete, and the participant was cued by the experimenter to provide the pain rating after each pulse”

Please explain how auditory masking was made during data collection.

Auditory masking noise was not played through the headphones, given that Experiment 2 controlled for auditory evoked potentials. We have made this clearer:

“Auditory masking was not used. Instead, auditory evoked potentials resulting from the TMS click sound were controlled for in Experiment 2”

Please explain if online TEP monitoring was used during data collection

Online TEP monitoring was not available with our EEG software. We have made this clearer in the manuscript:

“Online TEP monitoring was not available with the EEG software”

Line 499: what is subthreshold TMS here? You are measuring TEPs, and not MEPs initially, so you may have a threshold for MEPs and TEPs, which are not the same.

The intensity was calibrated relative to the MEP response (rather than TEP response) - this has now been clarified:

“… and the inclusion of a subthreshold TMS (90% of resting motor threshold) condition intermixed within both the pre-pain and pain blocks.”

Please provide a reference and a figure to illustrate the electric stimulation used in the sham procedure in Study 2

The apparatus for the electrical stimulation is shown in Figure 7A, and was based on previous papers using electrical stimulation over motor cortex to simulate the somatosensory aspect of real TMS (Chowdhury et al., 2022; Gordon et al., 2022; Rocchi et al., 2021). We have made this clearer:

“Electrical stimulation was based on previous studies attempting to simulate the somatosensory component of active TMS (Chowdhury et al., 2022; Gordon et al., 2022; Rocchi et al., 2021)”

Gordon, P. C., Jovellar, D. B., Song, Y., Zrenner, C., Belardinelli, P., Siebner, H. R., & Ziemann, U. (2021). Recording brain responses to TMS of primary motor cortex by EEG–utility of an optimized sham procedure. Neuroimage, 245, 118708.

Chowdhury, N. S., Rogasch, N. C., Chiang, A. K., Millard, S. K., Skippen, P., Chang, W. J., ... & Schabrun, S. M. (2022). The influence of sensory potentials on transcranial magnetic stimulation–Electroencephalography recordings. Clinical Neurophysiology, 140, 98-109.

Rocchi, L., Di Santo, A., Brown, K., Ibánez, J., Casula, E., Rawji, V., ... & Rothwell, J. (2021). Disentangling EEG responses to TMS due to cortical and peripheral activations. Brain stimulation, 14(1), 4-18.

It is not so common to use active electrodes for TMS-EEG. Please confirm the electrodes used and if they are c-ring TMS compatible and provide reference if otherwise (or actual papers recommending active ones)

To be more specific about the electrode type we have indicated:

“Signals were recorded from 63 TMS-compatible active electrodes (6mm height, 13mm width), embedded in an elastic cap (ActiCap, Brain Products, Germany), in line with the international 10-10 system”

A paper directly comparing TEPs between active and passive electrodes found no difference between the two and concluded TEPs can be reliably obtained using active electrodes (Mancuso et al., 2021). There is also evidence that active electrodes have better signal quality than passive electrodes at higher impedance levels (Laszlo et al., 2014).

This information has now been added to the paper:

“Active electrodes result in similar TEPs (both magnitude and peaks) to more commonly used passive electrodes (Mancuso et al., 2021). There is also evidence that active electrodes have higher signal quality than passive electrodes at higher impedance levels (Laszlo, Ruiz-Blondet, Khalifian, Chu, & Jin, 2014).”

There is a growing literature showing that monophonic pulses are not reliable for TEPs when compared to biphasic ones, please provide references. https://doi.org/10.1016/j.brs.2023.02.009

The reference provided by the reviewer states that biphasic and monophasic pulses both have advantages and disadvantages, rather than stating “monophonic pulses are not reliable for TEPs”. While there is some evidence that the artefacts resulting from monophasic pulses are larger than biphasic pulses, the EEG signal still returns to baseline levels within 5ms of the TMS pulse (Rogasch et al., 2013). Moreover, one paper (Casula et al. 2018) found that the resultant TEPs evoked by monophasic pulses are larger than those resulting from biphasic pulses. The authors postulated that monophasic pulses are more effective at activating widespread cortical areas than biphasic pulses. Ultimately the reference provided by the reviewer concludes that “effect of pulse shape on TEPs has not been systematically investigated and more studies are needed”.

Rogasch, N. C., Thomson, R. H., Daskalakis, Z. J., & Fitzgerald, P. B. (2013). Short-latency artifacts associated with concurrent TMS–EEG. Brain stimulation, 6(6), 868-876.

Casula, E. P., Rocchi, L., Hannah, R., & Rothwell, J. C. (2018). Effects of pulse width, waveform and current direction in the cortex: A combined cTMS-EEG study. Brain stimulation, 11(5), 1063-1070.

In most heads, a pulse in the PA direction is not obtained by a coil oriented 45o to the midline. The later induced later-medial pulses, good to obtain MEPs

We followed previous studies measuring MEPs from the ECRB elbow muscle (Schabrun et al., 2016; de Martino et al., 2019) whereby the TMS coil handle was angled at 45 degrees relative to the midline in order to induce a posterior-anterior current. We are not aware of literature that shows that the 45 degrees orientation does not induce a posterior anterior current in most heads.

Schabrun, S. M., Christensen, S. W., Mrachacz-Kersting, N., & Graven-Nielsen, T. (2016). Motor cortex reorganization and impaired function in the transition to sustained muscle pain. Cerebral Cortex, 26(5), 1878-1890.

De Martino, E., Seminowicz, D. A., Schabrun, S. M., Petrini, L., & Graven-Nielsen, T. (2019). High frequency repetitive transcranial magnetic stimulation to the left dorsolateral prefrontal cortex modulates sensorimotor cortex function in the transition to sustained muscle pain. Neuroimage, 186, 93-102.

The definition of RMT is (very) unusual. RMT provides small 50microV MEPs in 50% of times. If you obtain MEPs at 50microV you are supra threshold!

The TMS motor threshold assessment tool calculates threshold in the same manner as other threshold tools – it calculates the intensity that elicits an MEP of 50 microvolts, 50% of the time. We have made this clearer:

“The RMT was determined using the TMS motor thresholding assessment tool, which estimates the TMS intensity required to induce an MEP of 50 microvolts with a 50% probability using maximum likelihood parametric estimation by sequential testing (Awiszus and Borckardt, 2011). This method has been shown to achieve the accuracy of methods such as the Rossini-Rothwell method (Rossini et al., 1994; Rothwell et al., 1999) but with fewer pulses (Qi et al., 2011; Silbert et al., 2013).”

Please inform the inter TMS pulse interval used of TEPs and whether they were randomly generated.

The pulses were delivered manually – the interval was not randomly generated – as stated:

“As TMS was delivered manually, there was no set interpulse interval. However, the 40 second stimulus duration allowed for 11 pulses for each heat stimulus …. (~ 4 seconds in between …)”

Why have you stimulated suprathreshold on M1 when assessing TEP´s? The whole idea is that large TEPs can be obtained at lower intensities below real RMT and that prevents re-entering loops of somatosensory and joint movement inputs that insert "noise" to the TEPs.

The suprathreshold intensity was used to concurrently measure MEPs during pre-pain, pain and post-pain blocks.

We have made this clearer:

“The test stimulus intensity was set at 110% RMT to concurrently measure MEPs and TEPs during pre-pain, pain and post-pain blocks.”

The influence of re-afferent muscle activity was controlled for in Experiment 3.

Did you assess pain intensity after each of the TEP pulses? Please discuss how such a cognitive task may have influenced results

Pain intensity was assessed after each TMS pulse, as stated:

“TMS pulses were manually delivered, with a verbal pain rating score (0 = no pain, and 10 = most pain imaginable) obtained between pulses”

Reviewer 3 also brought up a concern of whether the verbal rating task might have influenced the TEPs. However, it is unlikely that the task contaminated the TEP response as the subsequent TMS pulse was not delivered until the verbal rating was complete and given that each participant was cued by the experimenter to provide the pain rating after each pulse (rather than the participant giving the rating at any time). We have made this clearer where we state:

“To avoid contamination of TEPs by verbal ratings, the subsequent TMS pulse was not delivered until the verbal rating was complete, and the participant was cued by the experimenter to provide the pain rating after each pulse”

The QST approach is unusual. Please confirm the sequence of CDT, WDT and HPT were not randomised and that no interval beyond 6sec were used. Proper references are welcome.

In line with a previous study (Furman et al., 2020), the sequence of the CPT, WDT and HPT were not randomized, and the interval was not more than 6 seconds.

We have made this clearer:

“A total of three trials was conducted for each test to obtain an average, with an interstimulus interval of six seconds. The sequence of cold, warmth and pain threshold was the same for all participants (Furman et al. 2020)”

Performing 60 pulses for TEPs is unusual, and against the minimum number in recommendations

Please explain and comment.https://doi.org/10.1016/j.brs.2023.02.009

Please refer to our previous response to this concern in the public reviews.

Line 578: when you refer to "heat" the reader may confound warm/heat with heat meaning suprathreshold. Please revise the wording.

We have now replaced the word heat stimulus with thermal stimulus.

Why were Bayesian statistics used instead as frequentist ones?

We have made this clearer:

“Given we were interested in determining the evidence for pain altering TEP peaks in certain conditions (e.g., active TMS) and pain not altering TEP peaks in other conditions (sham TMS), we used a Bayesian approach as opposed to a frequentist approach, which considers the strength of the evidence for the alternative vs. null hypothesis”

RESULTS

There is a huge response with high power after 100ms- Please discuss if you believe auditory potentials may have influenced it.

It is indeed possible that auditory potentials were present at 100ms. We now state:

“Indeed, the signal at ~100ms post-TMS from Experiment 1 may reflect an auditory N100 response”

The presence of auditory contamination does not impact the main conclusions of the paper given this was controlled for in Experiment 2.

Please discuss how pain ranging from 3-10 may have influenced results in the "PAIN" situation,

It is anticipated that the fixed thermal stimulus intensity approach would lead to large variations in pain ratings (Adamczyk et al., 2022). This is a recommended approach when the aim of the research is to determine relationships between neurophysiological measures and individual differences in pain sensitivity (Adamczyk et al., 2022). Indeed, we were interested in whether alterations in neurophysiological measures were associated with pain intensity, and we found that higher pain ratings were associated with smaller reductions in MEP amplitude and larger increases in N45 amplitude.

Adamczyk, W. M., Szikszay, T. M., Nahman-Averbuch, H., Skalski, J., Nastaj, J., Gouverneur, P., & Luedtke, K. (2022). To calibrate or not to calibrate? A methodological dilemma in experimental pain research. The Journal of Pain, 23(11), 1823-1832.

Please indicate if any participants offered pain after warm stimulation ( possible given secondary hyperalgesia after so many plateaux of heat stimulation).

As stated in the results “All participants reported 0/10 pain during the pre-pain and post-pain blocks”.

Please discuss the potential effects of having around 10% of "bad channels) In average per experiment per participants, its impacts in source localisation and in TEP measurement. Same for >5 epochs excluded by participant.

The number of bad channels has been incorrectly stated by the reviewer as being 10% on average per experiment per participant, whereas the correct number of reported bad channels was 3%, 4.7% and 9.8% for Experiment 1, 2 and 3 respectively (see supplementary material). These numbers are below the accepted number of bad channels to interpolate (10%) in EEG pipelines (e.g., Debnath et al., 2020; Kayhan et al., 2022), so it is unlikely that our channel exclusions significantly influenced the quality of our source localization an TEP data.

Debnath, R., Buzzell, G. A., Morales, S., Bowers, M. E., Leach, S. C., & Fox, N. A. (2020). The Maryland analysis of developmental EEG (MADE) pipeline. Psychophysiology, 57(6), e13580.

Kayhan, E., Matthes, D., Haresign, I. M., Bánki, A., Michel, C., Langeloh, M., ... & Hoehl, S. (2022). DEEP: A dual EEG pipeline for developmental hyperscanning studies. Developmental cognitive neuroscience, 54, 101104.

The number of excluded epochs is unlikely to have influenced the results given there was evidence for no difference in the number of rejected epochs between conditions (E1 BF10 = 0.145, E2 BF10 = 0.27, E3 BF10 = 0.169 – these BFs have now been reported in the supplementary material), and given the reliability of the N45 was high (see response to previous comment on the number of trials per condition).

HPT of 42.9 {plus minus} 2.5{degree sign}C means many participants had HPT close to 46oC. Please discuss

While some participants did indeed have pain thresholds close to 46 degrees, they nonetheless reported pain during the test blocks. While such participants may have reported less pain compared to others, we aimed for larger variations in pain ratings, given one of the research questions was to determine why pain intensity differs between individuals (given the same noxious stimulus). Indeed, we showed that this variation was meaningful (pain intensity was related to alterations in N45 and MEP amplitude).

Please explain the sentence : line 139 "As such, if we had used an alternative design with blocks of warm stimuli intermixed with blocks of painful stimuli, the warm stimuli blocks would not serve as a valid non-painful baseline." I cannot see why.

Please refer to our previous point on why the fixed sequence was included.

And on the top of that heat was not individualised according to HPT.

Please refer to our previous point on why we used a fixed stimulus approach.

Sequences of warm/heat were not randomised. Please refer to our previous point on the why the sequence of blocks was not randomized.

Line 197: "However, as this is the first study investigating the effects of experimental pain on TEPsamplitude, there were no a priori regions or timepoints of interest to compare betweenconditions". This is not clear. It means you have not measured the activity (size of the N45) under the electrode closest to the TMS coil? The TEP is supposed to by higher under the stimulated target/respective corresponding electrode…

We are not aware of any current recommendations that state that the region of interest should be based on the site of stimulation. The advantage of TMS-EEG is that it allows characterisation of cortical excitability changes throughout the brain, not just the site of stimulation. We based our region of interest on a cluster-based permutation analysis, as recommended by Frömer, Maier, & Abdel Rahman, (2018)

Frömer, R., Maier, M., & Abdel Rahman, R. (2018). Group-level EEG-processing pipeline for flexible single trial-based analyses including linear mixed models. Frontiers in neuroscience, 12, 48.

Please explain where N45 values came from.

The N45 was calculated using the TESA peak function (Rogasch et al., 2017) which identifies a data point which is larger/smaller than +/- 5 data points within a specified time window (e,g, 40-70ms post-TMS as in the present study). Where multiple peaks are found, the amplitude of the largest peak is returned. Where no peak is found, the amplitude at the specified latency is returned.

Rogasch, N. C., Sullivan, C., Thomson, R. H., Rose, N. S., Bailey, N. W., Fitzgerald, P. B., ... & Hernandez-Pavon, J. C. (2017). Analysing concurrent transcranial magnetic stimulation and electroencephalographic data: A review and introduction to the open-source TESA software. Neuroimage, 147, 934-951.

If only the cluster assessment was made please provide the comparison between P45 from the target TMS channel location in pre pain vs pain.

We assume the reviewer is referring to the N45 rather than P45, and that by “target” TMS channel they are referring to the stimulated region.

We first clarify that there is no “target” channel given the motor hotspot differs between individuals and so the channel that is closest to the site of stimulation will always differ.

Secondly, as stated above, we are not aware of any current recommendations in TMS-EEG research that states that the region of interest for TEP analysis should be based on the site of stimulation. The advantage of TMS-EEG is that it allows characterisation of cortical excitability throughout the brain, not just the site of stimulation. If we based our ROI on the target channel only, we would lose valuable information about excitability changes occurring in other brain regions.

Lastly, the N45 was localized at frontocentral electrodes, which is also where the cluster differences emerged. As such, we do not believe it would be informative to compare N45 peak amplitude at the region of stimulation.

Also explain how correction for multiple comparisons was made

Please refer to our response to the public review related to this issue.

And report data from pain vs post-pain.

The pain vs. post-pain comparisons are now reported in the Supplementary material.

There is a strong possibility the response at N85 is an auditory /muscle signal. Please provide the location of this response.

We have opted not to include the topography at 85ms in the main paper as it would introduce too much clutter into the figures (which are already very dense), and because the topography was very similar to the topography at 100ms. As an example, for the reviewer, in Author response image 1 we have shown the topography for the pre-pain condition of Experiment 1.

Author response image 1.

Experiment 2: I have a strong impression both active TEPs and sham TEPs were contaminated by auditory (and muscle) noise. Please explain.

While it possible that auditory noise may have influenced TEPs in the active and sham groups, it does not impact the main conclusions of the paper, given that the purpose of the sham condition was to control for auditory and somatosensory stimulation resulting from TMS.

While muscle activity may also affect have influenced the TEPs in active and sham conditions, we used fastICA in all conditions to suppress muscle activity. The fastICA algorithm (Rogasch et al., 2017) runs an independent component analysis on the data, and classifies components as neural, TMS-evoked muscle, eye movements and electrode noise, based on a set of heuristic thresholding rules (e.g., amplitude, frequency and topography of the components). Components classified as TMS-evoked muscle/other muscle artefacts are then removed. In the supplementary material, we further report that the number of components removed did not differ between conditions, suggesting the impact of muscle artefacts are not larger in some conditions vs. others.

Rogasch, N. C., Sullivan, C., Thomson, R. H., Rose, N. S., Bailey, N. W., Fitzgerald, P. B., ... & Hernandez-Pavon, J. C. (2017). Analysing concurrent transcranial magnetic stimulation and electroencephalographic data: A review and introduction to the open-source TESA software. Neuroimage, 147, 934-951.

Experiment 3: One interpretation can be that both supra and sub-threshold TMS were leading to somatosensory re-afferent responses, based on the way RMT was calculated, which hyper estimate the RMT and delivers in reality 2 types of supra-threshold stimulations. Please discuss

Please refer to our response to the public review related to this issue.

Please provide correlation between N45 size and MEPs amplitudes.

This has now been included:

“There was no conclusive evidence of any relationship between alterations in MEP amplitude during pain, and alterations in N100, N45 and P60 amplitude during pain (see supplementary material).”<br /> The supporting statistics for these analyses have been included in the supplementary material.

DISCUSSION

Line 303: " The present study determined whether acute experimental pain induces alterations in cortical inhibitory and/or facilitatory activity observed in TMS-evoked potentials".

Well, no. The study assessed the N45, and was based on it. It did not really explore other metrics in a systematic fashion. P60 and N100 changes were not replicated in experiments 2 and 3..

We assume the reviewer is stating that we did not assess other TEP peaks (such as the N15, P30 and P180). However, we did indeed assess these peaks in a systematic fashion. First, we identified the ROI by using a cluster-based analysis. This is a recommended approach when the ROI is unclear (Frömer, Maier, & Abdel Rahman, 2018). We then analysed the TEP representing the mean voltage across the electrodes within the cluster, and then identified any differences in all peaks between conditions (not just the N45). This has been made clearer in the manuscript.

This has now been included:

“For all experiments, the mean TEP waveform of any identified clusters from Experiment 1 were plotted, and peaks (e.g., N15, P30, N45, P60, N100) were identified using the TESA peak function (Rogasch et al., 2017)”

Frömer, R., Maier, M., & Abdel Rahman, R. (2018). Group-level EEG-processing pipeline for flexible single trial-based analyses including linear mixed models. Frontiers in neuroscience, 12, 48.

And the N45 is not related to facilitatory or inhibitory activity, it is a measure of an evoked response indicating excitability

Evidence suggests the N45 is mediated by GABAAergic neurotransmission (inhibitory activity), as drugs which increase GABAA receptor activity increase the amplitude of the N45 (Premoli et al., 2014) and drugs which decrease GABAA receptor activity decrease the amplitude of the N45 (Darmani et al., 2016). As such, we and various other empirical papers (e.g., Bellardinelli et al., 2021; Noda et al., 2021; Opie at 2019 ) and review papers (Farzan & Bortoletto, 2022; Tremblay et al., 2019) have interpreted changes in the N45 peak as reflecting changes in cortical inhibitory/GABAA mediated activity.

Premoli, I., Castellanos, N., Rivolta, D., Belardinelli, P., Bajo, R., Zipser, C., ... & Ziemann, U. (2014). TMS-EEG signatures of GABAergic neurotransmission in the human cortex. Journal of Neuroscience, 34(16), 5603-5612.

Belardinelli, P., König, F., Liang, C., Premoli, I., Desideri, D., Müller-Dahlhaus, F., ... & Ziemann, U. (2021). TMS-EEG signatures of glutamatergic neurotransmission in human cortex. Scientific reports, 11(1), 8159.

Darmani, G., Zipser, C. M., Böhmer, G. M., Deschet, K., Müller-Dahlhaus, F., Belardinelli, P., ... & Ziemann, U. (2016). Effects of the selective α5-GABAAR antagonist S44819 on excitability in the human brain: a TMS–EMG and TMS–EEG phase I study. Journal of Neuroscience, 36(49), 12312-12320.

Noda, Y., Barr, M. S., Zomorrodi, R., Cash, R. F., Lioumis, P., Chen, R., ... & Blumberger, D. M. (2021). Single-pulse transcranial magnetic stimulation-evoked potential amplitudes and latencies in the motor and dorsolateral prefrontal cortex among young, older healthy participants, and schizophrenia patients. Journal of Personalized Medicine, 11(1), 54.

Farzan, F., & Bortoletto, M. (2022). Identification and verification of a'true'TMS evoked potential in TMS-EEG. Journal of neuroscience methods, 378, 109651.

Opie, G. M., Foo, N., Killington, M., Ridding, M. C., & Semmler, J. G. (2019). Transcranial magnetic stimulation-electroencephalography measures of cortical neuroplasticity are altered after mild traumatic brain injury. Journal of Neurotrauma, 36(19), 2774-2784.

Tremblay, S., Rogasch, N. C., Premoli, I., Blumberger, D. M., Casarotto, S., Chen, R., ... & Daskalakis, Z. J. (2019). Clinical utility and prospective of TMS–EEG. Clinical Neurophysiology, 130(5), 802-844.

Line 321: why have you not measured SEPs in experiment 3?

It is not possible to directly measure the somatosensory evoked potentials resulting from a TMS pulse, given that the TMS pulse produces a range of signals including cortical activity, muscle/eye blink responses, auditory responses, somatosensory responses and other artefacts. While some researchers attempt to isolate the SEP from TMS using pre-processing methods such as ICA, others use control conditions such as sensory sham conditions (to control for the “tapping” artefact) or subthreshold intensity conditions (to control for reafferent muscle activity), as we have done in Experiment 2 and 3 of our study.

We have now stated this in the manuscript:

“As it is extremely challenging to isolate and filter these auditory and somatosensory evoked potentials using pre-processing pipelines, masking methods have been used to suppress these sensory inputs, (Ilmoniemi and Kičić, 2010; Massimini et al., 2005). However recent studies have shown that even when these methods are used, sensory contamination of TEPs is still present, as shown by commonalities in the signal between active and sensory sham conditions that mimic the auditory/somatosensory aspects of real TMS (Biabani et al., 2019; Conde et al., 2019; Rocchi et al., 2021). This has led many leading authors (Biabani et al., 2019; Conde et al., 2019) to recommend the use of sham conditions to control for sensory contamination”

Line 365: SICI is dependent on GABAa activity. But the way the text is written if conveys the idea that TMS pulses "activate" GABA receptors, which is weird...Please rephrase.

This has now been reworded.

“SICI refers to the reduction in MEP amplitude to a TMS pulse that is preceded 1-5ms by a subthreshold pulse, with this reduction believed to be mediated by GABAA neurotransmission (Chowdhury et al., 2022)”

Reviewer #3 (Recommendations For The Authors):

-Key references Ye et al., 2022 and Che et al., 2019 need to be included in the reference list.

These references have now been included in the reference list.

-Heat pain stimuli and TMS stimuli are applied simultaneously. Sometimes the term "stimulus" is used without specifying whether it refers to TMS pulses or heat pain stimuli. Clarifying this whenever the word "stimulus" is used would enhance clarity for the reader.

We have now clarified the use of the word “stimulus” throughout the paper.

-Panels A-D in Figure 6 should be correctly labeled in the text and the figure legend.

Figure 6 Panel labels have now been amended.

eLife assessment

This valuable study provides convincing evidence that acute experimental pain induces changes of cortical excitability. Although the modality specificity of the findings is not fully clear, the findings will be of interest to researchers interested in the brain mechanisms of pain.

Reviewer #1 (Public Review):

The objective of this investigation was to determine whether experimental pain could induce alterations in cortical inhibitory / facilitatory activity observed in TMS-evoked potentials (TEPs). Previous TMS investigations of pain perception had focused on motor evoked potentials (MEPs), which reflect a combination of cortical, spinal, and peripheral activity, as well as restricting the focus to M1. The main strength of this investigation is the combined use of TMS and EEG in the context of experimental pain. More specifically, Experiment 1 investigated whether acute pain altered cortical excitability, reflected in the modulation of TEPs. The main outcome of this study is that relative to non-painful warm stimuli, painful thermal stimuli led to an increase on the amplitude of the TEP N45, with a larger increase associated with higher pain ratings. Because it has been argued that a significant portion of TEPs could reflect auditory potentials elicited by the sound (click) of the TMS, Experiment 2 constituted a control study that aimed to disentangle the cortical response related to TMS and auditory activity. Finally, Experiment 3 aimed to disentangle the cortical response to TMS and reafferent feedback from muscular activity elicited by suprathreshold TMS applied over M1. The fact that the authors accompanied their main experiment with two control experiments strengthens the conclusion that the N45 TEP peak could be implicated in the perception of painful stimuli. Perhaps, the addition of a highly salient but non-painful stimulus (i.e. from another modality) would have further ruled out that the effects on the N45 are not predominantly related to intensity / saliency of the stimulus rather than to pain per se.

Reviewer #3 (Public Review):

The present study aims to investigate whether pain influences cortical excitability. To this end, heat pain stimuli are applied to healthy human participants. Simultaneously, TMS pulses are applied to M1 and TMS-evoked potentials (TEPs) and pain ratings are assessed after each TMS pulse. TEPs are used as measures of cortical excitability. The results show that TEP amplitudes at 45 msec (N45) after TMS pulses are higher during painful stimulation than during non-painful warm stimulation. Control experiments indicate that auditory, somatosensory, or proprioceptive effects cannot explain this effect. Considering that the N45 might reflect GABAergic activity, the results suggest that pain changes GABAergic activity. The authors conclude that TEP indices of GABAergic transmission might be useful as biomarkers of pain sensitivity.

Pain-induced cortical excitability changes is an interesting, timely, and potentially clinically relevant topic. The paradigm and the analysis are sound, the results are convincing, and the interpretation is adequate. The findings will be of interest to researchers interested in the brain mechanisms of pain.

eLife assessment

This study presents valuable findings on the presence of 6mA in the Drosophila genome and challenges previous findings regarding the role of TET in 6mA modification. The evidence supporting the claims is solid, and the paper has the potential to stimulate re-evaluations of the significance and regulatory mechanisms of 6mA DNA modifications in Drosophila.

Reviewer #1 (Public Review):

This work challenges previously published results regarding the presence and abundance of 6mA in the Drosophila genome, as well as the claim that the TET or DMAD enzyme serves as the "eraser" of this DNA methylation mark and its roles in development. This information is needed to clarify these questions in the field. I am less familiar with the biochemical approaches in this work, so my comments are mainly on the genetic analyses. Generally speaking, the methods for fly husbandry and treatment seem to be in accordance with those established in the field.

Reviewer #2 (Public Review):

DNA adenine methylation (6mA) is a rediscovered modification that has been described in a wide range of eukaryotes. However, 6mA presence in eukaryote remains controversial due to the low abundance of its modification in eukaryotic genome. In this manuscript, Boulet et al. re-investigate 6mA presence in drosophila using axenic or conventional fly to avoid contaminants from feeding bacteria. By using these flies, they find that 6mA is rare but present in the drosophila genome by performing LC/MS/MS. They also find that the loss of TET (also known as DMAD) does not impact 6mA levels in drosophila, contrary to previous studies. In addition, the authors find that TET is required for fly development in its enzymatic activity-independent manner.

The strength of this study is, that compared to previous studies of 6mA in drosophila, the authors employed axenic or conventional fly for 6mA analysis. These fly strains make it possible to analyze 6mA presence in drosophila without bacterial contaminant. Therefore, showing data of 6mA abundance in drosophila by performing LC-MS/MS in this manuscript is more convincing as compared with previous studies. Intriguingly, the authors find that the conserved iron-binding motif required for the catalytic activity of TET is dispensable for its function. This finding could be important to reveal TET function in organisms whose genomic 5mC levels are very low.

The manuscript in this paper is well written but some aspects of data analysis and discussion need to be clarified and extended.<br /> 1) It is convincing that an increase in 6mA levels is not observed in TETnull presented in Fig1. But it seems 6mA levels are altered in Ax.TET1/2 compared with Ax.TETwt and Ax.TETnull presented in Fig1f (and also WT vs TET1/2 presented in Fig1g). Is it sure that no statistically significant were not observed between Ax.TET1/2 and Ax.TETwt?<br /> 2) The representing data of in vitro demethylation assay presented in Fig.3 is convincing, but it is not well discussed and analyzed why these results are contrary to previous reports (Yao et al., 2018 and Zhang et al., 2015).

eLife assessment

This fundamental study advances our understanding of how Notch signaling activates transcription by analyzing the dynamics of the Mastermind transcriptional co-activator and its role in the activation complex. The evidence is compelling with precise quantitative measurements. The evidence presented by the authors features methods, data, and analyses that are currently state-of-the-art but have not previously been applied to how transcription is regulated in the Notch pathway.

Reviewer #1 (Public Review):

In this manuscript by DeHaro-Arbona et al., the authors wish to understand how a signaling pathway (Notch) is dynamically decoded to elicit a specific transcriptional output. In particular, they investigate the kinetic properties of Notch-responsive nuclear complexes (the DNA binding factor CSL and its co-activator Mastermind (mam) along with several candidate interacting partners). Their experimental model is the polytene chromosome of the Drosophila salivary gland, in which the naturally inactive Notch can be artificially induced through the expression of a constitutively active form of Notch.

The authors develop a series of CRISPR and transgenic lines enabling the live imaging of these complexes at a specific locus and in various backgrounds (genetic perturbations/drug treatments). This quantitative live imaging data suggests that Notch nuclear complexes form hubs and the authors characterize their binding dynamics. Interestingly, they elegantly demonstrate that the content of these hubs and their kinetic properties can evolve, even within Notch ON cells. Hence, they propose the existence of distinct hubs, distinguishing an open (CSL), engaged (CSK-Mam), or active (CSL-Mam-Med-PolII) configuration in Notch ON cells and an inactive hub (in Notch OFF having previously been exposed to Notch) state, that would explain the surprising transcriptional memory that the authors observe hours after Notch withdrawal.

Reviewer #2 (Public Review):

The manuscript from deHaro-Arbona et al, entitled "Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind", uses single molecule microscopy imaging in live tissues to understand the dynamics and molecular determinants of transcription factor recruitment to the E(spl)-C locus in Drosophila salivary gland cells under Notch-ON and -OFF conditions. Previous studies have identified the major players that are involved in transcription regulation in the Notch pathway, as well as the importance of general transcriptional coregulators, such as CBP/P300 and the Mediator CDK module, but the detailed steps and dynamics involved in these processes are poorly defined. The authors present a wealth of single molecule data that provides significant insights into Notch pathway activation, including:

1. Activation complexes, containing CSL and Mam, have slower dynamics than the repressor complexes, containing CSL and Hairless.

2. Contribution of CSL, NICD, and Mam IDRs to recruitment.

3. CSL-Mam slow-diffusing complexes are recruited and form a hub of high protein concentrations around the target locus in Notch-ON conditions.

4. Mam recruitment is not dependent on transcription initiation or RNA production.

5. CBP/P300 or its associated HAT activity is not required for Mam recruitment.

6. Mediator CDK module and CDK8 activity are required for Mam recruitment, and vice-versa, but not CSL recruitment.

7. Mam is not required for chromatin accessibility but is dependent on CSL and NICD.

8. CSL recruitment and increased chromatin accessibility persist after NICD removal and loss of Mam, which confers a memory state that enables rapid re-activation in response to subsequent Notch activation.

9. Differences in the proportions of nuclei with both Pol II and with Mam enrichment, which results in transcription being probabilistic/stochastic. These data demonstrate that the presence of Mam-complexes is not sufficient to drive all the steps required for transcription in every Notch-ON nucleus.

10. The switch from more stochastic to robust transcription initiation was elicited when ecdysone was added.

Overall, the manuscript is well written, concise, and clear, and makes significant contributions to the Notch field, which are also important for a general understanding of transcription factor regulation and behavior in the nucleus. I recommend that the authors address my relatively minor criticisms detailed below.

Page 7, bottom. The authors speculate, "It is possible therefore that, once recruited, Mam can be retained at target loci independently of CSL by interactions with other factors so that it resides for longer." Is it possible that another interpretation of that data is that Mam is a limiting factor?

Page 9. The authors write, "A very low level of enrichment was evident for... for the CSL C-terminus..". The recruitment of CSL ct IDR does not appear to be statistically significant or there is no apparent difference (Figure S2C), suggesting the CSL ct IDR does not play a role in enrichment.

Page 9. The authors write, "Notably, MamnIDR::GFP fusion was present in droplets, suggesting it can self-associate when present in a high local concentration (Figure S2B)." Is this result only valid for Mam nIDR or does full-length Mam also localize into droplets, as has been previously observed for full-length mammalian Maml1 in transfected cells?

Previous studies in mammalian cells suggest that Maml1 is a high-confidence target for phosphorylation by CDK8, see Poss et al 2016 Cell Reports https://doi.org/10.1016/j.celrep.2016.03.030. By sequence comparison, does fly Mam have similar potential phosphorylation sites, and might these be critical for Mam/CDK module recruitment?

Page 11: The authors write, "The differences in the effects on Mam and CSL imply that the CDK module is specifically involved in retaining Mam in the hub, and that in its absence other CSL complexes "win-out", either because the altered conditions favour them and/or because they are the more abundant." Are the "other" complexes the authors are referring to Hairless-containing complexes? With the reagents the authors have in hand couldn't this be explicitly shown for CSL-complexes rather than speculated upon?

Page 12/13: The authors write, "Based on these results we propose that, after Notch activity decays, the locus remains accessible because when Mam-containing complexes are lost they are replaced by other CSL complexes (e.g. co-repressor complexes)." Again, why not actually test this hypothesis rather than speculate? The dynamics of Hairless complexes following the removal of Notch would be very interesting and build upon previously published results from the Bray lab.

Page 13: The authors write, "As Notch removal leads to a loss of Mam, but not CSL, from the hub, it should recapitulate the effects of MamDN." While the data in Figure 5B seem to support this hypothesis, it's not clear to me that the loss of Mam and MamDN should phenocopy each other, bc in the case of MamDN, NICD would still be present.

The temporal dynamics for Mam recruitment using the temperature- and optogenetic-paradigms are quite different. For example, in the optogenetic time course experiments, the preactivated cells are in the dark for 4 hours, while in the temperature-controlled experiments, there is still considerable enrichment of Mam at 4 hours. For the preactivated optogenetic experiments, how sure are the authors that Mam is completely gone from the locus, and alternatively, can the optogenetic experimental results be replicated in the temperature-controlled assays? My concern is whether the putative "memory" observation is just due to incomplete Mam removal from the previous activation event.

Reviewer #3 (Public Review):

Summary:<br /> DeHaro-Arbona and colleagues investigate the in vivo dynamics of Notch-dependent transcriptional activation with a focus on the role of the Mastermind (MAM) transcriptional co-activator. They use GFP and HALO-tagged versions of the CSL DNA-binding protein and MAM to visualize the complex, and Int/ParB to visualize the site of Notch-dependent E(Spl)-C transcription. They make several conclusions. First, MAM accumulates at E(Spl)-C when Notch signaling is active, just like CSL. Second, MAM recruits the CDK module of Mediator but does not initiate chromatin accessibility. Third, after signaling is turned off, MAM leaves the site quickly but CSL and chromatin accessibility are retained. Fourth, RNA pol II recruitment, Mediator recruitment, and active transcription were similar and stochastic. Fifth, ecdysone enhances the probability of transcriptional initiation.

Strengths:<br /> The conclusions are well supported by multiple lines of extensive data that are carefully executed and controlled. A major strength is the strategic combination of Drosophila genetics, imaging, and quantitative analyses to conduct compelling and easily interpretable experiments. A second major strength is the focus on MAM to gain insights into the dynamics of transcriptional activation specifically.

Weaknesses:<br /> Weaknesses are minor. There were no p-values reported for data presented in Figure S1D and no indication of how variable measurements were. In addition, the discussion of stochasticity was not integrated optimally with relevant literature.

eLife assessment

This valuable work on the paleovegetation history of Iceland has implications for the field of paleoecology, and the deglaciation history of Iceland and additional localities in Northern America and Europe via woody shrub colonization. The study uses a sedimentary ancient DNA metabarcoding approach to study this historic process. The strength of evidence is solid, with the methods (analysis of sedimentary DNA) and data analyses broadly supporting the claims.

Joint Public Review:

The revised version of the manuscript "Delayed postglacial colonization of Betula in Iceland and the circum North Atlantic" by Harning et al. investigates the colonization of shrubs during the Late Pleistocene/Holocene in Northern America and Europe by comparing published sedimentary ancient DNA (sedaDNA) records (and pollen data) with a new sedaDNA record from Island. The manuscript aims to identify shrub colonization patterns, discusses their drivers and evaluates the importance of shrubification under future warming.

The revised version improved the clarity of methods and discussion and results presented are more convincing.

However, parts of the methods (e.g. assessment of blanks and data filtering) and results (e.g. visualization of plant community data) could still be polished, and the figures should be improved to increase the clarity of the manuscript.

eLife assessment

This study presents initial findings in the generation of 3D cell constructs from endometrial cell mixtures seeded in Matrigel scaffold and treated with hormones as a proof of concept. While the study findings are valuable, functional validation to demonstrate its robustness is lacking, and therefore the strength of evidence is incomplete. The term organoids might not be appropriate to describe this in vitro model.

Reviewer #1 (Public Review):

Summary:

This study generated 3D cell constructs from endometrial cell mixtures that were seeded in the Matrigel scaffold. The cell assemblies were treated with hormones to induce a "window of implantation" (WOI) state. Although many bioinformatic analyses point in this direction, there are major concerns that must be addressed.

Strengths:

The addition of 3 hormones to enhance the WOI state (although not clearly supported in comparison to the secretory state).

Weaknesses:

First of all, the term organoid must be discarded. The authors just seed the endometrial cell mixture which assembles and aggregates into a 3D structure which is then immediately used for analysis. Organoids grow from tissue stem cells and must be passage-able (see their own description in lines 69-71). So, the term organoid must be removed everywhere, to not confuse the organoid field. It is not shown that the whole 3D assembly is passageable, which would be very surprising given the fact that immune and stromal cells do not grow in Matrigel because of the unfavorable growing conditions (which are targeted to epithelial cell growth).

Second, the study remains fully descriptive, bombing the reader with a mass of bioinformatic analyses without clear descriptions and take-home messages. The paper is very dense, meaning readers may give up. Moreover, functional validation, except for morphological and immunostaining analyses (which are posed as "functional" but actually are only again expression) is missing, such as in vivo functionality (after transplantation e.g.) and embryo interaction. Importantly, the 3D structure misses the right architecture with a lining luminal epithelium which is present in the receptive endometrium in vivo and needed as the first contact site with the embryo. So, in contrast to what the authors claim, this is not the best model to study embryo interaction, or the closest model to the in vivo state (line 318, line 326).

Third, receptive endometrial organoids (assembloids; Rawlings et al., eLife 2021) and receptive organoid-derived "open-faced endometrial layer" (Kagawa et al., Nature 2022) have already been described, which is in contrast to what the authors claim in several places that "they are the first" (e.g. lines 87-88, 316-319, etc). These studies used real organoids to achieve their model (and even showed embryo interaction), while in the present study, different cell types are just seeded and assembled. Hence, logically, immune cells are present which are never found in real organoid models. The only original aspect in the present study is the use of hormones to enhance the WOI phenotype. However, crucial information on this original aspect is missing such as concentration of the hormones, refreshment schedule, all 3 hormones added together or separately, and all 3 required?

Moreover, it is not a "robust" model at all as the authors claim, given the variability of the initial cell mixture (varying from patient to patient). Actually, the reproducibility is not shown. The proportions of the different cell types seeded in the Matrigel droplet will be different with every endometrial biopsy. It would be much better to recombine epithelial (passageable) organoids with stromal and immune cells in a quantified, standardized manner to establish a "robust" model.