Reviewer #3 (Public Review):

Seeking a selective inhibitor that precisely inhibits on-target activities and avoids side effects is a major challenge in the field of drug discovery and therapeutics. The authors proposed an alternative method that combines multiple inhibitors to maximize on-target inhibition and minimize off-target inhibition. Focusing on the kinase-inhibitor interaction dataset, the authors developed a quantitative way to measure the selectivity for mixtures of inhibitors by using the Jenson-Sahannon distance metric. The method sounds technical.

From their computation and assays, the multi-compound-multitarget scoring (MMS) method framework was validated to be able to select a combination of inhibitors that is more selective than a single highly selective inhibitor for one kinase target, or for multiple targets. The MMS method is a promising solution to reduce off-target effects and could be applicable to other inhibitor-target interactions. My suggestion is that a comparative analysis of MMS with other similar methods can be conducted to highlight the advantage of MMS over others.

The paper is not well organized and not easily readable. For example, first, the captions of the figures are two long; some of these texts could be moved to methods or results sections. Second, the concept of "penalty distribution" or "penalty prior" is vital to understand the MMS method, thus, at least a brief definition and introduction should be put in the main text rather than supporting method, as well as the rationale to use it. Third, the method section can be divided into several subsections with clear organizations and connections. Fourth, what is the difference between "a less selective inhibitor profile" and "an even less selective inhibitor profile" in Figure 3? Overall, the details of the paper are difficult to understand in the current version. I suggest rewriting<br /> the paper in a more concise and logical style.

Joint Public Review

In this manuscript, the authors develop a multi-scale agent-based model (ABM) capable of reproducing the self-organizing behavior observed in the intestinal crypt. By considering just the signaling pathways -previously reported as regulatory in the intestinal crypt- and local physical cell-to-cell interactions, the proposed model not only explains the emergence of the spatial organization, but also recapitulates cell composition dynamics in the crypt (proliferation, migration, and differentiation of cells), as previously characterized in the complex tissue of the small intestine epithelium in mice. The authors show that the self-organized system resulting from the model displays a stable composition over time. Additionally, the authors show how this model can be effectively used to test different conditions, such as biomedically relevant perturbations (e.g. stem cell ablation, cell cycle inhibition, and toxicity of particular drug treatments) and the posterior recovery, allowing to predict the safety of potential oncotherapies.

In summary, the authors provide a powerful and versatile model, which can be applied to better understand the formation and response of the intestinal crypt, as well as the functional heterogeneity of the intestinal epithelium at multiple scales. The proposed mathematical model simulates features across scales in the intestinal crypt such as multiple signaling pathways, the mechanical environment and its forces, and cell cycle regulation. The model demonstrates the stability of the homeostatic crypt and recovery following stem cell ablation. The model also simulates the cell cycle protein network and demonstrates that CDK1 inhibition creates oversized cells. In sum, the model generated by the authors increases the understanding of how these biological processes take place in vivo, exploring not only healthy cell behavior but also cell response to injury by oncotherapies or other external factors. Additionally, the authors provide a series of fascinating movies that show the spatial organization of the crypt during these processes, and the manuscript has clear applications for the clinics.

Nevertheless, in its current form, the manuscript has some weaknesses that are worth mentioning:

(1) The developed model considers the interaction of multiple signaling networks that are essential for morphogenesis and homeostasis in the intestinal tissue, as well as other elements that had been proposed as relevant in the literature. Nevertheless, the details of how these interactions are modeled couldn't be evaluated in the current revision as the model was not shared with the reviewers and it is not available yet online, nor specified in any detail in the current manuscript. Additionally, how quantitative information from Wnt and BMP signaling pathways is incorporated in a quantitative way in the model is not clear.

(2) Some conclusions by the authors are not properly justified in the text, as "Paneth cells are the main driver behind the differential mechanical environment in the niche", "Wnt-mediated feedback loop prevents the uncontrolled expansion of the niche", the specific effect of p27 in contrast with Wee1 phosphorylation over the cell cycle length, and "their recovery [absorptive progenitors] started before the end of the treatment, driven by a negative feedback loop from mature enterocytes to their progenitors".

(3) Only the results of the "main" model are shown, with no information about its sensitivity to parameter values, and how their conclusions depend on specific decisions on the model. For example, the authors said that "an optimal crypt cell composition is achieved when BMP and Wnt differentiation thresholds result in progenitors dividing approximately four times before differentiating into enterocytes", but the results of alternative scenarios are not shown.

(4) Regarding the construction of the model, the authors used "counts of Ki-67 positive cells recorded by position" while the original data reported "overall cell counts per crypt and villus". Some explanation about how this conversion was made, why it is valid, as well as any potential problems, is needed. Additionally, the model is based on experiments done by others in mouse models; the similarity to the response in human intestinal crypts is not discussed.

(5) The authors imply that their mathematical model of the intestinal crypt is an improvement over those already published but there is no direct comparison or review of the literature to substantiate this claim.

(6) The authors claim that the simulated data and the available mouse data match up. Nevertheless, the data vs the model still appear both quantitatively and qualitatively different (as presented in Figures 2E, F, and 5C, D). This puts in doubt how much the model can actually reproduce the experimental data. In conclusion, the model would benefit from further refinement, particularly if the goal is to use the model for predicting the dynamics of oncogenic drug candidates.

Reviewer #2 (Public Review):

Respiratory chain complexes assemble in higher-ordered structures termed supercomplexes or respirasomes. The functional significance of these assemblies is currently investigated, there are two main hypothesis tested, namely that supercomplexes provide kinetic advantages or structural stability. Here, the authors use the fruitfly to reveal that, while the respiratory chain in the organism normally does not form higher-order assemblies, it does so under conditions when their assembly is impaired. Because the rather moderate increase in supercomplex formation does not change oxygen consumption stimulated by CI or CII substrate, the authors conclude that supercomplex formation has more a structural than a functional role. The main strength of this work is that the technical quality of the experiments is high and that the authors induced defects in respiratory chain assembly through sets of well-controlled genetic models. The obtained data are mostly descriptive using standard approaches and are very well executed. The authors claim that their experiments allow to conclude that the role of supercomplex formation is restricted to a structural role and, hence, exclude a function directly related to electron transport efficiency. However, while the authors can show convincingly that supercomplexes form in the mutants, but not in the wild type, the main questions still remain, namely what is the structural mechanism of supercomplex formation and what is the significance of their formation. Given that the fly system does not show supercomplex formation under normal conditions, it is likely that it evolved functionally to work different than systems having supercomplexes. Because these differences are yet unknown, it remains questionable whether the fly system can be used to inform about the general significance of supercomplexes found in the other systems.

Reviewer #1 (Public Review):

In their manuscript, Brischigliaro et al. show that the disruption of respiratory complex assembly results in Drosophila melanogaster results in the accumulation of respiratory supercomplexes. Further, they show that the change in the supercomplex abundance does not impact respiratory function suggesting that the main role of supercomplex formation is structural. Overall, the manuscript is well written and the results and conclusion are supported. The D. melanogaster system, in which the abundance of supercomplexes can be altered through the genetic disruption of the assembly of the individual complexes, will be important for the field to discover the role of the supercomplexes. This manuscript will be of broad interest to the field of mitochondrial bioenergetics. The findings are valuable and the evidence is convincing.

Strengths<br /> The system developed in which the relative levels of SCs can be varied will be extremely useful for studying SC physiology.

The experiments are clearly described and interpreted.

Weaknesses<br /> The previous weaknesses identified have been addressed.

Reviewer #1 (Public Review):

DMRT1 is essential in testis development in different species. While Dmrt1 is the testis-determining factor in chicken and deletion encompassing this gene lead to gonadal dysgenesis in human, the role of DMRT1 in testis development remains to be clarified. Despite an early expression of Dmrt1 in the mouse gonad and a potential function as a pioneer factor, DMRT1 is only required for the maintenance of the Sertoli cell identity in the postnatal testis. The use of a new animal model could provide new insights into the role of this factor in humans. Here the authors have generated a knockout model of DMRT1 in rabbits. They show that the XY mutant gonads differentiate as ovary indicating that DMRT1 is required for testis differentiation in rabbits. In addition, most of the germ cells remain pluripotent as evidenced by the maintenance of POU5F1 in both XY and XX mutant gonads. These are very important results potentially explaining gonadal dysgenesis associated with the DMRT1 locus in disorders of sex development in humans.

The experiments are meticulous and convincing. I find the arguments of the authors about the role of DMRT1 in germ cells in addition to its function in Sertoli cell differentiation, both comprehensible and compelling. Clearly, this is an important insight in sex determination and gametogenesis.

Reviewer #2 (Public Review):

It is well known that DMRT proteins and more specifically, DMRT1 plays a key role in the sex determination processes of many species. While DMRT1 has been shown to be critical for the sex determination of fish, birds, and reptiles, it seems less crucial at the sex determination stages of the mice. It is important though for adult sex maintenance in mice.

Unlike its minor role in mouse sex determination, it seems that variants in DMRT1 in humans cause 46, XY DSD and sex reversal.

The paper by Dujardin et al. is a beautiful study that provides an answer to this long-lasting discrepancy of the difference between the two common mammal species: human and mouse. It is a really nice example of how working with other mammal species, like the rabbit, could serve as a nice model for understanding mammalian sex determination.

In this study the researchers first described the expression patterns of DMRT1 in the rabbit XY and XX gonads throughout the window of sex determination.

They then used CRISPR/Cas9 to generate DMRT1 KO rabbits and analysed the phenotype in XY and XX rabbits. They show that XY rabbits present with complete XY male-to-female sex reversal, very similar to what observed in human 46, XY DSD patients (but not the mice model). They further show that in the XY sex reversed gonads, germ cells fail to enter meiosis. They next analysed XX gonads and while there is no major effect on sex determination (as expected), the germ cells in these ovaries fail to enter meiosis, highlighting the critical role that DMRT1 has in germ cells.

I think it is really important that we start to embrace other mammal models that are not the mouse as we find many instances that the mouse is not the optimal system for understanding human sex determination.

The study is well explained and presented. The data is clear, and the paper is fluent to read.

Reviewer #3 (Public Review):

This manuscript deal with the sex-related gene, DMRT1, showing that is has a testis-promoting function in the rabbit. Loss-of-function studies the mouse and human, DMRT1 has a role in testis maintenance after birth, although forced expression in mouse can induce testis formation.

The authors used CRISPR/Cas9 genome editing to generate DMRT1-/- rabbit embryos. The gonads of these embryos developed as ovaries. Interestingly, in addition Y-linked SRY, DMRT1 is required for timely up-regulation of SOX9 during Sertoli cell differentiation in the male gonad. This is quite different to the situation in mouse, where Dmrt1 is not required in the testis until after birth (and Sry induced up-regulation of Sox9 hence does not require Dmrt1).

The work adds to the field of sex determination by further broadening our understanding of the DMRT1 gene and the evolution of gonadal sex determination.

In the Discussion section, it is suggested that DMRT1 could act as a pioneering factor to allow SRY action upon Sox9 in the rabbit model. The data show that DMRT1 may be more central to testis formation in mammals than previously considered. The work supports the notion that our understanding that the genetics of gonadal development (and indeed development more generally) should not rest solely on findings in the mouse.

Reviewer #2 (Public Review):

This manuscript by Martin-Flores et al. has examined the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic numbers. By using human AD brain databases and tissue samples, the authors showed that DKK3 protein and mRNA levels are increased in the brains of AD patients. DKK3 is expressed in the excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, secretion of DKK3 appears to be regulated by NMDAR antagonist as well as chemical LTD. Through gain and loss of function studies, the authors showed that DKK3 regulates the number of excitatory as well as inhibitory synapses with distinct downstream pathways. Finally, the authors investigated the contribution of DKK3 to synaptic changes in AD and found that DKK3 loss of function rescues both the excitatory and inhibitory synaptic defects, resulting in the improvement of memory function in J20 mice.

Overall, the data is clearly presented and deals with novel roles of DKK3 in controlling excitatory and inhibitory synapses. The finding that shRNA expression of DKK3 in AD model mice rescues synaptic phenotypes and memory impairment is potentially interesting and may provide a new strategy for AD treatment.

Reviewer #1 (Public Review):

In this study, Nuria Martin-Flores, Marina Podpolny and colleagues investigate the role of Dickkopf-3 (DKK3), a Wnt antagonist in synaptic dysfunction in Alzheimer's disease. Loss of synapses is a feature of Alzheimer's and other forms of dementia such as frontotemporal dementia and linked amyotrophic lateral sclerosis (FTD). The authors utilise a broad range of experimental approaches. They show that DKK3 levels are increased in Alzheimer's disease and that this occurs early in disease. This is an important finding since early disease changes are believed to be the most important. They also show increases in DKK3 in transgenic mouse models of Alzheimer's disease and that DKK3 knockdown restores synapse number and memory in one such model. Finally, they link these DKK3 increases to loss of excitatory synapses via the blockade of the Wnt pathway and subsequent activation of GSK3B; GSK3B is strongly linked to both Alzheimer's disease and FTD. The quality of the data is good and the conclusions well supported by these data. There are no major weaknesses. The findings support studies that target the Wnt pathway as a potential therapeutic for Alzheimer's disease.

Reviewer #3 (Public Review):

This study investigated cognitive mechanisms underlying approach-avoidance behavior using a novel reinforcement learning task and computational modelling. Participants could select a risky "conflict" option (latent, fluctuating probabilities of monetary reward and/or unpleasant sound [punishment]) or a safe option (separate, generally lower probability of reward). Overall, participant choices were skewed towards more rewarded options, but were also repelled by increasing probability of punishment. Individual patterns of behavior were well-captured by a reinforcement learning model that included parameters for reward and punishment sensitivity, and learning rates for reward and punishment. This is a nice replication of existing findings suggesting reward and punishment have opposing effects on behavior through dissociated sensitivity to reward versus punishment.

Interestingly, avoidance of the conflict option was predicted by self-reported task-induced anxiety. Importantly, when a subset of participants were retested over 1 week later, most behavioral tendencies and model parameters were recapitulated, suggesting the task may capture stable traits relevant to approach-avoidance decision-making.

The revised paper commendably adds important additional information and analyses to support these claims. The initial concern that not accounting for participant control over punisher intensity confounded interpretation of effects has been largely addressed in follow-up analyses and discussion.

This study complements and sits within a broad translational literature investigating interactions between reward/punishers and psychological processes in approach-avoidance decisions.

Reviewer #1 (Public Review):

This paper describes the development and initial validation of an approach-avoidance task and its relationship to anxiety. The task is a two-armed bandit where one choice is 'safer' - has no probability of punishment, delivered as an aversive sound, but also lower probability of reward - and the other choice involves a reward-punishment conflict. The authors fit a computational model of reinforcement learning to this task and found that self-reported state anxiety during the task was related to a greater likelihood of choosing the safe stimulus when the other (conflict) stimulus had a higher likelihood of punishment. Computationally, this was represented by a smaller value for the ratio of reward to punishment sensitivity in people with higher task-induced anxiety. They replicated this finding, but not another finding that this behavior was related to a measure of psychopathology (experiential avoidance), in a second sample. They also tested test-retest reliability in a sub-sample tested twice, one week apart and found that some aspects of task behavior had acceptable levels of reliability. The introduction makes a strong appeal to back-translation and computational validity. The task design is clever and most methods are solid - it is encouraging to see attempts to validate tasks as they are developed. The lack of replicated effects with psychopathology may mean that this task is better suited to assess state anxiety, or to serve as a foundation for additional task development.

Reviewer #2 (Public Review):

Summary:

The authors develop a computational approach-avoidance-conflict (AAC) task, designed to overcome the limitations of existing offer based AAC tasks. The task incorporated likelihoods of receiving rewards/ punishments that would be learned by the participants to ensure computational validity and estimated model parameters related to reward/punishment and task induced anxiety. Two independent samples of online participants were tested. In both samples participants who experienced greater task induced anxiety avoided choices associated with greater probability of punishment. Computational modelling revealed that this effect was explained by greater individual sensitivities to punishment relative to rewards.

Strengths:

Large internet-based samples, with discovery sample (n = 369), pre-registered replication sample (n = 629) and test-retest sub group (n = 57). Extensive compliance measures (e.g. audio checks) seek to improve adherence.

There is a great need for RL tasks that model threatening outcomes rather than simply loss of reward. The main model parameters show strong effects and the additional indices with task based anxiety are a useful extension. Associations were broadly replicated across samples. Fair to excellent reliability of model parameters is encouraging and badly needed for behavioral tasks of threat sensitivity.

The task seems to have lower approach bias than some other AAC tasks in the literature.

Appraisal and impact:<br /> Overall this is a very strong paper, describing a novel task that could help move the field of RL forward to take account of threat processing more fully. The large sample size with discovery, replication and test-retest gives confidence in the findings. The task has good ecological validity and associations with task-based anxiety and clinical self-report demonstrate clinical relevance. Test-retest of the punishment learning parameter is the only real concern. Overall this task provides an exciting new probe of reward/threat that could be used in mechanistic disease models.

Additional context:

The sex differences between the samples are interesting as effects of sex are commonly found in AAC tasks. It would be interesting to look at the main model comparison with sex included as a covariate.

Reviewer #1 (Public Review):

This study assesses the volatile profiles from the hair and bodies of 64 vertebrate species to compare odor constituents across taxa. Compared to a similar data set for floral volatiles, the study suggests that vertebrate odors are significantly less diverse and show little phylogenetic relationship regarding profile similarity. Human odors were particularly unique from other species. It is concluded that this may influence the odor coding of organisms (like vectors) who respond to these odors compared to plant-feeding organisms like most other insects. While the study is compelling, several methodological issues leave the conclusions less convincing. It is suggested that the paper be tempered accordingly with these issues mentioned.<br /> The study makes several assumptions about the methodology to be considered when interpreting the results:

Major Concerns:

1) Body hair as a proxy for animals. "Hair odour is likely a reasonable proxy for mammalian body odour, but may lose some volatile compounds during storage. Live-animal odour, on the other hand, can be contaminated with compounds from faeces or urine occasionally excreted during sampling." The study has addressed this by testing hair against the bodies of 4 humans, two rats, and one guinea pig (Figure S1). However, the results show that there are both quantitative and qualitative differences among all the samples. While the presence of waste accounts for some of this variation, this, too, is a natural response of the animal and could be present in natural settings. Also would not body heat in mammals have an impact on odors? The authors should support this. While this does not require reanalysis, the authors should address these differences, particularly when qualitative and quantitative differences are discussed heavily in the results.

2) Sampling medium: Tenax TA was used to sample the vertebrate odors. Please note that any sorbent will exhibit specificity regarding selectivity and sensitivity to VOCs. See https://www.eva.mpg.de/documents/Elsevier/Marcillo_Comparison_JChromA_2017_2452774.pdf for one comparison. For example, it is not surprising that "Aldehydes, ketones, alcohols, aromatics, terpenes, and hydrocarbons dominated" the samples given that these types of compounds are well retained by the Tenax polymer:<br /> https://www.sisweb.com/index/referenc/tenaxta.htm<br /> Many chemical ecology studies will employ multiple polar and non-polar polymers to retain different VOCs for better profile comparison.<br /> By itself, this limitation must be noted. However, it becomes even more relevant when compared to the floral volatile study, which used a different sorbent (Poropak) which is less hydrophobic and may retain more polar compounds than Tenax: https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/2859526<br /> Such differences must be considered when comparing these two datasets, particularly when the study makes conclusions about their differences. Alternatively, a small set of experiments with poropak and a few species (like for the hair vs. body control experiments) could clarify the effect of sorbent type on VOC retention.

3) Sampling quantification: The methods note that " All extractions were run for 5-80 minutes depending on the expected odour concentration of the sample." What does this mean? Such differences in sampling timing in our lab have shown profound differences in the type and amount of volatiles collected. Generally, it is best to sample for as long as possible to ensure that the most volatiles are collected (up to 24 hours if possible). The compounds will eventually come into equilibrium with the sorbent. However, for quantification, the timing must be calibrated carefully, usually by using a representative set of likely compounds with different functional groups to determine the optimal length of sampling time. Was this done in this case? If not, how can one account for the significant variation in sampling time regarding quantification?<br /> A second issue with quantification is the need for an internal standard. Even with robotic assistance, slight variations in processing can significantly affect the quantity of volatile retained through detection at the MS. This is generally avoided by using an internal standard in the sampling arena. See this example with multiple sampling techniques (also employing TD-GCMS): https://www.frontiersin.org/articles/10.3389/fevo.2021.607555/full<br /> Without these methodological controls, it is unclear how effective quantification can be performed. It might be more prudent to confine the results to qualitative discussions.

Reviewer #2 (Public Review):

Summary:<br /> Zung et al. use a comparative approach to examine the volatile headspace of diverse mammals and host species to understand the differences in chemical profiles that may provide mosquitoes with signatures of appropriate hosts. The authors collect the volatiles from hair samples and conduct qualitative analyses of the headspace composition. The authors' results suggest that mammals share overlapping volatile signatures, although the sampling method and statistical approaches reduce the veracity of the authors' findings. Additional comparisons between mammalian and floral odours were conducted, although the datasets were limited.

The inter-species comparisons will be helpful in the field, although the data pipeline and approaches may underestimate the headspace chemical diversity, and sampling artifacts and contaminants occur in the datasets, which further weakens the study's findings.

Strengths:<br /> The comparative approach is a strength of the manuscript. The authors identify an important gap in mosquito natural history by attempting to characterize the odours from various mammalian, bird, and reptile species that mosquitoes may use as blood hosts. Although others have compared the skin volatiles of humans, apes, and ungulates (Verhulst et al., 2018, not cited in the current manuscript), Zung and coworkers expand this sampling by using hair samples from collections and zoos. Unfortunately, the sampling approach leads to potential artifacts associated with the collected volatiles and statistical analyses.

Weaknesses:<br /> There are three major points of weakness associated with the manuscript: (1) sampling approach and analysis pipeline; (2) statistical analyses; and (3) premise and prior work.

1. Sampling approach and pipeline<br /> A. The authors have described their sampling and analysis as quantitative, but they use a qualitative approach by not quantifying their samples and using a low-res MS. I outline several approaches that would allow the authors to quantitate their samples. The authors must run synthetic standards for peak verification (the mass spectra alone are insufficient for compound identification). The authors are also encouraged to run the standards in a concentration curve to allow quantification of the compounds. The authors have only tentatively identified 120 compounds. Using an autosampler and standard analyses in the software, the authors could easily quantify their samples which would take less than a week's time (this is not impossible, as the authors state in the methods). Based on the volatile fragmentation and the MS detector, the compounds will differ in their relative abundances - running calibration curves, co-injection of authentic standards, and using multiple column types are necessary for the resulting statistical analyses to prevent mischaracterization of the abundances in the hair samples. Using an internal standard, by spiking the Tenax before collection, would also allow determination if column conditions change over the course of the experiment. These measurements would provide some quantitative measures to explore the differences in host odors. Details on these approaches can be found in Methods in Chemical Ecology, Techniques in Pheromone Research, and article reviews that describe more recent approaches and analyses (Tholl and Rose, 2006; Stashenko and Martínez, 2008; Spicer et al., 2017; Tholl et al., 2020; Eisen et al., 2021; Schulz and Mollerke, 2022).

B. Abundant contaminants in the samples. In the supplemental table of partially identified compounds, many contaminants are associated with the headspace collection method and environmental contaminants. Under thermal deadsorption, Tenax degradation produces many compounds, including quinolones and benzenoid compounds. Phenyl-substituted carbonyl compounds (benzaldehyde, acetophenone, benzene acetaldehyde) are formed as artifacts from the oxidation of Tenax with environmental contaminants. Other compounds, like phenol or -ethyl and methylated benzene compounds, are known to be released from the Tenax traps. The authors' pipeline and blank subtraction should have identified these compounds.

C. Hair and live headspace volatiles. I appreciate the authors' experiments comparing the composition and abundance of volatiles from live collections and hair samples. However, the results demonstrate that the hair does not always match the volatiles from the live animal. Humans 1, 3, and 4 differ significantly in their aldehyde abundances, especially nonanal. The hamster and mice samples also differ significantly. The matrix of the hair will adsorb and modify the emissions and ratios of compounds, which makes the inter-species comparisons difficult if not impossible if the headspace collection approaches differ. The authors need to change their phrasing of the host odours to "hair odours", and soften their statements associated with the complete host odour profile, and use hair samples as a standard matrix for the headspace collections. The comparison of human odour collections relative to hair samples is like the comparison of apples and oranges.

D. The authors need to use another column type to characterize their peaks further. Some of the compounds are enantiomers or closely elute from the column. Although the authors suggest their methods may separate these compounds, they may be misidentified without a different GC temperature ramp or column.

E. The authors should replace their retention indices with KRI values to further identify their compounds. The methods section does not describe whether the alkane standards were run parallel to the hair samples, and the manuscript's retention indices do not match published KRI values.

F. The number of compounds across species (including flower compounds) is very low (approximately 120 compounds) and surprising. This suggests that the analysis pipeline and thresholding may miss many compounds in the headspace. I would encourage the authors to lower their threshold to 10^-5 AU, or to perform a sensitivity analysis on their ability to identify the peaks. Running authentic standards would also allow the identification of compounds missed in the analysis.

G. I understand the difficulty in obtaining these samples across the different species. However, additional information is needed for those species that are limited in the number of replicates (individuals). Sampling the individual multiple times may indicate the variability in the hair volatiles. Although the authors and many others have shown the reproducibility of human skin volatiles through time, additional sampling would indicate this also occurs for other mammals while strengthening the authors' approach.

H. An important measure of natural odour statistics is the odor emission rates, and normalizing across samples by the sample mass. More information on the methods would have clarified these aspects. It needs to be clarified why the samples were collected for different time periods (5 to 80 minutes). The sample mass for each specimen should also be included as this would allow normalization by time and mass, and should be described in the methods. This would allow quantitative measurements of the samples.

I. A critical missing component in the headspace is the acids. Tenax does not perform well at collecting these compounds. However, Gerstel Twisters and other collection matrices can capture those compounds. The authors must use these other collection methods to sample the hair specimens and identify those compounds to include in their table and analyses. Without this information, the manuscript lacks a critical dimension in the human odour landscape that is critical for mosquito attraction.

2. Statistical Analyses<br /> A. Sampling effort and the replicate numbers used in the analyses is an important consideration that the authors do not address, but should be discussed in more detail. In many subfields of chemical ecology, a minimum of ten replicates per species has been suggested to accurately identify the composition of compounds, and even with ten samples, this may not be enough to characterize the volatile profile (Raguso and Pellmyr, 1998; Campbell et al 2019). The authors could perform a power analysis, or an accumulation curve to represent the needed sample number to identify the number of compounds in the hair headspace accurately.

B. It would be worthwhile for the authors to provide more detail on their supervised and unsupervised approaches, and how their data fits the assumptions of the analyses. The PCA parametric method may require log or square root transformation of the data to make residuals fit the normality assumption, but it's unclear if this was the case with the authors' datasets.

C. PCA is also not appropriate when many samples have zero values in the data matrix, which occurs in the authors' data. In such a case, the approaches of NMDS or canonical analysis of principal coordinates would be more appropriate, and allow distance measures (the Bray-Curtis distance) to define dissimilarity of different groups. An analysis of similarity (ANOSIM) could be used to determine if the data clustered significantly by species or by mosquito host.

D. The authors are encouraged to use alternate approaches, such as random forest (ML) approach, to determine if the odor classification is based on host or non-host. This method has been used for the last fifteen years in chemical ecology and human odor analysis (Cutler et al, 2007, Kwak et al 2008).

E. The authors use a phylogenetic framework for their analyses. Multivariate methods are now available to test evolutionary hypotheses about scent composition in a phylogenetic framework (Goolsby, 2017), and the authors are encouraged to use these approaches.

F. Comparison to floral odour space section. I would encourage the authors to examine other datasets of plant headspace samples, including plants used by mosquitoes. There are many datasets out there that the authors could use (El-Sayed 2021, Farré-Armengol et al 2020). Expanding the authors' dataset would provide more statistical power, and provide control of differences in plant visitor and plant phylogenetic relatedness.

G. Adding context related to mosquito olfaction. The authors describe how their work could provide insight into the coding of olfactory information by the mosquito. I would encourage the authors to analyze their data further by collapsing the host volatiles into groups based on biochemical pathways, or knowledge of the detection of the volatiles by the mosquitoes (such as using electroantennogram responses) to filter and identify only those responsive volatiles to keep in their dataset.

Premise and Background Knowledge<br /> A. Analyses of odour headspace have been known for the last three decades, e.g. (Methods in Chemical Ecology, Techniques in Pheromone Research, George Petri's work, Tholl and Rose, 2006; Stashenko and Martínez, 2008; Spicer et al., 2017; Tholl et al., 2020; Eisen et al., 2021; Schulz and Mollerke, 2022). But in many places, the paper conveys the impression that these are new discoveries and analyses. For example,<br /> -"Yet we remain remarkably ignorant of the composition of the chemical world."<br /> -"Our work provides one of the first quantitative descriptions of a natural odour space"<br /> -"Progress in understanding natural odours has also been hindered by the technical challenges of capturing and analyzing odour, especially the complex blends that constitute most natural odours"<br /> The Introduction and Discussion are rife with these overblown statements. I found this frustrating as the authors were not giving due credit to prior work on that topic while (maybe unintentionally) giving an impression that this specific idea was a new contribution. More care is needed to delineate which aspects of the study are 1) based on prior understanding, or 2) totally new). The authors are adding to an already extensive field of chemical ecology and olfactory processing of mixtures, and are contributing to this knowledge by adding datasets related to mammalian odor. I plead that the authors clearly describe these gaps, and place their results into proper context.

B. Similarly to the above statements relating to chemical ecology, the authors have numerous statements about gaps in odour processing. Mixture processing has been an important topic of study for the last forty years (Shorey, 1973, Caprio, 1988, Riffell et al 2009, Su et al 2009, Rokni et al 2014, Mathis et al 2016), which is based on encoding the temporal and concentration-dependent statistics of the odour.<br /> -"Yet compared to visual and auditory scenes, we know very little about the statistics of natural olfactory scenes"<br /> As described above, this is surprising and frustrating because of the rich literature on these topics (searching for "odour mixtures" provides 32,000 articles). In their manuscript, the authors are providing a strawman argument for their analyses by focusing on single odorant signatures, when the literature has repeatedly demonstrated the importance of odour mixtures for behavior and combinatorial processing.

C. There are increasing studies examining the mosquito behavioral and electrophysiological responses to hosts and other odours. However, this literature is not cited or included in the authors' analyses. The chemical ecology of mosquito attractants and natural odours has been studied in the Carde, Leal, Ignell, Carlson, Kline, Riffell, Takken, Torto, Verlhurst, Vosshall labs, and many others. The authors could use this information in their analyses and cite the literature.

Reviewer #3 (Public Review):

This study focused on collecting and analyzing odour samples from a wide range of vertebrate species to understand the composition and characteristics of vertebrate body odours. The researchers used dynamic headspace sampling to collect odour samples from 120 individual animals representing 64 vertebrate species. They collected odour from both live animals and hair samples, with hair being a reasonable proxy for mammalian body odour.

The odour samples were analyzed using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) to identify compounds and estimate their abundance. They identified a total of 116 compounds in the vertebrate odour extracts, including aldehydes, ketones, alcohols, aromatics, terpenes, and hydrocarbons. The compounds varied in prevalence across species, but a large number of compounds were found in at least 15 samples, indicating a broad overlap in odour composition among vertebrates.

The study compared the vertebrate odour space to floral odour space and found that vertebrate odours shared more compounds compared to floral odours. Floral odours tended to be less complex and more likely to contain unique compounds found only in a single species. The analysis also revealed that odour profiles did not show strong phylogenetic signals, indicating that closely related species did not necessarily have similar odour profiles. However, within-species clustering was observed, suggesting that body odour composition may be species-specific.

The researchers also investigated specific compounds that could serve as host-seeking cues for animals. They compared the odour of live vertebrate hosts to non-host stimuli and identified straight-chain aldehydes as abundant compounds in vertebrate odours. These aldehydes were found at substantially lower levels in non-host stimuli. Additionally, when comparing human odour to other vertebrate species and non-host stimuli, several compounds, including decanal, sulcatone, geranylacetone, and undecanal, emerged as strong predictors of human hosts.

Three shortcomings of the study can be highlighted:<br /> 1. Undersampling of certain compound classes: The study acknowledged that they undersampled carboxylic acids, which are generally too polar or non-volatile to be analyzed without a special derivatization step. This limitation could have resulted in an incomplete understanding of the full range of compounds present in vertebrate odours.<br /> 2. Missing highly volatile compounds: The study mentioned the difficulty of capturing and quantifying highly volatile compounds reliably. This limitation suggests that certain compounds with high volatility may not have been adequately represented in the analysis, potentially impacting the comprehensiveness of the odour space.<br /> 3. Lack of controlled experiment for species replicates: Although the study observed strong within-species clustering for some species in their dataset, they cautioned that many of the species replicates came from the same farm or zoo, which could confound the results with sample origin. The lack of a well-controlled experiment limits the generalizability of the findings regarding consistent and characteristic odour profiles across animals.

These shortcomings should be considered when interpreting the results of the study and could be addressed in future research to further advance our understanding of vertebrate body odours.

The manuscript highlights three open questions. First, the authors discuss the implications of the differences between vertebrate and floral odors for olfactory coding in blood feeders and floral visitors. Specialist mosquitoes require odor blends to detect hosts, while honeybees can generalize from attractive mixtures to individual components. The authors suggest that these differences may be influenced by the different odor spaces mosquitoes and bees inhabit.

Second, the authors note that although compounds in vertebrate odor are shared broadly across species, they are also common in other natural odors. This poses a challenge for generalist blood feeders, but the study suggests that straight-chain, saturated aldehydes, which are highly abundant in vertebrate odors, may still serve as useful indicators. These aldehydes have been shown to enhance host-seeking in mosquitoes and are even used by malaria parasites and orchids to attract mosquitoes. However, the study did not capture highly volatile or polar compounds that may also indicate the presence of a vertebrate host.

Third, the manuscript discusses the lack of phylogenetic signal in the odors of mammals, which make up the majority of the sampled species. This may explain why few mosquitoes exhibit preferences for taxonomic groups at the family or order level. The study suggests that within a species, there is high consistency in odor-blend composition, which may mediate species-specific host preference through olfactory cues.

The authors also focus on odor features that may serve as valuable cues for human specialists. They find that certain components of human odor, such as sulcatone, geranylacetone, decanal, and undecanal, are distinctive and enriched in human odor. Undecanal, despite being less common across non-human animals and in nature overall, is a more reliable indicator of human odor than decanal. The two ketones are even more reliable indicators. The authors speculate that the reliance on aldehydes by human-specialist mosquitoes may be due to the evolutionary history of these mosquitoes, which arose from an ancestral generalist subspecies.

In conclusion, this manuscript presents a quantitative study of vertebrate animal odors, highlighting the differences between vertebrate and floral odors. It raises questions about olfactory coding in blood feeders and floral visitors, the challenges faced by generalist blood feeders, and the lack of phylogenetic signal in mammalian odors. The study also explores odor features that may be valuable cues for human specialists and discusses the evolutionary implications of these findings.

Reviewer #1 (Public Review):

This study seeks to understand how selective mRNA translation informs cellular identity using the Drosophila brain as a model. Using drivers specific for either neurons or glia, the authors express a tagged large ribosomal subunit protein, which they then use as a handle for isolating total mRNA and ribosome footprints. Throughout the study, they compare these data sets to transcriptional and ribosome profiles from the whole fly head, which contains multiple cell types including fat tissue, pigment cells, and others, in addition to neurons and glia. Using GO term analyses, they demonstrate the specificity of their cell-type-based ribosome profiling: known glial mRNAs are efficiently translated in glia and likewise in neurons as well. In further examining their RNAseq data set, they find that "neuronal" mRNAs, such as ion channels, are expressed in both neurons and glia, but are translated at higher rates in neurons. Based on this, they hypothesize that neuronal mRNAs are actively suppressed in glia, and next seek to determine the underlying mechanism. By meta-analysis of all mapped ribosome footprints, they find that glia have higher ribosome occupancies in the 5' leader of neuronal mRNAs. This is corroborated by individual ribosome occupancy profiles for several neuronal mRNAs. In 5'leaders containing upstream AUG codons, they find that the glial data sets show enrichment of ribosomes at these upstream start sites. They thus conclude that 5' leaders containing upstream AUGs confer translational suppression in glia.

Overall, the sequencing data sets generated in this study and their subsequent bioinformatic analyses seem robust and reliable. Their data echo the trends of cell-type specific translational profiles seen in previous studies (e.g. 27380875, 30650354), and making their data sets and analyses accessible to the broader scientific community would be quite helpful. The findings are presented in a logical and methodical manner, and the data are depicted clearly. The authors' results that 5' leaders facilitate translation suppression is well-supported in literature. However, they overinterpret their data by claiming that such suppression is key for maintaining glial/neuronal identity (it is even featured in their title), but do not present any evidence that loss of such regulation has any impact on cellular identity. In many places, the authors do not acknowledge possible biases in their analytical methods, or consider alternate explanations for their data. These weaken the manuscript in its current form, but many of these issues which I describe below, are rectifiable with modest effort.

1. The authors' data in Fig. 2-S1A-B shows substantial cell-to-cell variation in RpL3::FLAG expression. The authors do not consider that this variation may cause certain neuronal/glial types to be overrepresented in their datasets. A related point is that the authors do not discuss whether RpL3::FLAG is only present in the cell body or if it is also trafficked to the neuronal/glial processes where localized translation is known to occur (reviewed in 31270476).

2. The RNA-seq data set that they use to calculate translation efficiency (TE) only represents mRNAs associated with RpL3::FLAG, which is part of the large ribosome subunit. As the authors are likely aware, there are mRNAs on which the full ribosome moiety does not assemble and these are effectively excluded from this data set. Ideally, a more complete picture of the mRNA landscape can be obtained by 40S subunit profiling but I appreciate that this is technically very challenging. At a minimum, this caveat needs to be acknowledged.

How does the TPM of differentially regulated transcripts (such as those in Fig. 2H) compare between whole heads, neurons, and glia? Since the whole head RNA-seq data was not from an enriched sample, this might serve as a decent proxy for showing that the neuron/glia RNA-seq data sets are representative of RNA abundance.

3. The analysis in Fig. 2F shows that low abundance mRNAs in glia are further translationally suppressed, which the authors point out in lines 151-152. However, this data also shows that mRNAs with a 1:1 ratio in neuron:glia (which fall in the 0.5-1 and 1-2 bin) have a TE1; this suggests that on average, mRNAs that are equally abundant are translated equally efficiently. This is the opposite of the thesis presented in Fig. 2G-H where many mRNAs of equal abundance in neurons and glia are actually poorly translated in glia. How do the authors reconcile these observations?<br /> It is also unclear from the manuscript whether all mRNAs were considered for the analysis in Fig. 2F or if some cutoff was employed.

4. Throughout the manuscript the authors favor a "translation suppression" model wherein glia (for example) actively suppress neuronal mRNAs, and this is substantiated in Fig. 3C showing higher ribosome occupancy on 5' leaders than in coding regions. However, they show no evidence that glial mRNAs (such as those indicated in Fig. 2B and 2-S2B) present a different pattern, say that of higher ribosome occupancy in CDS vs. 5' leaders. This type of positive control is a glaring omission from many of their analyses, including ribosome occupancy at upstream AUG codons (Fig. 4).<br /> In order to make a broad case (as they do in the title) that differential translation regulation specifies multiple cell types, it is necessary to show the corollary: that glial mRNAs (repo, bnb, pnt, etc) are suppressed in neurons. There is an inkling of this evidence in Fig. 3-S1 where fat body mRNAs in neurons are shown to have low ribosome occupancy in the CDS regions and enhanced occupancy in the 5' leader region. This data is not quantified, nor is a control neuron mRNA shown as a reference for what the ribosome occupancy profile of an actively translated mRNA looks like in a neuron.

5. The cell-type specific ribosome profiling data sets in the manuscript are from mRNAs associated with 80s subunits that have been treated with cycloheximide during sample preparation. Cycloheximide, and many other translation inhibitors, are known to non-uniformly bias reads towards start codons (PMID: 22056041,22927429). This important caveat and its implications on the start-codon occupancy analysis in Fig. 4 are not acknowledged in the manuscript.<br /> Again, the ideal resolution would be a ribosome profiling data set from 40S footprinting or harringtonine-treated samples (PMIDs: 32589966, 27487212, 32589964) to show the true accumulation of ribosomes at AUG codons. In the absence of such a data set, a comparative meta-analysis of the ribosome distribution around upstream and initiation AUG codons of differentially translated transcripts from neurons would be a useful control.

6. The authors chose Rhodopsin 1 (Rh1) as a model mRNA which is translated efficiently in neurons but suppressed in glia. Though the data in Fig. 2-S3B shows higher TE for Rh1 in neurons, the data in 5A show lower ribosome occupancy in the Rh1 CDS in neuron samples (at least in the fragment of the CDS visible). These data are somewhat contradictory.<br /> Further, given that the neuron data are from all nsyb-positive cells but that Rh1 is expressed only in R1-R6 photoreceptors, it is unclear what motivated them to choose Rh1 as opposed to an mRNA that is more broadly expressed in neurons.

7. Similar to the heterogeneity in nsyb- and repo-GAL4 expression in Fig. 2-S1A-B, Fig. 5C shows substantial variation in the expression of the UAS-GFP reporter driven by tub-GAL4. This variable GAL4 activity makes the mRNA abundance data difficult to interpret. Also, since the authors presume that Rh1 mRNA is expressed in glia (it is not annotated in the RNA-seq analysis in Fig. 2-S2B), would Rh1-GAL4 not be a more apt driver?<br /> These issues are further compounded by the lack of a cellular compartment marker (repo marks glial nuclei) which makes it impossible to determine which cell the mRNA signal is in. There are also no negative controls presented for the mRNA probes.<br /> Most confoundingly though, the control reporter itself seems to show variable translation efficiencies from one cell to another, with high-GFP protein cells showing lower GFP mRNA and vice versa.<br /> The mRNA:protein ratio may be easier to examine by using repo-GAL4 to specifically drive the Rh1-reporter expression in glia (such as in Fig. 5-S1A) rather than simultaneous expression in both neurons and glia using tub-GAL4.

Reviewer #2 (Public Review):

Summary:<br /> The authors used next-generation sequencing approaches combined with ribosome trapping to investigate gene expression in neurons and glia in the heads of adult fruit flies. Ribosome footprinting was further used to investigate the translational efficiency (TE) of particular RNAs in these two tissues. The evidence convincingly demonstrated that translation of specific messages is repressed in glia while others are repressed in neurons. Further evidence suggests that cis-acting elements within the 5'UTR of neuronal transcripts cause the repression of translation in glia. For instance, a fluorescent reporter using the 5'UTR of Rhodopsin-1 is highly translated in neurons but fluorescence from this reporter is nearly undetectable in glia. Furthermore, pausing of ribosomes on start codons of upstream Open Reading Frames (uORFs) is seen on the 5'UTR of this and other messages in glia but not in neurons.

Strengths:<br /> The main strength of the manuscript is its use of cutting-edge next-generation sequencing and bioinformatic approaches to investigate the tissue-specific translatome of Drosophila.

Weaknesses:<br /> A minor weakness is that little insight is provided into the mechanism that leads to ribosome stalling on uORFs in glia but not in neurons. The manuscript could be improved by some discussion on potential pathways that might control the differential TE through uORF pausing.

Reviewer #3 (Public Review):

It is well established that there is extensive post-transcriptional gene regulation in nervous systems, including the fly brain. For example, dynamic regulation of hundreds of genes during photoreceptor development could only be observed at the level of translated mRNAs, but not the entire transcriptomes. The present study instead addresses the role of differential translational regulation between cell types (or rather classes: neurons and glia, as both are still highly heterogenous groups) in the adult fly brain. By performing bulk RNA-seq and Ribo-seq on the same lysates, the authors are able to compare the translation efficiency (TE) of all transcripts between neurons and glia. Many genes display differential TE, but interestingly, they tend to be the genes that already show strong differences at their mRNA level. The most striking observation is the finding that neuronal transcripts in glia display increased ribosome stalling at their 5' UTR, and in particular at the start codons of short "upstream ORFs". This could suggest that glia specifically employ a mechanism to upregulate upstream ORF translation, enabling them to better suppress the expression of the genes that have them. And neuronal genes tend to have longer 5' UTRs, perhaps to facilitate this type of regulation.

However, it is difficult to evaluate the functional significance of these differences because the authors provide only one follow-up experiment to their RNA-seq analysis. Venus expressed with the Rh1 UTR sequences may be displaying differential levels between glia and neurons, but I find this image (Fig. 5C) rather unconvincing to support that conclusion. There are no quantifications of colocalization or even sample size information provided for this experiment. And if there is indeed a difference, it would still be difficult to argue this is because of the 5' stalling phenomenon authors observe with Rh1, because they switched both the 5' and 3' UTRs.

I also find it puzzling that the TE differences between the groups are mostly among the transcripts that are already strongly differentially expressed at the transcriptional level. The authors would like to frame this as a mechanism of 'contrast sharpening'; but it is unclear why that would be needed. Rh1, for instance, is not just differentially expressed between neurons and glia, but it is actually only expressed by a very specific neuronal type (photoreceptors). Thus it's not clear to me why the glia would need this 5' stalling mechanism to fully suppress Rh1 expression, while all the other neurons can apparently do so without it.

Reviewer #1 (Public Review):

In the article "Temporal transcriptomic dynamics in developing macaque neocortex", Xu et al. analyze the cellular composition and transcriptomic profiles of the developing macaque parietal cortex using single-cell RNA sequencing. The authors profiled eight prenatal rhesus macaque brains at five timepoints (E40, E50, E70, E80, and E90) and obtained a total of around 53,000 high-quality cells for downstream analysis. The dataset provides a high-resolution view into the developmental processes of early and mid-fetal macaque cortical development and will potentially be a valuable resource for future comparative studies of primate neurogenesis and neural stem cell fate specification. Their analysis of this dataset focused on the temporal gene expression profiles of outer and ventricular radial glia and utilized pesudotime trajectory analysis to characterize the genes associated with radial glial and neuronal differentiation. The rhesus macaque dataset presented in this study was then integrated with prenatal mouse and human scRNA-seq datasets to probe species differences in ventricular radial glia to intermediate progenitor cell trajectories. Additionally, the expression profile of macaque radial glia across time was compared to those of mouse apical progenitors to identify conserved and divergent expression patterns of transcription factors.

The main findings of this paper corroborate many previously reported and fundamental features of primate neurogenesis: deep layer neurons are generated before upper layer excitatory neurons, the expansion of outer radial glia in the primate lineage, conserved molecular markers of outer radial glia, and the early specification of progenitors. Furthermore, the authors show some interesting divergent features of macaque radial glial gene regulatory networks as compared to mouse. Overall, despite some uncertainties surrounding the clustering and annotations of certain cell types, the manuscript provides a valuable scRNA-seq dataset of early prenatal rhesus macaque brain development. The dynamic expression patterns and trajectory analysis of ventricular and outer radial glia provide valuable data and lists of differentially expressed genes (some consistent with previous studies, others reported for the first time here) for future studies.

The major weaknesses of this study are the inconsistent dissection of the targeted brain region and the loss of more mature excitatory neurons in samples from later developmental timepoint due to the use of single-cell RNA-seq. The authors mention that they could observe ventral progenitors and even midbrain neurons in their analyses. Ventral progenitors should not be present if the authors had properly dissected the parietal cortex. The fact that they obtained even midbrain cells point to an inadequate dissection or poor cell classification. If this is the result of poor classification, it could be easily fixed by using more markers with higher specificity. However, if it is the result of a poor dissection, some of the cells in other clusters could potentially be from midbrain as well. The loss of more mature excitatory neurons is also problematic because on top of hindering the analysis of these neurons in later developmental periods, it also affects the cell proportions the authors use to support some of their claims. The study could also benefit from the validation of some of the genes the authors uncovered to be specifically expressed in different populations of radial glia.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript by Xu et al., is an interesting study aiming to identify novel features of macaque cortical development. This study serves as a valuable atlas of single cell data during macaque neurogenesis, which extends the developmental stages previously explored. Overall, the authors have achieved their aim of collecting a comprehensive dataset of macaque cortical neurogenesis and have identified a few unknown features of macaque development.

Strengths:<br /> The authors have accumulated a robust dataset of developmental time points and have applied a variety of informatic approaches to interrogate this dataset. One interesting finding in this study is the expression of previously unknown receptors on macaque oRG cells. Another novel aspect of this paper is the temporal dissection of neocortical development across species. The identification that the regulome looks quite different, despite similar expression of transcription factors in discrete cell types, is intriguing.

Weaknesses:<br /> Due to the focus on demonstrating the robustness of the dataset, the novel findings in this manuscript are underdeveloped. There is also a lack of experimental validation. This is a particular weakness for newly identified features (like receptors in oRG cells). It's important to show expression in relevant cell types and, if possible, perform functional perturbations on these cell types. The presentation of the data highlighting novel findings could also be clarified at higher resolution, and dissected through additional informatic analyses. Additionally, the presentation of ideas and goals of this manuscript should be further clarified. A major gap in the study rationale and results is that the data was collected exclusively in the parietal lobe, yet the rationale and interpretation of what this data indicates about this specific cortical area was not discussed. Last, a few textual errors about neural development are also present and need to be corrected.

Reviewer #3 (Public Review):

Summary: The study adds to the existing data that have established that cortical development in rhesus macaque is known to recapitulate multiple facets cortical development in humans. The authors generate and analyze single cell transcriptomic data from the timecourse of embryonic neurogenesis.

Strengths:<br /> Studies of primate developmental biology are hindered by the limited availability and limit replication. In this regard, a new dataset is useful.

The study analyzes parietal cortex, while previous studies focused on frontal and motor cortex. This may be the first analysis of macaque parietal cortex and, as such, may provide important insights into arealization, which the authors have not addressed.

Weaknesses:<br /> The number of cells in the analysis is lower than recent published studies which may limit cell representation and potentially the discovery of subtle changes.

The macaque parietal cortex data is compared to human and mouse pre-frontal cortex. See data from PMCID: PMC8494648 that provides a better comparison.

A deeper assessment of these data in the context of existing studies would help others appreciate the significance of the work.

Reviewer #1 (Public Review):

The main goal of the study was to tease apart the associative and non-associative elements of cued fear conditioning that could influence which defensive behaviors are expressed. To do this, the authors compared groups conditioned with paired, unpaired, or shock only procedures followed by extinction of the cue. The cue used in the study was not typical; serial presentation of a tone followed by a white noise was used in order to assess switches in behavior across the transition from tone to white noise. Many defensive behaviors beyond the typical freezing assessments were measured, and both male and female mice were included throughout. The authors found changes in behavioral transitions from freezing to flight during conditioning as the tone transitioned into white noise, and a switch in freezing during extinction such that it became high during the white noise as flight behavior decreased. Overall, this was an interesting analysis of transitions in defensive behaviors to a serially presented cue consisting of two auditory stimuli during conditioning and then extinction. There are some concerns regarding the possibility that the white noise is more innately aversive than the tone, inducing more escape-like behaviors compared to a tone, especially since the shock only group also showed increased escape-like behaviors during the white noise versus tone. This issue would have been resolved by adding a control group where the order of the auditory stimuli was reversed (white noise->tone). While the more complete assessment of defensive behaviors beyond freezing is welcomed, the main conclusions in the discussion are overly focused on the paired group and the associative elements of conditioning, which would likely not be surprising to the field. If the goal, as indicated in the title, was to tease apart the associative and non-associative elements of conditioning and defensive behaviors, there needs to be a more emphasized discussion and explicit identification of the non-associative findings of their study, as this would be more impactful to the field.

Reviewer #2 (Public Review):

Summary:<br /> The authors examined several defensive responses elicited during Pavlovian conditioning using a serial compound stimulus (SCS) as the conditioned stimulus (CS) and a shock unconditioned stimulus (US) in male and female mice. The SCS consisted of tone pips followed by white noise. Their design included 3 treatment groups that were either exposed to the CS and US in a paired fashion, in an unpaired fashion, or only exposed to the shock US. They compared freezing, jumping, darting, and tail rattling across all groups during conditioning and extinction. During conditioning, strong freezing responses to the tone pips followed by strong jumping and darting responses to the white noise were present in the paired group but less robust or not present in the unpaired or shock only groups. During extinction, tone-induced freezing diminished while the jumping was replaced by freezing and darting in the paired group. Together, these findings support the idea that associative pairings are necessary for conditioned defensive responses.

Strengths:<br /> The study has strong control groups including a group that receives the same stimuli in an unpaired fashion and another control group that only receives the shock US and no CS to test the associative value of the SCS to the US. The authors examine a wide variety of defensive behaviors that emerge during conditioning and shift throughout extinction: in addition to the standard freezing response, jumping, darting, and tail rattling were also measured.

Weaknesses:<br /> This study could have greater impact and significance if additional conditions were added (e.g., using other stimuli of differing salience during the SCS), and determining the neural correlates or brain regions that are differentially recruited during different phases of the task across the different groups.

Reviewer #1 (Public Review):

The paper submitted by Yogesh and Keller explores the role of cholinergic input from the basal forebrain (BF) in the mouse primary visual cortex (V1). The study aims to understand the signals conveyed by BF cholinergic axons in the visual cortex, their impact on neurons in different cortical layers, and their computational significance in cortical visual processing. The authors employed two-photon calcium imaging to directly monitor cholinergic input from BF axons expressing GCaMP6 in mice running through a virtual corridor, revealing a strong correlation between BF axonal activity and locomotion. This persistent activation during locomotion suggests that BF input provides a binary locomotion state signal. To elucidate the impact of cholinergic input on cortical activity, the authors conducted optogenetic and chemogenetic manipulations, with a specific focus on L2/3 and L5 neurons. They found that cholinergic input modulates the responses of L5 neurons to visual stimuli and visuomotor mismatch, while not significantly affecting L2/3 neurons. Moreover, the study demonstrates that BF cholinergic input leads to decorrelation in the activity patterns of L2/3 and L5 neurons.

This topic has garnered significant attention in the field, drawing the interest of many researchers actively investigating the role of BF cholinergic input in cortical activity and sensory processing. The experiments and analyses were thoughtfully designed and conducted with rigorous standards, leading to convincing results which align well with findings in previous studies. In other words, some of the main findings, such as the correlation between cholinergic input and locomotor activity and the effects of cholinergic input on V1 cortical activity, have been previously demonstrated by other labs (Goard and Dan, 2009; Pinto et al., 2013; Reimer et al., 2016). However, the study by Yogesh and Keller stands out by combining cutting-edge calcium imaging and optogenetics to provide compelling evidence of layer-specific differences in the impact of cholinergic input on neuronal responses to bottom-up (visual stimuli) and top-down inputs (visuomotor mismatch).

Reviewer #2 (Public Review):

The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses are required to fully support several of the study's conclusions.

1) In experiments analysing the activity of V1 neurons, GCaMP6f was expressed using a ubiquitous Ef1a promoter, which is active in all neuronal cell types as well as potentially non-neuronal cells. The manuscript specifically refers to responses of excitatory neurons but it is unclear how excitatory neuron somata were identified and distinguished from that of inhibitory neurons or other cell types.

2) The manuscript concludes that cholinergic axons convey a binary locomotion signal and are not tuned to running speed. The average running velocity of mice in this study is very slow - slower than 15 cm/s in the example trace in Figure 1D and speeds <6 cm/s were quantified in Figure 2E. However, mice can run at much faster speeds both under head-fixed and freely moving conditions (see e.g. Jordan and Keller, 2020, where example running speeds are ~35 cm/s). Given that the data in the present manuscript cover such a narrow range of running speeds, it is not possible to determine whether cholinergic axons are tuned to running speed or convey a binary locomotion signal.

3) The analyses in Figure 4 only consider the average response to all grating orientations and directions. Without further analysing responses to individual grating directions it is unclear how stimulation of cholinergic inputs affects visual responses. Previous work (e.g. Datarlat and Stryker, 2017) has shown that locomotion can have both additive and multiplicative effects and it would be valuable to determine the type of modulation provided by cholinergic stimulation.

4) The difference between the effects of locomotion and optogenetic stimulation of cholinergic axons in Figure 5 may be confounded by differences in the visual stimulus. These experiments are carried out under open-loop conditions, where mice may adapt their locomotion based on the speed of the visual stimulus. Consequently, locomotion onsets are likely to occur during periods of higher visual flow. Since optogenetic stimulation is presented randomly, it is likely to occur during periods of lower visual flow speed. Consequently, the difference between the effect of locomotion and optogenetic stimulation may be explained by differences in visual flow speed and it is important to exclude this possibility.

5) It is unclear why chemogenetic manipulations of cholinergic inputs had no effect on pairwise correlations of L2/3 neuronal responses while optogenetic stimulation did.

6) The effects of locomotion and optogenetic stimulation on the latency of L5 responses in Figure 7 are very large - ~100 ms. Indeed, typical latencies in mouse V1 measured using electrophysiology are themselves shorter than 100 ms (see e.g. Durand et al., 2016). Visual response latencies in stationary conditions or without optogenetic stimulation appear surprisingly long - much longer than reported in previous studies even under anaesthesia. Such large and surprising results require careful analysis to ensure they are not confounded by artefacts. However, as in Figure 4, this analysis is based only on average responses across all gratings and no individual examples are shown.

Reviewer #1 (Public Review):

This manuscript by He et al. explores the molecular basis of the different stinging behaviors of two related anemones. The freshwater Nematostella which only stings when a food stimulus is presented with mechanical stimulation and the saltwater Exaiptasia which stings in response to mechanical stimuli. The authors had previously shown that Nematostella stinging is calcium-dependent and mediated by a voltage-gated calcium channel (VGCC) with very pronounced voltage-dependent inactivation, which gets removed upon hyperpolarization produced by taste receptors.

In this manuscript, they show that Exaiptacia and Nematostella differing stinging behavior is near optimal, according to their ecological niche, and conforms to predictions from a Markov decision model.

It is also shown that Exaiptacia stinging is also calcium-dependent, but the calcium channel responsible is much less inactivated at resting potential and can readily induce nematocyte discharge only in the presence of mechanical stimulation. To this end, the authors record calcium currents from Exaipacia nematocysts and discover that the VGCCs in this anemone are not strongly inactivated and thus are easily activated by mechanical stimuli-induced depolarization accounting for the different stinging behavior between species. The authors further explore the role of the auxiliary beta subunit in the modulation of VGCC inactivation and show that different n-terminal splice variants in Exaiptacia produce strong and weak voltage-dependent inactivation.

The manuscript is clear and well-written and the conclusions are in general supported by the experiments and analysis. The findings are very relevant to increase our understanding of the molecular basis of non-neural behavior and its evolutionary basis. This manuscript should be of general interest to biologists as well as to more specialized fields such as ion channel biophysics and physiology.

Reviewer #2 (Public Review):

This manuscript links the distinctive stinging behavior of sea anemones in different ecological niches to varying inactivation properties of voltage-gated calcium channels that are conferred by the identity of auxiliary Cavbeta subunits. Previous work from the Bellono lab established that the burrowing anemone, Nematostella vectensis, expresses a CaV channel that is strongly inactivated at rest which requires a simultaneous delivery of prey extract and touch to elicit a stinging response, reflecting a precise stinging control adapted for predation. They show here that by contrast, the anemone Exaiptasia diaphana which inhabits exposed environments, indiscriminately stings for defense even in the absence of prey chemicals, and that this is enabled by the expression of a CaVbeta splice variant that confers weak inactivation. They further use the heterologous expression of CaV channels with wild type and chimeric anemone Cavbeta subunits to infer that the variable N-termini are important determinants of Cav channel inactivation properties.

Reviewer #3 (Public Review):

Summary:<br /> The present article attempts to answer both the ultimate question of why different stinging behaviours have evolved in Cnidiarians with different ecological niches and shed light on the proximate question of which electro-physiological mechanisms underlie these distinct behaviours.

Account of major methods and results:<br /> In the first part of the paper, the authors try to answer the ultimate question of why distinct dependencies of the sting response on internal starvation levels have evolved. The premise of the article that Exaiptasia's nematocyte discharge is independent of the presence of prey (Artemia nauplii) as compared to Nematostella's significant dependence of the discharge on the presence of actual prey, is shown to be a robust phenomenon justified by the data in Figure 1.

The hypothesis that defensive vs. predatory stinging leads to different nematocyte discharge behaviours is analysed in mathematical models based on the suitable framework of optimal control/decision theory. By assuming functional relations between the:<br /> 1) cost of a full nematocyte discharge and the starvation level.<br /> 2) probability of successful predation/avoidance on the discharge level.<br /> 3) desirability/reward of the reached nutritional state.

Based on these assumptions of environmental and internal influences, the optimal choice of attack intensity is calculated using Bellman's equation for this problem. The model predictions are validated using counted nematocytes on a coverslip. The scaling of normalised nematocyte discharge numbers with scaled starvation time is qualitatively comparable to what is predicted from the models. The abundance of nematocytes in the tentacles was, on the other hand, independent of the starvation state of the animals.

Next, the authors turn to investigate the proximate cause of the differential stinging behaviour. The authors have previously reported convincing evidence that a strongly inactivating Cav2.1 channel ortholog (nCav) is used by Nematostella to prevent stinging in the absence of prey (Weir et al. 2020). This inactivation is released by hyperpolarising sensory inputs signalling the presence of prey. In this article, it is clearly shown by blocking respective currents that Exaiptasia, too, relies on extracellular Ca2+ influx to initiate stinging. Patch clamp data of the involved currents is provided in support. However, the authors find that in addition to the nCav with a low-inactivation threshold, Exaiptasia has a splice variant with a higher inactivation threshold expressed (Figure 3D).

The authors hypothesise that it is this high-threshold nCav channel population that amplifies any voltage depolarisation to release a sting irrespective of the presence of prey signals. They found that the β subunit that is responsible for Nematostella's unusually low inactivation threshold exists in Exaiptasia as two alternative splice isoforms. These N-terminus variants also showed the greatest variation in a phylogenetic comparison (Figure 5), rendering it a candidate target for mutations causing variation in stinging responses.

Appraisal of methodology in support of the conclusions:<br /> The authors base their inference on a normative model that yields quantitative predictions which is an exciting and challenging approach. The authors take care in stating the model assumptions as well as showing that the data indeed does not contradict their model predictions. The interesting comparative nature of the modelling part of the study is complicated by slightly different cost assumptions for the two scenarios. Hence, Figure 2 needs to be carefully digested by readers.

It would be even more prudent to analyse the same set of cost-of-discharge vs. starvation scenarios for both species. Specifically, for Nematostella the complete cost-of-discharge vs starvation-state curves as for Exaiptasia (Figure 2E, example 2-4) could be used. It is likely that the differential effect size of Nematostella and Exaiptasia behaviour is the strongest if only the flat cost-of-discharge vs starvation is used (Figure 2A) for Nematostella. But as a worst-case comparison the other curves, where the cost to the animal scales with starvation would be a good comparison. This could help the reader to understand when the different prediction of Nematostella's behaviour breaks down. In addition, this minor change could shed light on broader topics like common trade-offs in pursuit predation.

The qualitatively similar scaling of the model-derived relation between starvation and sting intensity with the counted nematocytes for different feeding pauses is evidence that feeding has indeed been optimised for the two distinct ecological niches.<br /> To prove that Exaiptasia uses a similar Ca2+ channel ortholog as well as a different splice variant, the authors employed both clean electrophysiological characterisation (Figure 3) as well as transcriptomics data (Figure 4S1).

To strengthen the authors' hypothesis that variation in the N-termini leads to changes in Ca2+ channel inactivation and hence altered stinging, the response sequence variability of 6 Cnidaria was analysed.

Additional context:<br /> Although, the present article focuses on nematocytes alone, currently, there has been a refocus in neurobiology on the nervous systems of more basal metazoans, which received much attention already in the works of Romanes (1885). In part, this is driven by the goal to understand the early evolution of nervous systems. Cnidarians and Ctenophors are exciting model organisms in this venture. This will hopefully be accompanied by more comparative studies like the present one. Some of the recent literature also uses computational models to understand mechanisms of motor behaviour using full-body simulations (Pallasdies et al. 2019; Wang et al. 2023), which can be thought of as complementary to the normative modelling provided by the authors.

Comparative studies of recent Cnidarians, such as the present article, can shed light on speculative ideas on the origin of nervous systems (Jékely, Keijzer, and Godfrey-Smith 2015). During a time (the Ediacarium/Cambrium transition) that has seen the genesis of complex trophic foodwebs with preditor-prey interaction, symbioses, but also an increase of body sizes and shapes, multiple ultimate causes can be envisioned that drove the increase in behavioural complexity. The authors show that not all of it needs to be implemented in dedicated nerve cells.

References:

Jékely, Gáspár, Fred Keijzer, and Peter Godfrey-Smith. 2015. "An Option Space for Early Neural Evolution." Philosophical Transactions of the Royal Society B: Biological Sciences 370 (December): 20150181. https://doi.org/10.1098/rstb.2015.0181.

Pallasdies, Fabian, Sven Goedeke, Wilhelm Braun, and Raoul-Martin Memmesheimer. 2019. "From Single Neurons to Behavior in the Jellyfish Aurelia Aurita." eLife 8 (December). https://doi.org/10.7554/elife.50084.

Romanes, G. J. 1885. Jelly-Fish, Star-Fish and Sea-Urchins: Being a Research on Primitive Nervous Systems. Appleton.

Wang, Hengji, Joshua Swore, Shashank Sharma, John R. Szymanski, Rafael Yuste, Thomas L. Daniel, Michael Regnier, Martha M. Bosma, and Adrienne L. Fairhall. 2023. "A Complete Biomechanical Model of hydra Contractile Behaviors, from Neural Drive to Muscle to Movement." Proceedings of the National Academy of Sciences 120 (March). https://doi.org/10.1073/pnas.2210439120.

Weir, Keiko, Christophe Dupre, Lena van Giesen, Amy S-Y Lee, and Nicholas W Bellono. 2020. "A Molecular Filter for the Cnidarian Stinging Response." eLife 9 (May). https://doi.org/10.7554/elife.57578.

Joint Public Review:

The authors have previously established that activation of dopamine inputs to prefrontal cortex during adolescence can drive increases in mPFC DA bouton number and enhanced mPFC activity in WT mice. The current study was designed to test the hypothesis that neural circuit plasticity during adolescence can be targeted to restore cortical function under conditions of developmental disruptions that are relevant to psychiatric disorders.

Specifically, the manuscript explores how transient adolescent stimulation of ventral midbrain neurons that project to the frontal cortex may help to improve performance on certain memory tasks. The authors used DREADDs to regulate the mesofrontal cortical dopamine system in two mouse models - one with a reporter replacing the Arc gene, and another with knockout of the schizophrenia-associated gene Disc1, both of which are thought to have reduced prefrontal cortical activity. The manuscript provides an interesting set of observations that DREADD-based activation over only 3 days during adolescence provides a fast-acting and long-lasting improvement in performance on Y-maze spontaneous alternation as well as aspects of neuronal function as assessed using in vivo imaging methods.

A strength of this study is that the authors performed key manipulations using age and dose/intensity as dependent variables to show that the level of neural circuit activation during adolescence follows an inverted U-shape pattern, though the precise postsynaptic mechanisms underlying the positive impact of adolescent mesofrontal dopamine neuron stimulation were not addressed.

One limitation discussed by the reviewers is that using TH-Cre mice (as compared with DAT-Cre) to drive transgene expression in VTA neurons could lead to expression outside the dopaminergic population of neurons, though in the revision the authors have provided additional lines of evidence to support their model of dopamine regulation of frontal cortex in this study.

Collectively, this is a well-design study with many strengths and novel findings that are likely to positively impact a widespread of disciplines within the biological psychiatry and neuroscience field.

Reviewer #3 (Public Review):

The authors present here a comparative meta-analysis analysis designed to detect evidence for a reproduction/ survival trade-off, central to expectations from life history theory. They present variation in clutch size within species as an observation in conflict with expectations of optimisation of clutch size and suggest that this may be accounted for from weak selection on clutch size. The results of their analyses support this explanation - they found little evidence of a reproduction - survival trade-off across birds. They extrapolated from this result to show in a mathematical model that the fitness consequences of enlarged clutch sizes would only be expected to have a significant effect on fitness in extreme cases, outside of normal species' clutch size ranges. Given the centrality of the reproduction-survival trade-off, the authors suggest that this result should encourage us to take a more cautious approach to applying concepts the trade-off in life history theory and optimisation in behavioural ecology more generally. While many of the findings are interesting, I don't think the argument for a major re-think of life history theory and the role of trade-offs in fitness maximisation is justified.

The interest of the paper, for me, comes from highlighting the complexities of the link between clutch size and fitness, and the challenges facing biologists who want to detect evidence for life history trade-offs. Their results highlight apparently contradictory results from observational and experimental studies on the reproduction-survival trade-off and show that species with smaller clutch sizes are under stronger selection to limit clutch size.

Unfortunately, the authors interpret the failure to detect a life history trade-off as evidence that there isn't one. The construction of a mathematical model based on this interpretation serves to give this possible conclusion perhaps more weight than is merited on the basis of the results, of this necessarily quite simple, meta-analysis. There are several potential complicating factors that could explain the lack of detection of a trade-off in these studies, which are mentioned and dismissed as unimportant (lines 248-250) without any helpful, rigorous discussion. I list below just a selection of complexities which perhaps deserve more careful consideration by the authors to help readers understand the implications of their results:

• Reproductive output is optimised for lifetime reproductive success and so the consequences of being pushed off the optimum for one breeding attempt are not necessarily detectable in survival but in future reproductive success (and, therefore, lifetime reproductive success).<br /> • The analyses include some species that hatch broods simultaneously and some that hatch sequentially (although this information is not explicitly provided (see below)). This is potentially relevant because species which have been favoured by selection to set up a size asymmetry among their broods often don't even try to raise their whole broods but only feed the biggest chicks until they are sated; any added chicks face a high probability of starvation. The first point this observation raises is that the expectation of more chicks= more cost, doesn't hold for all species. The second more general point is that the very existence of the sequential hatching strategy to produce size asymmetry in a brood is very difficult to explain if you reject the notion of a trade-off.<br /> • For your standard, pair-breeding passerine, there is an expectation that costs of raising chicks will increase linearly with clutch size. Each chick requires X feeding visits to reach the required fledge weight. But this is not the case for species which lay precocious chicks which are relatively independent and able to feed themselves straight after hatching - so again the relationship of care and survival is unlikely to be detectable by looking at the effect of clutch size but again, it doesn't mean there isn't a trade-off between breeding and survival.<br /> • The costs of raising a brood to adulthood for your standard pair-breeding passerine is bound to be extreme, simply by dint of the energy expenditure required. In fact, it was shown that the basal metabolic rate of breeding passerines was at the very edge of what is physiologically possible, the human equivalent being cycling the Tour de France (Nagy et al. 1990). If birds are at the very edge of what is physiologically possible, is it likely that clutch size is under weak selection?<br /> • Variation in clutch size is presented by the authors as inconsistent with the assumption that birds are under selection to lay the Lack clutch. Of course, this is absurd and makes me think that I have misunderstood the authors' intended point here. At any rate, the paper would benefit from more clarity about how variable clutch size has to be before it becomes a problem for optimality in the authors' view (lines 84-85; line 246). See Perrins (1965) for an exquisite example of how beautifully great tits optimise clutch size on average, despite laying between 5-12 eggs.

[Editors’ note: the authors had already made data files publicly available, available here, https://doi.org/10.5061/dryad.q83bk3jnk.]

Reviewer #1 (Public Review):

Summary:

The authors present a comprehensive meta-analysis of Clostridioides difficile (CD) occurrence across 42,900 metagenomes from 253 public studies, largely representing stool samples from human adults, infants, and with a smaller fraction of samples from non-gut body sites and from environmental samples (e.g., non-human animals, wastewater, soil, etc.). In particular, the authors looked at adults who were healthy, diseased (but not with C. diff), and with diagnosed C. diff infection (CDI) and found that CD occurrence was fairly low: ~30% in adult CDI samples, ~2% in adult diseased samples, and ~1% in healthy samples. CD was much more prevalent in infants (15 and 40% in healthy and diseased infants, respectively). These findings, if they hold true, would be significant because they would suggest an over-diagnosis of CDI and an under-diagnosis of other putative enteric pathogens (also enriched in CDI samples) across the population. Furthermore, these results suggest that the asymptomatic carriage of CD in adults (~1-5%, depending on demographics) may be much lower than some prior estimates (some as high as ~30-40%).

Strengths:

The authors have done an admirable job pulling down an enormous data set for this CD-focused meta-analysis, which is a valuable service to the field. The results push against some common wisdom in the field, in terms of the prevalence of CD in CDI patients, which will be impactful if they hold up to further scrutiny. Furthermore, the identification of commensal bacteria that are positively or negatively associated with CD presence in both healthy and diseased people at different periods of the lifespan (infant, child, and adult), is a valuable synthesis with potential translational value. The manuscript is clearly written and the figures are presented well. The methodology is robust, although I have a few suggestions for improvement.

Weaknesses:

My main critique relates to detection limitations, both in terms of sequencing depth and read-mapping. Given that CD detection is the root of the main conclusions reported here, this deserves some additional care. The authors have already done some work to address this by including sequencing depth in their linear mixed effects model, which is great. Furthermore, they were conservative with how they labeled CD positive/negative individuals with multiple time points (i.e., if you had CD detected at any point, this sample was selected for the cross-sectional analysis, and that individual was labeled as CD positive). I have a few additional suggestions to explore this issue, which I outline in the recommendations for authors.

Reviewer #2 (Public Review):

Summary:

C. difficile infection (CDI) is clinically important as a hospital-acquired infection and a frequent cause of antibiotic-associated diarrhea, which is associated with high morbidity and mortality and increases in prevalence. It is also the prime example of a disease that is associated with gut microbiome dysbiosis and successfully treated with fecal microbiota transfer, highlighting the important but unclear functional or structural role of this bacterial pathogen and the condition of CDI for the gut microbiome, which is the focus of this study.

Ferretti et al. assembled an impressive gut metagenome dataset from previous and ongoing microbiome studies, which involves a large number of samples from patients with CDI or other diarrheal and non-diarrheal diseases and from healthy individuals, as well as from infants, adolescents, and adults. The authors analyze the prevalence and relative abundance of C. difficile in this dataset in relation to CDI diagnosis, host age and disease background, and the composition of the remaining microbiota. They detect C. difficile only in a minority of samples labelled as originating from CDI patients but frequently identify other pathogens and their toxin genes in the same samples. In infants, they detect C. difficile at high frequency and relative abundance in samples without clinical symptoms. They associate C. difficile presence in infant samples with "multiple indicators of healthy gut microbiome maturation' and suggest 'distinct biotic and physiological contexts in infants and adults' for C. difficile.

Strengths:

The manuscript provides an important overview of the complex relationship of C. difficile with the gut microbiome of healthy and diseased infants and adults, mostly due to the large studied dataset and convincing applied analysis that underlies the presented findings. This includes a number of interesting findings including, for example, that CDI can be reliably predicted based on taxonomic microbiota compositions, without including C. difficile itself or that C. difficile in infants appears not to originate from maternal sources.

Weaknesses:

Inconsistent associations of C. difficile with what is clinically labeled CDI, as well as the frequent detection of C. difficile in healthy infants, have been reported before and the manuscript does not reveal to what extent this bacterium reflects or even directly influences the gut microbiome of infants and adults. Whether the increased microbiota diversity, richness, and compositional similarity of C. difficile-positive infants to their mothers is sufficient to associate this bacterium with "healthy gut microbiome maturation" seems questionable, since C. difficile was also found to be more prevalent in preterm infants, formula-fed or antibiotically treated infants, and infants born by C-section, all of which are typically considered detrimental influences on microbiota development. The conclusion that "C. difficile may be a transient hallmark of healthy gut microbiome maturation" therefore appears too strong.

In addition, the statement that "its asymptomatic carriage in adults depends on microbial context" is not sufficiently supported by the presented data. Apparently, the authors are unable to define or measure "asymptomatic carriage", as they convincingly show that many patients diagnosed with "CDI" appear not to carry C. difficile, suggesting that neither asymptomatic nor symptomatic "CDI" conditions are necessarily linked to C. difficile.

The manuscript includes a large number of samples from poorly defined, but diverse patient backgrounds. It might be helpful to better define these samples (e.g. fecal samples vs. other gut samples) and to specify subcategories for samples from "diseased control subjects without CDI". Maybe this information could help validate the interesting suggestion from the manuscript that C. difficile may be (one of several) dysbiosis marker rather than the cause of (CDI) dysbiosis.

The phylogenetic analysis of C. difficile from metagenomic sequence data seems to suggest that there is a large mostly toxin gene-free cluster that is only identified in infants (Supplementary Figure 13). Could this indicate that there are, in fact, less pathogenic C. difficile lineages that are more prevalent in infants?

The authors argue in the Discussion that "Differential diagnosis against multiple enteropathogens may therefore stratify patients with CDI-like symptoms, towards adapted therapeutic interventions." It might be helpful to expand this discussion of different clinical options that could be adapted to highlight the clinical applicability of the presented findings.

Reviewer #3 (Public Review):

This contribution focuses on the zinc(II) transporter YiiP, a widely used model system of the Cation Diffusion Facilitator (CDF) superfamily. CDF proteins function as dimers and are typically involved in the maintenance of homeostasis of transition metal ions in organisms from all kingdoms of life. The system investigated here, YiiP, is a prokaryotic zinc(II)/H+ antiporter that exports zinc(II) ions from the cytosol. The authors addressed multiple crucial questions related to the functioning of YiiP, namely the specific role of the three zinc(II) binding sites present in each protomer, the zinc(II):H+ stoichiometry of antiport, and the impact of protonation on the transport process. Clarity on all these aspects is required to reach a thorough understanding of the transport cycle.

The experimental approach implemented in this work consisted of a combination of site-directed mutagenesis, high-quality 3D structural determination by cryoEM, microscale electrophoresis, thermodynamic modeling and molecular dynamics. The mutants generated in this work removed one (for the structural characterization) or two (for microscale electrophoresis) of the three zinc(II) binding sites of YiiP, allowing the authors to unravel respectively the structural role of metal binding at each site and the metal affinity of every site individually. pH-dependent measurements and constant pH molecular dynamics simulations, together with the metal affinity data, provided a detailed per-site overview of dissociation constants and Ka values of the metal-binding residues, casting light on the interplay between protonation and metal binding along the transport cycle. This thermodynamic modeling constitutes an important contribution, with consistent experimental information gained from the various mutants.

Overall the authors were successful in providing a model of the transport cycle (Figure 5) that is convincing and well supported by the experimental data. The demonstration that two protomers act asymmetrically during the cycle is another nice achievement of this work, confirming previous suggestions. This novel overview of the cycle can constitute a basis for future work on other systems such as human ZnT transporters, also exploiting a methodological approach for the thermodynamic of these proteins similar to the one deployed here. The latter approach may be applicable also to other superfamilies of metal transporters.

Reviewer #1 (Public Review):

The manuscript by Hussein et al. uses cryoEM structure, microscale thermophoresis (MST), and molecular dynamics simulations (conventional and CpHMD) to unravel the Zn2+ and proton role in the function of the Cation Diffusion Facilitator YiiP. First, they generate mutants that abolish each of the three Zn2+ models to study the role of each of them separately, both structurally and functionally. Next, they used a Monte Carlo approach refining the CpHMD data with the MST points to establish the Zn2+ or proton binding state depending on the pH. That predicted a stoichiometry of one Zn2+ to 2 or 3 protons (1:3 under lower pH values). Finally, they proposed a mechanism that involves first the binding of Zn2+ to one low-affinity site and then, after the Zn2+ migrates to the highest affinity site in the transmembrane portion of the protein. The lack of Zn2+ in the low-affinity site might induce occlusion of the transporter.

The manuscript is well-written it is of interest to the field of Cation Facilitator Transporters. It is also an excellent example of a combination of different techniques to obtain relevant information on the mechanism of action of a transporter.

Reviewer #2 (Public Review):

In this work, the authors reported cryo-EM structures of four types of zinc-binding site mutants of a bacterial Zn2+/H+ antiporter YiiP, and proposed distinct structural/functional roles of each of the binding sites in the intramolecular Zn2+ relay and the integrity of the homodimeric structure of YiiP. MST analysis using the mutants with a single Zn2+-binding site at different pH further clarified the pH dependence of Zn2+ binding affinity of each site. Moreover, the inverse Multibind approach refined the CpHMD pKa values of the key Zn2+-binding residues so that they agreed with the MST data. Consequently, energetic coupling of Zn2+ export to the proton-motive force has been suggested. These findings definitely provide new mechanistic insight into this Zn2+/H+ antiporter.

Reviewer #3 (Public Review):

Summary<br /> CLC-2 channels play an important role in cellular homeostasis and electrical excitability, and dysfunctions are associated with aldosteronism and leukodystrophy. Structural insights into the functioning of CLC-2 are just emerging. CLC-2 channels are distinct among the members of the CLC family in that they are activated by hyperpolarization. Earlier studies have implicated channel regulation by a "ball-and-chain" type of channel block mechanism which underlies its strong rectification and use-dependent "run-up" properties. Structural insights into these mechanisms are currently lacking. In this manuscript, Xu et al present CryoEM structures of CLC-2 in the apo and inhibitor-bound conformations in the 2.5-2.7 A resolution range. Several novel structural features are presented that lend support to the "ball-and chain" model, identify an interesting role for the c-terminal domain in gating, and establish the interaction pocket for AK-42. Electrophysiology and simulations nicely support the structural work. Overall, an elegant study, with high-quality data, and a well-presented manuscript.

Strengths<br /> 1. The cryoEM data presented reveals that the channel is in a closed conformation at depolarizing potential (0 mv). Structures for the closed state of CLCs were not previously available. A strong density for Glu205, which constitutes the Egate, allows for an unambiguous assignment of its position at the Scen Cl-binding site, thereby establishing the basis for the block in the closed channel.<br /> 2. The apo state particles were sorted into two classes that differ in the conformation of the CTD. A previously unobserved rearrangement of the CBS region in the CTD is reported wherein the CTD is positioned closer to the TM region in one of the subunits, breaking the C2 symmetry. The data implicates a role for the conformational flexibility of CTD in gating.<br /> 3. The most interesting finding of this work is, perhaps, the presence of an additional density, corresponding to a hairpin-like structure, that is seen only at the subunit where the CTD is positioned away from the TMD. The authors propose that the additional density corresponds to a 13 aa stretch in the N-terminal region. The position of the hairpin at the intracellular mouth of the CL-permeation pathway is likely to impede ion conduction, by a mechanism analogous to the "ball-and-chain" proposed in other voltage-gated channels.<br /> 4. The structure of CLC-2 in complex with a selective inhibitor AK-42 is in a conformation very similar to that of the apo state, with a clear additional density for the AK-42 molecule. Binding site interaction provides insights into AK-42 selectivity for CLC-2 vs CLC-1.

Weaknesses<br /> Although the conformation-dependent placement of the hairpin loop is convincing based on the density, the sequence assigned to this region is not conclusive.

Reviewer #1 (Public Review):

This manuscript deftly combines cryo-EM and electrophysiology to investigate the gating mechanisms of human CLC-2. Although another structure of CLC-2 was recently reported, this is the first structure to report density for the absolutely critical gating glutamate, and - an even more exciting result - the first structure to identify the N-terminal gating peptide that is the heart of this manuscript. There has been previous controversy over such a gating peptide in CLC-2, but the combined structural/functional approach appears to establish a role for this peptide in gating and sets up exciting future experiments to understand why its effects might change under different physiological scenarios. The experiments reported here are thoughtful and well-controlled and the data presentation is excellent. For the electrophysiology experiments, the use of inhibitor AK-42 (developed by the current senior author's lab) to establish a zero current level is a welcome advance and should become standard for electrophysiological studies of CLC-2.

Reviewer #2 (Public Review):

This paper makes important and novel advances that significantly enhance our understanding of the ClC-2 channel. The EM data are of high quality, and the most important argument, concerning the role of the N-terminus of the protein as an occluding inactivation gate, is very well supported by structural, computational, and functional data (some of which is previously published). The proposal that the "run up" observed in patch clamp experiments represents relief of inactivation is interesting and compelling. The model predicts that mutations at the hairpin binding site should influence this "run up", which should be tested in the near future. Finally, the confirmation of the AK-42 binding site further solidifies evidence that this is a pore-blocking compound; the authors' argument about determinants of specificity is convincing.

Joint Public Review:

This study provides evidence of the ability of sublethal imidacloprid doses to affect growth and development of honeybee larva. While checking the effect of doses that do not impact survival or food intake, the authors found changes in the expression of genes related to energy metabolism, antioxidant response, and metabolism of xenobiotics. The authors also identified cell death in the alimentary canal, and disturbances in levels of ROS markers, molting hormones, weight and growth ratio. The study strengths come from exploring different aspects and impacts of imidacloprid exposure on honeybee juvenile stages and for that it demonstrates potential for assessing the risks posed by pesticides. The study weaknesses come from the lack of in depth investigation and an incomplete methodological design. For instance, many of the study conclusions are based on RT-qPCR, which show only a partial snapshot of gene expression, which was performed at a single time point and using whole larvae. There is no understanding of how different organs/tissues might respond to exposure and how they change over time. That creates a problem to understand the mechanisms of damage caused by the pesticide in the situation studied here. There is no investigation of what happens after pupation. The authors show that the doses tested have no impact on survival, food consumption and time to pupation, and the growth index drops from ~0.96 to ~0.92 in exposed larvae, raising the question of its biological significance. The origin of ROS are not investigated, nor do the authors investigate if the larvae recover from the damage observed in the gut after pupation. That is important as it could affect the adult workers' health. One of the study's central claims is that the reduced growth index is due to the extra energy used to overexpress P450s and antioxidant enzymes, but that is based on RT-qPCR only. Other options are not well explored and whether the gut damage could be causing nutrient absorption problems, or the oxidative stress could be impairing mitochondrial energy production is not investigated. These alternatives may also affect the growth index. The authors also state that the honeybee larvae has 7 instars, which is an incorrect as Apis mellifera have 5 larval instars. It is not clear from methods which precise stage of larval development was used for gut preparations. That information is important because prior to pupation larvae defecate and undergo shedding of gut lining. That could profoundly affect some of the results in case gut preparations for microscopy were made close to this stage. A more in-depth investigation and more complete methodological design that investigates the mechanisms of damage and whether the exposures tested could affect adult bees may demonstrate the damage of low insecticide doses to a vital pollinator insect species.

Joint Public Review:

The manuscript by Aguirre et al. describes an elegant approach for developing selective inhibitors of inositol hexakisphosphate kinases (IP6Ks). There are 3 IP6K isozymes (IP6K1-3) in humans, which catalyze the synthesis of inositol pyrophosphates. The lack of isozyme-selective inhibitors has hampered efforts to understand their individual physiological roles. While several inhibitors of IP6Ks have been described, they either lack isozyme selectivity or inhibit other kinases. To address this gap, Aguirre et al. used an analog-sensitive approach, which involves the identification of a mutant that, in an ideal world, doesn't impact the activity of the enzyme but renders it sensitive to an inhibitor that is absolutely selective for the engineered (analog-sensitive) enzyme. Initially, they generated the canonical gatekeeper (Leu210 in IP6K1) mutations (glycine and alanine); unfortunately, these mutations had a deleterious effect on the enzymatic activity of IP6K1. Interestingly, mutation of Leu210 to a valine, a subtly smaller amino acid, didn't affect enzymatic activity. The authors then designed a clever high-throughput assay to identify compounds that show selectivity for L210V IP6K1 versus WT IP6K1. The assay monitors the reverse reaction catalyzed by IP6Ks, monitoring the formation of ATP using a luminescence-based readout. After validating the screen, the authors screened 54,912 compounds. After culling the list of compounds using several criteria, the authors focused on one particular compound, referred to as FMP-201300. FMP-201300 was ~10-fold more potent against L210V IP6K1 compared to WT IP6K1. This selectivity was maintained for IP6K2. Mechanistic studies showed that FMP-201300 is an allosteric inhibitor of IP6K1. The authors also did a small SAR campaign to identify key functional groups required for inhibition.

Overall, this manuscript describes a unique and useful strategy for developing isozyme-selective inhibitors of IP6Ks. The serendipitous finding that subtle changes to the gatekeeper position can sensitize the IP6K1 mutant to allosteric inhibitors will undoubtedly inspire other analog-sensitive inhibitor studies. The manuscript is well-written and the experiments are generally well-controlled.

Reviewer #1 (Public Review):

In the "Drivers of species knowledge across the Tree of Life", Mammola and collaborators explore the determinants of scientific and societal interest across very wide taxonomic and spatial scales. Their work highlights our uneven knowledge of biodiversity and its potential causes.

Reviewer #2 (Public Review):

Using standard and widely used tools, the author revealed the factors (cultural, phenotypic, phylogenetic, etc.) shaping societal and scientific interest in natural species around the globe. The strength of this ms (and the authors) lies in its command of the available literature, database and variable management and analysis, and its solid discussion. The authors thus achieved a manuscript that was pleasant to read.

While I agree that doing a global study requires losing details of local patterns, maybe this is exactly the biggest shortcoming of the manuscript, oblivious to how different cultures (compare USA to PNG, for example) are reflected in these global patterns.

Joint Public Review:

When the left-right asymmetry of an animal body is established, a barrier that prevents the mixing of signals or cells across the midline is essential. Such a midline barrier preventing the spreading of asymmetric Nodal signaling during early left-right patterning has been identified. However, midline barriers during later asymmetric organogenesis have remained largely unknown, except in the brain. In this study, the authors discovered an unexpected structure in the midline of the developing midgut in the chick. Using immunofluorescence, they convincingly show the chemical composition of this midline structure as a double basement membrane and its transient existence during the left-right patterning of the dorsal mesentery, which authors showed previously to be essential for forming the gut loop and guiding local vasculogenesis. Labelling experiments suggest a physical barrier function, to cell mixing and signal diffusion in the dorsal mesentery. Cell labelling and graft experiments rule out a cellular composition of the midline from dorsal mesenchyme or endoderm origin and rule out an inducing role by the notochord. Based on laminin expression pattern and Ntn4 resistance, the authors propose a model, whereby the midline basement membrane is progressively deposited by the descending endoderm.

Laterality defects encompass severe malformations of visceral organs, with a heterogenous spectrum that remains poorly understood, by a lack of knowledge of the different players of left-right asymmetry. This fundamental work significantly advances our understanding of left-right asymmetric organogenesis, by identifying an organ-specific and stage-specific midline barrier. The complexities of basement membrane assembly, maintenance, and function are of importance in several other contexts, as for example in the kidney and brain. Thus, this original work is of broad interest.

Overall, reviewers refer to a strong and elegant paper discovering a novel midline structure, combining classic but challenging techniques, to show the dynamics, chemical, and physical properties of the midline. However, reviewers also indicate that further work will be necessary to conclude on the origin and impact of the midline for asymmetric organogenesis. Three issues have been raised to strengthen the claims:

1) The function of the midline as a physical barrier requires clarification. Dextran injection here seems to label cells and not the extracellular space. By counting the proportion of dextran-labeled cells rather than dextran intensity itself, the authors do not measure diffusion per se, but rather cell mixing.

2) The descending endoderm zippering model for the formation of the midline lacks direct evidence. The claim of an endoderm origin is based on laminin expression, but the laminin observed in the midline with an antibody may not necessarily correspond to the same subtype assessed by in situ hybridization. The midline may be Ntn4 resistant until it is injected in the relevant source cells. Alternative origins could be considered, from the bilateral dorsal aortae or the paraxial mesoderm, which would explain the double layer as a meeting point of two lateral tissues.

3) The title implies a role of the midline in left-right asymmetric gut development. However, the importance of the midline is currently inferred from previously published data and stage correlations and will require more direct evidence.

Reviewer #1 (Public Review):

This work is hugely significant in the context of the debates surrounding ancient Egyptian activity on the Red Sea and voyages to the Egyptian land of Punt. Using genetic studies to provenance baboon mummified baboons in Egypt with contemporary baboon populations in the Red Sea region, the study argues persuasively that Egyptians obtained Baboons from coastal Eritrea, and thus that Egyptian baboon trade involved this region. This of course brings up the larger issue of Egyptian trade with Punt and the southern Red Sea, a place known to have furnished Egyptians with baboons. The authors argue logically for the region of the ancient port-city of Adulis as being particularly important in this baboon trade, as the region around the harbour was said to have been a baboon habitat in the Graeco-Roman period.

Along with other previous geoprovenancing scientific studies relating to baboon isotopes and obsidian trace elements, this study provides a solid foundation for considering coastal Sudan and especially Eritrea as part of the land of Punt.

Reviewer #2 (Public Review):

The paper presents new mitochondrial sequence data from baboons from a museum collection and from one ancient Egyptian mummified baboon. By comparing the mitochondrial sequence of the mummified baboon with the new and existing data, they conclude that it originated from present-day Eritrea, specifically the ancient city of Adulis.

The paper is well-written and an interesting read. The background and details of the study are well-described and logical. Not knowing much about the history of the region I learned a lot. The data also seem sound and the analysis robust, with the exception of one check that should be added (in particular, to assess contamination by looking at mismatching reads).

The main limitation of the paper is just down to the N=1 sample and the limits of mitochondrial phylogeography. Based on the present-day distribution of hamadryas, the baboon must either come from the area of Africa around present-day Eritrea/Ethiopia/Sudan, or from Arabia. All the authors can really reasonably establish here is that this particular baboon did not come from Arabia. But beyond that, there is not much more they can say. Fig 2b makes it clear that the G3Y clade extends over a large range. Given the limited sampling, this is a minimum bound for the range, which probably includes most of the non-Arabian hamadryas range. The link to Adulis is speculative. There may be historical or archaeological evidence to support this but the genetic data really do not come close to establishing this. The authors do acknowledge this in the text, though the abstract makes a much stronger claim. And of course, it also remains possible that other baboons in the assemblage came from other places.

Reviewer #1 (Public Review):

This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such experiments and were unable to replicate their published results. It is imperative that the authors be forthcoming about whether they have conducted such experiments and what were the results. If they have not conducted such experiments, they should do them and include the results here. If they cannot replicate their results, then the reliability of the Neuron paper is in doubt.

How do the results presented here relate to the results published in the Neuron paper and why are they not definitive with respect to the utility of SiR? The original publication in Neuron presents results that do not appear to be plausible and are best explained by the possibility that some experiments described in that manuscript were conducted using mutated SiR. This became most apparent when shortly after the Neuron publication, the Tripodi lab shared SiR as well as TEV expressing cell lines for propagation with other labs. Several of those groups observed that when they progagated the SiR received from the Tripodi lab, there was a mutation that removed the linkage of the PEST targeting sequence to N. This would be expected to allow the virus to replicate and spread without the need for TEV protease to remove the PEST sequence - precisely the phenotype observed in the trans-synaptic tracing experiments described in the Neuron paper. In the Neuron paper, culture experiments showed that the N-PEST (SiR) rabies could not replicate in the absence of TEV. And additional experiments showed that the virus is not toxic to neurons directly infected. These are the same experiments that are replicated in this submission. But then (in the Neuron paper) comes the unlikely report that this virus can spread trans-synaptically in vivo, in the absence of TEV expression. An alternative explanation would be that the virus used for those experiments was mutated and that is why TEV expression was not needed. There are no experiments in the original Neuron paper that address this possibility. Specifically, the experiments in Neuron describing cell survival during trans-synaptic tracing are not adequate to rule this out. This is because the two timepoints during which neurons were counted correspond to an early time when labeled neurons would be expected to still be accumulating and a later time that might be past the peak and represent a time when many neurons have died. To quantify proportions of neurons that survive, it is necessary to follow the same neurons over time, as has been done to demonstrate that only about half of neurons infected with G-deleted rabies die (half survive). Until tests are conducted testing whether TEV expression is required to obtain trans-synaptic labeling with an SiR that is known to not be mutated, it is irrelevant whether mutations can be prevented under particular culture conditions. The utility of this virus depends on whether it can be used for trans-synaptic tracing without toxicity and this manuscript presents no experiments to address that. Further, the omission of such experiments is glaring, as it is difficult to imagine that they have not been attempted.

Other comments:

"A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, represents the first tool for open-ended genetic manipulation of neural circuits."<br /> It is not clear what the authors intend to be claiming with respect to "open-ended genetic manipulation of neural circuits" but it is clear that this assertion is overblown. There are numerous tools that are available for genetic manipulation of neural circuits. This is not the first, won't be the last, and it is arguably not the best.

"Interestingly, a fraction of tdTomato+ neurons survived in ΔG- Rab-CRE-injected brains, differing from what we observed when injecting ΔGRab-GFP, where no cells were detected at 3 weeks p.i. (Fig 3CD) (Ciabatti et al., 2017). " This is a known result (same as Chatterjee et al., 2018) with a known mechanism. GFP expression is not observed because the rabies virus transitions from transcription to replication resulting in the termination of GFP expression. But Cre-recombination of the genome permanently labels cells with TdTomato. This is how Chatterjee et al. demonstrated that half of the neurons infected with G-deleted rabies survive. They imaged cells and saw that the GFP disappeared but the cells marked by Cre-recombination and RFP expression remained healthy indefinitely. The consideration of this in the Introduction is strange. There is no reason to suppose that Cre expression would somehow protect cells from rabies infection and there is no need to propose any such mechanism to explain the observed results.

"Here we show that revertant-free SiR-CRE efficiently traces neurons in vivo without toxicity in cortical and subcortical regions for several months p.i.."<br /> This wording is disingenuous and appears to be intentionally misleading. "Trace" implies that circuits were traced by transynaptic labeling, which they were not.

Reviewer #2 (Public Review):

The study by Ciabatti et al examined the mutation issue for self-inactivating rabies (SiR), which was found by other labs. The authors identified the mutations in the rabies genome and showed that this mutation occurred more frequently after multiple passage of production cell lines with suboptimal TEVp expressions. The authors further showed that such mutation did not accumulate in vivo and that SiR-labeled cells remained alive across longitudinal imaging in vivo.

In this study, the rabies genome is rigorously examined by sequencing many viral particles from independent preparations. The rabies with point mutation in the PEST domain is directly engineered for sequencing and infection test. Overall, the mutation issue is well addressed by the authors and the conclusions are well supported, but some more aspects of discussion and data analysis need to be extended for an easier production of SiR in a condition not that optimal.

1) The authors stated that one should produce SiR from cDNA in order to avoid the potential mutation in SiR. From a practical point of view, it would be much better to amplify the rabies from a stock virus directly in the production cell lines. Any discussion or exploration on this direction would be appreciated in the field.

2) 6 passages of production cell lines are not that extensive. In Fig.2C, there was already some level of TEVp activity reduction at 2nd passage. It is not clear to me that how the TEVp activity reduction naturally happens. Is there some room to play around puromycin concentration to maintain high TEVp activity?

Reviewer #3 (Public Review):

This paper is a response to the report by Lin et al., bioRxiv 2022 (DOI: https://doi.org/10.1101/550640) that mutations in the genome of SiR were identified, which could result in a canonical G-deleted Rabies virus.

Strengths:

First, the authors found that SiR production from cDNA leads to revertant-free viruses by analyzing a total of 400 individual viral particles obtained from 8 independent viral productions with Sanger sequencing. Next, they identified the molecular mechanisms of mutations in the SiR; they found that extensive amplification of packaging cells HEK-TGG leads to the selection of clones with suboptimal TEVp expression level, which leads to the accumulation of revertant mutants, where, as the authors discuss, the revertant mutants have a specific replication advantage. Based on these observations, the authors recommend producing SiR freshly from cDNA with low passage packaging cells. Lastly, the authors observed that SiR-infected hippocampal and cortical neurons can survive for longer periods of time than the neurons infected with revertant mutants or a canonical G-deleted Rabies virus by combining next-generation sequencing of RNAs isolated from infected tissue and 2-photon in vivo longitudinal imaging of infected cortical neurons. Together, these findings support the idea that the degradation of N by PEST-mediated cellular mechanism results in the self-inactivation of SiR as suggested in the original SiR manuscript (Ciabatti et al., Cell 2017). Thus, SiR remains a powerful viral tool for the chronic investigation of neuronal circuitry and function as long as the virus is prepared in a way the authors recommend.

Weaknesses:

While most of the findings are solid, some conclusions are not fully supported by the data presented. The authors need to address the following points:

1. In Figure 3B-D, the authors concluded that SiR-CRE -infected cells did not show cell death in contrast to Rab-CRE and SiR-G453X, but it cannot be fully supported only by this experiment. The authors should consider the potential variance in infection efficiency in each experimental animal and show evidence of suppressed cell death. In addition, it needs to be confirmed that SiR-Cre is diminished in infected cells at later times. The authors should explain and address these concerns by conducting additional experiments, for example, cleaved caspase-3 staining and quantification of virus RNA levels in each time point as performed in their previous study Ciabatti et al., Cell 2017 (DOI: 10.1016/j.cell.2017.06.014).

2. In Figure 3E-F, to ensure the long-term stability of SiR-Cre in the vivo mouse brain, authors conducted SMRT sequencing 1 week after the virus infection. To test the potential slow accumulation of mutations at 1-month and 2-month, the authors should perform the same experiment at these time points. Only when SiR-Cre was undetected at 1-month and 2-month, would it be reasonable to show only 1-week data, however, such data is not presented.

3. In figure 4, the authors used only 2 mice for this experiment, although this is one of the most important experiments to ensure SiR-infected cells stay alive for the long term in vivo animals. It should be confirmed whether the conclusion remains the same by increasing the number of animals.

4. The legend in Table 3 doesn't match the contents.

Reviewer #1 (Public Review):

Wang and colleagues show that tree shrews can detect optogenetic stimulation of the lateral geniculate nucleus (LGN) using an AAV2-CamKIIα-ChR2 construct after training detection of visual stimuli. Solid evidence links optogenetic stimulation to behavioural detection and neurophysiological responses in LGN and local field potentials in V1.

The major strength is the carefully conducted optogenetic detection experiments showing that training of a visual detection task can be transferred to the the detection of focal optogenetic stimulation in the LGN. The optogenetic stimulation can evoke responses in LGN that can be transmitted to V1.

However, the behavioural results are highly variable between individual animals and different optogenetic stimulation frequencies. The nature of this variability remains unclear. A weakness of this complex in vivo study lies in the underspecified description of some of the details and the links between the histology, the neurophysiology and optogenetic results, in order to understand this variability better. The neurophysiological results are clear and important, but the distribution of significant results across the different animals studied is missing. The expression patterns across layers of the optogenetic viruses appear to differ in the histology of three different animals shown, but it is unclear except for one animal from which experimental individuals these results stem. While the methods of the behavioural and neurophysiological results are well described, the methods section is incomplete with regards to the very nice histology presented (perfusion, sectioning, staining).

The detection of optogenetic activation of LGN in this visual animal model suggests that LGN is a potential target for a neuroprosthetic device. This paper is potentially of interest to neuroscientists and clinicians working on the visual system and visual prostheses.

Reviewer #2 (Public Review):

Wang et al. investigate the LGN in the tree shrew as a potential target for artificial vision. They report that (a) animals pre-trained on a visual detection task can generalize from visual to optogenetic detection and (b) optogenetic activation of the LGN results in reliable field potential activity in V1.

In this revised version of the manuscript, the authors have done a commendable job of addressing the critiques from the previous round of reviews.

Among the new results, the analysis of V1 LFP entrainment with optogenetic stimulation in the LGN is quite interesting and convincing. However, I found the spiking results in V1 to be underwhelming (which the authors also acknowledge). I find this a little surprising, given the robustness of the LFP results. Was this a matter of finding a better alignment of LGN and V1 sites? Might the authors have found more convincing spiking activity results if they use laminar electrodes in V1 to find monosynaptic connectivity between the LGN injection sites and their targets in V1?

Reviewer #3 (Public Review):

The overarching goal of this study is to assess the feasibility of using optogenetic stimulation in the LGN for future visual neuroprostheses. This is an interesting and important research direction.

To address this goal, the author express ChR2 in the LGN of tree shrews, implant a wireless μ‐LED stimulation probe, and test for the ability of tree shrews to generalize from visual detection to detection of optogenetic stimulation. The authors provide compelling evidence that tree shrews can generalize from visual detection to the detection of optogenetic stimulation in the LGN. This is an important and novel finding which demonstrates that optogenetic stimulation in the LGN can lead to detectable percepts. While the basic finding seems to be robust, some aspects of the paper still need further attention.

Reviewer #1 (Public Review):

Dolgova et al present a well-written manuscript focused on the mechanism of MEMO1 function in tumor cells. They use genome-wide analyses to predict function based on MEMO1 structure in yeast, identify MEMO1 expression in a screen of cancer cell lines, and demonstrate a correlation between MEMO1 expression and severity of disease in primary breast cancer cells. The authors focus on a breast cancer model as it overexpresses MEMO1 and melanoma as a control and uses CRISPR-Cas9 knockdown of MEMO1 in breast and melanoma cell lines and concurrently knockdown selected genes in the iron homeostasis pathway. In this data, MEMO1 appears to interact with elements involved in iron trafficking and sensing and its overexpression leads to possible hypersensitivity while knockout/knockdown leads to resistance to lipid oxidation. They also interrogate the effect of iron chelation on mitochondrial morphology and ferroptosis. In addition, they evaluate iron and copper binding loci and resolve MEMO1 structure. The work is of high quality. However, there are some inaccuracies regarding the known function of some iron-related elements. Furthermore, it is unresolved whether controlling iron per se (by modulating other importers and transporters or limiting iron availability in culture) recapitulates or ameliorates their findings, currently attributed specifically to the mechanism of action of MEMO1. In addition, the authors make claims that they have not substantiated about overexpression of MEMO1 by extrapolating from data about MEMO1 knockdown or knockout. Finally, the results show only indirect evidence for a central role for MEMO1 via regulation of iron trafficking and more targeted approaches are necessary to increase confidence in the claims.

Reviewer #2 (Public Review):

While the hypothesis that MEMO1 plays a key role in cell iron homeostasis remains to be directly tested, the data presented herein clearly support further delineation of the underlying mechanisms. The key findings in this regard are the facts, as established herein, that: 1) MEMO1 binds ferrous iron (the appropriate valence state for cell iron) along with glutathione (Fig. 5A); 2) the structure of MEMO1 in complex with Fe(II)-GSH reveals the coordination site within the protein for this complex (Fig. 5B/c); 3) oxidative stress and sensitivity to ferroptosis correlate with MEMO1 protein abundance in a consistent fashion (Fig. 4); and 4) while the effect is limited, there are data that indicate a relation between cell iron content and MEMO1 abundance (Fig. 4A/B).

Experimentally, it is thorough and well-documented and offers a new look at a protein that has been at the edges of iron metabolism (and copper, but I agree with the authors that this is not likely to be the case). This work and its subject will stimulate much further research.

Reviewer #3 (Public Review):

The goal of this manuscript is to determine the function of MEMO1 (mediator of ERBB2-driven cell motility 1), an evolutionarily conserved protein with many putative functions but none that have been firmly established. The authors take an unbiased, bioinformatics approach to identify genetic interactions between MEMO1 and other genes in cancer cell lines. Notably, they uncovered multiple links to genes with relevance to cellular iron homeostasis. They then explore these genetic links through a variety of experiments. First, they use shRNA-mediated gene knockdown to confirm the functional interaction between MEMO1 and interacting genes at the level of protein expression and cell proliferation. Second, they analyze the impact of altered MEMO1 levels on iron levels, mitochondrial morphology, and sensitivity to ferroptosis. Third, they determine the crystal structure of MEMO1, both wild-type and mutant forms, and demonstrate that MEMO1 binds iron as well as copper.

There are notable strengths to this manuscript. I appreciated the unbiased, bioinformatics approach they took to identify genes that interact with MEMO1 and the ensuing approaches they took to explore the potential relevance of MEMO1 to cancer cell iron homeostasis. The methods employed are varied and state-of-the-art and address different aspects of MEMO1's potential role in cellular iron biology. There are some weaknesses. One is that direct protein-protein interactions are not assessed between MEMO1 and TFR2, one of the key genes shown to genetically interact with MEMO1 in cancer cell lines. This limits the authors' ability to more strongly assign a function for MEMO1 in cellular iron homeostasis. They do show that MEMO1 binds to iron, but how does this finding relate to the MEMO1-TFR2 interaction?

The authors conclude that MEMO1 is an iron-binding protein that regulates iron homeostasis in cancer cells. To this end, I agree that the authors have generated adequate evidence in support of this conclusion. The impact of this paper is that it will direct the field to focus on the relevance of MEMO1 to iron homeostasis. While this manuscript does not firmly establish the specific role of MEMO1 in iron homeostasis, future studies should be able to address that knowledge gap.

Joint Public Review:

In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript addresses the biological function of PfFKBP35 and the antimalarial activity of FK506.

The authors combine conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide conclusive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death-like phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors clearly demonstrate that FKBP35 is essential for parasite growth and that ribosome biogenesis is disrupted, but further insights into the pathway itself would be more convincing that this is a direct consequence rather than a secondary feature of parasite death.

The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death-like phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

The authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35, based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, indicating that FK506 activity is independent of FKBP35 levels. Using cellular thermal shift assays, the authors confirm the interaction between FK506 and FKBP35, and further identify candidate proteins bound by the compound, albeit at lower affinity. Further work is needed to validate whether these putative targets contribute to the FKBP35-independent antimalarial activity of FK506.

Reviewer #1 (Public Review):

In this study, Jiamin Lin et al. investigated the potential positive feedback loop between ZEB2 and ACSL4, which regulates lipid metabolism and breast cancer metastasis. They reported a correlation between high expression of ZEB2 and ACSL4 and poor survival of breast cancer patients, and showed that depletion of ZEB2 or ACSL4 significantly reduced lipid droplets abundance and cell migration in vitro. The authors also claimed that ZEB2 activated ACSL4 expression by directly binding to its promoter, while ACSL4 in turn stabilized ZEB2 by blocking its ubiquitination. While the topic is interesting, there are several concerns with the study:

1. My concern regarding the absence of appropriate thresholds or False Discovery Rate (FDR) adjustments for the RNA-seq analysis has not been addressed, leading to incorrect thresholds and erroneous identification of significant signals.

2. In Figure 3B and C, it appears that the knockdown efficiency of ACSL4 is inadequate in these cells, which contradicts the Western blot results presented in Figure 2F.

3. Regarding Figure 6, the discovery of consensus binding sequences (CACCT) for ZEB2 alone is insufficient evidence to support the direct binding of ZEB2 to the ACSL4 promoter.

4. For Figure 7E, there are multiple bands present, and it appears that ZEB2-HA has been cropped, which should ideally be presented with unaltered raw data. Please provide the uncropped raw data.

5. In Figure 7C, the author claimed to have used 293T cells for the ubiquitin assay, which are not breast cancer cells. Moreover, the efficiency of over-expression differs between ZEB2 and ACSL4 in 293T cell lines. Performing the experiment in an unrelated cell line to justify an important interaction is not acceptable.

Reviewer #2 (Public Review):

In this study, the authors validated a positive feedback loop between ZEB2 and ACSL4 in breast cancer, which regulates lipid metabolism to promote metastasis.

Overall, the study is original, well structured, and easy to read.

Reviewer #3 (Public Review):

The manuscript by Lin et al. reveals a novel positive regulatory loop between ZEB2 and ACSL4, which promotes lipid droplets storage to meet the energy needs of breast cancer metastasis.

Reviewer #1 (Public Review):

Summary:

Tiemann et al. have undertaken an original study on the availability of molecular dynamics (MD) simulation datasets across the Internet. There is a widespread belief that extensive, well-curated MD datasets would enable the development of novel classes of AI models for structural biology. However, currently, there is no standard for sharing MD datasets. As generating MD datasets is energy-intensive, it is also important to facilitate the reuse of MD datasets to minimize energy consumption. Developing a universally accepted standard for depositing and curating MD datasets is a huge undertaking. The study by Tiemann et al. will be very valuable in informing policy developments toward this goal.

Strengths:

The study presents an original approach to addressing a growing concern in the field. It is clear that adopting a more collaborative approach could significantly enhance the impact of MD simulations in modern molecular sciences.

The timing of the work is appropriate, given the current interest in developing AI models for describing biomolecular dynamics.

Weaknesses:

The study primarily focuses on one major MD engine (GROMACS), although this limitation is not significant considering the proof-of-concept nature of the study.

Reviewer #2 (Public Review):

Summary:

Molecular dynamics (MD) data is deposited in public, non-specialist repositories. This work starts from the premise that these data are a valuable resource as they could be used by other researchers to extract additional insights from these simulations; it could also potentially be used as training data for ML/AI approaches. The problem is that mining these data is difficult because they are not easy to find and work with. The primary goal of the authors was to discover and index these difficult-to-find MD datasets, which they call the "dark matter of the MD universe" (in contrast to data sets held in specialist databases).

The authors developed a search strategy that avoided the use of ill-defined metadata but instead relied on the knowledge of the restricted set of file formats used in MD simulations as a true marker for the data they were looking for. Detection of MD data marked a data set as relevant with a follow-up indexing strategy of all associated content. This "explore-and-expand" strategy allowed the authors for the first time to provide a realistic census of the MD data in non-specialist repositories.

As a proof of principle, they analyzed a subset of the data (primarily related to simulations with the popular Gromacs MD package) to summarize the types of simulated systems (primarily biomolecular systems) and commonly used simulation settings.

Based on their experience they propose best practices for metadata provision to make MD data FAIR (findable, accessible, interoperable, reusable).

A prototype search engine that works on the indexed datasets is made publicly available. All data and code are made freely available as open source/open data.

Strengths:

- The novel search strategy is based on relevant data to identify full datasets instead of relying on metadata and thus is likely to have many true positives and few false positives.

- The paper provides a first glimpse at the potential hidden treasures of MD simulations and force field parametrizations of molecules.

- Analysis of parameter settings of MD simulations from how researchers *actually* run simulations can provide valuable feedback to MD code developers for how to document/educate users. This approach is much better than analyzing what authors write in the Methods sections.

- The authors make a prototype search engine available.

- The guidelines for FAIR MD data are based on experience gained from trying to make sense of the data.

Weaknesses:

- So far the work is a proof-of-concept that focuses on MD data produced by Gromacs (which was prevalent under all indexed and identified packages).

As discussed in the manuscript, some types of biomolecules are likely underrepresented because different communities have different preferences for force fields/MD codes (for example: carbohydrates with AMBER/GLYCAM using AMBER MD instead of Gromacs).

- Materials sciences seem to be severely under-represented --- commonly used codes in this area such as LAMMPS are not even detected, and only very few examples could be identified. As it is, the paper primarily provides an insight into the *biomolecular* MD simulation world.

The authors succeed in providing a first realistic view on what MD data is available in public repositories. In particular, their explore-expand approach has the potential to be customized for all kinds of specialist simulation data, whereby specific artifacts are<br /> used as fiducial markers instead of metadata. The more detailed analysis is limited to Gromacs simulations and primarily biomolecular simulations (even though MD is also widely used in other fields such as the materials sciences). This restricted view may simply be correlated with the user community of Gromacs and hopefully, follow-up studies from this work will shed more light on this shortcoming.

The study quantified the number of trajectories currently held in structured databases as ~10k vs ~30k in generalist repositories. To go beyond the proof-of-principle analysis it would be interesting to analyze the data in specialist repositories in the same way as the one in the generalist ones, especially as there are now efforts underway to create a database for MD simulations (Grant 'Molecular dynamics simulation for biology and chemistry research' to establish MDDB' DOI 10.3030/101094651). One should note that structured databases do not invalidate the approach pioneered in this work; if anything they are orthogonal to each other and both will likely play an important role in growing the usefulness of MD simulations in the future.

Reviewer #3 (Public Review):

Molecular dynamics (MD) simulations nowadays are an essential element of structural biology investigations, complementing experiments and aiding their interpretation by revealing transient processes or details (such as the effects of glycosylation on the SARS-CoV-2 spike protein, for example (Casalino et al. ACS Cent. Sci. 2020; 6, 10, 1722-1734 https://doi.org/10.1021/acscentsci.0c01056) that cannot be observed directly. MD simulations can allow for the calculation of thermodynamic, kinetic, and other properties and the prediction of biological or chemical activity. MD simulations can now serve as "computational assays" (Huggins et al. WIREs Comput Mol Sci. 2019; 9:e1393. https://doi.org/10.1002/wcms.1393). Conceptually, MD simulations have played a crucial role in developing the understanding that the dynamics and conformational behaviour of biological macromolecules are essential to their function, and are shaped by evolution. Atomistic simulations range up to the billion atom scale with exascale resources (e.g. simulations of SARS-CoV-2 in a respiratory aerosol. Dommer et al. The International Journal of High Performance Computing Applications. 2023; 37:28-44. doi:10.1177/10943420221128233), while coarse-grained models allow simulations on even larger length- and timescales. Simulations with combined quantum mechanics/molecular mechanics (QM/MM) methods can investigate biochemical reactivity, and overcome limitations of empirical forcefields (Cui et al. J. Phys. Chem. B 2021; 125, 689 https://doi.org/10.1021/acs.jpcb.0c09898).

MD simulations generate large amounts of data (e.g. structures along the MD trajectory) and increasingly, e.g. because of funder mandates for open science, these data are deposited in publicly accessible repositories. There is real potential to learn from these data en masse, not only to understand biomolecular dynamics but also to explore methodological issues. Deposition of data is haphazard and lags far behind experimental structural biology, however, and it is also hard to answer the apparently simple question of "what is out there?". This is the question that Tiemann et al explore in this nice and important work, focusing on simulations run with the widely used GROMACS package. They develop a search strategy and identify almost 2,000 datasets from Zenodo, Figshare and Open Science Framework. This provides a very useful resource. For these datasets, they analyse features of the simulations (e.g. atomistic or coarse-grained), which provides a useful snapshot of current simulation approaches. The analysis is presented clearly and discussed insightfully. They also present a search engine to explore MD data, the MDverse data explorer, which promises to be a very useful tool.

As the authors state: "Eventually, front-end solutions such as the MDverse data explorer tool can evolve being more user-friendly by interfacing the structures and dynamics with interactive 3D molecular viewers". This will make MD simulations accessible to non-specialists and researchers in other areas. I would envisage that this will also include approaches using interactive virtual reality for an immersive exploration of structure and dynamics, and virtual collaboration (e.g. O'Connor et al., Sci. Adv.4, eaat2731 (2018). DOI:10.1126/sciadv.aat2731)

The need to share data effectively, and to compare simulations and test models, was illustrated clearly in the COVID-19 pandemic, which also demonstrated a willingness and commitment to data sharing across the international community (e.g. Amaro and Mulholland, J. Chem. Inf. Model. 2020, 60, 6, 2653-2656 https://doi.org/10.1021/acs.jcim.0c00319; Computing in Science & Engineering 2020, 22, 30-36 doi: 10.1109/MCSE.2020.3024155). There are important lessons to learn here, for simulations to be reproducible and reliable, for rapid testing, for exploiting data with machine learning, and for linking to data from other approaches. Tiemann et al. discuss how to develop these links, providing good perspectives and suggestions.

I agree completely with the statement of the authors that "Even if MD data represents only 1 % of the total volume of data stored in Zenodo, we believe it is our responsibility, as a community, to develop a better sharing and reuse of MD simulation files - and it will neither have to be particularly cumbersome nor expensive. To this end, we are proposing two solutions. First, improve practices for sharing and depositing MD data in data repositories. Second, improve the FAIRness of already available MD data notably by improving the quality of the current metadata."

This nicely states the challenge to the biomolecular simulation community. There is a clear need for standards for MD data and associated metadata. This will also help with the development of standards of best practice in simulations. The authors provide useful and detailed recommendations for MD metadata. These recommendations should contribute to discussions on the development of standards by researchers, funders, and publishers. Community organizations (such as CCP-BioSim and HECBioSim in the UK, BioExcel, CECAM, MolSSI, learned societies etc) have an important part to play in these developments, which are vital for the future of biomolecular simulation.

Reviewer #1 (Public Review):

Summary:<br /> Khaitova and co-workers present here an analysis of centromere composition and function during elevated temperatures in the plant Arabidopsis. The work relates to the ongoing climate change during which spikes in high temperatures will be found. Hence, the paper addresses a timely subject.

The authors start by confirming earlier studies that high temperatures reduce the fertility of Arabidopsis plants. Interestingly, a hypomorphic mutant of the centromeric histone variant CENH3 (CENP-A), which was previously described by the authors, sensitizes plants to heat and results in a drop in viable pollen and silique length. The drop in fertility coincides with the formation of micronuclei in meiosis and an extension of meiotic progression as revealed by live cell imaging. Based on this finding, the authors then show that at high temperatures, the fluorescence intensity of a YFP:CENH3 declines in meiosis but remarkably not in the surrounding cells (tapetum cells). In addition, the amount of BMF1 (a Bub1 homolog and part of the spindle assembly checkpoint) also appears to decline on the kinetochores of meiocytes as judged by BMF1 reporter line. However, whether this is dependent on a decline of CENH3 or represents a separate pathway is not clear. Finally, the authors measure the duration of the spindle checkpoint and find that it is extended under high temperatures from which they conclude that the attachment of spindle fibers to kinetochores is compromised under heat.

Strengths:<br /> This is an interesting and important paper as it links centromere organization/function to heat stress in plants. A major conclusion of the authors is that weakened centromeres, presumably by heat, may be less effective in establishing productive interactions with spindle microtubules.

Weaknesses:<br /> The paper does not explain the molecular reason why CENH3 levels in meiocyctes are reduced or why the attachment of spindle fibers to kinetochore is less efficient at high versus low temperatures.

Reviewer #2 (Public Review):

Summary:<br /> This work investigates how increased temperature affects pollen production and fertility of Arabidopsis thaliana plants grown at selected temperature conditions ranging from 16C to 30C. They report that pollen production and fertility decline with increasing temperature. To identify the cause of reduced pollen and fertility, they resort to living cell imaging of male meiotic cells to identify that the duration of meiosis increases with an increase in temperature. They also show that pollen sterility is associated with the increased presence of micronuclei likely originating from heat stress-induced impaired meiotic chromosome segregation. They correlate abnormal meiosis to weakened centromere caused by meiosis-specific defective loading of the centromere-specific histone H3 variant (CenH3) to the meiotic centromeres. Similar is the case with kinetochore-associated spindle assembly checkpoint(SAC) protein BMF1. Intriguingly, they observe a reverse trend of strong CENH3 presence in the somatic cells of the tapetum in contrast to reduced loading of CENH3 in male meiocytes with increasing temperature. In contrast to CENH3 and BMF1, the SAC protein BMF3 persists for longer periods than the WT control, based on which authors conclude that the heat stress prolongs the duration of SAC at metaphase I, which in turn extends the time of chromosome biorientation during meiosis I. The study provides preliminary insights into the processes that affect plant reproduction with increasing temperatures which may be relevant to develop climate-resilient cultivars.

Strengths:<br /> The authors have mastered the live cell imaging of male meiocytes which is a technically demanding exercise, which they have successfully employed to examine the time course of meiosis in Arabidopsis thaliana plants exposed to different temperature conditions. In continuation, they also monitor the loading dynamics and resident time of fluorescently tagged centromere/kinetochore proteins and spindle assembly checkpoint proteins to precisely measure the time duration of respective proteins to study their precise dynamics and function in male meiosis.

Weaknesses:<br /> Here the authors use only one representative centromere protein CENH3, one kinetochore-associated SAC protein BMF1, and the SAC protein BMF3 to conclude that heat stress impairs centromere function and prolongs SAC with increased temperatures. Centromere and its associated protein complex the kinetochores and the SAC contain a multitude of proteins, some of which are well characterized in Arabidopsis thaliana. Hence the authors could have used additional such tagged proteins to further strengthen their claim. Though the results presented here are interesting and solid, the study lacks a deeper mechanistic understanding of what causes the defective loading of CenH3 to the centromeres, and why the SAC protein BMF3 persists only at meiotic centromeres to prolong the spindle assembly checkpoint. Also, this observation should be interpreted in light of the fact that SAC is not that robust in plants as several null mutants of plant SAC components are known to grow as healthy as wild-type plants at normal growth conditions without any vegetative and reproductive defects. One of the immediate responses to heat stress is the production of heat shock proteins(Hsps), which act as molecular chaperones to safeguard the proteome. It will be interesting to see if the expression levels of known HsPs can be correlated with their role in stabilizing the structure of SAC proteins like BMF1 to prolong its presence at the meiotic kinetochores.

Reviewer #3 (Public Review):

Summary:<br /> Khaitova et al. report the formation of micronuclei during Arabidopsis meiosis under elevated temperatures. Micronuclei form when chromosomes are not correctly collected to the cellular poles in dividing cells. This happens when whole chromosomes or fragments are not properly attached to the kinetochore microtubules. The incidence of micronuclei formation is shown to increase at elevated temperatures in wild-type and more so in the weak centromere histone mutant cenH3-4. The number of micronuclei formed at high temperatures in the recombination mutant spo11 is like that in wild-type, indicating that the increased sensitivity of cenh3-4 is not related to the putative role of cenh3 in recombination. The abundance of CENH3-GFP at the centromere declines with higher temperature and correlates with a decline in spindle assembly checkpoint factor BMF1-GFP at the centromeres. The reduction in CENH3-GFP under heat is observed in meiocytes whereas CENH3-GFP abundance increases in the tapetum, suggesting there is a differential regulation of centromere loading in these two cell types. These observations are in line with previous reports on haploidization mutants and their hypersensitivity to heat stress.

Strengths:<br /> This paper is an important contribution to our insights into the impact of heat stress on sexual reproduction in plants.

Weaknesses:<br /> While it is highly significant, I struggled to interpret the results because of the poor quality of the figures and the videos.

Reviewer #1 (Public Review):

Su et al propose the existence of two mechanisms repressing SBF activity during entry into meiosis in budding yeast. First, a decrease in Swi4 protein levels by a LUTI-dependent mechanism where Ime1 would act closing a negative feedback loop. Second, the sustained presence of Whi5 would contribute to maintaining SBF inhibited under sporulation conditions. The article is clearly written and the experimental approaches used are adequate to the aims of this work. The results obtained are in line with the conclusions reached by the authors but, in my view, they could also be explained by the existing literature and, hence, would not represent a major advance in the field of meiosis regulation.

Regarding the first mechanism, Fig 1 shows that Swi4 decreases very little after 1-2h in sporulation medium, whereas G1-cyclin expression is strongly repressed very rapidly under these conditions (panel D and work by others). This fact dampens the functional relevance of Swi4 downregulation as a causal agent of G1 cyclin repression. The authors use overexpression of Swi4 in Figs 2 and 3 to test the relevance of Swi4 downregulation but the pATG8-SWI4 construct produces levels much higher (4-5 fold) than the wild-type gene at time 0, which may likely introduce artifactual effects in the resulting observations. In addition, the LUTI-deficient SWI4 mutant does not cause any noticeable relief in CLN2 repression, arguing against the relevance of this mechanism in the repression of G1-cyclin transcription during entry into meiosis.

The authors propose a second mechanism where Whi5 would maintain SBF inactive under sporulation conditions. The role of Whi5 as a negative regulator of the SBF regulon is well known. On the other hand, the double WHI5-AA SWI4-dLUTI mutant does not upregulate CLN2, the G1 cyclin with the strongest negative effect on sporulation, raising serious doubts on the functional relevance of this backup mechanism during entry into meiosis.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript highlights a mechanistic insight into meiotic initiation in budding yeast. In this study, the authors addressed a genetic link between mitotic cell cycle regulator SBF (the Swi4-Swi6 complex) and a meiosis inducing regulator Ime1 in the context of meiotic initiation. The authors' comprehensive analyses with cytology, imaging, RNA-seq using mutant strains lead the authors to conclude that Swi4 levels regulates Ime1-Ume6 interaction to activate expression of early meiosis genes for meiotic initiation. The major findings in this paper are that (1) the higher level of Swi4, a subunit of SBF transcription factor for mitotic cell cycle regulation, is the limiting factor for mitosis-to-meiosis transition; (2) G1 cyclins (Cln1, Cln2), that are expressed under SBF, inhibit Ime1-Ume6 interaction under overexpression of SWI4, which consequently leads to downregulation of early meiosis genes; (3) expression of SWI4 is regulated by LUTI-based transcription in the SWI4 locus that impedes expression of canonical SWI4 transcripts; (4) expression of SWI4 LUTI is likely negatively regulated by Ime1; (5) Action of Swi4 is negatively regulated by Whi5 (homologous to Rb)-mediated inhibition of SBF, which is required for meiotic initiation. Thus, the authors proposed that meiotic initiation is regulated under the balance of mitotic cell cycle regulator SBF and meiosis-specific transcription factor Ime1.

Strengths:<br /> The most significant implication in their paper is that meiotic initiation is regulated under the balance of mitotic cell cycle regulator and meiosis-specific transcription factor. This finding will provide a mechanistic insight in initiation of meiosis not only into the budding yeast also into mammals. The manuscript is overall well written, logically presented and raises several insights into meiotic initiation in budding yeast. Therefore, the manuscript should be open for the field. I would like to raise the following concerns, though they are not mandatory to address. However, it would strengthen their claims if the authors could technically address and revise the manuscript by putting more comprehensive discussion.

Weaknesses:<br /> The authors showed that increased expression of the SBF targets, and reciprocal decrease in expression of meiotic genes upon SWI4 overexpression at 2 h in SPO (Figure 2F). However, IME1 was not found as a DEG in Supplemental Table 1. Meanwhile, IME1 transcript level was decreased at 2 h SPO condition in pATG8-CLN2 cells in Fig S4C.

Now this reviewer still wonders with confusion whether expression of IME1 transcripts per se is directly or in directly suppressed under SBF-activated gene expression program at 2 h SPO in pATG8-SWI4 and pATG8-CLN2 cells. This reviewer wonders how Fig S4C data reconciles with the model summarized in Fig 6F.

One interpretation could be that persistent overexpression of G1 cyclin caused active mitotic cell cycle, and consequently delayed exit from mitotic cell cycle, which may have given rise to an apparent reduction of cell population that was expressing IME1. For readers to better understand, it would be better to explain comprehensively this issue in the main text.

The % of cells with nuclear Ime1 was much reduced in pATG8-CLN2 cells (Fig 2B) than in pATG8-SWI4 cells (Fig 4C). Is the Ime1 protein level comparable or different between pATG8-CLN2 strain and pATG8-SWI4 strain? Since it is difficult to compare the quantifications of Ime1 levels in Fig S1D and Fig S4B, it would be better to comparably show the Ime1 protein levels in pATG8-CLN2 and pATG8-SWI4 strains.<br /> Further, it is uncertain how pATG8-CLN2 cells mimics the phenotype of pATG8-SWI4 cells in terms of meiotic entry. It would be nice if the authors could show RNA-seq of pATG8-CLN2/WT and/or quantification of the % of cells that enter meiosis in pATG8-CLN2.

The authors stated that reduced Ime1-Ume6 interaction is a primary cause of meiotic entry defect by CLN2 overexpression (Line 320-322, Fig 4J-L). This data is convincing. However, the authors also showed that GFP-Ime1 protein level was decreased compared to WT in pATG8-CLN2 cells by WB (Fig S4A). Further, GFP-Ime1 signals were overall undetectable through nuclei and cytosol in pATG8-CLN2 cells (Fig 4B), and accordingly cells with nuclear Ime1 were reduced (Fig 4C). Although the authors raised a possibility that the meiotic entry defect in the pATG8-CLN2 mutant arises from downregulation of IME1 expression (Line 282-283), causal relationship between meiotic entry defect and CLN2 overexpression is still not clear. Is the Ime1 protein level reduced in the pATG8-CLN2;UME6-⍺GFP strain compared to WT? It would be better to comparably show the Ime1 protein levels in the pATG8-CLN2 strain and the pATG8-CLN2;UME6-⍺GFP strain by WB. Also, it would be nice if the authors could show quantification of the % of cells that enter meiosis in the pATG8-CLN2;UME6-⍺GFP strain to see how and whether artificial tethering of Ime1 to Ume6 rescued normal meiosis program rather than simply showing % sporulation in Fig4A.

The authors showed Ume6 binding at the SWI4LUTI promoter (Figure 5K). However, since Ume6 forms a repressive form with Rpd3 and Sin3a and binds to target genes independently of Ime1, Ume6 binding at the SWI4LUTI promoter bind does not necessarily represent Ime1-Ume6 binding there. Instead, it would be better to show Ime1 ChIP-seq at the SWI4LUTI promoter.

The authors showed ∆LUTI mutant and WHI5-AA mutant did not significantly change the expression of SBF targets nor early meiotic genes relative to wildtype (Figure 6A, C). Accordingly, they concluded that LUTI- or Whi5-based repression of SBF alone was not sufficient to cause a delay in meiotic entry (Line451-452), and perturbation of both pathways led to a significant delay in meiotic entry (Figure 6E). This reviewer wonders whether Ime1 expression level and nuclear localization of Ime1 was normal in ∆LUTI mutant and WHI5-AA mutant.

Reviewer #3 (Public Review):

The paper by Su, Yendluri and Unal reports several regulatory processes that control the activity of the SBF complex (Swi4/Swi6) in S. cerevisiae and its interaction with the meiotic inducer Ime1.

Entry into meiosis requires both the turning down of some components of the mitotic program and turning on meiotic genes. SBF (Swi4/Swi6) is an important player in entry in the mitotic cycle, acting at the G1/S transition. Previous data suggest the possibility that SBF may be differentially regulated during meiosis, potentially down-regulated. Here the authors first show a down regulation of Swi4 at the protein level, and then investigate downstream consequences. Overall the study is revealing several regulations of Swi4, with a repression of activity and a reduction of protein level by the Swi4-LUT1 transcript. The authors identify several components involved in this SWI4 pathway: 1) CLN1 and 2, which are targets of Swi4, and which mutation allows rescuing delay in meiotic entry when Swi4 is overexpressed; 2) Ime1 which activity is antigonized by Swi4, and more specifically its interaction with Ume6.

Reviewer #1 (Public Review):

Summary: The authors use the innovative CRISPRi method to uncover regulators of cell density and volume in neutrophils. The results show that cells require NHE activity during chemoattractant-driven cell migration. Before migration occurs, cells also undergo a rapid cell volume increase. These results indicate that water flux, driven by ion channels, appears to play a central role in neutrophil migration. The paper is very well written and clear. I suggest adding some discussion about the role of actin in the process, but this is not essential.

Strengths: The novel use of CRIPSPi to uncover cell density regulators is very novel. Some of the uncovered molecules were known before, e.g. discussed in Li & Sun, Frontiers in Cell and Developmental Biology, 2021. Others are more interesting, for example PI3K-gamma. The use of caged fMLP is also nice.

Weaknesses: One area of investigation that seems to be absent is mentioned in the introduction. I.e., actin is expected to play a role in regulating cell volume increase. Did the authors perform any experiments with LatA? What was seen there? Do cells still migrate with LatA, or is a different interplay seen? The role of PI3K is interesting, and maybe somewhat related to actin. But this may be a different line of inquiry for the future.

Reviewer #2 (Public Review):

Nagy et al investigated the role of volume increase and swelling in neutrophils in response to the chemoattractant. Authors show that following chemoattractant response cells lose their volume slightly owing to the cell spreading phase and then have a relatively rapid increase in the cell volume that is concomitant with cell migration. The authors performed an impressive genome-wide CRISPR screen and buoyant density assay to identify the regulators of neutrophil swelling. This assay showed that stimulating cells with chemoattractant fMLP led to an increase in the cell volume that was abrogated with the FPR1 receptor knockout. The screen revealed a cascade that could potentially be involved in cell swelling including NHE1 (sodium-proton antiporter) and PI3K. NHE1 and PI3K are required for chemoattractant-induced swelling in human primary neutrophils. Authors also suggest slightly different functions of NHE1 and PI3K activity where PI3K is also required to maintain chemoattractant-induced cell shape changes. The authors convincingly show that chemoattractant-induced cell swelling is linked to cell migration and NHE1 is required for swelling at the later stages of swelling since the cells at the early point work on low-volume and low-velocity regime. Interestingly, the authors also show that lack of swelling in NHE1-inhibited cells could be rescued by mild hypo-osmotic swelling strengthening the argument that water influx followed chemoattractant stimulation is important for potentiation for migration.

The conclusions of this paper are mostly well supported by data and are pretty convincing, but some aspects of image acquisition and data analysis need to be clarified and extended.

Weaknesses<br /> 1) It would really help if the authors could add the missing graph for the footprint area when cells are treated with Latranculin. Graph S1F for volume changes with Lat treatment should be compared with DMSO-treated controls.<br /> 2) The authors show inhibition of NHE1 blocked cell swelling using Coulter counter, a similar experiment should be done with PI3K inhibitions especially since they see PI3K inhibition impact chemoattractant-induced cell shape change.<br /> 3) It would be more convincing visually if the authors could also include the movie of cell spreading (footprint) and then mobility with PI3K inhibition.<br /> 4) It is not clear how cell spreading and later volume increase are linked to overall mobility of neutrophils. Are authors suggesting that cell spreading is not required for cell mobility in neutrophils?<br /> 5) Volume fluctuations associated with motility were impacted by NHE1 inhibition at the baselines, what about PI3K inhibitions? Does that impact the actual fluctuations?<br /> 6) It would really help if the authors compared similar analyses and drew conclusions from that, for example, it is unclear what the authors mean by they found no change in the angular persistence of WT and NHE1 inhibited cells which is in contrast to PI3K inhibition since they do not really have an analysis for angular persistence in PI3K inhibited cells. (S4A and S4B).

Reviewer #1 (Public Review):

In this work, Vezina et al. present Bactabolize, a rapid reconstruction tool for the generation of strain-specific metabolic models. Similar to other reconstruction pipelines such as CarveMe, Bactabolize builds a strain-specific draft reconstruction and subsequently gap-fills it. The model can afterwards be used to predict growth on carbon sources. The authors constructed a pan-model of the Klebsiella pneumoniae species complex (KpSC) and used it as input for Bactabolize to construct a genome-sale reconstruction of K. pneumoniae KPPR1. They compared the generated reconstruction with a reconstruction built through CarveMe as well as a manually curated reconstruction for the same strain. They then compared predictions of carbon, nitrogen, phosphor, and sulfur sources and found that the Bactabolize reconstruction had the overall highest accuracy. Finally, they built draft reconstructions for 10 clinical isolates of K. pneumoniae and evaluated their predictive performance. Overall, this is a useful tool, the data is well-presented, and the paper is well-written.

Reviewer #2 (Public Review):

The authors present a pipeline for generating strain-specific genome-scale metabolic models for bacteria using Klebsiella spp. as the demonstrative data. This paper claims to provide a high-throughput tool for generating strain-specific models for bacteria. However, in reality, the tool requires a reference pan-genome-based complete model to generate the strain-specific model of the species of interest, which in this study is Klebsiella pneumoniae. This requirement renders the tool redundant for high-throughput purposes since the process of building or generating the pan-genome reference model is performed separately. Additionally, the quality of the newly built strain-specific model will depend on the reference model used. Therefore, this tool, on its own, can only work specifically with the available pan-genome model of reference, which in this case is only applicable to Klebsiella pneumoniae. Its effectiveness with other bacteria has not been proven. I would suggest that the authors either reframe the performance and results to be applicable only to Klebsiella or consider adding more reference pan-genome models for the study.

Reviewer #1 (Public Review):

Summary:

Chartampila et al. describe the effect of early-life choline supplementation on cognitive functions and epileptic activity in a mouse model of Alzheimer's disease. The cognitive abilities are assessed by the novel object recognition test and the novel object location test, performed in the same cohort of mice at 3 months and 6 months of age. Neuronal loss was tested using NeuN immunoreactivity, and neuronal hyperexcitability was examined using FosB and video-EEG recordings.

Strengths:

The study was designed as a 6-month follow-up, with repeated behavioral and EEG measurements through disease development, providing valuable and interesting findings on AD progression and the effect of early-life choline supplantation. Moreover, the behavioral data that suggest an adverse effect of low choline in WT mice are interesting and important beyond the context of AD.

Weaknesses:

1. The multiple headings and subheadings, focusing on the experimental method rather than the narrative, reduce the readability.<br /> 2. Quantification of NeuN and FosB in WT littermates is needed to demonstrate rescue of neuronal death and hyperexcitability by high choline supplementation and also to gain further insights into the adverse effect of low choline on the performance of WT mice in the behavioral test.<br /> 3. Quantification of the discrimination ratio of the novel object and novel location tests can facilitate the comparison between the different genotypes and diets.<br /> 4. The longitudinal analyses enable the performance of multi-level correlations between the discrimination ratio in NOR and NOL, NeuN and Fos levels, multiple EEG parameters, and premature death. Such analysis can potentially identify biomarkers associated with AD progression. These can be interesting in different choline supplementation, but also in the standard choline diet.

Reviewer #1 (Public Review):

In this study, authors have investigated the effects of TMEM127 depletion on RET regulation and function that could potentially contribute to PCC pathogenesis. They have demonstrated that the loss of TMEM127 leads to cell surface accumulation and constitutive activation of RET due to membrane organization, leading to reduced efficiency of endocytosis, decreased internalization of RET, and a global impairment of membrane trafficking. TMEM127 depletion has contributed to increased RET half-life, constitutive RET-mediated signaling, increased membrane protein diffusibility, impaired normal membrane transitions, and inappropriate accumulation of actively signaling RET molecules at the cell membrane. Collectively, these findings have shown that the mis-localized RET is the pathogenic mechanism in TMEM127-mutant pheochromocytoma.

Experimental design and mechanistic studies are thorough and sound. The methodological weakness lies in the lack of pheochromocytoma cell line utility to reproduce novel findings observed in generated cell lines. This may represent a significant challenge that could undermine the inferred value of these potentially paradigm-changing findings. 3-dimensional patient-derived pheochromocytoma organoid in vitro model and/or patient-derived organoid xenograft in vivo model may aid in reconciling these exciting new findings and factoring in that the pheochromocytoma is a hormonally active tumor.

Fundamentally, the authors have successfully achieved all proposed aims supported by their conclusions.

These findings carry potentially significant clinical impact and may offer new therapeutic venues in patients with pheochromocytoma.

Reviewer #2 (Public Review):

Summary: Walker et al have proposed that the tumor suppressor TMEM127 converges with RET activation to drive adrenal phenochromocytoma. RET is a common oncogene both in familial and sporadic forms of this cancer, and TMEM127 has also been observed as a loss of function mutation in sporadic disease. The authors hypothesize that loss of the TMEM127 might signal stabilization of RET on the cell surface, mimicking an activating mutation. Through a nice set of experiments, they show that TMEM127 loss impairs endosome function and promotes RET surface accumulation. This expression was resistant to GDNF, suggesting that recycling via endosome recirculation was impaired such that the half-life of RET on the cell surface was extended. RET interaction with clathrin-coated pits was also disrupted, as the CCPs themselves were significantly smaller, and plasma membrane organization was affected by the impaired endosome recycling. Notably, a number of proteins were found to be accumulating on the cell surface via the purported mechanism, EGFR, TFR1, N cadherin, integrin beta 3. The authors applied a RET inhibitor to cells, showing decreased cellular proliferation.

Strengths: In summary, this is an interesting finding, that is preliminary in nature and is incompletely validated currently. It is certainly worth further investigation as a central feature linking TMEM127 mutations and pheochromocytoma through a common pathway of RET activation by fixing this factor in an active state on the cell surface.

Weaknesses: Although this is a provocative finding, and the authors test the interaction in a number of ways, there are several factors that limit the enthusiasm for this work as currently presented. The work is limited to one isogenic cell line with limited validation.

Reviewer #1 (Public Review):

In this study, the authors aim to understand why decision formation during behavioural tasks is distributed across multiple brain areas. They hypothesize that multiple areas are used in order to implement an information bottleneck (IB). Using neural activity recorded from monkey DLPFC and PMd performing a 2-AFC task, they show that DLPFC represents various task variables (decision, color, target configuration), while downstream PMd primarily represents decision information. Since decision information is the only information needed to make a decision, the authors point out that PMd has a minimal sufficient representation (as expected from an IB). They then train 3-area RNNs on the same task and show that activity in the first and third areas resemble the neural representations of DLPFC and PMd, respectively. In order to propose a mechanism, they analyse the RNN and find that area 3 ends up with primarily decision information because feedforward connections between areas primarily propagate decision information.

The paper addresses a deep, normative question, namely why task information is distributed across several areas.

Overall, it reads well and the analysis is well done and mostly correct (see below for some comments). My major problem with the paper is that I do not see that it actually provides an answer to the question posed (why is information distributed across areas?). I find that the core problem is that the information bottleneck method, which is evoked throughout the paper, is simply a generic compression method. Being a generic compressor, the IB does not make any statements about how a particular compression should be distributed across brain areas - see major points (1) and (2).

If I ignore the reference to the information bottleneck and the question of why pieces of information are distributed, I still see a more mechanistic study that proposes a neural mechanism of how decisions are formed, in the tradition of RNN-modelling of neural activity as in Mante et al 2013. Seen through this more limited sense, the present study succeeds at pointing out a good model-data match. I point out some suggestions for improvement below.

Major points<br /> (1) It seems to me that the author's use of the IB is based on the reasoning that deep neural networks form decisions by passing task information through a series of transformations/layers/areas and that these deep nets have been shown to implement an IB. Furthermore, these transformations are also loosely motivated by the data processing inequality.

However, assuming as a given that deep neural networks implement an IB does not mean that an IB can only be implemented through a deep neural network. In fact, IBs could be performed with a single transformation just as well. More formally, a task associates stimuli (X) with required responses (Y), and the IB principle states that X should be mapped to a representation Z, such that I(X;Z) is minimal and I(Y,Z) is maximal. Importantly, the form of the map Z=f(X) is not constrained by the IB. In other words, the IB does not impose that there needs to be a series of transformations. I therefore do not see how the IB by itself makes any statement about the distribution of information across various brain areas.

A related problem is that the authors really only evoke the IB to explain the representation in PMd: Fig 2 shows that PMd is almost only showing decision information, and thus one can call this a minimal sufficient representation of the decision (although ignoring substantial condition independent activity). However, there is no IB prediction about what the representation of DLPFC should look like. Consequently, there is no IB prediction about how information should be distributed across DLPFC and PMd.

(2) Now the authors could change their argument and state that what is really needed is an IB with the additional assumption that transformations go through a feedforward network. However, even in this case, I am not sure I understand the need for distributing information in this task. In fact, in both the data and the network model, there is a nice linear readout of the decision information in dPFC (data) or area 1 (network model). Accordingly, the decision readout could occur at this stage already, and there is absolutely no need to tag on another area (PMd, area 2+3).

Similarly, I noticed that the authors consider 2,3, and 4-area models, but they do not consider a 1-area model. It is not clear why the 1-area model is not considered. Given that e.g. Mante et al, 2013, manage to fit a 1-area model to a task of similar complexity, I would a priori assume that a 1-area RNN would do just as well in solving this task.

I think there are two more general problems with the author's approach. First, transformations or hierarchical representations are usually evoked to get information into the right format in a pure feedforward network. An RNN can be seen as an infinitely deep feedforward network, so even a single RNN has, at least in theory, and in contrast to feedforward layers, the power to do arbitrarily complex transformations. Second, the information coming into the network here (color + target) is a classical xor-task. While this task cannot be solved by a perceptron (=single neuron), it also is not that complex either, at least compared to, e.g., the task of distinguishing cats from dogs based on an incoming image in pixel format.

(3) I am convinced of the author's argument that the RNN reproduces key features of the neural data. However, there are some points where the analysis should be improved.

(a) It seems that dPCA was applied without regularization. Since dPCA can overfit the data, proper regularization is important, so that one can judge, e.g., whether the components of Fig.2g,h are significant, or whether the differences between DLPFC and PMd are significant.

(b) I would have assumed that the analyses performed on the neural data were identical to the ones performed on the RNN data. However, it looked to me like that was not the case. For instance, dPCA of the neural data is done by restretching randomly timed trials to a median trial. It seemed that this restretching was not performed on the RNN. Maybe that is just an oversight, but it should be clarified. Moreover, the decoding analyses used SVC for the neural data, but a neural-net-based approach for the RNN data. Why the differences?

(4) The RNN seems to fit the data quite nicely, so that is interesting. At the same time, the fit seems somewhat serendipitous, or at least, I did not get a good sense of what was needed to make the RNN fit the data. The authors did go to great lengths to fit various network models and turn several knobs on the fit. However, at least to me, there are a few (obvious) knobs that were not tested.

First, as already mentioned above, why not try to fit a single-area model? I would expect that a single area model could also learn the task - after all, that is what Mante et al did in their 2013 paper and the author's task does not seem any more complex than the task by Mante and colleagues.

Second, I noticed that the networks fitted are always feedforward-dominated. What happens when feedforward and feedback connections are on an equal footing? Do we still find that only the decision information propagates to the next area? Quite generally, when it comes to attenuating information that is fed into the network (e.g. color), then that is much easier done through feedforward connections (where it can be done in a single pass, through proper alignment or misalignment of the feedforward synapses) than through recurrent connections (where you need to actively cancel the incoming information). So it seems to me that the reason the attenuation occurs in the inter-area connections could simply be because the odds are a priori stacked against recurrent connections. In the real brain, of course, there is no clear evidence that feedforward connections dominate over feedback connections anatomically.

More generally, it would be useful to clarify what exactly is sufficient:

(a) the information distribution occurs in any RNN, i.e., also in one-area RNNs<br /> (b) the information distribution occurs when there are several, sparsely connected areas<br /> (c) the information distribution occurs when there are feedforward-dominated connections between areas

Reviewer #2 (Public Review):

Kleinman and colleagues conducted an analysis of two datasets, one recorded from DLPFC in one monkey and the other from PMD in two monkeys. They also performed similar analyses on trained RNNs with various architectures.

The study revealed four main findings. (1) All task variables (color coherence, target configuration, and choice direction) were found to be encoded in DLPFC. (2) PMD, an area downstream of PFC, only encoded choice direction. (3) These empirical findings align with the celebrated 'information bottleneck principle,' which suggests that FF networks progressively filter out task-irrelevant information. (4) Moreover, similar results were observed in RNNs with three modules.

While the analyses supporting results 1 and 2 were convincing and robust, I have some concerns and recommendations regarding findings 3 and 4, which I will elaborate on below. It is important to note that findings 2 and 4 had already been reported in a previous publication by the same authors (ref. 43).

Major recommendation/comments:<br /> The interpretation of the empirical findings regarding the communication subspace in relation to the information bottleneck theory is very interesting and novel. However, it may be a stretch to apply this interpretation directly to PFC-PMd, as was done with early vs. late areas of a FF neural network.

In the RNN simulations, the main finding indicates that a network with three or more modules lacks information about the stimulus in the third or subsequent modules. The authors draw a direct analogy between monkey PFC and PMd and Modules 1 and 3 of the RNNs, respectively. However, considering the model's architecture, it seems more appropriate to map Area 1 to regions upstream of PFC, such as the visual cortex, since Area 1 receives visual stimuli. Moreover, both PFC and PMd are deep within the brain hierarchy, suggesting a more natural mapping to later areas. This contradicts the CCA analysis in Figure 3e. It is recommended to either remap the areas or provide further support for the current mapping choice.

Reviewer #1 (Public Review):

Funabiki et al, performed a co-evolutionary analysis of Lsh/HELLS and CDCA7, two factors with links to DNA methylation pathways in mammals, amphibia and fish. The authors suggest that conserved roles for the two factors in DNA methylation maintenance pathways can be traced back to the last eukaryotic common ancestor. Overall, the findings are important and the results could be useful for researchers studying DNA methylation pathways in many different organisms.

Reviewer #2 (Public Review):

In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues - such as ICF related mutations for CDCA7 and SNF2 domains for HELLS - as well as maximum likelihood phylogenetic analyses. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

The data and analyses reported are significant and solid. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

Strengths:<br /> - The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.<br /> - Deep mapping of DNA methylation related process in eukaryotes.<br /> - Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.<br /> - Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

Reviewer #1 (Public Review):

This paper focuses on the effects of a L114P mutation in the TALK-1 channel on islet function and diabetes. This mutation is clinically relevant and a cause of MODY diabetes. This work employs a mouse model with heterozygous and homozygous mutants. The homozygous mice are homozygous lethal from severe hyperglycemia. The work shows that the mutation increases K+ currents and inhibits insulin secretion. This is a very nice paper with mechanistic insight and clear clinical importance. It is generally well-written and the data is well-presented.

Reviewer #2 (Public Review):

Summary:<br /> This work follows previous work from the group where they have demonstrated the role of TASK1 in the regulation of glucose-stimulated insulin secretion. Moreover, a recent study links a mutation in KCNK16, the gene encoding TALK-1 channels to MODY. Here the authors have constructed a mouse model with the specific mutation (TALK-1 L114P mutation) and investigated the phenotype. They have to perform a couple of breeding tricks to find a model that is lethal in adult which might complicate the conclusions, however, the phenotype of the heterozygote model used has a MODY-like phenotype. The study is convincing and solid.

Strengths:<br /> 1) The work is a natural follow-up from previous studies from the groups.

2) The authors present convincing and solid data that in the long perspective will help patients with these mutations.

3) Both in vivo and in vitro data are presented to give the full picture of the phenotype.

4) Data from both female and male mice are presented.

Weaknesses:<br /> 1) The authors perform an RNA-sequencing showing that the cAMP amplifying pathway is upregulated. A weakness is that this is not further followed up. The remaining questions include; Is this also true in humans with this mutation? Would treatment with incretins improve glucose-stimulated insulin secretion and and lower blood glucose?<br /> 2) The authors avoid further investigating what it means that the glucagon area and secretion are increased in the model.<br /> 3) The performance of measurements in both male and female mice is praiseworthy. However, despite differences in the response, the authors do not investigate the potential reason for this. Are hormonal differences of importance?

Reviewer #3 (Public Review):

Summary:<br /> The L114P gain of function mutation in the K2P channel TALK-1 encoded by KCNJ16 has been associated with MODY. In this study, Nakhe et al. generated mice carrying L114P TALK-1 and evaluated the impact of the mutation on glucose homeostasis. The authors report that the mutation increases neonatal lethality, owing to hyperglycemia caused by a lack of glucose-stimulated Ca2+ influx and insulin secretion. Adult mutant mice showed glucose intolerance and fasting hyperglycemia, which is attributed to blunted glucose-stimulated insulin secretion as well as increased glucagon secretion. Interestingly, male mice were more affected than female mice. Islets from adult mutant mice were found to have reduced Ca2+ entry upon glucose stimulation but also enhanced IP3-induced ER Ca2+ release, consistent with previous studies from the group showing a role of TALK-1 in ER Ca2+ homeostasis. Finally, a comparison of bulk RNA sequencing results from WT and mutant islets revealed altered expression of genes involved in β-cell identification, function, and signalling, which also contributes to the observed islet dysfunction.

The study is in general well designed and executed, and the conclusions are largely supported by the experimental evidence. The results confirm the pathogenic effect of L114P TALK-1 in human MODY. The findings that the mutation causes neonatal diabetes and affects male mice more than female mice have potential clinical implications with regard to genetic screening and diagnosis.

Strengths:<br /> A major strength of the study is the detailed characterization of the mutant mice in two different genetic backgrounds. The overall results provide compelling evidence that L114P TALK-1 disrupts glucose-stimulated insulin secretion and causes hyperglycemia. The neonatal diabetes phenotype and the gender difference in adults uncovered by the study are significant and should be considered in human patients. Results showing that the mutation not only attenuates membrane depolarization and Ca2+ entry upon glucose stimulation but also enhances IP3-induced ER Ca2+ release is consistent with the channel's dual role in membrane hyperpolarization and in providing counter currents to support ER Ca2+ release. The observed altered islet cell composition and the RNA seq data also add to the story and suggest the mutation has secondary effects that could explain the phenotypes observed.

Weaknesses:<br /> Some conclusions lack definitive evidence. For example, the authors conclude that L114P TALK-1 causes transient neonatal diabetes but there is no longitudinal glucose monitoring data to show remission of the diabetes. The contribution to defective insulin response from defects in plasma membrane depolarization relative to that from ER Ca2+ mishandling is not addressed. It is unclear whether the altered Ca2+ release in response to Ach is a direct result of GOF TALK-1 in the ER membrane or is due to the many transcriptional changes observed in the mutant islets.

Reviewer #1 (Public Review):

In this manuscript, the authors are developing a new protocol that aims at expanding pancreatic progenitors derived from human pluripotent stem cells under GMP-compliant conditions. The strategy is based on hypothesis-driven experiments that come from knowledge derived from pancreatic developmental biology.

The topic is of major interest in the view of the importance of amplifying human pancreatic progenitors (both for fundamental purposes and for future clinical applications). There is indeed currently a major lack of information on efficient conditions to reach this objective, despite major recurrent efforts by the scientific community.

Using their approach that combines stimulation of specific mitogenic pathways and inhibition of retinoic acid and specific branches of the TGF-beta and Wnt pathways, the authors claim to be able, in a highly robust and reproducible manner) to amplify in 10 passages the number of pancreatic progenitors (PP) by 2,000 folds, which is really an impressive breakthrough.

The work is globally well-performed and quite convincing. I have however some technical comments mainly related to the quantification of pancreatic progenitor amplification and to their differentiation into beta-like cells following amplification.

Reviewer #2 (Public Review):

Summary:

The paper presents a novel approach to expand iPSC-derived pdx1+/nkx6.1+ pancreas progenitors, making them potentially suitable for GMP-compatible protocols. This advancement represents a significant breakthrough for diabetes cell replacement therapies, as one of the current bottlenecks is the inability to expand PP without compromising their differentiation potential. The study employs a robust dataset and state-of-the-art methodology, unveiling crucial signaling pathways (eg TGF, Notch...) responsible for sustaining pancreas progenitors while preserving their differentiation potential in vitro.

Strengths:

This paper has strong data, guided omics technology, clear aims, applicability to current protocols, and beneficial implications for diabetes research. The discussion on challenges adds depth to the study and encourages future research to build upon these important findings.

Weaknesses:

The paper does have some weaknesses that could be addressed to improve its overall clarity and impact. The writing style could benefit from simplification, as certain sections are explained in a convoluted manner and difficult to follow, in some instances, redundancy is evident. Furthermore, the legends accompanying figures should be self-explanatory, ensuring that readers can easily understand the presented data without the need to be checking along the paper for information.

The culture conditions employed in the study might benefit from more systematic organization and documentation, making them easier to follow.

Another important aspect is the functionality of the expanded cells after differentiation. While the study provides valuable insights into the expansion of pancreas progenitors in vitro and does the basic tests to measure their functionality after differentiation the paper could be strengthened by exploring the behavior and efficacy of these cells deeper, and in an in vivo setting.

Quantifications for immunofluorescence (IF) data should be displayed.

Some claims made in the paper may come across as somewhat speculative.

Additionally, while the paper discusses the potential adaptability of the method to GMP-compatible protocols, there is limited elaboration on how this transition would occur practically or any discussion of the challenges it might entail.

Reviewer #3 (Public Review):

Summary:

In this work, Jarc et al. describe a method to decouple the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms promoting their differentiation.

The authors aimed at expanding pancreatic progenitor (PP) cells, strictly characterized as PDX1+/SOX9+/NKX6.1+ cells, for several rounds. This required finding the best cell culture conditions that allow sustaining PP cell proliferation along cell passages, while avoiding their further differentiation. They achieve this by comparing the transcriptome of PP cells that can be expanded for several passages against the transcriptome of unexpanded (just differentiated) PP cells.

The optimized culture conditions enabled the selection of PDX1+/SOX9+/NKX6.1+ PP cells and their consistent, 2000-fold, expansion over ten passages and 40-45 days. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. These optimized culture conditions consisted of substituting the Vitamin A containing B27 supplement with a B27 formulation devoid of vitamin A (to avoid retinoic acid (RA) signaling from an autocrine feed-forward loop), substituting A38-01 with the ALK5 II inhibitor (ALK5i II) that targets primarily ALK5, supplementation of medium with FGF18 (in addition to FGF2) and the canonical Wnt inhibitor IWR-1, and cell culture on vitronectin-N (VTN-N) as a substrate instead of Matrigel.

Strengths:

The strength of this work relies on a clever approach to identify cell culture modifications that allow expansion of PP cells (once differentiated) while maintaining, if not reinforcing, PP cell identity. Along the work, it is emphasized that PP cell identity is associated with the co-expression of PDX1, SOX9, and NKX6.1. The optimized protocol is unique (among the other datasets used in the comparison shown here) in inducing a strong upregulation of GP2, a unique marker of human fetal pancreas progenitors. Importantly GP2+ enriched hPS cell-derived PP cells are more efficiently differentiating into pancreatic endocrine cells (Aghazadeh et al., 2022; Ameri et al., 2017).

The unlimited expansion of PP cells reported here would allow scaling-up the generation of beta cells, for the cell therapy of diabetes, by eliminating a source of variability derived from the number of differentiation procedures to be carried out when starting at the hPS cell stage each time. The approach presented here would allow the selection of the most optimally differentiated PP cell population for subsequent expansion and storage. Among other conditions optimized, the authors report a role for Vitamin A in activating retinoic acid signaling in an autocrine feed-forward loop, and the supplementation with FGF18 to reinforce FGF2 signaling.

This is a relevant topic in the field of research, and some of the cell culture conditions reported here for PP expansion might have important implications in cell therapy approaches. Thus, the approach and results presented in this study could be of interest to researchers working in the field of in vitro pancreatic beta cell differentiation from hPSCs. Table S1 and Table S4 are clearly detailed and extremely instrumental to this aim.

Weaknesses:

The experiments performed and the methods used to evaluate the treatment effects are well-suited and state-of-the-art. However, further details on the characterization or the discussion of some of the results might help to more clearly contextualize their findings, and improve their impact on the field.

The authors strictly define PP cells as PDX1+/SOX9+/NKX6.1+ cells, and this phenotype was convincingly characterized by immunofluorescence, RT-qPCR, and FACS analysis along the work. However, broadly defined PDX1+/SOX9+/NKX6.1+ could include pancreatic multipotent progenitor cells (MPC, defined as PDX1+/SOX9+/NKX6.1+/PTF1A+ cells) or pancreatic bipotent progenitors (BP, defined as PDX1+/SOX9+/NKX6.1+/PTF1A-) cells. It has been indeed reported that Nkx6.1/Nkx6.2 and Ptf1a function as antagonistic lineage determinants in MPC (Schaffer, A.E. et al. PLoS Genet 9, e1003274, 2013), and that the Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. It would be important to define whether culturing PDX1+/SOX9+/NKX6.1+ PP (as defined in this work) in the best conditions allowing cell expansion is reinforcing either an MPC or BP phenotype. Data from Figure S2A (last paragraph of page 7) suggests that PTF1A expression is decreased in C5 culture conditions, thus more homogeneously keeping BP cells in this media composition. However, on page 15, 2nd paragraph it is stated that "the strong upregulation of NKX6.2 in our procedure suggested that our ePP cells may have retracted to an earlier PP stage". Evaluating the co-expression of the previously selected markers with PTF1A (or CPA2), or the more homogeneous expression of novel BP markers described, such as DCDC2A (Scavuzzo et al. Nat Commun 9, 3356, 2018), in the different culture conditions assayed would more shield light into this relevant aspect.

In line with the previous comment, it would be extremely insightful if the authors could characterize or at least discuss a potential role for YAP underlying the mechanistic effects observed after culturing PP in different media compositions. It is well known that the nuclear localization of the co-activator YAP broadly promotes cell proliferation, and it is a key regulator of organ growth during development. Importantly in this context, it has been reported that TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors and disruption of this interaction arrests the growth of the embryonic pancreas (Cebola, I. et al. Nat Cell Biol 17, 615-26, 2015). More recently, it has also been shown that a cell-extrinsic and intrinsic mechanotransduction pathway mediated by YAP acts as gatekeeper in the fate decisions of BP in the developing pancreas, whereby nuclear YAP in BPs allows proliferation in an uncommitted fate, while YAP silencing induces EP commitment (Mamidi, A. et al. Nature 564, 114-118, 2018; Rosado-Olivieri et al. Nature Communications 10, 1464, 2019). This mechanism was further exploited recently to improve the in vitro pancreatic beta cell differentiation protocol (Hogrebe et al., Nature Protocols 16, 4109-4143, 2021; Hogrebe et al, Nature Biotechnology 38, 460-470, 2020). Thus, YAP in the context of the findings described in this work could be a key player underlying the proliferation vs differentiation decisions in PP.

Regarding the improvements made in the PP cell culture medium composition to allow expansion while avoiding differentiation, some of the claims should be better discussed and contextualized with current state-of-the-art differentiation protocols. As an example, the use of ALK5 II inhibitor (ALK5i II) has been reported to induce EP commitment from PP, while RA was used to induce PP commitment from the primitive gut tube cell stage in recently reported in vitro differentiation protocols (Hogrebe et al., Nature Protocols 16, 4109-4143, 2021; Rosado-Olivieri et al. Nature Communications 10, 1464, 2019). In this context, and to the authors' knowledge, is Vitamin A (triggering autocrine RA signaling) usually included in the basal media formulations used in other recently reported state-of-the-art protocols? If so, at which stages? Would it be advisable to remove it?

In this line also, the supplementation of cell culture media with the canonical Wnt inhibitor IWR-1 is used in this work to allow the expansion of PP while avoiding differentiation. A role for Wnt pathway inhibition during endocrine differentiation using IWR1 has been previously reported (Sharon et al. Cell Reports 27, 2281-2291.e5, 2019). In that work, Wnt inhibition in vitro causes an increase in the proportion of differentiated endocrine cells. It would be advisable to discuss these previous findings with the results presented in the current work. Could Wnt inhibition have different effects depending on the differential modulation of the other signaling pathways?

Reviewer #1 (Public Review):

This study provides insights into the early detection of malignancies with noninvasive methods. The study contained a large sample size with an external validation cohort, which raises the credibility and universality of this model. The new model achieved high levels of AUC in discriminating malignancies from healthy controls, as well as the ability to distinguish tumor of origin. Based on these findings, prospective studies are needed to further confirm its predictive capacity.

Reviewer #2 (Public Review):

The authors tried to diagnose cancers and pinpoint tissues of origin using cfDNA. To achieve this goal, they developed a framework to assess methylation, CNA, and other genomic features. They established discovery and validation cohorts for systematic assessment and successfully achieved robust prediction power.

Reviewer #1 (Public Review):

Summary: The ciliary photoreceptor cells and its downstream neurons of larval annelid must be orchestrated in a specific pattern to promote downward swimming in response to long duration of UV exposure. The authors first conducted neuroanatomical examination of the circuit to identify NOS-expression neurons (INNOS) that are immediately downstream to the ciliary photoreceptor cells. The INNOS is activated by UV and produces NO. The NOS is required for UV avoidance by Platynereis larvae and neural dynamics of the photoreceptor cells and their downstream circuit. Following up the RNA-seq data with in situ hybridization experiments, the authors found that two unconventional guanylate cyclases, NIT-GC1 and NIT-GC2, are expressed and localized in different subcellular domain of the photoreceptor cells. Experiments using the culture cells and genetically encoded sensors demonstrated that NIT-GC1 can generate cGMP in response to nitric oxide. Finally, authors build a mathematical model that fit the live imaging data and used it to predict how the magnitude of the photoreceptor activation varied by intensity and duration of UV light.

Strengths: The authors conducted comprehensive interrogations of the UV avoidance pathway at the molecular and circuit levels, and constructed a mathematical model. The main conclusions are supported by layers of evidence from different assays.

Weaknesses: Statistics are missing in both figure legends and methods. The perturbations of genes and molecules were not cell-type-specific and therefore the observed behavioral defect could be attributed to the malfunction of the circuit elsewhere not examined in this study. I suggest adding more explanation about the functions of other NOS-expressing cells and conducting a control experiment to test behavioral response to a non-visual stimulus.

Reviewer #2 (Public Review):

Summary:<br /> This study is quite thorough, tackling this NO-dependent UV avoidance circuit with both breadth and depth. There are several novel discoveries throughout, but the whole package represents perhaps even more than the sum of these parts.

Strengths:<br /> The presentation of the work is compelling. The introduction sets up the question and the state of the field very nicely. The discovery of the non-canonical NO receptor pathway in the ciliary photoreceptors is fascinating and will likely open up new avenues for future research into NO-pathways in different species. The use of genetic and pharmacological manipulations of circuit components was well thought-out. The authors applied different experimental techniques expertly throughout the study so that they could develop a comprehensive view from the molecular to the behavioral levels.

Weaknesses:<br /> While I appreciate the intent of bringing together a large set of measurements from connectomics and calcium imaging in the framework of a model, the model seemed rather poorly constrained. How many parameters are in the model shown in Figure 6A? How many of them are well constrained by experimental measurements? The authors also don't perform sensitivity analysis on the parameters of the model. And ultimately, the conclusion over the model in Figure 7 is somewhat trivial within the unitless construction: larger amplitude and longer duration stimuli lead to increased activation of the downstream neuron thought to lead to the downward swim behavior. I could imagine that a large family of models would arrive at this same result, and without units, there is no way to really test it with new behavioral experiments.

Reviewer #3 (Public Review):

The transition from planktonic to benthic depends upon several physical and chemical cues. Nitric oxide (NO) is known as a critical player in the induction of larval metamorphosis in several invertebrates. Although NO is a widespread signalling molecule in a broad range of organisms regulating key physiological processes, internal regulatory mechanisms studies are scarce. While the UV sensing in larvae of the annelid Platynereis dumerilii using ciliary photoreceptors has been studied, the neuronal signalling mechanism remains unknown. In this study, Kei Jokura et al. investigated how annelid Platynereis dumerilii larvae detect UV sensing and modulate swimming behaviour through nitric oxide feedback. Using existing resources of Platynereis larval connectome/volume EM data, they identified NOS-expressing interneurons within the ciliary photoreceptors circuit (cPRCs). They demonstrated that NO is produced in cPRCs during UV/violet stimulation by using a fluorescent NO-reporter line. Further, they demonstrated that Nitric oxide signalling mediates UV-avoidance behaviour by using NOS-mutant larvae. Finally, they mapped out the signalled mechanisms of the cPRC circuit using published spatially mapped single-cell transcriptome data of Platynereis larvae, the Ca sensor lines, in situ HCR, and immunostaining. Additionally, by using their findings from Ca imagining data of cPRC, INNOS and INRGWa cells collected in wild-type, NOS knockout and NIT-GC2 morphant larvae, Kei Jokura et al. developed a mixed cellular-circuit-level mathematical model. However, my expertise in mathematical modelling is limited, so I cannot comment on this section.

No doubt, the study has been conducted extensively. However, I have a few comments, please see below.

Page 4: "In contrast, both two- and three-day-old homozygous NOS-mutant larvae showed a strongly diminished UV avoidance response (Figure 3A, B and Figure 3-figure supplement 1B, C)." Instead of using subjective terms like "strongly," it would be more relevant to provide statistical values. However, I could not locate any means of statistical analysis on larval behaviour. Can the authors indicate the statistical values for all behaviour studies?

Page 5: "(D) Vertical displacement in 30 sec bins of wild type and mutant (NOSΔ11/Δ11 and NOSΔ23/Δ23) three-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top." The error bars for WT are too long at the end of the experiment. It is not clear how the authors decided to use this time frame. Did the authors try carrying this out for an extended time period? How did the authors decide on 120 seconds as the time frame for exposure? Authors should provide data on larval behaviour for an extended time.

Page 13: "During the UV response, prototroch cilia beat slower than trunk cilia, resulting in a head-down stable state ('rear-wheel drive'). In contrast, during the pressure response prototroch cilia beat faster than trunk cilia, leading to a head-up orientation ('front-wheel drive'). Testing this hypothesis will require biophysical experiments and mathematical modelling." Authors should carry out ciliary beating analysis under UV light in the current study with NOS mutant larvae. Since the pressure and UV detection systems are closely related, comparing the difference in ciliary beating is important to demonstrate this hypothesis. Further, did the authors check the Ca sensor GCaMP6s under pressure conditions?

Page 18: "strips. One strip contained UV (395 nm) LEDs (SMB1W-395, Roithner Lasertechnik) and the other infrared (810 nm) LEDs (SMB1W-810NR-I, Roithner Lasertechnik)." Authors should test larval swimming behaviour at different wavelengths. Even though they are performed in previous work, the experiment with different wavelengths is necessary to be conducted in NOS mutant larvae in parallel with a control. This will confirm that NOS is principally associated with UV. Further, to demonstrate that this mechanism is associated with ciliary movement, authors need to provide this evidence.

Reviewer #1 (Public Review):

Summary<br /> In this manuscript, the authors generate an AAV-deliverable tool that generates action potentials in response to red light, but not blue light, when expressed in neurons. To do this, they screen some red light-excitatory/blue light-inhibitory opsin pairs to find ones that are spectrally and temporally matched. They first show that this works with Chrimson and GtACR2, however, they expand their search after finding that the tau-off (inactivation after light cessation) kinetics of these two opsins are not well-matched. They directly examine a small set of options based on a literature search and settle on a variant of red light-excitatory Chrimson and blue light-inhibitory ZipACR. To more closely match the kinetics of this pair, the authors create a structure homology model of the ZipACR retinal binding pocket and use this to guide the generation of a small mutagenesis panel, leading to a more optimized ZipACR mutant. They then show that a bicistronically expressed fusion arrangement of these opsins, plus some functional peptides, can drive action potentials up to 20hz with red light and does not do so with blue light, in hippocampal cells transduced by AAV. They also show function in vivo, in a mouse, using a physiological readout. They conclude that their new tool may be useful for complex experimental designs requiring multiple optical channels for write-in/read-out.

The major advantage claimed by the authors over existing tools is the temporal time-locking of their inhibitory opsin - this is driven by the contrast between the tau-off kinetics of their ZipACR variant compared to gtACR2, which is used by the leading competitor tool (BiPOLES).

Big thoughts<br /> While the authors were carefully thoughtful about the potential influence of temporal kinetics on the efficiency of a tool such as this one, there were no experiments conducted that made use of the unique properties of this molecular strategy. To understand why they embarked on this engineering program, I was required to put on my neuroscientist hat and contemplate this question myself:

First, experimental designs where I require multiple optical channels of control. This appears to be aligned with the author's thoughts, as they state, correctly, that opsins utilizing retinal as a light-sensing chromophore are universally activated by blue light (the so-called 'blue shoulder'). Therefore, their tool may be useful for stimulating multiple populations using a blue excitatory opsin in neuron A and their tool for red excitation of neuron B - or, in the author's own words, "A potential solution to the problem of cross-talk...". Yet, there are no data presented that showcases their new tool for this purpose (e.g. Vierock, Johannes, et al. "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons." Nature Communications 12.1 (2021): 4527. Figure 4f-I; 6). The same set-up could be imagined for green GECI (or equivalent) imaging of cells in the same volume that their tool is being used in - for instance, interleaving red stimulation light and blue imaging light, (perhaps) without the typical concern of imaging light bleed-through activating the opsin itself.

Second, for high-frequency temporal control over both excitation and inhibition in the same neuron. The red light turns the cell on, and blue light turns the cell off (see, for instance, Zhang, Feng, et al. "Multimodal fast optical interrogation of neural circuitry." Nature 446.7136 (2007): 633-639. Figure 2; Vierock as above, Figure 4a,b). Again, here the authors are long on theory ("The new system...can drive time-locked high-frequency action potentials in response to red pulses") and short on data. While they do show that red light = excitation and blue light = inhibition, they neither show 1) all-optical on/off modulation of the same cell; nor 2) high-frequency inhibition or excitation (max stim rate of 20hz, which is the same as the BiPOLES paper used for their LC stimulation paradigm; Vierock, as above, Figure 7a-d).

Despite these major shortcomings, the further development and characterization of tandem opsins, such as this one, is of interest to the community. There is ongoing work by the BiPOLES team to create new iterations (e.g. Wahid, J., et al. "P-15 BiPOLES2 is a bidirectional optogenetic tool with a narrow activation spectrum and low red-light excitability." Clinical Neurophysiology 148 (2023): e16.). To make the case that the tool described in this manuscript is worth the effort that the authors are requesting the neuroscience community invest in trialing it in their own hands, they must revise the manuscript to show that their approach is both 1) different in some way when compared to BiPOLES (it is my understanding that they did not do this, as per the supplementary alignment of the BiPOLES sequence and the sequence of the BiPOLES-like construct that they did test) and 2) that the properties that the investigators specifically tailored their construct to have confer some sort of experimental advantage when compared to the existing standard.

There are a number of additional concerns and clarifications that will strengthen the manuscript that are communicated directly to the authors through this peer-review process.

Reviewer #2 (Public Review):

Summary:<br /> One often wishes to combine activation of a neural population via red light with simultaneous modulation of a different population via blue light, or simultaneous imaging of a blue-excited fluorescent reporter. The problem is that all red-shifted opsins have an action spectrum with a long blue tail, leading to spurious opsin activation by blue light.

This valuable paper presents a clever solution to this problem, by pairing an engineered blue-shifted inhibitory chloride-conducting opsin with a red-shifted excitatory opsin. The combined effect is excitation by red light and shunting inhibition by blue light. The paper is very thorough, with convincing spectroscopic and patch clamp characterization of the tools, and tests in brain slices and in vivo. This tool is likely to be useful in the neuroscience community.

Strengths:<br /> The methods are solid, including the complete characterization of each tool separately, as well as the combination in vivo. The array of testing gives a strong degree of confidence that this tool will work as expected.

Weaknesses:<br /> There are two discussion points and one experimental point which would make the paper stronger.

1) In the Introduction or Discussion, the authors could better motivate the need for a red-shifted actuator that lacks blue crosstalk, by giving some specific examples of how the tool could be productively used, e.g. pairing with another blue-shifted excitatory opsin in a different population, or pairing with a GFP-based fluorescent indicator, e.g. GCaMP. The motivation for the current tool is not obvious to non-experts.

2) Simultaneous excitation and inhibition are not the same as non-excitation. The authors mentioned shunting briefly. Another possible issue is changes in osmotic balance. Activation of a Na+ channel and a Cl- channel will lead to net import of NaCl into the cell, possibly changing osmotic pressure. Please discuss.

3) The authors showed that in ZipT-IvfChr, orange light drives excitation and blue light does not. But what about simultaneous blue and orange light? Can the blue light overwhelm the effect of the orange light? Since the stated goal is to open the blue part of the spectrum for other applications, one is now worried about "negative" crosstalk. Please discuss and, ideally, characterize this phenomenon.<br /> 3.1) Does the use of the new tool require careful balancing of the expression levels of the ZipT and the IvfChr? Does it require careful balancing of blue and orange light intensities?<br /> 3.2) Also, many opsins show complex and nonlinear responses to dual-wavelength illumination, so each component should be characterized individually under simultaneous blue + orange light.<br /> 3.3) I was expecting to see photocurrents at different holding potentials as a function of illumination wavelength for the co-expressed construct (i.e. to see at what wavelength it switches from being excitatory to inhibitory); and also to see I-V curves of the photocurrent at blue and orange wavelengths for the co-expressed constructs (i.e. to see the reversal potential under blue excitation). Overall, the patch clamp and spectroscopic characterization of the individual constructs was stronger than that of the combined constructs.

Reviewer #3 (Public Review):

This study addresses the important topic of dual-color optogenetic control of neuronal activity, which is challenging due to significant optical crosstalk between channelrhodopsins of different absorption colors and ion selectivity. However, Mermet-Joret et al. demonstrate in flies that simple coexpression of a strong blue light-activated inhibitory opsin, such as the chloride-selective channelrhodopsin GtACR2, can suppress the blue light activity of a red-shifted excitatory opsin, such as Chrimson, and allow dual-color optogenetic control of the expressing neuron. The same concept was previously discussed by Vierock et al. and led to the generation of BiPOLES, which combines both channels in a single fusion protein. In the present manuscript, the authors introduce an alternative combination of channels with accelerated off-kinetics that are coexpressed by a bicistronic expression cassette. The goal is to better match the duration of illumination and optogenetic manipulation in order to reduce potential side effects induced by prolonged channel opening.

The major novelty of this work lies in the choice of the employed ion channels: the excitatory cation channel vf-Chrimson and the inhibitory anion channel ZipACR, alongside their subsequent modifications (Fig. 2 - 4). Both channels belong to the fastest known ChRs, but the choice of ZipACR raises questions. First, it has a peak absorption at 515 nm that is 40 nm further red-shifted than GtACR2 tested in Figure 1 and accordingly important optical cross-talk with the coexpressed Chrimson channel. Second, it was reported to have reduced chloride selectivity, first by Govorunova et al. in 2017 and later also by Kato et al. in 2018. Both of these aspects are also mentioned by the authors but were not resolved through molecular engineering. Instead site-directed mutagenesis primarily focused on membrane expression and photoreceptor kinetics of the employed channels. Nonetheless, improving the membrane targeting of the vf-Chrimson channel by exchange of the N-terminus finally provided sufficient red light activation at low light intensities to reliably activate expressing neurons and allowed in combination with the decelerated ZipACR mutants dual color optogenetic control with millisecond time resolution. At higher light intensities inactivation of Chrimson and the optical crosstalk of both channels seem to limit its performance.

The experimental results are well presented; but, certain questions persist:

1. The enhanced vf-Chrimson could potentially be a highlight of the manuscript, serving broader applications. Yet, gauging the overall improvements of ivf-Chrimson in comparison to other Chrimson variants remains intricate due to several reasons. First, photocurrents from ivf-Chrimson seem smaller than those from C-Chrimson (Supplemental Figure 3), and a direct comparison with standard vf-Chrimson is absent. Second, while membrane expression of ivf-Chrimson appears enhanced in provided bright-field recordings, the quantitative analysis would necessitate confocal microscopy and a membrane marker (Supplemental Figure 2). Finally, other N-terminal modified Chrimson variants, like CsChrimson by Klapoetke et al. in 2014 and C1Chrimson by Oda et al. in 2018, have been generated. Comparing ivf-Chrimson to vf-CsChrimson or vf-C1Chrimson would be important to evaluate the benefits of the applied N-terminal modification.

2. The action spectra of ZipACR suggest peak absorption of ZipACR WT and its mutant at 525 - 550 nm (Fig. 3). This is even further red-shifted than previously reported by Govorunova et al. Further action spectra recordings differ for all constructs between recordings initiated with blue or red light (Supplementary Fig. 5). This discrepancy is unexpected and should be discussed. Additionally, the representative photocurrents of Zip(151V) in Fig. 3D1 do not align with the corresponding action spectrum in Fig. 3D2 as they show maximal photocurrents for 400 nm excitation.

3. The authors introduce two different bicistronic expression cassettes-ZipT-IvfChR and ZipV-IvfChR-without providing clear guidelines on their conditions of use. Although the authors assert that ZipT is slower and further red-shifted than ZipV, the differences in the data for both ACR mutants are small and the benefits of the different final constructs should be explained.

4. The ZipT/V-IvfChRs are designed as bicistronic constructs; yet, disparities in membrane trafficking and protein degradation between the two channels could lead to divergences in blue and red light photoresponses. For future applicants, understanding the extent of expression ratio variations across cells using the presented expression cassettes could be of significance and should be discussed.

Reviewer #1 (Public Review):

Summary: By elevating Ca influx and inducing PTP, the authors have maximized the release probability. In this condition, the release probability is nearly one. Under such a condition, the release site can release another vesicle in a short time. By analyzing mean, variance, and covariance, the authors propose a release model that each release site contains a docking site and a replacement site. They excluded the LS-TS model (Neher and Brose) based on a discrepancy between the model and the data (mean and covariance).

Strengths: The authors have used minimal stimulation and modeling nicely to look into the stochastic nature of release sites with good resolution. This cannot be done at other synapses. Overall conclusions are reasonable and convincing.

Weaknesses: The interpretation is somewhat model-dependent, and it is unclear if the interpretation is unique. For example, it is unclear if the heterogeneous release probability among sites, silent sites, can explain the results. N estimates out of variance-mean analysis for example may be limited by the availability of postsynaptic receptors.

Reviewer #2 (Public Review):

Summary:<br /> Silva et al. describe an experimental study conducted on cerebellar parallel fiber-to-molecular interneuron synapses to investigate the size of the readily releasable pool (RRP) of synaptic vesicles (SVs) per docking site in response to trains of action potentials. The study aims to determine whether there are multiple binding sites for SVs at each docking site, which could lead to a higher RRP size than previously thought.

The researchers used this glutamatergic synapse to conduct their experiments. They employed various techniques and manipulations to enhance release probability, docking site occupancy, and synaptic depression. By counting the number of released SVs in response to action potential trains and normalizing the results based on the number of docking sites, they estimated the RRP size per docking site.

The key findings and observations in the manuscript are as follows:

Docking Site Occupancy and Release Probability Enhancement: The researchers used 4-amidopyridine (4-AP) and post-tetanic potentiation (PTP) protocols to enhance the release probability of docked SVs and the occupancy of docking sites, respectively.

Synchronous and Asynchronous Release: Synchronous release refers to SVs released in response to individual action potentials, while asynchronous release involves SVs released after the initial release response due to calcium elevation. The study observed changes in the balance between synchronous and asynchronous release under different conditions, revealing the degree of filling of the RRP.

Modeling of Release Dynamics: The researchers employed a modeling approach based on the "replacement site/docking site" (RS/DS) model, where SVs bind to a replacement site before moving to a docking site and eventually undergoing release. The model was adjusted to experimental conditions to estimate parameters like docking site occupancy and release probabilities.

Comparison of Different Models: The study compared the RS/DS model with an alternative model known as the "loosely docked/tightly docked" (LS/TS) model. The LS/TS model assumes that a docking site can only accommodate one SV at a time, while the RS/DS model considers the possibility of accommodating multiple SVs.

Maximum RRP Size: Through a combination of experimental results and model simulations, the study revealed that the maximum RRP size per docking site reached close to two SVs under certain conditions, supporting the idea that each docking site can accommodate multiple SVs.

Strengths:<br /> The study is rigorously conducted and takes into consideration the previous work on RRP size and SV docking site estimation. The study addresses a long-standing question in synaptic physiology.

Weaknesses:<br /> It remains unclear how generalizable the findings are to other types of synapses.

Reviewer #1 (Public Review):

This study explores the relationship between the most common spatial patterns of neurodegeneration and the density of different cell types in the cerebral cortex. The authors present data showing that atrophy patterns in Alzheimer's disease and Frontotemporal dementia strongly associate with the abundance of astrocytes and microglia. While the results here may be considered preliminary, this work takes a step in the right direction by emphasizing the critical role that cells other than neurons play in the degeneration patterns observable with neuroimaging.

I have two main comments:

1) The authors make an important innovation by applying the cellular deconvolution approach to create brain-wide maps of cellular abundance, and then comparing these maps to atrophy patterns from the most common neurodegenerative diseases and dementia syndromes.

2) I would have preferred to see more figures with brain images showing the cellular abundance maps and the atrophy maps. Without being able to see these figures, it's difficult for the reader to 1) validate the atrophy patterns or 2) gain intuition about how the cellular abundance maps vary across the brain. The images in Figure 1C give a small preview, but I'd like to see these maps in their entirety on the brain surface or axial image slices.

Reviewer #2 (Public Review):

Pak et al. report on a study using a computational method to assess differences in the relative proportion of six canonical brain cell types, across eleven neurodegenerative classes (defined as both clinical syndromes (e.g. FTD, PD), groups of neurogenerative diseases (e.g. 4-repeat tauopathies) or distinct neuropathological entities (e.g. FTLD-TDP type C), as they relate to a standard map of class-dependent volume loss. The study uses innovative methods and is commendable in its goal to highlight the contribution of non-neuronal cell types to the pathobiology of neurodegeneration. The findings of the study are in part contradicting expected results based on extensive literature on the biology of these diseases. The authors based their methodology on the use of a deconvolutional cell classifier; however, do not extensively recognize that their data on gene expression are based on normal brain levels rather than on diseased ones. Also, while predicted levels are uniquely based on patterns of brain atrophy, it is not possible to know whether this strategy is generalizable to all diseases (for instance, it is known that pure DLB, PD and ALS are not associated with extensive brain atrophy), or even adequately comparable between subtypes of diseases within the same class (e.g., different forms of FTLD). The authors do not acknowledge that only data based on true neuropathological assessment may prove whether their findings are true. Subject characteristics, numbers, and diagnostic criteria are hard to assess and only described in the methods section. This format prevents the reader from assessing data robustness while going through the results, especially when fundamental biological bases of nomenclature and differences between clinical syndromes and pathological entities are omitted or uncharacteristically provided.

Reviewer #3 (Public Review):

This study is a fine example of a recent productive trend in the integration of neuroimaging and molecular biology of the brain: in brief, overlaying some neuroimaging data (usually from a large cohort) onto the high spatial resolution gene expression in the Allen Human Brain Atlas data, derived from 6 individuals. By projecting structural MRI images over cell type proportions identified in the Allen data, the authors can represent various diseases in terms of their spatially-associated cell types. The result has implications for prioritizing the contributions of various cell types to each disease and creates an even-handed cell type profile through which the 11 diseases can be compared.

Reviewer #1 (Public Review):

Summary:<br /> In their manuscript, Chim et al. identify an association of rare loss-of-function (LOF) SLC39A5 variants with increased circulating zink levels and decreased T2D risk and complement these observations with a notably comprehensive analysis of metabolically challenged (genetically or diet-induced) Slc39a5-/- mice that demonstrate enhanced hepatic zinc levels, improved liver function, reduced hyperglycemia, partial resistance to NASH induction, and likely involvement of AMPK and AKT signaling.

Strengths:<br /> Overall, the work appears well designed, executed, clearly presented (although navigating the 16 supplementary figures and 6 supplementary tables can be a bit of a challenge), and supports the authors' main conclusions.

Weaknesses:<br /> Nevertheless, two major concerns pertain to the characterization of LOF SLC39A5 variants as well as the seeming absence of a "pancreatic phenotype" in Slc39a5-/- mice that contrasts with earlier reports including impaired glucose tolerance and glucose-stimulated insulin secretion in mice lacking Slc39a5 specifically in beta cells; these concerns should be addressed experimentally and by more extensive discussion of previously published Slc39a5-/- mouse models, respectively.

Reviewer #2 (Public Review):

Summary:<br /> This study links rare human loss of function mutations in the zinc transporter family member SLC39A5 to increased circulating and hepatic concentrations of this trace element. Beneficial metabolic changes were observed in a corresponding convincing mouse model relevant to the development of NASH.

Strengths:<br /> Authors combine human exome sequencing data, meta-analysis of four large European cohorts, and a patient recall approach to link the rare loss of function variants of SLC39A5 to the phenotype and protection from T2DM.

Using a SLC39A5-null mouse model challenged either by cross-breeding with Lepr-/- mice or diet-induced obesity they unravel the metabolic impact of elevated circulating and hepatic zinc concentration with respect to T2DM, glucose homeostasis, hepatic steatosis, and NASH development. Some mechanistic aspects and a remarkable sex difference in the outcome are identified from mouse ex vivo readouts and supported by in vitro hepatocyte cellular studies. Authors present evidence that increased hepatic zinc concentrations inhibit zinc-regulated phosphatases resulting in activation of AMPK and AKT signalling with consequences for lipid and glucose metabolism and insulin sensitivity.

Weaknesses:<br /> The reasons for the observed sex differences in the metabolic consequences of SLC39A5 inactivation in the mouse models remain unclear. While heterozygous rare SLC39A5 variants show distinct phenotypes only SLC39A5-null mice and no heterozygous mice are studied. The role of SLC39A5 in pancreatic islets and on insulin secretion remains unclear because authors do not address data published recently that claim a relevant role of SLC39A5 in b-cell function and glucose tolerance.

Reviewer #1 (Public Review):

Medwig-Kinney et al perform the latest in a series of studies unraveling the genetic and physical mechanisms involved in the formation of C. elegans gonad. They have paid particular attention to how two different cell fates are specified, the ventral uterine (VU) or anchor cell (AC), and the behaviors of these two cell types. This cell fate choice is interesting because the anchor cell performs an invasive migration through a basement membrane. A process that is required for correct C. elegans gonad formation and that can act as a model for other invasive processes, such as malignant cancer progression. The authors have identified a range of genes that are involved in the AC/VC fate choice, and that impart the AC cell with its ability to arrest the cell cycle and perform an invasive migration. Taking advantage of a range of genetic tools, the authors show that the transcription factor NHR-63 is strongly expressed in the AC cell. The authors also present evidence that NHR-63 is could function as a transcriptional repressor through interactions with a Groucho and also a TCF homolog, and they also suggest that these proteins are forming repressive condensates through phase separation.

The authors have produced an extensive dataset to support their two primary claims: that NHR-67 expression levels determine whether a cell is invasive or proliferative, and also that NHR-67 forms a repressive complex through interactions with other proteins. The authors should be commended for clearly and honestly conveying what is already known in this area of study with exhaustive references. Future data unambiguously linking the formation and dissolution of NHR-67 condensates with the activation of downstream genes that NHR-67 is actively repressing would be of great interest to the transcriptional research community.

Reviewer #2 (Public Review):

Medwig-Kinney et al. explore the role of the transcription factor NHR-67 in distinguishing between AC and VU cell identity in the C. elegans gonad. NHR-67 is expressed at high levels in AC cells where it induces G1 arrest, a requirement for the AC fate invasion program (Matus et al., 2015). NHR-67 is also present at low levels in the non-invasive VU cells and, in this new study, the authors suggest a role for this residual NHR-67 in maintaining VU cell fate. What this new role entails, however, is not clear.

The authors present two models: 1) That NHR-67 switches from a transcriptional activator in ACs to a transcriptional repressor in VUs by virtue of recruiting translational repressors, or 2) that these interactions sequester NHR-67 away from its transcription targets in VU cells. Neither model is fully supported by the data, leaving a paper with extensive data but no single compelling conclusions, and leaving open the question of what is the function, if any, of NHR-67 condensates in VU cells?

While the authors report on interesting observations, in particular the co-localization of NHR-67 with UNC-37/Groucho and POP-1 in nuclear puncta, the functional significance of these observations remains unclear. The authors have not demonstrated that the "repressive condensates" are functional and play a role in the suppression of AC fate in VU cells as claimed. The colocalization data suggest that NHR-67 interacts with repressors, but additional experiments are needed to demonstrate that these interactions are specific to VUs, impact VU fate, and sequester NHR-67 from its targets or transform NHR-67 into a transcriptional repressor.

[Editor's note: we feel that the current state of the data with respect to this question is best captured in the response by the authors to the original concerns expressed by reviewer 2, which we include in abbreviated form here]

1) The authors report that NHR-67 forms "repressive condensates" (aka. puncta) in the nuclei of VU cells and imply that these condensates prevent VU cells from becoming ACs. However, there are also examples of AC cells presented that have NHR-67 puncta (these are less obvious simply due to the higher levels of NHR-67 in ACs). Similarly, there also are UNC-37 and LSY-22 also puncta in ACs. The presence of NHR-67 puncta in the AC seems to directly contradict the author's assumption that the puncta repress the AC fate.

RESPONSE: The puncta formed by NHR-67 in the AC are different in appearance than those observed in the VU cells and furthermore do not exhibit strong colocalization with that of UNC-37 or LSY-22. The Manders' overlap coefficient between NHR-67 and UNC-37 is 0.181 in the AC, whereas it is 0.686 in the VU cells. Likewise, the Manders' overlap coefficient between NHR-67 and LSY-22 is 0.189 in the AC compared to 0.741 in the VU cells. We speculate that the areas of NHR-67 subnuclear enrichment in the AC may represent concentration around transcriptional targets, but testing this would require knowledge of direct targets of NHR-67.

2) While a pool of NHR-67 localizes to "repressive condensates", it appears that a substantial portion of NHR-67 also exists diffusively in the nucleoplasm. This would appear to contradict a "sequestration model" since, for such a model to work, a majority of NHR-67 should be in puncta? What proportion of NHR-67 is in puncta? Is the concentration of NHR-67 in the nucleoplasm lower in VUs compared to ACs and does this depend on the puncta?

RESPONSE: The proportion of NHR-67 localizing to puncta versus the nucleoplasm is dynamic, as these puncta form and dissolve over the course of the cell cycle. However, we estimate that approximately 25-40% of NHR-67 protein resides in puncta based on segmentation and quantification of fluorescent intensity. We also measured NHR-67 concentration in the nucleoplasm of VU cells and found that it is only 28% of what is observed in ACs (n = 10). We also disagree with the notion that the majority of NHR-67 protein should be located in puncta to support the sequestration model. As one example, previously published work examining phase separation of endogenous YAP shows that it is present in the nucleoplasm in addition to puncta (Cai et al., 2019, doi: 10.1038/s41556-019-0433-z). In our system, it is possible that the combination of transcriptional downregulation and partial sequestration away from DNA is sufficient to disrupt the normal activity of NHR-67.

3) The authors do not report whether NHR-67, UNC-37, LSY-22, or POP-1 localization to puncta is interdependent, as implied by their model.

RESPONSE: We based our model, shown in Fig. 7E, on known or predicted protein-protein interactions, which we confirmed through yeast two-hybrid analyses (Fig. 7D; Fig. 7-figure supplement 1). It is difficult to test whether localization of these proteins to puncta is interdependent, as a perturbation of UNC-37, LSY-22, and POP-1 result in ectopic ACs. Trying to determine if loss of puncta results in VU-to-AC transdifferentiation or vice versa becomes a chicken-egg argument. It is also possible that UNC-37 and LSY-22 are at least partially redundant in this context.

4) The evidence that the "repressor condensates" suppress AC fate in VUs is presented in Fig. 4D where the authors deplete the presumed repressor LSY-22. First, the authors do not examine whether NHR-67 forms puncta under these conditions. Second, the authors rely on a single marker (cdh-3p::mCherry::moeABD) to score AC fate: this marker shows weak expression in cells flanking one bright cell (presumably the AC) which the authors interpret as a VU AC transformation. The authors, however, do not identify the cells that express the marker by lineage analyses and dismiss the possibility that the marker-positive cells could arise from the division of an AC-committed cell. Finally, the authors did not test whether marker expression was dependent on NHR-67, as predicted by the model shown in Fig. 7.

RESPONSE: For the auxin-inducible degron experiments, strains contained labeled AID-tagged proteins, a labeled TIR1 transgene, and a labeled AC marker. Thus, we were limited by the number of fluorescent channels we could covisualize and therefore could not also visualize NHR-67 (to assess for puncta formation) or another AC marker (such as LAG-2). We could have generated an AID-tagged LSY-22 strain without a fluorescent protein, but then we would not be able to quantify its depletion, which this reviewer points out is important to measure. We did visualize NHR-67::GFP expression following RNAi-induced knockdown of POP-1 and observed consistent loss of puncta in ectopic ACs. However, it is unclear whether cell fate change causes loss of puncta or vice-versa.

5) Interaction between NHR-67 and UNC-37 is shown using Y2H, but not verified in vivo. Furthermore, the functional significance of the NHR-67/UNC-37 interaction is not tested.

We attempted to remove the intrinsically disordered region found at the C-terminus of the endogenous nhr-67 locus, using CRISPR/Cas9, as this would both confirm the NHR-67/UNC-37 interaction in vivo and allow us to determine the functional significance of this interaction. However, we were unable to recover a viable line after several attempts, suggesting that this region of the protein is vital.

6) Throughout the manuscript, the authors do not use lineage analysis to confirm fate transformation as is the standard in the field. There are 4 multipotential gonadal cells with the potential to differentiate into VUs or ACs. Which ones contribute to the extra ACs in the different genetic backgrounds examined was not determined, which complicates interpretation. The authors should consider and test the following possibilities: disruption of NHR-67 regulation causes 1) extra pluripotent cells to directly become ACs early in development, 2) causes VU cells to gradually trans-fate to an AC-like fate after VU fate specification (as implied by the authors), or 3) causes an AC to undergo extra cell division(s)? In Fig. 1F, 5 cells are designated as ACs, which is one more that the 4 precursors depicted in Fig. 1A, implying that some of the "ACs" were derived from progenitors that divided.

The timing between AC/VU cell fate specification and AC invasion (the point at which we look for differentiated ACs) is approximately 10-12 hours at 25 {degree sign}C. With our imaging setup, we are limited to approximately 3-4 hours of live-cell imaging. Therefore, lineage tracing was not feasible for our experiments. Instead, we relied on visualization of established markers of AC and VU cell fate to determine how ectopic ACs arose. In Fig. 6B,C we show that the expression of two AC markers (cdh-3 and lag-2) turn on while a VU marker (lag-1) gets downregulated within the same cell. In our opinion, live-imaging experiments that show in real time changes in cell fate via reporters was the most definitive way to observe the phenotype.

7) There are 4 multipotential gonadal cells with the potential to differentiate into VUs or ACs. Which ones contribute to the extra ACs in the different genetic backgrounds examined was not determined, which complicates interpretation. The authors should consider and test the following possibilities: disruption of NHR-67 regulation causes 1) extra pluripotent cells to directly become ACs early in development, 2) causes VU cells to gradually trans-fate to an AC-like fate after VU fate specification (as implied by the authors), or 3) causes an AC to undergo extra cell division(s)?? In Fig. 1F, 5 cells are designated as ACs, which is one more that the 4 precursors depicted in Fig. 1A, implying that some of the "ACs" were derived from progenitors that divided.

RESPONSE: When trying to determine the source of the ectopic ACs, we considered the three possibilities noted by the reviewer: (1) misspecification of AC/VU precursors, (2) VU-to-AC transdifferentiation, or (3) proliferation of the AC. We eliminated option 3 as a possibility, as the ectopic ACs we observed here were invasive and all of our previous work has shown that proliferating ACs cannot invade and that cell cycle exit is necessary for invasion (Matus et al., 2015; MedwigKinney & Smith et al., 2020; Smith et al., 2022). Specifically, NHR-67 is upstream of the cyclin dependent kinase CKI-1 and we found that induced expression of NHR-67 resulted in slow growth and developmental arrest, likely because of inducing cell cycle exit. For our experiment using hsp::NHR-67, we induced heat shock after AC/VU specification. For POP-1 perturbation, we explicitly acknowledged that misspecification of the AC/VU precursors could also contribute to ectopic ACs (Fig. 6A; lines 368-385). We could not achieve robust protein depletion through delayed RNAi treatment, so instead we utilized timelapse microscopy and quantification of AC and VU cell markers (Fig. 6B,C; see response 2.7 above).

Reviewer #1 (Public Review):

In this work, the authors investigate an important question - under what circumstances should a recurrent neural network optimised to produce motor control signals receive preparatory input before the initiation of a movement, even though it is possible to use inputs to drive activity just-in-time for movement?

This question is important because many studies across animal models have shown that preparatory activity is widespread in neural populations close to motor output (e.g. motor cortex / M1), but it isn't clear under what circumstances this preparation is advantageous for performance, especially since preparation could cause unwanted motor output during a delay.

They show that networks optimised under reasonable constraints (speed, accuracy, lack of pre-movement) will use input to seed the state of the network before movement and that these inputs reduce the need for ongoing input during the movement. By examining many different parameters in simplified models they identify a strong connection between the structure of the network and the amount of preparation that is optimal for control - namely, that preparation has the most value when nullspaces are highly observable relative to the readout dimension and when the controllability of readout dimensions is low. They conclude by showing that their model predictions are consistent with the observation in monkey motor cortex that even when a sequence of two movements is known in advance, preparatory activity only arises shortly before movement initiation.

Overall, this study provides valuable theoretical insight into the role of preparation in neural populations that generate motor output, and by treating input to motor cortex as a signal that is optimised directly this work is able to sidestep many of the problematic questions relating to estimating the potential inputs to motor cortex.

However, there are a number of issues regarding framing and technical limitations that would be useful for readers to keep in mind when interpreting the conclusions.

1) It's important to keep in mind that this work involves simplified models of the motor system, and often the terminology for 'motor cortex' and 'models of motor cortex' are used interchangeably, which may mislead some readers. Similarly, the introduction fails in many cases to state what model system is being discussed (e.g. line 14, line 29, line 31), even though these span humans, monkeys, mice, and simulations, which all differ in crucial ways that cannot always be lumped together.<br /> 2) At multiple points in the manuscript thalamic inputs during movement (in mice) is used as a motivation for examining the role of preparation. However, there are other more salient motivations, such as delayed sensory feedback from the limb and vision arriving in motor cortex, as well as ongoing control signals from other areas such as premotor cortex.<br /> 3) Describing the main task in this work as a delayed reaching task is not justified without caveats (by the authors' own admission: line 687), since each network is optimised with a fixed delay period length. Although this is mentioned to the reader, it's not clear enough that the dynamics observed during the delay period will not resemble those in the motor cortex for typical delayed reaching tasks.<br /> 4) A number of simplifications in the model may have crucial consequences for interpretation.<br /> a) Even following the toy examples in Figure 4, all the models in Figure 5 are linear, which may limit the generalisability of the findings.<br /> b) Crucially, there is no delayed sensory feedback in the model from the plant. Although this simplification is in some ways a strength, this decision allows networks to avoid having to deal with delayed feedback, which is a known component of closed-loop motor control and of motor cortex inputs and will have a large impact on the control policy.<br /> 5) A key feature determining the usefulness of preparation is the direction of the readout dimension. However, all readouts had a similar structure (random gaussian initialization). Therefore, it would be useful to have more discussion regarding how the structure of the output connectivity would affect preparation, since the motor cortex certainly does not follow this output scheme.

Reviewer #2 (Public Review):

This work clarifies neural mechanisms that can lead to a phenomenology consistent with motor preparation in its broader sense. In this context, motor preparation refers to an activity that occurs before the corresponding movement. Another property often associated with preparatory activity is a correlation with global movement characteristics such as reach speed (Churchland et al., Neuron 2006), reach angle (Sun et al., Nature 2022), or grasp type (Meirhaeghe et al., Cell Reports 2023). Such activity has notably been observed in premotor and primary motor cortices, and it has been hypothesized to serve as an input to a motor execution circuit. The timing and mechanisms by which such 'preparatory' inputs are made available to motor execution circuits remain however unclear in general, especially in light of the presence of a 'trigger-like' signal that appears to relate to the transition from preparatory dynamics to execution activity (Kaufman et al. eNeuron 2016, Iganaki et al., Cell 2022, Zimnik and Churchland, Nature Neuroscience 2021).

The preparatory inputs have been hypothesized to fulfill one or several (non-mutually-exclusive) possible objectives. Two notable hypotheses are that these inputs could be shaped to maximize output accuracy under regularization of the input magnitude; or that they may help the flexible re-use of the neural machinery involved in the control of movements in different contexts.

Here, the authors investigate in detail how the former hypothesis may be compatible with the presence of early inputs in recurrent network models driving arm movements, and compare models to data.

Strengths:

The authors are able to deploy an in-depth evaluation of inputs that are optimized for producing an accurate output at a pre-defined time while using a regularization term on the input magnitude, in the case of movements that are thought to be controlled in a quasi-open loop fashion such as reaches.

First, the authors have identified that optimal control theory is a great framework to study this question as it provides methods to find and analyze exact solutions to this cost function in the case of models with linear dynamics. The authors not only use this framework to get an exact assessment of how much activity before movement start happens in large recurrent networks, but also give insight into the mechanisms by which it happens by dissecting in detail low-dimensional networks. The authors find that two key network properties - observability of the readout's nullspace and limited controllability - give rise to optimal inputs that are large before the start of the movement (while the corresponding network activity lies in the nullspace of the readout). Further, the authors numerically investigate the timing of optimized inputs in models with nonlinear dynamics, and find that pre-movement inputs can also arise in these more general networks. Finally, the authors point out some coarse-grained similarities between the pre-movement activity driven by the optimized inputs in some of the models they studied, and the phenomenology of preparation observed in the brain during single reaches and reach sequences. Overall, the authors deploy an impressive arsenal of tools and a very in-depth analysis of their models.

Limitations:

1. Though the optimal control theory framework is ideal to determine inputs that minimize output error while regularizing the input norm, it however cannot easily account for some other varied types of objectives - especially those that may lead to a complex optimization landscape. For instance, the reusability of parts of the circuit, sparse use of additional neurons when learning many movements, and ease of planning (especially under uncertainty about when to start the movement), may be alternative or additional reasons that could help explain the preparatory activity observed in the brain. It is interesting to note that inputs that optimize the objective chosen by the authors arguably lead to a trade-off in terms of other desirable objectives. Specifically, the inputs the authors derive are time-dependent, so a recurrent network would be needed to produce them and it may not be easy to interpolate between them to drive new movement variants. In addition, these inputs depend on the desired time of output and therefore make it difficult to plan, e.g. in circumstances when timing should be decided depending on sensory signals. Finally, these inputs are specific to the full movement chain that will unfold, so they do not permit reuse of the inputs e.g. in movement sequences of different orders.

2. Relatedly, if the motor circuits were to balance different types of objectives, the activity and inputs occurring before each movement may be broken down into different categories that may each specialize into one objective. For instance, previous work (Kaufman et al. eNeuron 2016, Iganaki et al., Cell 2022, Zimnik and Churchland, Nature Neuroscience 2021) has suggested that inputs occurring before the movement could be broken down into preparatory inputs 'stricto sensu' - relating to the planned characteristics of the movement - and a trigger signal, relating to the transition from planning to execution - irrespective of whether the movement is internally timed or triggered by an external event. The current work does not address which type(s) of early input may be labeled as 'preparatory' or may be thought of as a part of 'planning' computations.

3. While the authors rightly point out some similarities between the inputs that they derive and observed preparatory activity in the brain, notably during motor sequences, there are also some differences. For instance, while both the derived inputs and the data show two peaks during sequences, the data reproduced from Zimnik and Churchland show preparatory inputs that have a very asymmetric shape that really plummets before the start of the next movement, whereas the derived inputs have larger amplitude during the movement period - especially for the second movement of the sequence. In addition, the data show trigger-like signals before each of the two reaches. Finally, while the data show a very high correlation between the pattern of preparatory activity of the second reach in the double reach and compound reach conditions, the derived inputs appear to be more different between the two conditions. Note that the data would be consistent with separate planning of the two reaches even in the compound reach condition, as well as the re-use of the preparatory input between the compound and double reach conditions. Therefore, different motor sequence datasets - notably, those that would show even more coarticulation between submovements - may be more promising to find a tight match between the data and the author's inputs. Further analyses in these datasets could help determine whether the coarticulation could be due to simple filtering by the circuits and muscles downstream of M1, planning of movements with adjusted curvature to mitigate the work performed by the muscles while permitting some amount of re-use across different sequences, or - as suggested by the authors - inputs fully tailored to one specific movement sequence that maximize accuracy and minimize the M1 input magnitude.

4. Though iLQR is a powerful optimization method to find inputs optimizing the author's cost function, it also has some limitations. First, given that it relies on a linearization of the dynamics at each timestep, it has a limited ability to leverage potential advantages of nonlinearities in the dynamics. Second, the iLQR algorithm is not a biologically plausible learning rule and therefore it might be difficult for the brain to learn to produce the inputs that it finds. It remains unclear whether using alternative algorithms with different limitations - for instance, using variants of BPTT to train a separate RNN to produce the inputs in question - could impact some of the results.

5. Under the objective considered by the authors, the amount of input occurring before the movement might be impacted by the presence of online sensory signals for closed-loop control. It is therefore an open question whether the objective and network characteristics suggested by the authors could also explain the presence of preparatory activity before e.g. grasping movements that are thought to be more sensory-driven (Meirhaeghe et al., Cell Reports 2023).

Reviewer #3 (Public Review):

This study tackles an interesting topic from a new perspective. The manuscript is well-written, logical, and conceptually clear. The central topic regards the purpose of preparatory activity in motor & premotor cortex. Preparatory activity has long captured the imaginations of experimentalists because it is a window on an unknown internal process - a process that is informed by sensation and related to action but tied directly to neither. Preparatory activity was the first truly 'internal' form of activity to be studied in awake behaving animals. The meaning and nature of the internal preparatory process has long been debated. In the 1960's, it was thought to reflect the priming of reflex circuits and motoneurons. By the 1980's, it was understood to reflect 'motor programming', i.e., the readying of cortical movement-generating machinery. But why programming was needed, and might be accomplished during preparation, remained unclear. By the 2000s, preparatory activity was seen as initializing movement-generating dynamics, much as the initial state of a dynamical system governs its future evolution. This provided a mechanistic purpose for preparation, but didn't answer a fundamental question: why use that strategy at all? Why indirectly influence execution by creating a preparatory state when you could send inputs during execution and accomplish the same thing directly?

The authors point out that the many neural network models presently in existence do not address this question because they already assume that preparatory inputs are used. Thus, those models show that the preparatory strategy works, and that it matches the data in multiple ways, but they don't reveal why it is the right strategy. An additional issue with existing networks is that they potentially create an artificial dichotomy where inputs are divided into two types: preparation-creating and movement-creating. It would be more elegant if one simply assumed that motor cortex receives inputs that attempt to serve the needs of the animal, with preparation being an emergent phenomenon rather than being baked in from the beginning. In some ways the field is already starting to shift in this direction, with preparation being seen as a special case of a general phenomenon: inputs that arrive in the null-space of network outputs. However, this shift is still nascent, and no paper to date has really addressed this issue. Thus, the present study can be seen as being the first to take a fully modern view of preparation, where it emerges as part of the solution to a more general problem.

The study is clearly written and clearly presented, and I found both the results and the reasoning to be compelling, with some exceptions noted below. The authors demonstrate that many aspects of the empirical data can be accounted for as natural outcomes of a very simple assumption: that the inputs to motor cortex are optimized to create accurate motor-cortex output while being 'well-behaved' in the sense of remaining modest in magnitude. More broadly, the idea is that preparation emerges as a consequence of constraints on motor-cortex inputs. If upstream areas could magically control motor cortex any way they wanted, then there would be no need for preparation. The necessary patterns of execution activity could just be created directly by inputs at that time. However, when there exist constraints on inputs (i.e., on what upstream areas can do) preparation becomes a useful - perhaps necessary - strategy. By sending inputs early, upstream areas can leverage the dynamics of motor cortex in ways that would be harder to accomplish during movement.

The authors illustrate how a very simple constraint on inputs - a high 'cost' to large inputs - makes preparation a good strategy. Preparation isn't strictly necessary, but it produces a lower-cost solution (reduced input magnitude for a given level of accuracy). Consequently, preparation appears naturally, with a time-course of ~300 ms before movement onset. This late rise in preparation doesn't match the longer plateau most people are used to from studies that use a randomized instructed delay, but that actually makes sense. In those studies, the animal does not know when the go cue will be given, and must be ready for it to occur at any time. In contrast, the present study considers the situation where the time of future movement is known internally and is part of the optimization process. This more closely matches situations where the animal chooses when to move, and in those situations, preparation does indeed appear late in most cases. So the predictions of their simulations are qualitatively correct (which is all that is desired, given uncertainty regarding things like the right internal time-constants). Their simulations also successfully predict two bouts of preparation during sequence tasks, matching recent empirical findings.

The main strength of the study is its ability to elegantly explain well-known features of data in terms of simple normative principles. The study is thorough and careful in key ways. For example, they show that the emergence of preparation, in the service of satisfying the cost function, is a very general property that holds across a broad range of network types (including very simple toy networks and a variety of larger networks of different types). They also go to considerable trouble to show why cost is reduced by preparatory inputs, including illustrating different scenarios with different readout-vector orientations. The result is a conceptually clear study that conveys a fresh perspective on what preparation is and why it exists.

The main limitation of the study is that it focuses exclusively on one specific constraint - magnitude - that could limit motor-cortex inputs. This isn't unreasonable, but other constraints are at least as likely, if less mathematically tractable. The basic results of this study will probably be robust with regard such issues - generally speaking, any constraint on what can be delivered during execution will favor the strategy of preparing - but this robustness cuts both ways. It isn't clear that the constraint used in the present study - minimizing upstream energy costs - is the one that really matters. Upstream areas are likely to be limited in a variety of ways, including the complexity of inputs they can deliver. Indeed, one generally assumes that there are things that motor cortex can do that upstream areas can't do, which is where the real limitations should come from. Yet in the interest of a tractable cost function, the authors have built a system where motor cortex actually doesn't do anything that couldn't be done equally well by its inputs. The system might actually be better off if motor cortex were removed. About the only thing that motor cortex appears to contribute is some amplification, which is 'good' from the standpoint of the cost function (inputs can be smaller) but hardly satisfying from a scientific standpoint.

The use of a term that punishes the squared magnitude of control signals has a long history, both because it creates mathematical tractability and because it (somewhat) maps onto the idea that one should minimize the energy expended by muscles and the possibility of damaging them with large inputs. One could make a case that those things apply to neural activity as well, and while that isn't unreasonable, it is far from clear whether this is actually true (and if it were, why punish the square if you are concerned about ATP expenditure?). Even if neural activity magnitude an important cost, any costs should pertain not just to inputs but to motor cortex activity itself. I don't think the authors really wish to propose that squared input magnitude is the key thing to be regularized. Instead, this is simply an easily imposed constraint that is tractable and acts as a stand-in for other forms of regularization / other types of constraints. Put differently, if one could write down the 'true' cost function, it might contain a term related to squared magnitude, but other regularizing terms would by very likely to dominate. Using only squared magnitude is a reasonable way to get started, but there are also ways in which it appears to be limiting the results (see below).

I would suggest that the study explore this topic a bit. Is it possible to use other forms of regularization? One appealing option is to constrain the complexity of inputs; a long-standing idea is that the role of motor cortex is to take relatively simple inputs and convert them to complex time-evolving inputs suitable for driving outputs. I realize that exploring this idea is not necessarily trivial. The right cost-function term is not clear (should it relate to low-dimensionality across conditions, or to smoothness across time?) and even if it were, it might not produce a convex cost function. Yet while exploring this possibility might be difficult, I think it is important for two reasons. First, this study is an elegant exploration of how preparation emerges due to constraints on inputs, but at present that exploration focuses exclusively on one constraint. Second, at present there are a variety of aspects of the model responses that appear somewhat unrealistic. I suspect most of these flow from the fact that while the magnitude of inputs is constrained, their complexity is not (they can control every motor cortex neuron at both low and high frequencies). Because inputs are not complexity-constrained, preparatory activity appears overly complex and never 'settles' into the plateaus that one often sees in data. To be fair, even in data these plateaus are often imperfect, but they are still a very noticeable feature in the response of many neurons. Furthermore, the top PCs usually contain a nice plateau. Yet we never get to see this in the present study. In part this is because the authors never simulate the situation of an unpredictable delay (more on this below) but it also seems to be because preparatory inputs are themselves strongly time-varying. More realistic forms of regularization would likely remedy this.

At present, it is also not clear whether preparation always occurs even with no delay. Given only magnitude-based regularization, it wouldn't necessarily have to be. The authors should perform a subspace-based analysis like that in Figure 6, but for different delay durations. I think it is critical to explore whether the model, like monkeys, uses preparation even for zero-delay trials. At present it might or might not. If not, it may be because of the lack of more realistic constraints on inputs. One might then either need to include more realistic constraints to induce zero-delay preparation, or propose that the brain basically never uses a zero delay (it always delays the internal go cue after the preparatory inputs) and that this is a mechanism separate from that being modeled.

I agree with the authors that the present version of the model, where optimization knows the exact time of movement onset, produces a reasonably realistic timecourse of preparation when compared to data from self-paced movements. At the same time, most readers will want to see that the model can produce realistic looking preparatory activity when presented with an unpredictable delay. I realize this may be an optimization nightmare, but there are probably ways to trick the model into optimizing to move soon, but then forcing it to wait (which is actually what monkeys are probably doing). Doing so would allow the model to produce preparation under the circumstances where most studies have examined it. In some ways this is just window-dressing (showing people something in a format they are used to and can digest) but it is actually more that than, because it would show that the model can produce a reasonable plateau of sustained preparation. At present it isn't clear it can do this, for the reasons noted above. If it can't, regularizing complexity might help (and even if this can't be shown, it could be discussed).

In summary, I found this to be a very strong study overall, with a conceptually timely message that was well-explained and nicely documented by thorough simulations. I think it is critical to perform the test, noted above, of examining preparatory subspace activity across a range of delay durations (including zero) to see whether preparation endures as it does empirically. I think the issue of a more realistic cost function is also important, both in terms of the conceptual message and in terms of inducing the model to produce more realistic activity. Conceptually it matters because I don't think the central message should be 'preparation reduces upstream ATP usage by allowing motor cortex to be an amplifier'. I think the central message the authors wish to convey is that constraints on inputs make preparation a good strategy. Many of those constraints likely relate to the fact that upstream areas can't do things that motor cortex can do (else you wouldn't need a motor cortex) and it would be good if regularization reflected that assumption. Furthermore, additional forms of regularization would likely improve the realism of model responses, in ways that matter both aesthetically and conceptually. Yet while I think this is an important issue, it is also a deep and tricky one, and I think the authors need considerable leeway in how they address it. Many of the cost-function terms one might want to use may be intractable. The authors may have to do what makes sense given technical limitations. If some things can't be done technically, they may need to be addressed in words or via some other sort of non-optimization-based simulation.

Specific comments

As noted above, it would be good to show that preparatory subspace activity occurs similarly across delay durations. It actually might not, at present. For a zero ms delay, the simple magnitude-based regularization may be insufficient to induce preparation. If so, then the authors would either have to argue that a zero delay is actually never used internally (which is a reasonable argument) or show that other forms of regularization can induce zero-delay preparation.

I agree with the authors that prior modeling work was limited by assuming the inputs to M1, which meant that prior work couldn't address the deep issue (tackled here) of why there should be any preparatory inputs at all. At the same time, the ability to hand-select inputs did provide some advantages. A strong assumption of prior work is that the inputs are 'simple', such that motor cortex must perform meaningful computations to convert them to outputs. This matters because if inputs can be anything, then they can just be the final outputs themselves, and motor cortex would have no job to do. Thus, prior work tried to assume the simplest inputs possible to motor cortex that could still explain the data. Most likely this went too far in the 'simple' direction, yet aspects of the simplicity were important for endowing responses with realistic properties. One such property is a large condition-invariant response just before movement onset. This is a very robust aspect of the data, and is explained by the assumption of a simple trigger signal that conveys information about when to move but is otherwise invariant to condition. Note that this is an implicit form of regularization, and one very different from that used in the present study: the input is allowed to be large, but constrained to be simple. Preparatory inputs are similarly constrained to be simple in the sense that they carry only information about which condition should be executed, but otherwise have little temporal structure. Arguably this produces slightly too simple preparatory-period responses, but the present study appears to go too far in the opposite direction. I would suggest that the authors do what they can to address these issue via simulations and/or discussion. I think it is fine if the conclusion is that there exist many constraints that tend to favor preparation, and that regularizing magnitude is just one easy way of demonstrating that. Ideally, other constraints would be explored. But even if they can't be, there should be some discussion of what is missing - preparatory plateaus, a realistic condition-invariant signal tied to movement onset - under the present modeling assumptions.

On line 161, and in a few other places, the authors cite prior work as arguing for "autonomous internal dynamics in M1". I think it is worth being careful here because most of that work specifically stated that the dynamics are likely not internal to M1, and presumably involve inter-area loops and (at some latency) sensory feedback. The real claim of such work is that one can observe most of the key state variables in M1, such that there are periods of time where the dynamics are reasonably approximated as autonomous from a mathematical standpoint. This means that you can estimate the state from M1, and then there is some function that predicts the future state. This formal definition of autonomous shouldn't be conflated with an anatomical definition.

Reviewer #1 (Public Review):

The authors aimed to develop a whole-brain multivariate pattern predicting decisions to trust and to use this pattern to assess the construct validity of the concept of trust. To this end, they used machine learning to develop and validate a whole-brain pattern capable of predicting decisions to trust in three previously published fMRI datasets in which participants played an economic trust game. They then assessed how this pattern was expressed in several other published fMRI datasets operationalizing various psychological concepts. They observed that the trust pattern could discriminate between risky or safe economic decisions and different emotional states but could not discriminate between several other concepts such as reward/losses, famous/unfamiliar face perception, etc. Spatial similarity analyses across datasets showed converging results.

This study adopts a rigorous analytical approach, examining fMRI data from thousands of participants spanning fifteen datasets to investigate the relationship between the multivariate pattern of trust and other psychological concepts. Researchers interested in the concept of trust will find this work valuable. More importantly, it exemplifies the potential of using brain data to explore the construct validity of psychological concepts through this methodological approach.

Despite the strengths of this study, there are several points that, in my view, need further attention:

1. The trust pattern developed and validated by the authors is based on one type of task, the economic trust game. This means that the multivariate trust pattern developed by the authors is heavily dependent on how trust is specifically defined and operationalized within this task, which may limit its generalizability. Without evidence that the model generalizes to other operationalizations of trust, the authors should interpret their results more conservatively. Unless additional evidence is given, this should be presented as a pattern of the "decision to trust in an economic context".

2. In datasets 1-1 and 1-2, trust is operationalized as a form of social gambling, where participants choose to share money (trust) with someone else, hoping to triple their investment but risk losing it all, with the alternative being to keep the money (distrust). However, these datasets also include non-social control conditions (the lottery condition in Fareri et al., 2012, and the computer condition in Fareri et al., 2015), which are not discussed in this paper. Evaluating how the trust model behaves in these control conditions seems crucial, as they provide the closest comparison to similar tasks that exclude the trust component. If the trust model is not specific to social decisions in the original datasets (i.e., it cannot distinguish between gambling and not gambling), this significant limitation should be addressed and discussed.

3. The analytical strategy used to establish convergent and discriminant validity is based on the significance of the average group accuracy of forced-choice tests to assess the capacity of the model to discriminate between different concepts (e.g. rewards vs. loss, safety vs. risk). The model is assumed to be specific to trust when the accuracy is not significantly different from chance and related to the other construct when the accuracy is significantly above chance. However, the absence of an effect is related to the power of the test, and in several cases, the sample sizes were relatively small. The use of one-tailed tests also exacerbates this issue since only effects in the hypothesized directions can be significant. These analyses could be improved by adopting a different approach to evaluate support for the null effect, by setting a higher bar for what is considered a generalization of the model, or by interpreting the results more carefully to recognize that lack of evidence isn't necessarily evidence of absence.

Reviewer #2 (Public Review):

Summary:<br /> The authors set out to characterise "trust" in terms of a spatial pattern of neural responses, and then validate whether different tasks, in different datasets, express this pattern or do not express it, according to their hypotheses. They based their approach on linear classifiers (Support Vector Machines), which they trained to distinguish trust from distrust in an investment game, and then applied the classifier to other datasets. Additionally, they performed visualisations of the similarity among participants and among tasks in their neural responses, using dimensionality reduction techniques.

Strengths:<br /> The key strength of this study is the use of multiple datasets to test whether a single study's characterisation of trust, in terms of a spatial pattern of neural responses, generalises to other tasks and populations. This is a nice use for existing data, which bolsters the interpretation of fMRI results, demonstrating that they are generalisable. While I am not a specialist in decoding methods, the analyses appear to have been performed conscientiously and to a high standard. The manuscript is also clearly written.

Weaknesses:<br /> It's worth noting an obvious but important statistical point. In this study, the *inability* of a classifier to distinguish between conditions in particular datasets is taken as evidence that those conditions do not differ in terms of the effect of interest (trust). In this case, these results make sense, in that they are consistent with the authors' hypotheses. However, there are various reasons why the classifier may not work well on particular datasets - e.g. differences in noise, or a lack of linear separability between patterns (which might mandate a non-linear classifier or a different SVM kernel). Therefore, any null result obtained with classical statistics should be interpreted with caution.

Reviewer #3 (Public Review):

Summary:<br /> This is a timely and impressive study that applies a neuroscientific approach to provide an objective measurement of the psychological construct of trust. Drawing links from psychometrics, the presented neurometric approach will be beneficial to many open research questions within and beyond the field.

Strengths:<br /> There are multiple strengths to highlight. First, the study followed and moved beyond best practices in psychometrics research to establish the neurometrics of trust. Second, it made use of multiple datasets to rigorously validate the model and tested its specificity and generalizability. The choice of these datasets was well justified and informed by previous studies. Third, the study combined a series of data-driven approaches to provide converging and complementary evidence of their neurometric model, and this sets an excellent example for future work in similar veins.

Weaknesses:<br /> There were a few things that would be helpful to clarify, on top of the already comprehensive paper. First, it will be helpful to draw an even closer side-by-side analogy between neurometrics and psychometrics. Imaginably this work will benefit both psychology and neuroscience; using an illustration (such as a box) detailing the counterpart of neurometrics with respect to psychometrics will be very helpful for many researchers. Relatedly, I am curious about what the "end product" will be by using the neurometrics approach. In psychometrics, the product will naturally be the scale/questionnaire, and then there is the related validity & reliability check, etc. So is the multivariate pattern map the product, or something else? Practically, how can users make use of the maps as easily as using a questionnaire? Second, the relationship between trust and no-reward (and similarly between distrust and reward) is indeed puzzling. The authors attributed that to the non-linear nature of the methodology. But if this is true, does the non-linear nature of the methods also hamper the other results? It is perhaps worth checking the reward-related maps at the decision stage (to reflect the anticipation) rather than the outcome state (where participants actually saw the win/loss). Lastly, the measurement of "pattern expression" and the associated "expression difference" lacks detailed explanations, as in, what do the magnitude and sign mean? How to interpret them?

Reviewer #2 (Public Review):

Summary<br /> In this experiment, Voltage Sensitive Dye Imaging (VSDI) was used to measure neural activity in macaque primary visual cortex in monkeys trained to detect an oriented grating target that was presented either alone or against an oriented mask. Monkeys' ability to detect the target (indicated by a saccade to its location) was impaired by the mask, with the greatest impairment observed when the mask was matched in orientation to the target, as is also the case in human observers. VSDI signals were examined to test the hypothesis that the target-evoked response would be maximally suppressed by the mask when it matched the orientation of the target. In each recording session, fixation trials were used to map out the spatial response profile and orientation domains that would then be used to decode the responses on detection trials. VSDI signals were analyzed at two different scales: a coarse scale of the retinotopic response to the target and a finer scale of orientation domains within the stimulus-evoked response. Responses were recorded in three conditions: target alone, mask alone, and target presented with mask. Analyses were focused on the target evoked response in the presence of the mask, defined to be the difference in response evoked by the mask with target (target present) versus the mask alone (target absent). These were computed across five 50 msec bins (total, 250 msec, which was the duration of the mask (target present trials, 50% of trials) / mask + target (target present trials, 50% of trials). Analyses revealed that in an initial (transient) phase the target evoked response increased with similarity between target and mask orientation. As the authors note, this is surprising given that this was the condition where the mask maximally impaired detection of the target in behavior. Target evoked responses in a later ('sustained') phase fell off with orientation similarity, consistent with the behavioral effect. When analyzed at the coarser scale the target evoked response, integrated over the full 250 msec period showed a very modest dependence on mask orientation. The same pattern held when the data were analyzed on the finer orientation domain scale, with the effect of the mask in the transient phase running counter to the perceptual effect of the mask and the sustained response correlating the perceptual effect. The effect of the mask was more pronounced when analyzed at the scale.

Strengths<br /> The work is on the whole very strong. The experiments are thoughtfully designed, the data collection methods are good, and the results are interesting. The separate analyses of data at a coarse scale that aggregates across orientation domains and a more local scale of orientation domains is a strength and it is reassuring that the effects at the more localized scale are more clearly related to behavior, as one would hope and expect. The results are strengthened by modeling work shown in Figure 8, which provides a sensible account of the population dynamics. The analyses of the relationship between VSDI data and behavior are well thought out and the apparent paradox of the anti-correlation between VSDI and behavior in the initial period of response, followed by a positive correlation in the sustained response period is intriguing.

Points to Consider / Possible Improvements<br /> The biphasic nature of the relationship between neural and behavioral modulation by the mask and the surprising finding that the two are anticorrelated in the initial phase are left as a mystery. The paper would be more impactful if this mystery could be resolved.

The finding is based on analyses of the correlation between behavior and neural responses. This appears in the main body of the manuscript and is detailed in Figures S1 and S2, which show the correlation over time between behavior and target response for the retinotopic and columnar scale.

One possible way of thinking of this transition from anti- to positive correlation with behavior is that it might reflect the dynamics of a competitive interaction between mask and target, with the initial phase reflecting predominantly the mask response, with the target emerging, on some trials, in the latter phase. On trials when the mask response is stronger, the probability of the target emerging in the latter phase, and triggering a hit, might be lower, potentially explaining the anticorrelation in the initial phase. The sustained response may be a mixture of trials on which the target response is or is not strong enough to overcome the effect of the mask sufficiently to trigger target detection.

It would, I think, be worth examining this by testing whether target dynamics may vary, depending on whether the monkey detected the target (hit trials) or failed to detect the target (miss trials). Unless I missed it I do not think this analysis was done. Consistent with this possibility, the authors do note (lines 226-229) that "The trajectories in the target plus mask conditions are more complex. For example, when mask orientation is at +/- 45 deg to the target, the population response is initially dominated by the mask, but then in mid-flight, the population response changes direction and turns toward the direction of the target orientation." This suggests (to this reviewer, at least) that the emergence of a positive correlation between behavioral and neural effects in the latter phase of the response could reflect either a perceptual decision that the target is present or perhaps deployment of attention to the location of the target.

It may be that this transition reflected detection, in which it might be more likely on hit trials than miss trials. Given the SNR it would presumably be difficult to do this analysis on a trial-by-trial basis, but the hit and miss trials (which make each make up about 1/2 of all trials) could be averaged separately to see if the mid-flight transition is more prominent on hit trials. If this is so for the +/- 45 degree case it would be good to see the same analysis for other combinations of target and mask. It would also be interesting to separate correct reject trials from false alarms, to determine whether the mid-flight transition tends to occur on false alarm trials.

If these analyses do not reveal the predicted pattern, they might still merit a supplemental figure, for the sake of completeness.

Reviewer #1 (Public Review):

This is a clear account of some interesting work. The experiments and analyses seem well done and the data are useful. It is nice to see that VSDI results square well with those from prior extracellular recordings. But the work may be less original than the authors propose, and their overall framing strikes me as odd. Some additional clarifications could make the contribution more clear.

My reading is that this is primarily a study of surround suppression with results that follow pretty directly from what we already know from that literature, and although they engage with some of the literature they do not directly mention surround suppression in the text. Their major effect - what they repeatedly describe as a "paradoxical" result in which the responses initially show a stronger response to matched targets and backgrounds and then reverse - seems to pretty clearly match the expected outcome of a stimulus that initially evokes additional excitation due to increased center contrast followed by slightly delayed surround suppression tuned to the same peak orientation. Their dynamics result seems entirely consistent with previous work, e.g. Henry et al 2020, particularly their Fig. 3 https://elifesciences.org/articles/54264, so it seems like a major oversight to not engage with that work at all, and to explain what exactly is new here.

- In the discussion (lines 315-316), they state "in order to account for the reduced neural sensitivity with target-background similarity in the second phase of the response, the divisive normalization signal has to be orientation selective." I wonder whether they observed this in their modeling. That is, how robust were the normalization model results to the values of sigma_e and sigma_n? It would be useful to know how critical their various model parameters were for replicating the experimental effects, rather than just showing that a good account is possible.

- The majority of their target/background contrast conditions were collected only in one animal. This is a minor limitation for work of this kind, but it might be an issue for some.

- The authors point out (line 193-195) that "Because the first phase of the response is shorter than the second phase, when V1 response is integrated over both phases, the overall response is positively correlated with the behavioral masking effect." I wonder if this could be explored a bit more at the behavioral level - i.e. does the "similarity masking" they are trying to explain show sensitivity to presentation time?

- From Fig. 3 it looks like the imaging ROI may include some opercular V2. If so, it's plausible that something about the retinotopic or columnar windowing they used in analysis may remove V2 signals, but they don't comment. Maybe they could tell us how they ensured they only included V1?

- In the discussion (lines 278-283) they say "The positive correlation between the neural and behavioral masking effects occurred earlier and was more robust at the columnar scale than at the retinotopic scale, suggesting that behavioral performance in our task is dominated by columnar scale signals in the second phase of the response. To the best of our knowledge, this is the first demonstration of such decoupling between V1 responses at the retinotopic and columnar scales, and the first demonstration that columnar scale signals are a better predictor of behavioral performance in a detection task." I am having trouble finding where exactly they demonstrate this in the results. Is this just by comparison of Figs. 4E,K and 5E,K? I may just be missing something here, but the argument needs to be made more clearly since much of their claim to originality rests on it.

Reviewer #1 (Public Review):

It has been shown that there are relationships between a transdiagnostic construct of anxious-depression, and average confidence rating in a perceptual decision task. This study sought to investigate these results, which have been replicated several times but only in cross-sectional studies. This work applies a perceptual decision-making task with confidence ratings and a transdiagnostic psychometric questionnaire battery to participants before and after an iCBT course. The iCBT course reduced AD scores in participants, and their mean confidence ratings increased without a change in performance. Participants with larger AD changes had larger confidence changes. These results were also shown in a separate smaller group receiving antidepressant medication. A similar sized control group with no intervention did not show changes.

The major strength of the study is the elegant and well-powered data set. Longitudinal data on this scale is very difficult to collect, especially with patient cohorts, so this represents an exciting breakthrough. Analysis is straightforward and clearly presented. No multiple comparison correction is applied despite many different tests. While in general I am not convinced of the argument in the citation provided to justify this, I think in this case the key results are not borderline (p<0.001) and many of the key effects are replications, so there are not so many novel/exploratory hypothesis and in my opinion the results are convincing and robust as they are. The supplemental material is a comprehensive description of the data set, which is a useful resource.

The authors achieved their aims, and the results clearly support the conclusion that the AD and mean confidence in a perceptual task covary longitudinally.

I think this provides an important impact to the project of computational psychiatry, specifically, it shows the relationship between transdiagnostic symptom dimensions and behaviour is meaningful within as well as across individuals.

Reviewer #2 (Public Review):

The authors of this study investigated the relationship between (under)confidence and the anxious-depressive symptom dimension in a longitudinal intervention design. The aim was to determine whether confidence bias improves in a state-like manner when symptoms improve. The primary focus was on patients receiving internet-based CBT (iCBT; n=649), while secondary aims compared these changes to patients receiving antidepressants (n=82) and a control group (n=88).

The results support the authors' conclusions, and the authors convincingly demonstrated a weak link between changes in confidence bias and anxious-depressive symptoms (not specific to the intervention arm)

The major strength and contribution of this study is the use of a longitudinal intervention design, allowing the investigation of how the well-established link between underconfidence and anxious-depressive symptoms changes after treatment. Furthermore, the large sample size of the iCBT group is commendable. The authors employed well-established measures of metacognition and clinical symptoms, used appropriate analyses, and thoroughly examined the specificity of the observed effects.

However, due to the small expected effect sizes, the comparisons with the antidepressant and control groups were underpowered, reducing comparability between interventions and the generalizability of the results. The lack of interaction effect with treatment makes it harder to interpret the observed differences in confidence.

Reviewer #3 (Public Review):

This study reports data collected across time and treatment modalities (internet CBT or iCBT, pharmacological intervention, and control), with a particularly large sample in the iCBT group. This study addresses the question of whether metacognitive confidence is related to mental health symptoms in a trait-like manner, or whether it shows state dependency. The authors report an increase in metacognitive confidence as anxious-depression symptoms improve with iCBT (and the extent to which confidence increases is related to the magnitude of symptom improvement), a finding that is largely mirrored in those who receive antidepressants (without the correlation between symptom change and confidence change). I think these findings are exciting because they directly relate to one of the big assumptions when relating cognition to mental health - are we measuring something that changes with treatment (is malleable), so might be mechanistically relevant, or even useful as a biomarker?

This work is also useful in that it replicates a finding of heightened confidence in those with compulsivity, and lowered confidence in those with elevated anxious-depression.

One caveat to the interest of this work is that it doesn't allow any causal conclusions to be drawn, and only measures two timepoints, so it's hard to tell if changes in confidence might drive treatment effects (but this would be another study). The authors do mention this in the limitations section of the paper.

Another caveat is the small sample in the antidepressant group.

I appreciate the authors' efforts to respond to queries I had about this paper: including the addition of a sensitivity analysis to examine whether excluding 'inattentive' participants made a difference to results.

I am still not fully convinced by the argument that these results are specific to metacognition, given that task difficulty significantly increased in the antidepressant group but not the control group. Whilst there is a lack of association between this change and symptom change, this 'null result' is not the same as showing there is no relationship and therefore that increased general performance in specific groups might drive increased confidence (though accuracy is the same). The authors' argument is strengthened by the lack of group*time interaction in dot difficulty, but individual tests (e.g. of change in antidepressant arm; and change in control arm) showed differing significance. This is a minor point, but could point to an alternative explanation of the results.

Reviewer #1 (Public Review):

Secondary cell walls support vascular plants and conduct water throughout the plant body, but are also important resources for lignocellulosic feedstocks. Secondary cell wall synthesis is under complex transcriptional control, presumably because it must only be initiated after cell growth is complete. Here, the authors found that two Musashi-type RNA-binding proteins, MSIL2 and MSIL4 are redundantly required for secondary cell wall development in Arabidopsis. The plant phenotypes could be complemented by the wild-type version of either protein, but not by a MSIL4 version that carries mutations in the conserved RNA-binding domains, and the authors localized MSIL2 & 4 to stress granules, implicating the RNA-binding function of MSIL4 in the cell wall phenotype. Upon closer inspection, the secondary cell wall phenotypes included changes in vasculature morphology, and minor changes to lignin and hemicellulose (glucuronoxylan). While there were no changes to likely cell wall target genes in the transcriptome of msil2msil4 plants, proteomics experiments found glucuronoxylan biosynthesis components were upregulated in the mutants, and they detected an increase in substituted xylan via several methods. Finally, they documented MSIL4 binding to RNA encoding one of these targets, suggesting that MSIL2 and MSIL4 act to post-transcriptionally regulate glucuronoxylan modification. Altogether, this is a new mechanism by which cell wall composition could be regulated.

Overall, the manuscript is well-written, the data are generally high-quality, and the authors typically use several independent methods to support each claim. However, several important questions remain unanswered by this work in its current state and the model presented in Figure 7 is quite speculative. For example, the link between the striking plant phenotype and GXM misregulation is unclear since GXM overexpression doesn't alter plant phenotypes or lignin content (Yuan et al 2014 Plant Science), so misregulation of GXMs in msil2msil4 mutants clearly is not the whole story. It also remains to be determined why one particular secondary cell wall synthesis enzyme is regulated likely post-transcriptionally, while so much of the pathway is regulated at the transcriptional level. There are likely other targets for MSIL2- and MSIL4-mediated regulation since it seems that MSIL2 and MSIL4 are expressed in tissues that are not synthesizing secondary cell walls.

Reviewer #2 (Public Review):

This work explored the biological functions of a small family of RNA-binding proteins that was previously studied in animals, but was uncharacterized in plants. Combinatorial T-DNA insertional mutants disrupting the expression of the four Mushashi-like (MSIL) genes in Arabidopsis revealed that only the msil2 msil4 double mutant visibly alters plant development. The msil2/4 plants produced stems that could not stand upright. Transgene complementation, site-directed mutagenesis of MSIL4 conserved RNA-binding motifs, and in vitro RNA binding assays support the conclusion that the loss of MSIL2 and MISL4 function is responsible for the observed morphological defects. MSIL2/4 interact with proteins associated with mRNA 3'UTR binding and translational regulation.

The authors present compelling biochemical evidence that Mushashi-like2 (MSIL2) and MSIL4 jointly regulate secondary cell wall biosynthesis in the Arabidopsis stem. Quantitative analyses of proteins and transcripts in msil2/4 stems uncovered transcriptional upregulation of several xylan-related enzymes (despite WT-like RNA levels). Consistent with MALDI-TOF data for released xylan oligosaccharides, the authors propose a model in which MSIL2/4 negatively regulate the translation of GXM (glucuronoxylan methyltransferase), a presumed rate-limiting step. The molecular links between overmethylated xylans and the observed stem defects (which include subtle reductions in lignin and increases beta-glucan polymer distribution) warrants further investigation in future studies. Similarly, as the authors point out, it is intriguing that the loss of the broadly expressed MSIL2/4 genes only significantly affects specific cell types in the stem.

Reviewer #3 (Public Review):

The manuscript by Kairouani et al. investigates the function of a small family of plant RNA binding proteins with similarity to the well-studied Musashi protein in animals, and, therefore, called MUSASHI-LIKE1-4 (MSL1-4). Studies on the biological importance of post-transcriptional control of gene expression via RNA-binding proteins in plants are not numerous, and advances in this important field are much needed. The thorough work presented in this manuscript is such an advance.

The central observations of the paper are<br /> - Knockout of any MSL gene alone does not produce a phenotype.<br /> It is of note that basic characterization of knockout mutations is really well done - for example, the authors have taken care to raise specific antibodies to each of the MSL proteins and use them to demonstrate that each of the T-DNA insertion mutants used actually does knock out protein production from the corresponding gene.

- Knockout of MSL2/4 (but no other double mutant) produces a clear leaf phenotype, and a remarkable stem phenotype in which the mutants collapse as they are unable to support upright growth

- The phenotypes of knockout mutants persist in point mutants defective in RNA-binding, indicating that RNA-binding is required for biological activity. Consistent with this, and associate physically with other RNA-binding proteins and translation factors.

- MSL proteins are cytoplasmic

- The msl2/4 mutants present multiple defects in secondary cell wall composition and structure, probably explaining their inability to grow upright. I did not examine the cell wall analyses in detail as I am no specialist in this field.

- Msl2/4 mutants show transcriptomic changes with at large two big categories of differentially expressed genes compared to wild type.<br /> (1) Genes related to cell wall metabolism<br /> (2) Genes associated with defense against herbivores and pathogens

- Two of the mRNAs encoding cell wall factors with significant upregulation in msl2/4 mutants compared to wild type also associate physically with MSL4 as judged by RNA-immunoprecipitation-RT-PCR assays, and this physical association is abrogated in the RNA-binding deficient MSL4 mutant.

Altogether, the study shows clear biological relevance of the MSL family of RNA-binding proteins and provides good arguments that the underlying mechanism is control of mRNAs encoding enzymes involved in secondary cell wall metabolism (although concluding on translational control in the abstract is perhaps saying too much - post-transcriptional control will do given the evidence presented). One observation reported in the study makes it vulnerable to alternative interpretation, however, and I think this should be explicitly treated in the discussion:

The fact that immune responses are switched on in msl2/4 mutants could also mean that MSL2/4 have biological functions unrelated to cell wall metabolism in wild type plants, and that cell wall defects arise solely as an indirect effect of immune activation (that is known to involve changes in expression of many cell wall-modifying enzymes and components such as pectin methylesterases, xyloglucan endotransglycosylases, arabinogalactan proteins etc. Indeed, the literature is rich in examples of gene functions that have been misinterpreted on the basis of knockout studies because constitutive defense activation mediated by immune receptors was not taken into account (see for example Lolle et al., 2017, Cell Host & Microbe 21, 518-529).

With the evidence presented here, I am actually close to being convinced that the primary defect of msl2/msl4 mutants is directly related to altered cell wall metabolism, and that defense responses arise as a consequence of that, not the other way round. But I do not think that the reverse scenario can be formally excluded with the evidence at hand, and a discussion listing arguments in favor of the direct effect proposed here would be appropriate. Elements that the authors could consider to include would be the isolation of a cellulose synthase mutant as a constitutive expressor of jasmonic acid responses (cev1) as a clear example that a primary defect in cell wall metabolism can produce defense activation as secondary effect. The interaction of MSL4 with GXM1/3 mRNAs is also helpful to argue for a direct effect, and it would strengthen the argument if more examples of this kind could be included.

Reviewer #1 (Public Review):

Summary:<br /> Authors propose mathematical methods for inferring evolutionary parameters of interest from bulk/single cell sequencing data in healthy tissue and hematopoiesis. In general, the introduction is well-written and adequately references the relevant and important previous literature and findings in this field (e.g. the power laws for well-mixed exponentially growing populations). The authors consider 3 phases of human development: early development, growth and maintenance, and mature phase. In particular, time-dependent mutation rates in Figure 2d is an intriguing and strong result, and the process underlying Figures 3 and 4 are generally well-explained and convincing.

Notes & suggestions:<br /> 1. The explanation of Figure 2 in Lines 101 - 111 should be expanded for clarity. First, is Figure 2a derived from stochastic simulation (line 101 suggests) or some theoretical analysis? Second, the gradual transition from f-2 to f-1 is appreciated, but the shape of the intermediates is not addressed in detail. The power laws are straight lines, and the simulations provide curved lines -- please expand in what range (low or high frequency variants) the power law approximations apply.

Additionally, I do not understand the claim in line 108, that the transition is fast for low frequency variants, as the low frequency (on the left of the graph) lines are all close together, whereas the high frequency lines are far apart.

It would be helpful to reiterate in this paragraph that these power laws are derived based on exponentially growing populations and are expected to break down under homeostatic conditions.

2. The sample vs population (blue vs orange) in Figure 3 is under-explained. How is it that the mutational burden and inferred mutation rate in A and B roughly match, but the VAF distributions in C are so different? How was the sampled set chosen? Perhaps this is an unimportant distinction based on the particular sample set, but the divergence of the two in C may serve as a distraction, here.

3. The comparison of results herein to claims by Mitchell (ref. 12) are quite important results within the paper. I appreciate the note in the final paragraph of the discussion, and I suggest adding a sentence referencing the result noted in line 248-249 to the abstract, as well.

Reviewer #2 (Public Review):

Summary: The authors provide a nice summary on the possibility to study genetic heterogeneity and how to measure the dynamics of stem cells. By combining single cell and bulk sequencing analyses, they aim to use a stochastic process and inform on different aspects of genetic heterogeneity.

Strengths: Well designed study and strong methods

Weaknesses: Minor<br /> Further clarification to Figure 3 legend would be good to explain the 'no association' of number of samples and mutational burden estimate as per line 180-182 p.8

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes a study in which younger, normal-hearing adults listened to two concurrent speech streams (audio-visual presentation) while magnetoencephalography (MEG) was recorded. They were asked to attend to one and ignore the other speech stream. Speech materials were processed using natural language processing (NLP) model approaches to categorize speech chunks of about 3.5 s duration as being of either high or low probability based on topic modeling. MEG results show that decoding performance (reconstruction of speech) was high for the high-probability speech chunks under both the attend and ignore conditions, suggesting that semantic information in the unattended speech was still processed. The conclusions of this paper are mostly well supported by the data.

Strengths:<br /> 1) The authors use sophisticated analyses using natural language processing models - that are beyond the state-of-the-art - to make inferences about semantic speech processing in the brain. The analytic methods are well described, enabling readers to possibly implement the approach for their own analyses.

2) The study shows that highly salient semantic information of speech is processed in the brain even when a listener attends to something different. The work has implications for selective attention models that are concerned with how individuals process speech.

Weaknesses:<br /> 1) The title of the manuscript may be a bit misleading: "Get the gist of the story: Neural map of topic keywords in multi-speaker environment". The study was not about the gist of the story but about the gist of speech chunks of about 3.5 s. The study shows important evidence that neural activity is sensitive to the gist of short speech segments, even in unattended speech, but the gist of the story is a yet more abstract level that cannot be reduced to the gist of short speech chunks.

2) The calculations of t-values for the spatial maps showing significant clusters were non-standard, which makes interpreting the magnitude of the t-values difficult. Better motivation for why the specific approach was chosen would be important, or perhaps replacing it with a more standard approach. It further appears that the region of interest analyses were carried out without multiple comparison corrections, possibly suggesting a note of caution about some of the source-localization results.

Reviewer #2 (Public Review):

Summary:

This study by Park and Gross investigates the spatiotemporal neural representation of semantic information most pertinent to the gist of speech materials presented to subjects as magnetoencephalography was recorded. Participants heard and saw naturalistic continuous speech recordings (with the auditory component presented to one ear), while also presented with distractor auditory speech (presented in the other ear). Participants were instructed to attend to the speech stream that matched the video of the speaker. The stimuli were semantically parsed to create short segments to which topic probabilities were assigned. These segments were then organized into high and low topic probabilities for each of the four topics (determined using Latent Dirichlet Allocation (LDA) analysis). The results suggest clear differences in the fidelity of neural encoding of the speech envelope during high-topic probability segments, which is interpreted as the brain representing key information for a story whether that information is explicitly attended to.

Strengths:<br /> The use of LDA analysis makes possible the quantification of whether a particular speech segment is relevant to a particular topic and enables analysis based on this high-temporal resolution of semantic salience. The authors show clear differences between attended and unattended speech conditions, as well as, surprisingly, differences between semantically salient unattended speech and attended, less semantically relevant speech.

Weaknesses:<br /> Though the effect sizes of the results of this study show clear differences between stimulus conditions, clarification of the experimental methods is needed to appreciate their interpretation. Broadly, I would suggest adding a clearer description of the task during data collection, even though it has been published elsewhere.

One key piece of information that is missing is how semantically relevant topics are assigned, so that salient semantic information can be compared between attended and unattended stories. It's unclear to me how results are combined across topics and stories. If a particular speech segment is assigned 4 topic probabilities, that segment has both a high probability of belonging to one topic and a low probability of belonging to another. I understand how this can be used to create the experimental conditions for a single topic, but how are results combined across topics?

I think some discussion of using the encoding and decoding of the speech envelope as a measure of what is semantically relevant is warranted. The fidelity with which the speech envelop is represented has been used as a proxy for how well that speech is attended to, but it is unclear to me whether we should expect to see high-fidelity encoding of speech envelop outside of the primary and secondary auditory regions of the brain, or how it relates to the semantic information contained in the speech signal.

Additionally, I wonder if it might be more informative to decode the topic labels themselves directly by building a model to predict the topic probabilities from the neural data? This might give a more direct measure of where and when semantically relevant information is represented.

Reviewer #1 (Public Review):

This manuscript by Leibinger et al describes their results from testing an interesting hypothesis that microtubule detyrosination inhibits axon regeneration and its inhibitor parthenolide could facilitate axon regeneration and perhaps functional recovery. Overall, the results from in vitro studies are largely well performed. However, the in vivo data are less convincing.

Interpretation of the findings in this study are limited by several gaps:<br /> 1. It is unclear whether microtubule detyrosination a primary effect of hIL-6 and PTEN deletion or secondary to the increased axon growth?

2. Is there any direct evidence for Akt and/or JAK/Stat3 to promote microtubule detyrosination?

3. What is the impact of parthenolide on cell soma of neurons and other cell types?

4. Direct evidence that parthenolide augments PTEN deletion in optic nerve or spinal cord is not provided.

5. Serotonergic neurotoxin DHT ablates both regenerating and non-regenerating serotonergic axons, which makes spinal cord findings it difficult to interpret.

6. DMAPT was given by i.p. injection. What happens to microtubule detyrosination in other cells within and outside of CNS?

Reviewer #2 (Public Review):

In the current study, Fischer and colleagues extensively examined the role of parthenolide in inhibiting microtubule detyrosination and making the mechanistic link for the compound to facilitate the role of IL6 and PTEN/KO in promoting neurite outgrowth and axon regeneration. The in vitro and mechanistic work laid the foundation for the authors to reach several key predictions that such detyrosination can be applied for in vivo applications. Thus the authors extended the work to optic nerve regeneration and spinal cord recovery. The in vivo compound that the authors utilized is DMAPT, which plays a synergistic role with existing pro-regeneration therapies, such as Il6 treatment.

The major strength of the work is the first half of the mechanistic inquiries, where the authors combined cell biology and biochemistry approaches to dissect the mechanistic link from parthenolide to microtube dynamics. The shortcoming is that the in vivo data is limited, and the effects might be considered mild, especially by benchmarking with other established and effective strategies.

The work is solid and prepares a basis for others to test the role of DMAPT in other settings, especially in the setting of other effective pro-regenerative approaches. With the goal of comprehensive and functional recovery in vivo, the impact of the work and the utilities of the methods remain to be tested broadly in other models in vivo.

Reviewer #3 (Public Review):

The primary goal of this paper is to examine microtubule detyrosination as a potential therapeutic target for axon regeneration. Using dimethylamino-parthenolide (DMAPT), this study extensively examines mechanistic links between microtubule detyrosination, interleukin-6 (IL-6), and PTEN in neurite outgrowth in retinal ganglion cells in vitro. These findings provide convincing evidence that parthenolide has a synergistic effect on IL-6- and PTEN-related mechanisms of neurite outgrowth in vitro. The potential efficacy of systemic DMAPT treatment to promote axon regeneration in mouse models of optic nerve crush and spinal cord injury was also examined.

Strengths<br /> 1. The examination of synergistic activities between parthenolide, hyperIL-6, and PTEN knockout is leveraged not only for potential therapeutic value, but also to validate and delineate mechanism of action.<br /> 2. The in vitro studies, including primary human retinal ganglion cells, utilize a multi-level approach to dissect the mechanistic link from parthenolide to microtubule dynamics.<br /> 3. The studies provide a basis for others to test the role of DMAPT in other settings, particularly in the context of other effective pro-regenerative approaches.

Weaknesses<br /> 1. In vivo studies are limited to select outcomes of recovery and do not validate or address mechanism of action in vivo.

Reviewer #1 (Public Review):

In the manuscript entitled "A theory of hippocampal theta correlations", the authors propose a new mechanism for phase precession and theta-time scale generation, as well as their interpretation in terms of navigation and neural coding. The authors propose the existence of extrinsic and intrinsic sequences during exploration, which may have complementary functions. These two types of sequences depend on external input and network interactions, but differ on the extent to which they depend on movement direction. Moreover, the authors propose a novel interpretation for intrinsic sequences, namely to signal a landmark cue that is independent of direction of traversal. Finally, a readout neuron can be trained to distinguish extrinsic from intrinsic sequences.

The study puts forward novel computational ideas related to neural coding, partly based on previous work from the authors, including published (Leibold, 2020, Yiu et al., 2022) and unpublished (Ahmedi et al., 2022. bioRxiv) work. The manuscript will contribute to the understanding of the mechanisms behind phase precession, as well as to how we interpret hippocampal temporal coding for navigation and memory.

Reviewer #2 (Public Review):

Place cells fire sequentially during hippocampal theta oscillations, forming a spatial representation of behavioral experiences in a temporally-compressed manner. The firing sequences during theta cycles are widely considered as essential assemblies for learning, memory, and planning. Many theoretical studies have investigated the mechanism of hippocampal theta firing sequences; however, they are either entirely extrinsic or intrinsic. In other words, they attribute the theta sequences to external sensorimotor drives or focus exclusively on the inherent firing patterns facilitated by the recurrent network architectures. Both types of theories are inadequate for explaining the complexity of the phenomena, particularly considering the observations in a previous paper by the authors: theta sequences independent of animal movement trajectories may occur simultaneously with sensorimotor inputs (Yiu et al., 2022).

In this manuscript, the authors concentrate on the CA3 area of the hippocampus and develop a model that accounts for both mechanisms. Specifically, the model generates extrinsic sequences through the short-term facilitation of CA3 cell activities, and intrinsic sequences via recurrent projections from the dentate gyrus. The model demonstrates how the phase precession of place cells in theta sequences is modulated by running direction and the recurrent DG-CA3 network architecture. To evaluate the extent to which firing sequences are induced by sensorimotor inputs and recurrent network architecture, the authors use the Pearson correlation coefficient to measure the "intrinsicity" and "extrinsicity" of spike pairs in their simulations.

I find this research topic to be both important and interesting, and I appreciate the clarity of the paper. The idea of combining intrinsic and extrinsic mechanisms for theta sequences is novel, and the model effectively incorporates two crucial phenomena: phase precession and directionality of theta sequences. I particularly commend the authors' efforts to integrate previous theories into their model and conduct a systematic comparison. This is exactly what our community needs: not only the development of new models, but also understanding the critical relationships between different models.

Reviewer #1 (Public Review):

The authors present a study of visuo-motor coupling primarily using wide-field calcium imaging to measure activity across the dorsal visual cortex. They used different mouse lines or systemically injected viral vectors to allow imaging of calcium activity from specific cell-types with a particular focus on a mouse-line that expresses GCaMP in layer 5 IT (intratelencephalic) neurons. They examined the question of how the neural response to predictable visual input, as a consequence of self-motion, differed from responses to unpredictable input. They identify layer 5 IT cells as having a different response pattern to other cell-types/layers in that they show differences in their response to closed-loop (i.e. predictable) vs open-loop (i.e. unpredictable) stimulation whereas other cell-types showed similar activity patterns between these two conditions. Surprisingly, they find that presentation of a visual grating actually decreases the responses of L5 IT cells in V1. They interpret their results within a predictive coding framework that the last author has previously proposed. The response pattern of the L5 IT cells leads them to propose that these cells may act as 'internal representation' neurons that carry a representation of the brain's model of its environment. Though this is rather speculative. They subsequently examine the responses of these cells to anti-psychotic drugs (e.g. clozapine) with the reasoning that a leading theory of schizophrenia is a disturbance of the brain's internal model and/or a failure to correctly predict the sensory consequences of self-movement. They find that anti-psychotic drugs strongly enhance responses of L5 IT cells to locomotion while having little effect on other cell-types. Finally, they suggest that anti-psychotics reduce long-range correlations between (predominantly) L5 cells and reduce the propagation of prediction errors to higher visual areas and suggest this maybe a mechanism by which these drugs reduce hallucinations/psychosis.

This is a large study containing a screening of many mouse-lines/expression profiles using wide-field calcium imaging. Wide-field imaging has its caveats, including a broad point-spread function of the signal and susceptibility to hemodynamic artifacts, which can make interpretation of results difficult. The authors acknowledge these problems and directly address the hemodynamic occlusion problem. It was reassuring to see supplementary 2-photon imaging of soma to complement this data-set, even though this is rather briefly described in the paper. Overall the paper's strengths are its identification of a very different response profile in the L5 IT cells compared other layers/cell-types which suggests an important role for these cells in handling integration of self-motion generated sensory predictions with sensory input. The interpretation of the responses to anti-psychotic drugs is more speculative but the result appears robust and provides an interesting basis for further studies of this effect with more specific recording techniques and possibly behavioral measures.

Reviewer #2 (Public Review):

Summary:<br /> This work investigates the effects of various antipsychotic drugs on cortical responses during visuomotor integration. Using wide-field calcium imaging in a virtual reality setup, the researchers compare neuronal responses to self-generated movement during locomotion-congruent (closed loop) or locomotion-incongruent (open loop) visual stimulation. Moreover, they probe responses to unexpected visual events (halt of visual flow, sudden-onset drifting grating). The researchers find that, in contrast to a variety of excitatory and inhibitory cell types, genetically defined layer 5 excitatory neurons distinguish between the closed and the open loop condition and exhibit activity patterns in visual cortex in response to unexpected events, consistent with unsigned prediction error coding. Motivated by the idea that prediction error coding is aberrant in psychosis, the authors then inject the antipsychotic drug clozapine, and observe that this intervention specifically affects closed loop responses of layer 5 excitatory neurons, blunting the distinction between the open and closed loop conditions. Clozapine also leads to a decrease in long-range correlations between L5 activity in different brain regions, and similar effects are observed for two other antipsychotics, aripripazole and haloperidol, but not for saline or the stimulant amphetamine. The authors suggest that altered prediction error coding in layer 5 excitatory neurons due to reduced long-range correlations in L5 neurons might be a major effect of antipsychotic drugs and speculate that this might serve as a new biomarker for drug development.

Strengths:<br /> - Relevant and interesting research question:<br /> The distinction between expected and unexpected stimuli is blunted in psychosis but the neural mechanisms remain unclear. Therefore, it is critical to understand whether and how antipsychotic drugs used to treat psychosis affect cortical responses to expected and unexpected stimuli. This study provides important insights into this question by identifying a specific cortical cell type and long-range interactions as potential targets. The authors identify layer 5 excitatory neurons as a site where functional effects of antipsychotic drugs manifest. This is particularly interesting as these deep layer neurons have been proposed to play a crucial role in computing the integration of predictions, which is thought to be disrupted in psychosis. This work therefore has the potential to guide future investigations on psychosis and predictive coding towards these layer 5 neurons, and ultimately improve our understanding of the neural basis of psychotic symptoms.

- Broad investigation of different cell types and cortical regions:<br /> One of the major strengths of this study is quasi-systematic approach towards cell types and cortical regions. By analysing a wide range of genetically defined excitatory and inhibitory cell types, the authors were able to identify layer 5 excitatory neurons as exhibiting the strongest responses to unexpected vs. expected stimuli and being the most affected by antipsychotic drugs. Hence, this quasi-systematic approach provides valuable insights into the functional effects of antipsychotic drugs on the brain, and can guide future investigations towards the mechanisms by which these medications affect cortical neurons.

- Bridging theory with experiments<br /> Another strength of this study is its theoretical framework, which is grounded in the predictive coding theory. The authors use this theory as a guiding principle to motivate their experimental approach connecting visual responses in different layers with psychosis and antipsychotic drugs. This integration of theory and experimentation is a powerful approach to tie together the various findings the authors present and to contribute to the development of a coherent model of how the brain processes visual information both in health and in disease.

Weaknesses:<br /> - Unclear relevance for psychosis research<br /> From the study, it remains unclear whether the findings might indeed be able to normalise altered predictive coding in psychosis. Psychosis is characterised by a blunted distinction between predicted and unpredicted stimuli. The main results of this study indicate that antipsychotic drugs further blunt the distinction between predicted and unpredicted stimuli, which would suggest that antipsychotic drugs would deteriorate rather than ameliorate the predictive coding deficit found in psychosis. However, these findings were based on observations in wild-type mice at baseline. Given that antipsychotics are thought to have little effects in health but potent antipsychotic effects in psychosis, it seems possible that the presented results might be different in a condition modelling a psychotic state, for example after a dopamine-agonistic or a NMDA-antagonistic challenge. Therefore, future work in models of psychotic states is needed to further investigate the translational relevance of these findings.

- Incomplete testing of predictive coding interpretation<br /> While the investigation of neuronal responses to different visual flow stimuli is interesting, it remains open whether these responses indeed reflect internal representations in the framework of predictive coding. While the responses are consistent with internal representation as defined by the researchers, i.e., unsigned prediction error signals, an alternative interpretation might be that responses simply reflect sensory bottom-up signals that are more related to some low-level stimulus characteristics than to prediction errors. Moreover, this interpretational uncertainty is compounded by the fact that the used experimental paradigms were not suited to test whether behaviour is impacted as a function of the visual stimulation which makes it difficult to assess what the internal representation of the animal actually was. For these reasons, the observed effects might reflect simple bottom-up sensory processing alterations and not necessarily have any functional consequences. While this potential alternative explanation does not detract from the value of the study, future work would be needed to explain the effect of antipsychotic drugs on responses to visual flow. For example, experimental designs that systematically vary the predictive strength of coupled events or that include a behavioural readout might be more suited to draw from conclusions about whether antipsychotic drugs indeed alter internal representations.

Conclusion:<br /> Overall, the results support the idea that antipsychotic drugs affect neural responses to predicted and unpredicted stimuli in deep layers of cortex. Although some future work is required to establish whether this observation can indeed be explained by a drug-specific effect on predictive coding, the study provides important insights into the neural underpinnings of visual processing and antipsychotic drugs, which is expected to guide future investigations on the predictive coding hypothesis of psychosis. This will be of broad interest to neuroscientists working on predictive coding in health and disease.

Reviewer #3 (Public Review):

The study examines how different cell types in various regions of the mouse dorsal cortex respond to visuomotor integration and how antipsychotic drugs impacts these responses. Specifically, in contrast to most cell types, the authors found that activity in Layer 5 intratelencephalic neurons (Tlx3+) and Layer 6 neurons (Ntsr1+) differentiated between open loop and closed loop visuomotor conditions. Focussing on Layer 5 neurons, they found that the activity of these neurons also differentiated between negative and positive prediction errors during visuomotor integration. The authors further demonstrated that the antipsychotic drugs reduced the correlation of Layer 5 neuronal activity across regions of the cortex, and impaired the propagation of visuomotor mismatch responses (specifically, negative prediction errors) across Layer 5 neurons of the cortex, suggesting a decoupling of long-range cortical interactions.<br /> The data when taken as a whole demonstrate that visuomotor integration in deeper cortical layers is different than in superficial layers and is more susceptible to disruption by antipsychotics. Whilst it is already known that deep layers integrate information differently from superficial layers, this study provides more specific insight into these differences. Moreover, this study provides a first step into understanding the potential mechanism by which antipsychotics may exert their effect.<br /> Whilst the paper has several strengths, the robustness of its conclusions is limited by weaknesses in statistical analyses. A summary of the paper's strengths and weaknesses follow.

Strengths:

The authors perform an extensive investigation of how different cortical cell types (including Layer 2/3, 4 , 5, and 6 excitatory neurons, as well as PV, VIP, and SST inhibitory interneurons) in different cortical areas (including primary and secondary visual areas as well as motor and premotor areas), respond to visuomotor integration. This investigation provides strong support to the idea that deep layer neurons are indeed unique in their computational properties. This large data set will be of considerable interest to neuroscientists interested in cortical processing.<br /> The authors also provide several lines of evidence that visuomotor information is differentially integrated in deep vs. superficial layers. They show that this is true across experimental paradigms of visuomotor processing (open loop, closed loop, mismatch, drifting grating conditions) and experimental manipulations, with the demonstration that Layer 5 visuomotor integration is more sensitive to disruption by the antipsychotic drug clozapine, compared with cortex as a whole.

The study further uses multiple drugs (clozapine, aripiprazole and haloperidol) to bolster its conclusion that antipsychotic drugs disrupt correlated cortical activity in Layer 5 neurons, and further demonstrates that this disruption is specific to antipsychotics, as the psychostimulant amphetamine shows no such effect.

In widefield calcium imaging experiments, the authors effectively control for the impact of hemodynamic occlusions in their results, and try to minimize this impact using a crystal skull preparation, which performs better than traditional glass windows. Moreover, they examine key findings in widefield calcium imaging experiments with two-photon imaging.

Weaknesses:

A critical weakness of the paper is its statistical analysis and data representations. The study does not use mice as its independent unit for statistical comparisons but rather relies on other definitions (see authors' Tabe S1), without appropriate justification, which results in an inflation of sample sizes. For example, in Figure 2, the independent statistical unit is defined as sessions instead of mice, and in Figures 6 and 7 its pairs of cortical regions of interest. This greatly inflates N by at least 1-2 orders of magnitude compared to using N = number of mice. With such inflated sample sizes, it becomes more likely to find spurious differences between groups as significant.

It should be noted, however, that the authors have redone some analyses in their revision, specifically for Figure 1L, in which mice are used as independent units (shown in Figure S4) without any change in conclusion. However, this is not done for all other problematic figures in the manuscript.

Furthermore - and related to the previous comment - trace averages and SEMs across the figures of the manuscript come from hundreds to thousands of data points (e.g. locomotion onsets or cells) repeatedly measured from only a handful of mice. This can be visually misleading for the reader (even if statistics are not being formally performed on these traces) as it artificially reduces the size of the SEM masking the true variability (and size) of the effects portrayed in the paper. Again, this practice is only justified if the data (e.g. locomotion onsets) within a mouse is actually statistically independent, which the authors do not test for or justify.

It should be noted that the authors do show some trace averages and SEMs for a some of their data (Figure S2), in which N = individual mice, without any change in conclusion. However, this is not done for all other problematic figures in the manuscript.

The above statistical problems are apparent throughout the manuscript. The more disciplined approach would be to average the data within a mouse, and then use the mouse as an independent unit for statistical comparison and/or for the purposes of presenting means and SEMs for aggregate data. Alternatively, the authors should provide clear justification in the manuscript for opting for other definitions of N.

Finally, it is important to note that whilst the study demonstrates that antipsychotics may selectively impact visuomotor integration in L5 neurons, it does not show that this effect is necessary or sufficient for the action of antipsychotics; though this is likely beyond the scope of the study it is something for readers to keep in mind.

Reviewer #1 (Public Review):

In this manuscript, Bilgic et al aim to identify the progenitor types (and their specific progeny) that underlie the expanded nature of gyrencephalic brains. To do this, they take a comparative scRNAseq (single cell transcriptomics) approach between neurodevelopment of the gyrencephalic ferret, and previously published primary human brain and organoid data.

They first improve gene annotations of the ferret genome and then collect a time series of scRNAseq data of 6 stages of the developing ferret brain spanning both embryonic and post-natal development. Among the various cell types they identify are a small proportion of truncated radial glial cells (tRGs), a population known to be enriched in humans and macaques that emerges late in neurogenesis as the RGC scaffold splits into an oRGC that contact the pial surface and a tRG that contacts the ventricular surface. They find that the tRGs consist of three distinct subpopulations two of which are committed to ependymal and astroglial fates.

By integrating these data with publicly available data of developing human brains and human brain organoids they make some important observations. Human and ferret tRGs have very similar transcriptional states, suggesting that the human tRGs too give rise to ependymal and astroglial fates. They also find that the current culture conditions of human brain organoids seem to lack tRGs, something that will need to be addressed if they are to be used to study tRGs. While the primary human data set did contain tRGs, the stage or the region sampled were likely not appropriate, and therefore, the number of cells they could retrieve was low.

The authors have spent considerable efforts in improving gene modeling of the ferret genome, which will be important for the field. They've generated valuable time series data for the developing ferret brain, and have proposed the lineal progeny for the tRGs in the human brain. Whether tRGs actually do give rise to the ependymal and astrogial fates needs to be validated in future studies.

Reviewer #2 (Public Review):

Bilgic et al first explored cellular diversity in the developing cerebral cortex of ferret, honing in on progenitor cell diversity by employing FACS sorting of HES5-positive cells. They have generated a novel single cell transcriptomic dataset capturing the diversity of cells in the developing ferret cerebral cortex, including diverse radial glial and excitatory neuron populations. Unexpectedly, this analysis revealed the presence of CRYAB-positive truncated radial glia previously described only in humans. Using bioinformatic analyses, the investigators proposed that truncated radial glia produce ependymal cells, astrocytes, and to a lesser degree, neurons. Of particular interest to the field, they identify enriched expression of FOXJ1 in late truncated radial glia strongly indicating that towards the end of neurogenesis, these cells likely give rise to ependymal cells. This study represents a major advancement in the field of cortical development and a valuable dataset for future studies of ferret cortical development.

Reviewer #2 (Public Review):

Summary:<br /> In this article, the authors provide a method of evaluating the safety of orthopedic implants in relation to radiofrequency-induced heating issues. The authors provide an open-source computational heterogeneous human model and explain computational techniques in a finite element method solver to predict the RF-induced temperature increase due to an orthopedic implant while being exposed to MRI RF fields at 1.5 T.

Strengths:<br /> The open-access computational human model along with their semiautomatic algorithm to position the implant can help realistically model the implant RF exposure in patients avoiding over- or under-estimation of RF heating measured using rectangular box phantoms such as ASTM phantom. Additionally, using numerical simulation to predict radiofrequency-induced heating will be much easier compared to the experimental measurements in an MRI scanner, especially when the scanner availability is limited.

Weaknesses:<br /> The proposed method only used radiofrequency (RF) field exposure to evaluate the heating around the implant. However, in the case of bulky implants, the rapidly changing gradient field can also produce significant heating due to large eddy currents. So the gradient-induced heating still remains an issue to be evaluated to decide on the safety of the patient. Moreover, the method is limited to a single human model and might not be representative of patients with different age, sex, and body weights. Additionally, the authors compare the temperature rise predicted by their method to an earlier study. However, there is no information about how they controlled the input power in their simulation testbed compared to the earlier study in showing validation of the method.

Reviewer #1 (Public Review):

Summary:<br /> In this work, the authors are trying to satisfy a real need in MR safety, when concerns can arise about the thermal increase due to metallic materials in patients carrying orthopedic implants. The "MR conditional" labeling of the implant obtained by ASTM in-vitro tests may help to plan the MR scan, but it is normally limited to a single specific MR sequence and a B0 value, and it is not always available. The adoption of an in-silico simulation testbed overcomes this limitation, providing a fast and reliable prediction of temperature increase from RF, in real-life scan conditions on human-like digital models. The FDA is pushing this approach.

Strengths:<br /> The presented in-silico testbed looks valuable and validated. It is based on the widely available Visible Human Project (VHP) datasets, and the testbed is available online. The approval of the testbed by the FDA as a medical device development tool (MDDT) is a good premise for the large-scale adoption of this kind of solution.

Weaknesses:<br /> There are a couple of limitations in the study that must be clearly highlighted to the readers.

While the RF-related heating is very well modeled, the gradients-related heating is out of the scope of this paper and not considered. Readers must be warned that RF causes only a part of the heating, and literature is reporting cases where also gradient switching can contribute, as correctly mentioned in this work. A cautious attitude should consider this as a significant limitation of the study.

Moreover, the way the implant is embedded in the VHP model is shortly documented in the materials and methods and mostly focuses on implant registration on bone tissue. It is not clear how to manage the empty space and the soft tissue stretching/reshaping generated by the simulated surgery (for example, by the cut of the femoral head in total hip arthroplasty). It is reported by literature that the level of accuracy in the simulated surgery can impact in some cases (RF vs. gradients heating, massive vs. thin or elongated implants) on temperature predictions.

Reviewer #2 (Public Review):

This paper illustrates that PSCs can model myogenesis in vitro by mimicking the in vivo development of the somite and dermomyotome. The advantages of this 3D system include (1) better structural distinctions, (2) the persistence of progenitors, and (3) the spatial distribution (e.g. migration, confinement) of progenitors. The finding is important with the implication in disease modeling. Indeed the authors tried DMD model although it suffered the lack of deeper characterization.

The differentiation protocol is based on a current understanding of myogenesis and is compelling. They characterized the organoids in depth (e.g. many time points and immunofluorescence). The evidence is solid.

Reviewer #1 (Public Review):

The authors aimed to establish a cell culture system to investigate muscle tissue development and homeostasis. They successfully developed a complex 3D cell model and conducted a comprehensive molecular and functional characterization. This approach represents a critical initial step towards using human cells, rather than animals, to study muscular disorders in vitro. Although the current protocol is time-consuming and the fetal cell model may not be mature enough to study adult-onset diseases, it nonetheless provides a valuable foundation for future disease modeling studies using isogenic iPSC lines or patient-derived cells with specific mutations. The manuscript does not explore whether or how this stem cell model can advance our understanding of muscular diseases, which would be an exciting avenue for future research. Overall, the detailed protocol presented in this paper will be useful for informing future studies and provide a valuable resource to the stem cells community. Future work could focus on disease modeling using isogenic iPSC lines or patient-derived cells.

Reviewer #1 (Public Review):

The authors investigate the roles of ACOT12/8 in the production of acetate by the liver. They observe that acetate concentration parallels ketone concentrations during fasting and T1DM. They show that acetate is produced from fatty acids in hepatocytes, but though described as a novel "ketone body", this acetate is not a product of ketogenesis or acetoacetate. They also provide serum acetate data from human subjects who were classified as either "healthy" or "diabetic,". These subjects are noted as T2DM patients, but there is no other characterization or description, making it difficult to ascertain the context in which they were studied or their relevance to the mouse studies. Although the function of ACOT12/8 is reported in the literature, they are not widely studied, and there also remains surprising uncertainties regarding the mechanism of acetate production by the liver. In this regard, the manuscript provides some important insight. The authors use ShACOT12/8 and ACOT12/8 knockout mice to demonstrate that these acetyl-CoA hydrolases are largely necessary for acetate production. Using a 3H-palmitate assay, the authors then find that loss of these ACOTs inhibit fatty acid oxidation and propose that the mechanism involves scavenging CoA, analogous to the canonical role of ketogenesis. The idea is plausible but not proven. A related finding is that loss of these ACOTs inhibit ketogenesis, which the authors attribute to the loss of function of HMGC2S, partially through acetylation. These mechanisms suffer some limitations based on the cytosolic and mitochondrial compartmentation of the two processes, but the observations appear sound. Interestingly, the loss of the ACOTs have a more profound effect on lowering ketones than acetate, which may have parallel effects but they are not investigated. Finally, the authors try to demonstrate that hepatic ACOT-mediated acetate production is necessary for normal motor function in STZ treated mice, ostensibly as compensation for impaired glucose utilization by the CNS. Injections of 13C acetate and 13C enrichment in downstream metabolites of brain are used to support the importance of acetate metabolism, but the experiment was not performed in loss of function models. In addition, the resulting 13C enrichment data is reported generically as "relative intensity" without further elaboration on how this data was generated and should not be taken at face value by the reader. Conceptually, one may also be skeptical of the rather dramatic loss of motor function in the context of a relatively minor circulating nutrient. Nevertheless, this finding may be important if more supporting evidence with proper controls for ketone concentrations can be provided. Overall, there are important data in the manuscript, but the reader may find it difficult to navigate the 20+ figure panels. The most important findings are that ACOT12/8 are critical for hepatic acetate production in mice, which will be helpful for the field, but the ramifications require more rigorous investigation.

Reviewer #2 (Public Review):

Catabolic conditions lead to increased formation of ketone bodies in the liver, which under these conditions play an important role in supplying energy to metabolically active organs. In this manuscript, the authors explore the concept of whether and to what extent hepatic formation of acetate might contribute to energy supply under metabolic stress conditions. The authors show that patients with diabetes have increased acetate levels, which is explained as a consequence of the increased fatty acid flux from adipose tissue to the liver. This is confirmed in a preclinical model for type 1 diabetes, where acetate concentrations are in a similar range to ketone bodies. Acetate concentrations also increase under physiological conditions of fasting. Using stable isotopes, the authors show that palmitate is used as the primary source for acetate production in primary hepatocytes. Using cell culture studies and adenoviral-mediated knockdown in mice, it can be shown that the conversion of acetyl-CoA to acetate is catalyzed in peroxisomes by acyl-CoA thioesterase8 (ACOT8) and after transport of citrate from mitochondria and subsequent conversion to acetyl-CoA in the cytosol by ACOT12. Remarkably, ACOT8/12 not only regulates the formation of acetate but plays a crucial role in the maintenance of cellular CoA concentration. Accordingly, depletion of ACOT8/12 activity leads to a reduction of other CoA derivatives such as HMG-CoA, which resulted in the inhibition of ketone body synthesis. In diabetic mice, ACOT 8 or ACOT12 knockdown appears to lead to some limitations in strength and behavior.

In summary, the authors clearly demonstrate that hepatic release-mediated by ACOT8 and ACOT12-determines the plasma concentration of acetate. This is a very remarkable observation since most studies assume that short-chain fatty acids in plasma are primarily generated by fermentation of dietary fiber by intestinal bacteria. The authors demonstrate in very well performed studies the metabolic changes that result from impaired thiolysis. On the other hand, the ACOT12 phenotype has been demonstrated in a recently published study (PMID: 34285335). In this study, ACOT12 deficiency caused NAFLD, thus it would be worth determining whether deficiency of ACOT12 and/or ACOT8 promotes de novo lipogenesis under the conditions of the present study. As a further limitation, it should be noted that the relevance of acetate production for the energy supply of peripheral organs including the central nervous system could not be clearly demonstrated. For instance, impaired ketone body production due to impaired CoA availability could affect the metabolic activity of various organs. Moreover, the human cohort is not very well described, e.g. it is unclear whether the patients have type 1 or type 2 diabetes.

Reviewer #3 (Public Review):

Wang et al. investigated the role of acetate production, a byproduct of fatty acid oxidation, in the context of metabolic stressors, including diabetes mellitus and prolonged fasting. Mechanistically, they show the importance of the liver enzymes ACOT8 (peroxisome) and ACOT12 (cytoplasm) in converting FFA-derived acetyl-CoA into acetate and CoA. The regeneration of CoA allows for subsequent fatty acid oxidation. Inhibiting the generation of acetate has negative motor consequences in streptozocin-treated mice, which are mitigated with acetate injection.

This paper's strengths include using multiple mouse models, metabolic stressors (db/db-/-, streptozocin, and prolonged starvation), numerous cell lines, precise knockout and rescue experiments, and complimentary use of mass spectrometry and nuclear magnetic resonance analytical platforms. The presented data support the conclusions of this paper and highlight the role of acetate in energy stress conditions.

In clinical medicine, common ketones that are measured are acetoacetate, beta-hydroxybutyrate, and acetone which can help determine the severity of illness. However, the data presented here suggest the potential importance of measuring acetate as another biomarker when patients present with ketoacidosis in uncontrolled diabetes or starvation. This requires further investigation.

Reviewer #1 (Public Review):

Summary:<br /> This is a follow-up study to the authors' previous report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:<br /> Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study.

Weaknesses:<br /> I have just one concern that I would like the authors to address. It is about the text that begins at line 133: "We assayed their ability to clear GLUT1 from the RPE surface (Figure 2A)". Please provide more details about this. From the figure it appears that n = 1 for this experiment, but given how careful the authors are with these types of studies that seems unlikely. How did the authors quantify the ability to clear GLUT1 from the surface? Was it cleared from both the apical and basal surface? (It is hard to resolve the apical and basal surfaces in the images provided). The experiments shown in Fig. 1H and Fig. 1I of PMID 31365873 shows how GLUT1 disappears only from the apical surface (under the conditions of that experiment and through the mechanism described in their text). It would be helpful for the authors to discuss their current results in the context of that experiment.

Reviewer #2 (Public Review):

The hard work of the authors is much appreciated. With overexpression of a-arrestin Txnip in RPE, cones and the combined respectively, the authors show a potential gene agnostic treatment that can be applied to retinitis pigmentosa. Furthermore, since Txnip is related to multiple intracellular signaling pathway, this study is of value for research in the mechanism of secondary cone dystrophy as well.

There are a few areas in which the article may be improved through further analysis and application of the data, as well as some adjustments that should be made in to clarify specific points in the article.

Reviewer #3 (Public Review):

Summary:

Xue et al. extended their groundbreaking discovery demonstrating the protective effect of Txnip on cone photoreceptor survival. This was achieved by investigating the protection of cone degeneration through the overexpression of five distinct mutated variants of Txnip within the retinal pigment epithelium (RPE). Moreover, the study explored the roles of two proteins, HSP90AB1 and Arrdc4, which share similarities or associations with Txnip. They found the protection of Txnip in RPE cells and its mechanism is different from its protection in cone cells. These discoveries have significant implications for advancing our understanding of the mechanisms underlying Txnip's protection on cone cells.

Strengths:<br /> 1. Identify the roles of different Txnip mutations in RPE and their effects on the expression of glucose transporter<br /> 2. Dissect the mechanism of Txnip in RPE vs Cone photoreceptors in retinal degeneration models.<br /> 3. Explore the functions of ARrdc4, a protein similar to Txnip and HSP90AB1 in cone degeneration.

Weaknesses:<br /> 1. Arrdc4 has deleterious effect on cone survival but no discussion on its mechanism.<br /> 2. Inhibition of HSP90 is known to cause retinal generation. It is unclear why inhibition enhances the protection of Txnip.

Reviewer #1 (Public Review):

Summary:

This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.

Strengths:

Host blood meal source, temperature, and photoperiod were all examined together.

Weaknesses:

The study was conducted in laboratory conditions with a local population of Cx. quinquefasciatus from Argentina. I'm not sure if there is any evidence for a seasonal shift in the host use pattern in Cx. quinquefasciatus populations from the southern latitudes.

Reviewer #2 (Public Review):

Summary:

Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness in birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used a generalized linear mixed model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity and fertility and a null model analysis via data randomization for hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite of that hypothesized. While this finding is interesting and worth reporting, there are significant issues with the experimental design and the conclusions that are drawn from the results, which are described below. These issues should be addressed to make the findings trustworthy.

Strengths:

1. Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.<br /> 2. The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field.

Weaknesses:

1. There is no replication built into this study. Egg lay is a highly variable trait, even within treatments, so it is important to see replication of the effects of treatment across multiple discrete replicates. It is standard practice to replicate mosquito fitness experiments for this reason. Furthermore, the sample size was particularly small for some groups (e.g. 15 egg rafts for the second gonotrophic cycle of mice in the autumn, which was the only group for which a decrease in fecundity and fertility was detected between 1st and 2nd gonotrophic cycles). Replicates also allow investigators to change around other variables that might impact the results for unknown reasons; for example, the incubators used for fall/summer conditions can be swapped, ensuring that the observed effects are not artifacts of other differences between treatments. While most groups had robust sample sizes, I do not trust the replicability of the results without experimental replication within the study.<br /> 2. Considering the hypothesis is driven by the host switching observed in the field, this phenomenon is discussed very little. I do not believe Cx. quinquefasciatus host switching has been observed in Argentina, only in the northern hemisphere, so it is possible that the species could have an entirely different ecology in Argentina. It would have been helpful to conduct a blood meal analysis prior to this experiment to determine whether using an Argentinian population was appropriate to assess this question. If the Argentinian populations don't experience host switching, then an Argentinian colony would not be the appropriate colony to use to assess this question. Given that this experiment has already been conducted with this population, this possibility should at least be acknowledged in the discussion. Or if a study showing host switching in Argentina has been conducted, it would be helpful to highlight this in the introduction and discussion.<br /> 3. The impacts of certain experimental design decisions are not acknowledged in the manuscript and warrant discussion. For example, the larvae were reared under the same conditions to ensure adults of similar sizes and development timing, but this also prevents mechanisms of action that could occur as a result of seasonality experienced by mothers, eggs, and larvae.<br /> 4. There are aspects of the data analysis that are not fully explained and should be further clarified. For example, there is no explanation of how the levels of categorical variables were compared.<br /> 5. The results show the opposite trend as was predicted by the authors based on observed feeding switches from birds to mammals in the autumn. However, they only state this once at the end of the discussion and never address why they might have observed the opposite trend as was hypothesized.<br /> 6. Generally speaking, the discussion has information that isn't directly related to the results and/or is too detailed in certain parts. Meanwhile, it doesn't dig into the meaning of the results or the ways in which the experimental design could have influenced results.<br /> 7. Beyond the issue of lack of replication limiting trust in the conclusions in general, there is one conclusion reached at the end of the discussion that would not be supported, even if additional replicates are conducted. The results do not show that physiological changes in mosquitoes trigger the selection of new hosts. Host selection is never measured, so this claim cannot be made. The results don't even suggest that fitness might trigger selection because the results show that physiological changes are in the opposite direction as what would be hypothesized to produce observed host switches. Similarly, the last sentence of the abstract is not supported by the results.<br /> 8. Throughout the manuscript, there are grammatical errors that make it difficult to understand certain sentences, especially for the results.

This study is driven by an interesting question and has the potential to be a valuable contribution to the literature.

Reviewer #1 (Public Review):

Summary:<br /> The goal of this study was to develop and validate novel molecules to selectively activate a cell signaling pathway, the Wnt pathway in this case, in target cells expressing a specific receptor. This was achieved through a two-component system that the authors call BRAID, where each component simultaneously binds the target cell-specific marker BKlotho and a Wnt co-receptor. These components, called SWIFT molecules, bring together the Wnt co-receptors LRP and FZD, activating the pathway specifically in cells that express BKlotho. Results presented in the study demonstrate the desired activity of SWIFT molecules; the binding assays support the simultaneous association of SWIFT with BKlotho and a Wnt co-receptor, and the Wnt reporter and qPCR assays support pathway activation in cell lines and primary cells in a BKlotho-dependent manner. In the future, the BRAID approach could be applied to activate Wnt signaling or another pathway initiated by a co-receptor complex in a cell type-specific manner, and/or in a FZD subtype-specific manner to activate distinct branches of Wnt signaling.

Strengths:<br /> • This study successfully demonstrates a novel way to activate Wnt signaling in target cells expressing a specific marker. Given the role of the Wnt signaling pathway in key processes such as cell proliferation and tissue renewal and the value of modulating cell signaling in a cell type-specific manner, the cell targeting system developed here holds great therapeutic and research potential. It will be curious to see whether the BRAID design can be applied to other cell surface markers for Wnt activation, or for activation of other signaling pathways that require co-receptor association.

• Octet assay results show simultaneous binding of SWIFT molecules to both the Wnt co-receptor FZD/LRP and BKlotho, while negative control molecules without the FZD/LRP or BKlotho-binding module show neither receptor binding nor Wnt pathway activation. These results indicate that SWIFT molecules function through the intended mechanism.

Weaknesses:<br /> • Here, the activity of SWIFT molecules was assessed in single cell types with or without BKlotho expression. Ultimately, the ability of the SWIFT molecules to activate Wnt signaling in a cell type-specific manner should be tested in the context of many different cellular identities that express BKlotho to different extents. It would be good to demonstrate that Wnt activation by SWIFT correlates with BKlotho expression level in multiple cell types - such data would strengthen the claim of cell-type specificity.

• The study does not address whether the targeted cells express FGFR1c/2c/3c and whether the FGF21 full-length moiety or the 39F7 IgG moiety of SWIFT molecules could unintentionally activate FGF signaling in these cells.

Reviewer #2 (Public Review):

Summary:<br /> The study introduces BRAID, a novel approach for targeting drugs to specific cell types, addressing the challenges of pleiotropic drug actions. Unlike existing methods, this one involves breaking a protein drug molecule into inactive parts that are then put back together using a bridging receptor on the target cell. The individual components of this assembly are not required to be together, thereby affording it a degree of flexibility. The authors applied this idea to the WNT/-catenin signaling pathway by splitting a WNT mimic into two parts with FZD and LRP binding domains and bridging receptors. This combined method, which is called SWIFT, showed that WNT signaling was turned on in target cells, showing cell-specific targeting. The technique shows promise for the development of therapeutics, as it provides a way to more precisely target signaling pathways.

The authors have effectively elucidated their strategy through visually appealing diagrams, providing clear and thorough visual aids that facilitate comprehension of the concept. In addition, the authors have provided convincing evidence that the C-terminal region of FGF21 is essential for the binding process. Their meticulous and thorough presentation of experimental results emphasizes the significance of this specific binding domain and validates their findings.

Strengths:<br /> BRAID, a novel cell targeting method, divides an active drug molecule into inactive components formed by a bridging receptor. This novel approach to cell-specific drug action may reduce systemic toxicity.

The SWIFT approach successfully targets cells in the WNT/β-catenin signaling pathway. The approach activates WNT signaling only in target cells (hepatocytes), proving its specificity.

The study indicates that the BRAID approach can target various signaling systems beyond WNT/β-catenin, indicating its versatility. Therapeutic development may benefit from this adaptability.

Weaknesses:<br /> The study shows the SWIFT approach works in vitro using cell lines, primary human hepatocytes, and human intestinal organoids, but it lacks an in vivo animal model or clinical validation. The applicability of this approach to therapy is still unknown.

The success of SWIFT depends on the presence and expression of the bridging receptor (βKlotho) on target cells. The approach may fail if the target receptor is not expressed or available.

Reviewer #1 (Public Review):

This work continues a series of recent publications from the Grigorieff lab (https://doi.org/10.7554/eLife.25648, https://doi.org/10.7554/eLife.68946, https://doi.org/10.7554/eLife.79272, https://doi.org/10.1073/pnas.2301852120) showcasing the development of high-resolution 2D template matching (2DTM) for detection and reconstruction of macromolecules in cryo-electron microscopy (cryo-EM) images of crowded cellular environments. It is well known in the field of cryo-EM that searching noisy images with a template can result in retrieval of the template itself when averaging the candidate particles detected, an effect known as "Einstein-from-noise" (https://doi.org/10.1073/pnas.1314449110). Briefly, this occurs because it is statistically likely to find a match to an arbitrary motif over a large noisy dataset just by chance. The effect can be mitigated for example by limiting the resolution of the template, but this prevents the accurate detection of macromolecules in a crowded environment, as their "fingerprint" lies in the high-resolution range (https://doi.org/10.7554/eLife.25648). Here, the authors show through several experiments on in vitro and in situ data that features as small as drug compounds and water molecules can be reliably retrieved by 2DTM if they are searched by a template (the "bait") that contains expected neighboring features but not the targets themselves.

The ideas are generally clearly presented with appropriate references to related work, and claims are well supported by the data. In particular, the experiments for verifying the density of the ribosomal protein L7A as well as the systematic removal of residuals from the template model to assess bias are particularly clever.

One key point that could use further clarification is how to interpret densities in the reconstruction that do overlap with the template. If the omitted regions can be reliably reconstructed, and the density is smooth throughout, it implies the detected particles are not only (mostly) true positives but also their poses must be essentially correct. Therefore, why cannot the entire reconstruction be trusted, including portions overlapping with the template? In the "Future applications" section, the authors state that in order to obtain a reconstruction that is entirely devoid of template bias, it would be necessary to successively omit parts of the template structure through its entirety. I wonder if that is really necessary and if the presented approach of omitting template portions could be better framed as a "gold-standard" validation procedure.

In other words, given the compelling evidence provided by the reconstructions in the omitted areas, I find it hard to imagine how the procedure would be "hallucinating" features in the rest of the structure, as the entire reconstruction depends on the same pose and defocus parameters. A possible experiment to test this hypothesis would be to go the opposite way, deliberately adding an unrealistic feature to the bait and checking whether it comes up in the reconstruction, while at the same time checking how it behaves in omitted parts.

When assessing their approach to in situ data (the yeast ribosome), it is intriguing to see that the resolution downgraded from 3.1 to 8 Å when refinement of the particle poses against the current reconstruction was attempted. The authors do provide some possible explanations, such as the reduced signal of the reconstruction at high resolution and the crowded background, but it leaves one to wonder if this means that a 3.1 Å reconstruction could never be obtained from these data by conventional single-particle analysis procedures.

Furthermore, in the section "Quantifying template bias", the authors make the intriguing statement that there can still be some overfitting of noise even in true positives. I understand this overfitting would occur in the form of errors in the pose and defocus estimation, but a clarification would be helpful.

In the Discussion, the claim that "it is not necessary to use tomography to generate high-resolution reconstructions of macromolecular complexes in cells" is a misconception, at least in part. As demonstrated in works by the same group and others (https://doi.org/10.1016/j.xinn.2021.100166, https://doi.org/10.1038/s41467-023-36175-y, https://doi.org/10.1038/s41586-023-05831-0), 2D imaging of native cellular environments does offer a faster and better way to obtain high-resolution reconstructions compared to tomography. However, tomography provides the entire 3D context of the macromolecules, such as their localization to membranes and the cellular architecture, which can be readily visualized in a tomogram even at low resolution, so methods for structure determination from tilt series data such as subtomogram averaging remain of paramount importance. Most likely, a combination of 2D and 3D imaging approaches will be necessary to retrieve both the highest structural resolution and their cellular context to address biological questions.

The "Materials and Methods" section lacks a description of transmission electron microscopy data collection.

Finally, the preprint version of this work posted on bioRxiv (https://doi.org/10.1101/2023.07.03.547552) contains the following competing interests statement, which is missing from the submitted version:<br /> "The authors are listed as inventors on a closely related patent application named "Methods and Systems for Imaging Interactions Between Particles and Fragments", filed on behalf of the University of Massachusetts."

Reviewer #2 (Public Review):

This paper by Lucas et al follows on from earlier work by the same group. They use high-resolution 2D template matching (2DTM) to find particles of a given target structure in 2D cryo-EM images, either of in vitro single-particle samples or of more complicated samples, such as FIB-milled cells (which would otherwise perhaps be used for 3D electron tomography). One major concern for high-resolution template matching has been the amount of model bias that gets introduced into a reconstruction that is calculated straight from the orientations and positions identified by the projection matching algorithm. This paper assesses the amount of model bias that gets introduced in high-resolution features of such maps.

For a high-signal-to-noise in vitro single-particle cryo-EM data set, the authors show that their approach does not yield much model bias. This is probably not very surprising, as their method is basically a low false-positive particle picker, which works very well on such data. Still, I guess that is the whole point of it, and it is good to see that they can reconstruct density for a small-molecule compound that was not present in the original template.

For FIB-milled lamella of yeast cells with stalled ribosomes, the SNR is much lower and the dangers of model bias will be higher. This is also evidenced by the observation that further refinement of initial 2DTM-identified orientations and positions worsens the map. This is obviously a more relevant SNR regime to assess their method. Still, they show convincing density for the GHX compound that was not present in the template but was there in the reconstruction from the identified particles.

Quantification of the amount of model bias is then performed using omit maps, where every 20th residue is removed from the template and corresponding reconstructions are compared (for those residues) with the full-template reconstructions. As expected, model bias increases with lower thresholds for the picking. Some model bias (Omega=8%) remains even for very high thresholds. The authors state this may be due to overfitting of noise when template-matching true particles, instead of introducing false positives. Probably, that still represents some sort of problem. Especially because the authors then go on to show that their expectation of the number of false positives does not always match the correct number of false positives, probably due to inaccuracies in the noise model for more complicated images. This may warrant further in-depth discussion in a revised manuscript.

Overall, I think this paper is well written and it has made me think differently (again) about the 2DTM technique and its usefulness in various applications, as outlined in the Discussion. Therefore, it will be a constructive contribution to the field.

Reviewer #3 (Public Review):

The authors evaluate the effect of high-resolution 2D template matching on template bias in reconstructions, and provide a quantitative metric for overfitting. It is an interesting manuscript that made me reevaluate and correct some mistakes in my understanding of overfitting and template bias, and I'm sure it will be of great use to others in the field. However, its main point is to promote high-resolution 2D template matching (2DTM) as a more universal analysis method for in vitro and, more importantly, in situ data. While the experiments performed to that end are sound and well-executed in principle, I fail to make that specific conclusion from their results.

The authors correctly point out that overfitting is largely enabled by the presence of false-positives in the data set. They go on to perform their in situ experiments with ribosomes, which provide an extremely favorable amount of signal that is unrealistic for the vast majority of the proteome. This seems cherry-picked to keep the number of false-positives and false-negatives low. The relationship between overfitting/false-positive rate and the picking threshold will remain the same for smaller proteins (which is a very useful piece of knowledge from this study). However, the false-negative rate will increase a lot compared to ribosomes if the same high picking threshold is maintained. This will limit the applicability of 2DTM, especially for less-abundant proteins.

I would like to see an ablation study: Take significantly smaller segments of the ribosome (for which the authors already have particle positions from full-template matching, which are reasonably close to the ground-truth), e.g. 50 kDa, 100 kDa, 200 kDa etc., and calculate the false-negative rate for the same picking threshold. If the resulting number of particles does plummet, it would be very helpful to discuss how that affects the utility of 2DTM for non-ribosomes in situ.

Another point of concern is the dramatic resolution decrease to 8 A after multiple iterations of refinement against experimental reconstructions described in line 159. Was this a local search from the poses provided by 2DTM, or something more global? While this is not a manifestation of overfitting as the authors have conclusively shown, I think it adds an important point to the ongoing "But do we really need tomograms, or can we just 2D everything?" debate in the field, which is also central to the 2D part of 2DTM. Reaching 8 A with 12k ribosome particles would be considered a rather poor subtomogram averaging result these days. Being in the "we need tilt series to be less affected by non-Gaussian noise" camp myself, I wonder if this indicates 2D images are inherently worse for in situ samples. If they are, the same limitations would extend to template matching. In that case, shouldn't the authors advocate for 3DTM instead of 2DTM? It may not be needed for ribosomes, but could give smaller proteins the necessary edge.

Right now, this study is also an invitation to practitioners who do not understand the picking threshold used here and cannot relate it to other template-matching programs to do a lot of questionable template matching and claim that the results are true because templates are "unoverfittable". I think such undesirable consequences should be discussed prominently.

Reviewer #1 (Public Review):

Koesters and colleagues investigated the role of the presynaptic small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed an increase in GluA2 puncta size and intensity in wild type, but not Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which presynaptic Rab3A is required for homeostatic scaling of synaptic transmission through GluA2-dependent and independent mechanisms.

While the title of the manuscript is mostly supported by data of solid quality, many conclusions, as well as the final model, cannot be derived from the results presented. Importantly, the results do not indicate that Rab3A modulates quantal size on both sides of the synapse. Moreover, several analysis approaches seem inappropriate.

The following points should be addressed:

1. The model shown in Figure 10 is not supported by the data. The authors neither provide evidence for two different functional states of Rab3A being involved in mEPSC amplitude modulation, nor for a change in glutamate content of vesicles. Furthermore, the data do not fully support the conclusion of a presynaptic role for Rab3A in homeostatic scaling.<br /> 2. The analysis of mEPSC data using quantile sampling followed by ratio calculation is not meaningful under the tested experimental conditions because of the following reasons: (i) The analysis implicitly assumes that all events have been detected. The prominent mEPSC frequency increase after TTX suggests that this is not the case, i.e., many (small) mEPSCs are likely missed under control conditions. (ii) The analysis is used to conclude how events of a certain size are altered by TTX treatment. However, this analysis compares the smallest mEPSCs of the TTX condition with the smallest control mEPSCs, but this is not a pre-post experimental design. Variation between cells and between coverslips will markedly affect the results and lead to misleading interpretations. (iii) The ratio (TTX/control) vs. control plots seem to suffer from a division by small value artifact (see Figure 6F). Correspondingly, ratio-analysis differs considerably for different control conditions (Fig. 1Giii, Fig. 2Giii, Fig. 6C, Fig. 9A).<br /> 3. As noted by the authors in a previous publication (Hanes et al. 2020), statistical analysis of CDFs suffers from n-inflation. In addition, the quantile sampling method chosen violates an important assumption of the K-S test. Indeed, p-values for these comparisons are typically several orders of magnitude smaller. Given that the statistical N most likely corresponds to the number of cultures (see, e.g., https://doi.org/10.1371/journal.pbio.2005282), CDF comparisons are not informative and should thus not be used to draw conclusions from the data. The plots can be informative, though.<br /> 4. How does recoding noise and the mEPSC amplitude threshold affect "divergent scaling"?<br /> 5. What is the justification for the line fits of the ratio data/how was the fit range chosen?<br /> 6. TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data.<br /> 7. Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.<br /> 8. There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3Aebd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+, and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?<br /> 9. Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs. -15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.<br /> 10. The authors discuss previous papers showing changes in VGLUT1 intensity. Was VGLUT intensity altered in the stainings presented in the manuscript?<br /> 11. The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?<br /> 12. The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold? What was the rationale for a threshold of 400 a.u. or 450 a.u.? How does this threshold compare to the mEPSC threshold of 3 pA? The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size. (ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 18-20%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)? Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors. (iv) It is not entirely clear if their imaging experiments will sample from all synapses. Other AMPAR subtypes than GluA2 could contribute, as could kainate or NMDA receptors.<br /> Furthermore, the statement "complete lack of correspondence of TTX/CON ratios" is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/- , the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2), particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.<br /> 13. Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?

Reviewer #2 (Public Review):

In this study, Koesters et al. investigated whether Rab3A, a small GTPase that regulates synaptic vesicle fusion pore opening, is required for excitatory synaptic scaling in response to TTX-induced activity suppression in dissociated mouse cortical neuronal culture. They first show that, while pyramidal neurons from wild-type (WT) littermates show normal synaptic scaling in response to 48h of TTX treatment (~30% increase in the mean mEPSC amplitude), those from two different mouse lines with either deletion (Rab3A-/-) or loss-of-function mutation of Rab3A (Rab3AEbd/Ebd) fail to engage this homeostatic compensation. They perform cumulative distribution analysis to show that the mEPSC population has gone through divergent scaling in WT neurons. Similarly, this phenomenon is absent in neurons from the two Rab3A mouse lines. They further demonstrate that GluA2-containing AMPARs likely account for the increase in mEPSC amplitudes by comparing measurements before and after washing in blockers specific for GluA2-lacking AMPARs. Subsequently, they perform electrophysiology and immunohistochemistry side by side for WT neurons from the same culture following TTX treatment, and find that both mEPSC amplitudes and GluA2 cluster sizes have shifted towards higher values, while GluA2 cluster intensity remains unchanged. Importantly, all these homeostatic compensations are absent in Rab3A-/- neurons. Finally, they mix neurons and astrocyte feeders either from WT or Rab3A-/- mice, which reveals that neuronal but not astrocytic Rab3A knockout leads to impaired scaling up of mEPSCs. They conclude that Rab3A is required for homeostatic scaling up of mEPSC amplitude in cortical neurons, most likely from the presynaptic side.

Although the authors have raised an interesting question, their conclusion is not well supported by the data presented. I list my technical and conceptual concerns below.

Technical concerns:

1. The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006). It is important to ensure there is enough spiking activity before doing any activity manipulation. Similarly, it is also unknown whether spiking activity is normal in Rab3A KO/Ebd neurons.

2. Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.

3. Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapses on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.

Conceptual concerns:

The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The author claims that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study's contribution to the field is very limited.

1. Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019). Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.

2. The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.

3. The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the Naspm wash-in experiments clearly show that GluA1 homomer contributions have not changed before and after TTX treatment.

Ibata K, Sun Q, Turrigiano GG (2008) Rapid synaptic scaling induced by changes in postsynaptic firing. Neuron 57:819-826.

Jakawich SK, Nasser HB, Strong MJ, McCartney AJ, Perez AS, Rakesh N, Carruthers CJL, Sutton MA (2010) Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis. Neuron 68:1143-1158.

Pozo K, Cingolani LA, Bassani S, Laurent F, Passafaro M, Goda Y (2012) β3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons. Proceedings of the National Academy of Sciences 109:1323-1328.

Silva MM, Rodrigues B, Fernandes J, Santos SD, Carreto L, Santos MAS, Pinheiro P, Carvalho AL (2019) MicroRNA-186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons. Proceedings of the National Academy of Sciences 116:5727-5736.

Sutton MA, Ito HT, Cressy P, Kempf C, Woo JC, Schuman EM (2006) Miniature Neurotransmission Stabilizes Synaptic Function via Tonic Suppression of Local Dendritic Protein Synthesis. Cell 125:785-799.

Tan HL, Queenan BN, Huganir RL (2015) GRIP1 is required for homeostatic regulation of AMPAR trafficking. Proceedings of the National Academy of Sciences 112:10026-10031.

Thapliyal S, Arendt KL, Lau AG, Chen L (2022) Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity. eLife 11:e79863.

Watt AJ, Rossum MCW van, MacLeod KM, Nelson SB, Turrigiano GG (2000) Activity Coregulates Quantal AMPA and NMDA Currents at Neocortical Synapses. Neuron 26:659-670.

Reviewer #3 (Public Review):

Summary: The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength seems already elevated. In this context, it is unclear if the plasticity is absent or just occluded by a ceiling effect due the synapses already being strengthened. The authors do appropriately discuss both options. There are also differences in genetic background between the Rab3A KO and Earlybird mutants that could also impact the results, which are also noted. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between synaptic strength during HSP and AMPA receptor trafficking, and conclude that trafficking is largely not responsible for the changes in synaptic strength.

Strengths: This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms.

Weaknesses: However, the rather strong conclusions on the dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. The key issue is the GluA2 immunostaining in comparison with the mESPC recordings. Their imaging method involves only assessing puncta clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, judging from the sample micrographs (Fig 5). To my knowledge, this is a new and unvalidated approach that could represent a particular subset of synapses not representative of the synapses contributing to the mEPSC change (they are also sampling different neurons for the two measurements; an additional unknown detail is how far from the cell body were the analyzed dendrites for immunostaining). While the authors acknowledge that a sampling issue could explain the data, they still use this data to draw strong conclusions about the lack of AMPAR trafficking contribution to the mEPSC amplitude change. This apparent difference may be a methodological issue rather than a biological one, and at this point it is impossible to differentiate these. It will unfortunately be difficult to validate their approach. Perhaps if they were to drive NMDA-dependent LTD or chemLTP, and show alignment of the imaging and ephys, that would help. More helpful would be recordings and imaging from the same neurons but this is challenging. Sampling from identified synapses would of course be ideal, perhaps from 2P uncaging combined with SEP-labeled AMPARs, but this is more challenging still. But without data to validate the method, it seems unwarranted to make such strong conclusions such as that AMPAR trafficking does not underlie the increase in mEPSC amplitude, given the previous data supporting such a model.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is quite unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. It is also unclear why the authors argue this proves that the NASPM was at an effective concentration (lines 399-400). Further, the amplitude data show a strong trend towards smaller amplitude. The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. And the decrease is larger in the TTX neurons. Considering the strong claims for a pre-synaptic locus and the use of this data to justify only looking at GluA2 by immunostaining, these data do not offer much support of the conclusions. Between the sampling issues and perhaps looking at the wrong GluA subunit, it seems premature to argue that trafficking is not a contributor to the mEPSC amplitude change, especially given the substantial support for that hypothesis. Further, even if trafficking is not the major contributor, there could be shifts in conductance (perhaps due to regulation of auxiliary subunits) that does not necessitate a pre-synaptic locus. While the authors are free to hypothesize such a mechanism, it would be prudent to acknowledge other options and explanations.

The frequency data are missing from the paper, with the exception of the NASPM dataset. The mEPSC frequencies should be reported for all experiments, particularly given that Rab3A is generally viewed as a pre-synaptic protein regulating release. Also, in the NASPM experiments, the average frequency is much higher in the TTX treated cultures. Is this statistically above control values?

Unaddressed issues that would greatly increase the impact of the paper:<br /> 1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role (and particularly the hypothesized and somewhat novel idea that the amount of glutamate released per vesicle is altered in HSP). They could use sparse knock-down of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs and/or a decrease of GABA-packaging in vesicles (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at these synapses.

Reviewer #1 (Public Review):

Summary:

This valuable study analyzes the contribution of fungal and bacterial microbiota species to the growth and development of Drosophila. The authors use bacterial and fungal species associated with Drosophila in the wild to analyze their respective contributions in promoting larval growth in a decaying banana, mimicking the natural niche of fruit flies. They found that some fungal species and some fungus/bacteria combinations effectively promote growth by supplementing key branched amino acids in the food substratum. Production of these amino acids by Drosophila itself is not sufficient, and only fungal species that secrete these amino acids into the medium can sustain Drosophila growth. Thus, the authors clarify how facultative symbionts contribute to host growth in a natural setting by changing the food substratum in a dynamic manner.

Strengths:

The natural setting developed by the authors to analyze the impact of the microbiota is clearly valuable, as is the focus on the role of fungal microbiota species. This complements studies of Drosophila microbiota that have previously focused on bacterial species and used a lab setting. While there has been an extensive focus on obligate endosymbionts or gut symbionts, this study analyzes how facultative symbionts shape the food substratum and influence host growth. A last strength of this study is that it analyzes the contribution of Drosophila microbiota over a dynamic timeframe, analyzing how microbial species change in decaying fruit over time.

Weaknesses:

1) The authors should better review what we know of fungal Drosophila microbiota species as well as the ecology of rotting fruit. Are the microbiota species described in this article specific to their location/setting? It would have been interesting to know if similar species can be retrieved in other locations using other decaying fruits. The term 'core' in the title suggests that these species are generally found associated with Drosophila but this is not demonstrated. The paper is written in a way that implies the microbiota members they have found are universal. What is the evidence for this? Have the fungal species described in this paper been found in other studies? Even if this is not the case, the paper is interesting, but there should be a discussion of how generalizable the findings are.

2) Can the authors clearly demonstrate that the microbiota species that develop in the banana trap are derived from flies? Are these species found in flies in the wild? Did the authors check that the flies belong to the D. melanogaster species and not to the sister group D. simulans?

3) Did the microarrays highlight a change in immune genes (ex. antibacterial peptide genes)? Whatever the answer, this would be worth mentioning. The authors described their microarray data in terms of fed/starved in relation to the Finke article. They should clarify if they observed significant differences between species (differences between species within bacteria or fungi, and more generally differences between bacteria versus fungi).

4) The whole paper - and this is one of its merits - points to a role of the Drosophila larval microbiota in processing the fly food. Are these bacterial and fungal species found in the gut of larvae/adults? Are these species capable of establishing a niche in the cardia of adults as shown recently in the Ludington lab (Dodge et al.,)? Previous studies have suggested that microbiota members stimulate the Imd pathway leading to an increase in digestive proteases (Erkosar/Leulier). Are the microbiota species studied here affecting gut signaling pathways beyond providing branched amino acids?

Reviewer #2 (Public Review):

Summary:

In this manuscript, Mure et al investigated host-microbe interactions in wild-mimicked settings. They analyzed microbiome composition using bananas that had been fed on by wild larvae and found that the microbiota composition shifted from the early stage of feeding to the later stage of the fermentation process. They isolated several yeast and bacterial species from the food, and examined larval growth on banana-based food, mimicking a natural setting where germ-free larvae cannot grow on it. The authors found that a yeast, Hanseniaspora uvarum, can support larval growth sufficiently, and insisted that branched-chain amino acids (BCAAs) provided by the yeast may partly account for the growth support. Interestingly, in other isolated yeast species, some were non-supportive strains in terms of larval growth, which can assist larval development when they are heat-killed. Besides, they showed that acetic acid bacteria, isolated from well-fermented banana (later-stage food), is sufficiently supportive but their presence depended on other microbes, lactic acid bacteria or yeast.

Strengths:

So far, host-microbe studies using Drosophila melanogaster have focused relatively less on the roles of fungi, and many studies used only "model" yeasts. In the experimental setting where natural conditions may be well mimicked, the authors successfully isolated wild yeast species and convincingly showed that wild yeast plays a critical role in promoting host growth. In addition, the authors provided intriguing observations that all of the heat-killed yeast promoted larval growth even though some of the yeast never supported the development when they were alive, suggesting that wild yeasts produce the necessary nutrients for larval development, but the nutrients of non-supportive yeasts are not accessible to the host. This might be an interesting indication for further studies revealing host-fungi interactions.

Weaknesses:

The experimental setting that, the authors think, reflects host-microbe interactions in nature is one of the key points. However, it is not explicitly mentioned whether isolated microbes are indeed colonized in wild larvae of Drosophila melanogaster who eat bananas. Another matter is that this work is rather descriptive and a few mechanical insights are presented. The evidence that the nutritional role of BCAAs is incomplete, and molecular level explanation is missing in "interspecies interactions" between lactic acid bacteria (or yeast) and acetic acid bacteria that assure their inhabitation. Apart from these matters, the future directions or significance of this work could be discussed more in the manuscript.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Mure et al. describe interactions between diet, microbiome, and host development using Drosophila as a model. By characterizing microbial communities in food sources and animals, the authors showed that microbial community dynamics in the food source are critical for host development.

Strengths:

This is a very interesting study where the authors managed to tackle a difficult question in an elegant manner. How the interactions between different microbial species within the microbiome shape host physiology is an area of great interest but equally challenging due to the complexity of intercellular interactions in complex, host-associated microbial communities. By using a simplified model and interrogating not only microbe-microbe and host-microbe interactions, but also the role played by diet, authors were able to identify significant interactions during fly development.

Weaknesses:

Despite describing important findings, I believe that a more thorough explanation of the experimental setup and the steps expected to occur in the exposed diet over time, starting with natural "inoculation" could help the reader, in particular the non-specialist, grasp the rationale and main findings of the manuscript. When exactly was the decision to collect early-stage samples made? Was it when embryos were detected in some of the samples? What are the implications of bacterial presence in the no-fly traps? These samples also harbored complex microbial communities, as revealed by sequencing. Were these samples colonized by microbes deposited with air currents? Were they the result of flies that touched the material but did not lay eggs? Could the traps have been visited by other insects? Another interesting observation that could be better discussed is the fact that adult flies showed a microbiome that more closely resembles that of the early-stage diet, whereas larvae have a more late-stage-like microbiome. It is easy to understand why the microbiome of the larvae would resemble that of the late-stage foods, but what about the adult microbiome? Authors should discuss or at least acknowledge the fact that there must be a microbiome shift once adults leave their food source. Lastly, the authors should provide more details about the metabolomics experiments. For instance, how were peaks assigned to leucine/isoleucine (as well as other compounds)? Were both retention times and MS2 spectra always used? Were standard curves produced? Were internal, deuterated controls used?

Reviewer #1 (Public Review):

Taking advantage of a publicly available dataset, neuronal responses in both the visual and hippocampal areas to passive presentation of a movie are analyzed in this manuscript. Since the visual responses have been described in a number of previous studies (e.g., see Refs. 11-13), the value of this manuscript lies mostly on the hippocampal responses, especially in the context of how hippocampal neurons encode episodic memories. Previous human studies show that hippocampal neurons display selective responses to short (5 s) video clips (e.g. see Gelbard-Sagiv et al, Science 322: 96-101, 2008). The hippocampal responses in head-fixed mice to a longer (30 s) movie as studied in this manuscript could potentially offer important evidence that the rodent hippocampus encodes visual episodes.

The analysis strategy is mostly well designed and executed. A number of factors and controls, including baseline firing, locomotion, frame-to-frame visual content variation, are carefully considered. The inclusion of neuronal responses to scrambled movie frames in the analysis is a powerful method to reveal the modulation of a key element in episodic events, temporal continuity, on the hippocampal activity. The properties of movie fields are comprehensively characterized in the manuscript.

Comments on latest version:

The new analysis on how behavioral states and hippocampal ripples impacted the tuning of movie fields makes the main finding substantially more convincing. Other relatively minor concerns on the methodology and interpretation are also improved. I do not have further concerns.

Reviewer #3 (Public Review):

In their study, Purandare & Mehta analyze large-scale single unit recordings from the visual system (LGN, V1, extrastriate regions AM and PM) and hippocampal system (DG, CA3, CA1 and subiculum) while mice monocularly viewed repeats of a 30s movie clip. The data were part of a larger release of publicly available recordings from the Allen Brian Observatory. The authors found that cells in all regions exhibited tuning to specific segments of the movie (i.e. "movie fields") ranging in duration from 20ms to 20s. The largest fractions of movie-responsive cells were in visual regions, though analyses of scrambled movie frames indicated that visual neurons were driven more strongly by visual features of the movie images themselves. Cells in the hippocampal system, on the other hand, tended to exhibit fewer "movie fields", which on average were a few seconds in duration, but could range from >50ms to as long as 20s. Unlike the visual system "movie fields" in the hippocampal system disappeared when the frames of the movie were scrambled, indicating that the cells encoded more complex (episodic) content, rather than merely passively reading out visual input.

The paper is conceptually novel since it specifically aims to remove any behavioral or task engagement whatsoever in the head-fixed mice, a setup typically used as an open-loop control condition in virtual reality-based navigational or decision making tasks (e.g. Harvey et al., 2012). Because the study specifically addresses this aspect of encoding (i.e. exploring effects of pure visual content rather than something task-related), and because of the widespread use of video-based virtual reality paradigms in different sub-fields, the paper should be of interest to those studying visual processing as well as those studying visual and spatial coding in the hippocampal system.

Comments on latest version:

The revised manuscript by Purandare et al. has been improved with the inclusion of additional analyses and discussion, and the changes mainly satisfy the concerns raised in the initial version of the manuscript.

Regarding the methods, it was particularly helpful that the authors took measures to consider the impact of different states of arousal (pupil diameter), mobility, and SWRs on the expression and significance of movie field tuning, considering the lack of a task structure or behavioral report. Relatedly, the additional metrics applied (information rate and depth of movie field modulation) substantiate the results as based on z-scored sparsity. The explanation of lifetime sparseness as used here vs. in the work of de Vries et al. 2020 was also helpful.

The addition of more clearly tuned cells also helps the study feel more rooted in solid ground. For clarity, and consistency with the rest of the paper, it would be helpful to add the sparseness metrics above the newly added neural data in the Figure supplements.

The Discussion also contains elements that help balance both it and the paper as a whole. It draws a clearer distinction between the representation of visual scenes rather than encoding the contents of episodic memory, clarifying that hippocampal neurons were more likely doing the former than the latter. It is also appreciated that the authors added discussion acknowledging that the cortical processing did not quite follow an apparent hierarchical order.

As a last observation, though the authors assert in their rebuttal that analysis of the visual content encoded in the movie fields is beyond the scope of the study, this would add an interesting dimension to the work. Because, to my awareness, much less is known regarding how the visual and hippocampal systems in rodents encode visual information when the visual input is dynamic and chunked, as with movies. It would prove an interesting addition to the more extensive work on the processing of static visual scenes.

Reviewer #1 (Public Review):

With this work, the authors address a central question regarding the potential consequences of post-translational modifications for the pathogenesis of neurodegenerative diseases. Phosphorylation and mislocalization of the RNA binding protein TDP43 are characteristic of ~50% of frontotemporal lobar degeneration (FTLD), as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.

This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.

Comments on the latest version:

The authors have addressed most concerns. The additional analysis demonstrating a lack of neuron loss is quite different from the original study -- it is good that the authors pursued this question. In addition, new data focusing on native TDP-43 splice targets, rather than the splicing reporter, are excellent.

Reviewer #2 (Public Review):

This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field.

Reviewer #3 (Public Review):

Numerous experimental models are phenotyped in this manuscript including mouse neurons, iPSC-derived human neurons, knock-in mice, and knock-in iPSCs. Expression of acetylation-mimic or acetylation-null TDP-43 protein is achieved either with overexpression or CRISPR-Cas9-based knock-in. A complex phenotype is observed including loss of TDP-43 function (reduced autoregulation, increased cryptic splicing) and a gain of TDP-43 (increased insoluble TDP-43 protein). These correlate with downstream neurobehavioral changes which are most consistent with a cortical/hippocampal phenotype without a motor phenotype. Post-translational modifications of disease-associated proteins are thought to contribute to neurodegenerative disease pathogenesis, and this study succeeds in demonstrating that TDP-43 acetylation results in downstream molecular and behavioral phenotypes.

TDP-43 acetylation is a post-translational modification that is known to be associated with TDP-43 inclusions that are characteristic of human diseases. An important strength is the rigorous use of multiple different experimental models (rodent cells, iPSC-derived neurons, mice, overexpression, knock-in) with overall consistent results. Moreover, multiple orthogonal endpoints are presented including histology/cytology/immunostaining, biochemistry, molecular biology, and neurobehavioral assays. As TDP-43 acetylation is known to block RNA binding, these novel cellular and mouse models represent interesting albeit complex tools to study the functional consequences of a partial loss of function. As TDP-43 regulates its own expression (i.e. autoregulation), the complexity lies at least in part due to the loss of RNA binding leading to a functional loss of TDP-43 function which includes the increased expression of the TARDBP transcript and TDP-43 protein.

Conceptually, there is a disconnect in that the mouse model exhibits primarily a cortical/hippocampal phenotype more akin to frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), while TDP-43 acetylation is only seen in ALS tissues and not in FTLD-TDP tissues because most of the pathologic protein in the latter is N-terminally truncated (i.e. the acetylation site is not present). That being said, there is no mouse model which completely and faithfully recapitulates the human disease, and this mouse model avoids overt overexpression (increased TDP-43 protein expression stemming from altered autoregulation) and avoids the use of synthetic/artificial mutation (such as mutation of the TDP-43 nuclear localization signal).

This revision addresses most of my prior comments including documenting the lack of neurodegeneration in this model, the use of more appropriate statistical methods, and the use of more robust/quantitative aberrant splicing measures. The one thing which would still be helpful is sequencing the top predicted off target genomic loci for their various CRISPR'd models irrespective of whether these loci are exonic or noncoding. Having actual sequencing verification of the lack of mutations at these loci is preferable over relying only on computational likelihood estimates.

Reviewer #1 (Public Review):

Summary:

This is an important study that tests the effects of using neurofeedback, in the form of reward delivery when large sharp wave-ripples (SWRs) are detected, on neurophysiological and behavioral measures. The authors report that the rate of SWRs ripples increased prior to reward delivery, but this increased rate of SWRs had no significant effect on memory performance. They also found that compensatory decreases in SWR rate occurred in the period after reward delivery such that the overall SWR rate remained stable.

Strengths:

The study has many strengths. The paradigm of closed loop detection of SWRs and reward delivery is powerful and provides an innovative way to causally test the effects of increasing SWR rates. Other studies could adopt this method to test other hypotheses or to assess the effects of increasing SWR rates prior to reward delivery in rodent models of brain disorders. The methods and results are clearly explained. The results are presented in a transparent way.

Weaknesses:

In the linear mixed effects model analysis used in Figure 2, and statistics reported in the figure legend, an interaction effect showing that neurofeedback differentially affected the SWR rate and count pre- and post-award seems to be missing in the reported statistics.

In the Discussion, the authors write, "Further, because subjects learn to modulate SWR rate, rather than simply generating a single suprathreshold event on command, it is likely that they learn to engage a SWR-permissive state during the targeted interval in which brain-wide neural activity and neuromodulatory tone also enter a SWR-permissive realm". This seems to imply that the neurofeedback is directly modulating neural activity. However, it is unclear from the paper exactly how the neurofeedback is modulating the SWR rate. Considering that SWRs occur during immobility, is it possible that the animals are learning to remain more immobile and modulating the SWR rate in that way?

Reviewer #2 (Public Review):

Gillespie et al. introduced a novel neurofeedback (NF) procedure to train rats in enhancing their sharp-wave ripple (SWR) rate within a short duration, a key neural mechanism associated with memory consolidation. The training, embedded within a spatial memory task, spanned 20-30 days and utilized food rewards as positive reinforcement upon SWR detection. Rats were categorized into NF and control groups, with the NF group further divided into NF and delay trials for within-subject control. While single trial differences were elusive due to the variability of SWR occurrence, the study revealed that statistically rats in NF trials exhibited a notably higher SWR rate before receiving rewards compared to delay trials. This difference was even more pronounced when juxtaposed with rats not exposed to NF training (control group). The unique design of blending the NF phase with the memory dependent spatial task enabled the authors to analyze whether the NF training influence the task performance and replay content during SWRs across three different conditions (NF trials, delay trials and control group). Interestingly, despite the NF training, there was no significant improvement or decline in the performance of the spatial memory task, and the replay content remained consistent across all three conditions. Hence, the operant conditioning only amplified the SWR rate before reward in NF trials without altering the task performance and the replay content during SWR. Moreover, considering the post-reward period, the total SWR count was consistent across all conditions as well, meaning the NF training also do not affect the total SWR count. The study concludes with the hypothesis of a potential homeostatic mechanism governing the total SWR production in rats. This research significantly extends previous work by Ishikawa et al. (2014), offering insights into the NF training with external reward on the SWR rate/counts, replay content and task performance.

Strengths:

- Integration of NF task and spatial memory task in a single trial<br /> The integration of NF training within a spatial memory task poses significant challenges. Gillespie and colleagues overcame this by seamlessly blending the NF task and the spatial memory task into a single trial. Each trial involved a rat undergoing three steps: First, initiating a trial. Second, moving to either the NF port or the delay trial port, as indicated by an LED, and then maintaining a nosepoke at one of the center ports. During this step, the rat had to keep its nose (in the NF port) until a sharp-wave ripple (SWR) exceeding a set threshold was detected, which then triggered a reward, or until a variable time elapsed (in the delay port). Third, the rat would choose one of eight arms to explore before starting the next trial. This integration of the two tasks (step two as the NF task and step three as the spatial memory task) facilitated a direct analysis of the impact of NF training on behaviorally relevant replay content during SWRs and the performance in the spatial memory task.

- Clear Group Separation<br /> A robust study design necessitates clear distinctions between experimental conditions to ensure that observed differences can be attributed to the variable under investigation. This study meticulously categorized rats into three distinct conditions: NF trials, delay trials (for within-subject control), and a control group (for across-subject control). Furthermore, for each trial, the times of interest (TOI) were separated into pre-reward and post-reward periods. This clear separation ensures that any observed differences in SWR rates and other outcomes can be confidently attributed to the effects of neurofeedback training during specific time periods, minimizing potential confounding factors.

- Evidence of SWR rate modulation<br /> The study's results offer compelling evidence that rats can be trained to modulate their SWR rates during the pre-reward period. This is evident from the observation that rats in the NF trials consistently displayed a higher SWR rate before receiving rewards compared to those in delay trials or the control group (Fig. 2). Such findings not only validate the efficacy of the NF paradigm but also underscore the potential of operant conditioning in influencing neural mechanisms. The observation that rats were able to produce larger SWR events by modulating their occurrence rate, rather than merely waiting for these events, suggests a learned strategy to generate them more efficiently.

- Evidence of SWR count homeostasis<br /> A notable finding from the study was the observation of a consistent total SWR count during both pre-reward and post-reward periods across all conditions, despite the evident increase in SWR rates during the pre-reward period in NF trials. This points to a potential homeostatic mechanism governing SWR production in rats. This balance suggests that while NF training can modulate the timing and rate of SWRs over a short duration, it doesn't influence the overall count of SWRs over a longer period. Such a mechanism might be essential in ensuring that the brain neither overcompensates nor depletes its capacity for SWRs, maintaining the overall neural balance and functionality. This discovery deepens our understanding of neural mechanisms and highlights potential avenues for future research into the regulatory processes governing neural activity.

Weaknesses:

- Misleading Title<br /> The title, "Neurofeedback training can modulate task-relevant memory replay in rats," implies that through neurofeedback training, rats can learn to modulate the content of their memory replay. However, the study's findings contradict this implication. Particularly, one of the subtitles of this paper is "Neurofeedback training preserves replay content during SWRs," which directly contrasts with the main title's suggestion. The authors conclusively demonstrated that there was no discernible difference in the replay content between animals that underwent NF training and those that did not. The current title easily leads to misinterpretations about the study's primary outcomes, especially for readers who might not delve into the detailed findings.

- Lack of control analysis baseline for each animal<br /> While the authors meticulously categorized trial types into three distinct conditions: NF trials, delay trials, and control groups, they did not clearly establish a baseline for each animal. The animal could have a total different baseline SWR rates. The paper appears to operate under the assumption that each animal possesses a consistent SWR rate baseline, leading to only the final comparisons being presented.

- Vagueness of what animal really control during NF trials after training<br /> The authors state that, "Moreover, although we did observe a slightly lower mean speed during the pre-reward period on neurofeedback trials compared to delay trials and trials from the control cohort (Supplementary Figure 2F), movement differences could not explain the difference in SWR rates (Supplementary Figure 2G, H)." This assertion raises questions about the underlying mechanisms at play. In a typical operant conditioning scenario, training could result in direct neural modulation, behavioral changes, or a combination of both. For instance, rats might adopt a more stationary posture during the pre-reward period on NF trials compared to other conditions, or they might actively influence the occurrence rate of SWRs during this period. The paper would benefit from a clearer delineation of what the animals are specifically controlling or modulating during the NF trials, ensuring a more comprehensive understanding of the observed effects.

- Clinical Implications<br /> The study was conducted on healthy, young animals but suggests potential benefits for older, cognitively impaired animals. However, it's possible that older or deficit animals might not respond to the NF protocol in the same way.

Reviewer #3 (Public Review):

Summary:

This study implements an innovative neurofeedback procedure in rats, providing food reward upon detection of a sharp wave-ripple event (SWR) in the hippocampus. The elegant experimental design enables a within-animal comparison of the effects of this neurofeedback procedure as compared to a control condition in which an equivalent reward is provided in a non-contingent manner. The neurofeedback procedure was found to increase SWR rate, followed by a compensatory reduction in SWR rate. These changes in SWR rate were not accompanied by any changes in memory performance on the memory-guided task.

Strengths:

The scientific premise for the study is outstanding. It addresses an issue of high importance, of developing ways to not merely describe correlations between SWRs (and their content) and memory performance, but to manipulate them. The authors argue clearly and convincingly that even studies that have performed causal manipulations of SWRs have important confounds and limitations, and most importantly for translational purposes, they are all invasive. So, the idea of developing a potentially non-invasive neurofeedback procedure for modulating SWRs is compelling both as an innovative new experimental manipulation in studies of SWRs, and as a potentially impactful therapeutic avenue.

In addition to addressing an important issue with an innovative approach, the study has many other strengths. The data unambiguously show that the method is effective at increasing SWR rate in each individual subject. The experimental design allows within-subject comparison of neurofeedback and control trials, where the subjects wait an equivalent amount of time. The careful analyses of SWR properties and their content establish that neurofeedback SWRs are comparable to control SWRs. The data add further evidence to the notion that SWR rate is subject to homeostatic control. The paper is also exceptionally well written, and was a pleasure to read. So, there is a clear technical advance, in that there is now a method for increasing SWR rate non-invasively, which is rigorously established and characterized.

Weaknesses:

The one overall limitation I find with this study is that it is unclear to what extent the same (or better) results could have been obtained using behavior-feedback instead of neuro-feedback. Because SWR rates are generally higher during states of quiescence compared to active movement or task engagement, it is possible that reinforcing behaviorally detected quiescent states (e.g. low movement) would indirectly increase SWR rates. The observation that all 4 subjects had lower movement speeds during neurofeedback compared to control trials supports this interpretation. This is an important issue that would help clarify whether the neurofeedback approach is worth the additional effort and expense compared to behavioral feedback.

Reviewer #1 (Public Review):

Summary:<br /> The paper presents a nice study investigating differences in biological motion perception in participants with ADHD in comparison with controls. Motivated by the idea that there is a relationship between biological motion perception and social capabilities, the authors investigated local and global (holistic) biological motion perception, the group, and several additional behavioral variables that are affected in ADHS (IQ, social responsiveness, and attention/impulsivity). As well as local global biological motion perception is reduced in ADHD participants. In addition, the study demonstrates a significant correlation between local biological motion perception skills and the social responsiveness score in the ADHD group, but not the controls. A path analysis in the ADHD data suggests that general performance in biological motion perception is influenced mainly by global biological motion perception performance and attentional and perceptual reasoning skills.

Strengths:<br /> It is true that there exists not much work on biological motion perception and ADHD. Therefore, the presented study contributes an interesting new result to the biological motion literature and adds potentially also new behavioral markers for this clinical condition. The design of the study is straightforward and technically sound, and the drawn conclusions are supported by the presented results.

Weaknesses:<br /> Some of the claims about the relationship between genetic factors and ADHD and the components of biological motion processing have to remain speculative at this point because genetic influences were not explicitly tested in this paper.

Reviewer #2 (Public Review):

Summary:<br /> Tian et al. aimed to assess differences in biological motion (BM) perception between children with and without ADHD, as well as relationships to indices of social functioning and possible predictors of BM perception (including demographics, reasoning ability and inattention). In their study, children with ADHD showed poorer performance relative to typically developing children in three tasks measuring local, global, and general BM perception. The authors further observed that across the whole sample, performance in all three BM tasks was negatively correlated with scores on the social responsiveness scale (SRS), whereas within groups a significant relationship to SRS scores was only observed in the ADHD group and for the local BM task. Local and global BM perception showed a dissociation in that global BM processing was predicted by age, while local BM perception was not. Finally, general (local & global combined) BM processing was predicted by age and global BM processing, while reasoning ability mediated the effect of inattention on BM processing.

Strengths:<br /> Overall, the manuscript is presented in a relatively clear fashion and methods and materials are presented with sufficient detail so the study could be reproduced by independent researchers. The study uses an innovative, albeit not novel, paradigm to investigate two independent processes underlying BM perception. The results are novel and have the potential to have wide-reaching impact on multiple fields.

Weaknesses:<br /> Except for the main analysis, it is unclear what the authors' specific predictions are regarding the three different tasks they employ. The three BM tasks are used to probe different processes underlying BM perception, but it is difficult to gather from the introduction why these three specific tasks were chosen and what predictions the authors have about the performance of the ADHD group in these tasks. Relatedly, the authors do not report whether (and if so, how) they corrected for multiple comparisons in their analyses. As the number of tests one should control for depends on the theoretical predictions (http://daniellakens.blogspot.com/2016/02/why-you-dont-need-to-adjust-you-alpha.html), both are necessary for the reader to assess the statistical validity of the results and any inferences drawn from them. The same is the case for the secondary analyses exploring relationships between the 3 individual BM tasks and social function measured by the social responsivity scale (SRS).

In relation to my prior point, the authors could provide more clarity on how the conclusions drawn from the results relate to their predictions. For example, it is unclear what specific conclusions the authors draw based on their findings that ADHD show performance differences in all three BM perception tasks, but only local BM is related to social function within this group. Here, the claim is made that their results support a specific hypothesis, but it is unclear to me what hypothesis they are actually referring to (see line 343 & following). This lack of clarity is aggravated by the fact that throughout the rest of the discussion, in particular when discussing other findings to support their own conclusions, the authors often make no distinction between the two processes of interest. Lastly, some of the authors' conclusions related to their findings on local vs global BM processing are not logically following from the evidence: For instance, the authors conclude that their data supports the idea that social atypicalities are likely to reduce with age in ADHD individuals. However, according to their own account, local BM perception - the only measure that was related to social function in their study - is understood to be age invariant (and was indeed not predicted by age in the present study).

Results reported are incomplete, making it hard for the reader to comprehensively interpret the findings and assess whether the conclusions drawn are valid. Whenever the authors report negative results (p-values > 0.05), the relevant statistics are not reported, and the data not plotted. In addition, summary statistics (group means) are missing for the main analysis.

Some of the conclusions/statements in the article are too strong and should be rephrased to indicate hypotheses and speculations rather than facts. For example, in lines 97-99 the authors state that the finding of poor BM performance in TD children in a prior study 'indicated inferior applicability' or 'inapplicable experimental design'. While this is one possibility, a perhaps more plausible interpretation could be that TD children show 'poor' performance due to outstanding maturation of the underlying (global) BM processes (as the authors suggest themselves that BM perception can improve with age). There are several other examples where statements are too strong or misleading, which need attention.

Reviewer #3 (Public Review):

Summary:<br /> The authors presented point light displays of human walkers to children (mean = 9 years) with and without ADHD to compare their biological motion perception abilities and relate them to IQ, social responsiveness scale (SRS) scores and age. They report that children with ADHD were worse at all three biological motion tasks, but that those loading more heavily on local processing related to social interaction skills and global processing to age. The important and solid findings are informative for understanding this complex condition, as well as biological motion processing mechanisms in general. However, I am unsure that these differences between local and global skills are truly supported by the data and suggest some further analyses.

Strengths:<br /> The authors present clear differences between the ADHD and TD children in biological motion processing, and this question has not received as much attention as equivalent processing capabilities in autism. They use a task that appears well controlled. They raise some interesting mechanistic possibilities for differences in local and global motion processing, which are distinctions worth exploring. The group differences will therefore be of interest to those studying ADHD, as well as other developmental conditions, and those examining biological motion processing mechanisms in general.

Weaknesses:<br /> I am unsure that the data are strong enough to support claims about differences between global and local processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but do not seem so plausible to me. I am also concerned about gender, and possible autism, confounds when examining the effect of ADHD. Specifics:

Gender confound. There are proportionally more boys in the ADHD than TD group. The authors appear to attempt to overcome this issue by including gender as a covariate. I am unsure if this addresses the problem. The vast majority of participants in the ADHD group are male, and gender is categorically, not continuously, defined. I'm pretty sure this violates the assumptions of ANCOVA.

Autism. Autism and ADHD are highly comorbid. The authors state that the TD children did not have an autism or ADHD diagnosis, but they do not state that the ADHD children did not have an autism diagnosis. Given the nature of the claims, this seems crucial information for the reader.

Conclusions. The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. I think that if the authors wish to make strong claims here they must show inferential stats supporting (1) a difference between ADHD and TD SRS-Task 1 correlations, and (2) a difference in those differences for Task 2 and 3 relative to Task 1. I think they should also show a scatterplot of this correlation, with separate lines of best fit for the two groups, for Tasks 2 and 3 as well. I.e. Figure 4 should have 3 panels. I would recommend the same type of approach for age. Currently, they have small samples for correlations, and are reading much of theoretical significance between some correlations passing significance threshold and others not. It would be incredibly interesting if the social skills (as measured by SRS) only relate to local BM abilities, and age only to global, but I think the data are not so clear with the current information. I would be surprised if all BM abilities did not improve with age. Even if there is some genetic starter kit (and that this differs according to particular BM component), most abilities improve with learning/experience/age.

Theoretical assumptions. The authors make some sweeping statements about local vs global biological motion processing that need to be toned down. They assume that local processing is specifically genetically whereas global processing is a product of experience. The fact their global, but not local, task performance improves with age would tend to suggest there could be some difference here, but the existing literature does not allow for this certainty. The chick studies showing a neonatal preference are controversial and confounded - I cannot remember the specifics but I think there an upper vs lower visual field complexity difference here.

Reviewer #1 (Public Review):

This work describes a new and powerful approach to a central question in ecology: what are the relative contributions of resource utilisation vs interactions between individuals in the shaping of an ecosystem? This approach relies on a very original quantitative experimental set-up whose power lies in its simplicity, allowing an exceptional level of control over ecological parameters and of measurement accuracy.

In this experimental system, the shared resource corresponds to 10^12 copies of a fixed single-stranded target DNA molecule to which 10^15 random single-stranded DNA molecules (the individuals populating the ecosystem) can bind. The binding process is cycled, with a 1000x-PCR amplification step between successive binding steps. The composition of the population is monitored via high-throughput DNA sequencing. Sequence data analysis describes the change in population diversity over cycles. The results are interpreted using estimated binding interactions of individuals with the target resource, as well as estimated binding interactions between individuals and also self-interactions (that can all be directly predicted as they correspond to DNA-DNA interactions). A simple model provides a framework to account for ecosystem dynamics over cycles. Finally, the trajectory of some individuals with high frequency in late cycles is traced back to the earliest cycles at which they are detected by sequencing. Their propensities to bind the resource, to form hairpins, or to form homodimers suggest how different interaction modes shape the composition of the population over cycles.

The authors report a shift from selection for binding to the resource to interactions between individuals and self-interactions over the course of cycles as the main drivers of their ecosystem. The outcome of the experiment is far from trivial as the individual-resource binding energy initially determines the relative enrichment of individuals, and then seems to saturate. The richness of the population dynamics observed with this simple system is thus comparable to that found in some natural ecosystems. The findings obtained with this new approach will likely guide the exploration of natural ecosystems in which parameters and observables are much less accessible.

My review focuses mainly on the experimental aspects of this work given my own expertise. The introduction exposes very convincingly the scientific context of this work, justifying the need for such an approach to address questions pertaining to ecology. The manuscript describes very clearly and rigorously the experimental set-up. The main strengths of this work are (i) the outstanding originality of the experimental approach and (ii) its simplicity. With this setup, central questions in ecology can be addressed in a quantitative manner, including the possibility of running trajectories in parallel to generalize the findings, as reported here. Technical aspects have been carefully implemented, from the design of random individuals bearing flanking regions for PCR amplification, binding selection and (low error) amplification protocols, and sequencing read-out whose depth is sufficient to capture the relevant dynamics. One missing aspect in the data analysis is the quantification of the effect of PCR amplification steps in shaping the ecosystem (to be modeled if significant). In addition, as it stands the current work does not fully harness the power of the approach. For instance, with this setup, one can tune the relative contributions of binding selection vs amplification for instance (to disentangle forces that shape the ecosystem). One can also run cycles with new DNA individuals, designed with arbitrarily chosen resource binding vs self-binding, that are predicted to dominate depending on chosen ecological parameters.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors introduced ADSE, a SELEX-based protocol to explore the mechanism of emergency of species. They used DNA hybridization (to the bait pool, "resources") as the driving force for selection and quantitatively investigated the factors that may contribute to the survival during generation evolution (progress of SELEX cycle), revealing that besides individual-resource binding, the inter- and intra-individual interactions were also important features along with mutualism and parasitism.

Strengths:<br /> The design of using pure biochemical affinity assay to study eco-evolution is interesting, providing an important viewpoint to partly explain the molecular mechanism of evolution.

Weaknesses:<br /> Though the evidence of the study is somewhat convincing, some aspects still need to be improved, mostly technical issues.

Reviewer #1 (Public Review):

In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, the authors also make some overly broad conclusions, for which additional experiments/data analyses should be explored to improve confidence in their findings. Furthermore, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and weaknesses of the manuscript.

Strengths<br /> 1. The authors report high-quality NRPE1 ChIP-seq data, allowing them to directly test how and where Pol V occupancy depends on histone H1 function in Arabidopsis.<br /> 2. nrpe1 mutants generated via CRISPR/Cas9 in the h1 mutant background (nrpe1 h1.1-1 h1.2-1 triple mutants), allow the authors to study the role of Pol V in ectopic DNA methylation in H1-deficient plants.<br /> 3. Pol V recruitment via ZincFinger-DMS3 expression (a modified version of Pol V's DMS3 recruitment factor) sends Pol V to new genomic loci and thus provides the authors with an innovative dataset for understanding H1 function at these sites.

Weaknesses<br /> 1. The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background.<br /> 2. Statistical tests are missing throughout and are needed to support several of the authors' claims.<br /> 3. The SUVH1-3xFLAG ChIP-seq analyses in Fig. 6 require additional controls and are not fully explained in the results. The broad conclusions drawn (based on those experiments) are thus not convincing.

Previous studies have charted the relationship between H1 function and RNA-directed DNA methylation (RdDM) via analyses of Pol IV-dependent 24 nt siRNAs and factors that recruit Pol IV (Choi et al., 2021 and Papareddy et al., 2020). Harris and colleagues have extended this work and shown that histone H1 function also antagonizes Pol V occupancy in the context of constitutive heterochromatin. The authors thus provide important evidence to show that H1 limits the encroachment of both polymerases Pol IV and Pol V into plant heterochromatin.

Reviewer #2 (Public Review):

Summary:<br /> The main conclusion of the manuscript is that the presence of linker Histone H1 protects Arabidopsis pericentromeric heterochromatic regions and longer transposable elements via chromatin compaction from encroachment by other repressive pathways. The manuscript focuses on the RNA-dependent DNA-methylation (RdDM) pathway but indirectly finds that other pathways must also be ectopically enriched.

Strengths:<br /> The authors present diverse sets of genomic data comparing Arabidopsis wild-type and h1 mutant background allowing an analysis of differential recruitment of RdDM component NPRE1, which is related to changes in DNA methylation and H1 coverage. As an addendum, the manuscript also contains recruitment data for SUVH1 in wild-type and h1 mutant backgrounds.

Furthermore, the authors make use of a line that recruits NRPE1 ectopically to show that H1 occupancy is not altered because of this recruitment. These are negative data, but well supported.

Weaknesses:<br /> The manuscript mostly confirms earlier observations but shows very limited novelty. It has already been reported that different classes of TEs show a differential response with respect to DNA methylation in absence of H1. Furthermore, the fact that loss of H1 affect global chromatin accessibility was recently published by Teano et al. in Cell reports (Volume 42, Issue 8, 29 August 2023). The authors have neither cited this report (that had been available since 2021 in BioRxiv), nor set their work in context to this study. The study by Teano showed that for some TEs, loss of H1 is related to a switch from DNA-methylation dependent repressive pathways to Polycomb Group-dependent pathways. The current manuscript could have looked at overlapping classes and integrated information from both studies, which would be particularly interesting for the examples illustrated in Figure 5b, showing examples of TEs that lose NRPE1 targeting and methylation in all contexts in H1 deletion mutants.

The proposed mechanism is that RdDM along with many other chromatin factors re-distribute to heterochromatic regions in h1 mutants because these regions are more accessible. There is a general problem with measuring the "difference in chromatin compaction" with methods that mostly resolve highly accessible chromatin in contrast to any other chromatin, such as ATAC-seq or DNAse-seq (employed in this manuscript). The changes in the regions of interest are so subtle that they are not easily detected at the level of individual genes, although they become usually more obvious in metagene plots. The general question is if this inadequate method is sufficient to draw strong conclusions on chromatin compaction, but to be fair, the current manuscript is not alone in using this method without pointing out certain caveats.

As a consequence of redistribution to heterochromatic sites, the authors postulate that there are also sites that lose RdDM coverage in h1, but these sites are not really evidenced in the report.<br /> Unfortunately, another weakness is that it is not possible to make easy use of the analysis from the available material as the current manuscript does not contain supplemental data indicating which TEs were and DMRs were considered in classes such as "long", "short", "heterochromatic", "euchromatic", "Class A", "Class B", "CMT2 dependent hypo-CHH", "DRM2 dependent CHH", "dynamic RdDM" etc. Since the bioinformatics pipelines are poorly documented (absence of dedicated script archive), the analysis cannot be easily recapitulated.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

Concerns<br /> 1. The evidence from this previous work for mixtures between different conflict sources makes the framing of 'infinite possible types of conflict' feel like a strawman. The authors cite classic work (e.g., Kornblum et al., 1990) that develops a typology for conflict which is far from infinite. I think few people would argue that every possible source and level of difficulty will have to be learned separately. This work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with the n-back, MOT, and random dot motion literature).

2. The degree of Stroop vs Simon conflict is perfectly negatively correlated across conditions. This limits their interpretation of an integrated cognitive space, as they cannot separately measure Stroop and Simon effects. The author's control analyses have limited ability to overcome this task limitation. While these results are consistent with parametric encoding, they cannot adjudicate between combined vs separated representations.

Reviewer #2 (Public Review):

This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors use a novel experimental design in which different types of response conflict (spatial Stroop, Simon) are parametrically manipulated. These conflict types are hypothesized to be encoded jointly, within an abstract "cognitive space", in which distances between task conditions depend only on the similarity of conflict types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon conflicts are represented with similar activity patterns). Authors contrast such a representational scheme for conflict with several other conceptually distinct schemes, including a domain-general, domain-specific, and two task-specific schemes. The authors conduct a behavioral and fMRI study to test which of these coding schemes is used by prefrontal cortex. Replicating the authors' prior work, this study demonstrates that sequential behavioral adjustments (the congruency sequence effect) are modulated as a function of the similarity between conflict types. In fMRI data, univariate analyses identified activation in left prefrontal and dorsomedial frontal cortex that was modulated by the amount of Stroop or Simon conflict present, and representational similarity analyses (RSA) that identified coding of conflict similarity, as predicted under the cognitive space model, in right lateral prefrontal cortex.

This study tackles an important question regarding how distinct types of conflict might be encoded in the brain within a computationally efficient representational format. The ideas postulated by the authors are interesting ones and the statistical methods are generally rigorous. The evidence supporting the authors claims, however, is limited by confounds in the experimental design and by lack of clarity in reporting the testing of alternative hypotheses within the method and results.

(1) Model comparison

The authors commendably performed a model comparison within their study, in which they formalized alternative hypotheses to their cognitive space hypothesis. We greatly appreciate the motivation for this idea and think that it strengthened the manuscript. Nevertheless, some details of this model comparison were difficult for us to understand, which in turn has limited our understanding of the strength of the findings.

The text indicates the domain-general model was computed by taking the difference in congruency effects per conflict condition. Does this refer to the "absolute difference" between congruency effects? In the rest of this review, we assume that the absolute difference was indeed used, as using a signed difference would not make sense in this setting. Nevertheless, it may help readers to add this information to the text.

Regarding the Stroop-Only and Simon-Only models, the motivation for using the Jaccard metric was unclear. From our reading, it seems that all of the other models --- the cognitive space model, the domain-general model, and the domain-specific model --- effectively use a Euclidean distance metric. (Although the cognitive space model is parameterized with cosine similarities, these similarity values are proportional to Euclidean distances because the points all lie on a circle. And, although the domain-general model is parameterized with absolute differences, the absolute difference is equivalent to Euclidean distance in 1D.) Given these considerations, the use of Jaccard seems to differ from the other models, in terms of parameterization, and thus potentially also in terms of underlying assumptions. Could authors help us understand why this distance metric was used instead of Euclidean distance? Additionally, if Jaccard must be used because this metric seems to be non-standard in the use of RSA, it would likely be helpful for many readers to give a little more explanation about how it was calculated.

When considering parameterizing the Stroop-Only and Simon-Only models with Euclidean distances, one concern we had is that the joint inclusion of these models might render the cognitive space model unidentifiable due to collinearity (i.e., the sum of the Stroop-Only and Simon-Only models could be collinear with the cognitive space model). Could the authors determine whether this is the case? This issue seems to be important, as the presence of such collinearity would suggest to us that the design is incapable of discriminating those hypotheses as parameterized.

(2) Issue of uniquely identifying conflict coding

We certainly appreciate the efforts that authors have taken to address potential confounders for encoding of conflict in their original submission. We broach this question not because we wish authors to conduct additional control analyses, but because this issue seems to be central to the thesis of the manuscript and we would value reading the authors' thoughts on this issue in the discussion.

To summarize our concerns, conflict seems to be a difficult variable to isolate within aggregate neural activity, at least relative to other variables typically studied in cognitive control, such as task-set or rule coding. This is because it seems reasonable to expect that many more nuisance factors covary with conflict --- such as univariate activation, level of cortical recruitment, performance measures, arousal --- than in comparison with, for example, a well-designed rule manipulation. Controlling for some of these factors post-hoc through regression is commendable (as authors have done here), but such a method will likely be incomplete and can provide no guarantees on the false positive rate.

Relatedly, the neural correlates of conflict coding in fMRI and other aggregate measures of neural activity are likely of heterogeneous provenance, potentially including rate coding (Fu et al., 2022), temporal coding (Smith et al., 2019), modulation of coding of other more concrete variables (Ebitz et al., 2020, 10.1101/2020.03.14.991745; see also discussion and reviews of Tang et al., 2016, 10.7554/eLife.12352), or neuromodulatory effects (e.g., Aston-Jones & Cohen, 2005). Some of these origins would seem to be consistent with "explicit" coding of conflict (conflict as a representation), but others would seem to be more consistent with epiphenomenal coding of conflict (i.e., conflict as an emergent process). Again, these concerns could apply to many variables as measured via fMRI, but at the same time, they seem to be more pernicious in the case of conflict. So, if authors consider these issues to be germane, perhaps they could explicitly state in the discussion whether adopting their cognitive space perspective implies a particular stance on these issues, how they interpret their results with respect to these issues, and if relevant, qualify their conclusions with uncertainty on these issues.

(3) Interpretation of measured geometry in 8C

We appreciate the inclusion of the measured similarity matrices of area 8C, the key area the results focus on, to the supplemental, as this allows for a relatively model-agnostic look at a portion of the data. Interestingly, the measured similarity matrix seems to mismatch the cognitive space model in a potentially substantive way. Although the model predicts that the "pure" Stroop and Simon conditions will have maximal self-similarity (i.e., the Stroop-Stroop and Simon-Simon cells on the diagonal), these correlations actually seem to be the lowest, by what appears to be a substantial margin (particularly the Stroop-Stroop similarities). What should readers make of this apparent mismatch? Perhaps authors could offer their interpretation on how this mismatch could fit with their conclusions.

Reviewer #3 (Public Review):

Yang and colleagues investigated whether information on two task-irrelevant features that induce response conflict is represented in a common cognitive space. To test this, the authors used a task that combines the spatial Stroop conflict and the Simon effect. This task reliably produces a beautiful graded congruency sequence effect (CSE), where the cost of congruency is reduced after incongruent trials. The authors measured fMRI to identify brain regions that represent the graded similarity of conflict types, the congruency of responses, and the visual features that induce conflicts. They applied univariate, multivariate, and connectivity analyses to fMRI data to identify brain regions that represent the graded similarity of conflict types, the congruency of responses, and the visual features that induce conflicts. They further directly assessed the dimensionality of represented conflict space.

The authors identified the right dlPFC (right 8C), which shows 1) stronger encoding of graded similarity of conflicts in incongruent trials and 2) a positive correlation between the strength of conflict similarity type and the CSE on behavior. The dlPFC has been shown to be important for cognitive control tasks. As the dlPFC did not show a univariate parametric modulation based on the higher or lower component of one type of conflict (e.g., having more spatial Stroop conflict or less Simon conflict), it implies that dissimilarity of conflicts is represented by a linear increase or decrease of neural responses. Therefore, the similarity of conflict is represented in multivariate neural responses that combine two sources of conflict.

The strength of the current approach lies in the clear effect of parametric modulation of conflict similarity across different conflict types. The authors employed a clever cross-subject RSA that counterbalanced and isolated the targeted effect of conflict similarity, decorrelating orientation similarity of stimulus positions that would otherwise be correlated with conflict similarity. A pattern of neural response seems to exist that maps different types of conflict, where each type is defined by the parametric gradation of the yoked spatial Stroop conflict and the Simon conflict on a similarity scale. The similarity of patterns increases in incongruent trials and is correlated with CSE modulation of behavior.

The main significance of the paper lies in the evidence supporting the use of an organized "cognitive space" to represent conflict information as a general control strategy. The authors thoroughly test this idea using multiple approaches and provide convincing support for their findings. However, the universality of this cognitive strategy remains an open question.

The task presented in the study involved two sources of conflict information through a single salient visual input, which might have encouraged the utilization of a common space. The similarity space was analyzed at the level of between-individuals (i.e., cross-subject RSA) to mitigate potential confounds in the design, such as congruency and the orientation of stimulus positions. This approach makes it challenging to establish a direct link between the quality of conflict space representation and the patterns of behavioral adaptations within individuals.

Furthermore, it remains unclear at which cognitive stages during response selection such a unified space is recruited. Can we effectively map any sources of conflict into a single scale? Is this unified space adaptively adjusted within the same brain region? Additionally, does the amount of conflict solely define the dimensions of this unified space across many conflict-inducing tasks? These questions remain open for future studies to address.

Taken together, this study presents an exciting possibility that information requiring high levels of cognitive control could be flexibly mapped into cognitive map-like representations that both benefit and bias our behavior. Further characterization of the representational geometry and generalization of the current results look promising ways to understand representations for cognitive control.

Reviewer #1 (Public Review):

Summary:<br /> The authors developed computational models that capture the electrical and Ca2+ signaling behavior in mesenteric arterial cells from male and female mice. A baseline model was first formulated with eleven transmembrane currents and three calcium compartments. Sex-specific differences in the L-type calcium channel and two voltage-gated potassium channels were then tuned based on experimental measurements. To incorporate the stochastic ion channel openings seen in smooth muscle cells under physiological conditions, noise was added to the membrane potential and the sarcoplasmic Ca2+ concentration equations. Finally, the models were assembled into 1D vessel representations and used to investigate the tissue-level electrical response to an L-type calcium channel blocker.

Strengths:<br /> A major strength of the paper is that the modeling studies were performed on three different scales: individual ionic currents, whole-cell, and 1D tissue. This comprehensive computational framework can help provide mechanistic insight into arterial myocyte function that might be difficult to achieve through traditional experimental methods.

The authors aimed to develop sex-specific computational models of mesenteric arterial myocytes and demonstrate their use in drug-testing applications. Throughout the paper, model behavior was both validated by experimental recordings and supported by previously published data. The main findings from the models suggested that sex-specific differences in membrane potential and Ca2+ handling are attributable to variability in the gating of a small number of voltage-gated potassium channels and L-type calcium channels. This variability contributes to a higher Ca2+ channel blocker sensitivity in female arterial vessels. Overall, the study successfully met the aims of the paper.

Weaknesses:<br /> A main weakness of the paper, as addressed by the authors, is the simplicity of the 1D vessel model; it does not take into account various signaling pathways or interactions with other cell types which could impact smooth muscle electrophysiology. Another potential shortcoming is the use of mouse data for optimizing the model, as there could be discrepancies in signaling behavior that limit the translatability to human myocyte predictions.

Reviewer #2 (Public Review):

In this study, Hernandez-Hernandez et al developed a gender-dependent mathematical model of arterial myocytes based on a previous model and new experimental data. The ionic currents of the model and its sex difference were formulated based on patch-clamp experimental data, and the model properties were compared with single-cell and tissue scale experimental results. This is a study that is of importance for the modeling field as well as for experimental physiology.

Reviewer #3 (Public Review):

Summary:<br /> This hybrid experimental/computational study by Hernandez-Hernandez sheds new light on sex-specific differences between male and female arterial myocytes from resistance arteries. The authors conduct careful experiments in isolated myocytes from male and female mice to obtain the data needed to parameterize sex-specific models of two important ionic currents (i.e., those mediated by CaV1.2 and KV2.1). Available experimental data suggest that KV1.5 channel currents from male and female myocytes are similar, but simulations conducted in the novel Hernandez-Hernandez sex-specific models provide a more nuanced view. This gives rise to the first of the authors' three key scientific claims: (1) In males, KV1.5 is the dominant current regulating membrane potential; whereas, in females, KV2.1 plays a primary role in voltage regulation. They further show that this (2) the latter distinction drives drive sex-specific differences in intracellular Ca2+ and cellular excitability. Finally, working with one-dimensional models comprising several copies of the male/female myocyte models linked by resistive junctions, they use simulations to (3) predict that the sensitivity of arterial smooth muscle to Ca2+ channel-blocking drugs commonly used to treat hypertension is heightened in female compared to male cells.

Strengths:<br /> • The Methodology is described in exquisite detail in straightforward language that will be easy to understand for most if not all peer groups working in computational physiology. The authors have deployed standard protocols (e.g., parameter fitting as described by Kernik et al., sensitivity analysis as described by Sobie et al.) and appropriate brief explanations of these techniques are provided. The manoeuvre used to represent stochastic effects on voltage dynamics is particularly clever and something I have not personally encountered before. Collectively, these strengthen the credibility of the model and greatly enrich the manuscript.

• Broadly speaking, the Results section describes findings that robustly support the three key scientific claims outlined in my summary. While there is certainly room for further discussion of some nuanced points as outlined below, it is evident these experiments were carefully designed and carried out with care and intentionality. In the present version of the manuscript, there are a few figures in which experimental data is shown side-by-side with outputs from the corresponding models. These are an excellent illustration of the power of the authors' novel sex-specific computational simulation platform. I think these figures will benefit from some modest additional quantitative analysis to substantiate the similarities between experimental and computational data, but there is already clear evidence of a good match.

Areas for Improvement:<br /> • The authors used experimental data from a prior publication to calibrate their model of the BKCa current. As indicated in the manuscript, these data are for channel activity measured in a heterologous expression system (Xenopus oocytes). A similar principle applies to other major ion channels/pumps/etc. Is it possible there might be relevant sex-specific differences in these players as well? In the context of the present work, this feels like an important potential caveat to highlight, in case male/female differences in the activity of BKCa or other currents might influence model-predicted differences (e.g., the relative importance of KV1.5 and KV2.1). This should be discussed, and, if possible, related to the elegant sensitivity analysis presented in Fig. 5C (which shows, for example, that the models are relatively insensitive to variation in GBK).

• The authors state that their model can be expanded to 2D/3D applications, "transitioning seamlessly from single-cell to tissue-level simulations". I would like to see more discussion of this. For example, given the modest complexity of the cell-scale model, how considerable would the computational burden be to implement a large network model of a subset of the human female or male arterial system? Are there sex-specific differences in vessel and/or network macro-structure that would need to be considered? How would this influence feasibility? Rather than a 1D cable as implemented here, I imagine a multi-scale implementation would involve the representation of myocytes wrapped around vessels. How would the behaviour of such a system differ from the authors' presented work using a 1D representation of 100 myocytes coupled end-to-end? Could these differences partially explain why the traces in Fig. 8D are smoother than those in Fig. 8C? From my standpoint, discussing these points would enrich the paper.

• The nifedipine data presented in Fig. 9 are quite compelling, and a nice demonstration of the potential power of the new models. How does this relate to what is known about the clinical male/female responses to nifedipine? Are there sex differences in drug efficacy?

Reviewer #1 (Public Review):

The authors previously showed in cell culture that Su(H), the transcription factor mediating Notch pathway activity, was phosphorylated on S269 and they found that a phospho-deficient Su(H) allele behaves as a moderate gain of Notch activity in flies, notably during blood cell development. Since a downregulation of Notch signaling was proposed to be important for the production of a specialized blood cell types (lamellocytes) in response to wasp parasitism, the authors hypothesized that Su(H) phosphorylation might be involved in this cellular immune response.

Consistent with their hypothesis, the authors show that Su(H)S269A knock-in flies display a reduced response to wasp parasitism and that Su(H) is phosphorylated upon infestation. Using in vitro kinase assays and a genetic screen, they identify the PKCa family member Pkc53E as the putative kinase involved in Su(H) phosphorylation and they show that Pkc53E can bind Su(H). They further show that Pkc53E deficit or its knock-down in larval blood cells results in similar blood cell phenotypes as Su(H)S269A, including a reduced response to wasp parasitism, and their epistatic analyses indicate that Pkc53E acts upstream of Su(H).

Strengths<br /> The manuscript is well presented and the experiments are sound, with a good combination of genetic and biochemical approaches and several clear phenotypes which back the main conclusions. Notably Su(H)S269A mutation or Pkc53E deficiency strongly reduces lamellocyte production and the epistatic data are convincing.

Weaknesses<br /> The phenotypic analysis of larval blood cells remains rather superficial. Looking at melanized cells is a crude surrogate to quantify crystal cell numbers as it is biased toward sessile cells (with specific location) and does not bring information concerning the percentage of blood cells differentiated along this lineage.

In Su(H)S269A knock-in or Pkc53E zygotic mutants, the increase in crystal cells in uninfected conditions and the decreased capacity to induce lamellocytes following infection could have many origins which are not investigated. For instance, premature blood cell differentiation could promote crystal cell differentiation and reduce the pool of lamellocytes progenitors. These mutations could also affect the development and function of the posterior signaling center in the lymph gland, which plays a key role in lamellocyte induction. Similarly, the mild decrease on resistance to wasp infestation (Fig. 2A) could reflect a constitutive reduction in blood cell numbers in Su(H)S269A larvae rather than a defective down-regulation of Notch activity.<br /> Whereas the authors also present targeted-knock down/inhibition of Pkc53E suggesting that this enzyme is required in blood cells to control crystal cell fate (Fig. 6), it is somehow misleading to use lz-GAL4 as a driver in the lymph gland and hml-GAL4 in circulating hemocytes as these two drivers do not target the same blood cell populations/steps in the crystal cell development process.

In addition, the authors do not present evidence that Pkc55E function (and Su(H) phosphorylation) is required specifically in blood cells to promote lamellocyte production in response to infestation.

Finally, the conclusion that Pkc53E is (directly) responsible for Su(H) phosophorylation needs to be strengthened. Most importantly, the authors do not demonstrate that Pkc53E is required for Su(H) phosphorylation in vivo (i.e. that Su(H) is not phosphorylated in the absence of Pkc53E following infestation). In addition, the in vitro kinase assays with bacterially purified Pkc53E (in the presence of PMA or using an activated variant of Pkc53E) only reveal a weak activity on a Su(H) peptide encompassing S269 (Fig. 4). Moreover, while the authors show a coIP between an overexpressed Pkc53E and endogenous Su(H) (Fig. 7) (in the absence of infestation), it has recently been reported that Pkc53E is a cytoplasmic protein in the eye (Shieh et al. 2023), calling for a direct assessment of Pkc53E expression and localization in larval blood cells under normal conditions and upon infestation. Furthermore, the effect of the PKCa agonist PMA on Su(H)-induced reporter gene expression in cell culture and crystal cell number in vivo is somehow consistent with the authors hypothesis, but some controls are missing (notably western blots to show that PMA/Staurosporine treatment does not affect Su(H)-VP16 level) and it is unclear why STAU treatment alone promotes Su(H)-VP16 activity (in their previous reports, the authors found no difference between Su(H)S269A-VP16 and Su(H)-VP16) or why PMA treatment still has a strong impact on crystal cell number in Su(H)S269A larvae.

Reviewer #2 (Public Review):

Summary: The current draft by Deischel et.al., entitled "Inhibition of Notch activity by phosphorylation of CSL in response to parasitization in Drosophila" decribes the role of Pkc53E in the phosphorylation of Su(H) to downregulate its transcriptional activity to mount a successful immune response upon parasitic wasp-infection. Overall, I find the study interesting and relevant especially the identification of Pkc53E in phosphorylation of Su(H) is very nice. However, I have a number of concerns with the manuscript which are central to the idea that link the phosphorylation of Su(H) via Pkc53E to implying its modulation of Notch activity. I enlist them one by one subsequently.

Strengths: I find the study interesting and relevant especially because of the following:<br /> 1. The identification of Pkc53E in phosphorylation of Su(H) is very interesting.<br /> 2. The role of this interaction in modulating Notch signaling and thereafter its requirement in mounting a strong immune response to wasp infection is also another strong highlight of this study.

Weaknesses:1. Epistatic interaction with Notch is needed: In the entire draft, the authors claim Pkc53E role in the phosphorylation of Su(H) is down-stream of notch activity. Given the paper title also invokes Notch, I would suggest authors show this in a direct epistatic interaction using a Notch condition. If loss of Notch function makes many more lamellocytes and GOF makes less, then would modulating Pkc53E (and SuH)) in this manifest any change? In homeostasis as well, given gain of Notch function leads to increased crystal cells the same genetic combinations in homeostasis will be nice to see.<br /> While I understand that Su(H) functions downstream of Notch, but it is now increasingly evident that Su(H) also functions independent of Notch. An epistatic relationship between Notch and Pkc will clarify if this phosphorylation event of Su(H) via Pkc is part of the canonical interaction being proposed in the manuscript and not a non-canoncial/Notch pathway independent role of Su(H).

This is important, as I worry that in the current state, while the data are all discussed inlight of Notch activity, any direct data to show this affirmatively is missing. In our hands we do find Notch independent Su(H) function in immune cells, hence this is a suggestion that stems from our own personal experience.

2. Temporal regulation of Notch activity in response to wasp-infection and its overlapping dynamics of Su(H) phosphorylation via Pkc is needed: First, I suggest the authors to show how Notch activity post infection in a time course dependent manner is altered. A RT-PCR profile of Notch target genes in hemocytes from infected animals at 6, 12, 24, 48 HPI, to gauge an understanding of dynamics in Notch activity will set the tone for when and how it is being modulated. In parallel, this response in phospho mutant of Su(H) will be good to see and will support the requirement for phosphorylation of Su(H) to manifest a strong immune response. Second, is the dynamics of phosphorylation in a time course experiment is missing. While the increased phosphorylation of Su(H) in response to wasp-infestation shown in Fig.2B is using whole animal, this implies a global down-regulation of Su(H)/Notch activity. The authors need to show this response specifically in immune cells. The reader is left to the assumption that this is also true in immune cells. Given the authors have a good antibody, characterizing this same in circulating immune cells in response to infection will be needed. A time course of the phosphorylation state at 6, 12, 24, 48 HPI, to guage an understanding of this dynamics is needed. The authors suggest, this mechanism may be a quick way to down-regulate Notch, hence a side by side comparison of the dynamics of Notch down-regulation (such as by doing RT-PCR of Notch target genes following different time point post infection) alongside the levels of pS269 will strengthen the central point being proposed. Last, in Fig7. the authors show Co-immuno-precipitation of Pkc53EHA with Su(H)gwt-mCh 994 protein from Hml-gal4 hemocytes. I understand this is in homeostasis but since this interaction is proposed to be sensitive to infection, then a Co-IP of the two in immune cells, upon infection should be incorporated to strengthen their point.

3. In Fig 5B, the authors show the change in crystal cell numbers as read out of PMA induced activation of Pkc53E and subsequent inhibition of Su(H) transcriptional activity, I would suggest the authors use more direct measures of this read out. RT-PCR of Su(H) target genes, in circulating immune cells, will strengthen this point. Formation of crystal cells is not just limited to Notch, I am not convinced that this treatment or the conditions have other affect on immune cells, such as any impact on Hif expression may also lead to lowering of CC numbers. Hence, the authors need to strengthen this point by showing that effects are direct to Notch and Su(H) and not non-specific to any other pathway also shown to be important for CC development.

4. In addition to the above mentioned points, the data needs to be strengthened to further support the main conclusions of the manuscript. I would suggest the authors present the infection response with details on the timing of the immune response. Characterization of the immune responses at respective time points (as above or at least 24 and 48 HPI, as norms in the field) will be important. Also, any change in overall cell numbers, other immune cells, plasmatocytes or CC post infection is missing and is needed to present the specificity of the impact. The addition of these will present the data with more rigor in their analysis.

5. Finally, what is the view of the authors on what leads to activation of Pkc53E, any upstream input is not presented. It will be good to see if wasp infection leads to increased Pkc53 kinase activity.

Overall, I think the findings in the current state are interesting and fill an important gap, but the authors will need to strengthen the point with more detailed analysis that includes generating new data and also presenting the current data with more rigor in their approach. The data have to showcase the relationship with Notch pathway modulation upon phosphorylation of CSL in a much more comprehensive way, both in homeostasis and in response to infection which is entirely missing in the current draft.

Reviewer #3 (Public Review):

Diechsel et al. provide important and valuable insights into how Notch signalling is shut down in response to parasitic wasp infestation in order to suppress crystal cell fate and favour lamellocyte production. The study shows that CSL transcription factor Su(H) is phosphorylated at S269A in response to parasitic wasp infestation and this inhibitory phosphorylation is critical for shutting down Notch. The authors go on to perform a screen for kinases responsible for this phosphorylation and have identified Pkc53E as the specific kinase acting on Su(H) at S269A. Using analysis of mutants, RNAi and biochemistry-based approaches the authors convincingly show how Pkc53E-Su(H) interaction is critical for remodelling hematopoiesis upon wasp challenge. The data presented supports the overall conclusions made by the authors. There are a few points below that need to be addressed by the authors to strengthen the conclusions:

1) The authors should check melanized crystal cells in Su(H)gwt and Su(H)S269A in presence of PMA and Staurosporine?<br /> 2) Data for number of dead pupae, flies eclosed, wasps emerged post infestation should be monitored for the following genotypes and should be included: Pkc53EΔ28, Su(H)S269A, Pkc53EΔ28 Su(H)S269A, Su(H)S269D, Su(H)S269D Pkc53EΔ28<br /> 3) The exact molecular trigger for activation of Pkc53E upon wasp infestation is not clear.<br /> 4) The authors should check if activating ROS alone or induction of Calcium pulses/DUOX activation can mimic this condition and can trigger activation of Pkc53E and thereby cause phosphorylation of Su(H) at S269<br /> 5) Does Pkc53E get activated during sterile inflammation?

Joint Public Review:

The study has many limitations which need to be addressed and there is a lack of functional explanation of carriage. These limitations are: a) the lack of inclusion of non-Black patients; and b) the lack of appropriate explanation if results are false-positive since APOL1 provides risk for chronic renal disease (CRD) and patients with CRD are predisposed to sepsis. Sepsis occurred in 565 Black subjects, of whom 105 (29% ) had APOL1 high-risk genotype and 460 had-low risk genotype. Importantly, the risk for sepsis associated with APOL1 HR variants was no longer significant after adjusting for subjects pre-existing severe renal disease or after excluding these subjects. Thus, the susceptibility pathway seems to be: APOL1 variants > CKD > sepsis diathesis.