Reviewer #1 (Public review):

This work introduces and describes a useful curation pipeline of antibody-antigen structures downloaded from the PDB database. The antibody-antigen structures are presented in a new database called AACDB - with associated website - alongside annotations that were either corrected from those present in the PDB database, or added de-novo with solid methodology. Sequences, structures and annotations can be very easily downloaded from the AACDB website, speeding up the development of structure-based algorithms and analysis pipelines to characterize antibody-antigen interactions. However, AACDB is missing some important annotations that I believe would greatly enhance its usefulness, such as binding affinity annotations.

I think the potentially most significant contribution of this database is the manual data curation to fix errors present in the PDB entries, by cross-referencing with the literature. The authors also seem to describe, whenever possible, the procedures they took to correct the annotations.

I have personally verified some of the examples presented by the authors, and found that SAbDab appears to fix the mistakes related to mis-identification of antibody chains, but not other annotations.

"(1) the species of the antibody in 7WRL was incorrectly labeled as "SARS coronavirus B012" in both PDB and SabDab" → I have verified the mistake and fix, and that SAbDab does not fix is, just uses the pdb annotation.<br /> "(2) 1NSN, the resolution should be 2.9 , but it was incorrectly labeled as 2.8" → I have verified the mistake and fix, and that saabdab does not fix it, just uses the PDB annotation.<br /> "(3) mislabeling of antibody chains as other proteins (e.g. in 3KS0, the light chain of B2B4 antibody was misnamed as heme domain of flavocytochrome b2)" → SAbDab fixes this as well in this case.<br /> "(4) misidentification of heavy chains as light chains (e.g. both two chains of antibody were labeled as light chain in 5EBW)" → SAbDab fixes this as well in this case.

I believe the splitting of the pdb files is a valuable contribution as it standardizes the distribution of antibody-antigen complexes. Indeed, there is great heterogeneity in how many copies of the same structure are present in the structure uploaded to the PDB, generating potential artifacts for machine learning applications to pick up on. That being said, I have two thoughts both for the authors and the broader community. First, in the case of multiple antibodies binding to different epitopes on the same antigen, one should not ignore the potentially stabilizing effect that the binding of one antibody has on the complex, thereby enabling the binding of the second antibody. In general, I urge the community to think about what is the most appropriate spatial context to consider when modeling the stability of interactions from crystal structure data. Second, and in a similar vein, some antigens occur naturally as homomultimers - e.g. influenza hemagglutinin is a homotrimer. Therefore, to analyze the stability of a full-antigen-antibody structure, I believe it would be necessary to consider the full homo-trimer, whereas in the current curation of AACDB with the proposed data splitting, only the monomers are present.

I think the annotation of interface residues is a very useful addition to structural datasets.

I am, however, not convinced of the utility of *change* in SASA as a useful metric for identifying interacting residues, beyond what is already identified via pairwise distances between the antibody and antigen residues. If we had access to the unbound conformation of most antibodies and antigens, then we could analyze the differences in structural conformations upon binding, which can be in part quantified by change in SASA. However, as only bound structures are usually available, one is usually force to approximate a protein's unbound structure by computationally removing its binding partner - as it seems to me the authors of this work are doing.

Some obvious limitations of AACDB in its current form include:

AACDB only contains entries with protein-based antigens of at most 50 amino-acids in length. This excludes non-protein-based antigens, such as carbohydrate- and nucleotide-based, as well as short peptide antigens.<br /> AACDB does not include annotations of binding affinity, which are present in SAbDab and have been proven useful both for characterizing drivers of antibody-antigen interactions (cite https://www.sciencedirect.com/science/article/pii/S0969212624004362?via%3Dihub) and for benchmarking antigen-specific antibody-design algorithms (cite https://www.biorxiv.org/content/10.1101/2023.12.10.570461v1))

Reviewer #2 (Public review):

Summary:

Antibodies, thanks to their high binding affinity and specificity to cognate protein targets, are increasingly used as research and therapeutic tools. In this work, Zhou et al. have created, curated and made publicly available a new database of antibody-antigen complexes to support research in the field of antibody modelling, development and engineering.

Strengths:

The authors have performed a manual curation of antibody-antigen complexes from the Protein Data Bank, rectifying annotation errors; they have added two methods to estimate paratope-epitope interfaces; they have produced a web interface capable of effective visualisation and of summarising the key useful information in one page. The database is also cross-linked to other databases that contain information relevant to antibody developability and therapeutic applications.

Weaknesses:

The database does not import all the experimental information from PDB and contains only complexes with large protein targets.

Comments on revisions: I thank the authors for having incorporated my feedback and I look forward to the next releases of this database.

Reviewer #1 (Public review):

In their manuscript, Papadopoli et al explore the role of ETFDH in transformation. They note that ETFDH protein levels are decreased in cancer, and that deletion of ETFDH in cancer cell lines results in increased tumorigenesis, elevated OXPHOS and glycolysis, and a reduction in lipid and amino acid oxidation. The authors attribute these effects to increased amino acid levels stimulating mTORC1 signaling and driving alterations in BCL6 and EIF4EBP1. They conclude that ETFDH1 is epigenetically silenced in a proportion of neoplasms, suggesting a tumor-suppressive function. Overall, the authors logically present clear data and perform appropriate experiments to support their hypotheses. I only have a few minor points related to the semantics of a few of the author's statements.

Minor Points

Authors state, "we identified ETF dehydrogenase (ETFDH) as one of the most dispensable metabolic genes in neoplasia." Surely there are thousands of genes that are dispensable for neoplasia. Perhaps the authors can revise this sentence and similar sentiments in the text.

Authors state, " These findings show that ETFDH loss elevates glutamine utilization in the CAC to support mitochondrial metabolism." While elevated glutamine to CAC flux is consistent with the statement that increased glutamine, the authors have not measured the effect of restoring glutamine utilization to baseline on mitochondrial metabolism. Thus, the causality implied by the authors can only be inferred based on the data presented. Indeed, the increased glutamine consumption may be linked to the increase in ROS, as glutamate efflux via system xCT is a major determinant of glutamine catabolism in vitro.

Authors state that the mechanism described is an example of "retrograde signaling". However, the mechanism seems to be related to a reduction in BCAA catabolism, suggesting that the observed effects may be a consequence of altered metabolic flux rather than a direct signaling pathway. The data presented do not delineate whether the observed effects stem from disrupted mitochondrial communication or from shifts in nutrient availability and metabolic regulation.

The authors should discuss which amino acids that are ETFDH substrates might affect mTORC1 activity, or consider whether other ETFDH substrates might also affect mTORC1 in their discussion. Along these lines, the authors might consider discussing why amino acids that are not ETFDH substrates are increased upon ETFDH loss.

Reviewer #2 (Public review):

Summary:

The altered metabolism of tumors enables their growth and survival. Classically, tumor metabolism often involves increased activity of a given pathway in intermediary metabolism to provide energy or substrates needed for growth. Papadopoli et al. investigate the converse - the role of mitochondrial electron transfer flavoprotein dehydrogenase (ETFDH) in cancer metabolism and growth. The authors present compelling evidence that ETFDH insufficiency, which is detrimental in non-malignant tissues, paradoxically enhances bioenergetic capacity and accelerates neoplastic growth in cancer cells in spite of the decreased metabolic fuel flexibility that this affords tumor cells. This is achieved through the retrograde activation of the mTORC1/BCL-6/4E-BP1 axis, leading to metabolic and signaling reprogramming that favors tumor progression.

Strengths:

This review focuses primarily on the cancer metabolism aspects of the manuscript.

The study provides robust evidence linking ETFDH insufficiency to enhanced cancer cell bioenergetics and tumor growth.

The use of multiple cancer cell lines and in vivo models strengthens the generalizability of the findings.

The mechanistic insights into the mTORC1/BCL-6/4E-BP1 axis and its role in metabolic reprogramming are of general interest within and outside the immediate field of tumor metabolism.

Weaknesses:

The ETFDH knockout experiments are well-controlled by the addback of sgRNA-resistant ETFDH, but do not determine if the catalytic activity of this enzyme is required for the phenotypes induced by ETFDH loss.

Although this is not critical, it would be nice to see if the increased labeled aspartate pools result in higher nucleotide pools to support tumor growth.

Conclusion:

This manuscript provides significant insights into the role of ETFDH insufficiency in cancer metabolism and growth. The findings highlight the potential of targeting the mTORC1/BCL-6/4E-BP1 axis in ETFDH-deficient cancers. The compelling data support the conclusions presented in the manuscript, which will be valuable to the cancer metabolism community.

Reviewer #1 (Public review):

To elucidate the mechanisms and evolution of animal biomineralization, Voigt et al. focused on the sponge phylum - the earliest branching extant metazoan lineages exhibiting biomineralized structures - with a particular emphasis on deciphering the molecular underpinnings of spicule formation. This study centered on calcareous sponges, specifically Sycon ciliatum, as characterized in previous work by Voigt et al. In S. ciliatum, two morphologically distinct spicule types are produced by a set of two different types of cells that secrete extracellular matrix proteins, onto which calcium carbonate is subsequently deposited. Comparative transcriptomic analysis between a region with active spicule formation and other body regions identified 829 candidate genes involved in this process. Among these, the authors focused on the calcarine gene family, which is analogous to the Galaxins, the matrix proteins known to participate in coral calcification. The authors performed three-dimensional structure prediction using AlphaFold, examined mRNA expression of Calcarin genes in spicule-forming cell types via in situ hybridization, conducted proteomic analysis of matrix proteins isolated from purified spicules, and carried out chromosome arrangement analysis of the Calcarin genes.

Based on these analyses, it was revealed that the combination of Calcarin genes expressed during spicule formation differs between the founder cells-responsible for producing diactines and triactines-and the thickener cells that differentiate from them, underscoring the necessity for precise regulation of Calcarin gene expression in proper biomineralization. Furthermore, the observation that 4 Calcarin genes are arranged in tandem arrays on the chromosome suggests that two rounds of gene duplication followed by neofunctionalization have contributed to the intricate formation of S. ciliatum spicules. Additionally, similar subtle spatiotemporal expression patterns and tandem chromosomal arrangements of Galaxins during coral calcification indicate parallel evolution of biomineralization genes between S. ciliatum and aragonitic corals.

Strengths:

(1) An integrative research approach, encompassing transcriptomic, genomic, and proteomic analyses as well as detailed FISH.

(2) High-quality FISH images of Calcarin genes, along with a concise summary clearly illustrating their expression patterns, is appreciated.

(3) It was suggested that thickener cells originate from founder cells. To the best of my knowledge, this is the first study to demonstrate trans-differentiation of sponge cells based on the cell-type-specific gene expression, as determined by in situ hybridization.

(4) The comparison between Calcarins of Calcite sponge and Galaxins of aragonitic corals from various perspective-including protein tertiary structure predictions, gene expression profiling during calcification, and chromosomal sequence analysis to reveal significant similarities between them.

(5) The conclusions of this paper are generally well supported by the data; however, some FISH images require clearer indication or explanation.

(6) Figure S2 (B, C, D): The fluorescent signals in these images are difficult to discern. If the authors choose to present signals at such low magnification, enhancing the fluorescence signals would improve clarity. Additionally, incorporating Figure S2A as an inset within Figure S2E may be sufficient to convey the necessary information about signal localization.

(7) Figure S3A: The claim that Cal2-expressing spherical cells are closely associated with the choanoderm at the distal end of the radial tube is difficult to follow. Are these Cal2-expressing spherical cells interspersed among choanoderm cells, or are they positioned along the basal surface of the choanoderm? Clarifying their precise localization and indicating it in the image would strengthen the interpretation.

(8) To further highlight the similarities between S.ciliatum and aragonitic corals in the molecular mechanisms of calcification, consider including a supplementary figure providing a concise depiction of the coral calcification process. This would offer valuable context for readers.

Reviewer #2 (Public review):

Summary:

This paper reports on the discovery of calcarins, a protein family that seems involved in calcification in the sponge Sycon ciliatum, based on specific expression in sclerocytes and detection by mass spectrometry within spicules. Two aspects stand out: (1) the unexpected similarity between Sycon calcarins and the galaxins of stony corals, which are also involved in mineralization, suggesting a surprising, parallel co-option of similar genes for mineralization in these two groups; (2) the impressively cell-type-specific expression of specific calcarins, many of which are restricted to either founder or thickener cells, and to either diactines, triactines, or tetractines. The finding that calcarins likely diversified at least partly by tandem duplications (giving rise to gene clusters) is a nice bonus.

Strengths:

I enjoyed the thoroughness of the paper, with multiple lines of evidence supporting the hypothesized role of calcarins: spatially and temporally resolved RNAseq, mass spectrometry, and whole-mount in situ hybridization using CISH and HCR-FISH (the images are really beautiful and very convincing). The structural predictions and the similarity to galaxins are very surprising and extremely interesting, as they suggest parallel evolution of biomineralization in sponges and cnidarians during the Cambrian explosion by co-option of the same "molecular bricks".

Weaknesses:

I did not detect any major weakness, beyond those inherent to working with sponges (lack of direct functional inhibition of these genes) or with fast-evolving gene families with complex evolutionary histories (lack of a phylogenetic tree that would clarify the history of galaxins/calcarins and related proteins).

Reviewer #3 (Public review):

Summary:

The study explores the extent to which the biomineralization process in the calcitic sponge Sycon ciliatum resembles aragonitic skeleton formation in stony corals. To investigate this, the authors performed transcriptomic, genomic, and proteomic analyses on S. ciliatum and examined the expression patterns of biomineralization-related genes using in situ hybridization. Among the 829 differentially expressed genes identified in sponge regions associated with spicule formation, the authors focused on calcarin genes, which encode matrix proteins analogous to coral galaxins. The expression patterns of calcarins were found to be diverse but specific to particular spicule types. Notably, these patterns resemble those of galaxins in stony corals. Moreover, the genomic organization of calcarine genes in S. ciliatum closely mirrors that of galaxin genes in corals, suggesting a case of parallel evolution in carbonate biomineralization between calcitic sponges and aragonitic corals.

Strengths:

The manuscript is well written, and the figures are of high quality. The study design and methodologies are clearly described and well-suited to addressing the central research question. Particularly noteworthy is the authors´ integration of various omics approaches with molecular and cell biology techniques. Their results support the intriguing conclusion that there is a case of parallel evolution in skeleton-building gene sets between calcitic sponges and aragonitic corals. The conclusions are well supported by the data and analyses presented.

Weaknesses:

The manuscript is strong, and I have not identified any significant weaknesses in its current form.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigated factors required for neural progenitors to exit the cell cycle before the adult stage. They first show that Kr is turned on in pupal stage MBNBs, and depletion of Kr from pupal stage NBs leads to retention of MBNBs into the adult stage. Then they demonstrate that these retained NBs maintain the expression of Imp, and co-depletion of Imp abolishes the extended neurogenesis. Further, they show that co-depletion of kr-h1 significantly reduces the retained MBNBs caused by loss of kr, suggesting antagonistic genetic interactions between these two. In addition, they demonstrate that over-expressing Kr-h1 leads to the striking phenotype of tumor-like neuroblast overgrowth in adult brains.

Strengths:

(1) The authors leveraged well-controlled, powerful genetic tools (including temporal control of RNAi knockdown using the Gal80ts system), and provided strong evidence that Kr expression in pupal stage MBNBs is required to repress Imp and promote the end of neurogenesis. Similarly, the experimental result of co-depleting Kr-h1 and Kr, and the striking phenotype upon Kr-h1 mis-expression, support the antagonistic roles played by Kr-h1 and Kr in this process.

(2) The sample sizes, quantification methods, and p-values are well documented for all experiments. In most parts, the data presented strongly support their conclusions.

(3) Identification of two transcription factors with opposite roles in controlling cell cycle exit, and their possible interactions with the Imp/Syp axis, is highly significant for the study on how the proliferation of neural progenitors is regulated and limited before the adult stage.

Weaknesses:

(1) The nature of the KrIf-1 allele is not clear. It is mentioned that this allele leads to misexpression of Kr in various tissues. However, it is not clear if Kr is mis-expressed or lost in MBNBs in the KrIf-1 mutant. If Kr is mis-expressed in MBNBs in the KrIf-1 mutant, then it would be difficult to explain why both loss of Kr and mis-expression of Kr in MBNBs lead to the same NB retention phenotype. The authors should examine Kr expression in MBNBs in the KrIf-1 mutant.

(2) Some parts of the regulations and interactions between Kr, Kr-h1, Imp, Syp, and E93 are not well-defined. For example, the data suggest that Kr is turned on in the pupal stage MBNBs, and is required to end neurogenesis through repressing Imp and Kr-h1. To further support this conclusion, the authors can examine if Kr-h1 expression is up-regulated in kr-RNAi. The authors suggested that Kr-h1 may act upstream or in parallel to Imp/Syp, but also suggested that Kr-h1 may repress E93. The expression of Imp, Syp, and E93 can be examined in brains with Kr-h1 mis-expression to determine where Kr-h1 acts. If Imp expression is elevated when Kr-h1 is mis-expressed, then Kr-h1 may act upstream of Imp. If Imp/Syp expression does not change, then Kr-h1 may act on the E93 level.

Reviewer #2 (Public review):

Summary:

In this paper, the authors study the role of Kruppel in regulating the survival of mushroom body neuroblasts. They first confirm that adult wild-type brains have no proliferation and report that Kruppel mutants and Kruppel RNAi in neuroblasts show a few proliferative clones; they show that these proliferative clones are localized in the mushroom body. They then show that Kruppel is expressed mostly during pupal stages and acts by downregulating the expression of Imp, which has been shown to positively regulate neuroblast proliferation and survival. Expectedly, this also affects neuronal diversity in the mushroom body, which is enriched in gamma neurons that are born during the Imp-expression window. Finally, they show that Kr acts antagonistically to Kr-h1, which is expressed predominantly in larval stages.

Strengths:

The main strength of this paper is that it identified a novel regulator of Imp expression in the mushroom body neuroblasts. Imp is a conserved RNA-binding protein that has been shown to regulate neural stem cell proliferation and survival in different animals.

Weaknesses:

(1) The main weakness of the paper is that the authors want to test adult neurogenesis in a system where no adult neurogenesis exists. To achieve this, they force neuroblasts to survive in adulthood by altering the genetic program that prevents them from terminating their proliferation. If this was reminiscing about "adult neurogenesis", the authors should at least show how adult neurons incorporate into the mushroom body even if they are born much later. On the contrary, this more likely resembles a tumorigenic phenotype, when stem cells divide way past their appropriate timing.

(2) Moreover, the figures are, in many cases, hard to understand, and the interpretation of the figures doesn't always match what one sees. The manuscript would benefit from better figures; for example, in Figure 2C, Miranda expression in insc>GFP in Kr-IF-1 is not visible.

(3) The authors describe a targeted genetic screen, but they don't describe which genes were tested, how they were chosen, and why Kruppel was finally selected.

(4) The authors argue that Kr does not behave as a typical tTF in MBNBs. However, they show no expression in the embryo, limited expression in the larva and early pupa, and a peak around P24-P48. This sounds like a temporally regulated expression of a transcription factor. Importantly, they mentioned that they tested their observations against different datasets (FlyAtlas2, modENCODE, and MBNB-lineage-specific RNA-seq data), but they don't provide the data.

(5) Finally, the contribution of Kr to the neuronal composition of the mushroom body is expected (since Imp is known to regulate neuronal diversity in the MB), but the presentation in the paper is very incomplete.

Unfortunately, based on the above, I am not convinced that the authors can use this framework to infer anything about adult neurogenesis. Therefore, the impact of this work is limited to the role of Kruppel in regulating Imp, which has already been shown to regulate the extent of neuroblast division, as well as the neuronal types that are born at different temporal windows.

Reviewer #3 (Public review):

Summary:

Drosophila neuroblasts (NBs) serve as a well-established model for studying neural stem cell biology. The intrinsic genetic programs that control their mitotic potential throughout development have been described in remarkable detail, highlighting a series of sequentially expressed transcription factors and RNA-binding proteins that together constitute the temporal patterning system.

However, the mechanisms that limit the number of NB divisions remain largely unknown in a specific subset of NBs known as mushroom body neuroblasts (MB NBs). Unlike other NBs, which terminate proliferation before or shortly after the onset of metamorphosis, MB NBs continue dividing until the end of metamorphosis, ceasing only just before adulthood.<br /> In this study, the authors identify the transcription factor Krüppel (Kr), a member of the conserved Krüppel-like family, as temporally regulated in MB NBs. They demonstrate that Kr knockdown during pupal stages maintains expression of the RNA-binding protein Imp and results in prolonged MB NB proliferation into adulthood. Their data suggest that Kr contributes to the timely silencing of Imp during metamorphosis. The authors further identify Kr-h1, a related transcription factor, as a potential antagonist. While Kr-h1 appears dispensable for the timely termination of MB NBs under normal conditions, its overexpression leads to their continued proliferation and tumor-like expansion in adults.

This work provides the first evidence for a transcription factor-driven temporal regulation mechanism in MB NBs, offering new insight into the control of neural stem cell self-renewal. Given the evolutionary conservation of Krüppel-like factors, this study may have broader implications for the neural stem cell field.

Strengths:

(1) The study possibly identifies a new series of temporal transcription factors that are specific for mushroom body neuroblasts.

(2) The mechanism could be conserved in vertebrates.

Weaknesses:

Some proposed regulatory interactions, particularly between Kr, Kr-h1, and other temporal factors like Imp, Chinmo, and E93, have not been thoroughly investigated, which weakens the support for the proposed model. Additional experimental validation is needed to confirm these relationships and strengthen the mechanistic framework.

Reviewer #1 (Public review):

Summary:

The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.

Strengths:

The observations of differences in growth, body mass and food intake in the wt D. melanogaster larvae vs. those expressing the AOX transgene are solid. The evidence that mild uncoupling of the ETS might accelerate development of the fly larvae is convincing.

Weaknesses:

Some of the observations, especially those concerning the origin of the metabolic remodelling in AOX-expressing larvae, are left unexplained, and the argumentation is somewhat speculative. What the authors mean by "reconfiguration" of the mitochondrial electron transfer system is not clear. If this implies that there is an actual change in ETS function and/or structural organisation in the presence of AOX, this conclusion is not supported by the experimental data. In addition, the influence of AOX activity in the mitochondrial ETS system is tested in vitro in the presence of saturating concentrations of substrates. The real degree to which AOX activity is actually influencing ETS activity in vivo remains unknown.

Reviewer #2 (Public review):

Summary:

This manuscript presents intriguing findings about the role of alternative oxidase (AOX) from the tunicate Ciona intestinalis in accelerating growth and development when expressed in Drosophila melanogaster.

Strengths:

The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. The present study provides valuable insights into AOX's role in Drosophila development. The paper attempts to explore a unique mechanism by which AOX influences Drosophila development, providing insights into mitochondrial respiration and its physiological effects. This is relevant for understanding mitochondrial dysfunction and potential therapeutic applications. The study employs a variety of approaches, including calorimetry, infrared thermography, and genetic analyses, to investigate AOX's impact on metabolism and development.

Weaknesses:

There are a number of methodological limitations and substantial gaps in the interpretation of the data presented, which reduces the strength of its conclusions. For instance, there is a misunderstanding of the non-proton motive nature of the AOX - it does not uncouple respiration, merely decouple it as it neither contributes to nor dissipates the proton motive force, in contrast to chemical uncouplers or proton uncouplers such as UCPs. The authors need to reassess their data in light of the above.

Reviewer #1 (Public review):

Summary:

The authors have used gene deletion approaches in zebrafish to investigate the function of genes of the hox clusters in pectoral fin "positioning" (but perhaps more accurately pectoral fin "formation").

Strengths:

The authors have employed a robust and extensive genetic approach to tackle an important and unresolved question.

The results are largely presented in a very clear way.

Weaknesses:

The Abstract suggests that no genetic evidence exists in model organisms for a role of Hox genes in limb positioning. There are, however, several examples in mouse and other models (both classical genetic and other) providing evidence for a role of Hox genes in limb position, which is elaborated on in the Introduction.

It would perhaps be more accurate to state that several lines of evidence in a range of model organisms (including the mouse) support a role for Hox genes in limb positioning. The author's work is not weakened by a more inclusive introduction that cites the current literature more comprehensively.

It would be helpful for the authors to make a clear distinction between "positioning" of the limb/fin and whether a limb/fin "forms" at all, independent of the relative position of this event along the body axis.

Discussion of why the zebrafish is sensitive to Hoxb loss with reference to the fin, but mouse Hoxb mutants do make a limb?

Is this down to exclusive expression of Hoxbs in the zebrafish pectoral fin forming region rather than a specific functional role of the protein? This is important as it has implications for the interpretation of results throughout the paper and could explain some apparently conflicting results.

Why is Hoxba more potent than Hoxbb? Is this because Hoxba has Hox4/5 present, while Hoxbb has only Hoxb5? Hoxba locus has retained many more Hox genes in cluster than hoxbb; therefore, one might expect to see greater redundancy in this locus).

Deletion of either Hoxa or Hoxd in the background of the Hoxba mutant does have some effect. Is this a reflection of protein function or expression dynamics of Hoxa/Hoxd genes?

Can we really be confident that there is a "transformation of pectoral fin progenitor cells into cardiac cells"?

The failure to repress Nkx2.5 in the posterior (pelvic fin) domain is clear, but have these cells actually acquired cardiac identity? They would be expected to express Tbx5a (or b) as cardiac precursors, but this domain does not broaden. There is no apparent expansion of the heart (field)/domain or progenitors beyond the 16 somite stage. The claimed "migration" of heart precursors in the mutant is not clear. The heart/cardiac domain that does form in the mutant is not clearly expanded in the mutant. The domain of cmlc2 looks abnormal in the mutant, but I am not convinced it is "enlarged" as claimed by the authors. The authors have not convincingly shown that "the cells that should form the pectoral fin instead differentiate into cardiac cells."

The only clear conclusion is the loss of pectoral fin-forming cells rather than these fin-forming cells being "transformed" into a new identity. It would be interesting to know what has happened to the cells of the pectoral fin-forming region in these double mutants.

It is not clear what the authors mean by a "converse" relationship between forelimb/pectoral fin and heart formation. The embryological relationship between these two populations is distinct in amniotes.

The authors show convincing data that RA cannot induce Tbx5a in the absence of Hob clusters, but I am not convinced by the interpretation of this result. The results shown would still be consistent with RA acting directly upstream of tbx5a, but merely that RA acts in concert with hox genes to activate tbx5a. In the absence of one or the other, Tbx5a would not be expressed. It is not necessary that RA and hoxbs act exclusively in a linear manner (i.e., RA regulates hoxb that in turn regulates tbx5a).

The authors have carried out a functional test for the function of hoxb6 and hoxb8 in the hemizygous hoxb mutant background. What is lacking is any expression analysis to demonstrate whether Hoxb6b or Hoxb8b are even expressed in the appropriate pectoral fin territory to be able to contribute to pectoral fin development, either in this assay or in normal pectoral fin development.

(The term "compensate" used in this section is confusing/misleading.)

The authors' confounding results described in Figures 6-7 are consistent with the challenges faced in other model organisms in trying to explore the function of genes in the hox cluster and the known redundancy that exists across paralogous groups and across individual clusters.

Given the experimental challenges in deciphering the actual functions of individual or groups of hox genes, a discussion of the normal expression pattern of individual and groups of hox genes (and how this may change in different mutant backgrounds) could be helpful to make conclusions about likely normal function of these genes and compensation/redundancy in different mutant scenarios.

Reviewer #2 (Public review):

Summary:

The authors of this manuscript performed a fascinating set of zebrafish mutant analyses on hox cluster deletion and pinpointed the cause of the pectoral fin loss in one combinatorial hox cluster mutant of Hoxba and Hoxbb.

Strengths:

The study is based on a variety of existing experimental tools that enabled the authors' past construction of hox cluster mutants, and is well-designed. The manuscript is well written to report the authors' findings on the mechanism that positions the pectoral fin.

Weaknesses:

The study does not focus on the other hox clusters other than ba and bb, and is confined to the use of zebrafish, as well as the comparison with existing reports from mouse experiments.

Reviewer #1 (Public review):

The manuscript by Ivan et al aimed to identify epitopes on the Abeta peptide for a large set of anti-Abeta antibodies, including clinically relevant antibodies. The experimental work was well done and required a major experimental effort, including peptide mutational scanning, affinity determinations, molecular dynamics simulations, IP-MS, WB, and IHC. Therefore, it is of clear interest to the field. The first part of the work is mainly based on an assay in which peptides (15-18-mers) based on the human Abeta sequence, including some containing known PTMs, are immobilized, thus preventing aggregation. Although some results are in agreement with previous experimental structural data (e.g. for 3D6), and some responses to disease-associated mutations were different when compared to wild-type sequences (e.g. in the case of Aducanumab) - which may have implications for personalized treatment - I have concerns about the lack of consideration of the contribution of conformation (as in small oligomers and large aggregates) in antibody recognition patterns. The second part of the study used full-length Abeta in monomeric or aggregated forms to further investigate the differential epitope interaction between Aducanumab, donanemab, and lecanemab (Figures 5-7). Interestingly, these results confirmed the expected preference of these antibodies for aggregated Abeta, thus reinforcing my concerns about the conclusions drawn from the results obtained using shorter and immobilized forms of Abeta. Overall, I understand that the work is of interest to the field and should be published without the need for additional experimental data. However, I recommend a thorough revision of the structure of the manuscript in order to make it more focused on the results with the highest impact (second part).

Reviewer #2 (Public review):

This paper investigates binding epitopes of different anti-Abeta antibodies. Background information on the clinical outcome of some of the antibodies in the paper, which might be important for readers to know, is lacking. There are no references to clinical outcomes from antibodies that have been in clinical trials. This paper would be much more complete if the status of the antibodies were included. The binding characteristics of aducanumab, donanemab, and lecanemab should be compared with data from clinical phase 3 studies.

Aducanumab was identified at Neurimmune in Switzerland and licensed to Biogen and Eisai. Aducanumab was retracted from the market due to a very high frequency of the side-effect amyloid-related imaging abnormalities-edema (ARIA-E). Gantenerumab was developed by Roche and had two failed phase 3 studies, mainly due to a high frequency of ARIA-E and low efficacy of Abeta clearance. Lecanemab was identified at Uppsala University, humanized by BioArctic, and licensed to Eisai, who performed the clinical studies. Eisai and Biogen are now marketing lecanemab as Leqembi on the world market. Donanemab was developed by Ely Lilly and is sold in the US as Kisunla.

Limitations:

(1) Conclusions are based on Abeta antigens that may not be the primary targets for some conformational antibodies like aducanumab and lecanemab. There is an absence of binding data for soluble aggregated species.

(2) Quality controls and characterization of different Abeta species are missing. The authors need to verify if monomers remain monomeric in the blocking studies for Figures 5 and 6.

(3) The authors should discuss the limitations of studying synthetic Abeta species and how aggregation might hide or reveal different epitopes.

(4) The authors should elaborate on the differences between synthetic Abeta and patient-derived Abeta. There is a potential for different epitopes to be available.

Reviewer #1 (Public review):

Summary:

The manuscript "Targeted Protein Degradation by KLHDC2 Ligands Identified by High Throughput Screening" by Zhou, H. et al. describes the development of a high-throughput FP-based screen and the identification of a KLHDC2 ligand from a small molecule library. A counter screen and other filtering criteria led to the identification of lead compounds that contained a tetrahydroquinoline scaffold. Commercially available analogs (52 compounds) that shared this scaffold were characterized by a KLHDC2 competitive binding assay. Optimized compounds were obtained that demonstrated improved potency and increased binding affinity by SPR. Docking of a lead candidate (compound 6) suggested it bound at a distal lipophilic site within the SelK binding pocket of KLHDC2. Based on this model, the authors then synthesized PROTACs that linked the KLHDC2 binder to a BRD4-binding molecule, JQ1. These PROTAC candidates possessed different linker configurations, and PROTAC 8 was able to cause BRD4 degradation in cells, with a half-maximal degradation concentration (DC50) of 80 nM. The authors demonstrate the identification and characterization of small-molecule KLHDC2 ligands that can be used to generate PROTACs that result in BRD4 degradation in cells.

Strengths:

The study by Zhou, H. et al. expands the E3 ligase toolkit by targeting KLHDC2 to identify ligands for PROTAC development, which has predominantly relied on VHL and CRBN. This was accomplished using a described FP-based high-throughput screening strategy (high Z' values in 1536 well format). Both target-specific and counter-specific assays were performed, along with subsequent stringent follow-up assays designed to address non-specific binding/specificity concerns. Label-free direct binding validations by SPR were used to determine binding affinity/kinetics. A strength of the study is the characterization of the interaction between candidate compounds and KLHDC2 versus related KEAP1.

Structural insight into the potential mode of binding was inferred by computational docking studies of the newly discovered KLHDC2 ligands. This was performed to identify where the identified scaffolds could be modified by linker incorporation for the design of PROTACs. The computational predictions were evaluated by linking a solvent-exposed site on the KLHDC2 ligand to JQ1. Three linkers were tested, and two compounds were found to result in BRD4 degradation in cells by HiBiT degradation assay and western blot. These findings demonstrate the feasibility of these compounds for the design of PROTAC-based degraders.

The authors present compelling KLHDC2 binding data for their lead compounds and demonstrate degradation of a target using a PROTAC strategy. Accordingly, the screening approach and compounds identified are likely to be of interest to the field and are likely to be generalizable to other PROTAC targets of interest.

Weaknesses:

The specificity of compounds for KLHDC2 was assessed by using a counter screen against KEAP1 and in vitro binding assays. However, off-target effects might occur in a cellular context, which weren't fully explored in the study. Notably, the authors do not demonstrate that the degradation induced by their PROTACs in cells is KLHDC2-dependent. A requirement for KLHDC2-mediated degradation could be evaluated, for example, by using knockout/knockdown of KLHDC2, or other means, to demonstrate specificity. Addressing specificity is deemed important to evaluate the proposed PROTAC mechanism of action in a cellular context that results in the degradation of BRD4. Specificity is important when considering the utility of these new compounds for PROTAC design.

Additional rationale behind the selection of linkers used to generate candidate PROTACs would be informative and would benefit from additional discussion and/or citation. The reasons for the lack of activity, such as for compound 9, were not fully explored or discussed, such as whether complex assembly is potentially affected by linker choice. Perhaps related to this point, the authors note that a trifluoromethoxy group increased the binding affinity of compound 6. However, the subsequent docking analysis revealed this moiety to be solvent-exposed. The relationship between this site of functionalization, linker selection, and the resulting binding affinity or effect on DC50 was not clear and/or could be developed further.

Minor issues related to the presentation of the manuscript include sections that would benefit from either additional citation and/or description, such as the KI-696 inhibitor used and the BRD4 HiBiT degradation assay that was used to assess PROTAC potency. Figure captions should be reviewed to ensure that the number of independent experiments is indicated, and what data points and error bars represent, as these are not indicated in several figures. BRD4 levels were quantified in 4E; however, error/reproducibility (n) is not indicated.

Reviewer #2 (Public review):

PROTACs are a class of small molecules that induce an interaction between a target protein and a ubiquitin ligase, thereby leading to the target protein's ubiquitination and subsequent proteasomal degradation. Given that the vast majority of PROTACs rely on the cereblon and VHL ubiquitin ligases, a major goal within this field has been to identify and develop ligands for additional ubiquitin ligases, in particular those whose expression affords tissue or subcellular specificity or those whose structure allows them to degrade targets that are otherwise incompatible with cereblon or VHL.

In this work, Zhou and colleagues from the Bollong group at Scripps utilize a high-throughput fluorescence polarization screen of >350,000 compounds to identify and optimize a novel ligand for KLHDC2, a ubiquitin ligase which had previously been discovered to be capable of proximity-induced degradation of target proteins. Zhou et al go on to show that this ligand can be used as the basis for PROTACs capable of degrading BRD4 in a cell line. Of note, prior to this paper, three other groups had also developed ligands to KLHDC2 and used them to generate active PROTACs. Interestingly, docking studies by Zhou suggest that their compound may bind to a different region of the KLHDC2's kelch domain.

The major strengths of this work are its brevity and the clarity of the writing and figures. Their claim that they have discovered a ligand for KLHDC2, which can be used to develop BRD4-degrading PROTACs, is well-supported by their findings from the screen, SPR, and cellular assays. The weakness of the work then, is not so much relevant to the paper at hand but rather stems from the fact that their story leaves me wanting to know more. Indeed, there are a number of interesting experiments that we need as a field in order to assess 1) how generalizable their findings are across cell lines and targets, and 2) how this new KLHDC2 ligand stacks up against the other recently discovered ligands for KLDHC2 as well as the existing standards, cereblon and VHL.

Nonetheless, Zhou and colleagues provide a valuable addition to the emerging repertoire of KLHDC2 ligands, and I'm certain that with time, we will come to understand what ligands work best for KLHDC2-based PROTACs and how they compare to the growing set of ubiquitin ligases in our armamentarium.

Reviewer #1 (Public review):

The study aims to determine the role of Slit-Robo signaling in the development and patterning of cardiac innervation, a key process in heart development. Despite the well-studied roles of Slit axon guidance molecules in the development of the central nervous system, their roles in the peripheral nervous system are less clear. Thus, the present study addresses an important question. The study uses genetic knockout models to investigate how Slit2, Slit3, Robo1, and Robo2 contribute to cardiac innervation.

Using constitutive and cell type-specific knockout mouse models, they show that the loss of endothelial-derived Slit2 reduces cardiac innervation. Additionally, Robo1 knockout, but not Robo2 knockout, recapitulated the Slit2 knockout effect on cardiac innervation, leading to the conclusion that Slit2-Robo1 signaling drives sympathetic innervation in the heart. Finally, the authors also show a reduction in isoproterenol-stimulated heart rate but not basal heart rate in the absence of endothelial Slit2.

The conclusions of this paper are mostly well supported by the data, but some should be modified to account for the study's limitations and discussed in the context of previous literature.

(1) It is well established that Slit ligands undergo proteolytic cleavage, generating N- and C-terminal fragments with distinct biological functions. Full-length Slit proteins and their fragments differ in cell association, with the N-terminal fragment typically remaining membrane-bound, while the C-terminal fragment is more diffusible. This distinction is crucial when evaluating the role of Slit proteins secreted by different cell types in the heart. However, this study does not examine or discuss the specific contributions of different Slit2 fragments, limiting its mechanistic insight into how Slit2 regulates cardiac innervation.

(2) The endothelial-specific deletion of Slit2 leads to its loss in endothelial cells across various organs and tissues in the developing embryo. Therefore, the phenotypes observed in the heart may be influenced by defects in other parts of the embryo, such as the CNS or sympathetic ganglia, and this possibility cannot be ruled out.

Reviewer #2 (Public review):

The aims of investigating Slit-Robo signaling in cardiac innervation were achieved by the experiments designed. While questions remain regarding signal regulation and interplay between established axon guidance signals and further role of other Slit ligands and Robo expression in endothelium, the results strongly support the conclusions drawn.

Writing and presentation are easy to follow and well structured, Appropriate controls are used, statistical analysis applied appropriately, and experiments directly test aims following a logical story.

The authors demonstrate a novel mechanism for Slit-Robo signaling in cardiac sympathetic innervation. The data establishes a framework for future studies.

Recommendations:

Further assessment of interplay between Slit ligands as well as other signaling pathways (Semaphorin, NGF, etc) could be investigated. Is it possible to rescue the phenotype by modulation of other signaling pathways? Can combined Slit2/Slit3 KO rescue? Additionally, as the authors state, conditional Robo1 knockouts will be important to validate the findings of constitutive knockout.

Reviewer #1 (Public review):

Summary:

Lysosomal damage is commonly found in many diseases including normal aging and age-related disease. However, the transcriptional programs activated by lysosomal damage have not been thoroughly characterized. This study aimed to investigate lysosome damage-induced major transcriptional responses and the underlying signaling basis. The authors have convincingly shown that lysosomal damage activates a ubiquitination-dependent signaling axis involving TAB, TAK1, and IKK, which culminates in the activation of NF-kB and subsequent transcriptional upregulation of pro-inflammatory genes and pro-survival genes. Overall, the major aims of this study were successfully achieved.

Strengths:

This study is well-conceived and strictly executed, leading to clear and well-supported conclusions. Through unbiased transcriptomics and proteomics screens, the authors identified NF-kB as a major transcriptional program activated upon lysosome damage. TAK1 activation by lysosome damage-induced ubiquitination was found to be essential for NF-kB activation and MAP kinase signaling. The transcriptional and proteomic changes were shown to be largely driven by TAK1 signaling. Finally, the TAK1-IKK signaling was shown to provide resistance to apoptosis during lysosomal damage response. The main signaling axis of this pathway was convincingly demonstrated.

Weaknesses:

One weakness was the claim of K63-linked ubiquitination in lysosomal damage-induced NF-kB activation. While it was clear that K63 ubiquitin chains were present on damaged lysosomes, no evidence was shown in the current study to demonstrate the specific requirement of K63 ubiquitin chains in the signaling axis being studied. Clarifying the roles of K63-linked versus other types of ubiquitin chains in lysosomal damage-induced NF-kB activation may improve the mechanistic insights and overall impact of this study.

Another weakness was that the main conclusions of this study were all dependent on an artificial lysosomal damage agent. It will be beneficial to confirm key findings in other contexts involving lysosomal damage.

Reviewer #2 (Public review):

Summary:

Endo et al. investigate the novel role of ubiquitin response upon lysosomal damage in activating cellular signaling for cell survival. The authors provide a comprehensive transcriptome and proteome analysis of aging-related cells experiencing lysosomal damage, identifying transcription factors involved in transcriptome and proteome remodeling with a focus on the NF-κB signaling pathway. They further characterized the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis in controlling gene expression, inflammatory responses, and apoptotic processes.

Strengths:

In the aging-related model, the authors provide a comprehensive transcriptome and characterize the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis. Through compelling experiments and advanced tools, they elucidate its critical role in controlling gene expression, inflammatory responses, and apoptotic processes.

Weaknesses:

The study lacks deeper connections with previous research, particularly:<br /> • The established role of TAB-TAK1 in AMPK activation during lysosomal damage<br /> • The potential significance of TBK1 in NF-κB signaling pathways

Reviewer #3 (Public review):

Summary:

The response to lysosomal damage is a fast-moving and timely field. Besides repair and degradation pathways, increasing interest has been focusing on damaged-induced signaling. The authors conducted both transcriptomics and proteomics to characterize the cellular response to lysosomal damage. They identify a signaling pathway leading to activation of NFkappaB. Based on this and supported by Western blot and microscopy data, the authors nicely show that TAB2/3 and TAK1 are activated at damaged lysosomes and kick off the pathway to alter gene expression, which induces cytokines and protect from cell death. TAB2/3 activation is proposed to occur through K63 ubiquitin chain formation. Generally, this is a careful and well conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves as a valuable resource for the field. More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports.

Strengths:

Generally, this is a careful and well-conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves as a valuable resource for the field.

Weaknesses:

More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports. Moreover, different damage types should be tested to probe relevance for different pathophysiological conditions.

Suggestions:

(1) A recent paper claims that NFkappaB is activated by Otulin/M1 chains upon lysosome damage through TBK1 (PMID: 39744815). In contrast, Endo et al. nicely show that ubiquitylation is needed (shown by TAK-243) for NFkB activation but only have correlative data to link it specifically to K63 chains. On page 15, line 11, the authors even argue a "potential" involvement of K63. This point should be better dealt with. Can the authors specifically block K63 formation? K63R overexpression or swapping would be one way. Is the K63 ligase ITCH involved (PMID: 38503285) or any other NEDD4-like ligase? This could be compared to LUBAC inhibition. Also, the point needs to be dealt with more controversially in the discussion as these are alternative claims (M1 vs K63, TAB vs TBK1).

(2) It would be interesting to know what the trigger is that induces the pathway. Lipid perturbation by LLOMe is a good model, but does activation also occur with GPN (osmotic swelling) or lipid peroxidation (oxidative stress) that may be more broadly relevant in a pathophysiological way? Moreover, what damage threshold is needed? Does loss of protons suffice? Can activation be induced with a Ca2+ agonist in the absence of damage?

(3) The authors nicely define JNK and p38 activation. This should be emphasized more, possibly also in the abstract, as it may contribute to the claim of increased survival fitness.

Reviewer #1 (Public review):

Summary:

This work by Govorunova et al. identified three naturally blue-shifted channelrhodopsins (ChRs) from ancyromonads, namely AnsACR, FtACR, and NlCCR. The phylogenetic analysis places the ancyromonad ChRs in a distinct branch, highlighting their unique evolutionary origin and potential for novel applications in optogenetics. Further characterization revealed the spectral sensitivity, ionic selectivity, and kinetics of the newly discovered AnsACR, FtACR, and NlCCR. This study also offers valuable insights into the molecular mechanism underlying the function of these ChRs, including the roles of specific residues in the retinal-binding pocket. Finally, this study validated the functionality of these ChRs in both mouse brain slices (for AnsACR and FtACR) and in vivo in Caenorhabditis elegans (for AnsACR), demonstrating the versatility of these tools across different experimental systems.

In summary, this work provides a potentially valuable addition to the optogenetic toolkit by identifying and characterizing novel blue-shifted ChRs with unique properties.

Strengths:

This study provides a thorough characterization of the biophysical properties of the ChRs and demonstrates the versatility of these tools in different ex vivo and in vivo experimental systems. The mutagenesis experiments also revealed the roles of key residues in the photoactive site that can affect the spectral and kinetic properties of the channel.

Weaknesses:

While the novel ChRs identified in this work are spectrally blue-shifted, there still seems to be some spectral overlap with other optogenetic tools. The authors should provide more evidence to support the claim that they can be used for multiplex optogenetics and help potential end-users assess if they can be used together with other commonly applied ChRs. Additionally, further engineering or combination with other tools may be required to achieve truly orthogonal control in multiplexed experiments.

In the C. elegans experiments, partial recovery of pharyngeal pumping was observed after prolonged illumination, indicating potential adaptation. This suggests that the effectiveness of these ChRs may be limited by cellular adaptation mechanisms, which could be a drawback in long-term experiments. A thorough discussion of this challenge in the application of optogenetics tools would prove very valuable to the readership.

Reviewer #2 (Public review):

Summary:

Govorunova et al present three new anion opsins that have potential applications in silencing neurons. They identify new opsins by scanning numerous databases for sequence homology to known opsins, focusing on anion opsins. The three opsins identified are uncommonly fast, potent, and are able to silence neuronal activity. The authors characterize numerous parameters of the opsins.

Strengths:

This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. The opsins AnsACR and FtACR are particularly notable, having extraordinarily fast onset kinetics that could have utility in many domains. Furthermore, the authors show that AnsACR is usable in multiphoton experiments having a peak photocurrent in a commonly used wavelength. Overall, the author's detailed measurements and characterization make for an important resource, both presenting new opsins that may be important for future experiments, and providing characterizations to expand our understanding of opsin biophysics in general.

Weaknesses:

First, while the authors frequently reference GtACR1, a well-used anion opsin, there is no side-by-side data comparing these new opsins to the existing state-of-the-art. Such comparisons are very useful to adopt new opsins.

Next, multiphoton optogenetics is a promising emerging field in neuroscience, and I appreciate that the authors began to evaluate this approach with these opsins. However, a few additional comparisons are needed to establish the user viability of this approach, principally the photocurrent evoked using the 2p process, for given power densities. Comparison across the presented opsins and GtACR1 would allow readers to asses if these opsins are meaningfully activated by 2P.

Reviewer #3 (Public review):

Summary:

The authors aimed to develop Channelrhodopsins (ChRs), light-gated ion channels, with high potency and blue action spectra for use in multicolor (multiplex) optogenetics applications. To achieve this, they performed a bioinformatics analysis to identify ChR homologues in several protist species, focusing on ChRs from ancyromonads, which exhibited the highest photocurrents and the most blue-shifted action spectra among the tested candidates. Within the ancyromonad clade, the authors identified two new anion-conducting ChRs and one cation-conducting ChR. These were characterized in detail using a combination of manual and automated patch-clamp electrophysiology, absorption spectroscopy, and flash photolysis. The authors also explored sequence features that may explain the blue-shifted action spectra and differences in ion selectivity among closely related ChRs.

Strengths:

A key strength of this study is the high-quality experimental data, which were obtained using well-established techniques such as manual patch-clamp and absorption spectroscopy, complemented by modern automated patch-clamp approaches. These data convincingly support most of the claims. The newly characterized ChRs expand the optogenetics toolkit and will be of significant interest to researchers working with microbial rhodopsins, those developing new optogenetic tools, as well as neuro- and cardioscientists employing optogenetic methods.

Weaknesses:

This study does not exhibit major methodological weaknesses. The primary limitation of the study is that it includes only a limited number of comparisons to known ChRs, which makes it difficult to assess whether these newly discovered tools offer significant advantages over currently available options. Additionally, although the study aims to present ChRs suitable for multiplex optogenetics, the new ChRs were not tested in combination with other tools. A key requirement for multiplexed applications is not just spectral separation of the blue-shifted ChR from the red-shifted tool of interest but also sufficient sensitivity and potency under low blue-light conditions to avoid cross-activation of the respective red-shifted tool. Future work directly comparing these new ChRs with existing tools in optogenetic applications and further evaluating their multiplexing potential would help clarify their impact.

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors (Fig. 1), ectopic neurons and glands along the AP axis (Fig. 2) and pharynx in ectopic anterior positions (Fig. 3). The rest of the study show that positional information is largely unaffected by loss of map3k1 (Fig. 4,5). However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route (Fig. 6). They also show that an ill-defined "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.<br /> (2) The work appears to be high-quality.<br /> (3) The conclusions are supported by the data.<br /> (4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not in other invertebrates.

Weaknesses:

(1) The paper is largely descriptive with no mechanistic insights.<br /> (2) Given the severe phenotypes of long-term depletion of map3k1, it is important that this exact timepoint is provided in the methods, figures, figure legends and results.<br /> (3) Fig. 1C, the ectopic eyes are difficult to see, please add arrows.<br /> (4) line 217 - why does the n=2/12 animals not match the values in Fig. 3B, which is 11/12 and 12/12. The numbers don't add up. Please correct/explain.<br /> (5) Figure panels do not match what is written in the results section. There is no Fig. 6E. Please correct.

Reviewer #2 (Public review):

Summary:<br /> The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Weaknesses:

The article presents a provocative idea related to the importance of positional control for organs and cells, which is at least in part regulated by map3k1. Nonetheless, the role of map3k1 or its potential interaction with regulators of the anterior-posterior, mediolateral axes, and PCGs is somewhat superficial. The authors could elaborate or even speculate more in the discussion section and the different scenarios incorporating these axial modulators into the map3k1 model presented in Figure 8.

The article can be improved by addressing inconsistencies and adding details to the results, including the main figures and supplements. This represents one of the most significant weaknesses of this otherwise intriguing manuscript. Below are some examples of a few figures, but the authors are expected to pay close attention to the remaining figures in the paper.

Details associated with the number of animals per experiment, statistical methods used, and detailed descriptions of figures appear inconsistent or lacking in almost all figures. In some instances, the percentage of animals affected by the phenotype is shown without detailing the number of animals in the experiment or the number of repeats. Figures and their legends throughout the paper lack details on what is represented and sometimes are mislabeled or unrelated. Specifically, the arrows in Figure 1A are different colors. Still, no reasoning is given for this, and in the exact figure, the top side (1A) shows the percentages and the number of animals below. Conversely, in Figures 1B, C, and D, no details on the number of animals or percentages are shown, nor an explanation of why opsin was used in Figure 1A but not 1B. Is Figure 1B missing an image for the respective control? Figure 1C needs details regarding what the two smaller boxes underneath are. Figure 1C could use an AP labeling map in 10 days (e.g., AP6 has one optic cup present). Figure 1C and F counts do not match. In Figure 1C, we do not know the number of animals tested, controls used, the scale bar sizes in the first two images, nor the degree of magnification used despite the pharynx region appearing magnified in the second image. Figure 1C is also shown out of chronological order; 36 days post RNAi is shown before 10 days post RNAi. Moreover, the legends for Figures 1C and 1D are swapped.

Additionally, Figure 1F and many other figures throughout the paper lack overall statistical considerations. Furthermore, Figure 1F has three components, but only one is labeled. Labeling each of them individually and describing them in the corresponding figure legend may be more appropriate.

Figure 2C shows images of gene expression for two genes, but the counts are shown for only one in Figure 2D. It is challenging to follow the author's conclusions without apparent reasoning and by only displaying quantitative considerations for one case but not the other. These inconsistencies are also observed in different figures. In Figure 2D, 24/24 animals were reported to show the phenotype, but only eight were counted (is there a reason for this?). In Figure 2E, the expression for three genes is shown, with some displaying anterior and posterior regions while others only show the anterior picture. Is there a particular reason for this? Also, in Figure 2F, the counts are shown for only the posterior region of two genes out of the three displayed in Figure 2E. It is unclear why the authors do not show counts for the anterior areas considered in Figure 2E. Furthermore, the legend for Figure 2D is missing, and the legend for 2F is mislabeled as a description for Figure 2D.

Supplement Figure 1 B reports data up to 6 weeks, but no text in the manuscript or supplement mentions any experiment going up to 6 weeks. There are no statistics for data in Supplement Figure 1E. Any significance between groups is unclear.

Reviewer #1 (Public review):

Summary:

This manuscript investigated the mechanism underlying boundary formation necessary for proper separation of vestibular sensory end organs. In both chick and mouse embryos, it was shown that a population of cells abutting the sensory (marked by high Sox2 expression) /nonsensory cell populations (marked by Lmx1a expression) undergo apical expansion, elongation, alignment and basal constriction to separate the lateral crista (LC) from the utricle. Using Lmx1a mouse mutant, organ cultures, pharmacological and viral-mediated Rock inhibition, it was demonstrated that the Lmx1a transcription factor and Rock-mediated actomyosin contractility is required for boundary formation and LC-utricle separation.

Strengths:

Overall, the morphometric analyses were done rigorously and revealed novel boundary cell behaviors. The requirement of Lmx1a and Rock activity in boundary formation was convincingly demonstrated.

Weaknesses:

However, the precise roles of Lmx1a and Rock in regulating cell behaviors during boundary formation were not clearly fleshed out. For example, phenotypic analysis of Lmx1a was rather cursory; it is unclear how Lmx1a, expressed in half of the boundary domain, control boundary cell behaviors and prevent cell mixing between Lmx1a+ and Lmx1a- compartments? Well-established mechanisms and molecules for boundary formation were not investigated (e.g. differential adhesion via cadherins, cell repulsion via ephrin-Eph signaling). Moreover, within the boundary domain, it is unclear whether apical multicellular rosettes and basal constrictions are drivers of boundary formation, as boundary can still form when these cell behaviors were inhibited. Involvement of other cell behaviors, such as radial cell intercalation and oriented cell division, also warrant consideration. With these lingering questions, the mechanistic advance of the present study is somewhat incremental.

Reviewer #2 (Public review):

Summary:

Chen et al. describe the mechanisms that separate the common pan-sensory progenitor region into individual sensory patches, which presage the formation of the sensory epithelium in each of the inner ear organs. By focusing on the separation of the anterior and then lateral cristae, they find that long supra-cellular cables form at the interface of the pan-sensory domain and the forming cristae. They find that at these interfaces, the cells have a larger apical surface area, due to basal constriction, and Sox2 is down-regulated. Through analysis of Lmx1 mutants, the authors suggest that while Lmx1 is necessary for the complete segregation of the sensory organs, it is likely not necessary for the initial boundary formation, and the down-regulation of Sox2.

Strengths:

The manuscript adds to our knowledge and provides valuable mechanistic insight into sensory organ segregation. Of particular interest are the cell biological mechanisms: The authors show that contractility directed by ROCK is important for the maintenance of the boundary and segregation of sensory organs.

Weaknesses:

The manuscript would benefit from a more in-depth look at contractility - the current images of PMLC are not too convincing. Can the authors look at p or ppMLC expression in an apical view? Are they expressed in the boundary along the actin cables? Does Y-27362 inhibit this expression?

The authors suggest that one role for ROCK is the basal constriction. I was a little confused about basal constriction. Are these the initial steps in the thinning of the intervening non-sensory regions between the sensory organs? What happens to the basally constricted cells as this process continues?

The steps the authors explore happen after boundaries are established. This correlates with a down-regulation of Sox2, and the formation of a boundary. What is known about the expression of molecules that may underlie the apparent interfacial tension at the boundaries? Is there any evidence for differential adhesion or for Eph-Ephrin signalling? Is there a role for Notch signalling or a role for Jag1 as detailed in the group's 2017 paper?

A comment on whether cellular intercalation/rearrangements may underlie some of the observed tissue changes.

The change in the long axis appears to correlate with the expression of Lmx1a (Fig 5d). The authors could discuss this more. Are these changes associated with altered PCP/Vangl2 expression?

Reviewer #3 (Public review):

Summary:

Lmx1a is an orthologue of apterous in flies, which is important for dorsal-ventral border formation in the wing disc. Previously, this research group has described the importance of the chicken Lmx1b in establishing the boundary between sensory and non-sensory domains in the chicken inner ear. Here, the authors described a series of cellular changes during border formation in the chicken inner ear, including alignment of cells at the apical border and concomitant constriction basally. The authors extended these observations to the mouse inner ear and showed that these morphological changes occurred at the border of Lmx1a positive and negative regions, and these changes failed to develop in Lmx1a mutants. Furthermore, the authors demonstrated that the ROCK-dependent actomyosin contractility is important for this border formation and blocking ROCK function affected epithelial basal constriction and border formation in both in vitro and in vivo systems.

Strengths:

The morphological changes described during border formation in the developing inner ear are interesting. Linking these changes to the function of Lmx1a and ROCK dependent actomyosin contractile function are provocative.

Weaknesses:

There are several outstanding issues that need to be clarified before one could pin the morphological changes observed being causal to border formation and that Lmx1a and ROCK are involved.

Reviewer #1 (Public review):

Summary:

In this manuscript, Taujale et al describe an interdisciplinary approach to mine the human channelome and further discover orthologues across diverse organisms, culminating in delineating co-conserved patterns in an example ion channel: CALHM. Overall, this paper comes in two sections, one where 419 human ion channels and 48,000+ channels from diverse organisms are found through a multidisciplinary data mining approach, and a second where this data is used to find co-conserved sequences, whose functional significance is validated via experiments on CALHM1 and CALHM6. Overall, this is an intriguing data-first approach to better understand even understudied ion channels like CALHM6. However, more needs to be done to pull this story together into a single, coherent narrative.

Strengths:

This manuscript takes advantage of modern-day LLM tools to better mine the literature for ion channel sequences in humans and other species with orthologous ion channel sequences. They explore the 'dark channome' of understudied ion channels to better reveal the information evolution has to tell us about our own proteins, and illustrate the information this provides access to in experimental studies in the final section of the paper. Finally, they provide a wealth of information in the supplementary tables (in the form of Excel spreadsheets) for others to explore. Overall, this is a creative approach to a wide-reaching problem that can be applied to other families of proteins.

Weaknesses:

Overall, while a considerable amount of work has been done for this manuscript, the presentation, both in terms of writing and figures, leaves much to be desired. One can imagine a story that clearly describes the need for a better-curated sequence database of ion channels, and clearly describes how existing resources fall short, but here this is not very clearly illustrated.

One question that arises with the part of the manuscript that discusses the identification and classification of ion channels is whether they plan to make these sequences available to the wider public. For the 419 human sequences, making a small database to share this result so that these sequences can be easily searched and downloaded would be desirable. There are a variety of acceptable formats for this: GitHub/figshare/zenodo/university website that allows a wider community to access their hard work. The authors have included enough information in the supplementary tables that this could be done by a motivated reader, but providing such a resource would greatly expand the impact of this paper. The same question can be asked of the 48,000+ ion channels from diverse organisms. For these, one is even worried that these are not properly sequenced genes? What checks have been done to confirm this? Uniport contains a good deal of unreviewed sequences, especially from single-celled organisms. Potentially, this is covered in the sentence in the Methods: "Finally, the results obtained from both the full-length and pore domains were retained as true orthologous relationships to remove extraneous hits." But this process could be discussed in more detail, clearly illustrating that the risk of gene duplicates and fragments in this final set of ion channel orthologues has been avoided. Related to this, does this analysis include or exclude isoforms?

Another aspect of the identification and classification of ion channel genes that could be improved is the figures for this section. One is relatively used to seeing trees as shown in Figures 3 and 4, which show relationships between genes as distances or evolutionary relationships. The decision to show the families of ion channels in Figure 1 as pie charts within a UMAP embedding is intriguing but somewhat non-intuitive and difficult to understand. Illustrating these results with a standard tree-like visualization of the relationship of these channels to each other would be preferred.

One aspect of the pie-chart/UMAP visualization that works well is the highlighting of the 'dark' ion channels according to the status as designated by IDG, which highlights a strength of this whole paper. However, throughout the paper, this could be emphasized more as the key advantage of this approach and how this or similar approaches could be used for other families of proteins. Specifically, in the initial statement describing 'light' vs 'dark channels', the importance of this distinction and the historical preference in science to study that which has already been studied can be discussed more, even including references to other studies that take this kind of approach. An example of a relevant reference here is to the Structural Genomics Consortium and its goals to achieve structures of proteins for which functions may not be well-characterized. Furthermore, this initial statement mentioning 'light channels' was initially confusing -- does this mean light-sensing channels? As one reads on this is clearly not the case, but for such an important central focus of this paper, these kinds of misunderstandings do not serve the authors well. Clarifying these motivations throughout the entire paper would strengthen it considerably.

Additionally, since the authors have generated this UMAP visualization, it would be interesting to understand how the human vs orthologue gene sets compare in this space. Furthermore, Figure 1, for just the human analysis, should say more clearly that this is an analysis of the human gene set and include more of the information in the text: 419 human ion channel sequences, 75 sequences previously unidentified, 4 major groups and 55 families, 62 outliers, etc. Clearer visualizations of these categories and numbers within the UMAP (and newly included tree) visualization would help guide the reader to better understand these results.

One of the most peculiar aspects of this paper is that it feels like two papers, one about better documenting the ion channel genes across species, and another with well-executed experiments on CALHM channels. One suggestion for how to link these two sections together better is to show that previous methods to analyze conserved residues in CALHM were significantly lacking. What results would that give? Why was this not enough? Were there just not enough identified CALHM orthologues to give strong signals in conservation analysis?

Some of the analysis pipeline is unclear. Specifically, the RAG analysis seems critical, but it is unclear how this works - is it on top of the GPT framework and recursively inquires about the answer to prompts? Some example prompts would be useful to understand this. Furthermore, the existence of 76 auxiliary non-pore containing 'ion channel' genes in this analysis is a little confusing, as it seems a part of the pipeline is looking for pore-lining residues. Furthermore, how many of these are picked up in the larger orthologues search? Are these harder to perform checks on to ensure that they are indeed ion channel genes? A further discussion of the choice to include these auxiliary sequences would be relevant. This could just be further discussion of the literature that has decided to do this in the past.

Overall, this manuscript is a valuable contribution to the field, but it requires a few main things to make it truly useful. Namely, how has this approach really improved the ability to identify conserved residues over a less-involved approach? A better description of their methods and results is required in the first section of the paper, as well as some cosmetic improvements.

Reviewer #2 (Public review):

Summary:

In this paper, the authors defined the "channelome," consisting of 419 predicted human ion channels as well as 48,000 ion channel orthologs from other organisms. Using this information, the ion channels were clustered into groups, which can potentially be used to make predictions about understudied ion channels in the groups. The authors then focused on the CALHM ion channel family, mutating conserved residues and assessing channel function.

Strengths:

The curation of the channelome provides an excellent resource for researchers studying ion channels. Supplemental Table 1 is well organized with an abundance of useful information.

Weaknesses:

There are substantial concerns regarding the analysis of the CALHM channels as detailed below.

(1) There are significant problems with the methodology used for the electrophysiology studies. Pulse protocol is used to assess the current voltage relationship (-100 to +140 mV), which extends far beyond the physiological range; currents for the mutant channels were only assessed at +120 mV. It is also unclear why a holding potential of 0 mV was used for CALHM6 recordings; the channel is already open at this voltage (and in Figure 4, only n = 3 for CALHM6). Further, proper controls were not performed. Inhibitors such as Gd3+ can be used to ensure that only CALHM currents are being measured.

(2) In line 334, the authors state that "expression levels of wild-type proteins and mutants are comparable." However, Western blots showing CALHM protein abundance (Supplementary Figure 3) are not of acceptable quality - in the top blot, WT CALHM1 can't even be seen. Representative blots were not shown for all mutants, and there was no effort to determine if levels were statistically significant compared to the wild-type control. Even if there is more or less protein, what does this mean? The protein could be in an intracellular compartment and not at the plasma membrane. In mammalian cells, CALHM6 is localized to intracellular compartments and only translocates to the plasma membrane upon activating stimulus (Danielli et al, EMBO J, 2023). Thus, if CALHM6 is only intracellular, the protein amount would not change, but the measured current would. Abundant intracellular CALHM1 has also been observed in mammalian cells transfected with this protein (Dreses-Werringloer et al., Cell, 2008). The best way to determine if mutations impact CALHM channel localization is to express GFP-tagged constructs in Xenopus oocytes and look for surface expression.

(3) Since the authors have not definitively shown that there are no defects in localization, they cannot make the claim in lines 346-356 that the mutations "either abolished or markedly reduced channel activity." Further, from their data, there is speculation regarding how these residues impact conformational changes during channel opening and closing. Line 404 - again, there is no concrete evidence that any of these residues play a role in gating function. Lines 406-433 - this entire paragraph is speculation without data to back it up. There is also a lack of specificity with statements such as "all mutants showed either reduced or completely abolished activity." What is meant by activity? Do the authors mean conductance?

(4) Line 303 - 13 aligned amino acids were conserved across all CALHM homologs - are these also aligned in related connexin and pannexin families? It is likely that cysteines and proline in TM2 are since CALHM channels overall share a lot of similarities with connexins and pannexins (Siebert et al, JBC, 2013). As in line 207, it would be expected that pannexins, connexins, and CALHM channel families would group together. Related to this, see Line 406 - in connexins, there is also a proline kink in TM2 that may play a role in mediating conformational changes between channel states (Ri et al, Biophysical Journal, 1999).

Reviewer #1 (Public review):

Summary:

This useful work extends a prior study from the authors to observe distance changes within the CNBD domains of a full-length CNG channel based on changes in single photon lifetimes due to tmFRET between a metal at an introduced chelator site and a fluorescent non-canonical amino acid at another site. The data are excellent and convincingly support the authors' conclusions. The methodology is of general use for other proteins. The authors also show that coupling of the CNBDs to the rest of the channel stabilizes the CNBDs in their active state, relative to an isolated CNBD construct.

Strengths:

The manuscript is very well written and clear.

Reviewer #2 (Public review):

The manuscript "Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET" by Eggan et al. investigates the energetics of conformational transitions in the cyclic nucleotide-gated (CNG) channel SthK. This lab pioneered transition metal FRET (tmFRET), which has previously provided detailed insights into ion channel conformational changes. Here, the authors analyze tmFRET fluorescence lifetime measurements in the time domain, yielding detailed insights into conformational transitions within the cyclic nucleotide binding domains (CNBDs) of the channel. The integration of tmFRET with time-correlated single-photon counting (TCSPC) represents an advancement of this technique.

The results summarize known conformational transitions of the C-helix and provide distance distributions that agree with predicted values based on available structures. The authors first validated their TCSPC approach using the isolated CNBD construct previously employed for similar experiments. They then study the more complex full-length SthK channel protein. The findings agree with earlier results from this group, demonstrating that the C-helix is more mobile in the closed state than static structures reflect. Upon adding the activating ligand cAMP, the C-helix moves closer to the bound ligand, as indicated by a reduced fluorescence lifetime, suggesting a shorter distance between the donor and acceptor. The observed effects depend on the cAMP concentration, with affinities comparable to functional measurements. Interestingly, a substantial amount of CNBDs appear to be in the activated state even in the absence of cAMP (Figure 6E and F, fA2 ~ 0.4).

This may be attributed to cooperativity among the CNBDs, which the authors could elaborate on further. In this context, the major limitation of this study is that distance distributions are observed only in one domain. While inter-subunit FRET is detected and accounted for, the results focus exclusively on movements within one domain. Thus, the resulting energetic considerations must be assessed with caution. In the absence of the activator, the closed state is favored, while the presence of cAMP favors the open state. This quantifies the standard assumption; otherwise, an activator would not effectively activate the channel. However, the numerical values of approximately 3 kcal/mol are limited by the fact that only one domain is observed in the experiment, and only one distance (C- helix relative to the CNBD) is probed. Additional conformational changes leading to pore opening (including rotation and upward movement of the CNBD, and radial dilation of the tetrameric assembly) are not captured by the current experiments. These limitations should be taken into account when interpreting the results.

Reviewer #3 (Public review):

Summary:

This is a lucidly written manuscript describing the use of transition-metal FRET to assess distance changes during functional conformational changes in a CNG channel. The experiments were performed on an isolated C-terminal nucleotide binding domain (CNBD) and on a purified full-length channel, with FRET partners placed at two positions in the CNBD.

Strengths:

The data and quantitative analysis are exemplary, and they provide a roadmap for use of this powerful approach in other proteins.

Weaknesses/Comments:

A ~3x lower Kd for nucleotide is seen for the detergent-solubilized full-length channel, compared to electrophysiological experiments. This is worth a comment in the Discussion, particularly in the context of the effect of the pore domain on the CNBD energetics.

Reviewer #1 (Public review):

The authors investigate how the viscoelasticity of the fingertip skin can affect the firing of mechanoreceptive afferents and they find a clear effect of recent physical skin state (memory), which is different between afferents. The manuscript is extremely well-written and well-presented. It uses a large dataset of low threshold mechanoreceptive afferents in the fingertip, where it is particularly noteworthy that the SA-2s have been thoroughly analyzed and play an important role here. They point out in the introduction the importance of the non-linear dynamics of the event when an external stimulus contacts the skin, to the point at which this information is picked up by receptors. Although clearly correlated, these are different processes, and it has been very well-explained throughout. I have some comments and ideas that the authors could think about that could further improve their already very interesting paper. Overall, the authors have more than achieved their aims, where their results very much support the conclusions and provoke many further questions. This impact of the previous dynamics of skin affecting current state can be explored further in so many ways and may help us in understanding skin aging and the effects of anatomical changes of the skin better.

Comments on revised submission:

The authors have taken all my considerations into account and provided excellent responses to them. They have modified their paper accordingly, which improves its clarity even more. Very interesting work and I have no further comments.

Reviewer #2 (Public review):

Summary:

The authors sought to identify the impact skin viscoelasticity has on neural signalling of contact forces that are representative of those experienced during normal tactile behaviour. The evidence presented in the analyses indicate there is a clear effect of viscoelasticity on the imposed skin movements from a force-controlled stimulus. Both skin mechanics and evoked afferent firing were affected based on prior stimulation, which has not previously been thoroughly explored. This study outlines that viscoelastic effects have an important impact on encoding in the tactile system, which should be considered in the design and interpretation of future studies. Viscoelasticity was shown to affect the mechanical skin deflections and stresses/strains imposed by previous and current interaction force, and also the resultant neuronal signalling. The result of this was an impaired coding of contact forces based upon previous stimulation. The authors may be able to strengthen their findings, by using the existing data to further explore the link between skin mechanics and neural signalling, giving a clearer picture than demonstrating shared variability. This is not a critical addition, but I believe would strengthen the work and make it more generally applicable.

Strengths:

-Elegant design of the study. Direct measurements have been made from the tactile sensory neurons to give detailed information on touch encoding. Experiments have been well designed and the forces/displacements have been thoroughly controlled and measured to give accurate measurements of global skin mechanics during a set of controlled mechanical stimuli.<br /> -Analytical techniques used. Analysis of fundamental information coding and information representation in the sensory afferents reveals dynamic coding properties to develop putative models of the neural representation of force. This advanced analysis method has been applied to a large dataset to study neural encoding of force, the temporal dynamics of this, and the variability in this.

Weaknesses:<br /> -Lack of exploration of the variation in neural responses. Although there is a viscoelastic effect which produces variability in the stimulus effects based on prior stimulation, it is a shame that the variability in neural firing and force induced skin displacements have been presented, and are similarly variable, but there has been no investigation of a link between the two. I believe with these data the authors can go beyond demonstrating shared variability. The force per se is clearly not faithfully represented in the neural signal, being masked by stimulation history, and it is of interest if the underlying resultant contact mechanics are.

Validity of conclusions:

The authors have succeeded in demonstrating skin viscoelasticity has an impact on skin contact mechanics with a given force and that this impacts on the resultant neural coding of force. Their study has been well designed and the results support their conclusions. The importance and scope of the work is adequately outlined for readers to interpret the results and significance.

Impact:

This study will have important implications for future studies performing tactile stimulation and evaluating tactile feedback during motor control tasks. In detailed studies of tactile function, it illustrates the necessity to measure skin contact dynamics to properly understand the effects of a force stimulus on the skin and mechanoreceptors.

Reviewer #1 (Public review):

Summary:

The authors demonstrate that two human preproprotein human mutations in the BMP4 gene cause a defect in proprotein cleavage and BMP4 mature ligand formation, leading to hypomorphic phenotypes in mouse knock-in alleles and in Xenopus embryo assays.

Strengths:

They provide compelling biochemical and in vivo analyses supporting their conclusions, showing the reduced processing of the proprotein and concomitant reduced mature BMP4 ligand protein from impressively mouse embryonic lysates. They perform excellent analysis of the embryo and post-natal phenotypes demonstrating the hypomorphic nature of these alleles. Interesting phenotypic differences between the S91C and E93G mutants are shown with excellent hypotheses for the differences. Their results support that BMP4 heterodimers act predominantly throughout embryogenesis whereas BMP4 homodimers play essential roles at later developmental stages.

Weaknesses:

In the revision the authors have appropriately addressed the previous minor weaknesses.

Reviewer #2 (Public review):

Summary:

The revised paper by Kim et al. reports two disease mutations in proBMP4, S91C and E93G, disrupt the FAM20C phosphorylation site at Ser91, blocking the activation of proBMP4 homodimers, while still allowing BMP4/7 heterodimers to function. Analysis of DMZ explants from Xenopus embryos expressing the proBMP4 S91C or E93G mutants showed reduced expression of pSmad1 and tbxt1. The expert amphibian tissue transplant studies were expanded to in vivo studies in Bmp4S91C/+ and Bmp4E93G/+ mice, highlighting the impact of these mutations on embryonic development, particularly in female mice, consistent with patient studies. Additionally, studies in mouse embryonic fibroblasts (MEFs) demonstrated that the mutations did not affect proBMP4 glycosylation or ER-to-Golgi transport but appeared to inhibit the furin-dependent cleavage of proBMP4 to BMP4. Based on these findings and AI modeling using AlphaFold of proBMP4, the authors speculate that pSer91 influences access of furin to its cleavage site at Arg289AlaLysArg292 in a new "Ideas and Speculation" section. Overall, the authors addressed the reviewers' comments, improving the presentation.

Strengths:

The strengths of this work continue to lie in the elegant Xenopus and mouse studies that elucidate the impact of the S91C and E93G disease mutations on BMP signaling and embryonic development. Including an "Ideas and Speculation" subsection for mechanistic ideas reduces some shortcomings regarding the analysis of the underlying mechanisms.

Reviewer #3 (Public review):

Summary:

The authors describe important new biochemical elements in the synthesis of a class of critical developmental signaling molecules, BMP4. They also present a highly detailed description of developmental anomalies in mice bearing known human mutations at these specific elements.

Strengths:

This paper presents exceptionally detailed descriptions of pathologies occurring in BMP4 mutant mice. Novel findings are shown regarding the interaction of propeptide phosphorylation and convertase cleavage, both of which will move the field forward. Lastly, a provocative hypothesis regarding furin access to cleavage sites is presented, supported by Alphafold predictions.

Reviewer #1 (Public review):

This study investigates the role of microtubules (MT) in regulating insulin secretion from pancreatic islet beta cells. This is of great importance considering that controlled secretion of insulin is essential to prevent diabetes. Previously, it has been shown that KIF5B plays an essential role in insulin secretion by transporting insulin granules to the plasma membrane. High glucose activates KIF5B to increase insulin secretion resulting in cellular uptake of glucose. In order to prevent hypoglycemia, insulin secretion needs to be tightly controlled. Notably, it is known that KIF5B plays a role in MT sliding. This is important, as the authors described previously that beta cells establish a peripheral sub-membrane MT array, which is critical for withdrawal of excessive insulin granules from the secretion sites. At high glucose, the sub-membrane MT array is destabilized to allow for robust insulin secretion. Here the authors aim to answer the question how the peripheral array is formed. Based on the previously published data the authors hypothesize that KIF5B organizes the sub-membrane MT array via microtubule sliding.

General comment:<br /> This manuscript provides data that indicate that KIF5B, like in many other cells, mediates MT sliding in beta cells to establish a non-radial sub-membrane MT array. This study is based mainly on in vitro assays and one cell line. To demonstrate the importance of KIF5B in vivo/under physiological conditions, the MT pattern and directionality in beta cells within whole isolated pancreatic islets from KIF5B KO mice was analyzed in comparison to their WT littermates. While the presented effects appear often rather small, it is important to note that small changes in MT configuration can have strong effects. However, the authors provide no link to insulin secretion and glucose uptake. Finally, it remains unclear whether a KIF5B-dependent mechanism regulating microtubule sliding plays a major role in controlling insulin secretion.

Specific comments:<br /> (1) It is difficult to appreciate that there is a "peripheral sub-membrane microtubule array" as it is not well defined in the manuscript. This reviewer assumes that this is in the respective field clear. Yet, while it is appreciated that there is an increased amount of MTs close to the cytoplasmic membrane, the densities appear very variable along the membrane. Please provide a clear description in the Introduction what is meant with "peripheral sub-membrane microtubule array".<br /> (2) The authors described a "consistent presence of a significant peripheral array in the C57BL/6J control mice, while the KO counterparts exhibited a partial loss of this peripheral bundle. Specifically, the measured tubulin intensity at the cell periphery was significantly reduced in the KO mice compared to their wild-type counterparts". In vitro "control cells had convoluted non-radial MTs with a prominent sub-membrane array, typical for β cells (Fig. 2A), KIF5B-depleted cells featured extra-dense MTs in the cell center and sparse receding MTs at the periphery (Fig. 2B,C)". Please comment/discuss why in vivo there are no "extra-dense MTs in the cell center".<br /> (3) Authors should include in the Discussion a paragraph discussing the fact that small changes in MT configuration can have strong effects.

Reviewer #2 (Public review):

This elegant study provides significant and impactful insights into the factors contributing to the distinct arrangement of sub-membrane microtubules within mouse β-cells of the pancreas. The authors propose that in these cells, the motor protein KIF5B plays a crucial role in sliding existing microtubules toward the cell periphery and aligning them with one another along the plasma membrane. Furthermore, similar to other physiological features of β-cells, high glucose levels enhance this microtubule sliding process. A precise arrangement of microtubules beneath the cell membrane in β-cells is vital for the regulated secretion of pancreatic enzymes and hormones; thus, KIF5B has a significant role in pancreatic activity in both healthy conditions and diseases. The authors support their model by demonstrating that the levels of KIF5B mRNA in MIN6 cells are higher than those of other known kinesins. They show that microtubule sliding becomes less efficient when KIF5B is genetically silenced using two different short hairpin RNAs (shRNAs). Additionally, silencing of KIF5A in the same cells results in a general reorganization of microtubules throughout the cell. Specifically, while control cells exhibit a convoluted and non-radial arrangement of microtubules near the cell membrane, KIF5B-depleted cells display a sparse and less dense sub-membrane array of microtubules. Based on these findings, the authors conclude that the loss of KIF5B strongly affects the localization of microtubules to the cell periphery. Using a dominant-negative approach, the authors also demonstrate that KIF5B facilitates the sliding of microtubules by binding to cargo microtubules through the kinesin-1 tail binding domain. They present evidence suggesting that KIF5B-mediated microtubule sliding is glucose-dependent, similar to the activity levels of kinesin-1, which increase in the presence of glucose. Lastly, they show that this is glucose-dependent.

Strengths:

This study unveils a previously unexplained mechanism that regulates the specific rearrangement of microtubules beneath the cell membrane in pancreatic β-cells. The findings have significant implications because the precise regulation of the microtubule array at the secretion zone plays a critical role in controlling pancreatic function in both healthy and diseased states. The provided data supports the authors' conclusions well, and the study demonstrates the use of state-of-the-art methodologies, including quantification techniques and elegant dominant-negative experiments.

Weaknesses: None

Reviewer #1 (Public review):

Summary:

This short report shows that the transcription factor gene mirror is specifically expressed in the posterior region of the butterfly wing imaginal disk, and uses CRISPR mosaic knock-outs to show it is necessary to specify the morphological features (scales, veins, and surface) of this area.

Strengths:

The data and figures support the conclusions. The article is swiftly written and makes an interesting evolutionary comparison to the function of this gene in Drosophila. Based on the data presented, it can now be established that mirror likely has a similar selector function for posterior-wing identity in a plethora of insects.

Comments on revisions:

The revision is satisfactory. I agree with the authors that this article provides interesting insights on the evolution of insect wings. Of note, butterfly and fly wing imaginal disks differ in their mode of development: while fly wing disks grow as epithelial sacs that evaginate during metamorphosis, butterfly wing disks develop as relatively flat epithelial sheets that expand and differentiate progressively. This makes the similar role of mirror all the more interesting.

The revised text appropriately discuss how selector genes like mirror regionalize the wing during larval and pupal development. This article makes a reasonable use of CRISPR mosaic knock outs and uses contralateral controls to show the nature of the phenotypic transformations.

Reviewer #2 (Public review):

This is a short and unpretentious paper. It is an interesting area and therefore, although much of this area of research was pioneered in flies, extending basic findings to butterflies would be worthwhile. Indeed, there is an intriguing observation but it is technically flawed and these flaws are far too serious to allow us to recommend publication

The authors show that mirror is expressed at the back of the wing in butterflies (as in flies). They present some evidence that is required for the proper development of the back of the wing in butterflies (a region dubbed the vannus by the ancient guru Snodgrass). But there are problems with that evidence. First, concerning the method, using CRISP they treat embryos and the expectation is that the mirror gene will be damaged in groups of cell lineages, giving a mosaic animal in which some lines of cells are normal for mirror and others not. We do not know where the clones or patches of cells that are defective for mirror are because they are not marked. Also, we do not know what part of the wing is wildtype and what part is mutant for mirror. When the mirror mutant cells colonise the back of the wing and that butterfly survives (many butterflies fail to develop), the back of the wing is altered in some selected butterflies. This raises a second problem: we do not know whether the rear of the wing is missing or transformed. From the images the appearance of the back of the wing is clearly different from wild type, but is that due to transformation or not? And then I believe we need to know specifically what us difference between the rear of the wing and the main part. What we see is a silvery look at the back that is not present in the main part, is it the structure of the scales? We are not told. There are other problems. Mirror is only part of a group of genes in flies and in flies both iroquois and mirror are needed to make the back of the wing, the alula (Kehl et al). What is known about iro expression in butterflies?

In flies, mirror regulates a late and local expression of dpp that seems to be responsible of making the alula. What happens in butterflies? Would a study of expression of Dpp in wildtype and mirror compromised wings be useful?

Thus, I find the paper to be disappointing for a general journal as it does little more than claim what was discovered in Drosophila is at least partly true in butterflies. Also it fails to explain what the authors mean by "wing domains" and "domain specification". They are not alone, butterfly workers in general appear vague about these concepts, their vagueness allowing too much loose thinking. Since these matters are at the heart of the purpose and meaning of the work reported here, we readers need a paper containing more critical thought and information. I would like to have a better and more logical introduction and discussion.

They do define what they mean by the vannus of the wing. In flies the definition of compartments is clear and abundantly demonstrated, with gene expression and requirement being limited precisely to sets of cells that display lineage boundaries. It is true that domains of gene expression in flies, for example, of the iroquois complex, which includes mirror, can only be related to pattern with difficulty. Some recap of what is known plus the opinion of the authors on how they interpret papers on possible lineage domains in butterflies might also be useful as the reader, is no wiser about what the authors might mean at the end of it!

The references are sometimes inappropriate. The discovery of the AP compartments should not be referred to Guillen et al 1995, but to Morata and Lawrence 1975.

Comments on revisions:

Nearly all the previous criticisms remain valid and are not discussed or overcome in the revision. The authors wish to draw their conclusions and we think they can do that, but they should make clear that key evidence is lacking. Thus their conclusions are speculative. But they present them more or less as facts. This is not justified. Let us suppose that clones lacking mirror do not survive or do not develop properly in the rear part of the wing and what they are seeing is occasional damage due to incomplete regeneration or to regenerative duplication?

Many clones in flies only include parts of one surface of the wing, could this happen here and how would it affect interpretations?

The null phenotype in the wing is not known but deduced from abnormal wings which "even in mKO..... appeared to have a mutant phenotype across the entire posterior region", a nice example of circular logic.

We believe the authors should be more objective and explain that their interpretations are not solid and that they should ideally be tested by finding ways of independently marking the clones. Other clonal mosaic experiments in butterflies have been done (eg https://journals.biologists.com/dev/article/150/18/dev201868/329659/Frizzled2-receives-WntA-signaling-during-butterfly) without cell autonomous independent markers, but they are more solid as transformed spots are made visible cell by cell by scale colour changes etc.

Their deduction that "mirror acts as a selector gene necessary to define the far posterior wing domain" is a speculative hypothesis, not a deduction and the readers should be so informed.

Reviewer #3 (Public review):

Summary:

The manuscript by Chatterjee et al., examines the role of the mirror locus in patterning butterfly wings. The authors examine the pattern of mirror expression in the common buckeye butterfly, Junonia coenia and then employ CRISPR mutagenesis to generate mosaic butterflies carrying clones of mirror mutant cells. They find that mirror is expressed in a well-defined posterior sector of final-instar wing discs from both hindwings and forewings and that CRISPR-injected larvae display a loss of adult wing structures presumably derived from the mirror expressing region of hindwing primordium (the case for forewings is a bit less clear since the mirror domain is narrower than in the hindwing, but there also do seem to be some anomalies in posterior regions of forewings in adults derived from CRISPR injected larvae). The authors conclude that wings of these butterflies have at least three different fundamental wing compartments, the mirror domain, a posterior domain defined by engrailed expression, and an anterior domain expressing neither mirror or engrailed. They speculate that this most posterior compartment has been reduced to a rudiment in Drosophila and thus has not been adequately recognized as a such a primary regional specialization.

Critique: This is a very straight-forward study and the experimental results presented support the key claims that mirror is expressed in a restricted posterior section of the wing primordium and that mosaic wings from CRISPR injected larvae display loss of adult wing structures presumably derived from cells expressing mirror (or at least nearby). The major issue I have with this paper is the strong interpretation of these findings that lead the authors to conclude that mirror is acting as a high level gene akin to engrailed in defining a separate extreme posterior wing compartment. To place this claim in context, it is important in my view to consider what is known about engrailed, for which there is ample evidence to support the claim that this gene does play a very ancestral and conserved function in a defining posterior compartments of all body segments (including the wing) across arthropods.

(A) engrailed is expressed in a broad posterior domain with a sharp anterior border in all segments of virtually all arthropods examined (broad use of a very good pan-species anti-En antibody makes this case very strong).

(B) In Drosophila, marked clones of wing cells (generated during larval stages) strictly obey a straight anterior-posterior border indicating that cells in these two domains do not normally intermix, thus, supporting the claim that a clear A/P lineage compartment exists.

In my opinion, mirror does not seem to be in the same category of regulator as engrailed for the following reasons:

(1) There is no evidence that I am aware of, either from the current experiments, or others that the mirror expression domain corresponds to a clonal lineage compartment. It is also unclear from the data shown in this study whether engrailed is co-expressed with mirror in posterior most cells of J. coenia wing discs? If so, it does not seem justified to infer that mirror acts as an independent determinant of the region of the wing where it is expressed.

(2) The mirror is not only expressed in a posterior region of the wing in flies but also in the ventral region of the eye. In Drosophila, mirror mutants not only lack the alula (derived approximately from cells where mirror is expressed), but also lacks tissue derived from the ventral region of the eye disc (although this ventral tissue loss phenotype may extend beyond the cells expressing mirror).

In summary, it seems most reasonable to me to think of mirror as a transcription factor that provides important development information for a diverse set of cells in which it can be expressed (posterior wing cells and ventral eye cells) but not that it acts as a high level regulator as engrailed.

Recommendation:

While the data provided in this succinct study are solid and interesting, it is not clear to me that these findings support the major claim that mirror defines an extreme posterior compartment akin to that specified by engrailed. Minimally, the authors should address the points outlined above in their discussion section and greatly tone down their conclusion regarding mirror being a conserved selector-like gene dedicated to establishing posterior-most fates of the wing. They also should cite and discuss the original study in Drosophila describing the mirror expression pattern in the embryo and eye and the corresponding eye phenotype of mirror mutants: McNeill et al., Genes & Dev. 1997. 11: 1073-1082; doi:10.1101/gad.11.8.1073.

Reviewer #1 (Public review):

The study addresses how faces and bodies are integrated in two STS face areas revealed by fMRI in the primate brain. It is building upon recordings and analysis of the responses of large populations of neurons to three sets of images, that vary face and body positions. These sets allowed the author to thoroughly investigate invariance to position on the screen (MC HC), to pose (P1 P2), to rotation (0 45 90 135 180 225 270 315), to inversion, to possible and impossible postures (all vs straight), to presentation of head and body together or in isolation. By analyzing neuronal responses, they find that different neurons showed preferences for body orientation, or head orientation or for the interaction between the two. By using a linear support vector machine classifier, they show that the neuronal population can decode head-body angle presented across orientations, in the anterior aSTS patch (but not middle mSTS patch), except for mirror orientation. On the contrary, mSTS neurons show less invariance for head-body angle and more specialization for head or body orientation.

Strengths:

These results expand prior work on the role of Anterior STS fundus face area in face-body integration and its invariance to mirror symmetry, with a rigorous set of stimuli revealing the workings of these neuronal populations in processing individuals as a whole, in an important series of carefully designed conditions.

It also raises questions for future investigations comparing humans and monkeys expertise with upright and inverted configurations, in light of monkey-specific hanging upside-down behavior. Further, using two types of body postures (sitting, standing), they show a correlation in head-body angle between postures, suggesting that monkey orientation might be more meaningful to these neurons than precise posture.

Reviewer #2 (Public review):

Summary:

This paper investigates the neuronal encoding of the relationship between head and body orientations in the brain. Specifically, the authors focus on the angular relationship between the head and body by employing virtual avatars. Neuronal responses were recorded electrophysiologically from two fMRI-defined areas in the superior temporal sulcus and analyzed using decoding methods. They found that: (1) anterior STS neurons encode head-body angle configurations; (2) these neurons distinguish aligned and opposite head-body configurations effectively, whereas mirror-symmetric configurations are more difficult to differentiate; and (3) an upside-down inversion diminishes the encoding of head-body angles. These findings advance our understanding of how visual perception of individuals is mediated, providing a fundamental clue as to how the primate brain processes the relationship between head and body-a process that is crucial for social communication.

Strengths:

The paper is clearly written, and the experimental design is thoughtfully constructed and detailed. The use of electrophysiological recordings from fMRI-defined areas elucidated the mechanism of head-body angle encoding at the level of local neuronal populations. Multiple experiments, control conditions, and detailed analyses thoroughly examined various factors that could affect the decoding results. The decoding methods effectively and consistently revealed the encoding of head-body angles in the anterior STS neurons. Consequently, this study offers valuable insights into the neuronal mechanisms underlying our capacity to integrate head and body cues for social cognition-a topic that is likely to captivate readers in this field.

Weaknesses:

I did not identify any major weaknesses in this paper.

Reviewer #3 (Public review):

Summary:

Zafirova et al. investigated the interaction of head and body orientation in the macaque superior temporal sulcus (STS). Combining fMRI and electrophysiology, they recorded responses of visual neurons to a monkey avatar with varying head and body orientations. They found that STS neurons integrate head and body information in a nonlinear way, showing selectivity for specific combinations of head-body orientations. Head-body configuration angles can be reliably decoded, particularly for neurons in the anterior STS, suggesting a transformation of face/body orientation signals from the middle to the anterior STS. Furthermore, body inversion resulted in reduced decoding of head-body configuration angles. Compared to previous work that examined face or body alone, this study demonstrates how head and body information are integrated to compute a socially meaningful signal.

Strengths:

This work presents an elegant design of visual stimuli, with a monkey avatar of varying head and body orientations, making the analysis and interpretation straightforward. Together with several control experiments, the authors systematically investigated different aspects of head-body integration in the macaque STS. The results and analyses of the paper are convincing.

Weakness:

While this work has characterized the neural integration of head and body information in detail, it's unclear how the neural representation relates to the animal's perception. Behavioural experiments using the same set of stimuli could help address this question, but I agree that these additional experiments may be beyond the scope of the current paper.

for - book - Embracing Paradox, Evolving Language - book - review - Embracing Paradox, Evolving Language - adjacency - Lisa's conlanger - Deep Humanity BEing journeys - Indyweb - provenance - Deep Humanity - language BEing journey - author - Lisa E. Maroski - to - post - LinkedIn - Bayo Akomolafe - from 'belief' to 'apolief" - https://hyp.is/go?url=https%3A%2F%2Fwww.linkedin.com%2Fposts%2Fbayoakomolafe_i-am-against-worldview-the-term-seems-activity-7319799984663535616-fpVW%2F&group=world

new trailmark - summary to review - the word "review" may be a better trailmark word than "summary" - At this point, I will replace "summary" with "review" in the case of book or article reviews

review - Lisa's book is an insightful convergence of an important but ignored subject, the experiential intersection between language and consciousness. - Her understanding that language plays an important role in constructing our reality leads to a bold and novel proposal, especially salient at this time of global poly-meta-perma-meaning crisis. - She proposes that we individually and collectively experiment and explore creating new words and language structures that transcend the limitations of our existing language - If patterns of language usage traps us in outdated conceptual paradigms, then breaking out of these may be challenging, if not impossible, without the creation of new linguistic and language structures. - From a Stop Reset Go and Deep Humanity perpsective, Lisa's proposal for practical experimentation with constructing new languages to unleash new forms of expression is very aligned to Deep Humanity BEing journeys - As I read and annotate Lisa's book, any potential linguistic and language BEing journeys that her words inspired will be recorded for posterity

Addendum - note from journal - 2025, May 8 - reflections on Lisa's book - asynchronous communication is only one half of indyweb     - the other half is asynchronous REFLECTION AND SYNTHESIS - Effective timebinding requires both     - Annotation captures interpersonal shared ideas     - journalling captures ours own unique synthesis only emerges from asynchronous reflections of our existent associative network of ideas and the newly ingested interpersonal ones - Annotations capture the novel and newly inputted interpersonal ones     - but annotation currently only applies to hypothesis - it needs to expand to realtime meetings such as zoom calls, emails, socials media comments and socials media chats in order to be complete - Until now, there has not been a medium with sufficient set of affordances to unleash the affordances potential in language itself - While digital media has existed and rapidly developed for the past 5 decades,     - employing and leveraging it to unleash the full potential of language itself has not ever been conceived of until the concept of Indyweb arrived - Indeed, we could make the claim that the indyweb is a foundational human technology on the same order as language itself because it completes language, revealing its empty ( shunyata) quality, thereby     - uniting it with the universe itself -  From the unlimited potential of the tacit,     - the limited forms of words emerge, both are 2 sides off the same nondual coin     - and unleashing the full , unrealized potential of language - It is the provenance aspect of the indyweb that provides an automatic trail of all our learning journey, making both the     - individual and     - intertwingled collective evolution of ideas available as records for. timebinding posterity

• when we feel in a good state of health and wellbeing and absent of any disease
  • we feel when everything is within harmony in our temporary state of being alive
• Any disease shows us how the diseases-free state is so fragilely constructed
• disease-free is an and condition of many subsystems working together harmoniously -aspectualizing is creating
  • a perspective,
  • a word
  • an idea
• the greatest freedom of afforded when we are free of all perspectives
  • for that is when a new perspective can emerge
• When we cling to words and ideas, we cling to perspectives and aspects of the whole
• The teaching of one taste is the highest and most subtle teaching - equal taste - and easiest to be misinterpreted
  • because we are anchored in the world of many different tastes and of measurement and scale,
    • where some things are greater than others on our scale
• Bayo Akomolafe does some language construction - conlangering on his LinkedIn post on the derivation of the word "apolief" from "belief"

Reviewer #1 (Public review):

Summary:

Kv2 subfamily potassium channels contribute to delayed rectifier currents in virtually all mammalian neurons and are encoded by two distinct types of subunits: Kv2 alpha subunits that have the capacity to form homomeric channels (Kv2.1 and Kv2.2), and KvS or silent subunits (Kv5,6,8.9) that can assemble with Kv2.1 or Kv2.2 to form heteromeric channels with novel biophysical properties. Many neurons express both types of subunits and therefore have the capacity to make both homomeric Kv2 channels and heteromeric Kv2/KvS channels. Determining the contributions of each of these channel types to native potassium currents has been very difficult because the differences in biophysical properties are modest and there are no Kv2/KvS-specific pharmacological tools. The authors set out to design a strategy to separate Kv2 and Kv2/KvS currents in native neurons based on their observation that Kv2/KvS channels have little sensitivity to the Kv2 pore blocker RY785 but are blocked by the Kv2 VSD blocker GxTx. They clearly demonstrate that Kv2/KvS currents can be differentiated from Kv2 currents in native neurons using a two-step strategy to first selectively block Kv2 with RY785, and then block both with GxTx. The manuscript is beautifully written; takes a very complex problem and strategy and breaks it down so both channel experts and the broad neuroscience community can understand it.

Strengths:

The compounds the authors use are highly selective and unlikely to have significant confounding cross-reactivity to other channel types. The authors provide strong evidence that all Kv2/KvS channels are resistant to RY785. This is a strength of the strategy - it can likely identify Kv2/KvS channels containing any of the 10 mammalian KvS subunits and thus be used as a general reagent on all types of neurons. The limitation then of course is that it can't differentiate the subtypes, but at this stage, the field really just needs to know how much Kv2/KvS channels contribute to native currents and this strategy provides a sound way to do so.

Weaknesses:

The authors are very clear about the limitations of their strategy, the most important of which is that they can't differentiate different subunit combinations of Kv2/KvS heteromers. This study is meant to be a start to understanding the roles of Kv2/KvS channels in vivo. As such, this is a minor weakness, far outweighed by the potential of the strategy to move the field through a roadblock that has existed since its inception.

The study accomplishes exactly what it set out to do: provide a means to determine the relative contributions of homomeric Kv2 and heteromeric Kv2/KvS channels to native delayed rectifier K+ currents in neurons. It also does a fabulous job laying out the case for why this is important to do.

Comments on revisions:

I liked this manuscript the first time and thought it was a great attempt to address a difficult problem, made more difficult by confusing background literature and conventions. The authors have kept all the strong points I liked from the first round and made it even stronger with their thoughtful and substantive responses to reviews. My first review was strongly supportive, and my initial short assessment/public review was written with the assumption that they would be public and the paper would be published essentially in its original form. All those points still apply so I am going to leave the initial reviews as is. The paper is a pleasure to read and a nice contribution to the field.

Reviewer #2 (Public review):

The authors used combined blockers/modulators to dissect the potassium currents mediated by inter-subunit heteromeric Kv channels. The method is robust given that the researchers know their limitations. Nevertheless, the authors elegantly tested their hypotheses, making this manuscript friendly to read despite the depth of all aspects they dealt with.

The quality of the data presented will positively impact the science involved in the study heteromeric channels, with clear developments in the field. Finally, the approach presented may unlock new studies related to these channels.

Comments on revisions:

The authors clarified all my points and beyond, specifically by adding some computational work that will also contribute to the subfield of heteromeric Kv channels.

Reviewer #2 (Public review):

Summary:

I found this an interesting manuscript describing a study investigating the role of MC4R signalling on kisspeptin neurons. The initial question is a good one. Infertility associated with MC4 mutations in humans has typically been ascribed to the consequent obesity and impaired metabolic regulation. Whether there is a direct role for MC4 in regulating the HPG axis has not been thoroughly examined. Here, the researchers have put together an elegant combination of targeted loss of function and gain of function in vivo experiments, specifically targeting MC4 expression in kisspeptin neurons. This excellent experimental design should provide compelling evidence for whether melanocortin signalling has a direct role in arcuate kisspeptin neurons to support normal reproductive function. There were definite effects on reproductive function (irregular estrous cycle, reduced magnitude of LH surge induced by exogenous estradiol). However, the magnitude of these responses and the overall effect on fertility were relatively minor. The mice lacking MC4R in kisspeptin neurons remained fertile despite these irregularities. The second part of the manuscript describes a series of electrophysiological studies evaluating the pharmacological effects of melanocortin signalling in kisspeptin cells in ex-vivo brain slides. These studies characterised interesting differential actions of melanocortins in two different populations of kisspeptin neurons. Collectively, I think the study provides novel insights into how direct actions of melanocortin signalling, via the MC4 receptor in kisspeptin neurons, contribute to the metabolic regulation of the reproductive system. Importantly, however, it is clear that other mechanisms are also at play.

Strengths:

The loss of function/gain of function experiments provide a conceptually simple but hugely informative experimental design. This is the key strength of the current paper - especially the knock-in study that showed improved reproductive function even in the presence of ongoing obesity. This is a very convincing result that documents that reproductive deficits in MC4R knockout animals (and humans with deleterious variants of the MC4R gene) can be ascribed to impaired signalling in the hypothalamic kisspeptin neurons and not necessarily simply caused as a consequence of obesity. As concluded by the authors: "reproductive impairments observed in MC4R deficient mice, which replicate many of the conditions described in humans, are largely mediated by the direct action of melanocortins via MC4R on Kiss1 neurons and not to their obese phenotype." This is important, as it might change the way such fertility problems are treated.

Limitation:

The mechanistic studies evaluating melanocortin signalling in kisppetin neurons were all completed in ovariectomized animals (with and without exogenous hormones). This reductionist approach allowed a focus on the direct actions of estradiol to regulate responses but missed an opportunity to evaluate how cyclical changes in hormones might impact the system. Such cyclical changes are fundamental to how these neurons function in vivo and may dynamically alter the way they respond to hormones and neuropeptides. However, the inclusion of gonad-intact animals would have significantly increased the complexity of experiments and can reasonably be considered outside of the scope of the present study.

Reviewer #1 (Public review):

Summary:

The authors track the motion of multiple consortia of Multicellular Magnetotactic Bacteria moving through an artificial network of pores and report a discovery of a simple strategy for such consortia to move fast through the network: an optimum drift speed is attained for consortia that swim a distance comparable to the pore size in the time it takes to align the with an external magnetic field. The authors rationalize their observations using dimensional analysis and numerical simulations. Finally, they argue that the proposed strategy could generalize to other species by demonstrating the positive correlation between the swimming speed and alignment time based on theoretical analysis and parameters derived from literature.

Strengths:

The underlying dimensional analysis and model convincingly rationalize the experimental observation of an optimal drift velocity: the optimum balances the competition between the trapping in pores at large magnetic fields and random pore exploration for weak magnetic fields.

Weaknesses:

The convex pore geometry studied here creates convex traps for cells, which I expect enhances their trapping. Natural environments may create a much smaller concentration of such traps. In this case, whether a non-monotonic dependence of the drift velocity on the Scattering number would persist is unclear.

Comments on revisions:

Thank you very much for addressing my comments. I think the revisions have improved the paper.

Reviewer #2 (Public review):

Summary:

The authors have made microfluidic arrays of pores and obstacles with a complex shape and studied the swimming of multicellular magnetotactic bacteria through this system. They provide a comprehensive discussion of the relevant parameters of this system and identify one dimensionless parameter, which they call the scattering number and which depends on the swimming speed and magnetic moment of the bacteria as well as the magnetic field and the size of the pores, as the most relevant. They measure the effective speed through the array of pores and obstacles as a function of that parameter, both in their microfluidic experiments and in simulations, with good agreement between the two. They find an optimal scattering number, which they estimate to reflect the parameters of the studied multicellular bacteria in their natural environment. They finally use this knowledge to compare different species. Despite the variability of bacteria parameters, they estimate the scattering number to be rather narrowly distributed, suggesting that their results apply to a broad range of species.

Strengths:

This is a beautiful experimental approach and the observation of an optimal scattering number (likely reflecting an optimal magnetic moment) is very convincing. The results here improve on similar previous work in two respects: On the one hand, the tracking of bacteria does not have the limitations of previous work, and on the other hand, the effective motility is quantified. Both features are enabled by choices of the experimental system: the use the multicellular bacteria which are larger than the usual single-celled magnetotactic bacteria and the design of the obstacle array which allows the quantification of transition rates due to the regular organization as well as the controlled release of bacteria into this array through a clever mechanism.

Weaknesses:

Some of the key experimental choices on which the strength of the approach is based also come at a price and impose some limitations, namely the use of a non-culturable organism and the regular, somewhat unrealistic artificial obstacle array, but the advantages of these choices outweigh the drawbacks.

Comments on revisions:

The paper has been improved with respect to presentation and content. In particular, I appreciate the new plots comparing the simulation and experiments directly and the estimate of the scattering number for different species. In my opinion, all issues raised by the reviewers have been addressed in a productive way.

Reviewer #1 (Public review):

Summary:

This manuscript reports the investigation of PriC activity during DNA replication initiation in Escherichia coli. It is reported that PriC is necessary for growth and control of DNA replication initiation under diverse conditions where helicase loading is perturbed at the chromosome origin oriC. A model is proposed where PriC loads helicase onto ssDNA at the open complex formed by DnaA at oriC. Reconstituted helicase loading assays in vitro are consistent with the model.

Strengths:

The complementary combination of genetics in vivo and reconstituted assays in vitro provide solid evidence to support the role of PriC at a replication origin.

The manuscript is well written and has a logical narrative.

The data provide new insight to how bacteria might load helicase at the replication origin when the wild-type DnaA-dependent loading pathway is perturbed.

Weakness:

It has not yet been established whether PriC localises at oriC in vivo under the conditions tested.

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins is suboptimal. Here highlighted by mutations in dnaA or dnaC.

This a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second in loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.<br /> This puts into questions the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support initiation of replication and cell viability only in the presence of PriC. Such mutants are capable to generate single strand opening but may fail to load the helicase in absence of PriC. Similarly, dnaA mutants may generate aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

I have a few specific questions/comments

The partial complementation of the dnaC2 strain by PriC seems quite straightforward since this particular mutation leads to initiation arrest at the open complex stage and this sets the stage for PriC to load the helicase. The situation is somewhat different for dnaA46. Why is this mutation partly complemented by PriC at 37C? DnaA46 binds neither ATP nor ADP, yet it functions in initiation at permissive temperature. At nonpermssive temperature, it binds oriC as well but does not lead to initiation. Does the present data imply that the true initiation defect of DnaA46 lies in helicase loading? The authors need to comment on this in the text.

Relating to the above. In Fig. 3 it is shown that the pFH plasmid partly complement dnaA46 in a PriC dependent manner. Again, it would be nice to know the nature of the DnaA46 protein defect. It would be interesting to see how a pING1-dnaA46 plasmid performs in the experiment presented in Fig. 3.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. The conclusions drawn supported by the evidence provided are compelling.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Reviewer #1 (Public review):

Summary:

This paper focuses on understanding how covalent inhibitors of peroxisome proliferator-activated receptor-gamma (PPARg) show improved inverse agonist activities. This work is important because PPARg plays essential roles in metabolic regulation, insulin sensitization, and adipogenesis. Like other nuclear receptors, PPARg, is a ligand-responsive transcriptional regulator. Its important role, coupled with its ligand-sensitive transcriptional activities, makes it an attractive therapeutic target for diabetes, inflammation, fibrosis, and cancer. Traditional non-covalent ligands like thiazolininediones (TZDs) show clinical benefit in metabolic diseases, but utility is limited by off-target effects and transient receptor engagement. In previous studies, the authors characterized and developed covalent PPARg inhibitors with improved inverse agonist activities. They also showed that these molecules engage unique PPARg ligand binding domain (LBD) conformations whereby the c-terminal helix 12 penetrates into the orthosteric binding pocket to stabilize a repressive state. In the nuclear receptor superclass of proteins, helix 12 is an allosteric switch that governs pharmacologic responses, and this new conformation was highly novel. In this study, the authors did a more thorough analysis of how two covalent inhibitors, SR33065 and SR36708 influence the structural dynamics of PPARg LBD.

Strengths:

(1) The authors employed a compelling integrated biochemical and biophysical approach.

(2) The cobinding studies are unique for the field of nuclear receptor structural biology, and I'm not aware of any similar structural mechanism described for this class of proteins.

(3) Overall, the results support their conclusions.

(4) The results open up exciting possibilities for the development of new ligands that exploit the potential bidirectional relationship between the covalent versus non-covalent ligands studied here.

Weaknesses:

(1) The major weakness in this work is that it is hard to appreciate what these shifting allosteric ensembles actually look like on the protein structure. Additional graphical representations would really help convey the exciting results of this study.

Reviewer #2 (Public review):

Summary:

The authors use ligands (inverse agonists, partial agonists) for PPAR, and coactivators and corepressors, to investigate how ligands and cofactors interact in a complex manner to achieve functional outcomes (repressive vs. activating).

Strengths:<br /> The data (mostly biophysical data) are compelling from well-designed experiments. Figures are clearly illustrated. The conclusions are supported by these compelling data. These results contribute to our fundamental understanding of the complex ligand-cofactor-receptor interactions.

Weaknesses:

This is not the weakness of this particular paper, but the general limitation in using simplified models to study a complex system.

Reviewer #1 (Public review):

Summary:

This manuscript by Harris and Gallistel investigates how the rate of learning and strength of conditioned behavior post learning depend on the various temporal parameters of Pavlovian conditioning. They replicate results from Gibbon and Balsam (1981) in rats to show that the rate of learning is proportional to the ratio between the cycle duration and the cue duration. They further show that the strength of conditioned behavior post learning is proportional to the cue duration, and not the above ratio. The overall findings here are interesting, provide context to many conflicting recent results on this topic, and are supported by reasonably strong evidence. Nevertheless, there are some major weaknesses in the evidence presented for some of the stronger claims in the manuscript.

Strengths:

This manuscript has many strengths including a rigorous experimental design, several different approaches to data analysis, careful consideration of prior literature, and a thorough introduction and discussion. The central claim-that animals track the rates of events in their environment, and that the ratio of two rates determine the rate of learning-is supported with solid evidence.

Weaknesses:

Despite the above major strengths, some key aspects of the paper need major improvement. These are listed below.

(1) A key claim made here is that the same relationship (including the same parameter) describes data from pigeons by Gibbon and Balsam (1981) and the rats in this study. I think the evidence for this claim is weak as presented here. First, the exact measure used for identifying trials to criterion makes a big difference in Fig 3. As best as I understand, the authors do not make any claims about which of these approaches is the "best" way. Second, the measure used for identifying trials to criterion in Fig 1 appears different from any of the criteria used in Fig 3. If so, to make the claim that the quantitative relationship is one and the same in both datasets, the authors need to use the same measure of learning rate on both datasets and show that the resultant plots are statistically indistinguishable. Currently, the authors simply plot the dots from the current dataset on the plot in Fig 1 and ask the readers to notice the visual similarity. This is not at all enough to claim that both relationships are the same. In addition to the dependence of the numbers on the exact measure of learning rate used, the plots are in log-log axis. Slight visual changes can mean a big difference in actual numbers. For instance, between Fig 3 B and C, the highest information group moves up only "slightly" on the y-axis but the difference is a factor of 5. The authors need to perform much more rigorous quantification to make the strong claim that the quantitative relationships obtained here and in Gibbon and Balsam 1981 are identical.

(2) Another interesting claim here is that the rates of responding during ITI and the cue are proportional to the corresponding reward rates with the same proportionality constant. This too requires more quantification and conceptual explanation. For quantification, it would be more convincing to calculate the regression slope for the ITI data and the cue data separately and then show that the corresponding slopes are not statistically distinguishable from each other. Conceptually, I am confused why the data used to the test the ITI proportionality come from the last 5 sessions. Specifically, if the model is that animals produce response rates during the ITI (a period with no possible rewards) based on the overall rate of rewards in the context, wouldn't it be better to test this before the cue learning has occurred? Before cue learning, the animals would presumably only have attributed rewards in the context to the context and thus, produce overall response rates in proportion to the contextual reward rate. After cue learning, the animals could technically know that the rate of rewards during ITI is zero. Why wouldn't it be better to test the plotted relationship for ITI before cue learning has occurred? Further, based on Fig 1, it seems that the overall ITI response rate reduces considerably with cue learning. What is the expected ITI response rate prior to learning based on the authors' conceptual model? Why does this rate differ pre and post cue learning? Finally, if the authors' conceptual framework predicts that ITI response rate after cue learning should be proportional to contextual reward rate, why should the cue response rate be proportional to cue reward rate instead of cue reward rate plus contextual reward rate?

(3) I think there was a major conceptual disconnect between the gradual nature of learning shown in Figs 7 and 8 and the information theoretic model proposed by the authors. To the extent that I understand the model, the animals should simply learn the association once the evidence crosses a threshold (nDKL > threshold) and then produce behavior in proportion to the expected reward rate. If so, why should there be a gradual component of learning as shown in these figures? In terms of the proportional response rule to rate of rewards, why is it changing as animals go from 10% to 90% of peak response? I think the manuscript would be much strengthened if these results are explained within the authors' conceptual framework. If these results are not anticipated by the authors' conceptual framework, please do explicitly state this in the manuscript.

(4) I find the idea stated in the Conclusion section that any model considering probability of reinforcement cannot be correct because it doesn't have temporal units to be weak. I think the authors might mean that existing models based on probability do not work and not that no possible model can work. For any point process, the standard mathematical treatment of continuous time is to compute the expected count of events as p*dt where p is the probability of occurrence of the event in that time bin and dt is an infinitesimal time bin. There is obviously a one-to-one mapping between probability of an event in a point process and its rate. Existing models use an arbitrary time bin/trial and thus, I get the authors' argument in the discussion. However, I think their conclusion is overstated.

(5) The discussion states that the mutual information defined in equation 1 does not change during partial reinforcement. I am confused by this. The mean delay between reinforcements increases in inverse proportion to the probability of reinforcement, but doesn't the mean delay between cue and next reinforcement increase by more than this amount (next reinforcement is greater than or equal to the cue-to-cue interval away from the cue for many trials)? Why is this ratio invariant to partial reinforcement?

Comments on revisions:

Update following revision

(1) This point is discussed in more detail in the attached file, but there are some important details regarding the identification of the learned trial that require more clarification. For instance, isn't the original criterion by Gibbon et al. (1977) the first "sequence of three out of four trials in a row with at least one response"? The authors' provided code for the Wilcoxon signed rank test and nDkl thresholds looks for a permanent exceeding of the threshold. So, I am not yet convinced that the approaches used here and in prior papers are directly comparable. Also, there's still no regression line fitted to their data (Fig 3's black line is from Fig 1, according to the legends). Accordingly, I think the claim in the second paragraph of the Discussion that the old data and their data are explained by a model with "essentially the same parameter value" is not yet convincing without actually reporting the parameters of the regression. Related to this, the regression for their data based on my analysis appears to have a slope closer to -0.6, which does not support strict timescale invariance. I think that this point should be discussed as a caveat in the manuscript.

(2) The authors report in the response that the basis for the apparent gradual/multiple step-like increases after initial learning remains unclear within their framework. This would be important to point out in the actual manuscript. Further, the responses indicating the fact that there are some phenomena that are not captured by the current model would be important to state in the manuscript itself.

(3) There are several mismatches between results shown in figures and those produced by the authors' code, or other supplementary files. As one example, rat 3 results in Fig 11 and Supplementary Materials don't match and neither version is reproduced by the authors' code. There are more concerns like this, which are detailed in the attached review file.

Reviewer #2 (Public review):

A long-standing debate in the field of Pavlovian learning relates to the phenomenon of timescale invariance in learning i.e. that the rate at which an animal learns about a Pavlovian CS is driven by the relative rate of reinforcement of the cue (CS) to the background rate of reinforcement. In practice, if a CS is reinforced on every trial, then the rate of acquisition is determined by the relative duration of the CS (T) and the ITI (C = inter-US-interval = duration of CS + ITI), specifically the ratio of C/T. Therefore, the point of acquisition should be the same with a 10s CS and a 90s ITI (T = 10; C = 90 + 10 = 100, C/T = 100/10 = 10) and with a 100s CS and a 900s ITI (T = 100; C = 900 + 100 = 1000, C/T = 1000/100 = 10). That is to say, the rate of acquisition is invariant to the absolute timescale as long as this ratio is the same. This idea has many other consequences, but is also notably different from more popular prediction-error based associative learning models such as the Rescrola-Wagner model. The initial demonstrations that the ratio C/T predicts the point of acquisition across a wide range of parameters (both within and across multiple studies) was conducted in Pigeons using a Pavlovian autoshaping procedure. What has remained under contention is whether or not this relationship holds across species, particularly in the standard appetitive Pavlovian conditioning paradigms used in rodents. The results from rodent studies aimed at testing this have been mixed, and often the debate around the source of these inconsistent results focuses on the different statistical methods used to identify the point of acquisition for the highly variable trial-by-trial responses at the level of individual animals.<br /> The authors successfully replicate same effect found in pigeon autoshaping paradigms decades ago (with almost identical model parameters) in a standard Pavlovian appetitive paradigm in rats. They achieve this through a clever change the experimental design, using a convincingly wide range of parameters across 14 groups of rats, and by a thorough and meticulous analysis of these data. It is also interesting to note that the two author's have published on opposing sides of this debate for many years, and as a result have developed and refined many of the ideas in this manuscript through this process.

Main findings

(1) The present findings demonstrate that the point of initial acquisition of responding is predicted by the C/T ratio.

(2) The terminal rates of responding to the CS appears to be related to the reinforcement rate of the CS (T; specifically, 1/T) but not its relation to the reinforcement rate of the context (i.e. C or C/T). In the present experiment, all CS trials were reinforced so it is also the case that the terminal rate of responding was related to the duration of the CS.

(3) An unexpected finding was that responding during the ITI was similarly related to the rate of contextual reinforcement (1/C). This novel finding suggests that the terminal rate of responding during the ITI and the CS are related to their corresponding rates of reinforcement. This finding is surprising as it suggests that responding during the ITI is not being driven by the probability of reinforcement during the ITI.

(4) Finally, the authors characterised the nature of increased responding from the point of initial acquisition until responding peaks at a maximum. Their analyses suggest that nature of this increase was best described as linear in the majority of rats, as opposed to the non-linear increase that might be predicted by prediction error learning models (e.g. Rescorla-Wagner). However, more detailed analyses revealed that these changes can be quite variable across rats, and more variable when the CS had lower informativeness (defined as C/T).

Strengths and Weaknesses:

There is an inherent paradox regarding the consistency of the acquisition data from Gibbon & Balsam's (1981) meta-analysis of autoshaping in pigeons, and the present results in magazine response frequency in rats. This consistency is remarkable and impressive, and is suggestive of a relatively conserved or similar underlying learning principle. However, the consistency is also surprising given some significant differences in how these experiments were run. Some of these differences might reasonably be expected to lead to differences in how these different species respond. For example:

- The autoshaping procedure commonly used in the pigeons from these data were pretrained to retrieve rewards from a grain hopper with an instrumental contingency between head entry into the hopper and grain availability. During Pavlovian training, pecking the key light also elicited an auditory click feedback stimulus, and when the grain hopper was made available the hopper was also illuminated.

- In the present experimental procedure, the rats were not given contextual exposure to the pellet reinforcers in the magazine (e.g. a magazine training session is typically found in similar rodent procedures). The Pavlovian CS was a cue light within the magazine itself.

These design features in the present rodent experiment are clearly intentional. Pretraining with the reinforcer in the testing chambers would reasonably alter the background rate of reinforcement (parameter), so it make sense not to include this but differs from the paradigm used in pigeons. Having the CS inside the magazine where pellets are delivered provides an effective way to reduce any potential response competition between CS and US directed responding and combines these all into the same physical response. This makes the magazine approach response more like the pecking of the light stimulus in the pigeon autoshaping paradigm. However, the location of the CS and US is separated in pigeon autoshaping, raising questions about why the findings across species are consistent despite these differences.

Intriguingly, when the insertion of a lever is used as a Pavlovian cue in rodent studies, CS directed responding (sign-tracking) often develops over training such that eventually all animals bias their responding towards the lever than towards the US (goal-tracking at the magazine). However, the nature of this shift highlights the important point that these CS and US directed responses can be quite distinct physically as well as psychologically. Therefore, by conflating the development of these different forms of responding, it is not clear whether the relationship between C/T and the acquisition of responding describes the sum of all Pavlovian responding or predominantly CS or US directed responding.

Another interesting aspect of these findings is that there is a large amount of variability that scales inversely with C/T. A potential account of the source of this variability is related to the absence of preexposure to the reward pellets. This is normally done within the animals' homecage as a form of preexposure to reduce neophobia. If some rats take longer to notice and then approach and finally consume the reward pellets in the magazine, the impact of this would systematically differ depending on the length of the ITI. For animals presented with relatively short CSs and ITIs, they may essentially miss the first couple of trials and/or attribute uneaten pellets accumulating in the magazine to the background/contextual rate of reinforcement. What is not currently clear is whether this was accounted for in some way by confirming when the rats first started retrieving and consuming the rewards from the magazine.

While the generality of these findings across species is impressive, the very specific set of parameters employed to generate these data raise questions about the generality of these findings across other standard Pavlovian conditioning parameters. While this is obviously beyond the scope of the present experiment, it is important to consider that the present study explored a situation with 100% reinforcement on every trial, with a variable duration CS (drawn form a uniform distribution), with a single relatively brief CS (maximum of 122s) CS and a single US. Again, the choice of these parameters in the present experiment is appropriate and very deliberately based on refinements from many previous studies from the authors. This includes a number of criteria used to define magazine response frequency that includes discarding specific responses (discussed and reasonably justified clearly in the methods section). Similarly, the finding that terminal rates of responding are reliably related to 1/T is surprising, and it is not clear whether this might be a property specific to this form of variable duration CS, the use of a uniform sampling distribution, or the use of only a single CS. However, it is important to keeps these limitations in mind when considering some of the claims made in the discussion section of this manuscript that go beyond what these data can support.

The main finding demonstrating the consistent findings across species is presented in Figure 3. In the analysis of these data, it is not clear why the correlations between C, T, and C/T and the measure of acquisition in Figure 3A were presented as r values, whereas the r2 values were presented in the discussion of Figure 3B, and no values were provided in discussing Figure 3C. The measure of acquisition in Figure 3A is based on a previously established metric, whereas the measure in Figure 3B employs the relatively novel nDKL measure that is argued to be a better and theoretically based metric. Surprisingly, when r and r2 values are converted to the same metric across analyses, it appears that this new metric (Figure 3B) does well but not as well as the approach in Figure 3A. This raises questions about why a theoretically derived measure might not be performing as well on this analysis, and whether the more effective measure is either more reliable or tapping into some aspect of the processes that underlie acquisition that is not accounted for by the nDKL metric. Unfortunately, the new metric is discussed and defined at great length but its utility is not considered.<br /> An important analysis issue that is unclear in the present manuscript is exactly how the statistics were run (how the model was defined, were individual subjects or group medians used, what software was used etc...). For example, it is not clear whether the analyses conducted in relation to Figure 3 used the data from individual rats or the group medians. Similarly, it appears that each rat contributes four separate data points, and a single regression line was fit to all these data despite the highly likely violation of the assumption independent observations (or more precisely, the assumption of uncorrelated errors) in this analysis. Furthermore, it is claimed that the same regression line fit the IT and CS period data in this figure, however this<br /> If the data in figure 3 were analyzed with log(ITI) or log(C/ITI) i.e. log(C/(T-C)), would this be a better fit for these data? Is it the case that the ratio of C/T the best predictor of the trial/point of acquisition, or is it the case that another metric related to reinforcement rates provides a better fit?

Based on the variables provided in Supplementary file 3, containing the acquisition data, I was unable to reproduce the values reported in the analysis of Figure 3.<br /> In relation to Figure 3: I am curious about whether the authors would be able to comment on whether the individual variability in trials to acquisition would be expected to scale differently based on C/T, or C, or (if a less restricted range was used) T?<br /> It is not clear why Figure 3C is presented but not analyzed, and why the data presented in Figure 4 to clarify the spread of the distribution of the data observed across the plots in Figure 3 uses the data from Figure 3C. This would seem like the least representative data to illustrate the point of Figure 4. It also appears to my eye that the data actually plotted in Figure 4 correspond to Figure 3A and 3B rather than the odds 10:1 data indicated in text.

What was the decision criteria used to decide on averaging the final 5 conditioning sessions as terminal responding for the analyses in Figure 5? This is an oddly specific number. Was this based on consistency with previous work, or based on the greatest number of sessions where stable data for all animals could be extracted?<br /> In the analysis corresponding to Figures 7-8: If I understand the description of this analysis correctly, for each rat the data are the cumulative response data during the CS, starting from the trial on which responding to the CS > ITI (t = 1), and ending at the trial on which CS responding peaked (maximum over 3 session moving average window; t = end). This analysis does not seem to account for changes (decline) in the ITI response rates over this period of acquisition, and it is likely that responding during the ITI is still declining after t=1. Are the 4 functions that were fit to these data to discriminate between different underlying generative processes still appropriate on total CS responding instead of conditional CS responding after accounting for changes in baseline response rates during ITI?

Page 27, Procedure, final sentence: The magazine responding during the ITI is defined as the 20s period immediately before CS onset. The range of ITI values (Table 1) always starts as low as 15s in all 14 groups. Even in the case of an ITI on a trial that was exactly 20s, this would also mean that the start of this period overlaps with the termination of the CS from the previous trial and delivery (and presumably consumption) of a pellet. Please indicate if the definition of the ITI period was modified on trials where the preceding ITI was <20s, and if any other criteria were used to define the ITI.

Were the rats exposed to the reinforcers/pellets in their home cage prior to acquisition? Please indicate whether rats where pre-exposed to the reward pellets in their home cages e.g. as is often done to reduce neophobia. Given the deliberate absence of a magazine-training phase, this information is important when assessing the experienced contingency between the CS and the US.

For all the analyses, please provide the exact models that were fit and the software used. For example, it is not necessarily clear to the reader (particularly in the absence of degrees of freedom) that the model fits discussed in Figure 3 are fit on the individual subject data points or the group medians. Similarly, in Figure 6 there is no indication of whether a single regression model was fit to all the plotted data or whether tests of different slopes for each of the conditions were compared. With regards to the statistics in Figure 6, depending on how this was run, it is also a potential problem that the analyses does not correct for the potentially highly correlated multiple measurements from the same subjects i.e. each rat provides 4 data points which are very likely not to be independent observations.

A number of sections of the discussion are speculative or not directly supported by the present experimental data (but may well be supported by previous findings that are not the direct focus of the present experiment). For example, Page 19, Paragraph 2: this entire paragraph is not really clearly explained and is presenting an opinion rather than a strong conclusion that follows directly from the present findings. Evidence for an aspect of RET in the present paper (i.e. the prediction of time scale invariance on the initial point of acquisition, but not necessarily the findings regarding the rate of terminal acquisition) - while supportive - does not necessarily provide unconditional evidence for this theory over all the alternatives.

Similarly, the Conclusion section (Page 23) makes the claim that "the equations have at most one free parameter", which may be an oversimplification that is conditionally true in the narrow context of the present experiment where many things were kept constant between groups and run in a particular way to ensure this is the case. While the equations do well in this narrow case, it is unlikely that additional parameters would not need to be added to account for more general learning situations. To clarify, I am not contending that this kind of statement is necessarily untrue, merely that it is being presented in a narrow context and may require a deeper discussion of much more of the literature to qualify/support properly - and the discussion section of the present experiment/manuscript may not be the appropriate place for this.

- Consider taking advantage of an "Ideas and Speculation" subsection within the Discussion that is supported by eLife [ https://elifesciences.org/inside-elife/e3e52a93/elife-latest-including-ideas-and-speculation-in-elife-papers ]. This might be more appropriate to qualify the tone of much of the discussion from page 19 onwards.

It seems like there are entire analyses and new figures being presented in the discussion e.g. Page 20: Information-Theoretic Contingency. These sections might be better placed in the methods section or a supplementary section/discussion.

Reviewer #1 (Public review):

Summary:

Systemic and partial Tcf7l2 repression is effective in protecting cancer mice from cachexia-induced death. Hence, this is a promising treatment strategy for cancer patients suffering from cachexia.

Strengths:

The method is well-designed and clearly explained.

Weaknesses:

(1) Abbreviations should be mentioned in full terms for the first time.

(2) Relatively old or even very old references in the Introduction and Discussion.

(3) The result section contains discussion with references, as well.

(4) The number of mice in individual groups is relatively small (3 mice in some groups).

Reviewer #2 (Public review):

Summary:

This study by Leong and colleagues examines the role of the TCF7L2 transcription factor in the Wnt signaling pathway as a regulator of colon/small intestinal cancers and cachexia. Investigators utilize a Tet off repressor genetic system in mice under Dox regulation to silence TCF7L2. Results show DSS-treated APCMin/+ mice lose body weight that can be rapidly rescued by Dox treatment and suppression of TCF7L2 expression. Reduction of TCF7L2 rescues features of cachexia, including body weight, gastrocnemius muscle and adipose mass, as well as molecular markers of cachexia such as the E3 Ub ligases, MuRF1, and Atrogin-1. The most significant finding in the study is that loss of TCF7L2 reduces but does not eliminate tumor progression, as tumors go from adenomas to adenocarcinomas over time while mice are treated with Dox, yet cachexia persists. This implies that TCF7L2 has a direct effect on cachexia. Overall, the study provides insight into the role of TCFL2 in the development of intestinal cancers and muscle atrophy in cachexia.

Strengths:

The study uses an elegant genetic mouse model to provide significant new insight into the role of TCFL2 in colon and small intestinal cancers. In addition, the authors describe the role of TCF7L2 as a regulator of muscle wasting in cachexia. This, too, can be viewed as a new finding for the cachexia field.

Weaknesses:

However, in its current form, the study lacks sufficient data to support the authors' claim regarding the relevance of TCF7L2 as a regulator of cachexia.

Reviewer #1 (Public review):

This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

Reviewer #2 (Public review):

Summary:

The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

Major strengths and weaknesses of the methods and results:

The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

Reviewer #3 (Public review):

Summary:

Here, Guo et al. (2025) propose that the IFN-induced GTPase GVIN1 forms a coat on cytosolic Burkholderia thailandensis, blocking actin tail formation through a mechanism analogous to GBP1-mediated restriction of Shigella motility.

Their study was prompted by the intriguing observation that IFNγ priming and GBP1 coat formation fail to inhibit B. thailandensis actin-based motility in HeLa cells, yet IFNγ restricts the motility of Burkholderia in T24 cells. Further investigation revealed that IFNγ restricts B. thailandensis motility in T24 cells via both GBP1-dependent and -independent mechanisms, suggesting that HeLa cells lack a critical GBP1 co-factor required to inhibit actin tail formation.

To identify the GBP1-independent mechanism, the authors performed an siRNA screen of interferon-stimulated genes (ISGs) and identified GVIN1, a large IFN-induced GTPase, as essential for restricting B. thailandensis motility. To identify the GBP1-independent mechanism, perform a knock-down screen for ISGs and find that the loss of GVIN, a very large IFN-induced GTPase, results in higher actin tail-positive B. thailandensis in T24 cells. They further demonstrate that GVIN forms coats on the surface of B. thailandensis, which prevent the polar localization of BimA and thus actin tail formation. In summary, the data reveal two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent, each relying on distinct host co-factors.

Global assessment:

This is a well-executed study that convincingly demonstrates how GVIN1 restricts the actin-based motility of B. thailandensis through the assembly of coatomers. The results are clearly described, the manuscript is easy to follow, and the data are overall compelling and well presented. I have only a few suggestions on how the manuscript could be further improved.

Reviewer #1 (Public review):

Summary:

This manuscript describes a novel magnetic steering technique to target human adipose derived mesenchymal stem cells (hAMSC) or induce pluripotent stem cells to the TM (iPSC-TM). The authors show delivery of the stem cells lowered IOP, increased ouflow facility, and increased TM cellularity.

Strengths:

The technique is novel and shows promise as a novel therapeutic to lower in IOP in glaucoma. hAMSC are able to lower IOP below baseline as well as increase outflow facility above baseline with no tumorigenicity. These data will have a positive impact on the field and will guide further research using hAMSC in glaucoma models.

Weaknesses:

The transgenic mouse model of glaucoma the authors used did not show ocular hypertensive phenotypes as previously reported; therefore, the Tg-MYOCY437H model should be used with caution in the future. However, the results presented here clearly show magnetically steered cell therapy as a viable treatment strategy to lower intraocular pressure even from baseline. Future studies are needed to demonstrate the effects in ocular hypertensive eyes.

Reviewer #2 (Public review):

This observational study investigates the efficacy of intracameral injected human stems cells as a means to re-functionalize the trabecular meshwork for the restoration of intraocular pressure homeostasis. Using a murine model of glaucoma, human adipose-derived mesenchymal stem cells are shown to be biologically safer and functionally superior at eliciting a sustained reduction in intraocular pressure (IOP). The authors conclude that the use of magnetically-steered human adipose-derived mesenchymal stem cells has potential for long-term treatment of ocular hypertension in glaucoma.

Comments on revisions: Previously noted concerns have been thoughtfully and sincerely considered by the authors and are now clearly addressed in the revised manuscript. No further concerns/comments.

Reviewer #1 (Public review):

The authors attempted to replicate previous work showing that counterconditioning leads to more persistent reduction of threat responses, relative to extinction. They also aimed to examine the neural mechanisms underlying counterconditioning and extinction. They achieved both of these aims, and were able to provide some additional information, such as how counterconditioning impacts memory consolidation. Having a better understanding of which neural networks are engaged during counterconditioning may provide novel pharmacological targets to aid in therapies for traumatic memories. It will be interesting to follow up by examining the impact of varying amounts of time between acquisition and counterconditioning phases, to enhance replicability to real world therapeutic settings.

Major strengths

• This paper is very well written and attempts to comprehensively assess multiple aspects counterconditioning and extinction processes. For instance, the addition of memory retrieval tests is not core to the primary hypotheses, but provides additional mechanistic information on how episodic memory is impacted by counterconditioning. This methodical approach is commonly seen in animal literature, but less so in human studies.

• The Group x Cs-type x Phase repeated measure statistical tests with 'differentials' as outcome variables are quite complex, however the authors have generally done a good job of teasing out significant F test findings with post hoc tests and presenting the data well visually. It is reassuring that there is convergence between self-report data on arousal and valence and the pupil dilation response. Skin conductance is a notoriously challenging modality, so it is not too concerning that this was placed in the supplementary materials. Neural responses also occurred in logical regions with regards to reward learning.

• Strong methodology with regards to neuroimaging analysis, and physiological measures.

• The authors are very clear on documenting where there were discrepancies from their pre-registration and providing valid rationales for why.

Major Weaknesses

• The statistics showing that counterconditioning prevents differential spontaneous recovery are the weakest p values of the paper (and using one tailed tests, although this is valid due to directions being pre-hypothesised). This may be due to relatively small number of participants and some variability in responses.

Reviewer #2 (Public review):

Summary:

The present study sets out to examine the impact of counterconditioning (CC) and extinction on conditioned threat responses in humans, particularly looking at neural mechanisms involved in threat memory suppression. By combining behavioral, physiological, and neuroimaging (fMRI) data, the authors aim to provide a clear picture of how CC might engage unique neural circuits and coding dynamics, potentially offering a more robust reduction in threat responses compared to traditional extinction.

Strengths:

One major strength of this work lies in its thoughtful and unique design - integrating subjective, physiological, and neuroimaging measures to capture the variouse aspects of counterconditiong (CC) in humans. Additionally, the study is centered on a well-motivated hypothesis and the findings have potentials for improving the current understanding of pathways associated with emotional and cognitive control.

The data presentation is systematic, and the results on behavioral and physiological measures fit well with the hypothesized outcomes. The neuroimaging results also provide strong support for distinct neural mechanisms underlying CC versus extinction.

Weaknesses:

Overall, this study is a well-conducted and thought-provoking investigation into counterconditioning, with strong potential to advance our understanding of threat modulation mechanisms. Two minor weaknesses concern the scope and decisions regarding analysis choices. First, while the findings are solid, the topic of counterconditioning is relatively niche and may have limited appeal to a broader audience. Expanding the discussion to connect counterconditioning more explicitly to widely studied frameworks in emotional regulation or cognitive control would enhance the paper's accessibility and relevance to a wider range of readers. This broader framing could also underscore the generalizability and broader significance of the results. In addition, detailed steps in the statistical procedures and analysis parameters seem to be missing. This makes it challenging for readers to interpret the results in light of potential limitations given the data modality and/or analysis choices.

Comments on revisions: My previous concerns and questions have been sufficiently addressed.

Reviewer #3 (Public review):

In this manuscript, Wirz et al use neuroimaging (fMRI) to show that counterconditioning produces a longer lasting reduction in fear conditioning relative to extinction and appears to rely on the nucleus accumbens rather than the ventromedial prefrontal cortex. These important findings are supported by convincing evidence and will be of interest to researchers across multiple subfields, including neuroscientists, cognitive theory researchers, and clinicians.

In large part, the authors achieved their aims of giving a qualitative assessment of the behavioural mechanisms of counterconditioning versus extinction, as well as investigating the brain mechanisms. The results support their conclusions and give interesting insights into the psychological and neurobiological mechanisms of the processes that underlie the unlearning, or counteracting, of threat conditioning.

Strengths:

* Clearly written with interesting psychological insights<br /> * Excellent behavioural design, well-controlled and tests for a number of different psychological phenomena (e.g. extinction, recovery, reinstatement, etc).<br /> * Very interesting results regarding the neural mechanisms of each process.<br /> * Good acknowledgement of the limitations of the study.

Weaknesses:

* I am not sure that the memories tested were truly episodic<br /> * Twice as many female participants than males

Comments on revisions: I have no remaining concerns

Reviewer #1 (Public review):

Summary:

Audio et al. present an interesting study examining cerebral blood volume (CBV) across cortical areas and layers in non-human primates (NHPs) using high-resolution MRI. While with contrast agents are frequently employed to improve fMRI sensitivity in NHP research, its application for characterizing baseline CBV distribution is less common. This study quantifies large-vessel distribution as well as regional and laminar CBV variations, comparing them with other metrics.

Strengths:

(1) Noninvasive mapping of relative cerebral blood volume is novel for non-human primates.<br /> (2) A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV.<br /> (3) The measured relative CBV values correlated with previously reported neuronal and receptor densities, potentially providing valuable physiological insights.

Weaknesses:

(1) A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels is not fully validated, especially on a laminar scale.<br /> (2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels. However, this approach depends on multiple factors, including spatial resolution, contrast agent dosage, and data processing methods. This raises concerns about the generalizability of these findings to other experimental setups or populations.<br /> (3) Baseline R2* is sensitive to baseline R2, vascular volume, iron content, and susceptibility gradients. Additionally, it is sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Although baseline R2* correlates with several physiological parameters, drawing direct physiological inferences from it remains challenging.<br /> (4) CBV-weighted deltaR2*, which depends on both CBV and contrast agent dose, correlates with various metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While such correlations may be useful for exploratory analyses, all comparisons depend on measurement accuracy. A fundamental question remains whether CBV-weighted ΔR2* can provide reliable and biologically meaningful insights into these metrics, particularly in diseased or abnormal brain states.

Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based, measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with known distribution of different types of neurons, markers of metabolic load and others. While the presented methodology captures and estimated 30% of the vasculature, the authors corroborated previous findings regarding lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non-invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to captures the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work, begins by characterizing the spacing of penetrating arteries and ascending veins using vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 is computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to other brain areas is therefore not possible without further exploration with independent methodologies.

Please note that these are comments on the revised version.

Reviewer #1 (Public review):

Summary:

This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.

Strengths:

This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.

Reviewer #2 (Public review):

Summary:

The authors generated three mouse lines harboring ASD, Schizophrenia, and Bi-polar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bi-polar disorder development.

Strengths:

The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bi-polar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was carefully examined using an impressive host of methods. The authors achieved their goal of identifying behavioral, physiological, and molecular alterations that are disorder/variant specific. The impact of this work is extremely high given the growing appreciation of TRIO dysfunction in a large number of brain-related disorders. This work is very interesting in that it begins to identify the unique and subtle ways brain function is altered in ASD, Schizophrenia, and Bi-polar disorder.

Weaknesses:

(1) Most assays were performed in older animals and perhaps only capture alterations that result from homeostatic changes resulting from prodromal pathology that may look very different.

(2) Identification of upregulated (potentially compensating) genes in response to these disorder specific Trio variants is extremely interesting. However, a functional demonstration of compensation is not provided.

(3) There are instances where data is not shown in the manuscript. See "data not shown". All data collected should be provided even if significant differences are not observed.

I consider weaknesses 1 and 2 minor. While they would very interesting to explore, these experiments might be more appropriate for a follow up study. The missing data in 3 should be provided in the supplemental material.

Revised Manuscript:

All of my above concerns were well addressed by the authors in the revised submission.

Reviewer #2 (Public review):

Summary:

This manuscript is about using different analytical approaches to allow ancestry adjustments to GWAS analyses amongst admixed populations. This work is a follow-on from the recently published ITHGC multi-population GWAS (https://doi.org/10.7554/eLife.84394), with the focus on the admixed South African populations. Ancestry adjustment models detected a peak of SNPs in the class II HLA DPB1, distinct from the class II HLA DQA1 loci signficant in the ITHGC analysis.

Strengths:

Excellent demonstration of GWAS analytical pipelines in highly admixed populations. Particularly the utility of ancestry adjustment to improve study power to detect novel associations. Further confirmation of the importance of the HLA class II locus in genetic susceptibility to TB.

Weaknesses:

Limited novelty compared to the group's previous existing publications and the body of work linking HLA class II alleles with TB susceptibility in South Africa or other African populations. This work includes only ~100 new cases and controls from what has already been published. High resolution HLA typing has detected significant signals in both the DQA1 and DPB1 regions identified by the larger ITHGC and in this GWAS analysis respectively (Chihab L et al. HLA. 2023 Feb; 101(2): 124-137).<br /> Despite the availability of strong methods for imputing HLA from GWAS data (Karnes J et Plos One 2017), the authors did not confirm with HLA typing the importance of their SNP peak in the class II region. This would have supported the importance of this ancestry adjustment versus prior ITHGC analysis.

The populations consider active TB and healthy controls (from high-burden presumed exposed communities) and do not provide QFT or other data to identify latent TB infection.

Important methodological points for clarification and for readers to be aware of when reading this paper:

(1) One of the reasons cited for the lack of African ancestry-specific associations or suggestive peaks in the ITHGC study was the small African sample size. The current association test includes a larger African cohort and yields a near-genome-wide significant threshold in the HLA-DPB1 gene originating from the KhoeSan ancestry. Investigation is needed as to whether the increase in power is due to increased African samples and not necessarily the use of the LAAA model as stated on lines 295 and 296?

Authors response - The Manhattan plot in Figure 3 includes the results for all four models: the traditional GWAS model (GAO), the admixture mapping model (LAO), the ancestry plus allelic (APA) model and the LAAA model. In this figure, it is evident that only the LAAA model identified the association peak on chromosome 6, which lends support the argument that the increase in power is due to the use of the LAAA model and not solely due to the increase in sample size.<br /> Reviewer comment - This data supports the authors conclusions that increase power is related to the LAAA model application rather than simply increase sample size.

(2) In line 256, the number of SNPs included in the LAAA analysis was 784,557 autosomal markers; the number of SNPs after quality control of the imputed dataset was 7,510,051 SNPs (line 142). It is not clear how or why ~90% of the SNPs were removed. This needs clarification.

Authors response:<br /> In our manuscript (line 194), we mention that "...variants with minor allele frequency (MAF) < 1% were removed to improve the stability of the association tests." A large proportion of imputed variants fell below this MAF threshold and were subsequently excluded from this analysis.

Reviewers additional comment: The authors should specify the number of SNPs in the dataset before imputation and indicate what proportion of the 784,557 remaining SNPs were imputed. Providing this information might help the reader better understand the rationale behind the imputation process.

(3) The authors have used the significance threshold estimated by the STEAM p-value < 2.5x10-6 in the LAAA analysis. Grinde et al. (2019 implemented their significance threshold estimation approach tailored to admixture mapping (local ancestry (LA) model), where there is a reduction in testing burden. The authors should justify why this threshold would apply to the LAAA model (a joint genotype and ancestry approach).

Authors response: We describe in the methods (line 189 onwards) that the LAAA model is an extension of the APA model. Since the APA model itself simultaneously performs the null global ancestry only model and the local ancestry model (utilised in admixture mapping), we thus considered the use of a threshold tailored to admixture mapping appropriate for the LAAA model.

Reviewers additional comment: While the LAAA model is an extension of the APA model, the authors describe the LAAA test as 'models the combination of the minor allele and the ancestry of the minor allele at a specific locus, along with the effect of this interaction,' thus a joint allele and ancestry effects model. Grinde et al. (2019) proposed the significance threshold estimation approach, STEAM, specifically for the LA approach, which tests for ancestry effects alone and benefits from the reduced testing burden. However, it remains unclear why the authors found it appropriate to apply STEAM to the LAAA model, a joint test for both allele and ancestry effects, which does not benefit from the same reduction in testing burden.

(4) Batch effect screening and correction (line 174) is a quality control check. This section is discussed after global and local ancestry inferences in the methods. Was this QC step conducted after the inferencing? If so, the authors should justify how the removed SNPs due to the batch effect did not affect the global and local ancestry inferences or should order the methods section correctly to avoid confusion.

Authors response: The batch effect correction method utilised a pseudo-case-control comparison which included global ancestry proportions. Thus, batch effect correction was conducted after ancestry inference. We excluded 36 627 SNPs that were believed to have been affected by the batch effect. We have amended line 186 to include the exact number of SNPs excluded due to batch effect.<br /> The ancestry inference by RFMix utilised the entire merged dataset of 7 510 051 SNPs. Thus, the SNPs removed due to the batch effect make up a very small proportion of the SNPs used to conduct global and local ancestry inferences (less than 0.5%). As a result, we do not believe that the removed SNPs would have significantly affected the global and local ancestry inferences. However, we did conduct global ancestry inference with RFMix on each separate dataset as a sanity check. In the tables below, we show the average global ancestry proportions inferred for each separate dataset, the average global ancestry proportions across all datasets and the average global ancestry proportions inferred using the merged dataset. The SAC and Xhosa cohorts are shown in two separate tables due to the different number of contributing ancestral populations to each cohort. The differences between the combined average global ancestry proportions across the separate cohorts does not differ significantly to the global ancestry proportions inferred using the merged dataset.

This is an excellent response and should remain accessible to readers for clarifying this issue.

Comments on revisions:

Thank you for addressing my other recommendations to authors. These have all been satisfactorily addressed.

Reviewer #1 (Public review):

Summary:

This study reveals that TRPV1 signaling plays a key role in tympanic membrane (TM) healing by promoting macrophage recruitment and angiogenesis. Using a mouse TM perforation model, researchers found that blood-derived macrophages accumulated near the wound, driving angiogenesis and repair. TRPV1-expressing nerve fibers triggered neuroinflammatory responses, facilitating macrophage recruitment. Genetic Trpv1 mutation reduced macrophage infiltration, angiogenesis, and delayed healing. These findings suggest that targeting TRPV1 or stimulating sensory nerve fibers could enhance TM repair, improve blood flow, and prevent infections. This offers new therapeutic strategies for TM perforations and otitis media in clinical settings. This is an excellent and high-quality study that provides valuable insights into the mechanisms underlying TM wound healing.

Strengths:

The work is particularly important for elucidating the cellular and molecular processes involved in TM repair. However, there are several concerns about the current version.

Weaknesses:

Major concerns

(1) The method of administration will be a critical factor when considering potential therapeutic strategies to promote TM healing. It would be beneficial if the authors could discuss possible delivery methods, such as topical application, transtympanic injection, or systemic administration, and their respective advantages and limitations for targeting TRPV1 signaling. For example, Dr. Kanemaru and his colleagues have proposed the use of Trafermin and Spongel to regenerate the eardrum.

(2) The authors appear to have used surface imaging techniques to observe the TM. However, the TM consists of three distinct layers: the epithelial layer, the fibrous middle layer, and the inner mucosal layer. The authors should clarify whether the proposed mechanism involving TRPV1-mediated macrophage recruitment and angiogenesis is limited to the epithelial layer or if it extends to the deeper layers of the TM.

Minor concerns

In Figure 8, the schematic illustration presents a coronal section of the TM. However, based on the data provided in the manuscript, it is unclear whether the authors directly obtained coronal images in their study. To enhance the clarity and impact of the schematic, it would be helpful to include representative images of coronal sections showing macrophage infiltration, angiogenesis, and nerve fiber distribution in the TM.

Reviewer #2 (Public review):

Summary:

This study examines the role of TRPV1 signaling in the recruitment of monocyte-derived macrophages and the promotion of angiogenesis during tympanic membrane (TM) wound healing. The authors use a combination of genetic mouse models, macrophage depletion, and transcriptomic approaches to suggest that neuronal TRPV1 activity contributes to macrophage-driven vascular responses necessary for tissue repair.

Strengths:

(1) The topic of neuroimmune interactions in tissue regeneration is of interest and underexplored in the context of the TM, which presents a unique model due to its anatomical features.

(2) The use of reporter mice and bone marrow chimeras allows for some dissection of immune cell origin.

(3) The authors incorporate transcriptomic data to contextualize inflammatory and angiogenic processes during wound healing.

Weaknesses:

(1) The primary claims of the manuscript are not convincingly supported by the evidence presented. Most of the data are correlative in nature, and no direct mechanistic experiments are included to establish causality between TRPV1 signaling and macrophage recruitment or function.

(2) Functional validation of key molecular players (such as Tac1 or Spp1) is lacking, and their roles are inferred primarily from gene expression data rather than experimentally tested.

(3) The reuse of publicly available scRNA-seq data is not sufficiently integrated or extended to yield new biological insights, and it remains largely descriptive.

(4) The macrophage depletion model (CX3CR1CreER; iDTR) lacks specificity, and possible off-target or systemic effects are not addressed.

(5) Several interpretations of the data appear overstated, particularly regarding the necessity of TRPV1 for monocyte recruitment and wound healing.

(6) Overall, the study appears to apply known concepts - namely, TRPV1-mediated neurogenic inflammation and macrophage-driven angiogenesis - to a new anatomical site without providing new mechanistic insight or advancing the field substantially.

Overall:

While the study addresses an interesting topic, the current version does not provide sufficiently strong or novel evidence to support its major conclusions. Additional mechanistic experiments and more rigorous validation would be necessary to substantiate the proposed model and clarify the relevance of the findings beyond this specific tissue context.

Reviewer #1 (Public review):

Summary:

This paper presents results from four independent experiments, each of which tests for rhythmicity in auditory perception. The authors report rhythmic fluctuations in discrimination performance at frequencies between 2 and 6 Hz. The exact frequency depends on the ear and experimental paradigm, although some frequencies seem to be more common than others.

Strengths:

The first sentence in the abstract describes the state of the art perfectly: "Numerous studies advocate for a rhythmic mode of perception; however, the evidence in the context of auditory perception remains inconsistent". This is precisely why the data from the present study is so valuable. This is probably the study with the highest sample size (total of > 100 in 4 experiments) in the field. The analysis is very thorough and transparent, due to the comparison of several statistical approaches and simulations of their sensitivity. Each of the experiments differs from the others in a clearly defined experimental parameter, and the authors test how this impacts auditory rhythmicity, measured in pitch discrimination performance (accuracy, sensitivity, bias) of a target presented at various delays after noise onset.

Weaknesses:

(1) The authors find that the frequency of auditory perception changes between experiments. I think they could exploit differences between experiments better to interpret and understand the obtained results. These differences are very well described in the Introduction, but don't seem to be used for the interpretation of results. For instance, what does it mean if perceptual frequency changes from between- to within-trial pitch discrimination? Why did the authors choose this experimental manipulation? Based on differences between experiments, is there any systematic pattern in the results that allows conclusions about the roles of different frequencies? I think the Discussion would benefit from an extension to cover this aspect.

(2) The Results give the impression of clear-cut differences in relevant frequencies between experiments (e.g., 2 Hz in Experiment 1, 6 Hz in Exp 2, etc), but they might not be so different. For instance, a 6 Hz effect is also visible in Experiment 1, but it just does not reach conventional significance. The average across the three experiments is therefore very useful, and also seems to suggest that differences between experiments are not very pronounced (otherwise the average would not produce clear peaks in the spectrum). I suggest making this point clearer in the text.

(3) I struggle to understand the hypothesis that rhythmic sampling differs between ears. In most everyday scenarios, the same sounds arrive at both ears, and the time difference between the two is too small to play a role for the frequencies tested. If both ears operate at different frequencies, the effects of the rhythm on overall perception would then often cancel out. But if this is the case, why would the two ears have different rhythms to begin with? This could be described in more detail.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures. The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions.

Weaknesses:

(1) Conceptional concerns:

The authors place their research in the context of a rhythmic mode of perception. They also discuss continuous vs rhythmic mode processing. Their study further follows a design that seems to be based on paradigms that assume a recent phase in neural oscillations that subsequently influence perception (e.g., Fiebelkorn et al.; Landau & Fries). In my view, these are different facets in the neural oscillation research space that require a bit more nuanced separation. Continuous mode processing is associated with vigilance tasks (work by Schroeder and Lakatos; reduction of low frequency oscillations and sustained gamma activity), whereas the authors of this study seem to link it to hearing tasks specifically (e.g., line 694). Rhythmic mode processing is associated with rhythmic stimulation by which neural oscillations entrain and influence perception (also, Schroeder and Lakatos; greater low-frequency fluctuations and more rhythmic gamma activity). The current study mirrors the continuous rather than the rhythmic mode (i.e., there was no rhythmic stimulation), but even the former seems not fully fitting, because trials are 1.8 s short and do not really reflect a vigilance task. Finally, previous paradigms on phase-resetting reflect more closely the design of the current study (i.e., different times of a target stimulus relative to the reset of an oscillation). This is the work by Fiebelkorn et al., Landau & Fries, and others, which do not seem to be cited here, which I find surprising. Moreover, the authors would want to discuss the role of the background noise in resetting the phase of an oscillation, and the role of the fixation cross also possibly resetting the phase of an oscillation. Regardless, the conceptional mixture of all these facets makes interpretations really challenging. The phase-reset nature of the paradigm is not (or not well) explained, and the discussion mixes the different concepts and approaches. I recommend that the authors frame their work more clearly in the context of these different concepts (affecting large portions of the manuscript).

(2) Methodological concerns:

The authors use a relatively unorthodox approach to statistical testing. I understand that they try to capture and characterize the sensitivity of the different analysis approaches to rhythmic behavioral effects. However, it is a bit unclear what meaningful effects are in the study. For example, the bootstrapping approach that identifies the percentage of significant variations of sample selections is rather descriptive (Figures 5-7). The authors seem to suggest that 50% of the samples are meaningful (given the dashed line in the figure), even though this is rarely reached in any of the analyses. Perhaps >80% of samples should show a significant effect to be meaningful (at least to my subjective mind). To me, the low percentage rather suggests that there is not too much meaningful rhythmicity present. I suggest that the authors also present more traditional, perhaps multi-level, analyses: Calculation of spectra, binning, or single-trial analysis for each participant and condition, and the respective calculation of the surrogate data analysis, and then comparison of the surrogate data to the original data on the second (participant) level using t-tests. I also thought the statistical approach undertaken here could have been a bit more clearly/didactically described as well.

The authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. In practice, this can be a disadvantage relative to keeping the intensity constant throughout, because, on average, correct trials will be associated with a higher intensity than incorrect trials, potentially making observations of perceptual rhythmicity more challenging. The authors would want to discuss this potential issue. Intensity adjustments could perhaps contribute to the observed rhythmicity effects. Perhaps the rhythmicity of the stimulus intensity could be analyzed as well. In any case, the adaptive procedure may add variance to the data.

Additional methodological concerns relate to Figure 8. Figures 8A and C seem to indicate that a baseline correction for a very short time window was calculated (I could not find anything about this in the methods section). The data seem very variable and artificially constrained in the baseline time window. It was unclear what the reader might take from Figure 8.

Motivation and discussion of eye-movement/pupillometry and motor activity: The dual task paradigm of Experiment 4 and the reasons for assessing eye metrics in the current study could have been better motivated. The experiment somehow does not fit in very well. There is recent evidence that eye movements decrease during effortful tasks (e.g., Contadini-Wright et al. 2023 J Neurosci; Herrmann & Ryan 2024 J Cog Neurosci), which appears to contradict the results presented in the current study. Moreover, by appealing to active sensing frameworks, the authors suggest that active movements can facilitate listening outcomes (line 677; they should provide a reference for this claim), but it is unclear how this would relate to eye movements. Certainly, a person may move their head closer to a sound source in the presence of competing sound to increase the signal-to-noise ratio, but this is not really the active movements that are measured here. A more detailed discussion may be important. The authors further frame the difference between Experiments 1 and 2 as being related to participants' motor activity. However, there are other factors that could explain differences between experiments. Self-paced trials give participants the opportunity to rest more (inter-trial durations were likely longer in Experiment 2), perhaps affecting attentional engagement. I think a more nuanced discussion may be warranted.

Discussion:

The main data in Figure 3 showed little rhythmicity. The authors seem to glance over this fact by simply stating that the same phase is not necessary for their statistical analysis. Previous work, however, showed rhythmicity in the across-participant average (e.g., Fiebelkorn's and similar work). Moreover, one would expect that some of the effects in the low-frequency band (e.g., 2-4 Hz) are somewhat similar across participants. Conduction delays in the auditory system are much smaller than the 0.25-0.5 s associated with 2-4 Hz. The authors would want to discuss why different participants would express so vastly different phases that the across-participant average does not show any rhythmicity, and what this would mean neurophysiologically.

An additional point that may require more nuanced discussion is related to the rhythmicity of response bias versus sensitivity. The authors could discuss what the rhythmicity of these different measures in different frequency bands means, with respect to underlying neural oscillations.

Figures:

Much of the text in the figures seems really small. Perhaps the authors would want to ensure it is readable even for those with low vision abilities. Moreover, Figure 1A is not as intuitive as it could be and may perhaps be made clearer. I also suggest the authors discuss a bit more the potential monoaural vs binaural issues, because the perceptual rhythmicity is much slower than any conduction delays in the auditory system that could lead to interference.

Reviewer #3 (Public review):

Summary:

The finding of rhythmic activity in the brain has, for a long time, engendered the theory of rhythmic modes of perception, that humans might oscillate between improved and worse perception depending on states of our internal systems. However, experiments looking for such modes have resulted in conflicting findings, particularly in those where the stimulus itself is not rhythmic. This paper seeks to take a comprehensive look at the effect and various experimental parameters which might generate these competing findings: in particular, the presentation of the stimulus to one ear or the other, the relevance of motor involvement, attentional demands, and memory: each of which are revealed to effect the consistency of this rhythmicity.

The need the paper attempts to resolve is a critical one for the field. However, as presented, I remain unconvinced that the data would not be better interpreted as showing no consistent rhythmic mode effect. It lacks a conceptual framework to understand why effects might be consistent in each ear but at different frequencies and only for some tasks with slight variants, some affecting sensitivity and some affecting bias.

Strengths:

The paper is strong in its experimental protocol and its comprehensive analysis, which seeks to compare effects across several analysis types and slight experiment changes to investigate which parameters could affect the presence or absence of an effect of rhythmicity. The prescribed nature of its hypotheses and its manner of setting out to test them is very clear, which allows for a straightforward assessment of its results

Weaknesses:

There is a weakness throughout the paper in terms of establishing a conceptual framework both for the source of "rhythmic modes" and for the interpretation of the results. Before understanding the data on this matter, it would be useful to discuss why one would posit such a theory to begin with. From a perceptual side, rhythmic modes of processing in the absence of rhythmic stimuli would not appear to provide any benefit to processing. From a biological or homeostatic argument, it's unclear why we would expect such fluctuations to occur in such a narrow-band way when neither the stimulus nor the neurobiological circuits require it.

Secondly, for the analysis to detect a "rhythmic mode", it must assume that the phase of fluctuations across an experiment (i.e., whether fluctuations are in an up-state or down-state at onset) is constant at stimulus onset, whereas most oscillations do not have such a total phase-reset as a result of input. Therefore, some theoretical positing of what kind of mechanism could generate this fluctuation is critical toward understanding whether the analysis is well-suited to the studied mechanism.

Thirdly, an interpretation of why we should expect left and right ears to have distinct frequency ranges of fluctuations is required. There are a large number of statistical tests in this paper, and it's not clear how multiple comparisons are controlled for, apart from experiment 4 (which specifies B&H false discovery rate). As such, one critical method to identify whether the results are not the result of noise or sample-specific biases is the plausibility of the finding. On its face, maintaining distinct frequencies of perception in each ear does not fit an obvious conceptual framework.

Reviewer #1 (Public review):

Summary:

The study by Cao et al. provides a compelling investigation into the role of mutational input in the rapid evolution of pesticide resistance, focusing on the two-spotted spider mite's response to the recent introduction of the acaricide cyetpyrafen. This well-documented introduction of the pesticide - and thus a clearly defined history of selection - offers a powerful framework for studying the temporal dynamics of rapid adaptation. The authors combine resistance phenotyping across multiple populations, extensive resequencing to track the frequency of resistance alleles, and genomic analyses of selection in both contemporary and historical samples. These approaches are further complemented by laboratory-based experimental evolution, which serves as a baseline for understanding the genetic architecture of resistance across mite populations in China. Their analyses identify two key resistance-associated genes, sdhB and sdhD, within which they detect 15 mutations in wild-collected samples. Protein modeling reveals that these mutations cluster around the pesticide's binding site, suggesting a direct functional role in resistance. The authors further examine signatures of selective sweeps and their distribution across populations to infer the mechanisms - such as de novo mutation or gene flow-driving the spread of resistance, a crucial consideration for predicting evolutionary responses to extreme selection pressure. Overall, this is a well-rounded, thoughtfully designed, and well-written manuscript. It shows significant novelty, as it is relatively rare to integrate broad-scale evolutionary inference from natural populations with experimentally informed bioassays, however, some aspects of the methods and discussion have an opportunity to be clarified and strengthened.

Strengths:

One of the most compelling aspects of this study is its integration of genomic time-series data in natural populations with controlled experimental evolution. By coupling genome sequencing of resistant field populations with laboratory selection experiments, the authors tease apart the individual effects of resistance alleles along with regions of the genome where selection is expected to occur, and compare that to the observed frequency in the wild populations over space and time. Their temporal data clearly demonstrates the pace at which evolution can occur in response to extreme selection. This type of approach is a powerful roadmap for the rest of the field of rapid adaptation.

The study effectively links specific genetic changes to resistance phenotypes. The identification of sdhB and sdhD mutations as major drivers of cyetpyrafen resistance is well-supported by allele frequency shifts in both field and experimental populations. The scope of their sampling clearly facilitated the remarkable number of observed mutations within these target genes, and the authors provide a careful discussion of the likelihood of these mutations from de novo or standing variation. Furthermore, the discovered cross-resistance that these mutations confer to other mitochondrial complex II inhibitors highlights the potential for broader resistance management and evolution.

Weaknesses:

(1) Experimental Evolution:

- Additional information about the lab experimental evolution would be useful in the main text. Specifically, the dose of cyetpyrafen used should be clarified, especially with respect to the LD50 values. How does it compare to recommended field doses? This is expected to influence the architecture of resistance evolution. What was the sample size? This will help readers contextualize how the experimental design could influence the role of standing variation.

- The finding that lab-evolved strains show cross-resistance is interesting, but potentially complicates the story. It would help to know more about the other mitochondrial complex II inhibitors used across China and their impact on adaptive dynamics at these loci, particularly regarding pre-existing resistance alleles. For example, a comparison of usage data from 2013, 2017, and 2019 could help explain whether cyetpyrafen was the main driver of resistance or if previous pesticides played a role. What happened in 2020 that caused such rapid evolution 3 years after launch?

(2) Evolutionary history of resistance alleles:

- It would be beneficial to examine the population structure of the sampled populations, especially regarding the role of migration. Though resistance evolution appears to have had minimal impact on genome-wide diversity (as shown in Supplementary Figure 2), could admixture be influencing the results? An explicit multivariate regression framework could help to understand factors influencing diversity across populations, as right now much is left to the readers' visual acuity.

- It is unclear why lab populations were included in the migration/treemix analysis. We might suggest redoing the analysis without including the laboratory populations to reveal biologically plausible patterns of resistance evolution.

- Can the authors explore isolation by distance (IBD) in the frequency of resistance alleles?

- Given the claim regarding the novelty of the number of pesticide resistance mutations, it is important to acknowledge the evolution of resistance to all pesticides (antibiotics, herbicides, etc.). ALS-inhibiting herbicides have driven remarkable repeatability across species based on numerous SNPs within the target gene.

- Figure 5 A-B. Why not run a multivariate regression with status at each resistance mutation encoded as a separate predictor? It is interesting that focusing on the predominant mutation gives the strongest r2, but it is somewhat unintuitive and masks some interesting variation among populations.

(3) Haplotype Reconstruction (Line 271-):

- We are a bit sceptical of the methods taken to reconstruct these haplotypes. It seems as though the authors did so with Sanger sequencing (this should be mentioned in the text), focusing only on homozygous SNPs. How many such SNPs were used to reconstruct haplotypes, along what length of sequence? For how many individuals were haplotypes reconstructed? Nonetheless, I appreciated that the authors looked into the extent to which the reconstructed haplotypes could be driven by recombination. Can the authors elaborate on the calculations in line 296? Is that the census population size estimate or effective?

(4) Single Mutations and Their Effect (line 312-):

- It's not entirely clear how the breeding scheme resulted in near-isogenic lines. Could the authors provide a clearer explanation of the process and its biological implications?

- If they are indeed isogenic, it's interesting that individual resistance mutations have effects on resistance that vary considerably among lines. Could the authors run a multivariate analysis including all potential resistance SNPs to account for interactions between them? Given the variable effects of the D116G substitution (ranging from 4-25%), could polygenic or epistatic factors be influencing the evolution of resistance?

- Why are there some populations that segregate for resistance mutations but have no survival to pesticides (i.e., the green points in Figure 5)? Some discussion of this heterogeneity seems required in the absence of validation of the effects of these particular mutations. Could it be dominance playing a role, or do the authors have some other explanation?

- The authors mention that all resistance mutations co-localized to the Q-site. Is this where the pesticide binds? This seems like an important point to follow their argument for these being resistance-related.

(5) Statistical Considerations for Allele Frequency Changes (Figure 3):

- It might be helpful to use a logistic regression model to assess the rate of allele frequency changes and determine the strength of selection acting on these alleles (e.g., Kreiner et al. 2022; Patel et al. 2024). This approach could refine the interpretation of selection dynamics over time.

Reviewer #2 (Public review):

Summary:

This paper investigates the evolution of pesticide resistance in the two-spotted spider mite following the introduction of an SDHI acaricide, cyatpyrafen, in China. The authors make use of cyatpyrafen-naive populations collected before that pesticide was first used, as well as more recent populations (both sensitive and resistant) to conduct comparative population genomics. They report 15 different mutations in the insecticide target site from resistant populations, many reported here for the first time, and look at the mutation and selection processes underlying the evolution of resistance, through GWAS, haplotype mapping, and testing for loss of diversity indicating selective sweeps. None of the target site mutations found in resistant populations was found in pre-exposure populations, suggesting that the mutations may have arisen de novo rather than being present as standing variation, unless initially present at very low frequencies; a de novo origin is also supported by evidence of selective sweeps in some resistant populations. Furthermore, there is no significant evidence of migration of resistant genotypes between the sampled field populations, indicating multiple origins of common mutations. Overall, this indicates a very high mutation rate and a wide range of mutational pathways to resistance for this target site in this pest species. The series of population genomic analyses carried out here, in addition to the evolutionary processes that appear to underlie resistance development in this case, could have implications for the study of resistance evolution more widely.

Strengths:

This paper combines phenotypic characterisation with extensive comparative population genomics, made possible by the availability of multiple population samples (each with hundreds of individuals) collected before as well as after the introduction of the pesticide cyatpyrafen, as well as lab-evolved lines. This results in findings of mutation and selection processes that can be related back to the pesticide resistance trait of concern. Large numbers of mites were tested phenotypically to show the levels of resistance present, and the authors also made near-isogenic lines to confirm the phenotypic effects of key mutations. The population genomic analyses consider a range of alternative hypotheses, including mutations arising by de novo mutation or selection from standing genetic variation, and mutations in different populations arising independently or arriving by migration. The claim that mutations most likley arose by multiple repeated de novo mutations is therefore supported by multiple lines of evidence: the direct evidence of none of the mutations being found in over 2000 individuals from naive populations, and the indirect evidence from population genomics showing evidence of selective sweeps but not of significant migration between the sampled populations.

Weaknesses:

As acknowledged within the discussion, whilst evidence supports a de novo origin of the resistance-associated mutations, this cannot be proven definitively as mutations may have been present at a very low frequency and therefore not found within the tested pesticide-naive population samples.

Near-isofemale lines were made to confirm the resistance levels associated with five of the 15 mutations, but otherwise, the genotype-phenotype associations are correlative, as confirmation by functional genetics was beyond the scope of this study.

Reviewer #1 (Public review):

Summary:

Felipe and colleagues try to answer an important question in Sarbecovirus Orf9b-mediated interferon signaling suppression, given that this small viral protein adopts two distinct conformations, a dimeric β-sheet-rich fold and a helix-rich monomeric fold when bound by Tom70 protein. Two Orf9b structures determined by X-ray crystallography and Cryo-EM suggest an equilibrium between the two Orf9b conformations, and it is important to understand how this equilibrium relates to its functions. To answer these questions, the authors developed a series of ordinary differential equations (ODE) describing the Orf9b conformation equilibrium between homodimers and monomers binding to Tom70. They used SPR and a fluorescent polarization (FP) peptide displacement assay to identify parameters for the equilibrium and create a theoretical model. They then used the model to characterize the effect of lipid-binding and the effects of Orf9b mutations in homodimer stability, lipid binding, and dimer-monomer equilibrium. They used their model to further analyze dimerization, lipid binding, and Orf9b-Tom70 interactions for truncated Orf9b, Orf9b fusion mutant S53E (blocking Tom70 binding), and Orf9b from a set of Sars-CoV-2 VOCs. They evaluated the ability of different Orf9b variants for binding Tom70 using Co-IP experiments and assessed their activity in suppressing IFN signaling in cells.

Overall, this work is well designed, the results are of high quality and well-presented; the results support their conclusions.

Strengths:

(1) They developed a working biophysical model for analyzing Orf9b monomer-dimer equilibrium and Tom70 binding based on SPR and FP experiments; this is an important tool for future investigation.

(2) They prepared lipid-free Orf9b homodimer and determined its crystal structure.

(3) They designed and purified obligate Orf9b monomer, fused-dimer, etc., a very important Orf9b variant for further investigations.

(4) They identified the lipid bound by Orf9b homodimer using mass spectra data.

(5) They proposed a working model of Orf9b-Tom70 equilibrium.

Weaknesses:

(1) It is difficult to understand why the obligate Orf9b dimer has similar IFN inhibition activity as the WT protein and obligate Orf9b monomer truncations.

(2) The role of Orf9b homodimer and the role of Orf9b-bound lipid in virus infection, remains unknown.

Reviewer #2 (Public review):

Summary:

This study focuses on Orf9b, a SARS-COV1/2 protein that regulates innate signaling through interaction with Tom70. San Felipe et al use a combination of biophysical methods to characterize the coupling between lipid-binding, dimerization, conformational change, and protein-protein-interaction equilibria for the Orf9b-Tom70 system. Their analysis provides a detailed explanation for previous observations of Orf9b function. In a cellular context, they find other factors may also be important for the biological functioning of Orf9b.

Strengths:

San Felipe et al elegantly combine structural biology, biophysics, kinetic modelling, and cellular assays, allowing detailed analysis of the Orf9b-Tom70 system. Such complex systems involving coupled equilibria are prevalent in various aspects of biology, and a quantitative description of them, while challenging, provides a detailed understanding and prediction of biological outcomes. Using SPR to guide initial estimates of the rate constants for solution measurements is an interesting approach.

Weaknesses:

This study would benefit from a more quantitative description of uncertainties in the numerous rate constants of the models, either through a detailed presentation of the sensitivity analysis or another approach such as MCMC. Quantitative uncertainty analysis, such as MCMC is not trivial for ODEs, particularly when they involve many parameters and are to be fitted to numerous data points, as is the case for this study. The authors use sensitivity analysis as an alternative, however, the results of the sensitivity analysis are not presented in detail, and I believe the authors should consider whether there is a way to present this analysis more quantitatively. For example, could the residuals for each +/-10% parameter change for the peptide model be presented as a supplementary figure, and similarly for the more complex models? Further details of the range of rate constants tested would be useful, particularly for the ka and kB parameters.

The authors build a model that incorporates an α-helix-β-sheet conformational change, but the rate constant for the conversion to the α-helix conformation is required to be second order. Although the authors provide some rationale, I do not find this satisfactorily convincing given the large number of adjustable parameters in the model and the use of manual model fitting. The authors should discuss whether there is any precedence for second-order rate constants for conformational changes in the literature. On page 14, the authors state this rate constant "had to be non-linear in the monomer β-sheet concentration" - how many other models did the authors explore? For example, would αT↔α↔αα↔ββ (i.e., conformational change before dimer dissociation) or α↔βαT↔ββ (i.e., Tom70 binding driving dimer dissociation) be other plausible models for the conformational change that do not require assumptions of second-order rate constants for the conformational change?

Overall, this study progresses the analysis of coupled equilibria and provides insights into Orf9b function.

Reviewer #1 (Public review):

Summary:

In this study, Meunier et al. investigated the functional role of IL-10 in avian mucosal immunity. While the anti-inflammatory role of IL-10 is well established in mammals, and several confirmatory knockout models are available in mice, IL-10's role in avian mucosal immunity is so far correlative. In this study, the authors generated two different models of IL-10 ablation in Chickens. A whole body knock-out model and an enhancer KO model leading to reduced IL10 expression. The authors first performed in vitro LPS stimulation-based experiments, and then in vivo two different infection models employing C. jejuni and E. tenella, to demonstrate that complete ablation of IL10 leads to enhanced inflammation-related pathology and gene expression, and enhanced pathogen clearance. At a steady-state level, however, IL-10 ablation did not lead to spontaneous colitis.

Strengths:

Overall, the study is well executed and establishes an anti-inflammatory role of IL-10 in birds. While the results are expected and not surprising, this appears to be the first report to conclusively demonstrate IL-10's anti-inflammatory role upon its genetic ablation in the avian model. Provided this information is applicable in combating pathogen infection in livestock species in sustainable industries like poultry, the study will be of interest to the field.

Weaknesses:

The study is primarily a confirmation of the already established anti-inflammatory role of IL-10.

Reviewer #2 (Public review):

Summary:

The authors were to investigate the functional role of IL10 on mucosal immunity in chickens. CRISPR technology was employed to generate IL10 knock-out chickens in both exon and putative enhancer regions. IL10 expressions were either abolished (knockout in exon) or reduced (enhancer knock-out). IL-10 plays an important role in the composition of the caecal microbiome. Through various enteric pathogen challenges, deficient IL10 expression was associated with enhanced pathogen clearance, but with more severe lesion scores and body weight loss.

Strengths:

Both in vitro and in vivo knock-out abolished and reduced IL10 expression, and broad enteric pathogens were challenged in vivo, and various parameters were examined to evaluate the functional role of IL10 on mucosal immunity.

Weaknesses:

Overexpression of IL-10 either in vitro or in vivo may further support the findings from this study.

Reviewer #1 (Public review):

Summary:

By applying a laser scanning photostimulation (LSPS) approach to a novel slice preparation, the authors aimed to study the degree of convergence and divergence of cortical inputs to individual striatal projection neurons (SPNs).

Strengths:

The experiments were well-designed and conducted, and data analysis was thorough. The manuscript was well written, and related work in the literature was properly discussed. This work has the potential to advance our understanding of how sensory inputs are integrated into the striatal circuits.

Weaknesses:

This work focuses on the connection strength of the corticostriatal projections, without considering the involvement of synaptic plasticity in sensory integration.

Reviewer #2 (Public review):

Summary:

How corticostriatal synaptic connectivity gives rise to SPN encoding of sensory information is an important and currently unanswered question. The authors utilize a clever slice preparation in combination with electrophysiology and glutamate uncaging to dissect the synaptic connectivity between barrel cortex and individual striatal SPNs. In addition to mapping connectivity across major anatomical axes and cortical layers, the authors provide data showing that SPNs uniquely integrate sparse input from variable stretches across barrel cortex.

Strengths:

The methodology shows impressive rigor, and the data robustly support the authors' conclusions. Overall, the manuscript addresses its core question, provides valuable insights into corticostriatal architecture, and is a welcome addition to the field.

Weaknesses:

A few minor changes to the figures and text could be made to improve clarity.

Reviewer #3 (Public review):

Summary:

The authors explored how individual dorsolateral striatum (DLS) spiny projection neurons (SPNs) receive functional input from whisker-related cortical columns. The authors developed and validated a novel slice preparation and method to which they applied rigorous functional mapping and thorough analysis. They found that individual SPNs were driven by sparse, scattered cortical clusters. Interestingly, while the cortical input fields of nearby SPNs had some degree of overlap, connectivity per SPN was largely distinct. Despite sparse, heterogeneous connectivity, topographical organization was identified. The authors lastly compared direct (D1) vs. indirect (D2) pathway cells, concluding that overall connectivity patterns were the same, but D1 cells received stronger input from L6 and D2 cells from L2/3. The paper thoughtfully addresses the question of whether barrel cortex broadly or selectively innervates SPNs. Their results indicate selective input that is loosely topographic. Their work deepens the understanding of how whisker-related somatosensory signals can drive striatal neurons.

Strengths:

Overall, this is a carefully conducted study, and the major claims are well-supported. The use of a novel ex vivo slice prep that keeps relevant corticostriatal projections intact allows for careful mapping of the barrel cortex to dorsolateral striatum SPNs. Careful reporting of both columnar and layer position, as well as postsynaptic SPN type (D1 or D2), allows the authors to uncover novel details about how the dorsolateral striatum represents whisker-related sensory information.

Weaknesses:

(1) Several factors may contribute to an underestimation of barrel cortex inputs to SPNs (and thus an overestimate of the input heterogeneity among SPNs). First, by virtue of the experiments being performed in an acute slice prep, it is probable that portions of recorded SPN dendritic trees have been dissected (in an operationally consistent anatomical orientation). If afferents happen to systematically target the rostral/caudal projections of SPN dendritic fields, these inputs could be missed. Similarly, the dendritic locations of presynaptic cortical inputs remain unknown (e.g., do some inputs preferentially target distal vs proximal dendritic positions?). As synaptic connectivity was inferred from somatic recordings, it's likely that inputs targeting the proximal dendritic arbor are the ones most efficiently detected. Mapping the dendritic organization of synapses is beyond the scope of this work, but these points could be broached in the text.

(2) In general, how specific (or generalizable) is the observed SPN-specific convergence of cortical barrel cortex projections in the dorsolateral striatum? In other words, does a similar cortical stimulation protocol targeted to a non-barrel sensory (or motor) cortex region produce similar SPN-specific innervation patterns in the dorsolateral striatum?

(3) In general, some of the figure legends are extremely brief, making many details difficult to infer. Similarly, some statistical analyses were either not carried out or not consistently reported.

Joint Public Review:

Following up on their previous work, the authors investigated whether HIV-1 cell-to-cell transmission activates the CARD8 inflammasome in macrophages, a key question given that inflammasome activation in myeloid cells triggers proinflammatory cytokine release. Co-cultures of HIV-infected T cells with macrophages led to viral spreading, resulting in IL1β release and cell death, with CARD8 playing a crucial role in this inflammasome response, triggered by HIV protease. The authors also found that HIV isolates resistant to protease inhibitors showed differences in CARD8 activation and IL1β production, highlighting the clinical relevance of their findings. Overall, this well-written study provides strong evidence for the role of CARD8 in protease-dependent sensing of viral spread, with implications for understanding chronic inflammation in HIV infections and its potential contribution to systemic immune activation, especially under ART. The authors have addressed initial weaknesses and verified effects in cocultures of primary T cells and macrophages. They now also provide evidence that CARD8 is activated by protease from incoming viral particles. Further studies are needed to clarify how much this mechanism contributes to systemic immune activation in untreated infections and whether this mechanism drives chronic inflammation under ART.

Reviewer #1 (Public review):

Summary:

Gruskin and colleagues use twin data from a movie-watching fMRI paradigm to show how genetic control of cortical function intersects with the processing of naturalistic audiovisual stimuli. They use hyperalignment to dissect heritability into the components that can be explained by local differences in cortical-functional topography and those that cannot. They show that heritability is strongest at slower-evolving neural time scales and is more evident in functional connectivity estimates than in response time series.

Strengths:

This is a very thorough paper that tackles this question from several different angles. I very much appreciate the use of hyperalignment to factor out topographic differences, and I found the relationship between heritability and neural time scales very interesting. The writing is clear, and the results are compelling.

Weaknesses:

The only "weaknesses" I identified were some points where I think the methods, interpretation, or visualization could be clarified.

(1) On page 16, the authors compare heritability in functional connectivity (FC) and response time series, and find that the heritability effect is larger in FC. In general, I agree with your diagnosis that this is in large part due to the fact that FC captures the covariance structure across parcels, whereas response time series only diverge in terms of univariate time-point-by-time-point differences. Another important factor here is that (within-subject) FC can be driven by intrinsic fluctuations that occur with idiosyncratic timing across subjects and are unrelated to the stimulus (whereas time-locked metrics like ISC and time-series differences cannot, by definition). This makes me wonder how this connectivity result would change if the authors used intersubject functional connectivity (ISFC) analysis to specifically isolate the stimulus-driven components of functional connectivity (Simony et al., 2016). This, to me, would provide a closer comparison to the ISC and response time series results, and could allow the authors to quantify how much of the heritability in FC is intrinsic versus stimulus-driven. I'm not asking that the authors actually perform this analysis, as I don't think it's critical for the message of the manuscript, but it could be an interesting future direction. As the authors discuss on page 17, I also suspect there's something fundamentally shared between response time series and connectivity as they relate to functional topography (Busch et al., 2021) that drives part of the heritability effect.

(2) The observation that regions with intermediate ISC have the largest differences between MZ, DZ, and UR is very interesting, but it's kind of hard to see in Figure 1B. Is there any other way to plot this that might make the effect more obvious? For example, I could imagine three scatter plots where the x- and y-axes are, e.g., MZ ISC and UR ISC, and each data point is a parcel. In this kind of plot, I would expect to see the middle values lifted visibly off the diagonal/unity line toward MZ. The authors could even color the data points according to networks, like in Figure 3C. (They also might not need to scale the ISC axis all the way to r = 1, which would make the differences more visible.)

(3) On page 9, if I understand correctly, the authors regress the vector of ISC values across parcels out of the vector of heritability values across parcels, and then plot the residual heritability values. Do they center the heritability values (or include some kind of intercept) in the process? I'm trying to understand why the heritability values go from all positive (Figure 2A) to roughly balanced between positive and negative (Figure 2B). Important question for me: How should we interpret negative values in this plot? Can the authors explain this explicitly in the text? (I also wonder if there's a more intuitive way to control for ISC. For example, instead of regressing out ISC at the parcel/map level, could they go into a single parcel and then regress the subject-level pairwise ISC values out when computing the heritability score?).

(4) On page 4 (line 155), the authors say "we shuffled dyad labels"- is this equivalent to shuffling rows and columns of the pairwise subject-by-subject matrix combined across groups? I'm trying to make sure their approach here is consistent with recommendations by Chen et al., 2016. Is this the same kind of shuffling used for the kinship matrix mentioned in line 189?

(5) I found panel A in Figure 4 to be a little bit misleading because their parcel-wise approach to hyperalignment won't actually resolve topographic idiosyncrasies across a large cortical distance like what's depicted in the illustration (at the scale of the parcels they are performing hyperalignment within). Maybe just move the green and purple brain areas a bit closer to each other so they could feasibly be "aligned" within a large parcel. Worth keeping in mind when writing that hyperalignment is also not actually going to yield a one-to-one mapping of functionally homologous voxels across individuals: it's effectively going to model any given voxel time series as a linear combination of time series across other voxels in the parcel.

(6) I believe the subjects watched all different movies across the two days, however, for a moment I was wondering "are Day 1 and Day 2 repetitions of the same movies?" Given that Day 1 and Day 2 are an organizational feature of several figures, it might be worth making this very explicit in the Methods and reminding the reader in the Results section.

References:

Busch, E. L., Slipski, L., Feilong, M., Guntupalli, J. S., di Oleggio Castello, M. V., Huckins, J. F., Nastase, S. A., Gobbini, M. I., Wager, T. D., & Haxby, J. V. (2021). Hybrid hyperalignment: a single high-dimensional model of shared information embedded in cortical patterns of response and functional connectivity. NeuroImage, 233, 117975. https://doi.org/10.1016/j.neuroimage.2021.117975

Chen, G., Shin, Y. W., Taylor, P. A., Glen, D. R., Reynolds, R. C., Israel, R. B., & Cox, R. W. (2016). Untangling the relatedness among correlations, part I: nonparametric approaches to inter-subject correlation analysis at the group level. NeuroImage, 142, 248-259. https://doi.org/10.1016/j.neuroimage.2016.05.023

Simony, E., Honey, C. J., Chen, J., Lositsky, O., Yeshurun, Y., Wiesel, A., & Hasson, U. (2016). Dynamic reconfiguration of the default mode network during narrative comprehension. Nature Communications, 7, 12141. https://doi.org/10.1038/ncomms12141

Reviewer #2 (Public review):

Summary:

The authors attempt to estimate the heritability of brain activity evoked from a naturalistic fMRI paradigm. No new data were collected; the authors analyzed the publicly available and well-known data from the Human Connectome Project. The paper has 3 main pieces, as described in the Abstract:

(1) Heritability of movie-evoked brain activity and connectivity patterns across the cortex.

(2) Decomposition of this heritability into genetic similarity in "where" vs. "how" sensory information is processed.

(3) Heritability of brain activity patterns, as partially explained by the heritability of neural timescales.

Strengths:

The authors investigate a very relevant topic that concerns how heritable patterns of brain activity among individuals subjected to the same kind of naturalistic stimulation are. Notably, the authors complement their analysis of movie-watching data with resting-state data.

Weaknesses:

The paper has numerous problems, most of which stem from the statistical analyses. I also note the lack of mapping between the subsections within the Methods section and the subsections within the Results section. We can only assess results after understanding and confirming the methods are valid; here, however, Methods and Results, as written, are not aligned, so we can't always be sure which results are coming from which analysis.

(A) Intersubject correlation (ISC) (section that starts from line 143): "We used non-parametric permutation testing to quantify average differences in ISC for each parcel in the Schaefer 400 atlas for each day of data collection across three groups: MZ dyads, DZ dyads, and unrelated (UR) dyads, where all UR dyads were matched for gender and age in years." ... "some participants contributed to ISC values for multiple dyads (thus violating independence assumptions)"

This is an indirect attempt to demonstrate heritability. And it's also incorrect since, as the authors themselves point out, some subjects contribute to more than one dyad.

Permutation tests don't quantify "average differences", they provide a measure of evidence about whether differences observed are sufficient to reject a hypothesis of no difference.

Matching subjects is also incorrect as it artificially alters the sample; covarying for age and sex, as done in standard analyses of heritability, would have been appropriate.

It isn't clear why the authors went through the trouble of implementing their own non-parametric test if HCP recommends using PALM, which already contains the validated and documented methods for permutation tests developed precisely for HCP data.

The results from this analysis, in their current form, are likely incorrect.

(B) Functional connectivity (FC) (section that starts from line 159): Here the authors compute two 400x400 FC matrix for each subject, one for rest, one for movie-watching, then correlate the correlations within each dyad, then compared the average correlation of correlations for MZ, DZ, and UR. In addition to the same problems as the previous analysis, here it is not clear what is meant by "averaging correlations [...] within a network combination". What is a "network combination"? Further, to average correlations, they need to be r-to-z transformed first. As with the above, the results from this analysis in its current form are likely incorrect.

(C) ISC and FC profile heritability analyses (section that starts from line 175): Here, the authors use first a valid method remarkably similar to the old Haseman-Elston approach to compute heritability, complemented by a permutation test. That is fine. But then they proceed with two novel, ill-described, and likely invalid methods to (1) "compare the heritability of movie and rest FC profiles" and (2) to "determine the sample size necessary for stable multidimensional heritability results". For (1), they permute, seemingly under the alternative, rest and movie-watching timeseries, and (2), by dropping subjects and estimating changes in the distribution.

The (1) might be correct, but there are items that are not clearly described, so the reader cannot be sure of what was done. What are the "153 unique network combinations"? Why do the authors separate by day here, whereas the previous analyses concatenated both days? Were the correlations r-to-z transformed before averaging?

The (2) is also not well described, and in any case, power can be computed analytically; it isn't clear why the authors needed to resort to this ad hoc approach, the validity of which is unknown. If the issue is the possibility that the multidimensional phenotypic correlation matrix is rank-deficient, it suffices that there are more independent measurements per subject than the number of subjects.

(D) Frequency-dependent ISC heritability analysis (from line 216): Here, the authors decompose the timeseries into frequency bands, then repeat earlier analyses, thus bringing here the same earlier problems and questions of non-exchangability in the permutations given the dyads pattern, r-z transforms, and sex/age covariates.

(E) FC strength heritability analysis (from line 236): Here, the authors use the univariate FC to compute heritability using valid and well-established methods as implemented in SOLAR. There is no "linkage" being done here (thus, the statement in line 238 is incorrect in this application. SOLAR already produces SEs, so it's unclear why the authors went out of their way to obtain jackknife estimates. If the issue is non-normality, I note that the assumption of normality is present already at the stage in which parameters themselves are estimated, not just the standard errors; for non-normal data, a rank-based inverse-normal transformation could have been used. Moreover, typically, r-to-z transformed values tend to be fairly normally distributed. So, while the heritabilities might be correct, the standard errors may not be (the authors don't demonstrate that their jackknife SE estimator is valid). The comparison of h2 between dyads raises the same questions about permutations, age/sex covariates, and r-z transforms as above.

(F) Hyperalignment (from line 245): It isn't clear at this point in the manuscript in what way hyperalignment would help to decompose heritability in "where vs. how" (from the Abstract). That information and references are only described much later, from around line 459. The description itself provides no references, and one cannot even try to reproduce what is described here in the Methods section. Regardless, it isn't entirely clear why this analysis was done: by matching functional areas, all heritabilities are going to be reduced because there will be less variance between subjects. Perhaps studying the parameters that drive the alignment (akin to what is done in tensor-based and deformation-based morphometry) could have been more informative. Plus, the alignment process itself may introduce errors, which could also reduce heritability. This could be an alternative explanation for the reduced heritability after hyperalignment and should be discussed. An investigation of hyperaligment parameters, their heritability, and their co-heritability with the BOLD-phenotypes can inform on this.

(G) Relationships between parcel area and heritability (from line 270): As under F), how much the results are distorted likely depends on the accuracy of the alignment, and the error variance (vs heritable variance) introduced by this.

(H) Neural timescale analyses (from line 280): Here, a valid phenotype (NT) is assessed with statistical methods with the same limitations as those previously (exchangability of dyads, age/sex covariates, and r-z transforms). NT values are combined across space and used as covariates in "some multivariate analyses". As a reader, I really wanted to see the results related to NT, something as simple as its heritability, but these aren't clearly shown, only differences between types of dyads.

(I) Significance testing for autocorrelated brain maps and FC matrices (from line 310): Here, the authors suddenly bring up something entirely different: reliability of heritability maps, and then never return to the topic of reliability again. As a reader, I find this confusing. In any case, analyses with BrainSMASH with well-behaved, normally distributed data are ok. Whether their data is well behaved or whether they ensured that the data would be well behaved so that BrainSMASH is valid is not described. As to why Spearman correlations are needed here, Mantel tests, or whether the 1000 "surrogate" maps are valid realizations of the data under the null, remains undemonstrated.

(J) Global signal was removed, and the authors do not acknowledge that this could be a limitation in their analyses, nor offer a side analysis in which the global signal is preserved.

(K) FDR is used to control the error rate, but in many cases, as it's applied to multiple sets of p-values, the amount of false discoveries is only controlled across all tests, but not within each set. The number of errors within any set remains unknown.

(L) Generally, when studying the heritability of a trait, the trait must be defined first. Here, multiple traits are investigated, but are never rigorously defined. Worse, the trait being analyzed changes at every turn.

Reviewer #3 (Public review):

Strengths:

It's sort of novel to study the heritability of movie-watching fMRI data. The methodology the authors used in the paper is also supportive of their findings. Figures are nicely organized and plotted. They finally found that sensory processing in the human brain is under genetic control over stable aspects of brain function (here referring to neural timescale and resting state connectivity).

Weaknesses:

What I am worried about most is the sample size and interpretation of heritability.

(1) Figure 1. I assumed that the authors just calculated the ISC within each group (MZ, DZ, and UR). Of course, you can get different variations between each group. Therefore, there is heritability. Why not calculate ISC across the whole sample, then separate MZ, DZ, and UR?

(2) Heritability scores in the paper are sort of small. If the sample size is small, please consider p-values, which will tell more about the trustworthiness of your heritability.

(3) I don't understand the high-frequency signals in fMRI data. It's always regarded as noise, the band 1 here in particular.

(4) The statement "we show that the heritability of brain activity patterns can be partially explained by the heritability of the neural timescale" should come from Figure 5. However, after controlling for NT, the heritability decreased max. 0.025 in temporal areas. I am not sure this change supports the statement. If the visual cortex is outlined, and combining ISC changes in the visual cortex, I think this would somehow be answered. Instead of delta h2, adding a new model h2 would be obvious to the readers.

(5) Figures 7 and 8, when getting the difference of heritability, please also consider the standard errors of the heritability estimates. Then you can compare across networks/regions.

(6) I think movie VS resting state is a really important result in this paper. However, there is almost no discussion. Discussing this part would be more beneficial for understanding the genetic control over the neuron arousal and excitation circuits.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors report that activation of excitatory DREADDs in the mid-cervical spinal cord can increase inspiratory activity in mice and rats. This is an important first step toward an ultimate goal of using this, or similar, technology to drive breathing in disorders associated with decreased respiratory motor output, such as spinal injury or neurodegenerative disease. Strengths to this study include a comparison of non-specific DREADD expression in the mid-cervical spinal cord versus specific targeting to ChAT-positive neurons, and the measurement of multiple respiratory-related outcomes, including phrenic inspiratory output, diaphragm EMG activity and ventilation. The data show convincingly that DREADDs can be used to drive phrenic inspiratory activity, which in turn increases diaphragm EMG activity and ventilation.

Comments on revisions: All of my prior comments have been sufficiently addressed.

Reviewer #2 (Public review):

Summary:

This study shows that when excitatory DREADD receptors are expressed in the ventral area of the cervical spinal cord containing phrenic motoneurons, systemic administration of the DREADD ligand J60 increases diaphragm EMG activity without altering respiratory rate. The authors took a non-selective expression approach in wild-type mice, as well as a more selective Cre-dependent approach in Chat-Cre mice and Chat-Cre rats to stimulate cervical motoneurons in the spinal cord. This is a proof of principle study that supports the use of DREADD technology to stimulate the motor output to the diaphragm.

Strengths:

The strengths of the study lie in the use of both mice and rats to test whether the chomogenetic activation of phrenic motoneurons with multiple experimental approaches increases diaphragm EMG activity (both tonic and phasic) and tidal volume.

Comments on revisions:

Thanks for addressing my comments. One last comment that could be discussed or addressed is :

Line 295- was the time post-infection, which varies considerably between groups and across samples, taken into consideration when comparison of response was made between ChatCre mice (4-9 weeks post-infection) and WT mice (four to five weeks post-infection)?

Reviewer #1 (Public review):

Summary:

This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.

Strengths:

The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.

Weaknesses were addressed during the revision.

The authors effectively addressed my critiques in the rebuttal letter and made corresponding changes in the manuscript. Specifically, they: 1) emphasized the importance of TO orientation in influencing STB nuclear subtype differentiation by adding text to the introduction; 2) clarified the differences in cluster numbers and names between primary tissue and TO data, explaining that each dataset was analyzed independently with separate clustering algorithms and adding clarifying text to the results section; 3) included additional rationale for using SN over SC sequencing, particularly for studying the multinucleated STB; 4) acknowledged that their original evidence was insufficient to definitively determine STBout nuclei differentiation status and removed language suggesting STB-3 as a terminally differentiated subtype, presenting alternative hypotheses in the discussion; and 5) incorporated new figures and clarifications, including RNA-FISH experiments, to validate subtype-specific marker gene expression. Overall, the authors' revisions strengthened the manuscript and aligned well with my critiques.

Reviewer #1 (Public review):

Summary:

This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.

Strengths:

The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.

Weaknesses were addressed during the revision.

The authors effectively addressed my critiques in the rebuttal letter and made corresponding changes in the manuscript. Specifically, they: 1) emphasized the importance of TO orientation in influencing STB nuclear subtype differentiation by adding text to the introduction; 2) clarified the differences in cluster numbers and names between primary tissue and TO data, explaining that each dataset was analyzed independently with separate clustering algorithms and adding clarifying text to the results section; 3) included additional rationale for using SN over SC sequencing, particularly for studying the multinucleated STB; 4) acknowledged that their original evidence was insufficient to definitively determine STBout nuclei differentiation status and removed language suggesting STB-3 as a terminally differentiated subtype, presenting alternative hypotheses in the discussion; and 5) incorporated new figures and clarifications, including RNA-FISH experiments, to validate subtype-specific marker gene expression. Overall, the authors' revisions strengthened the manuscript and aligned well with my critiques.

Reviewer #3 (Public review):

In this report, Keenen et al. present a thoroughly characterized platform for identifying potential molecular mechanisms regulating syncytiotrophoblast cell functions in placental biology. Application of single cell assessments to identify developmental trajectories of this lineage have been challenging due to the complex, multinucleated structure of the syncytium. The authors provide a comprehensive comparative assessment of term placental tissue and three independent trophoblast organoid models. They use single cell and single nucleus RNA sequencing followed by differential gene expression and pseudotime analyses to identify subpopulations and differentiation trajectories. They further compare the datasets generated in this study to publicly available datasets from first trimester placental tissue. The work is timely as optimization of trophoblast organoids is an evolving topic in placental research. And careful characterization of in vitro models has been noted as essential for model selection and result interpretation in the field.

The study elucidates syncytiotrophoblast nucleus subtypes and proportions in three different organoid models and compares subtypes and gene expression signatures to placental tissues. This work advances the field by demonstrating the utility of different trophoblast organoids to model syncytiotrophoblast differentiation. The in-depth characterization of cell types comprising the different organoid models and how they compare to placental tissue will help to inform model selection for future experimentation in the field. Defining cell composition and cell differentiation trajectories will also aid in data interpretation for data generated by these tissue and model sources. Overall, the conclusions presented in the manuscript are well supported by the data. The figures, as presented, are informative and striking.

The authors present outstanding progress toward their overall aim of identifying, "the underlying control of the syncytiotrophoblast". They identify the chromatin remodeler, RYBP, as well as other regulatory networks that they propose are critical to syncytiotrophoblast development.

The initial study was limited in fully addressing the aim, however, as functional evidence for the contributions of the factors/pathways to syncytiotrophoblast cell development was absent. In a revised version of the manuscript, the authors report the first application of CRISPR-mediated gene silencing in a TO model. They use CRISPR-Cas9-mediated gene targeting to generate RYBP and AFF1 knockout models. Deletion of either RYBP or AFF1 increased STB-2 marker gene expression, as determined using bulk RNA-seq. Future experimentation will assess the distribution of STB nuclear subtypes in the RYBP and AFF1 knockout models and explore the essentiality of RYBP, AFF1, and other identified factors to syncyiotrophoblast development and function.

Localization and validation of the identified factors within tissue and at the protein level will also provide further contextual evidence to address the hypotheses generated. In a revised version of the manuscript, the authors localize STB markers PAPPA2 and ADAMTS6 in TOs using RNA-FISH. Future work will aim to further validate the markers and hypotheses generated from this study.

Reviewer #1 (Public review):

Summary:

The study characterises an RNA polymerase (Pol) I mutant (RPA135-F301S) named SuperPol. This mutant was previously shown to increase yeast ribosomal RNA (rRNA) production by Transcription Run-On (TRO). In this work, the authors confirm this mutation increases rRNA transcription using a slight variation of the TRO method, Transcriptional Monitoring Assay (TMA), which also allows the analysis of partially degraded RNA molecules. The authors show a reduction of abortive rRNA transcription in cells expressing the SuperPol mutant and a modest occupancy decrease at the 5' region of the rRNA genes compared to WT Pol I. These results suggest that the SuperPol mutant displays a lower frequency of premature termination. Using in vitro assays, the authors found that the mutation induces an enhanced elongation speed and a lower cleavage activity on mismatched nucleotides at the 3' end of the RNA. Finally, SuperPol mutant was found to be less sensitive to BMH-21, a DNA intercalating agent that blocks Pol I transcription and triggers the degradation of the Pol I subunit, Rpa190. Compared to WT Pol I, short BMH-21 treatment has little effect on SuperPol transcription activity, and consequently, SuperPol mutation decreases cell sensitivity to BMH-21.

I'd suggest the following points to be taken into consideration:

Major comments:

(1) The differences in the transcriptionally engaged WT Pol I and SuperPol profiles (Figure 2) are very modest, without any statistical analyses. What is the correlation between CRAC replicates? Are they separated in PCA analyses? Please, include more quality control information. In my opinion, these results are not very convincing. Similarly, the effect of BMH-21 on WT Pol I activity (Figure 7) is also very subtle and doesn't match the effect observed in a previous study [1]. Could the author comment on the reasons for these differences? These discrepancies raise concerns about the methodology. In addition, according to the laboratory's previous work [2], Pol I ChIP signal at rDNA is not significantly different in cells expressing WT Pol I and SuperPol. How can these two observations be reconciled? I would suggest using an independent methodology to analyse Pol I transcription, for example, GRO-seq or TT-seq.

(2) While the experiments clearly show SuperPol mutant increases nascent transcription and decreases the production of abortive promoter-proximal transcripts compared to WT Pol I. RPA135-F301S mutation has a minor impact on total rRNA levels, at least those shown in Figure 3B. Are steady-state rRNA levels higher in cells expressing SuperPol mutant? It would be interesting to know if SuperPol mutant produces more functional rRNAs.

Significance:

The work further characterises a single amino acid mutation of one of the largest yeast Pol I subunits (RPA135-F301S). While this mutation was previously shown to increase rRNA synthesis, the current work expands the SuperPol mutant characterisation, providing details of how RPA135-F301S modifies the enzymatic properties of yeast Pol I. In addition, their findings suggest that yeast Pol I transcription can be subjected to premature termination in vivo. The molecular basis and potential regulatory functions of this phenomenon could be explored in additional studies.

Our understanding of rRNA transcription is limited, and the findings of this work may be interesting to the transcription community. Moreover, targeting Pol I activity is an open strategy for cancer treatment. Thus, the resistance of SuperPol mutant to BMH-21 might also be of interest to a broader community, although these findings are yet to be confirmed in human Pol I and with more specific Pol I inhibitors in future.

Reviewer #2 (Public review):

Summary:

This article presents a study on a mutant form of RNA polymerase I (RNAPI) in yeast, referred to as SuperPol, which demonstrates increased rRNA production compared to the wild-type enzyme. While rRNA production levels are elevated in the mutant, RNAPI occupancy as detected by CRAC is reduced at the 5' end of rDNA transcription units. The authors interpret these findings by proposing that the wild-type RNAPI pauses in the external transcribed spacer (ETS), leading to premature transcription termination (PTT) and degradation of truncated rRNAs by the RNA exosome (Rrp6). They further show that SuperPol's enhanced activity is linked to a lower frequency of PTT events, likely due to altered elongation dynamics and reduced RNA cleavage activity, as supported by both in vivo and in vitro data.

The study also examines the impact of BMH-21, a drug known to inhibit Pol I elongation, and shows that SuperPol is less sensitive to this drug, as demonstrated through genetic, biochemical, and in vivo approaches. The authors show that BMH-21 treatment induces premature termination in wild-type Pol I, but only to a lesser extent in SuperPol. They suggest that BMH-21 promotes termination by targeting paused Pol I complexes and propose that PTT is an important regulatory mechanism for rRNA production in yeast.

The data presented are of high quality and support the notion that 1) premature transcription termination occurs at the 5' end of rDNA transcription units; 2) SuperPol has an increased elongation rate with reduced premature termination; and 3) BMH-21 promotes both pausing and termination. The authors employ several complementary methods, including in vitro transcription assays. These results are significant and of interest for a broad audience.

Beyond the minor points listed below, my main criticism concerns the interpretation of data in relation to termination. While it is possible that the SuperPol mutation affects the wild-type Pol I's natural propensity for termination, it is also possible that premature termination is simply a consequence of natural or BMH-21-induced Pol I pausing. SuperPol may elongate more efficiently than the wild-type enzyme, pause less frequently, and thus terminate less often. In this light, the notion that termination "regulates" rRNA production might be an overstatement, with pausing as the primary event. Claiming a direct effect on termination by both the mutation and BMH-21 would require showing that with equivalent levels of pausing, termination occurs more or less efficiently, which would be challenging and should not be expected in this study. The authors address this point in the last two paragraphs of the discussion. My suggestion is to temper the claims regarding termination as a regulatory mechanism.

Significance:

These results are significant and of interest for a basic research audience.

Reviewer #3 (Public review):

Summary:

In the manuscript "Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription", Azouzi and co-authors investigate the regulatory mechanisms of ribosomal RNA (rRNA) transcription by RNA Polymerase I (RNAPI) in the budding yeast S. cerevisiae. They follow up on exploring the molecular basis of a mutant allele of the second largest subunit of RNAPI, RPA135-F301S, also dubbed SuperPol, that they had previously reported (Darrière et al, 2019), and which was shown to rescue Rpa49-linked growth defects, possibly by increasing rRNA production.

Through a combination of genomic and in vitro approaches, the authors test the hypothesis that RNAPI activity could be subjected to a Premature Transcription Termination (PPT) mechanism, akin to what is observed for RNA Polymerase II (RNAPII), and which is suggested to be an important step for the quality control of rRNA transcripts. SuperPol is proposed to lack such a regulatory mechanism, due to an increased processivity. In agreement, SuperPol is shown to be resistant to BMH-21, a drug previously shown to impair RNAPI elongation.<br /> Overall, the experiments are performed with rigor and include the appropriate controls and statistical analysis. Both the figures and the text present the data clearly. The Material and Methods section is detailed enough. The reported results are interesting; however, I am not fully convinced of the existence of PPT of RNAPI, and even less of its utmost importance.

The existence of PPT of RNAPI would entail an intended regulatory mechanism. The authors propose that PPT could serve as quality control step for the UTP-A complex loading on the rRNA 5'-end. While this hypothesis is enticing and cautiously phrased by the authors, the lack of evidence showing a specific regulatory function (such as UTP-A loading checkpoint or else) limits these termination events to possibly abortive actions of unclear significance.

The authors may want to consider comparisons to other processive alleles, such as the rpb1-E1103G mutant of the RNAPII subunit (Malagon et al, 2006) or the G1136S allele of E. coli RNAP (Bar-Nahum et al., 2005). While clearly mechanistically distinct, these mutations result in similarly processive enzymes that achieve more robust transcription, possibly at the cost of decreased fidelity. Indeed, an alternative possibility explaining these transcripts could be that they originate from unsuccessful resumption of transcription after misincorporation (see below).

I suggest reconsidering the study's main conclusions by limiting claims about the regulatory function of these termination events (the title of the manuscript should be changed accordingly). Alternatively, the authors should provide additional investigation on their regulatory potential, for example by assessing if indeed this quality control is linked to the correct assembly of the UTP-A complex. The expectation would be that SuperPol should rescue at least to some extent the defects observed in the absence of UTP-A components.

Moreover, the results using the clv3 substrate suggest the possibility that SuperPol might simply be more able to tolerate mismatches, thus be more processive in transcribing, because not subjected to proof-reading mechanisms, similarly to what observed in Schwank et al., 2022. This could explain many of the observations, and I think it is worth exploring by assessing the fidelity of the enzyme, especially in the frame of suggesting a regulatory function for these termination events.

Significance:

Azouzi and co-authors' work builds on their previous study (Darrière et al, 2019) of RPA135-F301S (SuperPol), a mutant allele of the second largest RNAPI subunit, which was shown to compensate for Rpa49 loss, potentially by increasing rRNA production. The work advances the mechanistic understanding of the the SuperPol allele, demonstrating the increased processivity of this enzyme compared to its wild-type counterpart. Such increased processivity "desensitizes" RNAPI from abortive transcription cycles, the existence of which is clearly shown, though the biological significance of this phenomenon remains unclear. The lack of evidence for a regulatory mechanism behind these early termination events is, in my opinion, a limitation of this study, as it does not allow for differentiation between an intended regulatory process and a byproduct of an imperfect system.

This work is of interest for researchers studying transcription regulation, particularly those interested in understanding RNAPI's role and fidelity. Demonstrating PPT as a regulatory quality control for RNAPI could point to common strategies in between RNAPI and RNAPII regulation, where premature termination has been extensively documented. However, without evidence of a specific regulatory function, these findings may currently be limited to descriptive insights.

Joint public review:

Summary:

This study investigates the hypoxia rescue mechanisms of neurons by non-neuronal cells in the brain from the perspective of exosomal communication between brain cells. Through multi-omics combined analysis, the authors revealed this phenomenon and logically validated this intercellular rescue mechanism under hypoxic conditions through experiments. The study proposed a novel finding that hemoglobin maintains mitochondrial function, expanding the conventional understanding of hemoglobin. This research is highly innovative, providing new insights for the treatment of hypoxic encephalopathy.

Overall, the manuscript is well organized and written, however, the authors have only partially answered the reviewers comments.

Reviewer #1 (Public review):

In this study, the authors introduced an essential role of AARS2 in maintaining cardiac function. They also investigated the underlying mechanism that through regulating alanine and PKM2 translation are regulated by AARS2. Accordingly, a therapeutic strategy for cardiomyopathy and MI was provided.

Comments on revised version:

The authors have completely addressed my concerns.

Reviewer #3 (Public review):

In the present study, the author revealed that cardiomyocyte-specific deletion of mouse AARS2 exhibited evident cardiomyopathy with impaired cardiac function, notable cardiac fibrosis, and cardiomyocyte apoptosis. Cardiomyocyte-specific AARS2 overexpression in mice improved cardiac function and reduced cardiac fibrosis after myocardial infarction (MI), without affecting cardiomyocyte proliferation and coronary angiogenesis. Mechanistically, AARS2 overexpression suppressed cardiomyocyte apoptosis and mitochondrial reactive oxide species production, and changed cellular metabolism from oxidative phosphorylation toward glycolysis in cardiomyocytes, thus leading to cardiomyocyte survival from ischemia and hypoxia stress. Ribo-Seq revealed that AARS2 overexpression increased pyruvate kinase M2 (PKM2) protein translation and the ratio of PKM2 dimers to tetramers that promote glycolysis. Additionally, PKM2 activator TEPP-46 reversed cardiomyocyte apoptosis and cardiac fibrosis caused by AARS2 deficiency. Thus, this study demonstrates that AARS2 plays an essential role in protecting cardiomyocytes from ischemic pressure via fine-tuning PKM2-mediated energy metabolism, and presents a novel cardiac protective AARS2-PKM2 signaling during the pathogenesis of MI.

Comments on revised version:

The authors addressed all the issues, no more comments.

Reviewer #1 (Public review):

Summary:

In this paper, Manley and Vaziri investigate whole-brain neural activity underlying behavioural variability in zebrafish larvae. They combine whole brain (single cell level) calcium imaging during the presentation of visual stimuli, triggering either approach or avoidance, and carry out whole brain population analyses to identify whole brain population patterns responsible for behavioural variability. They show that similar visual inputs can trigger large variability in behavioural responses. Though visual neurons are also variable across trials, they demonstrate that this neural variability does not degrade population stimulus decodability. Instead, they find that the neural variability across trials is in orthogonal population dimensions to stimulus encoding and is correlated with motor output (e.g. tail vigor). They then show that behavioural variability across trials is largely captured by a brain-wide population state prior to the trial beginning, which biases choice - especially on ambiguous stimulus trials. This study suggests that parts of stimulus-driven behaviour can be captured by brain-wide population states that bias choice, independently of stimulus encoding.

Comments on revisions:

The authors have revised their manuscript and provided novel analyses and figures, as well as additions to the text based on our reviewer comments.

As stated in my first review, the strength of the paper principally resides in the whole brain cellular level imaging - using a novel fourier light field microscopy (Flfm) method - in a well-known but variable behaviour.

Many of the authors' answers have provided additional support for their interpretations of results, but the new analysis in Figure 3g - further exploring the orthogonality of e1 and wopt - puts into question the interpretation of a key result: that e1 and wopt are orthogonal in a non-arbitrary way. This needs to be addressed. I have made suggestions below to address this:

Reviewer 3 had correctly highlighted the issue that in high-dimensional data, there is an increasingly high chance of two vectors being orthogonal. The authors address this by shuffling the stimulus labels. They then state (and provide a new panel g in Fig. 3) that the shuffled distribution is wider than the actual distribution, and state that a wilcoxon rank-sum test shows this is significant. Given the centrality of this claim, I would like the authors to clarify what exactly is being done here, as it is not clear to me how this conclusion can be drawn from this analysis:

In lines 449:453 the authors state:<br /> 'While it is possible to observe shuffled vectors which are nearly orthogonal to e1, the shuffled distribution spans a significantly greater range of angles than the observed data (p<0.05, Wilcoxon rank- sum test), demonstrating that this orthogonality is not simply a consequence of analyzing multi-dimensional activity patterns. '<br /> I don't understand how the authors arrive at the p-value using a rank-sum test here. (a) What is the n in this test? Is n the number of shuffles? If so, this violates the assumptions of the test (as n must be the number of independent samples and not the arbitrary number of shuffles). (b) If the shuffling was done once for each animal and compared with actual data with a rank-sum test, how likely is that shuffling result to happen in 10000 shuffle comparisons?<br /> I am highlighting this, as it looks from Figure 3g that the shuffled distribution is substantially overlapping with the actual data (i.e., not outside of the 95 percentile of the shuffled distribution), which would suggest that the angle found between e1 and wept could happen by chance.

I would also suggest the authors instead test whether e1 is consistently aligned with itself when calculated on separate held out data-sets (for example by bootstrapping 50-50 splits of the data). If they can show that there is a close alignment between independently calculated e1's across separate data sets (and do the same for wopt), and then show e1 and wopt are orthogonal, then that supports their statement that e1 and wopt are orthogonal in a meaningful way. Given that e1 captures tail vigor variability (and Wopt appears to not) then I would think this could be the case. But the current answer the authors have given is not supporting their statement.

Reviewer #2 (Public review):

This work by Manley and Vaziri identify brain networks that are associated with trial-to-trial variability during prey-capture and predator avoidance behaviors. However, mixing of signals across space and time make it difficult to interpret the data generated and relate the data to findings from prior work.

Comments on revisions:'

In their response to prior reviewer comments, Manley and Vaziri have now provided helpful methodological clarity and additional analyses. The additional work makes clear that the claims of variability and mixing of sensory, motor, and internal variables at the single-cell level are not well supported.

RESOLUTION<br /> - The new information provided regarding resolution may not be very relevant as this was from an experiment in air. It would be much more informative to show how PSF degrades in the brain with depth.

DEPTH<br /> - It is helpful to see the registered light-field and confocal images. Both appear to provide poor or little information in regions >200 below the surface (like the hypothalamus), making the claim that whole brain data is being collected at cellular resolution difficult to justify.

MERGING<br /> - The typical soma at these ages has a radius of 2.5 microns, which corresponds to a volume of 65 microns^3. Given the close packing of most cells, this means that a typical ROI of 750 microns^3 contains more than 10 neurons. Therefore, the authors should not claim they are reporting activity at cellular resolution.<br /> - Furthermore, the fact that these ROIs contains tens of cells brings into question the degree of variability at the single-cell level. For example, if every cluster of 10 cells has one variable cell, then all clusters might be labeled as exhibiting variability even though only 10% of the cells show variability.

SLOW CALCIUM DYNAMICS<br /> - Convolution/Deconvolution with the inappropriate kernel both have problems, some of which the authors have noted. However, by not deconvolving, the authors are significantly obscuring the interpretation of their data by mixing together signals across time.<br /> - Also, the claim that "neurons highly tuned to a particular stimulus exhibited variability in their responses across multiple presentations of the same stimuli" should be clarified or qualified. It is not clear from what has been shown if the responses are indeed variable, or rather if there is additional activity (or apparent activity) occasionally present that shifts the pre-stimulus baseline around (for example, 3J suggests that in many cases the visual signal from the prior trial is still present when a new trial begins).<br /> - Figure 3A should show when the stimulus occurs, should show some of the prestimulus period, and ideally be off-set corrected so all traces in a given panel start at the same y-value at the beginning of the stimulus period.

ORTHOGONALITY<br /> - It is now clearer that the visual signal and noise vectors were determined for the entire time series with all trials. Therefore, the concern that sources of activation in advance of a given trial were being ignored is alleviated. The concern remains, however, that these sources are being properly accounted for given potential kernel variations and nonlinearity. Nonetheless, it is recognized that the GCaMP filtering most likely would lead to a decrease in the disparity between two populations.<br /> - The authors' clarification that the analyzed ROIs consist of cell clusters raises the trivial possibility that the observed orthogonality between the visual signal and leading noise vectors is explained by noise simply reflecting the activation of different motor or motor-planning related neurons in an ROI, neurons that are separate from visually-encoding neurons in the same cluster.

SOURCES of VARIABILITY<br /> - The data presented in Supplemental Figure 3Ei actually is suggestive that eye movements are a significant contributor to the reported variability. Notice how in (1 4) vs (1 5) and (4 7) vs (4 8) there is a notable difference in the distribution of responses. Adding eye kinematic variables to the analysis of Figure S4 could be clarifying.-

Reviewer #1 (Public review):

Summary:

In this manuscript, Jin et. al., describe SMARTTR, an image analysis strategy optimized for analysis of dual-activity ensemble tagging mouse reporter lines. The pipeline performs cell segmentation, then registers the location of these cells into an anatomical atlas, and finally, calculates the degree of co-expression of the reporters in cells across brain regions. The authors demonstrate the utility of the method by labeling two ensemble populations during two related experiences: inescapable shock and subsequent escapable shock as part of learned helplessness.

Strengths:

- We appreciated that the authors provided all documentation necessary to use their method, and that the scripts in their publicly available repository are well commented. Submission of the package to CRAN will, as the other reviewer pointed out, ensure that the package and its dependencies can be easily installed using few lines of code in the future. Additionally, we particularly appreciate the recently added documentation website and vignettes, which provide guidance on package installation and use cases.<br /> - The manuscript was well-written and very clear, and the methods were generally highly detailed.<br /> - The authors have addressed our previous concerns, and we appreciate their revised manuscript.

Reviewer #2 (Public review):

Summary:

This manuscript describes a workflow and software package, SMARTR, for mapping and analyzing neuronal ensembles tagged using activity-dependent methods. They showcase this pipeline by analyzing ensembles tagged during the learned helplessness paradigm. This is an impressive effort, and I commend the authors for developing open-source software to make whole-brain analyses more feasible for the community. After peer-review, the authors addressed reviewer suggestions and concerns regarding the usability and maintainability of the SMARTTR package, ensuring that the package will be published on CRAN, improving documentation, and including unit tests to ensure code stability. Overall, this software package will prove to have a broad impact on the field.

Reviewer #1 (Public review):

Summary:

This study provides new insights into the role of miR-19b, an oncogenic microRNA, in the developing chicken pallium. Dynamic expression pattern of miR-19b is associated with its role in regulating cell cycle progression in neural progenitor cells. Furthermore, miR-19b is involved in determining neuronal subtypes by regulating Fezf2 expression during pallial development. These findings suggest an important role for miR-19b in the coordinated spatio-temporal regulation of neural progenitor cell dynamics and its evolutionary conservation across vertebrate species.

Strengths:

The authors identified conserved roles of miR-19 in the regulation of neural progenitor maintenance between mouse and chick, and the latter is mediated by the repression of E2f8 and NeuroD1. Furthermore, the authors found that miR-19b-dependent cell cycle regulation is tightly associated with specification of Fezf1 or Mef2c-positive neurons, in spatio-temporal manners during chicken pallial development. These findings uncovered molecular mechanisms underlying microRNA-mediated neurogenic controls.

Weaknesses:

Although the authors in this study claimed striking similarities of miR-19a/b in neurogenesis between mouse and chick pallium, a previous study by Bian et al. revealed that miR-19a contributes the expansion of radial glial cells by suppressing PTEN expression in the developing mouse neocortex, while miR-19b maintains apical progenitors via inhibiting E2f2 and NeuroD1 in chicken pallium. Thus, it is still unclear whether the orthologous microRNAs regulate common or species-specific target genes.

The spatiotemporal expression patterns of miR-19b and several genes are not convincing. For example, the authors claim that NeuroD1 is initially expressed uniformly in the subventricular zone (SVZ) but disappears in the DVR region by HH29 and becomes detectable by HH35 (Figure 1). However, the in situ hybridization data revealed that NeuroD1 is highly expressed in the SVZ of the DVR at HH29 (Figure 4F). Thus, perhaps due to the problem of immunohistochemistry, the authors have not been able to detect NeuroD1 expression in Figure 1D, and the interpretation of the data may require significant modification.

It seems that miR-19b is also expressed in neurons (Figure 1), suggesting the role of miR19-b must be different in progenitors and differentiated neurons. The data on the gain- and loss-of-function analysis of miR-19b on the expression of Mef2c should be carefully considered, as it is possible that these experiments disturb the neuronal functions of miR19b rather than in the progenitors.

The regions of chicken pallium were not consistent among figures: in Figure 1, they showed caudal parts of the pallium (HH29 and 35), while the data in Figure 4 corresponded to the rostral part of the pallium (Figure 4B).

The neurons expressing Fezf2 and Mef2 in the chicken pallium are not homologous neuronal subtypes to mammalian deep and superficial cortical neurons. The authors must understand that chicken pallial development proceeds in an outside-in manner. Thus, Mef2c-postive neurons in a superficial part are early-born neurons, while FezF2-positive neurons residing in deep areas are later-born neurons. It should be noted that the expression of a single marker gene does not support cell type homology, and the authors' description "the possibility of primitive pallial lamina formation in common ancestors of birds and mammals" is misleading.

Overexpression of CDKN1A or Sponge-19b induced ectopic expression of Fezf2 in the ventricular zone (Figure 3C, E). Do these cells maintain progenitor statement or prematurely differentiate to neurons? In addition, the authors must explain that the induction of Fezf2 is also detected in GFP-negative cells.

Reviewer #2 (Public review):

Summary:

This paper investigates the general concept that avian and mammalian pallium specifications share similar mechanisms. To explore that idea, the authors focus their attention on the role of miR-19b as a key controlling factor in the neuronal proliferation/differentiation balance. To do so, the authors checked the expression and protein level of several genes involved in neuronal differentiation, such as NeuroD1 or E2f8, genes also expressed in mammals after conducting their functional gene manipulation experiments. The work also shows a dysregulation in the number of neurons from lower and upper layers when miR-19b expression is altered.

To test it, the authors conducted a series of functional experiments of gain and loss of function (G&LoF) and enhancer-reporter assays. The enhancer-reporter assays demonstrate a direct relationship between miR-19b and NeuroD1 and E2f8 which is also validated by the G&LoF experiments. It´s also noteworthy to mention that the way miR-19b acts is maintaining the progenitor cells from the ventricular zone in an undifferentiated stage, thus promoting them into a stage of cellular division.

Overall, the paper argues that the expression of miR-19b in the ventricular zone promotes the cells in a proliferative phase and inhibits the expression of differentiation genes such as E2f8 and NeurD1. The authors claim that a decrease in the progenitor cell pool leads to an increase and decrease in neurons in the lower and upper layers, respectively.

Strengths:

(1) Novelty Contribution<br /> The paper offers strong arguments to prove that the neurodevelopmental basis between mammals and birds is quite the same. Moreover, this work contributes to a better understanding of brain evolution along the animal evolutionary tree and will give us a clearer idea about the roots of how our brain has been developed. This stands in contrast to the conventional framing of mammal brain development as an independent subject unlinked to the "less evolved species". The authors also nicely show a concept that was previously restricted to mammals - the role of microRNAs in development.

(2) Right experimental approach<br /> The authors perform a set of functional experiments correctly adjusted to answer the role of miR-19b in the control of neuronal stem cell proliferation and differentiation. Their histological, functional, and genetic approach gives us a clear idea about the relations between several genes involved in the differentiation of the neurons in the avian pallium. In this idea, they maintain the role of miR-19b as a hub controller, keeping the ventricular zone cells in an undifferentiated stage to perpetuate the cellular pool.

(3) Future directions<br /> The findings open a door to future experiments, particularly to a better comprehension of the role of microRNAs and pallidal genetic connections. Furthermore, this work also proves the use of avians as a model to study cortical development due to the similarities with mammals.

Weaknesses:

While there are questions answered, there are still several that remain unsolved. The experiments analyzed here lead us to speculate that the early differentiation of the progenitor cells from the ventricular zone entails a reduction in the cellular pool, affecting thereafter the number of latter-born neurons (upper layers). The authors should explore that option by testing progenitor cell markers in the ventricular zone, such as Pax6. Even so, it remains possible that miR-19b is also changing the expression pattern of neurons that are going to populate the different layers, instead of their numbers, so the authors cannot rule that out or verify it. Since the paper focuses on the role of miR-19b in patterning, I think the authors should check the relationship and expression between progenitors (Pax6) and intermediate (Tbr2) cells when miR-19b is affected. Since neuronal expression markers change so fast within a few days (HH24-HH35), I don't understand why the authors stop the functional experiments at different time points.

Reviewer #3 (Public review):

Summary:

This is a timely article that focuses on the molecular machinery in charge of the proliferation of pallial neural stem cells in chicks, and aims to compare them to what is known in mammals. miR19b is related to controlling the expression of E2f8 and NeuroD1, and this leads to a proper balance of division/differentiation, required for the generation of the right number of neurons and their subtype proportions. In my opinion, many experiments do reflect an interaction between all these genes and transcription factors, which likely supports the role of miR19b in participating in the proliferation/differentiation balance.

Strengths:

Most of the methodologies employed are suitable for the research question, and present data to support their conclusions.

The authors were creative in their experimental design, in order to assess several aspects of pallial development.

Weaknesses:

However, there are several important issues that I think need to be addressed or clarified in order to provide a clearer main message for the article, as well as to clarify the tools employed. I consider it utterly important to review and reinterpret most of the anatomical concepts presented here. The way the are currently used is confusing and may mislead readers towards an understanding of the bird pallium that is no longer accepted by the community.

Major Concerns:

(1) Inaccurate use of neuroanatomy throughout the entire article. There are several aspects to it, that I will try to explain in the following paragraphs:

a) Figure 1 shows a dynamic and variable expression pattern of miR19b and its relation to NeuroD1. Regardless of the terms used in this figure, it shows that miR19b may be acting differently in various parts of the pallium and developmental stages. However, all the rest of the experiments in the article (except a few cases) abolish these anatomical differences. It is not clear, but it is very important, where in the pallium the experiments are performed. I refer here, at least, to Figures 2C, E, F, H, I; 3D, E; 4C, D, G, I. Regarding time, all experiments were done at HH22, and the article does not show the native expression at this stage. The sacrifice timing is variable, and this variability is not always justified. But more importantly, we don't know where those images were taken, or what part of the pallium is represented in the images. Is it always the same? Do results reflect differences between DVR and Wulst gene expression modifications? The authors should include low magnification images of the regions where experiments were performed. And they should consider the variable expression of all genes when interpreting results.

b) SVZ is not a postmitotic zone (as stated in line 123, and wrongly assigned throughout the text and figures). On the contrary, the SVZ is a secondary proliferative zone, organized in a layer, located in a basal position to the VZ. Both (VZ and SVZ) are germinative zones, containing mostly progenitors. The only postmitotic neurons in VZ and SVZ occupy them transiently when moving to the mantle zone, which is closer to the meninges and is the postmitotic territory. Please refer to the original Boulder committee articles to revise the SVZ definition. The authors, however, misinterpret this concept, and label the whole mantle zone as it this would be the SVZ. Indeed, the term "mantle zone" does not appear in the article. Please, revise and change the whole text and figures, as SVZ statements and photographs are nearly always misinterpreted. Indeed, SVZ is only labelled well in Figure 4F.

The two articles mentioning the expression of NeuroD1 in the SVZ (line 118) are research in Xenopus. Is there a proliferative SVZ in Xenopus?

For the actual existence of the SVZ in the chick pallium, please refer to the recent Rueda-Alaña et al., 2025 article that presents PH3 stainings at different timepoints and pallial areas.

c) What is the Wulst, according to the authors of the article? In many figures, the Wulst includes the medial pallium and hippocampus, whereas sometimes it is used as a synonym of the hyperpallium (which excludes the medial pallium and hippocampus). Please make it clear, as the addition or not of the hippocampus definitely changes some interpretations.

d) The authors compare the entirety of the chick pallium - including the hippocampus (see above), hyperpallium, mesopallium, nidopallium - to only the neocortex of mammals. This view - as shown in Suzuki et al., 2012 - forgets the specificity of pallial areas of the pallium and compares it to cortical cells. This is conceptually wrong, and leads to incorrect interpretations (please refer to Luis Puelles' commentaries on Suzuki et al results); there are incorrect conclusions about the existence of upper-layer-like and deep-layer-like neurons in the pallium of birds. The view is not only wrong according to the misinterpreted anatomical comparisons, but also according to novel scRNAseq data (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025). These articles show that many avian glutamatergic neurons of the pallium have highly diversified, and are not comparable to mammalian cortical cells. The authors should therefore avoid this incorrect use of terminology. There are not such upper-layer-like and deep-layer-like neurons in the pallium of birds.

(2) From introduction to discussion, the article uses misleading terms and outdated concepts of cell type homology and similarity between chick and pallial territories and cells. The authors must avoid this confusing terminology, as non-expert readers will come to evolutionary conclusions which are not supported by the data in this article; indeed, the article does not deal with those concepts.

a) Recent articles published in Science (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025) directly contradict some views presented in this article. These articles should be presented in the introduction as they are utterly important for the subject of this article and their results should be discussed in the light of the new findings of this article. Accordingly, the authors should avoid claiming any homology that is not currently supported. The expression of a single gene is not enough anymore to claim the homology of neuronal populations.

b) Auditory cortex is not an appropriate term, as there is no cortex in the pallium of birds. Cortical areas require the existence of neuronal arrangements in laminae that appear parallel to the ventricular surface. It is not the case of either hyperpallium or auditory DVR. The accepted term, according to the Avian Nomenclature forum, is Field L.

c) Forebrain, a term overused in the article, is very unspecific. It includes vast areas of the brain, from the pretectum and thalamus to the olfactory bulb. However the authors are not researching most of the forebrain here. They should be more specific throughout the text and title.

(3) In the last part of the results, the authors claim miR19b has a role in patterning the avian pallium. What they see is that modifying its expression induces changes in gene expression in certain neurons. Accordingly, the altered neurons would differentiate into other subtypes, not similar to the wild type example. In this sense, miR19b may have a role in cell specification or neuronal differentiation. However, patterning is a different developmental event, which refers to the determination of broad genetic areas and territories. I don't think miR19b has a role in patterning.

(4) Please add a scheme of the molecules described in this article and the suggested interaction between them.

(5) The methods section is way too brief to allow for repeatability of the procedures. This may be due to an editorial policy but if possible, please extend the details of the experimental procedures.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to understand the neural basis of implicit causal inference, specifically how people infer causes of illness. They use fMRI to explore whether these inferences rely on content-specific semantic networks or broader, domain-general neurocognitive mechanisms. The study explores two key hypotheses: first, that causal inferences about illness rely on semantic networks specific to living things, such as the 'animacy network,' given that illnesses affect only animate beings; and second, that there might be a common brain network supporting causal inferences across various domains, including illness, mental states, and mechanical failures. By examining these hypotheses, the authors aim to determine whether causal inferences are supported by specialized or generalized neural systems.

The authors observed that inferring illness causes selectively engaged a portion of the precuneus (PC) associated with the semantic representation of animate entities, such as people and animals. They found no cortical areas that responded to causal inferences across different domains, including illness and mechanical failures. Based on these findings, the authors concluded that implicit causal inferences are supported by content-specific semantic networks, rather than a domain-general neural system, indicating that the neural basis of causal inference is closely tied to the semantic representation of the specific content involved.

Strengths:

- The inclusion of the four conditions in the design is well thought out, allowing for the examination of the unique contribution of causal inference of illness compared to either a different type of causal inference (mechanical) or non-causal conditions. This design also has the potential to identify regions involved in a shared representation of inference across general domains.

- The presence of the three localizers for language, logic, and mentalizing, along with the selection of specific regions of interest (ROIs), such as the precuneus and anterior ventral occipitotemporal cortex (antVOTC), is a strong feature that supports a hypothesis-driven approach (although see below for a critical point related to the ROI selection).

- The univariate analysis pipeline is solid and well developed.

- The statistical analyses are a particularly strong aspect of the paper.

Weaknesses:

After carefully considering the authors' response, I believe that my primary concern has not been fully addressed. My main point remains unresolved:

The authors attempt to test for the presence of a shared network by performing only the Causal vs. Non-causal analysis. However, this approach is not sufficiently informative because it includes all conditions mixed together and does not clarify whether both the illness-causal and mechanical-causal conditions contribute to the observed results.

To address this limitation, I originally suggested an additional step: using as ROIs the different regions that emerged in the Causal vs. Non-causal decoding analysis and conducting four separate decoding analyses within these specific clusters:<br /> (1) Illness-Causal vs. Non-causal - Illness First<br /> (2) Illness-Causal vs. Non-causal - Mechanical First<br /> (3) Mechanical-Causal vs. Non-causal - Illness First<br /> (4) Mechanical-Causal vs. Non-causal - Mechanical First

This approach would allow the authors to determine whether any of these ROIs can decode both the illness-causal and mechanical-causal conditions against at least one non-causal condition. However, the authors did not conduct these analyses, citing an independence issue. I disagree with this reasoning because these analyses would serve to clarify their initial general analysis, in which multiple conditions were mixed together. As the results currently stand, it remains unclear which specific condition is driving the effects.

My suggestion was to select the ROIs from their general analysis (Causal vs. Non-causal) and then examine in more detail which conditions were driving these results. This is not a case of double-dipping from my perspective, but rather a necessary step to unpack the general findings. Moreover, using ROIs would actually reduce the number of multiple comparisons that need to be controlled for.

If the authors believe that this approach is methodologically incorrect, then they should instead conduct all possible analyses at the whole-brain level to examine the effects of the specific conditions independently.

Reviewer #2 (Public review):

Summary:

In this study, the authors test whether intuitive biological causal knowledge is embedded in domain-specific semantic networks, primarily focusing on the precuneus as part of the animacy semantic network. They do so tanks to an fMRI task, by comparing brain activity elicited by participants' exposure to written situations suggesting a plausible cause of illness with brain activity in linguistically equivalent situations suggesting a plausible cause of mechanical failure or damage and non-causal situations. These contrasts confirm the PC as the main "culprit" in whole-brain and fROIs univariate analyses. In turn, inferring causes of mechanical failure engages mostly the PPA. The authors further test whether the content-specificity has to do with inferences about animates in general, or if there are some distinctions between reasoning about people's bodies versus mental states. To answer this question, the authors localize the mentalizing network and study the relation between brain activity elicited by Illness-Causal > Mech-Causal and Mentalizing > Physical stories. They conclude that inferring about the causes of illness partially differentiates from reasoning about people's states of mind. The authors finally test the alternative yet non-mutually exclusive hypothesis that both types of implicit causal inferences (illness and mechanical) depend on shared neural machinery. Good candidates are language and logic, which justifies the use of a language/logic localizer. No evidence of commonalities across causal inferences versus non-causal situations are found.

Strengths:

(1) This study introduces a useful paradigm and well-designed set of stimuli to test for implicit causal inferences.<br /> (2) Another important methodological advance is the addition of physical stories to the original mentalizing protocol.<br /> These tools pave the way for further investigation of domain-specific causal inference.<br /> (3) The authors have significantly improved the manuscript, addressing previous concerns and incorporating additional analyses that strengthen their conclusions.

Key improvements:<br /> (1) The revised introduction makes the study's contribution more explicit and resolves initial ambiguities regarding its scope.<br /> (2) The rationale for focusing primarily on the precuneus is now clearer and the additional analysis in the fusiform face area provides a valuable comparison.<br /> (3) The revised manuscript now includes a more detailed examination of the searchlight MVPA results, showing that illness and mechanical inferences elicit spatially distinct neural patterns in key regions, including the left PC, anterior PPA, and lateral occipitotemporal cortex.<br /> (4) The authors' justification for using an implicit inference task, arguing that explicit tasks introduce executive function confounds, is convincing.<br /> (5) The authors now acknowledge that while their results support a content-specific neural basis for implicit causal inference, domain-general mechanisms may still play a role in other contexts.

I have no major remaining concerns.

Reviewer #3 (Public review):

Summary:

This study employed an implicit task, showing vignettes to participants while bold signal was acquired. The aim was to capture automatic causal inferences that emerge during language processing and comprehension. In particular, the authors compared causal inferences about illness with two control conditions, causal inferences about mechanical failures and non-causal phrases related to illnesses. All phrases that where employed described contexts with people, to avoid animacy/inanimate confound in the results. The authors had a specific hypothesis concerning the role of the precuneus (PC) being sensitive to causal inferences about illnesses (that was preregistered).<br /> Findings indicate that implicit causal inferences are facilitated by semantic networks specialized for encoding causal knowledge.

Strengths:

The major strength of the study is the clever design of the stimuli (which are nicely matched for a number of features) which can tease apart the role of the type of causal inference (illness-causal or mechanical-causal) and the use of two localizers (logic/language and mentalizing) to investigate the hypothesis that the language and/or logical reasoning networks preferentially respond to causal inference regardless of the content domain being tested (illnesses or mechanical).

I think that authors' revisions of the original manuscript have strengthened the study. Overall, the paper provides an interesting contribution to the (rather new) field of study concerning the neural basis of implicit causal inference.

I see two weaknesses concerning the visualization of the data (which could be improved)

(1) Measures of dispersion are now provided for the average PSC in the critical window. It would be more appropriate to show the variance of the data also for the percentage signal changes (PSC) figures (e.g., 1A by using shaded lines providing SE around the means or boxplots at each timepoint).

(2) The authors could consider showing in Figure 2 the data of supplementary Figure 3. It is not clear why the authors report in the main manuscript the results of a subsample of participants (and only for this figure).

Reviewer #1 (Public review):

Summary:

The paper addresses the knowledge gap between the representation of goal direction in the central complex and how motor systems stabilize movement toward that goal. The authors focused on two descending neurons, DNa01 and 02, and showed that they play different roles in steering the fly toward a goal. They also explored the connectome data to propose a model to explain how these DNs could mediate response to lateralized sensory inputs. They finally used lateralized optogenetic activation/inactivation experiments to test the roles of these neurons in mediating turnings in freely walking flies.

Strengths:

The experiments are well-designed and controlled. The experiment in Figure 4 is elegant, and the authors put a lot of effort into ensuring that ATP puffs do not accidentally activate the DNs. They also have explained complex experiments well. I only have minor comments for the authors.

Comments on revisions:

I am happy with the revised manuscript and authors' response to our concerns. The addition of Figure S8, makes it more transparent and the revised text is now more accessible to the non-experts.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but has minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs is stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals chose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

Comments on revisions:

I am happy that the revised manuscript addresses all reviewers' concerns.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit-flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and show functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1). In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies which have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Fig. 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In-line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2). Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble (PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02 (Fig. 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring-attractor returns to its previous stable point.

(3). The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus directed turns.

Weaknesses:

While this study makes a compelling case for the importance of DNa02 in steering control, the role of DNa01 on the other hand seems unclear based on physiology, optogenetics perturbations as well as connectome analysis. DNa01 still remains a bit mysterious regarding both its role in controlling steering maneuvers as well as what in behavioral context it would be relevant.

Reviewer #3 (Public review):

Summary:

The hippocampal CA3 region is generally considered to be the primary site of initiation of sharp wave ripples-highly synchronous population events involved in learning and memory-although the precise mechanism remains elusive. A recent study revealed that CA3 comprises two distinct pyramidal cell populations: thorny cells that receive mossy fiber input from the dentate gyrus, and athorny cells that do not. That study also showed that it is athorny cells in particular which play a key role in sharp wave initiation. In the present work, Sammons, Masserini and colleagues expand on this by examining the connectivity probabilities among and between thorny and athorny cells. Using whole-cell patch clamp recordings, they find an asymmetrical connectivity pattern, with athorny cells receiving the most synaptic connections from both athorny and thorny cells, and thorny cells receiving fewer.

The authors then use a spiking network model to show how this assymmetrical connectivity is consistent with a preferential role of athorny cells in sharp wave initiation. Essentially, thorny and athorny cells are put into a winner-takes-all scenario in which athorny cells always win initially. Thorny cells can only become active after athorny cells decrease their firing rate due to adaptation, leading to a delay between the activation of athorny and thorny cells. As far as I understand, the initial victory of athorny cells in the winner-takes-all is doubly determined: it is both due to their intrinsic properties (lower rheobase and steeper f-I curve), and due to the bias in connectivity towards them. It appears to me that either of these two mechanisms (i.e., different intrinsic properties and symmetrical self- and cross-connections, or the same intrinsic properties and asymmetrical connectivity) would suffice to explain the sequential activation of the two cell types. From a theoretician's perspective, this overdetermination is not very elegant, but biology often isn't...

Strengths:

The authors provide independent validation of some of the findings by Hunt et al. (2018) concerning the distinction between thorny and athorny pyramidal cells in CA3 and advance our understanding of their differential integration in CA3 microcircuits. The properties of excitatory connections among and between thorny and athorny cells described by the authors will be key in understanding CA3 functions including, but not limited to, sharp wave initiation.

As stated in the paper, the modeling results lend support to the idea that the increased excitatory connectivity towards athorny cells plays an important role in causing them to fire before thorny cells in sharp waves. More generally, the model adds to an expanding pool of models of sharp wave ripples which should prove useful in guiding and interpreting experimental research.

Reviewer #1 (Public review):

Summary:

This study utilises fNIRS to investigate the effects of undernutrition on functional connectivity patterns in infants from a rural population in Gambia. fNIRS resting-state data recording spanned ages 5 to 24 months, while growth measures were collected from birth to 24 months. Additionally, executive functioning tasks were administered at 3 or 5 years of age. The results show an increase in left and right frontal-middle and right frontal-posterior connections with age and, contrary to previous findings in high-income countries, a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months of age. Additionally, the study describes some connectivity patterns, including stronger frontal interhemispheric connectivity, which is associated with better cognitive flexibility at preschool age.

Strengths:

- The study analyses longitudinal data from a large cohort (n = 204) of infants living in a rural area of Gambia. This already represents a large sample for most infant studies, and it is impressive, considering it was collected outside the lab in a population that is underrepresented in the literature. The research question regarding the effect of early nutritional deficiency on brain development is highly relevant and may highlight the importance of early interventions. The study may also encourage further research on different underrepresented infant populations (i.e., infants not residing in Western high-income countries) or in settings where fMRI is not feasible.

- The preprocessing and analysis steps are carefully described, which is very welcome in the fNIRS field, where well-defined standards for preprocessing and analysis are still lacking.

Weaknesses:

- While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level and investigates how restricted growth influences connectivity patterns at 24 months, it does not explore the links between adverse situations and developmental trajectories for functional connectivity. Considering the longitudinal nature of the dataset, it would have been interesting to apply more sophisticated analytical tools to link undernutrition to specific developmental trajectories in functional connectivity. The authors mention that they lack the statistical power to separate infants into groups according to their growing profiles. However, I wonder if this aspect could not have been better explored using other modelling strategies and dimensional reduction techniques. I can think about methods such as partial least squares correlation, with age included as a numerical variable and measures of undernutrition.

- Connectivity was asses in 6 big ROIs. While the authors justify this choice to reduce variability due to head size and optode placement, this also implies a significant reduction in spatial resolution. Individual digitalisation and co-registration of the optodes to the head model, followed by image reconstruction, could have provided better spatial resolution. This is not a weakness specific to this study but rather a limitation common to most fNIRS studies, which typically analyse data at the channel level since digitalisation and co-registration can be challenging, especially in complex setups like this. However, the BRIGHT project has demonstrated that it is possible and that differences in placement affect activation patterns, which become more localised when data is co-registered at the subject level (Collins-Jones et al., 2021). Could the co-registration of individual data have increased sensitivity, particularly given that longitudinal effects are being investigated?

- I believe that a further discussion in the manuscript on the application of global signal regression and its effects could have been beneficial for future research and for readers to better understand the negative correlations described in the results. Since systemic physiological changes affect HbO/HbR concentrations, resulting in an overestimation of functional connectivity, regressing the global signal before connectivity computation is a common strategy in fNIRS and fMRI studies. However, the recommendation for this step remains controversial, likely depending on the case (Murphy & Fox, 2017). I understand that different reasons justify its application in the current study. In addition to systemic physiological changes originating from brain tissue, fNIRS recordings are contaminated by changes occurring in superficial layers (i.e., the scalp and skull). While having short-distance channels could have helped to quantify extracerebral changes, challenges exist in using them in infant populations, especially in a longitudinal study such as the one presented here. The optimal source-detector distance that minimises sensitivity to changes originating from the brain would increase with head size, and very young participants would require significantly shorter source-detector distances (Brigadoi & Cooper, 2015). Thus, having them would have been challenging. Under these circumstances (i.e., lack of short channels and external physiological measures), and considering that the amount the signal is affected by physiological noise (either coming from the brain or superficial tissue) might change through development, the choice of applying global signal regression is justified. Nevertheless, since the method introduces negative correlations in the data by forcing connectivity to average to zero, I believe a further discussion of these points would have enriched the interpretation of the results.

Reviewer #2 (Public review):

Strengths:

The article addresses a topic of significant importance, focusing on early life growth faltering in low-income countries-a key marker of undernutrition-and its impact on brain functional connectivity (FC) and cognitive development. The study's strengths include the laborious data collection process, as well as the rigorous data preprocessing methods employed to ensure high data quality. The use of cutting-edge preprocessing techniques further enhances the reliability and validity of the findings, making this a valuable contribution to the field of developmental neuroscience and global health.

Weaknesses:

The study fails to fully leverage its longitudinal design to explore neurodevelopmental changes or trajectories, as highlighted by all three reviewers. The revised manuscript still primarily focuses on FC values at a single age stage (i.e., 24 months) rather than utilizing the longitudinal data to investigate how FC evolves over time or predicts cognitive development. Although the authors acknowledge that analyzing changes in FC (ΔFC) would reduce degrees of freedom (to ~30) and risk interpretability, they do not report or discuss these results, even as exploratory findings.

Furthermore, the study lacks specificity in identifying which specific brain networks are affected by growth faltering, as the current exploratory analyses mainly provide an overall conclusion that infant brain network development is impacted without pinpointing the precise neural mechanisms or networks involved.

Reviewer #3 (Public review):

Summary

This study aimed to investigate whether the development of functional connectivity (FC) is modulated by early physical growth, and whether these might impact cognitive development in childhood. This question was investigated by studying a large group of infants (N=204) assessed in Gambia with fNIRS at 5 visits between 5 and 24 months of age. Given the complexity of data acquisition at these ages and following data processing, data could be analyzed for 53 to 97 infants per age group. FC was analyzed considering 6 ensembles of brain regions and thus 21 types of connections. Results suggested that: i) compared to previously studied groups, this group of Gambian infants have different FC trajectory, in particular with a change in frontal inter-hemispheric FC with age from positive to null values; ii) early physical growth, measured through weight-for-length z-scores from birth on, is associated with FC at 24 months. Some relationships were further observed between FC during the first two years and cognitive flexibility, in different ways between 4- and 5-year-old preschoolers, but results did not survive corrections for multiple comparisons.

Strengths

The question investigated in this article is important for understanding the role of early growth and undernutrition on brain and behavioral development in infants and children. The longitudinal approach considered is highly relevant to investigate neurodevelopmental trajectories. Furthermore, this study targets a little studied population from a low-/middle-income country, which was made possible by the use of fNIRS outside the lab environment. The collected dataset is thus impressive and it opens up a wide range of analytical possibilities.

Weaknesses

- Data analyses were constrained by the limited number of children with longitudinal data on NIRS functional connectivity. Nevertheless, considering more advanced statistical modeling approaches would be relevant to further explore neurodevelopmental trajectories as well as relationships with early growth and later cognitive development.<br /> - The abstract and end of the discussion should make it clearer that the associations between FC and cognitive flexibility are results that need to be confirmed, insofar as they did not survive correction for multiple comparisons.

Reviewer #1 (Public review):

Summary:

Building on previous in vitro synaptic circuit work (Yamawaki et al., eLife 10, 2021), Piña Novo et al. utilize an in vivo optogenetic-electrophysiological approach to characterize sensory-evoked spiking activity in the mouse's forelimb primary somatosensory (S1) and motor (M1) areas. Using a combination of a novel "phototactile" somatosensory stimuli to the mouse's hand and simultaneous high-density linear array recordings in both S1 and M1, the authors report evoked activity in S1 was biased to middle layers, whereas it was biased to upper layers in M1. They report that M1 responses are delayed and attenuated relative to S1. Further analysis revealed a 20-fold difference in subcortical versus corticocortical propagation speeds. They also find that PV interneurons in S1 are strongly recruited by hand stimulation, and their selective activation can produce a suppression and rebound response similar to "phototactile" stimuli. Silencing S1 through local PV cells was sufficient to reduce M1 response to hand stimulation, suggesting S1 may directly drive M1 responses.

Strengths:

The study was technically well done, with convincing results. The data presented are appropriately analyzed. The author's findings build on a growing body of both in vitro and in vivo work examining the synaptic circuits underlying the interactions between S1 and M1. The paper is well-written and illustrated. Overall, the study will be valuable to those interested in forelimb S1-M1 interactions.

Weaknesses:

The authors have addressed my concerns

Reviewer #2 (Public review):

Summary:

Communication between sensory and motor corticies is likely to be important for many aspects of behavior, and in this study the authors carefully analyse neuronal spiking activity in S1 and M1 evoked by peripheral paw stimulation finding clear evidence for sensory responses in both cortical regions

Strengths:

The experiments and data analyses appear to have been carefully carried out and clearly represented.

Weaknesses:

The revised manuscript addressed the minor weaknesses I noted relating to the first submission.

Reviewer #3 (Public review):

Summary:

This is a solid study of stimulus-evoked neural activity dynamics in the feedforward pathway from mouse hand/forelimb mechanoreceptor afferents to S1 and M1 cortex. The conclusions are generally well supported and match expectations from previous studies of hand/forelimb circuits by this same group (Yamawaki et al., 2021), from the well-studied whisker tactile pathway to whisker S1 and M1, and from the corresponding pathway in primates. The study uses the novel approach of optogenetic stimulation of PV afferents in the periphery, which provides an impulse-like volley of peripheral spikes, which is useful for studying feedforward circuit dynamics. These are primarily proprioceptors, so results could differ for specific mechanoreceptor populations, but this is a reasonable tool to probe basic circuit activation. Mice are awake but not engaged in a somatosensory task, which is sufficient for the study goals.

The main results are: 1) brief peripheral activation drives brief sensory-evoked responses at ~ 15 ms latency in S1 and ~25 ms latency in M1, which is consistent with classical fast propagation on the subcortical pathway to S1, followed by slow propagation on the polysynaptic, non-myelinated pathway from S1 to M1; 2) each peripheral impulse evokes a triphasic activation-suppression-rebound response in both S1 and M1; 3) PV interneurons carry the major component of spike modulation for each of these phases; 4) activation of PV neurons in each area (M1 or S1) drives suppression and rebound both in the local area and in the other downstream area; 5) peripheral-evoked neural activity in M1 is at least partially dependent on transmission through S1.

All conclusions are well-supported and reasonably interpreted. There are no major new findings that were not expected from standard models of somatosensory pathways or from prior work in the whisker system.

Strengths:

This is a well-conducted and analyzed study in which the findings are clearly presented. The optogenetic sensory afferent stimulation method is novel and is well-suited for studying feedforward circuit dynamics. This study provides important baseline knowledge from which studies of more complex sensorimotor processing can build.

There are no further recommendations for the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors discovered MYL3 of marine medaka (Oryzias melastigma) as a novel NNV entry receptor, elucidating its facilitation of RGNNV entry into host cells through macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 pathway.

Strengths:

In this manuscript, the authors have performed in vitro and in vivo experiments to prove that MnMYL3 may serve as a receptor for NNV via macropinocytosis pathway. These experiments with different methods include Co-IP, RNAi, pulldown, SPR, flow cytometry, immunofluorescence assays and so on. In general, the results are clearly presented in the manuscript.

Weaknesses:

For the writing in the introduction and discussion sections, the author Yao et al mainly focus on the viral pathogens and fish in Aquaculture, the meaning and novelty of results provided in this manuscript are limited, not broad in biology. The authors should improve the likely impact of their work on the viral infection field, maybe also in the evolutionary field with fish model.

Additionally, detailed comments are as follows:

(1) Myosin is a big family, why did authors choose MYL3 as a candidate receptor for NNV?

(2) What's the relationship between MmMYL3 and MmHSP90ab1 and other known NNV receptors? Why dose NNV have so many receptors? Which one is supposed to serve as the key entry receptor?

(3) In vivo knockout of MYL3 using CRISPR-Cas9 should be conducted to verify whether the absence of MYL3 really inhibits NNV infection. Although it might be difficult to do it in marine medaka as stated by authors, the introduce of zebrafish is highly recommended, since it has already been reported that zebrafish could be served as a vertebrate model to study NNV (doi: 10.3389/fimmu.2022.863096).

(4) The results shown in Figure 6 are not enough to support the conclusion that "RGNNV triggers macropinocytosis mediated by MmMYL3". Additional electron microscopy of macropinosomes (sizes, morphological characteristics, etc.) will be a more direct evidence.

(5) MYL3 is "predominantly found in muscle tissues, particularly the heart and skeletal muscles". However, NNV is a virus mainly causes necrosis of nervous tissues (brain and retina). If MYL3 really acts as a receptor for NNV, how does it balance this difference so that nervous tissues, rather than muscle tissues, have the highest viral titers?

Comments on revisions:

The authors have addressed most of my concerns in the revised manuscript, but still one question need to further improve to strengthen the study's rationale and conclusions.

Specificity of MYL3 Selection:<br /> My previous question focused on why MYL3 was prioritized over other myosin family members. While the response broadly implicates myosins in viral entry, it does not justify why MYL3 was specifically chosen. For clarity, the "Introduction sections" should explicitly state the unique features of MYL3 (e.g., domain structure, binding affinity, or prior evidence linking it to NNV) that distinguish it from other myosins.

Reviewer #2 (Public review):

Summary:

The manuscript offers an important contribution to the field of virology, especially concerning NNV entry mechanisms. The major strength of the study lies in the identification of MmMYL3 as a functional receptor for RGNNV and its role in macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 signaling axis. This represents a significant advance in understanding NNV entry mechanisms beyond previously known receptors such as HSP90ab1 and HSC70. The data, supported by comprehensive in vitro and in vivo experiments, strongly justify the authors' claims about MYL3's role in NNV infection in marine medaka.

Strengths:

(1) The identification of MmMYL3 as a functional receptor for RGNNV is a significant contribution to the field. The study fills a crucial gap in understanding the molecular mechanisms governing NNV entry into host cells.

(2) The work highlights the involvement of IGF1R in macropinocytosis-mediated NNV entry and downstream Rac1/Cdc42 activation, thus providing a thorough mechanistic understanding of NNV internalization process. This could pave the way for further exploration of antiviral targets.

Comments on revisions:

The authors have addressed the concerns from reviewers. This manuscript can be published in the current form.

Reviewer #3 (Public review):

Summary:

The manuscript presents a detailed study on the role of MmMYL3 in the viral entry of NNV, focusing on its function as a receptor that mediates viral internalization through the macropinocytosis pathway. The use of both in vitro assays (e.g., Co-IP, SPR, and GST pull-down) and in vivo experiments (such as infection assays in marine medaka) adds robustness to the evidence for MmMYL3 as a novel receptor for RGNNV. The findings have important implications for understanding NNV infection mechanisms, which could pave the way for new antiviral strategies in aquaculture.

Strengths:

The authors show that MmMYL3 directly binds the viral capsid protein, facilitates NNV entry via the IGF1R-Rac1/Cdc42 pathway, and can render otherwise resistant cells susceptible to infection. This multifaceted approach effectively demonstrates the central role of MmMYL3 in NNV entry.

Comments on revisions:

The implemented revisions have remarkably improved the manuscript's conceptual clarity, scientific depth, and methodological rigor. Through comprehensive addressing of issue with meticulous attention to detail, the authors have produced a substantially strengthened manuscript that demonstrates enhanced experimental validity and theoretical coherence. No additional revisions appear necessary at this stage.

Reviewer #1 (Public review):

Summary:

Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely to remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.

Strengths:

The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability the move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.

Weaknesses:

No major weaknesses noted.

Reviewer #2 (Public review):

Summary:

In this work, the investigators isolated one Lacticaseibacillus rhamnosus strain (P118), and determined this strain worked well against Salmonella Typhimurium infection. Then, further studies were performed to identify the mechanism of bacterial resistance, and a list of confirmatory assays were carried out to test the hypothesis.

Strengths:

The authors provided details regarding all assays performed in this work, and this reviewer trusted that the conclusion in this manuscript is solid. I appreciate the efforts of the authors to perform different types of in vivo and in vitro studies to confirm the hypothesis.

Weaknesses:

I have mainly two questions for this work.

Main point-1:<br /> The authors provided the below information about the sources from which Lacticaseibacillus rhamnosus was isolated. More details are needed. What are the criteria to choose these samples? Where were these samples originate from? How many strains of bacteria were obtained from which types of samples?

Lines 486-488: Lactic acid bacteria (LAB) and Enterococcus strains were isolated from the fermented yoghurts collected from families in multiple cities of China and the intestinal contents from healthy piglets without pathogen infection and diarrhoea by our lab.

Lines 129-133: A total of 290 bacterial strains were isolated and identified from 32 samples of the fermented yoghurt and piglet rectal contents collected across diverse regions within China using MRS and BHI medium , which consist s of 63 Streptococcus strains, 158 Lactobacillus/ Lacticaseibacillus Limosilactobacillus strains and 69 Enterococcus strains.

Main-point-2:<br /> As probiotics, Lacticaseibacillus rhamnosus has been widely studied. In fact, there are many commercially available products, and Lacticaseibacillus rhamnosus is the main bacteria in these products. There are also ATCC type strain such as 53103.

I am sure the authors are also interested to know if P118 is better as a probiotics candidate than other commercially available strains. Also, would the mechanism described for P118 apply to other Lacticaseibacillus rhamnosus strains?

It would be ideal if the authors could include one or two Lacticaseibacillus rhamnosus which are currently commercially used, or from the ATCC. Then, the authors can compare the efficacy and antibacterial mechanisms of their P118 with other strains. This would open the windows for future work.

Reviewer #1 (Public review):

Shin et al. conduct extensive electrophysiological and behavioral experiments to study the mechanisms of short-term synaptic plasticity at excitatory synapses in layer 2/3 of the rat medial prefrontal cortex. The authors interestingly find that short-term facilitation is driven by progressive overfilling of the readily releasable pool, and that this process is mediated by phospholipase C/diacylglycerol signaling and synaptotagmin-7 (Syt7). Specifically, knockdown of Syt7 not only abolishes the refilling rate of vesicles with high fusion probability, but it also impairs the acquisition of trace fear memory.

Overall, the authors offer novel insight to the field of synaptic plasticity through well-designed experiments that incorporate a range of techniques.

Comments on revisions:

The authors have adequately addressed my earlier comments and questions.

Reviewer #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model and is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks. The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths:

This is a very thorough study, addressing the research question from many different angles and the experimental execution is superb. The impact of the work is high, as it applies recent models of short term plasticity behavior to in vivo circuits further providing insights how synapses provide dynamic control to enable working memory related behavior through non-permanent changes in synaptic output.

Weaknesses:

While this work is carefully examined and the results are presented and discussed in a detailed manner, the reviewer is still not fully convinced that regulation of release probability is not a putative contributor to the observed behavior. No additional work is needed, but in the moment, I am not convinced that changes in release probability are not in play. One solution may be to extend the discussion of changes in rules probability as an alternative.

Fig 3. I am confused about the interpretation of the Mean Variance analysis outcome. Since the data points follow the curve during induction of short term plasticity, doesn't these suggests that release probability and not the pool size increases? Related, to measure the absolute release probability and failure rate using the optogenetic stimulation technique is not trivial as the experimental paradigm bias the experiment to a given output strength, and therefore a change in release probability cannot be excluded.

Fig. 4B interprets the phorbol ester stimulation to be the result of pool overfilling, however, phorbol ester stimulation has also been shown to increase release probability without changing the size of the readily releasable pool. The high frequency of stimulation may occlude a increased paired pulse depression in presence of OAG, that others have interpreted in mammalian synapses as an increase in release probability.

The literature on Syt7 function is still quite controversial. An observation in the literature that loss of Syt7 function in the fly synapse leads to an increase of release probability. Thus the observed changes in short term plasticity characteristics in the Syt7 KD experiments may contain a release probability component. Can the authors really exclude this possibility? Figure 5 shows for the Syt7 KD group a very prominent depression of the EPSC/IPSC with the second stimulus, particularly for the short interpulse intervals, usually a strong sign of increased release probability, as lack of pool refilling can unlikely explain the strong drop in synaptic output.

Comments on revisions:

I am satisfied with the reply of the authors and I do not have any further points of concern.

Reviewer #3 (Public review):

The results are consistent with the main claim that facilitation is caused by overfilling a readily releasable pool, but alternative interpretations continue to seem more likely, especially when the current results are taken together with previous studies. Key doubts could be resolved with a single straightforward experiment (see below).

The central issue is the interpretation of paired pulse depression that occurs when the interval between action potentials is 25 ms, but not when 50. To summarize: a similar phenomenon was observed at Schaffer collateral synapses (Dobrunz and Stevens, 1997), but was interpreted as evidence for a decrease in pv. Ca2+-channel inactivation was proposed as the mechanism, but this was not proven. The key point for evaluating the current study is that Dobrunz and Stevens specifically ruled out the kind of decrease in pocc that is the keystone premise of the current study because the depression occurred independently of whether or not the first action potential elicited exocytosis. Of course, the mechanism might be different at layer 2/3 cortical synapses. But, it seems reasonable to hope that the older hypothesis would be ruled out for the cortical synapses before concluding that the new hypothesis must be correct.

The old and new hypotheses could be distinguished from each other cleanly with a straightforward experiment. Most/maybe all central synapses strengthen a great amount when extracellular Ca2+ is increased from 1.3 to 2 mM, even when intracellular Ca2+ is buffered with EGTA. According to the authors' model, this is only possible when pv is low, and so could not occur at synapses between layer 2/3 neurons. Because of this, confirmation that increasing extracellular Ca2+ does not change synaptic strength would support the hypothesis that baseline pv is high, as the authors claim, and the support would be impressive because large changes have been seen at every other type of synapse where this has been studied (to my knowledge at least). In contrast, the Ca2+ imaging experiment that has been added to the new version of the manuscript does not address the central issue because a wide range of mechanisms could, in principle, decrease release without involving prior exocytosis or altering bulk Ca2+ signals, including: a small decrease in nano-domain Ca2+, which wouldn't be detected because nano-domains contribute a minuscule amount to the bulk signal during Ca2+-imaging; or even very fast activity-dependent undocking of synaptic vesicles, which was reported in the same Kusick et al, 2020 study that is central to the LS/TS terminology adopted by the authors.

Additional points:

(1) A new section in the Discussion (lines 458-475) suggests that previous techniques employed to show that augmentation and facilitation are caused by increases in pv did not have the resolution to distinguish between pv and pocc, but this is misleading. The confusion might be because the terminology has changed, but this is all the more reason to clarify this section. The previous evidence for increases in pv - and against increases in pocc - is as follows: The residual Ca2+ that drives augmentation decreases the latency between the onset of hypertonic solution and onset of the postsynaptic response by about 150 ms, which is large compared to the rise time of the response. The decrease indicates that the residual Ca2+ drives a decrease in the energy barrier that must be overcome before readily releasable vesicles can undergo exocytosis, which is precisely the type of mechanism that would enhance pv. In contrast, an increase in pocc could change the rise time, but not the latency. There is a small change in the rise time, but this could be caused by changes in either pv or pocc, and one of the studies (Garcia-Perez and Wesseling, 2008) showed that augmentation occluded facilitation, even at times when pocc was reduced by a factor of 3, which would seem to argue against parallel increases in both pv and pocc.

(2) Similar evidence from hypertonic stimulation indicates that Phorbol esters increase pv, but I am not aware of evidence ruling out a parallel increase in pocc.

Comments on revisions:

There are at least two straightforward ways to address the main concern.

The first would be experiments analogous to those in Dobrunz and Stevens that show that - unlike at Schaffer collateral synapses - paired pulse depression at L2/3 synapses requires neurotransmitter release. I proposed this in the first round, but realized since that a simpler and more powerful strategy would be to test directly that pv is/is-not near 1.0 in 1.2 mM Ca2+ simply by increasing to 2 mM Ca2+ (and showing that synaptic strength does-not/does change). This would be powerful because the increase in Ca2+ greatly increases synaptic strength at Schaffer collaterals by about 2.5-fold. Concerns about a confounding elevation in the basal intracellular Ca2+ concentration could be easily neutralized by pre-treating with EGTA-AM, which the authors have already done for other experiments.

Reviewer #1 (Public review):

Summary:

This study provides comprehensive instructions for using the chromatophore tracking software, Chromas, to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. This software addresses a long-standing challenge faced by many researchers who study these soft-bodied creatures, known for their remarkable ability to change colour rapidly. The updated software features a user-friendly interface that can be applied to a wide range of applications, making it an essential tool for biologists focused on animal dynamic signalling. It will also be of interest to professionals in the fields of computer vision and image analysis.

Strengths:

This work provides detailed instructions for this toolkit along with examples for potential users to try. The Gitlab inventory hosts the software package, installation documentation, and tutorials, further helping potential users with a less steep learning curve.

Weaknesses:

The evidence supporting the authors' claims is solid, particularly demonstrated through the use of cuttlefish and squid. However, it may not be applicable to all coleoid cephalopods yet, such as octopuses, which have an incredibly versatile ability to change their body forms.

Reviewer #2 (Public review):

Summary:

The authors developed a computational pipeline named CHROMAS to track and analyze chromatophore dynamics, which provides a wide range of biological analysis tools without requiring the user to write code.

Strengths:

(1) CHROMAS is an integrated toolbox that provides tools for different biological tasks such as: segment, classify, track and measure individual chromatophores, cluster small groups of chromatophores, analyze full-body patterns, etc.

(2) It could be used to investigate different species. The authors have already applied it to analyze the skin of the bobtail squid Euprymna berryi and the European cuttlefish Sepia officinalis.

(3) The tool is open-source and easy to install. The paper describes in detail the command format to complete each task and provides relevant sample figures.

Weaknesses:

(1) The generality and robustness of the proposed pipeline need to be verified through more experimental evaluations. For example, the implementation algorithm depends on relatively specific or obvious image features, clean backgrounds, and objects that do not move too fast.

(2) The pipeline lacks some kind of self-correction mechanism. If at one moment there is a conflicting match with the previous frames, how does the system automatically handle it to ensure that the tracking results are accurate over a long period of time?

Reviewer #1 (Public review):

This paper presents a set of tools that will pave the way for a comprehensive understanding of the circuits that control wing motion in flies during flight or courtship. These tools are mainly focused on wing motor neurons and interneurons, as well as a few motor neurons of the haltere. This paper and the library of driver lines described within it will serve as a crucial resource in the pursuit of understanding how neural circuits give rise to behavior. Overall, I found the paper well-written, the figures are quite nice, and the data from the functional experiments convincing. I do not have many major concerns, but a few suggestions that I think will make the paper easier to understand.

I think the introduction could use some reorganization, as right now I found it quite difficult to follow. For example, lines 85-88 seem to fit more naturally at the end of the next paragraph, compared to the current location of those sentences, which feels rather disjointed. I would suggest introducing the organization of the wing motor system (paragraphs 3 and 4) and then discussing the VNC (paragraph 2) before moving on to describe the neurons within the VNC that may control wing motion. Additionally, lines 141-144, which describe the broad subdivisions of the VNC, can be moved up to where the VNC is first introduced.

One of my major takeaways from the paper is the call to examine the premotor circuits that govern wing motion. For that reason, I was surprised that there was little mention of the role of sensory input to these circuits. As the authors point out in the discussion, the haltere, for example, provides important input to the wing steering system. I recognize that creating driver lines for the sensory neurons that innervate the VNC is well beyond the scope of this project. I would just like some clarification in the text of the role these inputs play in structuring wing motion, especially as some act at rapid timescales that possibly forgo processing by the very circuits detailed here. This brings up a related issue: if the roles of the interneurons that are presynaptic to the wing motor neurons are "largely unexplored," with how much confidence can we say that they are the key for controlling behavior? To be sure, this has been demonstrated quite nicely in the case of courtship, but in flight, I think the evidence supporting this argument is less clear. I suggest the authors rephrase their language here.

Reviewer #2 (Public review):

Summary:

In this study, the researchers generated an impressive collection of sparse split GAL4 driver lines that target wing-relevant cell types. They then characterized the cell types according to function, development, and morphology. This resource is a necessary companion to the fly ventral nerve cord connectomes. The fly connectomes enable biologically-constrained hypothesis generation, but we need genetic reagents like the ones generated here in order to test those hypotheses and understand the biological limits of what we can learn from connectomes. This project identifies wing-relevant cell types and generates a library of driver lines to provide genetic access to small populations of these cell types. The study also characterizes these cell types according to developmental lineage and morphology, and performs functional analyses on some of the cell types, including single pairs of motor neurons.

Strengths:

The genetic toolkit that the authors produce is rigorous and well-documented, and will be broadly useful. Further, they bolster the utility of the resource by characterizing cell types according to developmental lineage, morphology, and connectome nomenclature. The authors successfully produce a foundation for future studies of the functional organization of neural circuits. In particular, the driver lines created in this study match the specificity of the connectome, providing a necessary resource to functionally test predictions from the connectomes.

Weaknesses:

The manuscript includes several broad statements about certain questions being "unexplored" (e.g., lines 71, 129). However, the authors cite papers (e.g., Harris 2015 and Lillvis 2024) that directly address these topics. To better support the narrative, it would be helpful to more accurately summarize the key findings from these prior studies. For example, the Harris paper found behavioral correlates of hemilineage activation. By using the sparse toolkit you have created, it may be possible to dissect behavior into finer-grained modules or specific movements, providing deeper insight into how complex behaviors are produced by the nervous system.

There are a few places where the current manuscript does not acknowledge the post-connectome universe it now exists in. For example, line 600: the morphology of DVMns in Drosophila had never been described, and line 762: revealed diversity within hemilineages which had not previously been reported. Although this manuscript was in progress before the VNC connectomes were released, they are now published, and the current manuscript should reflect this development.

The authors focus on some well-characterized "Named Neurons" e.g., ChINs and the PSI. This focused approach makes sense, but the authors miss the opportunity to point out a major strength of the toolkit they have produced: we are now less constrained by studying only these Named Neurons. With this new resource, we have genetic access to sparse sets of neurons that are likely just as important but were previously inaccessible.

Reviewer #3 (Public review):

Summary:

This paper provides a catalogue of 195 well-documented Drosophila strains with sparse and cell-type-specific GAL-4 expression in the adult ventral nerve cord (VNC). The focus is on motor neurons, interneurons, and modulatory unpaired neurons in the dorsal VNC neuropils that drive motor control of the wings. Intersegmental sensory and interneurons are not included. The expression patterns of all 195 fly strains are exceptionally well-characterized and catalogized. Compelling links to hemi-lineages and connectomics data are well documented, and some solid functional data demonstrate the applicability of the GAL4 strains in genetic silencing and optogenetic activation experiments. In sum, this catalogue provides a fantastic toolkit for future functional analyses of motor control centers in the dorsal VNC.

Strengths:

Particularly noteworthy is that the authors did a tremendous job in identifying and catalogizing the correspondences between the neurons in their catalogue and the MANC connectomics dataset, as well as with the respective hemi-lineages. The catalogue has been generated with exquisite care and is impressive.

Weaknesses:

There are no significant weaknesses. I have only minor recommendations on definitions and naming of neuron types, the text on the optogenetic experiments, and the comparison to other insects.

Reviewer #1 (Public review):

Summary:

The authors validate the contribution of RAP2A to GB progression. RAp2A participates in asymmetric cell division, and the localization of several cell polarity markers, including cno and Numb.

Strengths:

The use of human data, Drosophila models, and cell culture or neurospheres is a good scenario to validate the hypothesis using complementary systems.

Moreover, the mechanisms that determine GB progression, and in particular glioma stem cells biology, are relevant for the knowledge on glioblastoma and opens new possibilities to future clinical strategies.

Weaknesses:

While the manuscript presents a well-supported investigation into RAP2A's role in GBM, several methodological aspects require further validation. The major concern is the reliance on a single GB cell line (GB5), which limits the generalizability of the findings. Including multiple GBM lines, particularly primary patient-derived 3D cultures with known stem-like properties, would significantly enhance the study's relevance.

Additionally, key mechanistic aspects remain underexplored. Further investigation into the conservation of the Rap2l-Cno/aPKC pathway in human cells through rescue experiments or protein interaction assays would be beneficial. Similarly, live imaging or lineage tracing would provide more direct evidence of ACD frequency, complementing the current indirect metrics (odd/even cell clusters, Numb asymmetry).

Several specific points raised in previous reviews still require attention:

(1) The specificity of Rap2l RNAi needs further confirmation. Is Rap2l expressed in neuroblasts or intermediate neural progenitors? Can alternative validation methods be employed?

(2) Quantification of phenotypic penetrance and survival rates in Rap2l mutants would help determine the consistency of ACD defects.

(3) The observations on neurosphere size and Ki-67 expression require normalization (e.g., Ki-67+ cells per total cell number or per neurosphere size). Additionally, apoptosis should be assessed using Annexin V or TUNEL assays.

(4) The discrepancy in Figures 6A and 6B requires further discussion.

(5) Live imaging of ACD events would provide more direct evidence.

(6) Clarification of terminology and statistical markers (e.g., p-values) in Figure 1A would improve clarity.

(7) Given the group's expertise, an alternative to mouse xenografts could be a Drosophila genetic model of glioblastoma, which would provide an in vivo validation system aligned with their research approach.

Reviewer #2 (Public review):

This study investigates the role of RAP2A in regulating asymmetric cell division (ACD) in glioblastoma stem cells (GSCs), bridging insights from Drosophila ACD mechanisms to human tumor biology. They focus on RAP2A, a human homolog of Drosophila Rap2l, as a novel ACD regulator in GBM is innovative, given its underexplored role in cancer stem cells (CSCs). The hypothesis that ACD imbalance (favoring symmetric divisions) drives GSC expansion and tumor progression introduces a fresh perspective on differentiation therapy. However, the dual role of ACD in tumor heterogeneity (potentially aiding therapy resistance) requires deeper discussion to clarify the study's unique contributions against existing controversies. Some limitations and questions need to be addressed.

(1) Validation of RAP2A's prognostic relevance using TCGA and Gravendeel cohorts strengthens clinical relevance. However, differential expression analysis across GBM subtypes (e.g., MES, DNA-methylation subtypes ) should be included to confirm specificity.

(2) Rap2l knockdown-induced ACD defects (e.g., mislocalization of Cno/Numb) are well-designed. However, phenotypic penetrance and survival rates of Rap2l mutants should be quantified to confirm consistency.

(3) While GB5 cells were effectively used, justification for selecting this line (e.g., representativeness of GBM heterogeneity) is needed. Experiments in additional GBM lines (especially the addition of 3D primary patient-derived cell lines with known stem cell phenotype) would enhance generalizability.

(4) Indirect metrics (odd/even cell clusters, NUMB asymmetry) are suggestive but insufficient. Live imaging or lineage tracing would directly validate ACD frequency.

(5) The initial microarray (n=7 GBM patients) is underpowered. While TCGA data mitigate this, the limitations of small cohorts should be explicitly addressed and need to be discussed.

(6) Conclusions rely heavily on neurosphere models. Xenograft experiments or patient-derived orthotopic models are critical to support translational relevance, and such basic research work needs to be included in journals.

(7) How does RAP2A regulate NUMB asymmetry? Is the Drosophila Rap2l-Cno/aPKC pathway conserved? Rescue experiments (e.g., Cno/aPKC knockdown with RAP2A overexpression) or interaction assays (e.g., Co-IP) are needed to establish molecular mechanisms.

(8) Reduced stemness markers (CD133/SOX2/NESTIN) and proliferation (Ki-67) align with increased ACD. However, alternative explanations (e.g., differentiation or apoptosis) must be ruled out via GFAP/Tuj1 staining or Annexin V assays.

(9) The link between low RAP2A and poor prognosis should be validated in multivariate analyses to exclude confounding factors (e.g., age, treatment history).

(10) The broader ACD regulatory network in GBM (e.g., roles of other homologs like NUMB) and potential synergies/independence from known suppressors (e.g., TRIM3) warrant exploration.

(11) The figures should be improved. Statistical significance markers (e.g., p-values) should be added to Figure 1A; timepoints/culture conditions should be clarified for Figure 6A.

(12) Redundant Drosophila background in the Discussion should be condensed; terminology should be unified (e.g., "neurosphere" vs. "cell cluster").

Reviewer #1 (Public review):

Summary:

Cording et al. investigated how deletion of CNTNAP2, a gene associated with autism spectrum disorder, alters corticostriatal engagement and behavior. Specifically, the authors present slice electrophysiology data showing that striatal projection neurons (SPNs) are more readily driven to fire action potentials in response to stimulation of corticostriatal afferents, and this is due to increases in SPN intrinsic excitability rather than changes in excitatory or inhibitory synaptic inputs. Specifically, these changes seem to be due to preferential reduction of Kv1.2 in dSPNs. The authors separately show that CNTNAP2 mice display repetitive behaviors, enhanced motor learning and cognitive inflexibility. Overall, the authors' conclusions are supported by their data, but a few claims could use some more evidence to be convincing.

Strengths:

The use of multiple behavioral techniques, both traditional and cutting-edge machine learning-based analyses, provides a powerful means of assessing repetitive behaviors and behavioral transitions/rigidity. Characterization of both excitatory and inhibitory synaptic responses in slice electrophysiology experiments offers a broad survey of the synaptic alterations that may lead to increased corticostriatal engagement of SPNs.

Weaknesses:

As it stands, the reported changes in dorsolateral striatum SPN excitability are only correlative with reported changes in repetitive behaviors, motor learning and cognitive flexibility. The authors do broach this in the text (particularly in "Limitations and future directions").

Reviewer #2 (Public review):

Summary:

This is an important study characterizing striatal dysfunction and behavioral deficits in Cntnap2-/- mice. There is growing evidence suggesting that striatal dysfunction underlies core symptoms of ASD but the specific cellular and circuit level abnormalities disrupted by different risk genes remain unclear. This study addresses how deletion of Cntnap2 affects the intrinsic properties and synaptic connectivity of striatal spiny projection neurons (SPN) of the direct (dSPN) and indirect (iSPN) pathways. Using Thy1-ChR2 mice and optogenetics the authors found increased firing of both types of SPNs in response to cortical afferent stimulation. However, there was no significant difference in the amplitude of optically-evoked excitatory postsynaptic currents (EPSCs) or spine density between Cntnap2-/- and WT SPNs, suggesting that the increased corticostriatal coupling might be due to changes in intrinsic excitability. Indeed, the authors found Cntnap2-/- SPNs, particularly dSPNs, exhibited higher intrinsic excitability, reduced rheobase current and increased membrane resistance compared to WT SPNs. The enhanced spiking probability in Cntnap2-/- SPNs is not due to reduced inhibition. Despite previous reports of decreased parvalbumin-expressing (PV) interneurons in various brain regions of Cntnap2-/- mice, the number and function (IPSC amplitude and intrinsic excitability) of these interneurons in the striatum were comparable to WT controls.

This study also includes a comprehensive behavioral analysis of striatal related behaviors. Cntnap2-/- mice demonstrated increased repetitive behaviors (RRBs), including more grooming bouts, increased marble burying, and increased nose poking in the holeboard assay. MoSeq analysis of behavior further showed signs of altered grooming behaviors and sequencing of behavioral syllables. Cntnap2-/- mice also displayed cognitive inflexibility in a four-choice odor-based reversal learning assay. While they performed similarly to WT controls during acquisition and recall phases, they required significantly more trials to learn a new odor-reward association during reversal, consistent with potential deficits in corticostriatal function.

Strengths:

This study provides significant contributions to the field. The finding of altered SPN excitability, the detailed characterization of striatal inhibition, and the comprehensive behavioral analysis are novel and valuable to understand the pathophysiology of Cntnap2-/- mice.

Weaknesses:

All my concerns were addressed in the revised version of the manuscript

Reviewer #3 (Public review):

Summary:

The authors analyzed Cntnap2 KO mice to determine whether loss of the ASD risk gene CNTNAP2 alters the dorsal striatum's function.

Strengths:

The results demonstrate that loss of Cntnap2 results in increased excitability of striatal projection neurons (SPNs) and altered striatal-dependent behaviors, such as repetitive, inflexible behaviors. Unlike other brain areas and cell types, synaptic inputs onto SPNs were normal in Cntnap2 KO mice. The experiments are well-designed, and the results support the authors' conclusions.

Weaknesses:

The mechanism underlying SPN hyperexcitability was not explored, and it is unclear whether this cellular phenotype alone can account for the behavioral alterations in Cntnap2 KO mice. No clear explanation emerges for the variable phenotype in different brain areas and cell types.

Comments on revisions:

The authors have appropriately addressed all my comments. In my opinion, no further changes are required.

Reviewer #1 (Public review):

I was glad to see that the other reviewer and I had similar takeaways on the subjects of historical literature and paedomorphism. While the authors have adequately considered a more historical body of literature, they have not addressed the concerns we had with statements about paedomorphism. I'm inclined to agree with the other reviewer that the discussion on paedomorphism should be cut entirely. My comments below are to seek clarity and make sure you are saying what you intend to say.

Strengths:

Table 1 is the beginning of a useful glossary and possible character definitions with character states that can be coded for phylogenetic analyses. This is particularly important because the goal of the paper is to define terms for chondrichthyan skeletal features in order to unify research questions in the field, and add novel data on how these features might be distributed among chondrichthyan clades, starting with ratfish and little skate.

Opportunities:

Table 1 should be translated into a format reflecting 0s and 1s etc that can be coded and referred back to the matrix that is in Figure 7 (or a standalone matrix as an appendix). Right now, they do not correspond and thus it is challenging to follow and interpret the mapped characters on the tree. You are presuming reversals when you could just list the state and let the data show you the possibility of character transitions.

Figure 1 essentially shows two datapoints Holocephali and Elasmobranchi where holocephali have low TMD and Elasmobranchi have high TMD, therefore nothing directional from an ancestral to derived state. Also, because you drop the catshark from later figures/analysis, you are treating the ratfish and the little skate as sister taxa so you cannot determine which is paedomorphic and which is peramorphic. Unfortunately, the position of where the characters were mapped on Figure 7 is not able to help you determine ancestral states and therefore actually test for paedomorphism. Two sister taxa with two different conditions and no outgroup doesn't explain the TMD in Ratfishes is statistically different from that of little skates. But there is no direction. So you need to be able to reconstruct that state.

Paedomorphosis implies juvenile ancestral organization in actual existing adult stages of modern descendants. You haven't shown that yet. Only that there might be different rates of mineralization in little skates. I suggested that you datamine the literature for other stages if you think you can fill in gaps.

In the response to reviewers, the authors stated that: "... we had reported that the TMD of centra from little skate did significantly increase between stage 32 and 33. Supporting our argument that ratfish had features of little skate embryos, TMD of adult ratfish centra was significantly lower than TMD of adult skate centra (Fig 1). Also, it was significantly higher than stage 33 skate centra, but it was statistically indistinguishable from that of stage 33 and juvenile stages of skate centra. While we do agree that more samples from these and additional groups would bolster these data, we feel they are sufficiently powered to support our conclusions for this current paper."

I will respond to that. In Figure 6L, yes, A little skate stage 33 is significantly different than stage 32, though the SD bars for ratfish appear to overlap with the range for little skate 32. Also, ratfish values are not significantly different than state 33 or juvenile ratfish. You can add the adult little skate data from figures 1 to 6L and then state, "centra of adult ratfish have a TDM within the range of juvenile LS33 little skates." As per conversation earlier, it still doesn't account for paedomorphism, however, it does indicate different amounts of mineralization, and could indicate you hypothesize about rates of mineralization. I think you have a different discussion waiting to replace this one.

Reviewer #2 (Public review):

General comment:

This is a very valuable and unique comparative study. An excellent combination of scanning and histological data from three different species is presented. Obtaining the material for such a comparative study is never trivial. The study presents new data and thus provides the basis for an in-depth discussion about chondrichthyan mineralised skeletal tissues.

Comments on revised version:

The manuscript has been revised and improved and can be published. A very nice manuscript, indeed. My only recommendation (point of discussion, not a requirement) would still be to think about the claim of paedomorphosis in a holocephalan.

Within the chondrichthyes, how distant holocephali are in relation to elasmobranchii remains uncertain, holocephali are quite a specialised group. Holocephali are also older than Batoidea and Selachii. As paedomorphosis is a derived character, I imagine it is difficult to establish that development in an extant holocephalan is derived compared to development in elasmobranchii. If this type of development would have been typical for the "older" holocephali it would not be paedomorphic. Also, the uncertainty how distant holocephali are from elasmobranchii makes it difficult to identify paedomorphosis with reference to chondrichthyes.

Joint Public Review:

Carabalona and colleagues investigated the role of the membrane-deforming cytoskeletal regulator protein Abba (MTSS1L/MTSS2) in cortical development to better understand the mechanisms of abnormal neural stem cell mitosis. The authors used short hairpin RNA targeting Abba20 with a fluorescent reporter coupled with in utero electroporation of E14 mice to show changes to neural progenitors. They performed flow cytometry for in-depth cell cycle analysis of Abba-shRNA impact to neural progenitors and determined an accumulation in S phase. Using culture rat glioma cells and live imaging from cortical organotypic slides from mice in utero electroporated with Abba-shRNA, the authors found Abba played a prominent role in cytokinesis. They then used a yeast-two-hybrid screen to identify three high confidence interactors: Beta-Trcp2, Nedd9, and Otx2. They used immunoprecipitation experiments from E18 cortical tissue coupled with C6 cells to show Abba requirement for Nedd9 localization to the cleavage furrow/cytokinetic bridge. The authors performed an shRNA knockdown of Nedd9 by in utero electroporation of E14 mice and observed similar results as with the Abba-shRNA. They tested a human variant of Abba using in utero electroporation of cDNA and found disorganized radial glial fibers and misplaced, multipolar neurons, but lacked the impact of cell division seen in the shRNA-Abba model.

[Editors' note: the authors have responded to two sets of reviews, which can be found here, https://doi.org/10.7554/eLife.92748.2, and here, https://doi.org/10.7554/eLife.92748.1]

Reviewer #1 (Public review):

Summary:

Qi and colleagues investigated the role of Kallistatin pathway in increasing hippocampal amyloid-β plaques accumulation and tau hyperpholphorylation in Alzheimer's disease, linking the increased Kallistatin level in diabetic patients with a higher risk of Alzheimer's disease development. A Kallistatin overexpressing animal model was utilized, and memory impairment was assessed using Morris water maze and Y-maze. Kallistatin-related pathway protein levels were measured in the hippocampus, and phenotypes were rescued using fenofibrate and rosiglitazone. The current study provides evidence of a novel molecular mechanism linking diabetes and Alzheimer's disease, and suggests the potential use of fenofibrate to alleviate memory impairment. However, several issues need to be addressed before further consideration.

Strengths:

The finding of this study is novel. The finding will have great impacts on diabetes and AD research. The studies were well conducted, and results convincing.

Weaknesses:

(1) The mechanism by which fenofibrate rescues memory loss in Kallistatin-transgenic mice is unclear. As a PPARα agonist, does fenofibrate target the Kallistatin pathway directly or indirectly? Please provide discussion based on literature supporting either possibility.<br /> (2) The current study exclusively investigated hippocampus. What about other cognitive memory-related regions, such as prefrontal cortex? Including data from these regions or discussing the possibility of their involvement could provide a more comprehensive understanding of the role of Kallistatin in memory impairment.<br /> (3) Fenofibrate rescued phenotypes in Kallistatin-transgenic mice while rosiglitazone, a PPARα agonist, did not. This result contradicts the manuscript's emphasis on a PPARα-associated mechanism. Please address this inconsistency.<br /> (4) Most of the immunohistochemistry images are unclear. Inserts have similar magnification to the original representative images, making judgments difficult. Please provide larger inserts with higher resolution.<br /> (5) The immunohistochemistry images in different figures were taken from different hippocampal subregions with different magnifications. Please maintain consistency, or explain why CA1, CA3 or DG was analyzed in each experiment.<br /> (6) Figure 5B is missing a title. Please add a title to maintain consistency with other graphs.<br /> (7) Please list statistical methods used in the figure legends, such as t-test or One way ANOVA with post-hoc tests.

Comments on revisions:

The authors have addressed the issues raised from the review. The manuscript has been revised accordingly.

Reviewer #2 (Public review):

Summary:

The study links Alzheimer's disease (AD) with metabolic disorders through elevated Kallistatin levels in AD patients. Kallistatin-overexpressing mice show cognitive decline, increased Aβ and tau pathology, and impaired hippocampal function. Mechanistically, Kallistatin enhances Aβ production via Notch1 and promotes tau phosphorylation through GSK-3β activation. Fenofibrate improves cognitive deficits by reducing Aβ and tau phosphorylation in these mice, suggesting therapeutic potential in AD linked to metabolic syndromes.

Strengths:

This study presents a novel insights into Alzheimer's disease (AD) pathogenesis and provides strong evidences about the mechanistic roles of Kallistatin and the therapeutic potential of fenofibrate in AD.

It was suggested that Kallistatin is primarily produced by the liver. The study demonstrates increased Kallistatin levels in the hippocampus tissue of AD mice. They also found that Kallistatin is also increased in the liver of AD mice.

They also showed that Kallistatin directly binds to Notch1 and contributes to the activation of the Noch1-HES1 signaling pathway

Reviewer #3 (Public review):

Summary:

The authors investigated the role of kallistatin in metabolic abnormalities associated with AD. They found that Kallistatin promotes Aβ production by binding to the Notch1 receptor and upregulating BACE1 expression. They identified that Kallistatin is a key player that mediates Aβ accumulation and tau hyperphosphorylation in AD.

Strengths:

This manuscript not only provides novel insights into the pathogenesis of AD, but also indicates that the hypolipidemic drug fenofibrate attenuates AD-like pathology in Kallistatin transgenic mice.

Weaknesses:

The authors did not illustrate whether the protective effect of fenofibrate against AD depends on kallistatin.

The conclusions are supported by the results.

Reviewer #1 (Public review):

The authors investigated the role of the C. elegans Flower protein, FLWR-1, in synaptic transmission, vesicle recycling, and neuronal excitability. They confirmed that FLWR-1 localizes to synaptic vesicles and the plasma membrane and facilitates synaptic vesicle recycling at neuromuscular junctions. They observed that hyperstimulation results in endosome accumulation in flwr-1 mutant synapses, suggesting that FLWR-1 facilitates the breakdown of endocytic endosomes. Using tissue-specific rescue experiments, the authors showed that expressing FLWR-1 in GABAergic neurons restored the aldicarb-resistant phenotype of flwr-1 mutants to wild-type levels. By contrast, cholinergic neuron expression did not rescue aldicarb sensitivity at all. They also showed that FLWR-1 removal leads to increased Ca2+ signaling in motor neurons upon photo-stimulation. From these findings, the authors conclude that FLWR-1 helps maintain the balance between excitation and inhibition (E/I) by preferentially regulating GABAergic neuronal excitability in a cell-autonomous manner.

Overall, the work presents solid data and interesting findings, however the proposed cell-autonomous model of GABAergic FLWR-1 function may be overly simplified in my opinion.

Most of my previous comments have been addressed; however, two issues remain.

(1) I appreciate the authors' efforts conducting additional aldicarb sensitivity assays that combine muscle-specific rescue with either cholinergic or GABergic neuron-specific expression of FLWR-1. In the revised manuscript, they conclude, "This did not show any additive effects to the pure neuronal rescues, thus FLWR-1 effects on muscle cell responses to cholinergic agonists must be cell-autonomous." However, I find this interpretation confusing for the reasons outlined below.

Figure 1 - Figure Supplement 3B shows that muscle-specific FLWR-1 expression in flwr-1 mutants significantly restores aldicarb sensitivity. However, when FLWR-1 is co-expressed in both cholinergic neurons and muscle, the worms behave like flwr-1 mutants and no rescue is observed. Similarly, cholinergic FLWR-1 alone fails to restore aldicarb sensitivity (shown in the previous manuscript). These observations indicate a non-cell-autonomous interaction between cholinergic neurons and muscle, rather than a strictly muscle cell-autonomous mechanism. In other words, FLWR-1 expressed in cholinergic neurons appears to negate or block the rescue effect of muscle-expressed FLWR-1. Therefore, FLWR-1 could play a more complex role in coordinating physiology across different tissues. This complexity may affect interpretations of Ca2+ dynamics and/or functional data, particularly in relation to E/I balance, and thus warrants careful discussion or further investigation.

[Editor's note: The authors edited the text of the manuscript to acknowledge potential complexities in the interpretations of these results.]

(2) The revised manuscript includes new GCaMP analyses restricted to synaptic puncta. The authors mention that "we compared Ca2+ signals in synaptic puncta versus axon shafts, and did not find any differences," concluding that "FLWR-1's impact is local, in synaptic boutons." This is puzzling: the similarity of Ca2+ signals in synaptic regions and axon shafts seems to indicate a more global effect on Ca2+ dynamics or may simply reflect limited temporal resolution in distinguishing local from global signals due to rapid Ca2+ diffusion. The authors should clarify how they reached the conclusion that FLWR-1 has a localized impact at synaptic boutons, given that synaptic and axonal signals appear similar. Based on the presented data, the evidence supporting a local effect of FLWR-1 on Ca2+ dynamics appears limited.

[Editor's note: The authors acknowledged that some wording in the previous version was misleading and inaccurate. In the revised version, the authors have withdrawn the conclusion that FLWR-1 function is local in synaptic boutons.]

Reviewer #2 (Public review):

Summary:

The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.

Strengths:

A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.

The authors have adequately addressed most of my previous concerns, however, I recommend minor revisions to further strengthen the study's rigor and interpretation:

Major suggestions

(1) This study relies heavily on aldicarb assays to support its conclusions. While these assays are valuable, their results may not fully align with direct assessment of neurotransmitter release from motor neurons. For instance, prior work has shown that two presynaptic modulators identified through aldicarb sensitivity assays exhibited no corresponding electrophysiological defects at the neuromuscular junction (Liu et al., J Neurosci 27: 10404-10413, 2007). Similarly, at least one study from the Kaplan lab has noted discrepancies between aldicarb assays and electrophysiological analyses. The authors should consider adding a few sentences in the Discussion to acknowledge this limitation and the potential caveats of using aldicarb assays, especially since some of the aldicarb assay results in this study are not easily interpretable.

[Editor's note: The authors added a sentence in the first paragraph of the Discussion to acknowledge these complexities.]

(2) The manuscript states, "Elevated Ca2+ levels were not further enhanced in a flwr-1;mca-3 double mutant." (lines 549-550). However, Figure 7C does not include statistical comparisons between the single and double mutants of flwr-1 and mca-3. Please add the necessary statistical analysis to support this statement.

[Editor's note: In response, the authors noted that these comparisons were indeed carried out. As mentioned in the figure legend, the graph shows only those comparisons that indicated statistical significance.]

(3) The term "Ca2+ influx" should be avoided, as this study does not provide direct evidence (e.g. voltage-clamp recordings of Ca2+ inward currents in motor neurons) for an effect of the flwr-1 mutation of Ca2+ influx. The observed increase in neuronal GCaMP signals in response to optogenetic activation of ChR2 may result from, or be influenced by, Ca2+ mobilization from of intracellular stores. For example, optogenetic stimulation could trigger ryanodine receptor-mediated Ca2+ release from the ER via calcium-induced calcium release (CICR) or depolarization-induced calcium release (DICR). It would be more appropriate to describe the observed increase in Ca2+ signal as "Ca2+ elevation" rather than increased "Ca2+ influx".

[Editor's note: The authors revised their terminology to avoid ambiguities associated with the word "influx".]

Reviewer #1 (Public review):

Summary:

In this study, Xiao et al. conducted a comprehensive analysis of retroperitoneal liposarcoma (RPLS) by classifying patients into two distinct molecular subgroups based on whole transcriptome sequencing data from 88 cases. The G1 subgroup demonstrated a metabolic activation signature, whereas the G2 subgroup was characterized by enhanced cell cycle regulation and DNA damage repair pathways. Notably, the G2 subgroup exhibited more aggressive molecular profiles and poorer clinical prognosis compared to the G1 subgroup. Through the application of machine learning algorithms, the authors established a streamlined classification system, identifying LEP and PTTG1 as pivotal molecular biomarkers for differentiating between these two RPLS subgroups. The manuscript presents a well-structured and methodologically sound study, with particular significance attributed to its substantial sample size and the development of a clinically applicable classification framework. This innovative model holds considerable promise for advancing personalized treatment strategies and improving clinical outcomes for RPLS patients.

Comments on revisions:

The authors have adequately addressed all my concerns, and I have no further comments.

Reviewer #1 (Public review):

Summary:

In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The migration of these cells which are found in two nearby groups of cells normally happens unidirectionally along the dorsal trunk towards the posterior. Here, the authors study how this directionality is regulated. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells plays a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest that trafficking and vesicular transport of Upd2 from the fat body towards the tracheal cells might be important.

Strengths:

The manuscript is well written. This novel work demonstrates a likely link between Upd2-JAK/STAT signalling in the fat body and tracheal stem cells and the control of unidirectional cell migration of tracheal stem cells. The authors show that hid+rpr or Upd2RNAi expression in a fat body or Dome RNAi, Hop RNAi, or STAT92E RNAi expression in tracheal stem cells results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. Moreover, the authors hypothesise and provide some supporting evidence that extracellular vesicle transport of Upd2 might be involved in this Upd2-JAK/STAT signalling in the fat body and tracheal stem cells, which is quite interesting. Overall, the work presented here provides some novel insights into the mechanism that ensures unidirectional migration of tracheal stem cells that prevents bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration.

Weaknesses:

It remains somewhat unclear how Upd2 transported in extracellular vesicles would bind to the Dome receptor found on the surface of the tracheal cells? How Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway?

Reviewer #1 (Public review):

Summary:

In their manuscript, Li and colleagues introduce a pioneering investigation into the molecular and epigenetic foundations of neuroendocrine transdifferentiation in prostate cancer. By employing a genetically engineered cellular reprogramming approach, they elucidate the pivotal roles of ASCL1 and NeuroD1 as pioneer transcription factors that suppress AR signaling and orchestrate lineage plasticity toward NEPC. Their integrative multi-omics methodology delineates dynamic transcriptional and chromatin reorganization processes, offering profound insights into mechanisms of therapeutic resistance.

Strengths:

(1) The development of a reproducible in vitro reprogramming platform to transition ARPC cells into NEPC represents a significant technical achievement. This model enables high-resolution temporal analysis of NEtD, addressing constraints inherent in traditional PDX systems.

(2) The authors reveal that ASCL1 and NeuroD1 suppress AR signaling through chromatin structural modifications at somatically amplified AR enhancers, a significant discovery that clarifies the longstanding ambiguity surrounding AR pathway inactivation during lineage plasticity.

(3) The integration of RNA sequencing, CUT&RUN, and single-cell multiomic profiling delivers a holistic perspective on dynamic epigenetic and transcriptional reprogramming during NEtD. Their observation that AR suppression precedes NE marker activation provides chronological insights into this process.

(4) By delineating the distinct roles of ASCL1/NeuroD1-driven NE lineage programs versus REST inactivation, the study critiques the excessive dependence on limited immunohistochemical indicators for NEPC classification, directly informing improvements in molecular diagnostics.

(5) The association of ASCL1/NeuroD1 with MHC class I suppression mediated by PRC2 unveils opportunities for combining agents targeting epigenetic modifiers with immune-based therapies to counteract immune evasion in NEPC.

Weaknesses:

While the study is methodologically robust, a modest limitation lies in its primary reliance on established cell lines for mechanistic exploration. Although key observations are corroborated with clinical samples, additional validation in PDX models or organoid systems could enhance translational applicability. Furthermore, while the role of ASCL1/NeuroD1 in AR enhancer silencing is convincingly demonstrated, the upstream regulatory mechanisms governing ASCL1/NeuroD1 induction under therapeutic stress remain unaddressed, a compelling avenue for future research.

Reviewer #2 (Public review):

Summary:

This manuscript reported that the pioneer factors ASCL1 and NeuroD1 in neuroendocrine transdifferentiation of Neuroendocrine prostate cancer (NEPC) and uncovered their abilities to silence AR expression by remodeling chromatin at the somatically acquired AR enhancer and global AR binding sites with enhancer activity. It also elucidated the dynamic temporal changes in the transcriptomic and epigenomic landscapes of cells undergoing acute lineage conversion from AR-active prostate cancer to NEPC which should inform future therapeutic development.

Strengths:

Data from cell lines is great and solid.

Weaknesses:

The paper would be better if some clinical data could be added.

Reviewer #3 (Public review):

Summary:

This study investigates the molecular mechanisms underlying the transdifferentiation of androgen receptor-active prostate cancer (ARPC) to neuroendocrine prostate cancer (NEPC) in prostate cancer (PC). Using a cellular reprogramming strategy, the research team successfully converted ARPC cell lines into NEPC cell lines and explored key molecular mechanisms driving this transformation. The work demonstrates the pivotal role of neurogenic pioneer transcription factors ASCL1 and NeuroD1 in NEPC transdifferentiation, which silence AR expression and signaling by remodeling chromatin architecture while inducing NEPC-associated gene programs. Additionally, the study reveals dynamic transcriptomic and epigenomic changes during NEPC transformation, as well as downregulation of the MHC class I antigen processing and presentation pathway in NEPC cell lines.

Strengths:

(1) The study introduces a novel genetically defined cellular reprogramming strategy to directly convert ARPC to NEPC. This approach circumvents previous limitations by starting from AR-active cells, thereby addressing a critical gap in the field.<br /> (2) The study provides a comprehensive characterization of the dynamic changes in the transcriptomic and epigenomic landscapes during the NEPC transdifferentiation process.

Weaknesses:

(1) What was the rationale for selecting these specific candidate factors (e.g., ASCL1, NeuroD1) to drive neuroendocrine transdifferentiation (NEtD)? Was a comprehensive screening process conducted to identify additional potential drivers of this phenotypic shift?

(‌2) The AR bypass assay employed an AR response element-driven FKBP-Casp8 fusion protein for negative selection. How was the specificity and efficiency of this system validated? Are there additional validation experiments (e.g., orthogonal AR activity assays) to confirm the complete bypass of AR signaling?

(3‌) While extensive omics data (RNA-seq, ATAC-seq, CUT&RUN) are presented, have these datasets been deposited in public repositories (e.g., GEO, SRA) to enable validation and reuse by the scientific community?

(‌4) What criteria guided the selection of time points for analyzing dynamic changes during NEtD? Would denser time-point sampling (e.g., intermediate time courses) enhance resolution of critical transitional events?

(5‌) Were multiple hypothesis testing corrections (e.g., Benjamini-Hochberg) applied during differential expression and pathway enrichment analyses? How was the statistical significance of chromatin accessibility changes and super-enhancer reconfiguration rigorously validated?

Reviewer #1 (Public review):

Summary:

This is an interesting manuscript by Kirk and colleagues describing a highly valuable knock-down system that leverages CRISPRi in order to further elucidate the role of the Kruppel-Like Factor (KLF) transcription factor family in regulating the maturation of postnatal cortical projection neurons. The authors firstly use RNA-Seq and ATAC-Seq data in order to identify the KLF TF family as a potential regulator of cortical neuron maturation in the postnatal brain and subsequently knock down four KLF family members; KLF9, KL13, KLF6 and KLF7, in order to ascertain the functions of specific KLF genes in the developing cortex. The described CRISPRi knock down strategy is highly robust and penetrant as evidenced by a KD efficiency > 95% (assessed by both qPCR and single molecule FISH) and demonstrates that KLF6 and KLF7 play an activating role in driving the expression of target genes relating to axonal growth whereas KLF9 and 13 play a repressive role that inhibits the expression of overlapping gene targets. Together, the authors propose a model where the KLF TF family acts as a regulatory "switch" from activation to repression in the postnatal cortex as a mechanism to control a shift in projection neuron function from axonal growth to circuit refinement. The findings and conclusions of the manuscript offer a valuable contribution to the field of postnatal cortical development and further our understanding of the regulatory mechanisms that govern neuron maturation.

The conclusions of this manuscript are generally supported by the data, but some aspects of the data collection and analysis require some further clarification. Specifically:

(1) The authors comprehensively assess the molecular effects of KLF TF knock-down, however, the authors do not deeply address the cellular effects of these knock-downs. The authors conclude that knockdown of KLF6/7 and KLF9/13 cause downregulation and upregulation, respectively, of a common set of genes involved in cytoskeletal or axon regulation such as Tubb2 and Dpysl3. How is the morphology of the cells affected by these knockdowns? For example, does KLF9/13 knockdown cause neurite/axonal outgrowth? The authors should perform some basic experiments to assess changes in cell morphology following KLF TF KD. This is the one key point that needs addressing, in my opinion.

(2) The authors identify 374 DEGs in P10 Klf6/7 KD neurons and 115 DEGs at P20 (figure 6B). Have the authors looked to see what proportion of these DEGs are upregulated in the KLF9/13 KDs in order to get a more global understanding of the degree of overlap in the genes regulated by the KLF family members? Along similar lines, the authors later indicate that there are 144 shared targets between the KLF activator and repressor pairs (Figure 7C). What percentage does this represent of the total number of DEGs between the KLF pairs. This could further illustrate the degree to which the KLF pairs regulate the same set of genes. If it is already indicated in the manuscript, it should be made a bit more clear to the reader.

(3) Figures 5B and 6D2 are very interesting as they relate the changes in gene expression over time in neurons from P2 to P30 to the functions of KLF9/13 and KLF6/7, respectively. I would be curious to see how these two forms of analyses overlap with one another. For example, in Figure 6D2, where would the KLF9/13 upregulated genes fall on the plot shown in Figure 6D2? And would those overlapping genes fit a similar correlation?

(4) Figure 7E shows expression levels of shared KLF TF targets in control or KD conditions. Interestingly, the expression of Tubb2b, shows higher expression in ScrGFP P10 when compared to KLF9/13 P20, suggesting that derepression of KLF9/13 does not fully restore the expression level of Tubb2b seen at P10. This may suggest that other repressive regulators may be involved in the downregulation of Tubb2b from P10 to P20. Can the authors further comment on this, perhaps in the discussion, and speculate if there are other regulatory factors at play that may be controlling some of the shared targets by KLF6/7 and KLF9/13?

Reviewer #2 (Public review):

Summary:

Kirk et al. use RNA-Seq and CRISPRi to provide evidence that KLF family transcription factors regulate postnatal neuronal maturation of pyramidal neurons. The genetic programs regulating postnatal neuronal maturation are not well understood. The authors first analyzed chromatin accessibility and gene expression data from layer 4 and 6 pyramidal neurons and found that KLF TFs are predicted regulators of postnatal neuronal maturation. They then use CRISPRi knockdown and find that KLF activators first activate genes and then this is followed by KLF repressors repressing genes. Interestingly, some genes, such as those with cytoskeletal functions, are shared targets of KLF activators and repressors.

Strengths:

The study is well-executed and the paper is well-written. A major strength of this study is the application of state-of-the-art transgenic approaches. The CRISPRi approach used to knock down multiple KLFs is compelling. The genomic data generated appears to be high quality and is carefully analyzed. The presented findings provide important insights into the genetic programs that regulate postnatal maturation in cortical pyramidal neurons. The discovery that KLF family activators/repressors regulate gene expression changes during this critical step of neuronal development fills an important gap in the field.

Weaknesses:

A limitation of the current study is that the functional importance of KLF for postnatal neuronal maturation is unclear. Although the authors find that KLFs regulate some of the gene expression changes during postnatal neuronal maturation, it is still unclear whether such gene expression changes mediate the postnatal changes in morphology and physiology. While beyond the scope of the current study, future studies should investigate the contributions of KLFs on postnatal morphological and physiological changes.

Reviewer #3 (Public review):

Summary:

In their manuscript "Multiplexed CRISPRi Reveals a Transcriptional Switch Between KLF Activators and Repressors in the Maturing Neocortex", Kirk and colleagues seek to dissect the developmentally regulated pan-neuronal gene programs that control the postnatal maturation of cortical neurons. For this, the authors analyzed newly generated and existing RNA-seq and ATAC-seq of Layer 4 and Layer 6 cortical pyramidal neurons at postnatal day 2 (P2) and day 30 (P30), and identified thousands of shared developmentally regulated genes and genomic (promoter) regions, including genes involved in axon growth (tend to be downregulated) and synaptic function (tend to be upregulated). Motif enrichment analysis of promoters of differentially regulated genes revealed a strong presence of KLF/Sp family binding motifs, pointing to Krüppel-Like Factors (KLFs) as key transcriptional regulators of cortical maturation. Expression profiling showed a developmental switch from activating KLFs (Klf6, Klf7) expressed neonatally to repressive KLFs (Klf9, Klf13) upregulated during maturation. Using an elegant in vivo multiplexed CRISPR interference (CRISPRi) system, the authors achieved efficient, cell-type-specific knockdown of these TFs and showed that Klf9 and Klf13 repress a set of genes that includes cytoskeletal regulators such as Tubb2b, Dpysl3, and Rac3. Conversely, Klf6 and Klf7 promoted the expression of these same genes in the early postnatal period, and their knockdown led to reduced expression of these genes, particularly at P10 when their activating influence is strongest. Since promoters of shared KLF targets were enriched for KLF/Sp motifs but showed little change in chromatin accessibility, the authors propose a model in which distinct KLF family members function either as transcriptional repressors and activators that compete at constitutively accessible promoters and thereby act as a developmental transcriptional switch that coordinates the downregulation of axon growth programs and upregulation of synaptic maturation genes during cortical development.

Strengths:

The study addresses an interesting question and advances our understanding of the transcriptional regulation underlying postnatal cortical development. A major strength of the study lies in the innovative use of in vivo multiplexed CRISPR interference (CRISPRi), which allows for cell-type-specific, combinatorial knockdown of redundant TFs - this an elegant solution to a long-standing challenge in transcription factor research, and should be useful also for other neuroscience studies that require local and cell-type-specific gene loss-of-function. Also, the integration of RNA-seq and ATAC-seq across developmental time points provides a robust foundation for identifying direct targets of the KLF family, and the findings are reinforced by cross-species conservation and the identification of targets with clear neurodevelopmental relevance.

Weaknesses:

The major weakness of the study lies in its relatively narrow scope: the study focuses primarily on transcriptional mechanisms and largely lacks functional validation of the neuronal phenotypes that are predicted by the gene expression data (e.g. axonal morphology). For example, the authors analyzed the effects of KLF9/13 KD on the neurons' excitability and excitatory inputs, but did not assess the effects on inhibitory inputs and E/I-ratio or morphological parameters such as axonal length and axonal target fields - the manuscript would be strengthened considerably by such analyses (axonal projections could be analyzed e.g. via local injections of the gRNA AAVs and subsequent immunolabeling of brain sections). Similarly, the chromatin-based mechanisms underlying KLF activity remain relatively speculative, and the transcriptional mechanisms upstream of the KLFs remain unexplored (this could be addressed by analyzing existing datasets; see "Additional Point 1" below). Finally, the manuscript is too long (e.g., nearly five pages in the Discussion section are devoted to discussing various misregulated genes) and would benefit from presenting the Results and Discussion sections more concisely. However, despite these limitations, the paper offers an interesting model for a transcriptional switch during neuronal maturation in the cortex and establishes a powerful methodological framework for dissecting redundant gene networks in vivo.