Reviewer #3 (Public Review):

The manuscript from Barekatain et al. is investigating heterogeneity within the population of insulin vesicles from an insulinoma cell line (INS-1E) in response to glucose stimulation. Prevailing dogma in the beta-cell field suggests that there are distinct pools of mature insulin granules, such as ready-releasable and a reserve pool, which contribute to distinct phases of insulin release in response to glucose stimulation. Whether these pools (and others) are distinct in protein/lipid composition or other aspects is not known, but has been suggested. In this manuscript, the authors use density gradient sedimentation to enrich for insulin vesicles, noting the existence of a number of co-purifying contaminants (ER and mitochondrial markers). Following immunolabeling with synaptotagmin V and fluorescent-conjugated secondary antibodies, insulin vesicles were applied to a microfluidic device and separated by dielectrophoretic and electrokinetic forces following an applied voltage. The equilibrium between these opposing forces was used to physically separate insulin granules. Here some differences were observed in the insulin (Syt V positive) granule populations, when isolated from cells that were either non-stimulated or stimulated with glucose, which has been suggested previously by other studies as noted by the authors; however in the current manuscript, the inclusion of a number of control experiments may provide a better context for what the data reveal about these changes.

The major strength of the paper is in the use of the novel, highly sophisticated methodology to examine physical attributes of insulin granules and thus begin to provide some insight into the existence of distinct insulin granule populations within a beta-cell -these include insulin granules that are maturing, membrane-docked (i.e. readily releasable), in reserve, newly-synthesized, aged, etc. Whether physical differences exist between these various granule pools is not known. In this capacity, the technical abilities of the current manuscript may begin to offer some insight into whether these perceived distinctions are physical.

The major weakness of the manuscript is that the study falls short in terms of linking the biology to the sophisticated changes observed and primarily focuses on differences in response to glucose. Without knowing what the various populations of granules are, it is challenging to understand what the changes in response to glucose mean.

Specific concerns are as follows:

(1) There is confusion on what the DC-iDEP separation between stimulated and stimulated cells reveals. Do these changes reflect maturation state of granules, nascent vs. old granules? Ready-releasable vs. reserve pool? The comments in the text seem to offer all possibilities.

(2) It is unclear what we can infer regarding the physical changes of granules between the stimulated states of the cells. Without an understanding of the magnitude of the effect, it is unclear how biologically significant these changes are. For example, what degree of lipid or protein remodeling would be necessary to give a similar change?

(3) The reliance on a single vesicle marker, Syt V, is concerning given that granule remodeling is the focus.

(4) Additional confirmation that the isolated vesicles are in fact insulin granules would be helpful. As noted, granules were gradient enriched, but did carry contaminants. Note that the microscopy image provided does not provide any real validation for this marker.

Further confirmation that the immune-isolated vesicles are in fact insulin granules should be included. EM with immunogold labeling post-SytV enrichment would be a potential methodology to confirm.

(5) It would be useful to understand if the observed effects are specific to the INS-1E cell line or are a more universal effect of glucose on beta-cells.

Reviewer #1 (Public Review):

Summary:

The manuscript describes a model based on 5-state cellular automata of development of an infection. The model is motivated and qualitatively justified by time-resolved measurements of expression levels of viral, interferon-producing, and antiviral genes. The model is set up in such a way that the crucial difference in outcomes (infection spreading vs. confinement) depends on the initial fraction of special virus-sensing cells. Those cells (denoted as 'type a') cannot be infected and do not support the propagation of infection, but rather inhibit it in a somewhat autocatalytic way. Presumably, such feedback makes the transition between two outcomes very sharp: a minor variation in concentration of 'a' cells results in qualitative change from one outcome to another. As in any percolation-like system, the transition between propagation and inhibition of infection goes through a critical state with all its attributes, including a power-law distribution of the cluster size (corresponding to the fraction of infected cells) with a fairly universal exponent and a cutoff at the upper limit of this distribution.

Strengths:

The proposed model suggests a well-justified explanation for the frequently observed yet puzzling diversity of outcomes of viral infections such as COVID.

Weaknesses:

None.

Reviewer #2 (Public Review):

Xu et al. introduce a cellular automaton model to investigate the spatiotemporal spreading of viral infection. In this study, the author first analyzes the single-cell RNA sequencing data from experiments and identifies four clusters of cells at 48 hours post-viral infection, including susceptible cells (O), infected cells (V), IFN-secreting cells (N), and antiviral cells (A). Next, a cellular automaton model (NOVAa model) is introduced by assuming the existence of a transient pre-antiviral state (a). The model consists of an LxL lattice; each site represents one cell. The cells change their state following the rules depending on the interaction of neighboring cells. The model introduces a key parameter, p_a, representing the fraction of pre-antiviral state cells. Cell apoptosis is omitted in the model. Model simulations show a threshold-like behavior of the final attack rate of the virus when p_a changes continuously. There is a critical value p_c, so that when p_a < p_c, infections typically spread to the entire system, while at a higher p_a > p_c, the propagation of the infected state is inhibited. Moreover, the radius R that quantifies the diffusion range of N cells may affect the critical value p_c; a larger R yields a smaller value of the critical value p_c. The authors further examine the result with stochastic version dynamics, and the main findings are unchanged upon stochastic dynamics. The structure of clusters is different for different values of R; greater R leads to a different microscopic structure with fewer A and N cells in the final state. Compared with the single-cell RNA seq data, which implies a low fraction of IFN-positive cells of around 1.7%, the model simulation suggests R=5. The authors also explored a simplified version of the model, the OVA model, with only three states. The OVA model also has an outbreak size. The OVA model shows dynamics similar to the NOVAa model. However, the change in microstructure as a function of the IFN range R observed in the NOVAa model is not observed in the OVA model.

Reviewer #3 (Public Review):

Summary:

This study considers how to model distinct host cell states that correspond to different stages of a viral infection: from naïve and susceptible cells to infected cells and a minority of important interferon-secreting cells that are the first line of defense against viral spread. The study first considers the distinct host cell states by analyzing previously published single-cell RNAseq data. Then an agent-based model on a square lattice is used to probe the dependence of the system on various parameters. Finally, a simplified version of the model is explored, and shown to have some similarity with the more complex model, yet lacks the dependence on the interferon range. By exploring these models one gains an intuitive understanding of the system, and the model may be used to generate hypotheses that could be tested experimentally, telling us "when to be surprised" if the biological system deviates from the model predictions.

Strengths:

- Clear presentation of the experimental findings and a clear logical progression from these experimental findings to the modeling.<br /> - The modeling results are easy to understand, revealing interesting behavior and percolation-like features.<br /> - The scaling results presented span several decades and are therefore compelling.<br /> - The results presented suggest several interesting directions for theoretical follow-up work, as well as possible experiments to probe the system (e.g. by stimulating or blocking IFN secretion).

Weaknesses:

- The fixed time-step of the agent-based modeling may introduce biases. I would consider simulating the system with Gillespie dynamics where the reaction rates depend on the ambient system parameters.<br /> - Single-cell RNAseq data requires careful handling or it may generate false leads. The strength of the RNAseq evidence presented is not clear.

Two places where the manuscript could be extended:

- Since the "range" of IFN is an important parameter, it makes sense to consider other lattice geometries other than the square lattice, which is somewhat pathological. Perhaps a hexagonal lattice would generalize better.<br /> - Tissues are typically three-dimensional, not two-dimensional. (Epithelium is an exception). It would be interesting to see how the modeling translates to the three-dimensional case. Percolations transitions are known to be very sensitive to the dimensionality of the system.

Justification of claims and conclusions:

The claims and conclusions are well justified.

Reviewer #1 (Public Review):

This paper can be seen as an extension of a recent study by two of the same authors [1]. In the previous paper, the authors considered two variants of the Moran process, labelled Model A and Model B, and examined differences between the evolutionary dynamics of these two models. They further described the site frequency spectra, expected allele counts, and expected singleton counts of these models, building on analytical results from prior studies, and used numerical simulations to investigate the models' evolutionary dynamics. Finally, they compared the site frequency spectra of the two models (using numerical simulations) to spectra derived from a small breast cancer data set (two sets of three samples).

In the new paper, the authors consider the same two Moran process variants (Model A and Model B) and some related branching processes. As before, they compare the site frequency spectra and various summary statistics of these models, but here they present only numerical simulations (except that some prior analytical results are summarized in Appendix A, which are never referred to in the main text and seem unconnected to the study). They then compare the site frequency spectra of these models (again using numerical simulations) to those derived from the same breast cancer samples as before and thus infer some evolutionary parameters.

The first main conclusion is that the critical branching process and the Moran process models behave similarly and generate similar site frequency spectra. This finding is unsurprising (indeed, the authors acknowledge that the result "has been expected"). For a reasonably large population size, the population size in the critical branching process has been shown to vary relatively little over time and the model is thus essentially a continuous time Moran process (see, for example, Equation 8.55 in ref 2). Nor is it surprising that the authors see stronger similarities when they select only the subset of branching process replicates in which the final population size is particularly close to the initial population size (this is because, in these replicates, the population size likely varies even less than usual).

The second main conclusion is that, although "the mutational SFS alone is not adequate" to quantify the strength of selection, "All fitted values for the selective disadvantage of passenger mutations are nonzero, supporting the view that they exert deleterious selection during tumorigenesis". Although the question of whether mildly deleterious mutations play an important role in cancer evolution is of considerable interest, it's debatable whether the results presented here help resolve the issue.

Many prominent researchers have called into question whether cancer evolutionary parameters can be reliably inferred from site frequency spectra (e.g., [3-7]), even using sophisticated statistical methods. The statistical approach used here (though not named as such in the paper) is a crude kind of approximate Bayesian computation. To improve the accuracy of the results, it would have been better to have set reasonably vague priors for the uncertain mutation rates, rather than fixing them arbitrarily. It would also have been better to have chosen a likelihood function explicitly based on an analysis of the sampling and error distributions, rather than just summing the absolute logged deviations. It is well known that "Checking the model is crucial to statistical analysis" and "A good Bayesian analysis, therefore, should include at least some check of the adequacy of the fit of the model to the data and the plausibility of the model for the purposes for which the model will be used" [8]. The authors' failure to describe any attempt to validate or check their model, using simulated data or otherwise, casts doubt on the reliability of their inferences.

Putting aside the potential biassing effects of sampling error, measurement error, and the limitations of the authors' statistical method, it is well established that both population growth and spatial structure profoundly alter the shape of site frequency spectra in ways that can mimic the effects of selection (e.g. [9-11]). Indeed, Figures 3, 4 and 5 show that the critical and super-critical branching processes generate markedly different site frequency spectra. It follows that if the population dynamics and spatial structure of the mathematical model used for inference don't match those of the biological process that produced the data then any inferred evolutionary parameter values will be unreliable. Breast cancer has two indisputable ecological features that shape its evolutionary dynamics: the cell population expands by many orders of magnitude from a single cell, and the population is spatially structured. In the authors' mathematical model, the population size is initially 100 cells and either remains constant or varies little, and there is no spatial structure. These profound mismatches between model and data cast further doubt on what is supposed to be the paper's most important biological finding.

In this paper the authors offer no justification for their decision to model breast cancer as a non-growing, non-spatial cell population. Nor do they engage with the extensive recent literature on the challenges of inferring evolutionary parameters from cancer site frequency spectra (they cite none of the many relevant papers listed at https://www.sottorivalab.org/neutral-evolution.html). Their 2022 paper [1] claims that, "it sometimes makes sense to consider cancer growth in the framework of constant-population models. Our models correspond to the situation in which a constant population of N "healthy" stem cells is gradually replaced by a growing clone of transformed cells with increasing fitness." No evidence was presented to support this hypothesis regarding breast cancer progression. On the other hand, a wealth of evidence supports the consensus view that, in breast cancer and other human solid tumours, the number of cells with unlimited proliferative potential is several orders of magnitude greater than 100 and grows over time (e.g. [12]).

Analytic expressions for the site frequency spectra with neutral mutations are already known. It is well known that the site frequency spectrum of an exponentially growing population has a tail following a power law S_k ~ k^(-2) [13, 14]. Similarly, it is known that for the critical branching process or the Moran process, the site frequency spectrum at equilibrium is S_k ~ k^(-1) [13, 15]. Especially noteworthy yet uncited studies that use those results about site frequency spectra to make inferences based on sequencing data include ref 16, in which selection is inferred, and ref 17, in which evolutionary parameters of constant populations (healthy cell populations) are inferred.

Although the paper is well written, the figures are ineffective in communicating the results. As others have put it, "A figure is meant to express an idea or introduce some facts or a result that would be too long (or nearly impossible) to explain only with words" and "If your figure is able to convey a striking message at first glance, chances are increased that your article will draw more attention from the community" [18]. On the contrary, Figures 3, 4, 5 and 6 are bewilderingly complicated, crowded, and repetitive. These figures comprise no fewer than fifty-six plots, each containing numerous curves or histograms, spread across four pages. To compare the results of different scenarios, the reader is presumably expected to put these figures side by side and try to spot the differences, hampered by inconsistent axis ranges, absence of axis labels, absence of titles, absence of legends, and unreliable captions ("cyan" seems to refer to pale blue, and "orange" to something closer to red). For example, the only notable difference between Figures 3 and 4 is in the shape of a single green curve in panel I. In the main text of a published paper, one would expect fewer, more carefully curated figures drawing attention to salient features, so that the reader can infer the main results with minimal effort. The rest can be put in supplementary figures.

In summary, this paper adds somewhat to our understanding of some standard mathematical models; whether it tells us anything new about cancer is open to debate.

References<br /> (1) Kurpas, Monika K., and Marek Kimmel. "Modes of selection in tumors as reflected by two mathematical models and site frequency spectra." Frontiers in Ecology and Evolution 10 (2022): 889438.<br /> (2) Bailey, Norman TJ. The elements of stochastic processes with applications to the natural sciences. John Wiley & Sons, 1964.<br /> (3) Tarabichi, Maxime, et al. "Neutral tumor evolution?." Nature Genetics 50.12 (2018): 1630-1633.<br /> (4) McDonald, Thomas O., Shaon Chakrabarti, and Franziska Michor. "Currently available bulk sequencing data do not necessarily support a model of neutral tumor evolution." Nature Genetics 50.12 (2018): 1620-1623.<br /> (5) Balaparya, Abdul, and Subhajyoti De. "Revisiting signatures of neutral tumor evolution in the light of complexity of cancer genomic data." Nature Genetics 50.12 (2018): 1626-1628.<br /> (6) Noorbakhsh, Javad, and Jeffrey H. Chuang. "Uncertainties in tumor allele frequencies limit power to infer evolutionary pressures." Nature Genetics 49.9 (2017): 1288-1289.<br /> (7) Bozic, Ivana, Chay Paterson, and Bartlomiej Waclaw. "On measuring selection in cancer from subclonal mutation frequencies." PLoS Computational Biology 15.9 (2019): e1007368.<br /> (8) Neher, Richard A., and Oskar Hallatschek. "Genealogies of rapidly adapting populations." Proceedings of the National Academy of Sciences 110.2 (2013): 437-442.<br /> (9) Gelman, Andrew, et al. Bayesian data analysis (Third Edition). Chapman and Hall/CRC, 2014.<br /> (10) Fusco, Diana, et al. "Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments." Nature Communications 7.1 (2016): 12760.<br /> (11) Noble, Robert, et al. "Spatial structure governs the mode of tumour evolution." Nature Ecology & Evolution 6.2 (2022): 207-217.<br /> (12) Lawson, Devon A., et al. "Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells." Nature 526.7571 (2015): 131-135.<br /> (13) Gunnarsson, Einar B., Leder, Kevin, and Foo Jasmine. "Exact site frequency spectra of neutrally evolving tumors: A transition between power laws reveals a signature of cell viability" Theoretical Population Biology 142 (2021) 67-90<br /> (14) Durrett, Richard "Branching Process Models of Cancer" Springer (2015)<br /> (15) Durrett, Richard "Probability Models for DNA Sequence Evolution" Springer Science & Business media (2008)<br /> (16) Williams, Mark J. et al. "Quantification of subclonal selection in cancer from bulk sequencing data." Nature Genetics 50 (6). 895-903 (2018)<br /> (17) Moeller, Marius E. et al. "Measures of genetic diversification in somatic tissues at bulk and single-cell resolution" eLife (2024) 12:RP89780<br /> (18) Rougier, Nicolas P., Michael Droettboom, and Philip E. Bourne. "Ten simple rules for better figures." PLoS Computational Biology 10.9 (2014): e1003833.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors present a comparison of two models of cancer evolution with advantageous drivers and deleterious passengers: a fixed-population "Moran" model, and a "Branching Process" (BP) model with dynamic population size. The Moran model is more mathematically-tractable, but since cancer is a disease of uncontrolled growth, it is unclear to me how clinically-relevant it is to consider a model with constant population size. Intriguingly, both models can explain observed Site Frequency Spectrums (SFSs) in three breast cancers, which suggests that the Moran model may have some value. This distinction between the two models is addressed well.

Strengths:

The comparisons of the various BP models (extinction/non-extinction, and balanced/supercritical) are very interesting. The survivability of rare, fitness-disadvantaged clones has huge implications for treatment resistance in general - drug resistant clones are very often disadvantaged in the absence of drug. Clinical sequencing is, most decidedly, investigating population dynamics conditioned on non-extinction, however most published models do not condition on non-extinction - an unfortunate community oversight that this publication rectifies.

Site Frequency Spectrums in three breast cancers are measured with unprecedented resolution to my knowledge (allele abundances below one in a thousand).

Detailed description of the behavior of the various models.

Weaknesses:

I do not believe Moran B is a useful theoretical distinction between Moran A. Incorporating fitness effects into the birth process, instead of the death process, is generally mathematically equivalent when time is measured in generations (or cell divisions). Visible differences in the two models in Figures 2-6 by all accounts seem to be due to the fact that Moran B experiences more evolution in the balanced/driver-dominated case, and less evolution in the passenger dominated case. We generally do not use arbitrary time steps for this reason - we quantify time in 'generations'.

Reviewer #1 (Public Review):

Summary:

This study offers a new perspective. ACTL7A and ACTL7B play roles in epigenetic regulation in spermiogenesis. Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization, and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. It has been reported that ACTL7A is localized on the acrosome in mouse sperms (Boëda et al., 2011). Previous studies have identified ACTL7A mutations (c.1118G>A:p.R373H; c.1204G>A:p.G402S, c.1117C>T:p.R373C), All these variants were located in the actin domain and were predicted to be pathogenic, affecting the number of hydrogen bonds or the arrangement of nearby protein structures (Wang et al., 2023; Xin et al., 2020; Zhao et al., 2023; Zhou et al., 2023). This work used AI to model the role of ACTL7A/B in the nucleosome remodeling complex and proposed a testis-specific conformation of SCRAP complex. This is different from previous studies.

Strengths:

This study provides a new perspective to reveal the additional roles of these proteins.

Weaknesses:

The results section contains a substantial background description. However, the results and discussion sections require streamlining. There is a lack of mutual support for data between the sections, and direct data to support the authors' conclusions are missing.

Reviewer #2 (Public Review):

Summary:

How dynamics of gene expression accompany cell fate and cellular morphological changes is important for our understanding of molecular mechanisms that govern development and diseases. The phenomenon is particularly prominent during spermatogenesis, the process which spermatogonia stem cells develop into sperm through a series of steps of cell division, differentiation, meiosis, and cellular morphogenesis. The intricacy of various aspects of cellular processes and gene expression during spermatogenesis remains to be fully understood. In this study, the authors found that testis-specific actin-related proteins (which usually participate in modifying cells' cytoskeletal systems) ACTL7A and ACTL7B were expressed and localized in the nuclei of mouse spermatocytes and spermatids. Based on this observation, the authors analyzed protein sequence conservations of ACTL7B across dozens of species and identified a putative nuclear localization sequence (NLS) that is often responsible for the nuclear import of proteins that carry them. Using molecular biology experiments in a heterologous cell system, the authors verified the potential role of this internal NLS and found it indeed could facilitate the nuclear localization of marker proteins when expressed in cells. Using gene-deleted mouse models they generated previously, the authors showed that deletion of Actl7b caused changes in gene expression and mis-localization of nucleosomal histone H3 and chromatin regulator histone deacetylase HDAC1 and 2, supporting their proposed roles of ACTL7B in regulating gene expression. The authors further used alpha-Fold 2 to model the potential protein complexes that could be formed between the ARPs (ACTL7A and ACTL7B) and known chromatin modifiers, such as INO80 and SWI/SNF complexes and found that consistent with previous findings, it is likely that ACTL7A and ACTL7B interact with the chromatin-modifying complexes through binding to their alpha-helical HSA domain cooperatively. These results suggest that ACTL7B possesses novel functions in regulating chromatin structure and thus gene expression beyond conventional roles of cytoskeleton regulation, providing alternative pathways for understanding how gene expression is regulated during spermatogenesis and the etiology of relevant infertility diseases.

Strengths:

The authors provided sufficient background to the study and discussions of the results. Based on their previous research, this study utilized numerous methods, including protein complex structural modeling method alpha-fold 2 Multimers, to further investigate the functional roles of ACTL7B. The results presented here are in general of good quality. The identification of a potential internal NLS in ACTL7B is mostly convincing, in line with the phenotypes presented in the gene deletion model.

Weaknesses:

While the study offered an interesting new look at the functions of ARP proteins during spermatogenesis, some of the study is mainly theoretical speculations, including the protein complex formation. Some of the results may need further experimental verifications, for example, differentially expressed genes that were found in potentially spermatogenic cells at different developmental stages, in order to support the conclusions and avoid undermining the significance of the study.

Reviewer #3 (Public Review):

In this manuscript, Pierre Ferrer and colleagues explore the exciting possibility that, in the male germ line, the composition and function of deeply conserved chromatin remodeling complexes is fine-tuned by the addition of testis-specific actin-related proteins (ARPs). In this regard, the Authors aim to extend previously reported non-canonical (transcriptional) roles of ARPs in somatic cells to the unique developmental context of the germ line. The manuscript is focused on the potential regulatory role in post-meiotic transcription of two ARPs: ACTL7A and ACTL7B (particularly the latter). The canonical function of both testis-specific ARPs in spermatogenesis is well established, as they have been previously shown to be required for the extensive cellular morphogenesis program driving post-meiotic development (spermiogenesis). Disentangling the actual functions of ACTL7A and ACTL7B as transcriptional regulators from their canonical role in the profound morphological reshaping of post-meiotic cells (a process that also deeply impacts nuclear architecture and regulation) represents a key challenge in terms of interpreting the reported findings (see below).

The authors begin by documenting, via fluorescence microscopy, the intranuclear localization of ACTL7B. This ARP is convincingly shown to accumulate in the nucleus of spermatocytes and spermatids. Using a series of elegant reporter-based experiments in a somatic cell line, the authors map the driver of this nuclear accumulation to a potential NLS sequence in the ACTL7B actin-like body domain. Ferrer and colleagues then performed a testicular RNA-seq analysis in ACTL7B KO mice to define the putative role of ACTL7B in male germ cell transcription. They report substantial changes to the testicular transcriptome - particularly the upregulation of several classes of genes - in ACTL7B KO mice. However, wild-type testes were used as controls for this experiment, thus introducing a clear confounding effect to the analysis (ACTL7B KO testes have extensive post-meiotic defects due to the canonical role of ACTL7B in spermatid development). Then, the authors employ cutting-edge AI-driven approaches to predict that both ACTL7A and ACTL7B are likely to bind to four key chromatin remodeling complexes. Although these predictions are based on a robust methodology, they would certainly benefit from experimental validation. Finally, the authors associate the loss of ACTL7B with decreased lysine acetylation and lower levels of the HDAC1 and HDAC3 chromatin remodelers in the nucleus of developing spermatids.

Globally, these data may provide important insight into the unique processes male germ cells employ to sustain their extraordinarily complex transcriptional program. Furthermore, the concept that (comparably younger) testis-specific proteins can be incorporated into ancient chromatin remodeling complexes to modulate their function in the germ line is timely and exciting.

It is my opinion that the manuscript would benefit from additional experimental validation to better support the authors' conclusions. In particular, I believe that addressing two critical points would substantially strengthen the message of the manuscript:

(1) The proposed role of ACTL7B in post-meiotic transcriptional regulation temporally overlaps with the protein's previously reported canonical functions in spermiogenesis (PMID: 36617158 and 37800308). Indeed, the canonical functions of ACTL7B have been shown to have a profound effect at the level of spermatid morphology and to impact nuclear organization. This potentially renders the observed transcriptional deregulation in ACTL7B KO testes an indirect consequence of spermatid morphology defects. I acknowledge that it is experimentally difficult to disentangle the proposed intranuclear roles of ACTL7B from the protein's well-documented cytoplasmic function. Perhaps the generation of a NLS-scrambled ACTL7B variant could offer some insight. In light of the substantial investment this approach would represent, I would suggest, as an alternative, that instead of using wild-type testes as controls for the transcriptome and chromatin localization assays, the authors consider the possibility of using testicular tissue from a mutant with similarly abnormal spermiogenesis but due to transcription-independent defects. This would, in my opinion, offer a more suitable baseline to compare ACTL7B KO testes with.

(2) The manuscript would greatly benefit if experimental validation of the AI-driven predictions were to be provided (in terms of the binding capacity of ACTL7A and ACTL7B to key chromatin remodeling complexes). More so it seems that the authors have the technical expertise / available mass spectrometry data required for this purpose (lines 664-665). Still on this topic, given the predicted interactions of ACTL7A and ACTL7B with the SRCAP, EP400, SMARCA2 and SMARCA4 complexes (Figure 7), it is rather counter-intuitive that the Authors chose for their immunofluorescence assays, in ACTL7B KO testes, to determine the chromatin localization of HDAC1 and HDAC3, rather than that of any of above four complexes.

Reviewer #2 (Public Review):

Summary:

The goals of this study were to develop a genetic approach that would specifically and comprehensively target axo-axonic cells (AACs) throughout the brain and then to describe the patterns and characteristics of the targeted AACs in multiple, selected brain regions. The investigators have been successful in providing the most complete description of the regional distribution of putative (pAACs) throughout the brain to date. The supporting evidence is convincing, and the findings should serve as a guide for more detailed studies of AACs within each brain region and lead to new insights into their connectivity and functional organization of this important group of GABAergic interneurons.

Strengths:

The study has numerous strengths. A major strength is the development of a unique intersectional genetic strategy that uses cell lineage (Nkx2.1) and molecular (Unc5b or Pthlh) markers to identify AACs specifically and, apparently, nearly completely throughout the mouse brain. While AACs have been described previously in the cerebral cortex, hippocampus and amygdala, there has been no specific genetic marker that selectively identifies all AACs in these regions.

Importantly, the current genetic strategy labels pAACs in additional brain regions, including the claustrum-insular complex, extended amygdala, and several olfactory centers in which AACs have not been previously recognized. In general, the findings provide support for the specificity of the methods for targeting AACs and include several examples of labeling near markers of axon initial segments, providing validation of their AAC identity.

The descriptions and numerous low magnification images of the brain provide a roadmap for subsequent, detailed studies of AACs in numerous brain regions. The overview and summaries of the findings in the Abstract, Introduction and Discussion are particularly clear and helpful in placing the extensive regional descriptions of AACs in context.

Weaknesses:

Considering the unique and striking characteristics of AACs, it would have been ideal to include a clear, high resolution confocal image of an AAC from the Unc5b;Nkx2.1 mouse that would display the beauty of these cells with their numerous cartridges of axon terminals, emanating from a single AAC. While several cells are illustrated, the processes are often obscured by other labeling or the background created by the blue Dapi labeling. A high-resolution image of an isolated cell would not only support the identity of the cells as AACs but also demonstrate the potential advantages of their labeling for more detailed anatomical and neurophysiological studies. High magnification views of the axon terminals adjacent to AnkG-labeled axon initial segments are included and provide strong support for the identity of the cells. However, they cannot convey the extensiveness and patterns of the axonal arborizations of these cells.

The intersectional genetic methods included use of the lineage marker Nkx2.1 with either Unc5b or Pthlh as the molecular marker. As described, the mice with intersectional targeting of Nkx2.1 and Unc5b appear to show the most specific brain-wide labeling for AACs, and the majority of the descriptions are from these mice. The targeting with Nkx2.1 and Pthlh is less convincing and there appears to be a disconnect between the descriptions and the images. While the descriptions emphasize that the labeling is very similar in the two types of mice, the images suggest distinct differences, including labeling of non AACs in striatum and layer 4 of the cortex in the Pthlh;Nkx2.1 mouse, as described in the manuscript. In addition, the Pthlh;Nkx2.1 mouse has higher cell targeting in some regions and fewer labeled cells in others. Perhaps it would be more accurate to present the Pthlh;Nkx2.1 mouse as differing from the Unc5b;Nkx2.1 mouse, but useful for AAC labeling in select regions and under some conditions, such as following tamoxifen administration at specific ages. As currently presented, the inclusion of the Pthlh;Nkx2.1 detracts from the otherwise convincing argument that the Unc5b;Nkx2.1 mouse provides a specific and comprehensive way to identify AACs.

Reviewer #3 (Public Review):

Summary:

Raudales et al. aimed at providing an insight into the brain-wide distribution and synaptic connectivity of bona fide GABAergic inhibitory interneuron subtypes focusing on the axo-axonic cell (AAC), one of the most distinctive interneuron subtypes, which innervates the axon initial segments of glutamatergic projection neurons. They establish intersectional genetic strategies that enable them to specifically and comprehensively capture AACs based on their lineage (Nkx2.1) and marker expression (Unc5b, Pthlh). They find that AACs are deployed across essentially all the pallium-derived brain structures as well as anterior olfactory nucleus, taenia tecta, and lateral septum. They show that AACs in distinct areas and layers of the neocortex as well as different subregions of the hippocampal formation display unique soma and synaptic density and morphological variations. Rabies virus-based retrograde monosynaptic input tracing reveals that AACs in the neocortex, the hippocampus, and the basolateral amygdala receive synaptic inputs from common as well as specific brain regions and supports the utility of this novel genetic approach. This study elucidates brain-wide neuroanatomical features and morphological variations of AACs with solid techniques and analysis. Their novel AAC-targeting strategies will facilitate the study of their development and function in different brain regions. The conclusions in this paper are well supported by the data. However, there are a few minor comments.

(1) The authors added a description about validation of ChCs in the method section: "Validation was conducted with high-magnification confocal microscopy and defined by a cell exhibiting at least two RFP-labelled axons colocalized with AIS labelled by AnkryinG or Phospho-IκBα". However, this does not clearly define pAACs themselves. If they follow this criteria, an RFP-labeled cell exhibiting only one synaptic cartridge that is colocalized with an AIS should be a pAAC. Is this what the authors are triying to say?

On the other hand, in the response to reviewers, the authors apparently define pAACs in a different way, in which they more focus on the number of cells exhibiting cartridges that are associated with AISs in a certain anatomical region rather than the number of cartridges per cell.

"For BNST we did not positively identify more than a few exhibiting overlap with AnkryinG/IκBα, so we currently leave them as pAACs"<br /> "Putative AAC (pAACs) refers to populations in which relatively few single cell examples of AACs exhibiting co-localized cartridges were found"

The authors need to directly define pAACs.

(2) In the response to reviewers, the authors claimed that both Pthlh and Unc5b mice are useful for studying developing AACs. It would be nice if they include this content in the text (e.g. Discussion).

Reviewer #1 (Public Review):

Summary:

In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with the sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of fractal and classical, manually defined sleep cycles in numerous datasets.

Strengths:

The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.

These results suggest that the fractal cycle methodology could provide a valuable new method to study sleep architecture and avoid the time-consuming steps of manual cycle identification. Moreover, it has the potential to be applied to animal studies which rarely deal with sleep cycle structure.

Weaknesses:

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method. This raises the question as to whether differences are due to one method being more reliable than another or whether they are also identifying different underlying biological differences. It is not clear for example whether the agreement between the two methods is better or worse than between two human scorers, which generally serve as a gold standard to validate novel methods. The authors provide some insight into differences between the methods that could account for differences in results. However, given that the fractal method is automatic it would be important to clearly identify criteria for recordings in which it will produce similar results to the classical method.

Reviewer #2 (Public Review):

Summary:

This study focused on using strictly the slope of the power spectral density (PSD) to perform automated sleep scoring and evaluation of the durations of sleep cycles. The method appears to work well because the slope of the PSD is highest during slow-wave sleep, and lowest during waking and REM sleep. Therefore, when smoothed and analyzed across time, there are cyclical variations in the slope of the PSD, fit using an IRASA (Irregularly resampled auto-spectral analysis) algorithm proposed by Wen & Liu (2016).

Strengths:

The main novelty of the study is that the non-fractal (oscillatory) components of the PSD that are more typically used during sleep scoring can be essentially ignored because the key information is already contained within the fractal (slope) component. The authors show that for the most part, results are fairly consistent between this and conventional sleep scoring, but in some cases show disagreements that may be scientifically interesting.

Weaknesses:

One weakness of the study, from my perspective, was that the IRASA fits to the data (e.g. the PSD, such as in Figure 1B), were not illustrated. One cannot get a sense of whether or not the algorithm is based entirely on the fractal component or whether the oscillatory component of the PSD also influences the slope calculations. This should be better illustrated, but I assume the fits are quite good.

The cycles detected using IRASA are called fractal cycles. I appreciate the use of a simple term for this, but I am also concerned whether it could be potentially misleading? The term suggests there is something fractal about the cycle, whereas it's really just that the fractal component of the PSD is used to detect the cycle. A more appropriate term could be "fractal-detected cycles" or "fractal-based cycle" perhaps?

The study performs various comparisons of the durations of sleep cycles evaluated by the IRASA-based algorithm vs. conventional sleep scoring. One concern I had was that it appears cycles were simply identified by their order (first, second, etc.) but were not otherwise matched. This is problematic because, as evident from examples such as Figure 3B, sometimes one cycle conventionally scored is matched onto two fractal-based cycles. In the case of the Figure 3B example, it would be more appropriate to compare the duration of conventional cycle 5 vs. fractal cycle 7, rather than 5 vs. 5, as it appears is currently being performed.

There are a few statements in the discussion that I felt were either not well-supported. L629: about the "little biological foundation" of categorical definitions, e.g. for REM sleep or wake? I cannot agree with this statement as written. Also about "the gradual nature of typical biological processes". Surely the action potential is not gradual and there are many other examples of all-or-none biological events.

The authors appear to acknowledge a key point, which is that their methods do not discriminate between awake and REM periods. Thus their algorithm essentially detected cycles of slow-wave sleep alternating with wake/REM. Judging by the examples provided this appears to account for both the correspondence between fractal-based and conventional cycles, as well as their disagreements during the early part of the sleep cycle. While this point is acknowledged in the discussion section around L686. I am surprised that the authors then argue against this correspondence on L695. I did not find the "not-a-number" controls to be convincing. No examples were provided of such cycles, and it's hard to understand how positive z-values of the slopes are possible without the presence of some wake unless N1 stages are sufficient to provide a detected cycle (in which case, then the argument still holds except that its alterations between slow-wave sleep and N1 that could be what drives the detection).

To me, it seems important to make clear whether the paper is proposing a different definition of cycles that could be easily detected without considering fractals or spectral slopes, but simply adjusting what one calls the onset/offset of a cycle, or whether there is something fundamentally important about measuring the PSD slope. The paper seems to be suggesting the latter but my sense from the results is that it's rather the former.

Reviewer #1 (Public Review):

Summary:

This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with amplitude comparable to aspartate, and cells can quickly adapt (and generally over-adapt). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

Strengths:

The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH. The updated manuscript controls for the impact of pH on the fluorescent protein brightness that can bias the measured FRET signal. After correction the response amplitude and sharpness (hill coefficient) are comparable to conventional chemoattractants (e.g. aspartate), indicating the general mechanisms underlying the response may be similar. The authors suggest that the biphasic response of Tar mutants may be due to pH influencing the activity of other enzymes (CheA, CheR or CheB), which will be an interesting direction for future study.

Weaknesses:

The measured response may be biased by adaptation, especially for weak potassium signals. For other attractant stimuli, the response typically shows a low plateau before it recovers (adapts). In the case of potassium, the FRET signal does not have an obvious plateau following the stimuli of small potassium concentrations, perhaps due to the faster adaptation compared to other chemoattractants. It is possible cells have already partially adapted when the response reaches its minimum, so the measured response may be a slight underestimate of the true response. Mutants without adaptation enzymes appear to be sensitive to potassium only at much larger concentrations, where the pH significantly disrupts the FRET signal; more accurate measurements would require the development of new mutants and/or measurement techniques.

Note added after the second revision: The authors made a reasonable argument regarding the effects of adaptation, which were estimated to be small.

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain-age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain-age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

Comments on revised version:

I thank the authors for the revision of the manuscript and for being more explicit about the inherent conceptual limitations of Brain Age / Brain Cognition. I have no further comments.

Reviewer #1 (Public Review):

In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.

Strengths:

The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.

Weaknesses:

There are seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression. However, the authors have addressed these issues in their revised manuscript, incorporating potential reasons that might explain such complexity.

Reviewer #2 (Public Review):

The manuscript by Okholm and colleagues identified an interesting new instance of ceRNA involving a circular RNA. The data are clearly presented and support the conclusions. Quantification of the copy number of circRNA and quantification of the protein were performed, and this is important to support the ceRNA mechanism.

This is the second rebuttal and the authors further improved the manuscript. The data are of interest for the large spectrum of readers of the journal.

Reviewer #3 (Public Review):

Summary:

In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking it down and performing an RNA-seq analysis, the authors found thousand deregulated genes which look unaffected by miRNAs sponging function and that are, instead, enriched for a 11-mer motif. Further investigations showed that the 11-mer motif is shared with the circHIPK3 and able to bind the IGF2BP2 protein. The authors validated the binding of IGF2BP2 and demonstrated that IGF2BP2 KD antagonizes the effect of circHIPK3 KD and leads to the upregulation of genes containing the 11-mer. Among the genes affected by circHIPK3 KD and IGF2BP2 KD, resulting in downregulation and upregulation respectively, the authors found STAT3 gene which also consistently leads to the concomitant upregulation of one of its targets TP53. The authors propose a mechanism of competition between circHIPK3 and IGF2BP2 triggered by IGF2BP2 nucleation, potentially via phase separation.

Strengths:

The number of circRNAs continues to drastically grow however the field lacks detailed molecular investigations. The presented work critically addresses some of the major pitfalls in the field of circRNAs and there has been a careful analysis of aspects frequently poorly investigated. The time-point KD followed by RNA-seq, investigation of miRNAs-sponge function of circHIPK3, identification of 11-mer motif, identification and validation of IGF2BP2, and the analysis of copy number ratio between circHIPK3 and IGF2BP2 in assessing the potential ceRNA mode of action have been extensively explored and, comprehensively convincing.

Weaknesses:

The authors addressed the majority of the weak points raised initially. However the role played by the circHIPK3 in cancer remains elusive and not elucidated in full in this study.

Overall, the presented study surely adds some further knowledge in describing circHIPK3 function, its capability to regulate some downstream genes, and its interaction and competition for IGF2BP2. However, whereas the experimental part sounds technically logical, it remains unclear the overall goal of this study and the achieved final conclusions.

This study is a promising step forward in the comprehension of the functional role of circHIPK3. These data could possibly help to better understand the circHIPK3 role in cancer

Reviewer #1 (Public Review):

This paper describes RNA-sensing guide RNAs for controlled activation of CRISPR modification. This works by having an extended guide RNA with a sequence that folds back onto the targeting sequence such that the guide RNA cannot hybridise to its genomic target. The CRISPR is "activated" by the introduction of another RNA, referred to as a trigger, that competes with this "back folding" to make the guide RNA available for genome targeting. The authors first confirm the efficacy of the approach using several RNA triggers and a GFP reporter that is activated by dCas9 fused to transcriptional activators. A major potential application of this technique is the activation of CRISPR in response to endogenous biomarkers. As these will typically be longer than the first generation triggers employed by the authors they test some extended triggers, which also work though not always to the same extent. They then introduce MODesign which may enable the design of bespoke or improved triggers. After that, they determine that the mode of activation by the RNA trigger involves cleavage of the RNA complexes. Finally, they test the potential for their system to work in a developmental setting - specifically zebrafish embryos. There is some encouraging evidence, though the effects appear more subtle than those originally obtained in cell culture.

Overall, the potential of a CRISPR system that can be activated upon sensing an RNA is high and there are a myriad of opportunities and applications for it. This paper represents a reasonable starting point having developed such a system in principle.<br /> The weakness of the study is that it does not demonstrate that the system can be used in a completely natural setting. This would require an endogenous transcript as the RNA trigger with a clear readout. The authors now acknowledge this limitation in their revised manuscript. Future studies and experiments should focus on these aspects in order for the system to be employed to its full and intended potential.

Reviewer #1 (Public Review):

In the presence of predators, animals display attenuated foraging responses and increased defensive behaviors that serve to protect them from potential predatory attacks. Previous studies have shown that the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray matter (PAG) are necessary for the acquisition and expression of conditioned fear responses. However, it remains unclear how BLA and PAG neurons respond to predatory threats when animals are foraging for food. To address this question, Kim and colleagues conducted in vivo electrophysiological recordings from BLA and PAG neurons and assessed approach-avoidance responses while rats search for food in the presence of a robotic predator.

The authors observed that rats exhibited a significant increase in the latency to obtain the food pellets and a reduction in the pellet success rate when the predator robot was activated. A subpopulation of PAG neurons showing increased firing rate in response to the robot activation didn't change their activity in response to food pellet retrieval during the pre- or post-robot sessions. Optogenetic stimulation of PAG neurons increased the latency to procure the food pellet in a frequency- and intensity-dependent manner, similar to what was observed during the robot test. Combining optogenetics with single-unit recordings, the authors demonstrated that photoactivation of PAG neurons increased the firing rate of 10% of BLA cells. A subsequent behavioral test in 3 of these same rats demonstrated that BLA neurons responsive to PAG stimulation displayed higher firing rates to the robot than BLA neurons nonresponsive to PAG stimulation. Next, because the PAG does not project monosynaptically to the BLA, the authors used a combination of retrograde and anterograde neural tracing to identify possible regions that could convey robot-related information from PAG to the BLA. They observed that neurons in specific areas of the paraventricular nucleus of the thalamus (PVT) that are innervated by PAG fibers contained neurons that were retrogradely labeled by the injection of CTB in the BLA. In addition, PVT neurons showed increased expression of the neural activity marker cFos after the robot test, suggesting that PVT may be a mediator of PAG signals to the BLA.

Overall, the idea that the PAG interacts with the BLA via the midline thalamus during a predator vs. foraging test is new and quite interesting. The authors have used appropriated tools to address their questions.

In this revised version of the manuscript, the authors have made important modifications in the text, inserted new data analyses, and incorporated additional references, as recommended by the reviewers. These modifications have significantly improved the quality of the manuscript.

Reviewer #2 (Public Review):

The authors characterized activity of the dorsal periaqueductal gray (dPAG) - basolateral amygdala (BLA) circuit. They show that BLA cells that are activated by dPAG stimulation are also more likely to be activated by a robot predator. These same cells are also more likely to display synchronous firing.

The authors also replicate prior results showing that dPAG stimulation evokes fear and the dPAG is activated by a predator.

Lastly, the report performs anatomical tracing to show that the dPAG may act on the BLA via the paraventricular thalamus (PVT). Indeed, the PVT receives dPAG projections and also projects to the BLA. However, the authors do not show if the PVT mediates dPAG to BLA communication with any functional behavioral assay. Furthermore, the authors also do not thoroughly characterize the activity of BLA cells during the predatory assay.

The major impact in the field would be to add evidence to their prior work, strengthening the view that the BLA can be downstream of the dPAG.

Reviewer #3 (Public Review):

In the present study, the authors examined how dPAG neurons respond to predatory threats and how dPAG and BLA communicate threat signals. The authors employed single-unit recording and optogenetics tools to address these issues in an 'approach food-avoid predator' paradigm. They characterized dPAG and BLA neurons responsive to a looming robot predator and found that dPAG opto-stimulation elicited fleeing and increased BLA activity. Importantly, they found that dPAG stimulation produces activity changes in subpopulations of BLA neurons related to predator detection, thus supporting the idea that dPAG conveys innate fear signals to the amygdala. In addition, injections of anterograde and retrograde tracers into the dPAG and BLA, respectively, along with the examination of c-FOS activity in midline thalamic relay stations, suggest that the paraventricular nucleus of the thalamus (PVT) may serve as a mediator of dPAG to BLA neurotransmission. Of relevance, the study helps to validate an important concept that dPAG mediates primal fear emotion and may engage upstream amygdalar targets to evoke defensive responses. The series of experiments provide a compelling case for supporting their conclusions. The study brings important concepts revealing dynamics of fear-related circuits particularly attractive to a broad audience, from basic scientists interested in neural circuits to psychiatrists.

Reviewer #1 (Public Review):

Summary:

This work describes a new protein factor required for filamentous phage assembly. The protein PSB15 binds to the packaging signal of the ssDNA, Trx and cardiolipin. A mechanism how the phage DNA is targeted to the assembly site in the bacterial inner membrane is discussed.

Strengths:

The work describes a clever way to detect factors required for phage propagation by looking at the plaque size of pseudorevertants that arise after infection of a phage with a directed mutation in the packaging signal. This led to the detection of a phage protein expressed from ORF9, the PSB15.

The authors convincingly show that PSB15 is expressed in infected cells and can complement a phage with a mutated orf9.

Weaknesses:

Given the fact that the phage LF-UK is not well explored, many open questions should be mentioned in the introduction. For the study, it is important to know if the phageLF-UK has a mimick or homolog of gV and gXI, and if not, whether PSB15 could take their role.

I am not convinced of the proposition of their term "checkpoint". The truth is that the authors do not know the real purpose of PSB15. I do not see an advantage for a checkpoint that only adds an additional step to enter the phage assembly site. There must be a biochemical reason for the action of PSB15. Looking at Figure 7, the step from "Release" to "Loading" is just adding many unknowns, e.g. how to transfer the DNA, how to dispose of PSB15 and Trx? Also, in the previous step are three question marks that do not add any solid information.

The in vivo study of subcellular localization is very questionable. Why is there a single fluorescent dot if there are thousands of PSB15 molecules expressed in the cell? I have my doubts that the conclusions the authors make here are correct and meaningful. The movies do not add anything significant.

Reviewer #2 (Public Review):

Secretion of the prototypical F-associated filamentous phage (Ff) of E. coli depends on the selective binding of a hairpin (the packaging signal, PS) by two phage encoded protein, pVII and pIX. PVII and pIX target the PS to IM channels formed by pI and pIV. However, integrative filamentous phages lack a homologue of pIX and pIV, and many of them also lack a homologue of pVII, raising questions on the assembly and secretion of new phages. In the manuscript, Yueh et al. present the identification of a phage-encoded protein, PSB15, which binds to the PS signal of a Xanthomonas integrative filamentous phage, ΦLf-UK. They showed that PSB15 is required for viral assembly and is conserved in several other integrative filamentous phages. They further analyzed how PSB15 binds to PS and demonstrated that it associates to the IM, which targets phage DNA to it. Finally, they show that thioredoxin, the only host protein that was found to be essential for Ff secretion, interacts with PSB15 and releases the PSB15-PS complex from the IM. These results are important because they elucidate a major step in the secretion of integrative filamentous phage, and the role of thioredoxin on filamentous phage secretion in general.

I found the data and interpretation convincing. However, the presentation and description are confusing in places because the reader has to juggle between figures. A scheme depicting what is known and unknown in the integration of Ff phages and interactive filamentous phages in the introduction would be useful to the general reader.

Joint Public Review:

Summary

This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.

This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.

Despite the breadth of information presented, however, further quantification of results and explanation of experimental approaches would be needed to support some of the authors' claims. Additionally, a more thorough discussion is needed to contextualize their findings relative to previous work.

(1) Important quantification is lacking for the data presented. For example, multiple figures include immunohistochemistry or immunocytochemistry data (Figures 1, 5, 6), but they are presented without accompanying measures like fractions of cells labeled or comparisons against controls. As a result, for axons projecting via OEC bridges in Figure 1, it is unclear how common these bridges are in the presence or absence of OECs. For Figure 6., it is unclear whether cells having an alternative OEC morphology coincide with progenitor OEC subtype marker genes to a statistically significant degree. Similar quantification is missing in other types of data such as Western blot images (Fig. 9) and OEC marker gene data (for which p-values are not reported; Table S2).

The addition of quantitative measures and, where appropriate, statistical comparisons with p-values or other significance measures, would be important for supporting the authors' claims and more rigorously conveying the results.

(2) Some aspects of the experimental design that are relevant to the interpretation of the results are not explained. For example, OECs appear to be collected from only female rats, but the potential implications of this factor are not discussed.

Additionally, it is unclear from the manuscript to what degree immunopurified cells are OECs as opposed to other cell types. The antibody used to retain OECs, nerve growth factor receptor p75 (Ngfr-p75), can also be expressed by non-OEC olfactory bulb cell types including astrocytes [1-3]. The possible inclusion of Ngfr-p75-positive but non-OEC cell types in the OEC culture is not sufficiently addressed. Such non-OEC cell types are also not distinguished in the analysis of single-cell RNA sequencing data (only microglia, fibroblasts, and OECs are identified; Figure 2). Thus, it is currently unclear whether results related to the OEC subtype may have been impacted by these experimental factors.

(3) The introduction, while well written, does not discuss studies showing no significant effect of OEC implantation after spinal cord injury. The discussion also fails to sufficiently acknowledge this variability in the efficacy of OEC implantation. This omission amplifies bias in the text, suggesting that OECs have significant effects that are not fully reflected in the literature. The introduction would need to be expanded to properly address the nuance suggested by the literature regarding the benefits of OECs after spinal cord injury. Additionally, in the discussion, relating the current study to previous work would help clarify how varying observations may relate to experimental or biological factors.

(a) Cragnolini, A.B. et al., Glia, (2009), doi: 10.1002/glia.20857.<br /> (b) Vickland H. et al., Brain Res., (1991), doi: 10.1016/0006-8993(91)91659-O.<br /> (c) Ung K. et al., Nat Commun., (2021), doi: 10.1038/s41467-021-25444-3.

Reviewer #1 (Public Review):

Summary:

It seems as if the main point of the paper is about the new data related to rat fish although your title is describing it as extant cartilaginous fishes and you bounce around between the little skate and ratfish. So here's an opportunity for you to adjust the title to emphasize ratfish is given the fact that leader you describe how this is your significant new data contribution. Either way, the organization of the paper can be adjusted so that the reader can follow along the same order for all sections so that it's very clear for comparative purposes of new data and what they mean. My opinion is that I want to read, for each subheading in the results, about the the ratfish first because this is your most interesting novel data. Then I want to know any confirmation about morphology in little skate. And then I want to know about any gaps you fill with the cat shark. (It is ok if you keep the order of "skate, ratfish, then shark, but I think it undersells the new data).

Strengths:

The imagery and new data availability for ratfish are valuable and may help to determine new phylogenetically informative characters for understanding the evolution of cartilaginous fishes. You also allude to the fossil record.

Opportunities:

I am concerned about the statement of ratfish paedomorphism because stage 32 and 33 were not statistically significantly different from one another (figure and prior sentences). So, these ratfish TMDs overlap the range of both 32 and 33. I think you need more specimens and stages to state this definitely based on TMD. What else leads you to think these are paedomorphic? Right now they are different, but it's unclear why. You need more outgroups.

Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth.

Historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology and development of these fishes.

I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies and I don't think your list is exhaustive. You need to expand this list and history which will help with your ultimate comparative analysis without you needed to sample too many new data yourself.

I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text. Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?

In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.

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. I have, however, some comments. Some information is lacking and should be added to the manuscript text. I also suggest changes in the result and the discussion section of the manuscript.

Introduction:

The reader gets the impression almost no research on chondrichthyan skeletal tissues was done before the 2010 ("last 15 years", L45). I suggest to correct that and to cite also previous studies on chondrichthyan skeletal tissues, this includes studies from before 1900.

Material and Methods:

Please complete L473-492: Three different Micro-CT scanners were used for three different species? ScyScan 117 for the skate samples. Catshark different scanner, please provide full details. Chimera Scncrotron Scan? Please provide full details for all scanning protocols.

TMD is established in the same way in all three scanners? Actually not possible. Or, all specimens were scanned with the same scanner to establish TMD? If so please provide the protocol.

Please complete L494 ff: Tissue embedding medium and embedding protocol is missing. Specimens have been decalcified, if yes how? Have specimens been sectioned non-decalcified or decalcified?

Please complete L506 ff: Tissue embedding medium and embedding protocol is missing. Description of controls are missing.

Results:

L147: It is valuable and interesting to compare the degree of mineralisation in individuals from the three different species. It appears, however, not possible to provide numerical data for Tissue Mineral Density (TMD). First requirement, all specimens must be scanned with the same scanner and the same calibration values. This in not stated in the M&M section. But even if this was the case, all specimens derive from different sample locations and have, been preserved differently. Type of fixation, extension of fixation time in formalin, frozen, unfrozen, conditions of sample storage, age of the samples, and many more parameters, all influence TMD values. Likewise the relative age of the animals (adult is not the same as adult) influences TMD. One must assume different sampling and storage conditions and different types of progression into adulthood. Thus, the observation of different degrees of mineralisation is very interesting but I suggest not to link this observation to numerical values.

Parts of the results are mixed with discussion. Sometimes, a result chapter also needs a few references but this result chapter is full of references.

Based on different protocols, the staining characteristics of the tissue are analysed. This is very good and provides valuable additional data. The authors should inform the not only about the staining (positive of negative) abut also about the histochemical characters of the staining. L218: "fast green positive" means what? L234: "marked by Trichrome acid fuchsin" means what? And so on, see also L237, L289, L291<br /> Discussion

Please completely remove figure 7, please adjust and severely downsize the discussion related to figure 7. It is very interesting and valuable to compare three species from three different groups of elasmobranchs. Results of this comparison also validate an interesting discussion about possible phylogenetic aspects. This is, however, not the basis for claims about the skeletal tissue organisation of all extinct and extant members of the groups to which the three species belong. The discussion refers to "selected representatives" (L364), but how representative are the selected species? Can there be a extant species that represents the entire large group, all sharks, rays or chimeras? Are the three selected species basal representatives with a generalist life style?

Please completely remove the discussion about paedomorphosis in chimeras (already in the result section). This discussion is based on a wrong idea about the definition of paedomorphosis. Paedomorphosis can occur in members of the same group. Humans have paedormorphic characters within the primates, Ambystoma mexicanum is paedormorphic within the urodeals. Paedomorphosis does not extend to members of different vertebrate branches. That elasmobranchs have a developmental stage that resembles chimera vertebra mineralisation does not define chimera vertebra centra as paedomorphic. Teleost have a herocercal caudal fin anlage during development, that does not mean the heterocercal fins in sturgeons or elasmobranchs are paedomorphic characters.

L432-435: In times of Gadow & Abott (1895) science had completely wrong ideas bout the phylogenic position of chondrichthyans within the gnathostomes. It is curious that Gadow & Abott (1895) are being cited in support of the paedomorphosis claim.

The SCPP part of the discussion is unrelated to the data obtained by this study. Kawaki & WEISS (2003) describe a gene family (called SCPP) that control Ca-binding extracellular phosphoproteins in enamel, in bone and dentine, in saliva and in milk. It evolved by gene duplication and differentiation. They date it back to a first enamel matrix protein in conodonts (Reif 2006). Conodonts, a group of enigmatic invertebrates have mineralised structures but these structure are neither bone nor mineralised cartilage. Cat fish (6 % of all vertebrate species) on the other hand, have bone but do not have SCPP genes (Lui et al. 206). Other calcium binding proteins, such as osteocalcin, were initially believed to be required for mineralisation. It turned out that osteocalcin is rather a mineralisation inhibitor, at best it regulates the arrangement collagen fiber bundles. The osteocalcin -/- mouse has fully mineralised bone. As the function of the SCPP gene product for bone formation is unknown, there is no need to discuss SCPP genes. It would perhaps be better to finish the manuscript with summery that focuses on the subject and the methodology of this nice study.

Reviewer #1 (Public Review):

Summary:

The main observation that the sperm from CRISP proteins 1 and 3 KO lines are post-fertilization less developmentally competent is convincing. However, the molecular characterization of the mechanism that leads to these defects and the temporal appearance of the defects requires additional studies.

Strengths:

The generation of these double mutant mice is valuable for the field. Moreover, the fact that the double mutant line of Crisp 1 and 3 is phenotypically different from the Crisp 1 and 4 line suggests different functions of these epididymis proteins. The methods used to demonstrate that developmental defects are largely due to post-fertilization defects are also a considerable strength. The initial characterization of these sperm has altered intracellular Ca2+ levels, and increased rates of DNA fragmentation are valuable.

Weaknesses:

The study is mechanistically incomplete because there is no direct demonstration that the absence of these proteins alters the epididymal environment and fluid, wherein during the passage through the epididymis the sperm become affected. Also, a direct demonstration of how the proteins in question cause or lead to DNA damage and increased Ca2+ requires further characterization.

Reviewer #2 (Public Review):

The authors showed that CRISP1 and CRISP3, secreted proteins in the epididymis, are required for early embryogenesis after fertilization through DNA integrity in cauda epididymal sperm. This paper is the first report showing that the epididymal proteins are required for embryogenesis after fertilization. However, some data in this paper (Table 1 and Figure 2A) are overlapped in a published paper (Curci et al., FASEB J, 34,15718-15733, 2020; PMID: 33037689). Furthermore, the authors did not address why the disruption of CRISP1/3 leads to these phenomena (the increased level of the intracellular Ca2+ level and impaired DNA integrity in sperm) with direct evidence. Therefore, if the authors can address the following comments to improve the paper's novelty and clarification, this paper may be worthwhile to readers.

Reviewer #1 (Public Review):

Summary:

Using a cross-modal sensory selection task in head-fixed mice, the authors attempted to characterize how different rules reconfigured representations of sensory stimuli and behavioral reports in sensory (S1, S2) and premotor cortical areas (medial motor cortex or MM, and ALM). They used silicon probe recordings during behavior, a combination of single-cell and population-level analyses of neural data, and optogenetic inhibition during the task.

Strengths:

A major strength of the manuscript was the clarity of the writing and motivation for experiments and analyses. The behavioral paradigm is somewhat simple but well-designed and well-controlled. The neural analyses were sophisticated, clearly presented, and generally supported the authors' interpretations. The statistics are clearly reported and easy to interpret. In general, my view is that the authors achieved their aims. They found that different rules affected preparatory activity in premotor areas, but not sensory areas, consistent with dynamical systems perspectives in the field that hold that initial conditions are important for determining trial-based dynamics.

I think this is a well-performed, well-written and interesting study that shows differences in rule representations in sensory and premotor areas, and finds that rules reconfigure preparatory activity in motor cortex to support flexible behavior.

Reviewer #2 (Public Review):

Summary:

Chang et al. investigated neuronal activity firing patterns across various cortical regions in an interesting context-dependent tactile vs visual detection task, developed previously by the authors (Chevee et al., 2021; doi: 10.1016/j.neuron.2021.11.013). The authors report the important involvement of a medial frontal cortical region (MM, probably a similar location to wM2 as described in Esmaeili et al., 2021 & 2022; doi: 10.1016/j.neuron.2021.05.005; doi: 10.1371/journal.pbio.3001667) in mice for determining task rules.

Strengths:

The experiments appear to have been well carried out and the data well analysed. The manuscript clearly describes the motivation for the analyses and reaches clear and well-justified conclusions. I find the manuscript interesting and exciting!

Weaknesses:

I did not find any major weaknesses.

Reviewer #3 (Public Review):

Summary:

This study examines context-dependent stimulus selection by recording neural activity from several sensory and motor cortical areas along a sensorimotor pathway, including S1, S2, MM, and ALM. Mice are trained to either withhold licking or perform directional licking in response to visual or tactile stimulus. Depending on the task rule, the mice must respond to one stimulus modality while ignoring the other. Neural activity to the same tactile stimulus is modulated by task in all the areas recorded, with significant activity changes in a subset of neurons and population activity occupying distinct activity subspaces. Recordings further reveal a contextual signal in the pre-stimulus baseline activity that differentiates task context. This signal is correlated with subsequent task modulation of neural activity. Comparison across brain areas shows that this contextual signal is stronger in frontal cortical regions than sensory regions. Analyses link this signal to behavior by showing that it tracks the behavioral performance switch during task rule transitions. Silencing activity in frontal cortical regions during the baseline period impairs behavioral performance.

Strengths:

This is a carefully done study with solid results and thorough controls. The authors identify a contextual signal in baseline neural activity that predicts rule-dependent decision-related activity. The comprehensive characterization across a sensorimotor pathway is another strength. Analyses and perturbation experiments link this contextual signal to animals' behavior. The results provide a neural substrate that will surely inspire follow-up mechanistic investigations.

Weaknesses:

None. The authors have further improved the manuscript during the revision with additional analyses.

Impact:

This study reports an important neural signature for context-dependent decision-making that has important implications for mechanisms of context-dependent neural computation in general.

Reviewer #1 (Public Review):

Summary:

The classical pro/antisaccade task has become a valuable diagnostic tool in neurology and psychiatry (Antoniades et al., 2013, Vision Res). Although it is well-established that antisaccades require substantially longer latencies than prosaccades, the exact attentional mechanisms underlying these differences are not yet fully elucidated. This study investigates the separate influences of exogenous and endogenous attention on saccade generation. These two mechanisms are often confounded in classical pro/antisaccade tasks. In the current study, the authors build on their previous work using an urgent choice task (Salinas et al., 2019, eLife) to time-resolve the influences of exogenous and endogenous factors on saccade execution. The key contribution of the current study is to show that, when controlling for exogenous capture, antisaccades continue to require longer processing times. This longer processing time may be explained by a coupling between endogenous attention and saccade motor plans.

Strengths:

In the classical pro/antisaccade task the direction of exogenous capture (caused by the presentation of the cue) is typically congruent with the direction of prosaccades and incongruent with antisaccades. A key strength of the current study is the introduction of different experimental conditions that control for the effects of exogenous capture on saccade generation. In particular, Experiments 3 and 4 provide strong evidence for two independent (exogenous and endogenous) mechanisms that guide saccadic choices, acting at different times. Differences in timing for pro and antisaccades during the endogenous phase were consistent and independent of whether the exogenous capture biased early saccades toward the correct prosaccade direction or toward the correct antisaccade directions.

As in previous studies by the same group (Salinas et al., 2019, eLife; Goldstein et al., 2023, eLife), the detailed analysis of the time course of goal-directed saccades allowed the authors to determine the exact, additional time of 30 ms that is necessary to generate a correct antisaccade versus prosaccade.

Overall, the manuscript is very well written, and the data are presented clearly.

Weaknesses:

The main research question could be defined more clearly. In the abstract and at some points throughout the manuscript, the authors indicate that the main purpose of the study was to assess whether the allocation of endogenous attention requires saccade planning [e.g., ll.3-5 or ll.247-248]. While the data show a coupling between endogenous attention and saccades, they do not point to a specific direction of this coupling (i.e., whether endogenous attention is necessary to successfully execute a saccade plan or whether a saccade plan necessarily accompanies endogenous attention).

Some of the analyses were performed only on subgroups of the participants. The reporting of these subgroup analyses is transparent and data from all participants are reported in the supplementary figures. Still, these subgroup analyses may make the data appear more consistent, compared to when data is considered across all participants. For instance, the exogenous capture in Experiments 1 and 2 appears much weaker in Figure 2 (subgroup) than Figure S3 (all participants). Moreover, because different subgroups were used for different analyses, it is often difficult to follow and evaluate the results. For instance, the tachometric curves in Figure 2 (see also Figure 3 and 4) show no motor bias towards the cue (i.e., performance was at ~50% for rPTs <75 ms). I assume that the subsequent analyses of the motor bias were based on a very different subgroup. In fact, based on Figure S2, it seems that the motor bias was predominantly seen in the unreliable participants. Therefore, I often found the figures that were based on data across all participants (Figures 7 and S3) more informative to evaluate the overall pattern of results.

Reviewer #2 (Public Review):

Goldstein et al. provide a thorough characterization of the interaction of attention and eye movement planning. These processes have been thought to be intertwined since at least the development of the Premotor Theory of Attention in 1987, and their relationship has been a continual source of debate and research for decades. Here, Goldstein et al. capitalize on their novel urgent saccade task to dissociate the effects of endogenous and exogenous attention on saccades towards and away from the cue. They find that attention and eye movements are, to some extent, linked to one another but that this link is transient and depends on the nature of the task. A primary strength of the work is that the researchers are able to carefully measure the timecourse of the interaction between attention and eye movements in various well-controlled experimental conditions. As a result, the behavioral interplay of two forms of attention (endogenous and exogenous) is illustrated at the level of tens of milliseconds as they interact with the planning and execution of saccades towards and away from the cued location. Overall, the results allow the authors to make meaningful claims about the time course of visual behavior, attention, and the potential neural mechanisms at a timescale relevant to everyday human behavior.

Reviewer #3 (Public Review):

Summary and overall evaluation:

Human vision is inherently limited so that only a small part of a visual scene can be perceived at a given moment. To address this limitation, the visual system has evolved a number of strategies and mechanisms that work in concert. First, humans move their eyes using saccadic eye movements. This allows us to place the high-resolution region in the center of the eye's retina (the fovea centralis) on objects of interest so that these are sampled with high acuity. Second, salient, conspicuous stimuli that appear abruptly and/or differ strongly from the other stimuli in the scene, seem to automatically attract ("exogenous") attention, so that a large share of the neuronal "resources" for visual processing is devoted to the stimuli, which improves the perception of the stimuli. Third, stimuli that are important for the current task and the current behavioral goals can be prioritized by attention mechanisms ("endogenous" attention), which also secures their allocated share of processing resources and helps them be perceived. It is well-established that eye movements are closely linked to the mechanisms of attention (for a review, see Carrasco, 2011, cited in the manuscript). However, it is still unclear what role voluntary, endogenous attention plays in the control of saccadic eye movements.

The present study used an experimental procedure involving time-pressure for responding, in order to uncover how the control of saccades by exogenous and endogenous attention unfolds over time. The findings of the study indicate that saccade planning was indeed influenced by the locus of endogenous attention, but that this influence was short-lasting and could be overcome quickly. Taken together, the present findings reveal new dynamics between endogenous attention and eye movement control, and lead the way for studying them using experiments under time pressure.

The results provided by the present study advance our understanding of vision, eye movements, and their control by brain mechanisms for attention. In addition, they demonstrate how tasks involving time pressure can be used to study the dynamics of cognitive processes. Therefore, the present study seems highly important not only for vision science, but also for psychology, (cognitive) neuroscience, and related research fields more generally.

Strengths:

The experiments of the study are performed with great care and rigor and the data is analyzed thoroughly and comprehensively. Overall, the results support the authors' conclusions, so I have only minor comments (see below). Taken together, the findings seem important for a wide community of researchers in vision science, psychology, and neuroscience.

Weaknesses (minor points):

(1) In this experimental paradigm, participants must decide where to saccade based on the color of the cue in the visual periphery (they should have made a prosaccade toward a green cue and an antisaccade away from a magenta cue). Thus, irrespective of whether the cue signaled that a prosaccade or an antisaccade was to be made, the identity of the cue was always essential for the task (as the authors explain on p. 5, lines 129-138). Also, the location where the cue appeared was blocked, and thus known to the participants in advance, so that endogenous attention could be directed to the cue at the beginning of a trial (e.g., p. 5, lines 129-132). These aspects of the experimental paradigm differ from the classic prosaccade/antisaccade paradigm (e.g. Antoniades et al., 2013, Vision Research). In the classic paradigm, the identity of the cues does not have to be distinguished to solve the task, since there is only one stimulus that should be looked at (prosaccade) or away from (antisaccade), and whether a prosaccade or antisaccade was required is constant across a block of trials. Thus, in contrast to the present paradigm, in the classic paradigm, the participants do not know where the cue is about to appear, but they know whether to perform a prosaccade or an antisaccade based on the location of the cue.

The present paradigm keeps the location of the cue constant in a block of trials by intention, because this ensures that endogenous attention is allocated to its location and is not overpowered by the exogenous capture of attention that would happen when a single stimulus appeared abruptly in the visual field. Thus, the reason for keeping the location of the cue constant seems convincing. However, I wondered what consequences the constant location would have for the task representations that persist across the task and govern how attention is allocated. In the classic paradigm, there is always a single stimulus that captures attention exogenously (as it appears abruptly). In a prosaccade block, participants can prioritize the visual transient caused by the stimulus, and follow it with a saccade to its coordinates. In an antisaccade block, following the transient with a saccade would always be wrong, so that participants could try to suppress the attention capture by the transient, and base their saccade on the coordinates of the opposite location. Thus, in prosaccade and antisaccade blocks, the task representations controlling how visual transients are processed to perform the task differ. In the present task, prosaccades and antisaccades cannot be distinguished by the visual transients. Thus, such a situation could favor endogenous attention and increase its influence on saccade planning, even though saccade planning under more naturalistic conditions would be dominated by visual transients. I suggest discussing how this (and vice versa the emphasis on visual transients in the classic paradigm) could affect the generality of the presented findings (e.g., how does this relate to the interpretation that saccade plans are obligatorily coupled to endogenous attention? See, Results, p. 10, lines 306-308, see also Deubel & Schneider, 1996, Vision Research).

(2) Discussion (p. 16, lines 472-475): The authors suppose that "It is as if the exogenous response was automatically followed by a motor bias in the opposite direction. Perhaps the oculomotor circuitry is such that an exogenous signal can rapidly trigger a saccade, but if it does not, then the corresponding motor plan is rapidly suppressed regardless of anything else.". I think this interesting point should be discussed in more detail. Could it also be that instead of suppression, other currently active motor plans were enhanced? Would this involve attention? Some attention models assume that attention works by distributing available (neuronal) processing resources (e.g., Desimone & Duncan, 1995, Annual Review of Neuroscience; Bundesen, 1990, Psychological Review; Bundesen et al., 2005, Psychological Review) so that the information receiving the largest share of resources results in perception and is used for action, but this happens without the active suppression of information.

(3) Methods, p. 19, lines 593-596: It is reported that saccades were scored based on their direction. I think more information should be provided to understand which eye movements entered the analysis. Was there a criterion for saccade amplitude? I think it would be very helpful to provide data on the distributions of saccade amplitudes or on their accuracy (e.g. average distance from target) or reliability (e.g. standard deviation of landing points). Also, it is reported that some data was excluded from the analysis, and I suggest reporting how much of the data was excluded. Was the exclusion of the data related to whether participants were "reliable" or "unreliable" performers?

(4) Results, p. 9, lines 262-266: Some data analyses are performed on a subset of participants that met certain performance criteria. The reasons for this data selection seem convincing (e.g. to ensure empirical curves were not flat, line 264). Nevertheless, I suggest to explain and justify this step in more detail. In addition, if not all participants achieved an acceptable performance and data quality, this could also speak to the experimental task and its difficulty. Thus, I suggest discussing the potential implications of this, in particular, how this could affect the studied mechanisms, and whether it could limit the presented findings to a special group within the studied population.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #3 (Public Review):

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been studying ALK signalling in flies to gain mechanistic insight into its various roles. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

Weaknesses:

The manuscript has improved very substantially in revision. The authors have clearly taken the comments on board in good faith.

Editors' note: The authors have satisfactorily addressed the concerns raised in the previous rounds of review. These were related to the unconventional analysis of the TaDa data, the addition of other means of down regulated gene function, and the nature of analyses of behavioural data.

Reviewer #1 (Public Review):

Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data.

The exclusive use of males is a major concern lacking adequate justification and should be disclosed in the title and abstract to ensure readers are aware of this limitation. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings. The occurrence of an estrus cycle in typical female rats is not justification for their exclusion. Note also that male rodents experience great variability in hormonal states as well, distinguishing between individuals and within individuals across time. The study of endocrinological influences on behavior can be separated from the study of said behavior itself, across all sexes. Similarly, concerns about needing to increase the number of animals when including all sexes are usually unwarranted (see Shansky [2019] and Phillips et al. [2023]).

Regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, my comment on the originally reviewed version stands. It seems that the calls are sorted by an (unbiased) algorithm into categories based on their frequency and duration, and because 44kHz calls differ by definition on frequency and duration the fact that the algorithm sorts them as a distinct category is not evidence that they are "new calls [that] form a separate, distinct group". I appreciate that the authors have softened their language regarding the novelty and distinctness of these calls, but the manuscript contains several instances where claims of novelty and specificity (e.g. the subtitle on line 193) is emphasized beyond what the data justifies.

The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.

I appreciate the amendment in discussing the idea of arousal being the key determinant for the increased emission of 44kHz, and the addition of other factors. Some of the items in this list, such as annoyance/anger and disgust/boredom, don't really seem to fit the data. I'm not sure I find the idea that rats become annoyed or disgusted during fear conditioning to be a particularly compelling argument. As such the list appears to be a collection of emotion-related words, with unclear potential associations with the 44kHz calls.

Later in the Discussion the authors argue that the 44kHz aversive calls signal an increased intensity of a negative valence emotional state. It is not clear how the presented arguments actually support this. For example, what does the elongation of fear conditioning to 10 trials have to do with increased negative emotionality? Is there data supporting this relationship between duration and emotion, outside anthropomorphism? Each of the 6 arguments presented seems quite distant from being able to support this conclusion.

In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.

[Editors' note: The reviewer agrees that the additional analysis has ruled out the possibility that the calls are due to fatigue.]

Reviewer #1 (Public Review):

Summary:

The authors assess the accuracy of short variant calling (SNPs and indels) in bacterial genomes using Oxford Nanopore reads generated on R10.4 flow cells from a very similar genome (99.5% ANI), examining the impact of variant caller choice (three traditional variant callers: bcftools, freebayes, and longshot, and three deep learning based variant callers: clair3, deep variant, and nano caller), base calling model (fast, hac and sup) and read depth (using both simplex and duplex reads).

Strengths:

Given the stated goal (analysis of variant calling for reads drawn from genomes very similar to the reference), the analysis is largely complete and results are compelling. The authors make the code and data used in their analysis available for re-use using current best practices (a computational workflow and data archived in INSDC databases or Zenodo as appropriate).

Weaknesses:

While the medaka variant caller is now deprecated for diploid calling, it is still widely used for haploid variant calling and should at least be mentioned (even if the mention is only to explain its exclusion from the analysis).

Appraisal:

The experiments the authors engaged in are well structured and the results are convincing. I expect that these results will be incorporated into "best practice" bacterial variant calling workflows in the future.

Reviewer #2 (Public Review):

Summary:

Hall et al describe the superiority of ONT sequencing and deep learning-based variant callers to deliver higher SNP and Indel accuracy compared to previous gold-standard Illumina short-read sequencing. Furthermore, they provide recommendations for read sequencing depth and computational requirements when performing variant calling.

Strengths:

The study describes compelling data showing ONT superiority when using deep learning-based variant callers, such as Clair3, compared to Illumina sequencing. This challenges the paradigm that Illumina sequencing is the gold standard for variant calling in bacterial genomes. The authors provide evidence that homopolymeric regions, a systematic and problematic issue with ONT data, are no longer a concern in ONT sequencing.

Weaknesses:

(1) The inclusion of a larger number of reference genomes would have strengthened the study to accommodate larger variability (a limitation mentioned by the authors).

(2) In Figure 2, there are clearly one or two samples that perform worse than others in all combinations (are always below the box plots). No information about species-specific variant calls is provided by the authors but one would like to know if those are recurrently associated with one or two species. Species-specific recommendations could also help the scientific community to choose the best sequencing/variant calling approaches.

(3) The authors support that a read depth of 10x is sufficient to achieve variant calls that match or exceed Illumina sequencing. However, the standard here should be the optimal discriminatory power for clinical and public health utility (namely outbreak analysis). In such scenarios, the highest discriminatory power is always desirable and as such an F1 score, Recall and Precision that is as close to 100% as possible should be maintained (which changes the minimum read sequencing depth to at least 25x, which is the inflection point).

(4) The sequencing of the samples was not performed with the same Illumina and ONT method/equipment, which could have introduced specific equipment/preparation artefacts that were not considered in the study. See for example https://academic.oup.com/nargab/article/3/1/lqab019/6193612.

Reviewer #3 (Public Review):

Hall et al. benchmarked different variant calling methods on Nanopore reads of bacterial samples and compared the performance of Nanopore to short reads produced with Illumina sequencing. To establish a common ground for comparison, the authors first generated a variant truth set for each sample and then projected this set to the reference sequence of the sample to obtain a mutated reference. Subsequently, Hall et al. called SNPs and small indels using commonly used deep learning and conventional variant callers and compared the precision and accuracy from reads produced with simplex and duplex Nanopore sequencing to Illumina data. The authors did not investigate large structural variation, which is a major limitation of the current manuscript. It will be very interesting to see a follow-up study covering this much more challenging type of variation.

In their comprehensive comparison of SNPs and small indels, the authors observed superior performance of deep learning over conventional variant callers when Nanopore reads were basecalled with the most accurate (but also computationally very expensive) model, even exceeding Illumina in some cases. Not surprisingly, Nanopore underperformed compared to Illumina when basecalled with the fastest (but computationally much less demanding) method with the lowest accuracy. The authors then investigated the surprisingly higher performance of Nanopore data in some cases and identified lower recall with Illumina short read data, particularly from repetitive regions and regions with high variant density, as the driver. Combining the most accurate Nanopore basecalling method with a deep learning variant caller resulted in low error rates in homopolymer regions, similar to Illumina data. This is remarkable, as homopolymer regions are (or, were) traditionally challenging for Nanopore sequencing.

Lastly, Hall et al. provided useful information on the required Nanopore read depth, which is surprisingly low, and the computational resources for variant calling with deep learning callers. With that, the authors established a new state-of-the-art for Nanopore-only variant, calling on bacterial sequencing data. Most likely these findings will be transferred to other organisms as well or at least provide a proof-of-concept that can be built upon.

As the authors mention multiple times throughout the manuscript, Nanopore can provide sequencing data in nearly real-time and in remote regions, therefore opening up a ton of new possibilities, for example for infectious disease surveillance.

However, the high-performing variant calling method as established in this study requires the computationally very expensive sup and/or duplex Nanopore basecalling, whereas the least computationally demanding method underperforms. Here, the manuscript would greatly benefit from extending the last section on computational requirements, as the authors determine the resources for the variant calling but do not cover the entire picture. This could even be misleading for less experienced researchers who want to perform bacterial sequencing at high performance but with low resources. The authors mention it in the discussion but do not make clear enough that the described computational resources are probably largely insufficient to perform the high-accuracy basecalling required.

Reviewer #1 (Public Review):

Summary:

Winged seeds or ovules from the Devonian are crucial to understanding the origin and early evolutionary history of wind dispersal strategy. Based on exceptionally well-preserved fossil specimens, the present manuscript documented a new fossil plant taxon (new genus and new species) from the Famennian Series of Upper Devonian in eastern China and demonstrated that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds by using mathematical analysis.

Strengths:

The manuscript is well organised and well presented, with superb illustrations. The methods used in the manuscript are appropriate.

Weaknesses:

I would only like to suggest moving the "Mathematical analysis of wind dispersal of ovules with 1-4 wings" section from the supplementary information to the main text, leaving the supplementary figures as supplementary materials.

Reviewer #2 (Public Review):

Summary:

This manuscript described the second earliest known winged ovule without a capule in the Famennian of Late Devonian. Using Mathematical analysis, the authors suggest that the integuments of the earliest ovules without a cupule, as in the new taxon and Guazia, evolved functions in wind dispersal.

Strengths:

The new ovule taxon's morphological part is convincing. It provides additional evidence for the earliest winged ovules, and the mathematical analysis helps to understand their function.

Weaknesses:

The discussion should be enhanced to clarify the significance of this finding. What is the new advance compared with the Guazia finding? The authors can illustrate the character transformations using a simplified cladogram. The present version of the main text looks flat.

Reviewer #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to identifying a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as species-specific gamete recognition. Additionally, using a TPM >1 threshold for protein-coding transcripts may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis.

Reviewer #2 (Public Review):

Summary:

This is a tour de force study that aims to understand the genetic basis of male germ cell development across three animal species (human, mouse, and flies) by performing a genetic program conservation analysis (using phylostratigraphy and network science) with a special emphasis on genes that peak or decline during mitosis-to-meiosis. This analysis, in agreement with previous findings, reveals that several genes active during and before meiosis are deeply conserved across species, suggesting ancient regulatory mechanisms. To identify critical genes in germ cell development, the investigators integrated clinical genetics data, performing gene knockdown and knockout experiments in both mice and flies. Specifically, over 900 conserved genes were investigated in flies, with three of these genes further studied in mice. Of the 900 genes in flies, ~250 RNAi knockdowns had fertility phenotypes. The fertility phenotypes for the fly data can be viewed using the following browser link: https://pages.igc.pt/meionav. The scope of target gene validation is impressive. Below are a few minor comments.

(1) In Supplemental Figure 2, it is notable that enterocyte transcriptomes are predominantly composed of younger genes, contrasting with the genetic age profile observed in brain and muscle cells. This difference is an intriguing observation and it would be curious to hear the author's comments.

(2) Regarding the document, the figures provided only include supplemental data; none of the main text figures are in the full PDF.

(3) Lastly, it would be great to section and stain mouse testis to classify the different stages of arrest during meiosis for each of the mouse mutants in order to compare more precisely to flies.

This paper serves as a vital resource, emphasizing that only through the analysis of hundreds of genes can we prioritize essential genes for germ cell development. its remarkable that about 60% of conserved genes have no apparent phenotype during germ cell development.

Strengths:

The high-throughput screening was conducted on a conserved network of 920 genes expressed during the mitosis-to-meiosis transition. Approximately 250 of these genes were associated with fertility phenotypes. Notably, mutations in 5 of the 250 genes have been identified in human male infertility patients. Furthermore, 3 of these genes were modeled in mice, where they were also linked to infertility. This study establishes a crucial groundwork for future investigations into germ cell development genes, aiming to delineate their essential roles and functions.

Weaknesses:

The fertility phenotyping in this study is limited, yet dissecting the mechanistic roles of these proteins falls beyond its scope. Nevertheless, this work serves as an invaluable resource for further exploration of specific genes of interest.

Reviewer #1 (Public Review):

Summary:

The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by (1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, (2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), (3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, (4) better defines how the vasculature, nervous, and immune system integrates with the OR.

Strengths:

The data is beautifully presented and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.

Weaknesses:

It is not always clear what the novel findings are that this manuscript is presenting. It appears to be largely similar to the analysis done by McKey et al. (2022) but with more time points and molecular markers. The novelty of the present study's findings needs to be better articulated.

Reviewer #2 (Public Review):

A large number of ovarian experiments have been conducted - especially in morphological and molecular biology studies - specifically removing the ovarian membrane. This experiment is a good supplement to existing knowledge and plays an important role in early ovarian development and the regulation of ovarian homeostasis during the estrous cycle. There are also innovations in research ideas and methods, which will meet the requirements of experimental design and provide inspiration for other researchers.

This reviewer did not identify any major issues with the article. However, the following points could be further clarified:

(1) Is there any comparative data on the proteomics of RO and rete testis in early development? With some molecular markers also derived from rete testis, it would be better to provide the data or references.

(2) Although the size of RO and its components is quite small and difficult to operate, the researchers in this article had already been able to perform intracavitary injection of EOR and extract EOR or CR for mass spectrometry analysis. Therefore, can EOR, CR, or IOR be damaged or removed, providing further strong evidence of ovarian development function?

(3) Although IOR is shown on the schematic diagram, it cannot be observed in the immunohistochemistry pictures in Figure 1 and Figure 3. The authors should provide a detailed explanation.

Reviewer #3 (Public Review):

Summary:

The rete ovarii (RO) has long been disregarded as a non-functional structure within the ovary. In their study, Anbarci and colleagues have delineated the markers and developmental dynamics of three distinct regions of the RO - the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). Notably focusing on the EOR, the authors presented evidence illustrating that the EOR forms a convoluted tubular structure culminating in a dilated tip. Intriguingly, microinjections into this tip revealed luminal flow towards the ovary containing potentially secreted functional proteins. Additionally, the EOR cells exhibit associations with vasculature, macrophages, and neuronal projections, proposing the notion that the RO may play a functional role in ovarian development during critical ovariogenesis stages. By identifying marker genes within the RO, the authors have also suggested that the RO could serve as a potential structure linking the ovary with the neuronal system.

Strengths:

Overall, the reviewer commends the authors for their systematic research on the RO, shedding light on this overlooked structure in developing ovaries. Furthermore, the authors have proposed a series of hypotheses that are both captivating and scientifically significant, with the potential to reshape our understanding of ovarian development through future investigations.

Weaknesses:

There is a lack of conclusive data supporting many conclusions in the manuscript. Therefore, the paper's overall conclusions should be moderated until functional validations are conducted.

Joint Public Review:

Summary:

Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identifies EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts the central pattern generator (CPG) function necessary for motor coordination.

Strengths:

The main strength of this manuscript is the human genetic data, which provides convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.

Weaknesses:

The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not well supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. At a minimum, the language of the manuscript could be toned down, with the CPG defects put forward as a potential explanation for scoliosis in the discussion rather than as something this manuscript has "shown". An additional weakness of the manuscript is that the zebrafish genetic tools are not sufficiently validated to provide full confidence in the data and conclusions.

Reviewer #1 (Public Review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

Reviewer #2 (Public Review):

Summary:

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications.

Strengths:

Algorithm is almost as accurate as manual tracking and importantly saves time for researchers.

Weaknesses:

Applicability can be questioned because the device used is 2D and physiological biology is in 3D. Comparisons to other automated tools was not performed by the authors.

Reviewer #3 (Public Review):

Summary:

The authors aimed to establish a faster and more efficient method of tracking steps of T-cell extravasation across the blood brain barrier. The authors developed a framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging. The authors succinctly describe the basic requirements for tracking in the introduction followed by an in-depth account of the execution.

Weaknesses and Strengths:

Materials & methods and results:

(1) The methods section also lacks details of the microfluidic device that the authors talk about in the paper. Under physiological sheer stress, the T-cells detach from the pMBMEC monolayer, and are hence unable to be detected; however, this observation requires an explanation pertaining to the reason of occurrence and potential solutions to circumvent it to ensure physiologically relevant experimental parameters.

(2) The author describes a method for debris exclusion using UFMTrack that eliminates objects of <30 pixels in size from analysis based on a mean pixel size of 400 for T lymphocytes. However, this mean pixel size appears to stem from in-vitro activated CD8 T cells, which rapidly grow and proliferate upon stimulation. In line with this, activated lymphocytes exhibit increased cytoplasmic area, making them appear less dense or "brighter" by phase microscopy compared to naïve lymphocytes, which are relatively compact and subsequently appear dimmer. Given this, it is not clear whether UFMTrack is sufficiently trained to identify naïve human lymphocytes in circulating blood, nor smaller, murine lymphocytes. Analysis of each lymphocyte subtype in terms of pixel size and intensity would be beneficial to strengthen the claim that UFMTrack can identify each of these populations. Additionally, demonstrating that UFMTrack can correctly characterize the behavior of naïve versus activated lymphocytes isolated from murine and human sources would strengthen the claim that UFMTrack can be broadly applied to study lymphocyte dynamics in diverse models without additional training

(3) Average precision was compared to the analysis of UFMTrack but it is unclear how average precision was calculated. This information should have been included in the methods section

(4) CD4 and CD8 T cells exhibit distinct biology and interaction kinetics driven in part by their MHC molecule affinity and distinct receptor expression profiles. Thus, it is unclear why two distinct mechanisms of endothelial cell activation are needed to see differences between the populations.

(5) The BMECs are barrier tissues but were cultured on µdishes in this study. To study the transmigration of T-cells across the endothelium, the model would have been more relevant on a semi-permeable membrane instead of a closed surface.

(6) Methods are provided for the isolation and expansion of human effector and memory CD4+ T cells. However, there is no mention of specific CD4+ T cell populations used for analysis with UFMTrack, nor a clear breakdown of tracking efficiency for each subpopulation. Further, there is no similar method for the isolation of CD8+ T cell compartments. A clear breakdown of the performance efficiency of UFMTrack with each cell population investigated in this study would provide greater insight into the software's performance with regard to tracking the behavior and movement of distinct immune populations.

(7) The results section is quite extensive and discusses details of establishment of the framework while highlighting both the pros and cons of the different aspects of the process, for example the limitation of the two models, 2D and 2D+T were highlighted well. However, the results section includes details which may be more fitting in the methods section.

(8) A few statements in the results section lacked literary support, which was not provided in the discussion either, such as support for increased variance of T-cell instantaneous speed on stimulated vs non-stimulated pMBMECs. Another example is the enhancement of cytokine stimulation directed T-cell movement on the pMBMECs that the authors observed but failed to relay the physiological relevance of it. The authors don't provide enough references for developments in the field prior to their work which form the basis and need for this technology.

(9) The rationale for use of OT-1 and 2D2-derived murine lymphocytes is unclear here. The OT-1 model has been generated to study antigen-specific CD8+ T cell responses, while the 2D2 model has been generated to recapitulate CD4 T cell-specific myelin oligodendrocyte glycoprotein (MOG) responses.

Figures and text:

(1) There are certain discrepancies and misarrangement of figures and text. For example, discussion of the effect of sheer flow on T cell attachment as part of the introduction in figure 1 and then mentioning it in the text again in the results section as part of figure 4 is repetitive.

(2) Section IV, subsection 1 of the results section, refers to 'data acquisition section above' in line 279, however the said section is part of materials and methods which is provided towards the end of the manuscript.

(3) There are figures in the manuscript that have not been referenced in the results section, for example, figure 3A and B. Figure 1 hasn't been addressed until subsection 7 of materials and methods

(4) A lack of significance but an observed trend of increased variance of T cell instantaneous speed is reported in line 296-298; however, the graph (figure 4G) shows a significant change in instantaneous speed between non-stimulated and TNFα-stimulated systems. This is misleading to the readers.

(5) The authors talk about three beginner experimentors testing the manual T cell tracking process but figure 5 only showcases data from two experimentors without stating the reason for excluding experimentor 1.

Discussion:

(1) While the discussion captures the major takeaways from the paper, it lacks relevant supporting references to relate the observation to physiological conditions and applicability.

(2) The discussion lacks connection to the results since the figures were not referenced while discussing an observed trend

(3) The authors briefly looked into mouse and human BMECs and their individual interaction with T-cells, but don't discuss the differences between the two, if any, that challenged their framework.

(4) Even though though the imaging tool relies on difference in appearance for detection, the authors talk about lack of feasibility in detecting transmigration of BMDMs due to their significantly different appearance. The statement lacks a problem solving approach to discuss how and why this was the case.

Relevance to the field:

Utilizing the framework provided by the authors, the application can be adapted and/or utilized for visualizing a range of different cell types, provided they are different in appearance. However, this would require extensive changes to the script and won't be adaptable in its current form.

Reviewer #1 (Public Review):

Summary:

The study seeks to establish accurate computational models to explore the role of hydrodynamic interactions on energy savings and spatial patterns in fish schools. Specifically, the authors consider a system of (one degree-of-freedom) flapping airfoils that passively position themselves with respect to the streamwise direction, while oscillating at the same frequency and amplitude, with a given phase lag and at a constant cross-stream distance. By parametrically varying the phase lag and the cross-stream distance, they systematically explore the stability and energy costs of emergent configurations. Computational findings are leveraged to distill insights into universal relationships and clarify the role of the wake of the leading foil.

Strengths:

(1) The use of multiple computational models (computational fluid dynamics, CFD, for full Navier-Stokes equations and computationally efficient inviscid vortex sheet, VS, model) offers an extra degree of reliability of the observed findings and backing to the use of simplified models for future research in more complex settings.

(2) The systematic assessment of the stability and energy savings in multiple configurations of pairs and larger ensembles of flapping foils is an important addition to the literature.

(3) The discovery of a linear phase-distance relationship in the formation attained by pairs of flapping foils is a significant contribution, which helps compare different experimental observations in the literature.

(4) The observation of a critical size effect for in-line formations of larger, above which cohesion and energetic benefits are lost at once, is a new discovery in the field.

Weaknesses:

(1) The extent to which observations on one-degree-of-freedom flapping foils could translate to real fish schools is presently unclear so some of the conclusions on live fish schools are likely to be overstated and would benefit from some more biological framing.

(2) The analysis of non-reciprocal coupling is not as novel as the rest of the study and potentially not as convincing due to the chosen linear metric of interaction (that is, the flow agreement).

Overall, this is a rigorous effort on a critical topic: findings of the research can offer important insight into the hydrodynamics of fish schooling, stimulating interdisciplinary research at the interface of computational fluid mechanics and biology.

Reviewer #2 (Public Review):

The document "Mapping spatial patterns to energetic benefits in groups of flow-coupled swimmers" by Heydari et al. uses several types of simulations and models to address aspects of stability of position and power consumption in few-body groups of pitching foils. I think the work has the potential to be a valuable and timely contribution to an important subject area. The supporting evidence is largely quite convincing, though some details could raise questions, and there is room for improvement in the presentation. My recommendations are focused on clarifying the presentation and perhaps spurring the authors to assess additional aspects:

(1) Why do the authors choose to set the swimmers free only in the propulsion direction? I can understand constraining all the positions/orientations for investigating the resulting forces and power, and I can also understand the value of allowing the bodies to be fully free in x, y, and their orientation angle to see if possible configurations spontaneously emerge from the flow interactions. But why constrain some degrees of freedom and not others? What's the motivation, and what's the relevance to animals, which are fully free?

(2) The model description in Eq. (1) and the surrounding text is confusing. Aren't the authors computing forces via CFD or the VS method and then simply driving the propulsive dynamics according to the net horizontal force? It seems then irrelevant to decompose things into thrust and drag, and it seems irrelevant to claim that the thrust comes from pressure and the drag from viscous effects. The latter claim may in fact be incorrect since the body has a shape and the normal and tangential components of the surface stress along the body may be complex.

(3) The parameter taudiss in the VS simulations takes on unusual values such as 2.45T, making it seem like this value is somehow very special, and perhaps 2.44 or 2.46 would lead to significantly different results. If the value is special, the authors should discuss and assess it. Otherwise, I recommend picking a round value, like 2 or 3, which would avoid distraction.

(4) Some of the COT plots/information were difficult to interpret because the correspondence of beneficial with the mathematical sign was changing. For example, DeltaCOT as introduced on p. 5 is such that negative indicates bad energetics as compared to a solo swimmer. But elsewhere, lower or more negative COT is good in terms of savings. Given the many plots, large amounts of data, and many quantities being assessed, the paper needs a highly uniform presentation to aid the reader.

(5) I didn't understand the value of the "flow agreement parameter," and I didn't understand the authors' interpretation of its significance. Firstly, it would help if this and all other quantities were given explicit definitions as complete equations (including normalization). As I understand it, the quantity indicates the match of the flow velocity at some location with the flapping velocity of a "ghost swimmer" at that location. This does not seem to be exactly relevant to the equilibrium locations. In particular, if the match were perfect, then the swimmer would generate no relative flow and thus no thrust, meaning such a location could not be an equilibrium. So, some degree of mismatch seems necessary. I believe such a mismatch is indeed present, but the plots such as those in Figure 4 may disguise the effect. The color bar is saturated to the point of essentially being three tones (blue, white, red), so we cannot see that the observed equilibria are likely between the max and min values of this parameter.

(6) More generally, and related to the above, I am favorable towards the authors' attempts to find approximate flow metrics that could be used to predict the equilibrium positions and their stability, but I think the reasoning needs to be more solid. It seems the authors are seeking a parameter that can indicate equilibrium and another that can indicate stability. Can they clearly lay out the motivation behind any proposed metrics, and clearly present complete equations for their definitions? Further, is there a related power metric that can be appropriately defined and which proves to be useful?

(7) Why do the authors not carry out CFD simulations on the larger groups? Some explanations should be given, or some corresponding CFD simulations should be carried out. It would be interesting if CFD simulations were done and included, especially for the in-line case of many swimmers. This is because the results seem to be quite nuanced and dependent on many-body effects beyond nearest-neighbor interactions. It would certainly be comforting to see something similar happen in CFD.

(8) Related to the above, the authors should discuss seemingly significant differences in their results for long in-line formations as compared to the CFD work of Peng et al. [48]. That work showed apparently stable groups for numbers of swimmers quite larger than that studied here. Why such a qualitatively different result, and how should we interpret these differences regarding the more general issue of the stability of tandem groups?

(9) The authors seem to have all the tools needed to address the general question about how dynamically stable configurations relate to those that are energetically optimal. Are stable solutions optimal, or not? This would seem to have very important implications for animal groups, and the work addresses closely related topics but seems to miss the opportunity to give a definitive answer to this big question.

(10) Time-delay particle model: This model seems to construct a simplified wake flow. But does the constructed flow satisfy basic properties that we demand of any flow, such as being divergence-free? If not, then the formulation may be troublesome.

Reviewer #1 (Public Review):

Summary:

This is an interesting study by Xu et al showing the effects of infection with the Treponema pallidum virus (which causes syphilis disease) on neuronal development using iPSC-derived human brain organoids as a model and single-cell RNA sequencing. This work provides an important insight into the impact of the virus on human development, bridging the gap between the phenomena observed in studies using animal models as well as non-invasive human studies showing developmental abnormalities in fetuses infected with the virus in utero through maternal vertical transmission.

Using single-cell RNAseq in combination with qPCR and immunofluorescence techniques, the authors show that T. pallidum infected organoids are smaller in size, in particular during later growth stages, contain a larger number of undifferentiated neuronal lineage cells, and exhibit decreased numbers of specific neuronal subcluster, which the authors have identified as undifferentiated hindbrain neurons.

The study is an important first step in understanding how T. pallidum affects human neuronal development and provides important insight into the potential mechanisms that underlie the neurodevelopmental abnormalities observed in infected human fetuses.

Strengths:

(1) The study is well written, and the data quality is good for the most part.

(2) The study provides an important first step in utilizing human brain organoids to study the impact of T. pallidum infection on neuronal development.

(3) The study's conclusions may provide important insight to other researchers focused on studying how viral infections impact neuronal development.

Reviewer #3 (Public Review):

This article is the first report to study the effects of T. pallidum on the neural development of an iSPC-derived brain organoid model. The study indicates that T. pallidum inhibits the differentiation of subNPC1B neurons into hindbrain neurons, hence affecting brain organoid neurodevelopment. Additionally, the TCF3 and notch signaling pathways may be involved in the inhibition of the subNPC1B-hindbrain neuron differentiation axis. While the majority of the data in this study support the conclusions, there are still some questions that need to be addressed and data quality needs to be improved. The study provides valuable insights for future investigations into the mechanisms underlying congenital neurodevelopment disability.

Reviewer #1 (Public Review):

The revised manuscript "Diffusive lensing as a mechanism of intracellular transport and compartmentalization" is very similar to the original manuscript. The main difference between the revised and the original manuscript is that the authors have removed the reference to viscosity gradient and instead talk of diffusivity gradient. With this change the manuscript the analysis and claims in the manuscript are much more aligned. The manuscript, as the original version, explores the role of spatially varying diffusion constant in three scenarios:

(i) Spatial localization of non-particles<br /> (ii) Clustering in presence of inter-particle interactions<br /> (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity.

Since the manuscript has not changed much the strengths and weaknesses, in my opinion, remain similar to that of the original manuscript.

Strengths: The implications of a heterogeneous environment on phase separation and reaction kinetics in cells are under-explored. This makes the general theme of this manuscript relevant and interesting.

Weaknesses: The central part of the paper "diffusive lensing", i.e., particles localizing in the region of low diffusion constant is not new. Some of the papers authors cite already show that. The parts on phase separation and frap analysis that could provide new results are not rigorous enough for a theory paper.

I reiterate some of my comments from the original version that are valid for the revised version as well.

My main criticism was not to say that some convention should be used or some not. But instead, the main point was to say that just because there is spatial diffusion constant that does not mean there will be a spatial gradient of particles. From the authors response to my comments, it is clear that they understand the subtilties around it and are aware of the relevant papers. However, a reader not familiar with this discussion may work under the impression that if there if there is a spatialy varying diffusion constant in cell there will be an accumulation of particles in the region of low diffusivity but that may not always be the case. Moreover, localisation of particles in the region of low diffusivity has been reported in many different context. Some of the papers that the author cite already show that. For example, in Rupprecht et al. 2018 non-isothermal interpretation is applied to the dynamics of objects inside cells.

Given that the central result is not new. The paper could still be of general interest to the biophysics community if the follow up sections (ii) Clustering in presence of inter-particle interactions and (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity were analysed rigorously.

Reviewer #2 (Public Review):

Summary:

The authors study through theory and simulations the diffusion of microscopic particles, and aim to account for the effects of inhomogeneous viscosity and diffusion - in particular regarding the intracellular environment. They propose a mechanism, termed "Diffusive lensing", by which particles are attracted towards low-diffusivity regions where they remain trapped. To obtain these results, the authors rely on agent-based simulations using custom rules performed within the Ito stochastic calculus convention, without drift. They acknowledge the fact that this convention does not describe equilibrium systems, and that their results would not hold at equilibrium - and discard these facts by invoking the facts that cells are out-of-equilibrium. Finally, they show some applications of their findings, in particular enhanced clustering in the low-diffusivity regions. The authors conclude that as inhomogeneous diffusion is ubiquitous in life, so must their mechanism be, and hence it must be important.

Strengths:

The article is well-written, clearly intelligible, its hypotheses are stated relatively clearly and the models and mathematical derivations are compatible with these hypotheses. In the appendices, the authors connect their findings to known results for classic stochastic differential equation formalisms.

Weaknesses:

This study is, in my opinion, deeply flawed. The main problem lies in the hypotheses, in particular the choice of considering drift-less dynamics in the Ito convention. It is regrettable that the authors choose to use agent-based custom simulations with little physical motivation, rather than a well-established stochastic differential equations framework.

Indeed, stochastic conventions are a notoriously tricky business, but they are both mathematically and physically well-understood and do not result in any "dilemma" [some citations in the article, such as (Lau and Lubensky) and (Volpe and Wehr), make an unambiguous resolution of these]. In the continuous-time limit, conventions are not an intrinsic, fixed property of a system, but a choice of writing; however, whenever going from one to another, one must include a corresponding "spurious drift" that compensates the effect of this change - a mathematical subtlety that is omitted in the article (except in a quick note in the appendix): in the presence of diffusive gradients, if the drift is zero in one convention, it will thus be non-zero in another. It is well established that for equilibrium systems obeying fluctuation-dissipation, the spurious drift vanishes in the anti-Ito stochastic convention; more precisely one can write in the anti-Ito convention

dx/dt = - D(x)/kT grad U(x) + sqrt(2D(x)) dW

with D(x) the diffusion, kT the thermal energy (which is space-independent at equilibrium), and dW a d-dimensional Wiener process. Equivalently one can write in the Ito convention:

dx/dt = - D(x)/kT grad U(x) + sqrt(2D(x)) dW + div D(x) (*)

where the latter term is the spurious drift arising from convention change. This ensures that the diffusion gradients do not induce currents and probability gradients, and thus that the steady-state PDF is the Gibbs measure (this form has been confirmed experimentally, for instance, for colloidal particles near walls, that have strong diffusivity gradients despite not having significant forces). It generalizes to near-equilibrium systems with non-conservative forces and/or temperature gradient in the form:

dx/dt = F(x) + sqrt(2D(x)) dW + div D(x) (**)

where the drift field F(x) encodes these forces. In some cases, it has been shown through careful microscopic analysis that one can have effectively a different form for the last term, namely

dx/dt = F(x) + sqrt(2D(x)) dW + alpha div D(x)

where alpha is a "convention parameter" that would be =1 at equilibrium. For instance, in the Volpe and Wehr review this can occur through memory effects in robotic dynamics, or through strong fluctuation-dissipation breakdown. In a near-equilibrium system, this should be strongly justified, as the continuous-time dynamics with alpha \neq 1 and drift F would be indistinguishable from one with alpha = 1 and drift F + (1-alpha) div D: the authors would have the burden of proving that the observed (absence of) drift is indeed due to alpha\neq 1, rather than to much more common force fields F(x).

Here, without further motivation than the statement that cells are out-of-equilibrium, drifts are arbitrarily set to zero in the Ito convention, which is in (**) the equivalent to adding a force with drift $-div D$ exactly compensating the spurious drift. It is the effects of this arbitrary force that are studied in the article. The fact that it results in probability gradients is trivial once formulated this way (and in no way is this new - many of the references, for instance Volpe and Wehr, mention this). Enhanced clustering is also a trivial effect of this probability gradient (the local concentration is increased by this force field, so phase separation can occur). As a side note the "neighbor sensing" scheme to describe interactions is itself very peculiar and not physically motivated - it violates stochastic thermodynamics laws too, as detailed balance is apparently not respected. There again, the authors have chosen to disregard a century of stochastic thermodynamics in favor of a non-justified unphysical custom rule.

The authors make no further justification of their choice of driftless Ito simulations than the fact that cells are out-of-equilibrium, leaving the feeling that this is a detail. They make mentions of systems (eg glycogen, prebiotic environment) for which (near-)equilibrium physics should mostly prevail, and of fluctuation dissipation ("Diffusivity varies inversely with viscosity", in the introduction). Yet the "phenomenon" they discuss is entirely reliant on an undiscussed mechanism by which these assumptions would be completely violated (the citations they make for this - Gnesotto '18 and Phillips '12 - are simply discussions of the fact that cells are out-of-equilibrium, not on any consequences on the convention).

Finally, while inhomogeneous diffusion is ubiquitous, the strength of this effect in realistic conditions is not discussed. Even in the most "optimistic" case where alpha=0 would make sense (knowing that in the cellular context we are discussing thermal systems immersed in water and if energy consumption and metabolism were stopped alpha would relax back to 1), the equation (*) above shows that having zero ito drift is equivalent to having a potential countering the spurious drift, with value

U(x) = kT log(D(x) / D0 )

[I have assumed isotropic diffusion for simplicity here, so the div is replaced by a grad]. This means that the diffusion contrasts logarithmically compare to the chemical potential ones -- for instance a major diffusion difference of 100x is equivalent to 4.6kT in potential energy, a relatively modest effect. To prove that the authors' effect of "diffusive lensing" is involved in such a system, one would thus have to<br /> 1) observe strong spatial variations of the diffusion coefficient (this is doable, and was done before), AND<br /> 2) show that there is an enrichment of the diffusing species in the low-diffusion region inversely proportional to the diffusion, AND<br /> 3) show that this enrichment cannot be attributed to mild differences in potential energy, for instance by showing that if nonequilibrium energy consumption stops, the concentration fully homogenizes while the diffusion gradients remain.

If the authors were to successfully show all that in an experimental system, or design a theoretical framework where these effects convincingly emerge from physically realistic microscopic dynamical rules, they would have indeed discovered a new phenomenon. In contrast, the current article only demonstrates the well-known fact that when using arbitrary dynamical rules in heterogeneous diffusion simulations, one can get concentration gradients.

Joint Public Review:

This manuscript by Yue et al. aims to understand the molecular mechanisms underlying the better reproductive outcomes of Tibetans at high altitude by characterizing the transcriptome and histology of full-term placenta of Tibetans and compare them to those Han Chinese at high elevations.

The approach is innovative, and the data collected are valuable for testing hypotheses regarding the contribution of the placenta to better reproductive success of populations that adapted to hypoxia. The authors identified hundreds of differentially expressed genes (DEGs) between Tibetans and Han, including the EPAS1 gene that harbors the strongest signals of genetic adaptation. The authors also found that such differential expression is more prevalent and pronounced in the placentas of male fetuses than those of female fetuses, which is particularly interesting, as it echoes with the more severe reduction in birth weight of male neonates at high elevation observed by the same group of researchers (He et al., 2022).

Comments on latest version:

The revised manuscript has incorporated the suggested changes and weakened conclusions regarding natural selection. Limitations of the study are also clearly stated in the Discussion section.

Reviewer #1 (Public Review):

This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.

Comments on revised version:

This is my second review of this article, now entitled "Multi-study fMRI outlooks on subcortical BOLD responses in the stop-signal paradigm" by Isherwood and colleagues.

The authors have been very responsive to the initial round of reviews.

I still think it would be helpful to see a combined investigation of the available 7T data, just to really drive the point home that even with the best parameters and a multi-study sample size, fMRI cannot detect any increases in BOLD activity on successful stop compared to go trials. However, I agree with the authors that these "sub samples still lack the temporal resolution seemingly required for looking at the processes in the SST."

As such, I don't have any more feedback.

Reviewer #2 (Public Review):

This work aggregates data across 5 openly available stopping studies (3 at 7 tesla and 2 at 3 tesla) to evaluate activity patterns across the common contrasts of Failed Stop (FS) > Go, FS > stop success (SS), and SS > Go. Previous work has implicated a set of regions that tend to be positively active in one or more of these contrasts, including the bilateral inferior frontal gyrus, preSMA, and multiple basal ganglia structures. However, the authors argue that upon closer examination, many previous papers have not found subcortical structures to be more active on SS than FS trials, bringing into question whether they play an essential role in (successful) inhibition. In order to evaluate this with more data and power, the authors aggregate across five datasets and find many areas that are *more* active for FS than SS, including bilateral preSMA, GPE, thalamus, and VTA. They argue that this brings into question the role of these areas in inhibition, based upon the assumption that areas involved in inhibition should be more active on successful stop than failed stop trials, not the opposite as they observed.

Since the initial submission, the authors have improved their theoretical synthesis and changed their SSRT calculation method to the more appropriate integration method with replacement for go omissions. They have also done a better job of explaining how these fMRI results situate within the broader response inhibition literature including work using other neuroscience methods.

They have also included a new Bayes Factor analysis. In the process of evaluating this new analysis, I recognized the following comments that I believe justify additional analyses and discussion:

First, if I understand the author's pipeline, for the ROI analyses it is not appropriate to run FSL's FILM method on the data that were generated by repeating the same time series across all voxels of an ROI. FSL's FILM uses neighboring voxels in parts of the estimation to stabilize temporal correlation and variance estimates and was intended and evaluated for use on voxelwise data. Instead, I believe it would be more appropriate to average the level 1 contrast estimates over the voxels of each ROI to serve as the dependent variables in the ROI analysis.

Second, for the group-level ROI analyses there seems to be inconsistencies when comparing the z-statistics (Figure 3) to the Bayes Factors (Figure 4) in that very similar z-statistics have very different Bayes Factors within the same contrast across different brain areas, which seemed surprising (e.g., a z of 6.64 has a BF of .858 while another with a z of 6.76 has a BF of 3.18). The authors do briefly discuss some instances in the frequentist and Bayesian results differ, but they do not ever explain by similar z-stats yield very different bayes factors for a given contrast across different brain areas. I believe a discussion of this would be useful.

Third, since the Bayes Factor analysis appears to be based on repeated measures ANOVA and the z-statistics are from Flame1+2, the BayesFactor analysis model does not pair with the frequentist analysis model very cleanly. To facilitate comparison, I would recommend that the same repeated measures ANOVA model should be used in both cases. My reading of the literature is that there is no need to be concerned about any benefits of using Flame being lost, since heteroscedasticity does not impact type I errors and will only potentially impact power (Mumford & Nichols, 2009 NeuroImage).

Fourth, though frequentist statistics suggest that many basal ganglia structures are significantly more active in the FS > SS contrast (see 2nd row of Figure 3), the Bayesian analyses are much more equivocal, with no basal ganglia areas showing Log10BF > 1 (which would be indicative of strong evidence). The authors suggest that "the frequentist and Bayesian analyses are monst in line with one another", but in my view, this frequentist vs. Bayesian analysis for the FS > SS contrast seems to suggest substantially different conclusions. More specifically, the frequentist analyses suggest greater activity in FS than SS in most basal ganglia ROIs (all but 2), but the Bayesian analysis did not find *any* basal ganglia ROIs with strong evidence for the alternative hypothesis (or a difference), and several with more evidence for the null than the alternative hypothesis. This difference between the frequentist and Bayesian analyses seems to warrant discussion, but unless I overlooked it, the Bayesian analyses are not mentioned in the Discussion at all. In my view, the frequentist analyses are treated as the results, and the Bayesian analyses were largely ignored.

Overall, I think this paper makes a useful and mostly solid contribution to the literature. I have made some suggestions for adjustments and clarification of the neuroimaging pipeline and Bayesian analyses that I believe would strengthen the work further.

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #2 (Public Review):

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

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

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

Strengths:

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

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

Limitations:

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

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

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

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

(5) Under the objective considered by the authors, the amount of input occurring before the movement might be impacted by the presence of online sensory signals for closed-loop control. Even if considering that the inputs could include some sensory activity and/or that the RNN activity could represent general variables whose states can be decoded from M1, the model would not include mechanisms that process imperfect (delayed, noisy) sensory feedback to adapt the output in a trial-specific manner. It is therefore an open question whether the objective and network characteristics suggested by the authors could also explain the presence of preparatory activity before e.g. grasping movements that are thought to be more sensory-driven (Meirhaeghe et al., Cell Reports 2023).

Reviewer #3 (Public Review):

I remain enthusiastic about this study. The manuscript is well-written, logical, and conceptually clear. To my knowledge, no prior modeling study has tackled the question of 'why prepare before executing, why not just execute?' Prior studies have simply assumed, to emulate empirical findings, that preparatory inputs precede execution. They never asked why. The authors show that, when there are constraints on inputs, preparation becomes a natural strategy. In contrast, with no constraint on inputs, there is no need for preparation as one could get anything one liked just via the inputs during movement. For the sake of tractability, the authors use a simple magnitude constraint: the cost function punishes the integral of the squared inputs. Thus, if small inputs before movement can reduce the size of the inputs needed during movement, preparation is a good strategy. This occurs if (and only if) the network has strong dynamics (otherwise feeding it preparatory activity would not produce anything interesting). All of this is sensible and clarifying.

As discussed in the prior round of reviews, the central constraint that the authors use is a mathematically tractable stand-in for a range of plausible (but often trickier to define and evaluate) constraints, such as simplicity of inputs (or inputs being things that other areas could provide). The manuscript now embraces this fact more explicitly, and also gives some results showing that other constraints (such as on the derivative of activity, which is one component of complexity) can have the same effect. The manuscript also now discusses and addresses a modest weakness of the previous manuscript: the preparatory activity in their simulations is often overly complex temporally, lacking the (rough) plateau typically seen for data. Depending on your point of view, this is simply 'window dressing', but from my perspective it was important to know that their approach could yield more realistic-looking preparatory activity. Both these additions (the new constraint, and the more realistic temporal profile of preparatory activity) are added simply as supplementary figures rather than in the main text, and are brought up only in the Discussion. At first this struck me as slightly odd, but in the end I think this is appropriate. These are really Discussion-type issues, and dealing with them there makes sense. The 'different constraints' issue in particular is deep, tricky to explore for technical reasons, and could thus support a small research program. I think it is fair to talk about it thoughtfully (as the Discussion now does) and then just mention some simple results.

My remaining comments largely pertain to some subtle (but to me important) nuances at a few locations in the text. These should be easy for the authors to address, in whatever way they see fit.

Specific comments:

(1) The authors state the following on line 56: "For preparatory processes to avoid triggering premature movement, any pre-movement activity in the motor and dorsal pre-motor (PMd) cortices must carefully exclude those pyramidal tract neurons."<br /> This constraint is overly restrictive. PT neurons absolutely can change their activity during preparation in principle (and appear to do so in practice). The key constraint is looser: those changes should have no net effect on the muscles. E.g., if d is the vector of changes in PT neuron firing rates, and b is the vector of weights, then the constraint is that b'd = 0. d = 0 is one good way of doing this, but only one. Half the d's could go up and half could go down. Or they all go up, but half the b's are negative. Put differently, there is no reason the null space has to be upstream of the PT neurons. It could be partly, or entirely, downstream.<br /> In the end, this doesn't change the point the authors are making. It is still the case that d has to be structured to avoid causing muscle activity, which raises exactly the point the authors care about: why risk this unless preparation brings benefits? However, this point can be made with a more accurate motivation. This matters, because people often think that a null-space is a tricky thing to engineer, when really it is quite natural. With enough neurons, preparing in the null space is quite simple.

(2) Line 167: 'near-autonomous internal dynamics in M1'.<br /> It would be good if such statements, early in the paper, could be modified to reflect the fact that the dynamics observed in M1 may depend on recurrence that is NOT purely internal to M1. A better phrase might be 'near-autonomous dynamics that can be observed in M1'. A similar point applies on line 13. This issue is handled very thoughtfully in the Discussion, starting on line 713. Obviously it is not sensible to also add multiple sentences making the same point early on. However, it is still worth phrasing things carefully, otherwise the reader may have the wrong impression up until the Discussion (i.e. they may think that both the authors, and prior studies, believe that all the relevant dynamics are internal to M1). If possible, it might also be worth adding one sentence, somewhere early, to keep readers from falling into this hole (and then being stuck there till the Discussion digs them out).

(3) The authors make the point, starting on line 815, that transient (but strong) preparatory activity empirically occurs without a delay. They note that their model will do this but only if 'no delay' means 'no external delay'. For their model to prepare, there still needs to be an internal delay between when the first inputs arrive and when movement generating inputs arrive.

This is not only a reasonable assumption, but is something that does indeed occur empirically. This can be seen in Figure 8c of Lara et al. Similarly, Kaufman et al. 2016 noted that "the sudden change in the CIS [the movement triggering event] occurred well after (~150 ms) the visual go cue... (~60 ms latency)" Behavioral experiments have also argued that internal movement-triggering events tend to be quite sluggish relative to the earliest they could be, causing RTs to be longer than they should be (Haith et al. Independence of Movement Preparation and Movement Initiation). Given this empirical support, the authors might wish to add a sentence indicating that the data tend to justify their assumption that the internal delay (separating the earliest response to sensory events from the events that actually cause movement to begin) never shrinks to zero.

While on this topic, the Haith and Krakauer paper mentioned above good to cite because it does ponder the question of whether preparation is really necessary. By showing that they could get RTs to shrink considerably before behavior became inaccurate, they showed that people normally (when not pressured) use more preparation time than they really need. Given Lara et al, we know that preparation does always occur, but Haith and Krakauer were quite right that it can be very brief. This helped -- along with neural results -- change our view of preparation from something more cognitive that had to occur, so something more mechanical that was simply a good network strategy, which is indeed the authors current point. Working a discussion of this into the current paper may or may not make sense, but if there is a place where it is easy to cite, it would be appropriate.

Reviewer #1 (Public Review):

Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure.

Reviewer #2 (Public Review):

In this work, Dasgupta et al. investigate the role of Sema7a in the formation of peripheral sensory circuit in the lateral line system of zebrafish. They show that Sema7a protein is present during neuromast maturation and localized, in part, to the base of hair cells (HCs). This would be consistent with pre-synaptic Sema7a mediating formation and/or stabilization of the synapse. They use sema7a loss-of-function strain to show that lateral line sensory terminals display abnormal arborization. They provide highly quantitative analysis of the lateral line terminal arborization to show that a number of specific topological parameters are affected in mutants. Next, they ectopically express a secreted form of Sema7a to show that lateral line terminals can be ectopically attracted to the source. Finally, they also demonstrate that the synaptic assembly is impaired in the sema7a mutant. Overall, the data are of high quality and properly controlled. The availability of Sema7a antibody is a big plus, as it allows to address the endogenous protein localization as well to show the signal absence in the sema7a mutant. The quantification of the arbor topology should be useful to people in the field who are looking at the lateral line as well as other axonal terminals.

Reviewer #3 (Public Review):

The data reported here demonstrate that Sema7a defines the local behavior of growing axons in the developing zebrafish lateral line. The analysis is sophisticated and convincingly demonstrates effects on axon growth and synapse architecture. Collectively, the findings point to the idea that the diffusible form of sema7a may influence how axons grow within the neuromast and that the GPI-linked form of sema7a may subsequently impact how synapses form, though additional work is needed to strongly link each form to its' proposed effect on circuit assembly.

Comments on latest version:

The authors comprehensively and appropriately addressed most of the reviewers' concerns. In particular, they added evidence that hair cells express both Sema7A isoforms, showed that membrane bound Sema7A does not have long range effects on guidance, demonstrated how axons behave close to ectopic Sema7A, and analyzed other features of the hair cells that revealed no strong phenotypes. The authors also softened the language in many, but not all places. Overall, I am satisfied with the study as a whole.

Reviewer #4 (Public Review):<br /> <br /> This study provides direct evidence showing that Sema7a plays a role in the axon growth during the formation of peripheral sensory circuits in the lateral-line system of zebrafish. This is a valuable finding because the molecules for axon growth in hair-cell sensory systems are not well understood. The majority of the experimental evidence is convincing, and the analysis is rigorous. The evidence supporting Sema7a's juxtracrine vs. secreted role and involvement in synapse formation in hair cells is less conclusive. The study will be of interest to cell, molecular and developmental biologists, and sensory neuroscientists.

Reviewer #1 (Public Review):

Summary:

People with Parkinson's disease often experience a variety of nonmotor symptoms, the biological bases of which remain poorly understood. Johansson et al began to study potential roles of the dorsal raphe nucleus (DRN) degeneration in the pathophysiology of neuropsychiatric symptoms in PD.

Strengths:

Boi et al validated a transgenic reporter mouse line that can reliably label dopaminergic neurons in the DRN. This brain region shows severe neurodegeneration and has been proposed to contribute to the manifestation of neuropsychiatric symptoms in PD. Using this mouse line (and others), Boi and colleagues characterized electrophysiological and morphological phenotypes of dopaminergic and serotoninergic neurons in the raphe nucleus. This study involved very careful topographical registration of recorded neurons to brain slices for post hoc immunohistochemical validation of cell identity, making it an elegant and thorough piece of work.

Of relevance to PD pathophysiology, the authors evaluated the physiological and morphological changes of DRN serotoninergic and dopaminergic neurons after a partial loss of nigrostriatal dopamine neurons, which serves as a mouse model of early parkinsonian pathology. Moreover, the authors identified a series of physiological and morphological changes of subtypes of DRN neurons that depend on nigral dopaminergic neurodegeneration, LC noradrenergic neurodegeneration, or both. Indeed this work highlights the importance of LC noradrenergic degeneration in PD pathophysiology.

Overall, this is a well-designed study with high significance to the Parkinson's research field.

Reviewer #2 (Public Review):

In this paper, Boi et al. thoroughly classified the electrophysiological and morphological characteristics of serotonergic and dopaminergic neurons in the DRN and examined the alterations of these neurons in the 6-OHDA-induced mouse PD model. Using whole-cell patch clamp recording, they found that 5-HT and dopamine (DA) neurons in the DRN are electrophysiologically distinct from each other. Additionally, they characterized distinct morphological features of 5-HT and DA neurons in the DRN. Notably, these specific features of 5-HT and DA neurons in the DRN exhibited different changes in the 6-OHDA-induced PD model. Then the authors utilized desipramine (DMI) to separate the effects of nigrostriatal DA depletion and noradrenaline (NA) depletion induced by 6-OHDA. Interestingly, protection from NA depletion by DMI pretreatment reversed the changes in 5-HT neurons, while having a minor impact on the changes in DA neurons in the DRN. These data indicate that the role of NA lesion in the altered properties of DRN 5-HT neurons by 6-OHDA is more critical than that of DA lesions.

Overall, this study provides foundational data on the 5-HT and DA neurons in the DRN and their potential involvement in PD symptoms. Given the deficits of the DRN in PD, this paper may offer insights into the cellular mechanisms underlying non-motor symptoms associated with PD.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

The manuscript has improved in clarity and conceptual and methodological considerations in response to the last review. However, the reported results still provide incomplete support for the claims the authors make in the paper.

In relation to other reviewers' earlier comments, the model notation used is still not consistent and model results are reported incompletely, which make it difficult to gain a full picture of the data and how they support the authors' secondary claims. For instance, across the models in the supplementary materials, ß coefficients are only reported selectively which makes it difficult to assess the model as a whole. Furthermore, different terms (task 1, task 2 vs. BM-Local, BM-global) are used to refer to the same levels of a variable, and it is unclear which levels of a dummy variable correspond to which task, making it overall very difficult to comprehend the modelling procedure.

Reviewer #3 (Public Review):

The authors presented point light displays of human walkers to children (mean = 9 years) with and without ADHD to compare their biological motion perception abilities, and relate them to IQ, social responsiveness scale (SRS) scores and age. They report that children with ADHD were worse at all three biological motion tasks, but that those loading more heavily on local processing related to social interaction skills and global processing to age. The valuable and solid findings are informative for understanding this complex condition, as well as biological motion processing mechanisms in general. However, the correlations present a pattern that needs further examination in future studies because many of the differences between correlations are not significant.

Strengths:

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

Weaknesses:

The data are not strong enough to support claims about differences between global and lobal processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but the crucial tests of differences between correlations do not present a clear picture. Further empirical work would be needed to test this further. Specifics:

The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. The supplementary materials demonstrate that tests of differences between correlations present an incomplete picture. Currently they have small samples for correlations, so this is unsurprising.

Theoretical assumptions. The authors make some statements about local vs global biological motion processing that may have been made in previous studies, but would appear controversial and not definitive. E.g., that local BM processing does not improve with age and is uninfluenced by attention.

Reviewer #3 (Public Review):

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

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

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

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

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

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

Reviewer #2 (Public Review):

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

The current version of the paper has improved, increasing the clarity and providing clear explanations to the comments raised regarding quantifications, functionality of the cells in vivo etc...

The discussion on challenges adds depth to the study and encourages future research to build upon these important findings

Reviewer #1 (Public Review):

Summary:

The authors use innovative CRISPRi method to uncover regulators of cell density and volume in neutrophils. The results show that cells require NHE activity during chemoattractant-driven cell migration. Before migration occurs, cells also undergo a rapid cell volume increase. These results indicate that water flux, driven by ion channels, appears to play a central role in neutrophil migration. The paper is very well written and clear. The revised version has addressed all of my questions.

Reviewer #2 (Public Review):

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

The conclusions of this paper are mostly well supported by data and is pretty convincing

Reviewer #1 (Public Review):

This study presents a novel application of the inverted encoding (i.e., decoding) approach to detect the correlates of crossmodal integration in the human EEG (electrophysiological) signal. The method is successfully applied to data from a group of 41 participants, performing a spatial localization task on auditory, visual, and audio-visual events. The analyses clearly show a behavioural superiority for audio-visual localization. Like previous studies, the results when using traditional univariate ERP analyses were inconclusive, showing once more the need for alternative, more sophisticated approaches. Instead, the principal approach of this study, harnessing the multivariate nature of the signal, captured clear signs of super-additive responses, considered by many as the hallmark of multisensory integration. Unfortunately, the manuscript lacks many important details in the descriptions of the methodology and analytical pipeline. Although some of these details can eventually be retrieved from the scripts that accompany this paper, the main text should be self-contained and sufficient to gain a clear understanding of what was done. (A list of some of these is included in the comments to the authors). Nevertheless, I believe the main weakness of this work is that the positive results obtained and reported in the results section are conditioned upon eye movements. When artifacts due to eye movements are removed, then the outcomes are no longer significant.

Therefore, whether the authors finally achieved the aims and showed that this method of analysis is truly a reliable way to assess crossmodal integration, does not stand on firm ground. The worst-case scenario is that the results are entirely accounted for by patterns of eye movements in the different conditions. In the best-case scenario, the method might truly work, but further experiments (and/or analyses) would be required to confirm the claims in a conclusive fashion.

If finally successful, this approach could bring important advances in the many fields where multisensory integration has been shown to play a role, by providing a way to bring much-needed coherence across levels of analysis, from behaviour to single-cell electrophysiology. To achieve this, one would have to make sure that the pattern of super-additive effects, the standard self-imposed by the authors as a proxy for multisensory integration, shows up reliably regardless of eye movement or artifact corrections. One first step toward this goal would be, perhaps, to facilitate the understanding of results in context by reporting both the uncorrected and corrected analyses in the main results section. Second, one could try to support the argument given in the discussion, pointing out the origin of the super-additive effects in posterior electrode sites, by also modelling frontal electrode clusters and showing they aren't informative as to the effect of interest.

Reviewer #2 (Public Review):

Summary:

This manuscript seeks to reconcile observations in multisensory perception - from behavior and neural responses. It is intuitively obvious that perceiving a stimulus via two senses results in better performance than one alone. In fact, it is not uncommon to observe that for a perceptual task, the percentage of correct responses seen with two senses is higher than the sum of the percentage correct obtained with each modality individually. i.e. the gains are "superadditive". The gains of adding a second sense are typically larger when the performance with the first sense is relatively poor - this effect is often called the principle of inverse effectiveness. More generally, what this tells us is that performance in a multisensory perceptual task is a non-linear sum of performance for each sensory modality alone.

Despite this abundant evidence of behavioral non-linearity in multisensory integration, evoked responses (EEG) to such sensory stimuli often show little evidence of it - and this is the problem this manuscript tackles. The key assertion made is that univariate analysis of the EEG signal is likely to average out the non-linear effects of integration. This is a reasonable assertion, and their analysis does indeed provide evidence that a multivariate approach can reveal non-linear interactions in the evoked responses.

Strengths:

It is of great value to understand how the process of multisensory integration occurs, and despite a wealth of observations of the benefits of perceiving the world with multiple senses, we still lack a reasonable understanding of how the brain integrates information. For example - what underlies the large individual differences in the benefits of two senses over one? One way to tackle this is via brain imaging, but this is problematic if important features of the processing - such as non-linear interactions are obscured by the lack of specificity of the measurements. The approach they take to the analysis of the EEG data allows the authors to look in more detail at the variation in activity across EEG electrodes, which averaging across electrodes cannot.

This version of the manuscript is well-written and for the most part clear. It shows a good understanding of the non-linear effects described above (where many studies show a poor understanding of "superadditivity" of perceptual performance) and the report of non-linear summation of neural responses is convincing.

A particular strength of the paper is their use of a statistical model of multisensory integration as their "null" model of neural responses, and the "inverted-encoder" which infers an internal representation of the stimulus which can explain the EEG responses. This encoder generates a prediction of decoding performance, which can be used to generate predictions of multisensory decoding from unisensory decoding, or from a sum of the unisensory internal representations.

In behavioural performance, it is frequently observed that the performance increase from two senses is close to what is expected from the optimal integration of information across the senses, in a statistical sense. It can be plausibly explained by assuming that people are able to weigh sensory inputs according to their reliability - and somewhat optimally. Critically the apparent "superadditive" effect on performance described above does not require any non-linearity in the sum of information across the senses but can arise from correctly weighting the information according to reliability.

The authors apply a similar model to predict the neural responses expected to audiovisual stimuli from the neural responses to audio and visual stimuli alone, assuming optimal statistical integration of information. The neural responses to audiovisual stimuli exceed the predictions of this model and this is the main evidence supporting their conclusion, and it is convincing.

Weaknesses:

The main weakness of the manuscript is that their behavioural data show no evidence of performance that exceeds the predictions of these statistical models. In fact, the models predict multisensory performance from unisensory performance pretty well. So this manuscript presents the opposite problem to that which motivated the study - neural interactions across the senses which appear to be more non-linear than perception. This makes it hard to interpret their results, as surely if these nonlinear neural interactions underlie the behaviour, then we should be able to see evidence of it in the behaviour? I cannot offer an easy explanation for this.

Overall, therefore, I applaud the motivation and the sophistication of the analysis method and think it shows great promise for tackling these problems, but the manuscript unfortunately brushes over an important problem specific to the results. It appeals to the higher-level reasoning - that non-linearity is a behavioural hallmark of integration and therefore we should see it in neural responses. Yet it ignores the fact that the behaviour observed here does not exceed the predictions of the "null" model applied to the neural response.

Part of the problem, I think, is that the authors never explain the difference between superadditivity of perceptual performance (proportion correct) and superadditivity of the underlying processing, which is implied by the EEG results but not their behavior. This is of course a difficult matter to describe succinctly or clearly (I somehow doubt I have). It is however worth addressing. The literature is full of confusing claims of superadditivity. I believe these authors understand this distinction and have an opportunity to represent it clearly for the benefit of all.

Joint Public Review:

Mahapatra and Takahashi report on the physiological consequences of pharmacologically blocking either clathrin and dynamin function during compensatory endocytosis or of the cortical actin scaffold both in the calyx of Held synapse and hippocampal boutons in acute slice preparations.

Although many aspects of these pharmacological interventions have been studied in detail during the past decades, this is a comprehensive and comparative study, which reveals some interesting differences between a fast synapse (Calyx of Held) tuned to reliably transmit at several 100 Hz and a more slow hippocampal CA1 synapse. In particular the authors find that acute disturbance of the synaptic actin network leads to a marked frequency-dependent enhancement of synaptic depression in the Calyx, but not in the hippocampal synapse. This striking difference between both preparations is the most interesting finding.

Comments on latest version:

The authors have done a great job revising the paper and only minor revisions are suggested to the Discussion of the paper.

Two quite relevant and recent papers should be cited and briefly discussed because they relate directly to Pitstop2 effects and actin-myosin-scaffold proteins in the calyx of Held synapse.

One is: Paksoy A et al, (2022) "Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo." Front. Synaptic Neurosci. 14:1056308. doi: 10.3389/fnsyn.2022.1056308. This paper shows with EM that changes caused by PitStop2 perturbation of "clathrin function suggest that clathrin plays a role in SV recycling from both, the plasma membrane and large endosomes, under physiological activity patterns, in vivo."

Second: A role for actin-myosin and MLCK in short-term plasticity has been shown by Srinivasan G., et al. (2008) "The Pool of Fast Releasing Vesicles Is Augmented by Myosin Light Chain Kinase Inhibition at the Calyx of Held Synapse." J Neurophysiol 99: 1810-1824, 2008. The data here suggests that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release but not the Pr of the synapse. In other words, MLCK inhibition augments super-priming of vesicles at the calyx of Held synapse.

Reviewer #1 (Public Review):

Summary:

The authors aim to test the sensory recruitment theory of visual memory, which assumes that visual sensory areas are recruited for working memory and that these sensory areas represent visual memories in a similar fashion to how perceptual inputs are represented. To test the overlap between working memory (WM) and perception, the authors use coarse stimulus (aperture) biases that are known to account for (some) orientation decoding in visual cortex (i.e., stimulus energy is higher for parts of an image where a grating orientation is perpendicular to an aperture edge, and stimulus energy drives decoding). Specifically, the authors show gratings (with a given "carrier" orientation) behind two different apertures: One is a radial modulator (with maximal energy aligned with the carrier orientation) and the other an angular modulator (with maximal energy orthogonal to the carrier orientation). When subject detect contrast changes in these stimuli (the perceptual task), orientation decoding only works when training and testing within each modulator, but not across modulators, showing the impact of stimulus energy on decoding performance. Instead, when subjects remember the orientation over a 12s delay, orientation decoding works irrespective of the modulator used. The authors conclude that representations during WM are therefore not "sensory-like", given that they are immune to aperture biases. This invalidates the sensory recruitment hypothesis, or at least the part assuming that when sensory areas that are recruited during WM, they are recruited in a manner that resembles how these areas are used during perception.

Strengths:

Duan and Curtis very convincingly show that aperture effects that are present during perception, do not appear to be present during the working memory delay. Especially when the debate about "why can we decode orientations from human visual cortex" was in full swing, many may have quietly assumed this to be true (e.g., "the memory delay has no stimuli, and ergo no stimulus aperture effects"), but it is definitely not self-evident and nobody ever thought to test it directly until now. In addition to the clear absence of aperture effects during the delay, Duan and Curtis also show that when stimulus energy aligns with the carrier orientation, cross-generalization between perception and memory does work (which could explain why perception-to-memory cross decoding also works). All in all, this is a clever manipulation, and I'm glad someone did it, and did it well.

Weaknesses:

There seems to be a major possible confound that prohibits strong conclusions about "abstractions" into "line-like" representation, which is spatial attention. What if subjects simply attend the end points of the carrier grating, or attend to the edge of the screen where the carrier orientation "intersects" in order to do the task? This may also result in reconstructions that have higher bold at areas close to the stimulus/screen edges along the carrier orientation. The question then would be if this is truly an "abstracted representation", or if subjects are merely using spatial attention to do the task.

Alternatively (and this reaches back to the "fine vs coarse" debate), another argument could be that during memory, what we are decoding is indeed fine-scale inhomogenous sampling of orientation preferences across many voxels. This is clearly not the most convincing argument, as the spatial reconstructions (e.g., Figure 3A and C) show higher BOLD for voxels with receptive fields that are aligned to the remembered orientation (which is in itself a form of coarse scale bias), but could still play a role.

To conclude that the spatial reconstruction from the data indeed comes from a line-like representation, you'd need to generate modeled reconstructions of all possible stimuli and representations. Yes, Figure 4 shows that a line results in a modeled spatial map that resembles the WM data, but many other stimuli might too, and some may better match the data. For example, the alternative hypothesis (attention to grating endpoints) may very well lead to a very comparable model output to the one from a line. But testing this would not suffice, as there may be an inherent inverse problem (with multiple stimuli that can lead to the same visual field model).

The main conclusion, and title of the paper, that visual working memories are abstractions of percepts, is therefore not supported. Subjects could be using spatial attention, for example. Furthermore, even if it is true that gratings are abstracted into lines, this form of abstraction would not generalize to any non-spatial feature (e.g., color cannot become a line, contrast cannot become a line, etc.), which means it has limited explanatory power.

Additional context:

The working memory and perception tasks are rather different. In this case, the perception task does not require the subject to process the carrier orientation (which is largely occluded, and possibly not that obvious without paying attention to it), but attention is paid to contrast. In this scenario, stimulus energy may dominate the signal. In the WM task, subjects have to work out what orientation is shown to do the task. Given that the sensory stimulus in both tasks is brief (1.5s during memory encoding, and 2.5s total in the perceptual task), it would be interesting to look at decoding (and reconstructions) for the WM stimulus epoch. If abstraction (into a line) happens in working memory, then this perceptual part of the task should still be susceptible to aperture biases. It allows the authors to show that it is indeed during memory (and not merely the task or attentional state of the subject) that abstraction occurs.

What's also interesting is what happens in the passive perceptual condition, and the fact that spatial reconstructions for areas beyond V1 and V2 (i.e., V3, V3AB, and IPS0-1) align with (implied) grating endpoints, even when an angular modulator is used (Figure 3C). Are these areas also "abstracting" the stimulus (in a line-like format)?

Review after revision:

(1) It's nice of the authors to simulate how a dot stimulus affects the image computable model, but this does not entirely address my concern about attention to endpoints. The assumption that attention can be used in the same manner as a physical stimulus to calculate stimulus energy is questionable. (also, why would a dot at 15º lead to high stimulus energy tangential to that orientation?). This simulation also does not at all address my concern about model mimicry (many possible inputs can lead to a line-like output).

(2) It's also nice that the authors agree that much more work needs to be done, and these results may not generalize to all forms of memory. Given this agreement, and until that "more work" is done, I strongly believe we should refrain from making hyperbolic claims that might preemptively imply all visual working memories are abstractions of percepts. Time (and much more work) will likely show things to be much more subtle and complex.

The work presented in this paper is cool, but it uses a specific case: spatial stimuli (gratings) with the task to remember orientation. This limits possible conclusions for several reasons (1) These results are specific to EVC, as visual maps are a prerequisite meaning that these results will not hold up in other, non-retinotopic areas. (2) The fact that subjects are "focusing" along the main stimulus axis (attention or not) can simply be a strategy employed by the majority of (but not all) subjects - a strategy that may not be necessary to do the task, and therefore not a canonical method of Abstraction. It may be a "shared preferred strategy" or something. (3) If subjects had to (for example) remember contrast, and not orientation, results may have been entirely different (I would hypothesize there is no line-like abstraction in this case). Vice versa, if the perceptual task would have been on orientation (instead of contrast), the authors admit that "participants would reformat the grating into a line-like representation to make the judgments" (quote from author's response under "Additional context"). Thus, the results may be entirely about the task/ cognitive state, and not about how perceptual information is abstracted into memory.

Instead of unveiling *the* working memory Abstraction, this work (very nicely) shows a specific instance of possible abstraction. A more correct (but admittedly, less "sexy") conclusion may be "Visual working memories of orientation can be abstracted into a line in early visual cortex". As it stands, the authors still do not acknowledge any of the alternatives that myself (see above) and the other reviewers have put forth, nor do they acknowledge recent work by Chunharas et al. (2023, BioRxiv), that directly applies principles of efficient coding to address the exact same question of working memory abstraction. The link between a "line-like" representation and efficient coding implied by the authors (in their response) is merely tentative to me, but it would be great if the authors could explain this further.

These were, and remain, the major weaknesses in the original submission, that in my view have not been adequately addressed by the authors, as many overly broad conclusions about abstractions are currently still present in the manuscript (in for example the title).

Reviewer #2 (Public Review):

Summary:

In this work, Duan and Curtis addressed an important issue related to the nature of working memory representations. This work is motivated by findings illustrating that orientation decoding performance for perceptual representations can be biased by the stimulus aperture (modulator). Here, the authors examined whether the decoding performance for working memory representations is similarly influenced by these aperture biases. The results provide convincing evidence that working memory representations have a different representational structure, as the decoding performance was not influenced by the type of stimulus aperture.

Strengths:

The strength of this work lies in the direct comparison of decoding performance for perceptual representations with working memory representations. The authors take well-motivated approach and illustrate that perceptual and working memory representations do not share a similar representational structure. The authors test a clear question, with a rigorous approach and provide compelling evidence. First, the presented oriented stimuli are carefully manipulated to create orthogonal biases introduced by the stimulus aperture (radial or angular modulator), regardless of the stimulus carrier orientation. Second, the authors implement advanced methods to decode the orientation information, in visual and parietal cortical regions, when directly perceiving or holding an oriented stimulus in memory. The data illustrates that working memory decoding is not influenced by the type of aperture, while this is the case in perception. In sum, the main claims are important and shed light on the nature of working memory representations.

Weaknesses:

After the authors revised the original manuscript, a few of my initial concerns remain.

(1) Theoretical framing in the introduction. The introduction proposes that decoding of orientation information during perception does not reflect orientation selectivity, and it is instead driven by coarse scale biases. This is an overstatement. Recent work shows that orientation decoding is indeed influenced by coarse biases, but also reflects orientation selectivity (Roth, Kay & Merriam, 2022).

(2) The description of the image computable V1 model remains incomplete. The steerable pyramid is a model that simulates the responses of V1 neurons. To do so, it incorporates a set of linear receptive fields with varying orientation and spatial frequency tuning. However, the information that is lacking in the Methods is whether the implemented pyramid also included two quadrature phase pairs (odd and even phase Gabor filters making the output phase invariant). The sum of the squares of the responses to these offset phase filters computes the stimulus energy within each orientation and spatial frequency channel. Without this description, it is unclear what the model output represents.

Reviewer #1 (Public Review):

In this study, Gonzalez Alam et al. report a series of functional MRI results about the neural processing from the visual cortex to high-order regions in the default-mode network (DMN), compiling evidence from task-based functional MRI, resting-state connectivity, and diffusion-weighted imaging. Their participants were first trained to learn the association between objects and rooms/buildings in a virtual reality experiment; after the training was completed, in the task-based MRI experiment, participants viewed the objects from the earlier training session and judged if the objects were in the semantic category (semantic task) or if they were previously shown in the same spatial context (spatial context task). Based on the task data, the authors utilised resting-state data from their previous studies, visual localiser data also from previous studies, as well as structural connectivity data from the Human Connectome Project, to perform various seed-based connectivity analysis. They found that the semantic task causes more activation of various regions involved in object perception while the spatial context task causes more activation in various regions for place perception, respectively. They further showed that those object perception regions are more connected with the frontotemporal subnetwork of the DMN while those place perception regions are more connected with the medial-temporal subnetwork of the DMN. Based on these results, the authors argue that there are two main pathways connecting the visual system to high-level regions in the DMN, one linking object perception regions (e.g., LOC) leading to semantic regions (e.g., IFG, pMTG), the other linking place perception regions (e.g., parahippocampal gyri) to the entorhinal cortex and hippocampus.

Below I provide my takes on (1) the significance of the findings and the strength of evidence, (2) my guidance for readers regarding how to interpret the data, as well as several caveats that apply to their results, and finally (3) my suggestions for the authors.

(1) Significance of the results and strength of the evidence

I would like to praise the authors for, first of all, trying to associate visual processing with high-order regions in the DMN. While many vision scientists focus specifically on the macroscale organisation of the visual cortex, relatively few efforts are made to unravel how neural processing in the visual system goes on to engage representations in regions higher up in the hierarchy (a nice precedent study that looks at this issue is by Konkle and Caramazza, 2017). We all know that visual processing goes beyond the visual cortex, potentially further into the DMN, but there's no direct evidence. So, in this regard, the authors made a nice try to look at this issue.

Having said this, the authors' characterisation of the organisation of the visual cortex (object perception/semantics vs. place perception/spatial contexts) does not go beyond what has been known for many decades by vision neuroscience. Specifically, over the past two decades, numerous proposals have been put forward to explain the macroscale organisation of the visual system, particularly the ventrolateral occipitotemporal cortex. A lateral-medial division has been reliably found in numerous studies. For example, some researchers found that the visual cortex is organised along the separation of foveal vision (lateral) vs. peripheral vision (medial), while others found that it is structured according to faces (lateral) vs. places (medial). Such a bipartite division is also found in animate (lateral) vs. inanimate (medial), small objects (lateral) vs. big objects (medial), as well as various cytoarchitectonic and connectomic differences between the medial side and the lateral side of the visual cortex. Some more recent studies even demonstrate a tripartite division (small objects, animals, big objects; see Konkle and Caramazza, 2013). So, in terms of their characterisation of the visual cortex, I think Gonzalez Alam et al. do not add any novel evidence to what the community of neuroscience has already known.

However, the authors' effort to link visual processing with various regions of the DMN is certainly novel, and their attempt to gather converging evidence with different methodologies is commendable. The authors are able to show that, in an independent sample of resting-state data, object-related regions are more connected with semantic regions in the DMN while place-related regions are more connected with navigation-related regions in the DMN, respectively. Such patterns reveal a consistent spatial overlap with their Kanwisher-type face/house localiser data and also concur with the HCP white-matter tractography data. Overall, I think the two pathways explanation that the authors seek to argue is backed by converging evidence. The lack of travelling wave type of analysis to show the spatiotemporal dynamics across the cortex from the visual cortex to high-level regions is disappointing though because I was expecting this type of analysis would provide the most convincing evidence of a 'pathway' going from one point to another. Dynamic caudal modelling or Granger causality may also buttress the authors' claim of pathway because many readers, like me, would feel that there is not enough evidence to convincingly prove the existence of a 'pathway'.

(2) Guidance to the readers about interpretation of the data

The organisation of the visual cortex and the organisation of the DMN historically have been studied in parallel with little crosstalk between different communities of researchers. Thus, the work by Gonzalez Alam et al. has made a nice attempt to look at how visual processing goes beyond the realm of the visual cortex and continues into different subregions of the DMN.

While the authors of this study have utilised multiple methods to obtain converging evidence, there are several important caveats in the interpretation of their results:

(1) While the authors choose to use the term 'pathway' to call the inter-dependence between a set of visual regions and default-mode regions, their results have not convincingly demonstrated a definitive route of neural processing or travelling. Instead, the findings reveal a set of DMN regions are functionally more connected with object-related regions compared to place-related regions. The results are very much dependent on masking and thresholding, and the patterns can change drastically if different masks or thresholds are used.

(2) Ideally, if the authors could demonstrate the dynamics between the visual cortex and DMN in the primary task data, it would be very convincing evidence for characterising the journey from the visual cortex to DMN. Instead, the current connectivity results are derived from a separate set of resting state data. While the advantage of the authors' approach is that they are able to verify certain visual regions are more connected with certain DMN regions even under a task-free situation, it falls short of explaining how these regions dynamically interact to convert vision into semantic/spatial decision.

(3) There are several results that are difficult to interpret, such as their psychophysiological interactions (PPI), representational similarity analysis, and gradient analysis. For example, typically for PPI analysis, researchers interrogate the whole brain to look for PPI connectivity. Their use of targeted ROI is unusual, and their use of spatially extensive clusters that encompass fairly large cortical zones in both occipital and temporal lobes as the PPI seeds is also an unusual approach. As for the gradient analysis, the argument that the semantic task is higher on Gradient 1 than the spatial task based on the statistics of p-value = 0.027 is not a very convincing claim (unhelpfully, the figure on the top just shows quite a few blue 'spatial dots' on the hetero-modal end which can make readers wonder if the spatial context task is really closer to the unimodal end or it is simply the authors' statistical luck that they get a p-value under 0.05). While it is statistically significant, it is weak evidence (and it is not pertinent to the main points the authors try to make).

(3) My suggestion for the authors

There are several conceptual-level suggestions that I would like to offer to the authors:

(1) If the pathway explanation is the key argument that you wish to convey to the readers, an effective connectivity type of analysis, such as Granger causality or dynamic caudal modelling, would be helpful in revealing there is a starting point and end point in the pathway as well as revealing the directionality of neural processing. While both of these methods have their issues (e.g., Granger causality is not suitable for haemodynamic data, DCM's selection of seeds is susceptible to bias, etc), they can help you get started to test if the path during task performance does exist. Alternatively, travelling wave type of analysis (such as the results by Raut et al. 2021 published in Science Advances) can also be useful to support your claims of the pathway.

(2) I think the thresholding for resting state data needs to be explained - by the look of Figure 2E and 3E, it looks like whole-brain un-thresholded results, and then you went on to compute the conjunction between these un-thresholded maps with network templates of the visual system and DMN. This does not seem statistically acceptable, and I wonder if the conjunction that you found would disappear and reappear if you used different thresholds. Thus, for example, if the left IFG cluster (which you have shown to be connected with the visual object regions) would disappear when you apply a conventional threshold, this means that you need to seriously consider the robustness of the pathway that you seek to claim... it may be just a wild goose that you are chasing.

(3) There are several analyses that are hard to interpret and you can consider only reporting them in the supplementary materials, such as the PPI results and representational similarity analysis, as none of these are convincing. These analyses do not seem to add much value to make your argument more convincing and may elicit more methodological critiques, such as statistical issues, the set-up of your representational theory matrix, and so on.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Alam et al. sought to understand how memory interacts with incoming visual information to effectively guide human behavior by using a task that combines spatial contexts (houses) with objects of one or multiple semantic categories. Three additional datasets (all from separate participants) were also employed: one that functionally localized regions of interest (ROIs) based on subtractions of different visually presented category types (in this case, scenes, objects, and scrambled objects); another consisting of resting-state functional connectivity scans, and a section of the Human Connectome Project that employed DTI data for structural connectivity analysis. Across multiple analyses, the authors identify dissociations between regions preferentially activated during scene or object judgments, between the functional connectivity of regions demonstrating such preferences, and in the anatomical connectivity of these same regions. The authors conclude that the processing streams that take in visual information and support semantic or spatial processing are largely parallel and distinct.

Strengths:

(1) Recent work has reconceptualized the classic default mode network as two parallel and interdigitated systems (e.g., Braga & Buckner, 2017; DiNicola et al., 2021). The current manuscript is timely in that it attempts to describe how information is differentially processed by two streams that appear to begin in visual cortex and connect to different default subnetworks. Even at a group level where neuroanatomy is necessarily blurred across individuals, these results provide clear evidence of stimulus-based dissociation.

(2) The manuscript contains a large number of analyses across multiple independent datasets. It is therefore unlikely that a single experimenter choice in any given analysis would spuriously produce the overall pattern of results reported in this work.

Weaknesses:

(1) Throughout the manuscript, a strong distinction is drawn between semantic and spatial processing. However, given that only objects and spatial contexts were employed in the primary experiment, it is not clear that a broader conceptual distinction is warranted between "semantic" and "spatial" cognition. There are multiple grounds for concern regarding this basic premise of the manuscript.<br /> a. One can have conceptual knowledge of different types of scenes or spatial contexts. A city street will consistently differ from a beach in predictable ways, and a kitchen context provides different expectations than a living room. Such distinctions reflect semantic knowledge of scene-related concepts, but in the present work spatial and "all other" semantic information are considered and discussed as distinct and separate.<br /> b. As a related question, are scenes uniquely different from all other types of semantic/category information? If faces were used instead of scenes, could one expect to see different regions of the visual cortex coupling with task-defined face > object ROIs? The current data do not speak to this possibility, but as written the manuscript suggests that all (non-spatial) semantic knowledge should be processed by the FT-DMN.<br /> c. Recent precision fMRI studies characterizing networks corresponding to the FT-DMN and MTL-DMN have associated the former with social cognition and the latter with scene construction/spatial processing (DiNicola et al., 2020; 2021; 2023). This is only briefly mentioned by the authors in the current manuscript (p. 28), and when discussed, the authors draw a distinction between semantic and social or emotional "codes" when noting that future work is necessary to support the generality of the current claims. However, if generality is a concern, then emphasizing the distinction between object-centric and spatial cognition, rather than semantic and spatial cognition, would represent a more conservative and better-supported theoretical point in the current manuscript.

(2) Both the retrosplenial/parieto-occipital sulcus and parahippocampal regions are adjacent to the visual network as defined using the Yeo et al. atlas, and spatial smoothness of the data could be impacting connectivity metrics here in a way that qualitatively differs from the (non-adjacent) FT-DMN ROIs. Although this proximity is a basic property of network locations on the cortical surface, the authors have several tools at their disposal that could be employed to help rule out this possibility. They might, for instance, reduce the smoothing in their multi-echo data, as the current 5 mm kernel is larger than the kernel used in Experiment 2's single-echo resting-state data. Spatial smoothing is less necessary in multi-echo data, as thermal noise can be attenuated by averaging over time (echoes) instead of space (see Gonzalez-Castillo et al., 2016 for discussion). Some multi-echo users have eschewed explicit spatial smoothing entirely (e.g., Ramot et al., 2021), just as the authors of the current paper did for their RSA analysis. Less smoothing of E1 data, combined with a local erosion of either the MTL-DMN and VIS masks (or both) near their points of overlap in the RSFC data, would improve confidence that the current results are not driven, at least in part, by spatial mixing of otherwise distinct network signals.

(3) The authors identify a region of the right angular gyrus as demonstrating a "potential role in integrating the visual-to-DMN pathways." This would seem to imply that lesion damage to right AG should produce difficulties in integrating "semantic" and "spatial" knowledge. Are the authors aware of such a literature? If so, this would be an important point to make in the manuscript as it would tie in yet another independent source of information relevant to the framework being presented. The closest of which I am aware involves deficits in cued recall performance when associates consisted of auditory-visual pairings (Ben-Zvi et al., 2015), but that form of multi-modal pairing is distinct from the "spatial-semantic" integration forwarded in the current manuscript.

Reviewer #1 (Public Review):

The study by Chikermane and colleagues investigates the functional, structural, and dopaminergic network substrates of cortical beta oscillations (13-30 Hz). The major strength of the work lies in the methodology taken by the authors, namely a multimodal lesion network mapping. First, using invasive electrophysiological recordings from healthy cortical territories of epileptic patients they identify regions with the highest beta power. Next, they leverage open-access MRI data and PET atlases and use the identified high-beta regions as seeds to find (1) the whole-brain functional and structural maps of regions that form the putative underlying network of high-beta regions and (2) the spatial distribution of dopaminergic receptors that show correlation with nodal connectivity of the identified networks. These steps are achieved by generating aggregate functional, structural, and dopaminergic network maps using lead-DBS toolbox, and by contrasting the results with those obtained from high-alpha regions.

The main findings are:<br /> (1) Beta power is strongest across frontal, cingulate, and insular regions in invasive electrophysiological data, and these regions map onto a shared functional and structural network.<br /> (2) The shared functional and structural networks show significant positive correlations with dopamine receptors across the cortex and basal ganglia (which is not the case for alpha, where correlations are found with GABA).

Nevertheless, a few clarifications regarding the choice of high-power electrodes and distributions of functional connectivity maps (i.e., strength and sign across cortex and sub-cortex) can help with understanding the results.

Reviewer #2 (Public Review):

Summary:

This is a very interesting paper that leveraged several publicly available datasets: invasive cortical recording in epilepsy patients, functional and structural connectomic data, and PET data related to dopaminergic and gaba-ergic synapses. These were combined to create a unified hypothesis of beta band oscillatory activity in the human brain. They show that beta frequency activity is ubiquitous, not just in sensorimotor areas, and cortical regions where beta predominated had high connectivity to regions high in dopamine re-update.

Strengths:

The authors leverage and integrate three publicly available human brain datasets in a creative way. While these public datasets are powerful tools for human neuroscience, it is innovative to combine these three types of data into a common brain space to generate novel findings and hypotheses. Findings are nicely controlled by separately examining cortical regions where alpha predominates (which have a different connectivity pattern). GABA uptake from PET studies is used as a control for the specificity of the relationship between beta activity and dopamine uptake. There is much interest in synchronized oscillatory activity as a mechanism of brain function and dysfunction, but the field is short on unifying hypotheses of why particular rhythms predominate in particular regions. This paper contributes nicely to that gap. It is ambitious in generating hypotheses, particularly that modulation of beta activity may be used as a "proxy" for modulating phasic dopamine release.

Weaknesses:

As the authors point out, the use of normative data is excellent for exploring hypotheses but does not address or explore individual variations which could lead to other insights. It is also biased to resting state activity; maps of task-related activity (if they were available) might show different findings.

The figures, results, introduction, and methods are admirably clear and succinct but the discussion could be both shorter and more convincing.

Reviewer #3 (Public Review):

Summary:

In this paper, Chikermane et al. leverages a large open dataset of intracranial recordings (sEEG or ECoG) to analyze resting state (eyes closed) oscillatory activity from a variety of human brain areas. The authors identify a dominant proportion of channels in which beta band activity (12-30Hz) is most prominent and subsequently seek to relate this to anatomical connectivity data by using the sEEG/ECoG electrodes as seeds in a large set of MRI data from the human connectome project. This reveals separate regions and white matter tracts for alpha (primarily occipital) and beta (prefrontal cortex and basal ganglia) oscillations. Finally, using a third available dataset of PET imaging, the authors relate the parcellated signals to dopamine signaling as estimated by spatial uptake patterns of dopamine, and reveal a significant correlation between the functional connectivity maps and the dopamine reuptake maps, suggesting a functional relationship between the two.

Strengths:

Overall, I found the paper well justified, focused on an important topic, and interesting. The authors' use of 3 different open datasets was creative and informative, and it significantly adds to our understanding of different oscillatory networks in the human brain, and their more elusive relation with neuromodulator signaling networks by adding to our knowledge of the association between beta oscillations and dopamine signaling. Even my main comments about the lack of a theta network analysis and discussion points are relatively minor, and I believe this paper is valuable and informative.

Weaknesses:

The analyses were adequate, and the authors cleverly leveraged these different datasets to build an interesting story. The main aspect I found missing (in addition to some discussion items, see below) was an examination of the theta network. Theta oscillations have been involved in a number of cognitive processes including spatial navigation and memory, and have been proposed to have different potential originating brain regions, and it would be informative to see how their anatomical networks (e.g. as in Figure 2) look like under the author's analyses.

The authors devote a significant portion of the discussion to relating their findings to a popular hypothesis for the function of beta oscillations, the maintenance of the "status quo", mostly in the context of motor control. As the authors acknowledge, given the static nature of the data and lack of behavior, this interpretation remains largely speculative and I found it a bit too far-reaching given the data shown in the paper. In contrast, I missed a more detailed discussion on the growing literature indicating a role for beta in mood (e.g. in Kirkby et al. 2018), especially given the apparent lack of hippocampal and amygdala involvement in the paper, which was surprising.

Major comment:

• Although the proportion of electrodes with theta-dominant oscillations was lower (~15%) than alpha (~22%) or beta (~57%), it would be very valuable to also see the same analyses the authors carried out in these frequency bands extended to theta oscillations.

Reviewer #1 (Public Review):

Vision is a highly active process. Humans move their eyes 3-4 times per second to sample information with high visual acuity from our environment, and where eye movements are directed is critical to our understanding of active vision. Here, the authors propose that the cost of making a saccade contributes critically to saccade selection (i.e., whether and where to move the eyes). The authors build on their own recent work that the effort (as measured by pupil size) that comes with planning and generating an eye movement varies with saccade direction. To do this, the authors first measured pupil size for different saccade directions for each participant. They then correlated the variations in pupil size obtained in the mapping task with the saccade decision in a free-choice task. The authors observed a striking correlation: pupil size in the mapping task predicted the decision of where to move the eyes in the free choice task. In this study, the authors provide a number of additional insightful analyses (e.g., based on saccade curvature, and saccade latency) and experiments that further support their claim that the decision to move the eyes is influenced by the effort to move the eyes in a particular direction. One experiment showed that the same influence of assumed saccade costs on saccade selection is observed during visual search in natural scenes. Moreover, increasing the cognitive load by adding an auditory counting task reduced the number of saccades, and in particular reduced the costly saccades. In sum, these experiments form a nice package that convincingly establishes the association between pupil size and saccade selection.

In my opinion, the causal structure underlying the observed results is not so clear. While the relationship between pupil size and saccade selection is compelling, it is not clear that saccade-related effort (i.e., the cost of a saccade) really drives saccade selection. Given the correlational nature of this relationship, there are other alternatives that could explain the finding. For example, saccade latency and the variance in landing positions also vary across saccade directions. This can be interpreted for instance that there are variations in oculomotor noise across saccade directions, and maybe the oculomotor system seeks to minimize that noise in a free-choice task. In fact, given such a correlational result, many other alternative mechanisms are possible. While I think the authors' approach of systematically exploring what we can learn about saccade selection using pupil size is interesting, it would be important to know what exactly pupil size can add that was not previously known by simply analyzing saccade latency. For example, saccade latency anisotropies across saccade directions are well known, and the authors also show here that saccade costs are related to saccade latency. An important question would be to compare how pupil size and saccade latency uniquely contribute to saccade selection. That is, the authors could apply the exact same logic to their analysis by first determining how saccade latencies (or variations in saccade landing positions; see Greenwood et al., 2017 PNAS) vary across saccade directions and how this saccade latency map explains saccade selection in subsequent tasks. Is it more advantageous to use one or the other saccade metric, and how well does a saccade latency map correlate with a pupil size map?

In addition to eye-movement-related anisotropies across the visual field, there are of course many studies reporting visual field anisotropies (see Himmelberg, Winawer & Carrasco, 2023, Trends in Neuroscience for a review). It would be interesting to understand how the authors think about visual field anisotropies in the context of their own study. Do they think that their results are (in)dependent on such visual field variations (see Greenwood et al., 2017, PNAS; Ohl, Kroell, & Rolfs, 2024, JEP:Gen for a similar discussion)?

Finally, the authors conclude that their results "suggests that the eye-movement system and other cognitive operations consume similar resources that are flexibly allocated among each other as cognitive demand changes. The authors should speculate what these similar resources could mean? What are the specific operations of the auditory task that overlap in terms of resources with the eye movement system?

Reviewer #2 (Public Review):

The authors attempt to establish presaccadic pupil size as an index of 'saccade effort' and propose this index as one new predictor of saccade target selection. They only partially achieved their aim: When choosing between two saccade directions, the less costly direction, according to preceding pupil size, is preferred. However, the claim that with increased cognitive demand participants would especially cut costly directions is not supported by the data. I would have expected to see a negative correlation between saccade effort and saccade direction 'change' under increased load. Yet participants mostly cut upwards saccades, but not other directions that, according to pupil size, are equally or even more costly (e.g. oblique saccades).

Strengths:

The paper is well-written, easy to understand, and nicely illustrated.

The sample size seems appropriate, and the data were collected and analyzed using solid and validated methodology.

Overall, I find the topic of investigating factors that drive saccade choices highly interesting and relevant.

Weaknesses:

The authors obtain pupil size and saccade preference measures in two separate tasks. Relating these two measures is problematic because the computations that underly saccade preparation differ. In Experiment 1, the saccade is cued centrally, and has to be delayed until a "go-signal" is presented; In Experiment 2, an immediate saccade is executed to an exogenously cued peripheral target. The 'costs' in Experiment 1 (computing the saccade target location from a central cue; withholding the saccade) do not relate to Experiment 2. It is unfortunate, that measuring presaccadic pupil size directly in the comparatively more 'natural' Experiment 2 (where saccades did not have to be artificially withheld) does not seem to be possible. This questions the practical application of pupil size as an index of saccade effort

The authors claim that the observed direction-specific 'saccade costs' obtained in Experiment 1 "were not mediated by differences in saccade properties, such as duration, amplitude, peak velocity, and landing precision (Figure 1e,f)". Saccade latency, however, was not taken into account here but is discussed for Experiment 2.

The apparent similarity of saccade latencies and pupil size, however, is striking. Previous work shows shorter latencies for cardinal than oblique saccades, and shorter latencies for horizontal and upward saccades than downward saccades - directly reflecting the pupil sizes obtained in Experiment 1 as well as in the authors' previous study (Koevoet et al., 2023, PsychScience).

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The authors state that "from a costs-perspective, it should be efficient to not only adjust the number of saccades (non-specific), but also by cutting especially expensive directions the most (specific)". However, saccade targets should be selected based on the maximum expected information gain. If cognitive load increases (due to an additional task) an effective strategy seems to be to perform less - but still meaningful - saccades. How would it help natural orienting to selectively cut saccades in certain (effortful) directions? Choosing saccade targets based on comfort, over information gain, would result in overall more saccades to be made - which is non-optimal, also from a cost perspective.

Overall, I am not sure what practical relevance the relation between pupil size (measured in a separate experiment) and saccade decisions has for eye movement research/vision science. Pupil size does not seem to be a straightforward measure of saccade effort. Saccade latency, instead, can be easily extracted in any eye movement experiment (no need to conduct a separate, delayed saccade task to measure pupil dilation), and seems to be an equally good index.

Reviewer #3 (Public Review):

This manuscript extends previous research by this group by relating variation in pupil size to the endpoints of saccades produced by human participants under various conditions including trial-based choices between pairs of spots and search for small items in natural scenes. Based on the premise that pupil size is a reliable proxy of "effort", the authors conclude that less costly saccade targets are preferred. Finding that this preference was influenced by the performance of a non-visual, attention-demanding task, the authors conclude that a common source of effort animates gaze behavior and other cognitive tasks.

Strengths:

Strengths of the manuscript include the novelty of the approach, the clarity of the findings, and the community interest in the problem.

Weaknesses:

Enthusiasm for this manuscript is reduced by the following weaknesses:

(1) A relationship between pupil size and saccade production seems clear based on the authors' previous and current work. What is at issue is the interpretation. The authors test one, preferred hypothesis, and the narrative of the manuscript treats the hypothesis that pupil size is a proxy of effort as beyond dispute or question. The stated elements of their argument seem to go like this:<br /> PROPOSITION 1: Pupil size varies systematically across task conditions, being larger when tasks are more demanding.<br /> PROPOSITION 2: Pupil size is related to the locus coeruleus.<br /> PROPOSITION 3: The locus coeruleus NE system modulates neural activity and interactions.<br /> CONCLUSION: Therefore, pupil size indexes the resource demand or "effort" associated with task conditions.<br /> How the conclusion follows from the propositions is not self-evident. Proposition 3, in particular, fails to establish the link that is supposed to lead to the conclusion.

(2) The authors test one, preferred hypothesis and do not consider plausible alternatives. Is "cost" the only conceivable hypothesis? The hypothesis is framed in very narrow terms. For example, the cholinergic and dopamine systems that have been featured in other researchers' consideration of pupil size modulation are missing here. Thus, because the authors do not rule out plausible alternative hypotheses, the logical structure of this manuscript can be criticized as committing the fallacy of affirming the consequent.

(3) The authors cite particular publications in support of the claim that saccade selection is influenced by an assessment of effort. Given the extensive work by others on this general topic, the skeptic could regard the theoretical perspective of this manuscript as too impoverished. Their work may be enhanced by consideration of other work on this general topic, e.g, (i) Shenhav A, Botvinick MM, Cohen JD. (2013) The expected value of control: an integrative theory of anterior cingulate cortex function. Neuron. 2013 Jul 24;79(2):217-40. (ii) Müller T, Husain M, Apps MAJ. (2022) Preferences for seeking effort or reward information bias the willingness to work. Sci Rep. 2022 Nov 14;12(1):19486. (iii) Bustamante LA, Oshinowo T, Lee JR, Tong E, Burton AR, Shenhav A, Cohen JD, Daw ND. (2023) Effort Foraging Task reveals a positive correlation between individual differences in the cost of cognitive and physical effort in humans. Proc Natl Acad Sci U S A. 2023 Dec 12;120(50):e2221510120.

(4) What is the source of cost in saccade production? What is the currency of that cost? The authors state (page 13), "... oblique saccades require more complex oculomotor programs than horizontal eye movements because more neuronal populations in the superior colliculus (SC) and frontal eye fields (FEF) [76-79], and more muscles are necessary to plan and execute the saccade [76, 80, 81]." This statement raises questions and concerns. First, the basis of the claim that more neurons in FEF and SC are needed for oblique versus cardinal saccades is not established in any of the publications cited. Second, the authors may be referring to the fact that oblique saccades require coordination between pontine and midbrain circuits. This must be clarified. Second, the cost is unlikely to originate in extraocular muscle fatigue because the muscle fibers are so different from skeletal muscles, being fundamentally less fatigable. Third, if net muscle contraction is the cost, then why are upward saccades, which require the eyelid, not more expensive than downward? Thus, just how some saccades are more effortful than others is not clear.

(5) The authors do not consider observations about variation in pupil size that seem to be incompatible with the preferred hypothesis. For example, at least two studies have described systematically larger pupil dilation associated with faster relative to accurate performance in manual and saccade tasks (e.g., Naber M, Murphy P. Pupillometric investigation into the speed-accuracy trade-off in a visuo-motor aiming task. Psychophysiology. 2020 Mar;57(3):e13499; Reppert TR, Heitz RP, Schall JD. Neural mechanisms for executive control of speed-accuracy trade-off. Cell Rep. 2023 Nov 28;42(11):113422). Is the fast relative to the accurate option necessarily more costly?

(6) The authors draw conclusions based on trends across participants, but they should be more transparent about variation that contradicts these trends. In Figures 3 and 4 we see many participants producing behavior unlike most others. Who are they? Why do they look so different? Is it just noise, or do different participants adopt different policies?

Reviewer #1 (Public Review):

In the manuscript "A microglia clonal inflammatory disorder in Alzheimer's Disease", Vicario et al. provide a compelling study elucidating a potential contribution of somatic mutations within the microglia population of the CNS that accelerates microglia activation and disease-associated gene signatures in Alzheimer's disease. Here they especially identified an "enrichment" of pathological SNVs in microglia, but not the peripheral blood, that are associated with clonal proliferative disorders and neurological diseases in a subset of patients with AD. Convincingly, they identified P-SNVs in microglia of AD patients located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in human microglia-like cells in vitro.

Overall, this study provides clear and detailed evidence from an AD patient cohort pointing to a potential contribution of microglia-specific somatic mutations to disease onset and/or progression in a subset of patients with Alzheimer's disease.

Strengths:<br /> As outlined above, the study identified P-SNVs in microglia of AD patients associated with clonal proliferative disorders, but also gave an in-depth analysis of re-occurring P-SNVs located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in HEK cells, BV2 cells, MAC cells, and human microglia-like cells in vitro.

Great care was taken here to validate their hypotheses at each step, as well as to identify the limitations of the possible conclusions. For example, they highlight that the pathway proposed to be affected may be an explanation for a subset of AD patients, and emphasize that it is yet unclear whether this accumulation of pathological SNVs is a cause or consequence of disease progression

The study clearly supports an enrichment of P-SNVs in several genes associated with clonal proliferative disorders in microglia and nicely separates this from SNVs associated with clonal hematopoiesis in the peripheral blood found in AD patients and controls.

The authors further acknowledged that several age-matched control patients were diagnosed with cancer or tumor-associated diseases and carefully dissected the occurring SNVs in these patients are not associated with the P-SNVs identified in the microglial compartment of the AD cohort.

Weaknesses:

Even though the study is overall very convincing, several points could help to connect the seen somatic variants in microglia more with a potential role in disease progression. The connection of P-SNVs in the genes chosen from neurological disorders was not further highlighted by the authors.

The authors show in snRNA-seq data that a disease-associated microglia state seems to be enriched in patients with somatic variants in the CBL ring domain, however, this analysis could be deepened. For example, how this knowledge may translate to patient benefits when the relevant cell populations appear concentrated in a single patient sample (Figure 5; AD52) is unclear; increasing the analyzed patient pool for Figure 5 and showcasing the presence of this microglia state of interest in a few more patients with driving mutations for CBL or other MAPK pathway associated mutations would lend their hypotheses further credibility.

A potential connection between P-SNVs in microglia and disease pathology and symptoms was not further explored by the authors.

A recent preprint (Huang et al., 2024) connected the occurrence of somatic variants in genes associated with clonal hematopoiesis in microglia in a large cohort of AD patients, this study is not further discussed or compared to the data in this manuscript.

Reviewer #2 (Public Review):

Summary:

In this study, Vicaro et al. aimed to quantify and characterize mosaic mutations in human sporadic Alzheimer's disease (AD) brain samples. They focused on three broad classes of brain cells, neurons that express the marker NeuN, microglia that express the marker PU.1, and double-negative cells that presumably comprise all other brain cell types, including astrocytes, oligodendrocytes, oligodendrocyte progenitor cells, and endothelial cells. The authors find an enrichment of potentially pathogenic somatic mutations in AD microglia compared to controls, with MAPK pathway genes being particularly enriched for somatic mutations in those cells. The authors report a striking enrichment for mutations in the gene CBL and use in vitro functional assays to show that these mutations indeed induce MAPK pathway activation.

The current state of the AD and somatic mutation fields puts this work into context. First, AD is a devastating disease whose prevalence is only increasing as the population of the U.S. is aging, necessitating the investigation of novel features of AD to identify new therapeutic opportunities. Second, microglia have recently come into focus as important players in AD pathogenesis. Many AD risk genes are selectively expressed in microglia, and microglia from AD brain samples show a distinct transcriptional profile indicating an inflammatory phenotype. The authors' previous work shows that a genetic mouse model of mosaic BRAF activation in macrophages (including microglia) displays a neurodegenerative phenotype similar to AD (Mass et al., 2017, doi:10.1038/nature23672). Third, new technological developments have allowed for identifying mosaic mutations present in only a small fraction of or even single cells. Together, these data form a rationale for studying mosaic mutations in microglia in AD. In light of the authors' findings regarding MAPK pathway gene somatic mutations, it is also important to note that MAPK has previously been implicated in AD neuroinflammation in the literature.

Strengths:

The study demonstrated several strengths.

Firstly, the authors used two methods to identify mosaic mutations:<br /> (1) deep (~1,100x) DNA sequencing of a targeted panel of 716 genes they hypothesized might, if mutated somatically, play a role in AD, and<br /> (2) deep (400x) whole-exome sequencing (WES) to identify clonal mosaics outside of those 716 genes.

A second strength is the agreement between these experiments, where WES found many variants identified in the panel experiment, and both experiments revealed somatic mutations in MAPK pathway genes.

Third, the authors demonstrated in several in vitro systems that many mutations they identified in MAPK genes activate MAPK signaling. Finally, the authors showed that in some human brain samples, single-cell gene expression analysis revealed that cells bearing a mosaic MAPK pathway mutation displayed dysregulated inflammatory signaling and dysregulation in other pathways. This single-cell analysis was in agreement with their in vitro analyses.

Weaknesses:

The study also showed some weaknesses. The sample size (45 AD donors and 44 controls) is small, reflected in the relatively modest effect sizes and p-values observed. This weakness is partially ameliorated by the authors' extensive molecular and functional validation of mutation candidates. Another weakness is the lack of discussion of whether the genes found to be mutated somatically in AD show any AD-risk alleles in the population. If they did, it would further support the authors' conclusions that they are playing a role in AD. Finally, as the authors point out, this study cannot conclude whether microglial mosaic mutations cause AD or are an effect of AD. Future studies may shed more light on this important question.

Conclusions and Impact:

Considering the study's aims, strengths, and weaknesses, I conclude that the authors achieved their goal of characterizing the role of mosaic mutations in human AD. Their data strongly suggest that mosaic MAPK mutations in microglia are associated with AD. The impacts of this study remain to be seen, but they could include attempts to target CBL or other mutated genes in the treatment of AD. This work also suggests a similar approach to identifying potentially causative somatic mutations in other neurodegenerative diseases.

Reviewer #1 (Public Review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used.

(2) This study combines the behavioral tests, MRS, and fMRI.

Weaknesses:

(1) In the intro, it seems to me that the multiple-demand (MD) regions are the key in this study. However, I didn't see any results associated with the MD regions. Did I miss something??

(2) How was the sample size determined? Is it sufficient??

(3) In Schallmo elife 2018, there was no correlation between GABA concentration and SI. How can we justify the different results different here?

(4) Basically this study contains the data of SI, BDT, GABA in MT+ and V1, Glu in MT+ and V1-all 6 measurements. There should be 6x5/2 = 15 pairwise correlations. However, not all of these results are included in Figure 1 and supplementary 1-3. I understand that it is not necessary to include all figures. But I suggest reporting all values in one Table.

(5) In Melnick (2013), the IQ scores were measured by the full set of WAIS-III, including all subtests. However, this study only used the visual spatial domain of gF. I wonder why only the visuo-spatial subtest was used not the full WAIS-III?

(6) In the functional connectivity part, there is no explanation as to why only the left MT+ was set to the seed region. What is the problem with the right MT+?

(7) In Melnick (2013), the authors also reported the correlation between IQ and absolute duration thresholds of small and large stimuli. Please include these analyses as well.

Reviewer #2 (Public Review):

Summary:

Recent studies have identified specific regions within the occipito-temporal cortex as part of a broader fronto-parietal, domain-general, or "multiple-demand" (MD) network that mediates fluid intelligence (gF). According to the abstract, the authors aim to explore the mechanistic roles of these occipito-temporal regions by examining GABA/glutamate concentrations. However, the introduction presents a different rationale: investigating whether area MT+ specifically, could be a core component of the MD network.

Strengths:

The authors provide evidence that GABA concentrations in MT+ and its functional connectivity with frontal areas significantly correlate with visuo-spatial intelligence performance. Additionally, serial mediation analysis suggests that inhibitory mechanisms in MT+ contribute to individual differences in a specific subtest of the Wechsler Adult Intelligence Scale, which assesses visuo-spatial aspects of gF.

Weaknesses:

While the findings are compelling and the analyses robust, the study's rationale and interpretations need strengthening. For instance, Assem et al. (2020) have previously defined the core and extended MD networks, identifying the occipito-temporal regions as TE1m and TE1p, which are located more rostrally than MT+. Area MT+ might overlap with brain regions identified previously in Fedorenko et al., 2013, however the authors attribute these activations to attentional enhancement of visual representations in the more difficult conditions of their tasks. For the aforementioned reasons, It is unclear why the authors chose MT+ as their focus. A stronger rationale for this selection is necessary and how it fits with the core/extended MD networks.

Moreover, although the study links MT+ inhibitory mechanisms to a visuo-spatial component of gF, this evidence alone may not suffice to position MT+ as a new core of the MD network. The MD network's definition typically encompasses a range of cognitive domains, including working memory, mathematics, language, and relational reasoning. Therefore, the claim that MT+ represents a new core of MD needs to be supported by more comprehensive evidence.

Reviewer #3 (Public Review):

Summary:

This manuscript aims to understand the role of GABA-ergic inhibition in the human MT+ region in predicting visuo-spatial intelligence through a combination of behavioral measures, fMRI (for functional connectivity measurement), and MRS (for GABA/glutamate concentration measurement). While this is a commendable goal, it becomes apparent that the authors lack fundamental understanding of vision, intelligence, or the relevant literature. As a result, the execution of the research is less coherent, dampening the enthusiasm of the review.

Strengths:

(1) Comprehensive Approach: The study adopts a multi-level approach, i.e., neurochemical analysis of GABA levels, functional connectivity, and behavioral measures to provide a holistic understanding of the relationship between GABA-ergic inhibition and visuo-spatial intelligence.

(2) Sophisticated Techniques: The use of ultra-high field magnetic resonance spectroscopy (MRS) technology for measuring GABA and glutamate concentrations in the MT+ region is a recent development.

Weaknesses:

Study Design and Hypothesis<br /> (1) The central hypothesis of the manuscript posits that "3D visuo-spatial intelligence (the performance of BDT) might be predicted by the inhibitory and/or excitation mechanisms in MT+ and the integrative functions connecting MT+ with the frontal cortex." However, several issues arise:<br /> 1.1 The Suppression Index depicted in Figure 1a, labeled as the "behavior circle," appears irrelevant to the central hypothesis.<br /> 1.2 The construct of 3D visuo-spatial intelligence, operationalized as the performance in the Block Design task, is inconsistently treated as another behavioral task throughout the manuscript, leading to confusion.<br /> 1.3 The schematics in Figure 1a and Figure 6 appear too high-level to be falsifiable. It is suggested that the authors formulate specific and testable hypotheses and preregister them before data collection.

(2) Central to the hypothesis and design of the manuscript is a misinterpretation of a prior study by Melnick et al. (2013). While the original study identified a strong correlation between WAIS (IQ) and the Suppression Index (SI), the current manuscript erroneously asserts a specific relationship between the block design test (from WAIS) and SI. It should be noted that in the original paper, WAIS comprises Similarities, Vocabulary, Block design, and Matrix reasoning tests in Study 1, while the complete WAIS is used in Study 2. Did the authors conduct other WAIS subtests other than the block design task?

(3) Additionally, there are numerous misleading references and unsubstantiated claims throughout the manuscript. As an example of misleading reference, "the human MT ... a key region in the multiple representations of sensory flows (including optic, tactile, and auditory flows) (Bedny et al., 2010; Ricciardi et al., 2007); this ideally suits it to be a new MD core." The two references in this sentence are claims about plasticity in the congenitally blind with sensory deprivation from birth, which is not really relevant to the proposal that hMT+ is a new MD core in healthy volunteers.<br /> Another example of unsubstantiated claim: the rationale for selecting V1 as the control region is based on the assertion that "it mediates the 2D rather than 3D visual domain (Born & Bradley, 2005)". That's not the point made in the Born & Bradley (2005) paper on MT. It's crucial to note that V1 is where the initial binocular convergence occurs in cortex, i.e., inputs from both the right and left eyes to generate a perception of depth.

Results & Discussion<br /> (1) The missing correlation between SI and BDT is crucial to the rest of the analysis. The authors should discuss whether they replicated the pattern of results from Melnick et al. (2013) despite using only one WAIS subtest.

(2) ROIs: can the authors clarify if the results are based on bilateral MT+/V1 or just those in the left hemisphere? Can the authors plot the MRS scan area in V1? I would be surprised if it's precise to V1 and doesn't spread to V2/3 (which is fine to report as early visual cortex).

(3) Did the authors examine V1 FC with either the frontal regions and/or whole brain, as a control analysis? If not, can the author justify why V1 serves as the control region only in the MRS but not in FC (Figure 4) or the mediation analysis (Figure 5)? That seems a little odd given that control analyses are needed to establish the specificity of the claim to MT+.

(4) It is not clear how to interpret the similarity or difference between panels a and b in Figure 4.

(5) SI is not relevant to the authors' priori hypothesis, but is included in several mediation analyses. Can the authors do model comparisons between the ones in Figure 5c, d, and Figure S6? In other words, is SI necessary in the mediation model? There seem discrepancies between the necessity of SI in Figures 5c/S6 vs. Figure 5d.

(6) The sudden appearance of "efficient information" in Figure 6, referring to the neural efficiency hypothesis, raises concerns. Efficient visual information processing occurs throughout the visual cortex, starting from V1. Thus, it appears somewhat selective to apply the neural efficiency hypothesis to MT+ in this context.

Transparency Issues:<br /> (1) Don't think it's acceptable to make the claim that "All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary information". It is the results or visualizations of data analysis, rather than the raw data themselves, that are presented in the paper/supp info.

(2) No GitHub link has been provided in the manuscript to access the source data, which limits the reproducibility and transparency of the study.

Minor:<br /> "Locates" should be replaced with "located" throughout the paper. For example: "To investigate this issue, this study selects the human MT complex (hMT+), a region located at the occipito-temporal border, which represents multiple sensory flows, as the target brain area."

Use "hMT+" instead of "MT+" to be consistent with the term in the literature.

"Green circle" in Figure 1 should be corrected to match its actual color.

The abbreviation for the Wechsler Adult Intelligence Scale should be "WAIS," not "WASI."

Reviewer #1 (Public Review):

Summary:

This is a fine paper that serves the purpose to show that the use of light sheet imaging may be used to provide whole brain imaging of axonal projections. The data provided suggest that at this point the technique provides lower resolution than with other techniques. Nonetheless, the technique does provide useful, if not novel, information about particular brain systems.

Strengths:

The manuscript is well written. In the introduction a clear description of the functional organization of the barrel cortex is provided provides the context for applying the use of specific Cre-driver lines to map the projections of the main cortical projection types using whole brain neuroanatomical tracing techniques. The results provided are also well written, with sufficient detail describing the specifics of how techniques were used to obtain relevant data. Appropriate controls were done, including the identification of whisker fields for viral injections and determination of the laminar pattern of Cre expression. The mapping of the data provides a good way to visualize low resolution patterns of projections.

Weaknesses:

(1) The results provided are, as stated in the discussion, "largely in agreement with previously reported studies of the major projection targets". However it must be stated that the study does not "extend current knowledge through the high sensitivity for detecting sparse axons, the high specificity of labeling of genetically defined classes of neurons and the brain wide analysis for assigning axons to detailed brain regions" which have all been published in numerous other studies. ( the allen connectivity project and related papers, along with others). If anything the labeling of axons obtained with light sheet imaging in this study does not provide as detailed mapping obtained with other techniques. Some detail is provided of how the raw images are processed to resolve labeled axons, but the images shown in the figures do not demonstrate how well individual axons may be resolved, of particular interest would be to see labeling in terminal areas such as other cortical areas, striatum and thalamus. As presented the light sheet imaging appears to be rather low resolution compared to the many studies that have used viral tracing to look at cortical projections from genetically identified cortical neurons.<br /> (2) Amongst the limitations of this study is the inability to resolve axons of passage and terminal fields. This has been done in other studies with viral constructs labeling synaptophysin. This should be mentioned.<br /> (3) There is no quantitative analysis of differences between the genetically defined neurons projecting to the striatum, what is the relative area innervated by, density of terminals, other measures.<br /> (4) Figure 5 is an example of the type of large sets of data that can be generated with whole brain mapping and registration to the Allen CCF that provides information of questionable value. Ordering the 50 plus structures by the density of labeling does not provide much in terms of relative input to different types of areas. There are multiple subregions for different functional types ( ie, different visual areas and different motor subregions are scattered not grouped together. Makes it difficult to understand any organizing principles.<br /> (5) The GENSAT Cre driver lines used must have the specific line name used, not just the gene name as the GENSAT BAC-Cre lines had multiple lines for each gene and often with very different expression patterns. Rbp4_KL100, Tlx3_PL56, Sim1_KJ18, Ntsr1_ GN220.

Reviewer #2 (Public Review):

Summary:

This study takes advantage of multiple methodological advances to perform layer-specific staining of cortical neurons and tracking of their axons to identify the pattern of their projections. This publication offers a mesoscale view of the projection patterns of neurons in the whisker primary and secondary somatosensory cortex. The authors report that, consistent with the literature, the pattern of projection is highly different across cortical layers and subtype, with targets being located around the whole brain. This was tested across 6 different mouse types that expressed a marker in layer 2/3, layer 4, layer 5 (3 sub-types) and layer 6.<br /> Looking more closely at the projections from primary somatosensory cortex into the primary motor cortex, they found that there was a significant spatial clustering of projections from topographically separated neurons across the primary somatosensory cortex. This was true for neurons with cell bodies located across all tested layers/types.

Strengths:

This study successfully looks at the relevant scale to study projection patterns, which is the whole brain. This is achieved thanks to an ambitious combination of mouse lines, immuno-histochemistry, imaging and image processing, which results in a standardized histological pipeline that processes the whole-brain projection patterns of layer-selected neurons of the primary and secondary somatosensory cortex.<br /> This standardization means that comparisons between cell-types projection patterns are possible and that both the large-scale structure of the pattern and the minute details of the intra-areas pattern are available.<br /> This reference dataset and the corresponding analysis code are made available to the research community.

Weaknesses:

One major question raised by this dataset is the risk of missing axons during the post-processing step. Indeed, it appears that the control and training efforts have focused on the risk of false positives (see Figure 1 supplementary panels). And indeed, the risk of overlooking existing axons in the raw fluorescence data id discussed in the article.

Based on the data reported in the article, this is more than a risk. In particular, Figure 2 shows an example Rbp4-L5 mouse where axonal spread seems massive in Hippocampus, while there is no mention of this area in the processed projection data for this mouse line.

Similarily, the Ntsr1-L6CT example shows a striking level of fluorescence in Striatum, that does not reflect in the amount of axons that are detected by the algorithms in the next figures.<br /> These apparent discrepancies may be due to non axonal-specific fluorescence in the samples. In any case, further analysis of such anatomical areas would be useful to consolidate the valuable dataset provided by the article.

Reviewer #3 (Public Review):

Summary:

-The paper offers a systematic and rigorous description of the layer-and sublayer specific outputs of the somatosensory cortex using a modern toolbox for the analysis of brain connectivity which combines: 1) Layer-specific genetic drivers for conditional viral tracing; 2) whole brain analyses of axon tracts using tissue clearing and imaging; 3) Segmentation and quantification of axons with normalization to the number of transduced neurons; 4) registration of connectivity to a widely used anatomical reference atlas; 5) functional validation of the connectivity using optogenetic approaches in vivo.

Strengths:

- Although the connectivity of the somatosensory cortex is already known, precise data are dispersed in different accounts (papers, online resources,) using different methods. So the present account has the merit of condensing this information in one very precisely documented report. It also brings new insights on the connectivity, such as the precise comparison of layer specific outputs, and of the primary and secondary somatosensory areas. It also shows a topographic organization of the circuits linking the somatosensory and motor cortices. The paper also offers a clear description of the methodology and of a rigorous approach to quantitative anatomy.

Weaknesses:

The weakness relates to the intrinsic limitations of the in toto approaches, that currently lack the precision and resolution allowing to identify single axons, axon branching or synaptic connectivity. These limitations are identified and discussed by the authors.

Reviewer #1 (Public Review):

Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.

The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.

These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.

(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.

(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.

(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).

(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.

(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?

References:<br /> - Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.<br /> - D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.<br /> - Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.<br /> - Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.<br /> - Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.<br /> - Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.<br /> - Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.<br /> - Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.<br /> - Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.<br /> - Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.<br /> - Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.<br /> - Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.

Reviewer #2 (Public Review):

Summary:

The authors re-analyse MEG data from a speech production and perception study and extend their previous Granger causality analysis to a larger number of cortical-cortical and in particular cortical-subcortical connections. Regions of interest were defined by means of a meta-analysis using Neurosynth.org and connectivity patterns were determined by calculating directed influence asymmetry indices from the Granger causality analysis results for each pair of brain regions. Abbasi et al. report feedforward signals communicated via fast rhythms and feedback signals via slow rhythms below 40 Hz, particularly during speaking. The authors highlight one of these connections between the right cerebellum lobule VI and auditory association area A5, where in addition the connection strength correlates negatively with the strength of speech tracking in the theta band during speaking (significant before multiple comparison correction). Results are interpreted within a framework of active inference by minimising prediction errors.

While I find investigating the role of cortical-subcortical connections in speech production and perception interesting and relevant to the field, I am not yet convinced that the methods employed are fully suitable to this endeavour or that the results provide sufficient evidence to make the strong claim of dissociation of bottom-up and top-down information flow during speaking in distinct frequency bands.

Strengths:

The investigation of electrophysiological cortical-subcortical connections in speech production and perception is interesting and relevant to the field. The authors analyse a valuable dataset, where they spent a considerable amount of effort to correct for speech production-related artefacts. Overall, the manuscript is well-written and clearly structured.

Weaknesses:

The description of the multivariate Granger causality analysis did not allow me to fully grasp how the analysis was performed and I hence struggled to evaluate its appropriateness.<br /> Knowing that (1) filtered Granger causality is prone to false positives and (2) recent work demonstrates that significant Granger causality can simply arise from frequency-specific activity being present in the source but not the target area without functional relevance for communication (Schneider et al. 2021) raises doubts about the validity of the results, in particular with respect to their frequency specificity. These doubts are reinforced by what I perceive as an overemphasis on results that support the assumption of specific frequencies for feedforward and top-down connections, while findings not aligning with this hypothesis appear to be underreported. Furthermore, the authors report some main findings that I found difficult to reconcile with the data presented in the figures. Overall, I feel the conclusions with respect to frequency-specific bottom-up and top-down information flow need to be moderated and that some of the reported findings need to be checked and if necessary corrected.

Major points

(1) I think more details on the multivariate GC approach are needed. I found the reference to Schaum et al., 2021 not sufficient to understand what has been done in this paper. Some questions that remained for me are:

(i) Does multivariate here refer to the use of the authors' three components per parcel or to the conditioning on the remaining twelve sources? I think the latter is implied when citing Schaum et al., but I'm not sure this is what was done here?

If it was not: how can we account for spurious results based on indirect effects?

(ii) Did the authors check whether the GC of the course-target pairs was reliably above the bias level (as Schaum et. al. did for each condition separately)? If not, can they argue why they think that their results would still be valid? Does it make sense to compute DAIs on connections that were below the bias level? Should the data be re-analysed to take this concern into account?

(iii) You may consider citing the paper that introduced the non-parametric GC analysis (which Schaum et al. then went on to apply): Dhamala M, Rangarajan G, Ding M. Analyzing Information Flow in Brain Networks with Nonparametric Granger Causality. Neuroimage. 2008; 41(2):354-362. https://doi.org/10.1016/j.neuroimage.2008.02. 020

(2) GC has been discouraged for filtered data as it gives rise to false positives due to phase distortions and the ineffectiveness of filtering in the information-theoretic setting as reducing the power of a signal does not reduce the information contained in it (Florin et al., 2010; Barnett and Seth, 2011; Weber et al. 2017; Pinzuti et al., 2020 - who also suggest an approach that would circumvent those filter-related issues). With this in mind, I am wondering whether the strong frequency-specific claims in this work still hold.

(3) I found it difficult to reconcile some statements in the manuscript with the data presented in the figures:

(i) Most notably, the considerable number of feedforward connections from A5 and STS that project to areas further up the hierarchy at slower rhythms (e.g. L-A5 to R-PEF, R-Crus2, L CB6 L-Tha, L-FOP and L-STS to R-PEF, L-FOP, L-TOPJ or R-A5 as well as R-STS both to R-Crus2, L-CB6, L-Th) contradict the authors' main message that 'feedback signals were communicated via slow rhythms below 40 Hz, whereas feedforward signals were communicated via faster rhythms'. I struggled to recognise a principled approach that determined which connections were highlighted and reported and which ones were not.

(ii) "Our analysis also revealed robust connectivity between the right cerebellum and the left parietal cortex, evident in both speaking and listening conditions, with stronger connectivity observed during speaking. Notably, Figure 4 depicts a prominent frequency peak in the alpha band, illustrating the specific frequency range through which information flows from the cerebellum to the parietal areas." There are two peaks discernible in Figure 4, one notably lower than the alpha band (rather theta or even delta), the other at around 30 Hz. Nevertheless, the authors report and discuss a peak in the alpha band.

(iii) In the abstract: "Notably, high-frequency connectivity was absent during the listening condition." and p.9 "In contrast with what we reported for the speaking condition, during listening, there is only a significant connectivity in low frequency to the left temporal area but not a reverse connection in the high frequencies."<br /> While Fig. 4 shows significant connectivity from R-CB6 to A5 in the gamma frequency range for the speaking, but not for the listening condition, interpreting comparisons between two effects without directly comparing them is a common statistical mistake (Makin and Orban de Xivry). The spectrally-resolved connectivity in the two conditions actually look remarkably similar and I would thus refrain from highlighting this statement and indicate clearly that there were no significant differences between the two conditions.

(iv) "This result indicates that in low frequencies, the sensory-motor area and cerebellum predominantly transmit information, while in higher frequencies, they are more involved in receiving it."<br /> I don't think that this statement holds in its generality: L-CB6 and R-3b both show strong output at high frequencies, particularly in the speaking condition. While they seem to transmit information mainly to areas outside A5 and STS these effects are strong and should be discussed.

(4) "However, definitive conclusions should be drawn with caution given recent studies raising concerns about the notion that top-down and bottom-up signals can only be transmitted via separate frequency channels (Ferro et al., 2021; Schneider et al., 2021; Vinck et al., 2023)."

I appreciate this note of caution and think it would be useful if it were spelled out to the reader why this is the case so that they would be better able to grasp the main concerns here. For example, Schneider et al. make a strong point that we expect to find Granger-causality with a peak in a specific frequency band for areas that are anatomically connected when the sending area shows stronger activity in that band than the receiving one, simply because of the coherence of a signal with its own linear projection onto the other area. The direction of a Granger causal connection would in that case only indicate that one area shows stronger activity than the other in the given frequency band. I am wondering to what degree the reported connectivity pattern can be traced back to regional differences in frequency-specific source strength or to differences in source strength across the two conditions.

Reviewer #3 (Public Review):

In the current paper, Abbasi et al. aimed to characterize and compare the patterns of functional connectivity across frequency bands (1 Hz - 90 Hz) between regions of a speech network derived from an online meta-analysis tool (Neurosynth.org) during speech production and perception. The authors present evidence for complex neural dynamics from which they highlight directional connectivity from the right cerebellum to left superior temporal areas in lower frequency bands (up to beta) and between the same regions in the opposite direction in the (lower) high gamma range (60-90 Hz). Abbasi et al. interpret their findings within the predictive coding framework, with the cerebellum and other "higher-order" (motor) regions transmitting top-down sensory predictions to "lower-order" (sensory) regions in the lower frequencies and prediction errors flowing in the opposite direction (i.e., bottom-up) from those sensory regions in the gamma band. They also report a negative correlation between the strength of this top-down functional connectivity and the alignment of superior temporal regions to the syllable rate of one's speech.

Strengths:

(1) The comprehensive characterization of functional connectivity during speaking and listening to speech may be valuable as a first step toward understanding the neural dynamics involved.

(2) The inclusion of subcortical regions and connectivity profiles up to 90Hz using MEG is interesting and relatively novel.

(3) The analysis pipeline is generally adequate for the exploratory nature of the work.

Weaknesses:

(1) The work is framed as a test of the predictive coding theory as it applies to speech production and perception, but the methodological approach is not suited to this endeavor.

(2) Because of their theoretical framework, the authors readily attribute roles or hierarchy to brain regions (e.g., higher- vs lower-order) and cognitive functions to observed connectivity patterns (e.g., feedforward vs feedback, predictions vs prediction errors) that cannot be determined from the data. Thus, many of the authors' claims are unsupported.

(3) The authors' theoretical stance seems to influence the presentation of the results, which may inadvertently misrepresent the (otherwise perfectly valid; cf. Abbasi et al., 2023) exploratory nature of the study. Thus, results about specific regions are often highlighted in figures (e.g., Figure 2 top row) and text without clear reasons.

(4) Some of the key findings (e.g., connectivity in opposite directions in distinct frequency bands) feature in a previous publication and are, therefore, interesting but not novel.

(5) The quantitative comparison between speech production and perception is interesting but insufficiently motivated.

(6) Details about the Neurosynth meta-analysis and subsequent selection of brain regions for the functional connectivity analyses are incomplete. Moreover, the use of the term 'Speech' in Neurosynth seems inappropriate (i.e., includes irrelevant works, yielding questionable results). The approach of using separate meta-analyses for 'Speech production' and 'Speech perception' taken by Abbasi et al. (2023) seems more principled. This approach would result, for example, in the inclusion of brain areas such as M1 and the BG that are relevant for speech production.

(7) The results involving subcortical regions are central to the paper, but no steps are taken to address the challenges involved in the analysis of subcortical activity using MEG. Additional methodological detail and analyses would be required to make these results more compelling. For example, it would be important to know what the coverage of the MEG system is, what head model was used for the source localization of cerebellar activity, and if specific preprocessing or additional analyses were performed to ensure that the localized subcortical activity (in particular) is valid.

(8) The results and methods are often detailed with important omissions (a speech-brain coupling analysis section is missing) and imprecisions (e.g., re: Figure 5; the Connectivity Analysis section is copy-pasted from their previous work), which makes it difficult to understand what is being examined and how. (It is also not good practice to refer the reader to previous publications for basic methodological details, for example, about the experimental paradigm and key analyses.) Conversely, some methodological details are given, e.g., the acquisition of EMG data, without further explanation of how those data were used in the current paper.

(9) The examination of gamma functional connectivity in the 60 - 90 Hz range could be better motivated. Although some citations involving short-range connectivity in these frequencies are given (e.g., within the visual system), a more compelling argument for looking at this frequency range for longer-range connectivity may be required.

(10) The choice of source localization method (linearly constrained minimum variance) could be explained, particularly given that other methods (e.g. dynamic imaging of coherent sources) were specifically designed and might potentially be a better alternative for the types of analyses performed in the study.

(11) The mGC analysis needs to be more comprehensively detailed for the reader to be able to assess what is being reported and the strength of the evidence. Relatedly, first-level statistics (e.g., via estimation of the noise level) would make the mGC and DAI results more compelling.

(12) Considering the exploratory nature of the study, it is essential for other researchers to continue investigating and validating the results presented in the current manuscript. Thus, it is concerning that data and scripts are not fully and openly available. Data need not be in its raw state to be shared and useful, which circumvents the stated data privacy concerns.

Reviewer #1 (Public Review):

Summary:

This paper reports the finding that less fat accumulates in C. elegans that are feeding on Comamonas aquatica DA1877 (DA) vs the standard lab diet of Escherichia coli OP50 (OP50). While these bacteria are likely to be different in many ways, the authors found that fat accumulation phenotype depends on the vitamin B12 content of the bacterial diet and the involvement of B12 in the methionine cycle, affecting SAMS-1 and phosphatidylcholine (PC) synthesis. They report that low PC levels activate SREBP-1 (SBP-1 in C. elegans) and that an important target of SBP-1 is the delta 9 desaturase FAT-7. Finally, they describe a role for ASM-3, an acid sphingomyelinase, in influencing PC synthesis and fat accumulation in the worm.

Strengths:

This is a comprehensive story about how a dietary change affects fat accumulation in C. elegans. Their experimental evidence is convincing. The most novel aspect of this paper is that the coelomecyte expression of asm-3 contributes to PC/TAG homeostasis in C. elegans, which most likely occurs through the production of phosphocholine by the enzymatic breakdown of sphingomyelin by ASM-3. The phosphocholine will provide precursors for phosphatidylcholine (PC) synthesis, contributing to the PC synthesis pathway.

Weaknesses:

In the way the story is presented, the authors tend to imply that they discovered the pathways of B12, PC, SBP-1, and FAT-7, ignoring some important studies describing the relationship between PC synthesis and TAG accumulation in both the mammalian lipid metabolism field (liver) as well as in C. elegans. Many previous studies with similar results are not cited appropriately. Thus, the pathways reported in the paper are not new, and in this sense, the work is mostly confirmatory.

Reviewer #2 (Public Review):

Summary:

Han et al. present a manuscript focusing on difference metabolism and the regulatory circuits controlling it in C. elegans fed two bacterial diets. In the first three figures and a half figures, using a combination of methods, they investigate lipid levels, changes in gene expression and genetic assays to come to the conclusion that vitamin B12 acts through the S-adenosylmethioine synthase sams-1 to perturb phosphatidylcholine levels, which in turn stimulate the C. elegans ortholog of the SREBP transcription factors to activate fatty acid synthesis genes such as fat-7/SCD1. Thus, while connections between diet, metabolic pathways and gene regulation is of general interest, this study largely confirms the work of others without direct credit in many instances, then fails to develop a more novel cell non-autonomous link between the pathways in the last two figures. Thus, this study would be expected to have a useful impact on the field, if it can be placed in context of previously published work.

Strengths:

(1) Connections between diet, metabolic pathways and gene regulation is of general interest<br /> (2) Figures 1-4 confirm data/observations from previously published work from MacNeil, et al. Cell 2015; Walker, et al. Cell 2011; Svensk, et al. PLoS Genetics 2013; Smulan, et al. Cell Reports, 2016; Giese, et al. eLife 2020 and Qin, et al. Cell Reports 2022..<br /> (3) The data in figures 5 and 6 showing importance of non-cell autonomous effects on metabolism.

Weaknesses:

(1) In order to differentiate their study from previous work, it seems that the authors try to make the argument that PC is higher in Comomonas than E. coli, therefore they are looking at repression of SBP-1-dependent function, however, the pairing of the diets is arbitrary, and the comparisons could easily be reversed. They are simply comparing a higher to a lower level of PC, rather than a basal to a lower, thus the concepts are the same. In addition, they fail to cite the larger body of literature linking phospholipid balance to SREBP function. For example, multiple studies in mammalian models link phospholipid balance, not just lowered PC, to SREBP function: Lim, Genes and Dev 2011; Wang, et al. Cell Stem Cell, 2018; Rong, et al. J Clin Invest 2017; Smulan et al, Cell Reports, 2016; Dobrosotskaya, Science. 2002 and recently, Rong, et al. Cell Met 2024.

(2) Figure 1: For example, the data in figure 1, shows measures of lipid content, RNA seq showing changes in metabolic enzymes such as fat-7/SCD-1 and lipid levels have already been shown in MacNeil, et al. Cell 2013 (lipid levels and gene expression changes) and the lipid levels in Comomonas vs E. coli were published in Ditot, et al. Nature Communications 2022 by Dr. Marian Walhout's lab.

(3) Figure 2/3: In Figure 2 and 3, they use a genetic screen to find regulators of fat-7/scd1 expression, and unsurprisingly, pull out genes with known to regulate this pathway. The authors go on to show that changes in SAM lead to changes in PC, and affect SBP-1/SREBP-1-dependent lipogenesis. This is a well described pathway from publications by the Walhout lab, Dr. Amy Walker's lab and Dr. Marc Pilon's lab (Walker, et al. Cell 2011; Svensk, et al. PLoS Genetics 2013; Smulan, et al. Cell Reports, 2016; Giese, et al. eLife 2020) in addition to a recent publication, Qin, et al. Cell Reports 2022. While some of these studies are cited in other places in the manuscript, the authors describe their results as "discovery", then fail to cite the relevant studies at those points (selected examples below

(4) Selected examples of citation issues:

a) Selected example: pg 6: "To understand the mechanism underlying the regulation of host lipid content triggered by DA, we examined the gene expression changes elicited by the two different bacterial diets in young adult animals by RNA-seq...In particular, genes related to the biosynthesis of unsaturated fatty acids showed a significant decrease in expression in DA-fed worms. For example, the delta-(9) fatty acid desaturases, fat-5 and fat-7, (which convert fatty acids 16:0 to 16:1n7 and 18:0 to 18:1n9, respectively32) decreased"

MacNeil et al Cell 2013 published a transcriptomics comparing young adult DA and Op50, which demonstrated decreases in fat-5 and fat-7. While MacNeil is cited in other parts of the paper, since the authors have performed a highly similar experiment and obtained similar results, this should be described as confirming the MacNeil study rather than as new data.

b) Selected Example: pg 10: "To determine whether PC levels have a causal effect on organismal lipid content, we supplemented worm diets with choline, the PC precursor, and uncovered a dose-dependent decrease in lipid content as measured by O.R.O staining (Figure 3B)."

Addition of choline to supplement defects in PC synthesis was first shown by Brendza, et al. Biochem J 2007. It was confirmed in Walker, et al. 2011, and further confirmation of PC rescue show in Ding, et al. 2015. The Brendza study is not cited at all and while studies from the Walker lab are cited in other places, the authors omit that changes in the DA diet are the same as changes seen when choline rescues PC loss from other perturbations.

c) Selected Example: pg 9: "Notably, DA has been reported as a B12-rich bacterium compared to OP16, hinting at the possibility that the DA diet might boost dietary B12 levels."

Reference 16 is Watson, et al. Cell 2015 where the Walhout lab demonstrates that DA does in fact act through the diet to alter the Met/SAM cycle and other B12 dependent processes in C. elegans. This paper, along with MacNeil above broke ground in linking B12 and the Met/SAM cycle to specific phenotypes in C. elegans, which was followed up by extensive work from the Walhout lab on this cycle, thus, it seems odd that the authors describe their own data as "hinting" at this connection.

d) Selected example: pg 17: "Indeed, this is further supported by our observation that mutants of histone methyltransferases SET-2 and SET-30 (which install H3K4me1 and H3K4me2, respectively) exhibited elevated lipid content on DA diet (data not shown). Notably, while both set-2 and set-30 mutants had this effect, only set-2 appears to control fat-7 expression (data not shown)". Extensive work from Dr. Anne Brunet's lab (Greer, et al. Nature 2010; Greer, et al. Nature 2011; Han, et al. Nature 2017) link set-2 and H3K4 methylation to lipid accumulation and fat-7. The authors fail to cite these studies.

Reviewer #3 (Public Review):

Summary:

The authors presented data that linked vitamin B12, S-adenosyl methionine (SAM), and phosphatidylcholine (PC) synthesis to lipid homeostasis in C. elegans. They confirmed mechanisms previously shown by other labs, including the regulation of FAT-7 expression by SBP-1, and the targeting of SEIP-1 by PC levels. The authors also attempted to link the synthesis of phospho-choline by the ASM-3 sphingomyelinase to PC synthesis and lipid homeostasis. However, the relative contribution of phospho-choline by ASM-3 versus the canonical Kennedy pathway was not elucidated. Therefore, the significance of the ASM-3-dependent mechanism to PC synthesis requires further investigation.

Strengths:

The authors used a wide range of biochemical and cell biological methods to measure fatty acid composition, neutral lipid levels, and lipid droplet dynamics in C. elegans. The quality of the data is generally high.

Weaknesses:

Data interpretation and the construction of the working model did not seem to take into account the two well-established pathways for PC synthesis. The Kennedy pathway generates PC from phospho-choline and DAG via a cytidine-based intermediate. The second PC synthesis pathway entails the methylation of PE by PEMT, with the donor methyl groups provided by the vitamin B12-dependent 1-carbon cycle. The authors' model seemed to overlook part of the Kennedy pathway that involves choline kinase (and not ASM-3) as the canonical enzyme that generates phospho-choline. The authors also did not explicitly consider DAG as a precursor of triacylglycerol (TAG), which was directly or indirectly measured as a readout of organismal fat content in the paper. Therefore, alternative models should be entertained. For example, the proposed genetic and dietary effects on lipid homeostasis could stem from the competition for a limiting pool of precursors that were shared by PC and TAG synthesis. PC itself may not have a deterministic role, as depicted by the authors' model. Finally, the claim that "coelomocytes regulate diets-induced lipid homeostasis through asm-3" was not well supported. In the absence of quantitative analysis of phospho-choline in mutants, it was unclear how much ASM-3 contributed to the overall phospho-choline, and ultimately PC level. The proposed inter-tissue regulation of PC synthesis also requires coelomocytes-specific knock-down/depletion of asm-3 for verification.

Reviewer #1 (Public Review):

Li et al. report here on the expression of a G-protein subunit Gng13 in ectopic tuft cells that develop after severe pulmonary injury in mice. By deleting this gene in ectopic tuft cells as they arise, the authors observed worsened lung injury and greater inflammation after influenza infection, as well as a decrease in the overall number of ectopic tuft cells. This was in stark contrast to deletion of Trpm5, a cation channel generally thought to be required for all functional gustatory signaling in tuft cells, where no phenotype is observed. Strengths here include a thorough assessment of lung injury via a number of different techniques. Weaknesses are notable: Confusingly, these findings are at odds with reports from other groups demonstrating no obvious phenotype upon influenza infection in mice lacking the transcription factor Pou2f3, which is essential for all tuft cell specification and development. The authors speculate that heterogeneity within nascent tuft cell populations, specifically the presence of pro- and anti-inflammatory tuft cells, may explain this difference, but they do not provide any data to support this idea.

Notes on revision: The authors provided responses to some of my critiques. I think the central discrepancy between the lack of a phenotype in Pou2f3 and Trpm5 KO mice compared to the stronger phenotype in the Chat-Cre / Gng13 KO mice remains unresolved and will require future work to provide a clear model. This may or may not ultimately involve tuft cell heterogeneity.

Reviewer #2 (Public Review):

Summary:

The study by Li et al. aimed to demonstrate the role of the G𝛾13-mediated signal transduction pathway in tuft cell-driven inflammation resolution and repairing injured lung tissue. The authors showed the reduced number of tuft cells in the parenchyma of G𝛾13 null lungs following viral infection. Mice with a G𝛾13 null mutation showed increased lung damage and heightened macrophage infiltration when exposed to the H1N1 virus. Their further findings suggested that lung inflammation resolution, epithelial barrier and fibrosis were worsen in G𝛾13 null mutants.

Strengths:

The revised study carefully analyzed phenotypes in mice lacking G𝛾13 in response to viral infection, providing further support that G𝛾13+ tuft cells play a role in the resolution of inflammation and injury repair.

Reviewer #3 (Public Review):

Summary:

Prior research on SCC3, a cohesin subunit protein, in yeast and Arabidopsis has underscored its vital role in cell division. This study investigated into the specific functions of SCC3 in rice mitosis and meiosis. In a weakened SCC3 mutant, sister chromatids separating was observed in anaphase I, resulting in 24 univalents and subsequent sterility. The authors meticulously documented SCC3's loading and degradation dynamics on chromosomes, noting its impact on DNA replication. Despite the loss of homologous chromosome pairing and synapsis in the mutant, chromosomes retained double-strand breaks without fragmenting. Consequently, the authors inferred that in the scc3 mutant, DNA repair more frequently relies on sister chromatids as templates compared to the wild type.

Strengths:

The study presents exceptionally well-executed research in the field of rice cytogenetics.

Weaknesses:

While the paper's conclusions are generally well-supported, further substantiation is needed for the claim that SCC3 inhibits template choice for sister chromatids. To bolster this conclusion, I recommend that the authors perform whole-genome sequencing on parental and F1 individuals from two rice variants, subsequently calculating the allele frequencies at heterozygous sites in the F1 individuals. If SCC3 indeed inhibits inter-sister chromatid repair in the wild type, we would anticipate a higher frequency of inter-homologous chromosome repair (i.e., gene conversion). This should be manifested as a bias away from the Mendelian inheritance ratio (50:50) in the offspring of the wild type compared to the offspring of the scc3+/- mutant.

Reviewer #1 (Public Review):

Summary:

The revised manuscript is much improved. As stated previously, it is on an interesting and important topic and provides many new potentially important findings. The manuscript contains a large amount of high-quality data. In the revised manuscript, the authors have done a nice job addressing the concerns raised in the previous review. They have refined their conclusions and the evidence provided supports conclusions drawn. Likewise, the writing and low of the manuscript is much improved.

Strengths:

The manuscript contains a large amount of high-quality data that is used to draw interesting and important conclusions.

Weaknesses:

There are still some issues with grammar and word usage, but these should be easily corrected with some additional minor editing. Other than some minor editing, my only real question/concern is whether the data presented shows that SCC3 is directly involved in gene regulation. It may well be that changes in chromatin structure caused by mutations in SCC3 and the axial element protein containing genes examined indirectly affect transcript levels for the genes examined.

Reviewer #2 (Public Review):

Summary:

This manuscript shows detailed evidence about the role of cohesin regulator in rice meiosis and mitosis

Strengths:

There is a very clear mechanism for its role during replication

Weaknesses:

The authors did not consider to create heterozygous mutants for the replication fork.

April 15. Revisions read.

Reviewer #1 (Public Review):

Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.

The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.

Reviewer #2 (Public Review):

The authors identify a third component in the interaction between myosin Va and melanophilin- an interaction between a 32-residue sequence encoded by exon-g in myosin Va and melanophilin's actin binding domain. This interaction has implications for how melanosome motility may be regulated.

The authors have now included some necessary controls that were requested. In terms of adding new information to increase the significance and impact of the paper, they added a single affinity measurement. Unfortunately, it did not involve Exon G specifically. Moreover, they did not add any new mechanistic or functional data to provide a more conceptual advance. For example, is the Exon G interaction regulated by phosphorylation? Is this what dictates the choice between Mlph's actin binding domain (ABD) binding to actin or to exon-G. How does local actin concentration influence this decision. What changes regarding melanosome dynamics in cells between these two alternatives? Do in vitro reconstitution assays show that binding to Exon-G instead of actin affects the processivity of a Rab27a/Myosin 5a/Mlph transport complex? Finally, while the authors make clear in the abstract and text that they are just identifying a third component that mediates the Melanophilin-dependent association of myosin-5a with melanosomes, the title gives the impression that they identified all three in this manuscript. I really think the title should be changed to something like Identification of a third component that mediates the Melanophilin-dependent association of myosin-5a with melanosomes, as this accurately reflects what is new in this work.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

Reviewer #2 (Public Review):

Summary:

In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibit form stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:

The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain, and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gβγ subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Weakness:

The set of experiments with the disulfide bond S235C/M244C caused a bit of confusion for interpretation, it should be moved into the supplement, and the text and Figure 4 were altered accordingly.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42. In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

Reviewer #1 (Public Review):

The comments below are from my review of the first submission of this article. I would now like to thank the authors for their hard work in responding to my comments. I am happy with the changes they have made, in particular the inclusion of further experimental evidence in Figures 2 and 4. I have no further comments to make.

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development timecourse. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.

I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3 and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.

I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?

I had a number of issues with the way in which some of the data is presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels?

Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.

Reviewer #2 (Public Review):

Summary:

This study represents an ambitious endeavor to comprehensively analyze the role of miR-199a/b-5p and its networks in cartilage formation. By conducting experiments that go beyond in vitro MSC differentiation models, more robust conclusions can be achieved.

Strengths:

This research investigates the role of miR-199a/b-5p during chondrogenesis using bioinformatics and in vitro experimental systems. The significance of miRNAs in chondrogenesis and OA is crucial, warranting further research, and this study contributes novel insights.

Weaknesses:

While miR-140 and miR-455 are used as controls, these miRNAs have been demonstrated to be more relevant to Cartilage Homeostasis than chondrogenesis itself. Their deficiency has been genetically proven to induce Osteoarthritis in mice. Therefore, the results of this study should be considered in comparison with these existing findings.

Reviewer #2 (Public Review):

Summary:

This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, authors showed that TNXB knockdown lead to chondrocyte apoptosis, matrix degeneration and subchondral bone loss in vivo/vitro. Furthermore, AKT agonist promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:

In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:

Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

Reviewer #1 (Public Review):

Summary:

Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

Strengths:

(1) Both human and mouse samples were examined.<br /> (2) The HA model was used.<br /> (3) genome-wide DNA methylation analysis was performed.

Weaknesses:

(1) In some experiments, the selection of the control groups was not ideal.<br /> (2) More details on analyzing methods and information on replicates need to be included.<br /> (3) Discussion can be improved by comparing findings to other relevant studies.<br /> (4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

Reviewer #1 (Public Review):

The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.

Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:

(1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.

(2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.

Here, the authors' goal is to directly test if the auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.

First the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.

The authors have revised their manuscript and adequately addressed the major concerns. Although more in depth analyses of these rich datasets are definitely possible, the current results nevertheless stand on their own. Indeed, the work serves as a beacon to move away from the idea that cortico-collicular projections function primarily to impart behavioral relevance upon auditory midbrain neurons. This knowledge inspires a search for alternative explanations as to the role of auditory cortico-collicular synapses in behavior.

Reviewer #2 (Public Review):

Summary:

This study takes a new approach to studying the role of corticofugal projections from auditory cortex to inferior colliculus. The authors performed two-photon imaging of cortico-recipient IC neurons during a click detection task in mice with and without lesions of auditory cortex. In both groups of animals, they observed similar task performance and relatively small differences in the encoding of task-response variables in the IC population. They conclude that non-cortical inputs to the IC provide can substantial task-related modulation, at least when AC is absent.

Strengths:

This study provides valuable new insight into big and challenging questions around top-down modulation of activity in the IC. The approach here is novel and appears to have been executed thoughtfully. Thus, it should be of interest to the community.

Weaknesses:

There are however, substantial concerns about the interpretation of the findings and limitations to the current analysis. In particular, Analysis of single unit activity is absent, making interpretation of population clusters and decoding less interpretable. These concerns should be addressed to make sure that the results can be interpreted clearly in an active field that already contains a number of confusing and possibly contradictory findings.

Reviewer #3 (Public Review):

Summary:

This study aims to demonstrate that cortical feedback is not necessary to signal behavioral outcome to shell neurons of the inferior colliculus during a sound detection task. The demonstration is achieved in a very clear manner by the observation of the activity of cortico-recepient neurons in animals which have received lesions of the auditory cortex. The experiment shows that neither behavior performance nor neuronal responses are significantly impacted by cortical lesions except for the case of partial lesions which seem to have a disruptive effect on behavioral outcome signaling.

Strengths:

The demonstration of the main conclusions is based on state-of-the-art, carefully controlled methods and is highly convincing. There is an in depth discussion of the different effects of auditory cortical lesions on sound detection behavior.

Weaknesses:

The description of feedback signals could be more detailed although it is difficult to achieve good temporal resolution with the calcium imaging technique necessary for targeting cortico-recipient neurons.

Reviewer #1 (Public Review):

Summary:<br /> This is an excellent study by a superb investigator who discovered and is championing the field of migrasomes. This study contains a hidden "gem" - the induction of migrasomes by hypotonicity and how that happens. In summary, an outstanding fundamental phenomenon (migrasomes) en route to becoming transitionally highly significant.

Strengths:

Innovative approach at several levels. Migrasomes - discovered by Dr Yu's group - are an outstanding biological phenomenon of fundamental interest and now of potentially practical value.

Weaknesses:

I feel that the overemphasis on practical aspects (vaccine), however important, eclipses some of the fundamental aspects that may be just as important and actually more interesting. If this can be expanded, the study would be outstanding.

Reviewer #2 (Public Review):

Summary:

The authors' report describes a novel vaccine platform derived from a newly discovered organelle called a migrasome. First, the authors address a technical hurdle in using migrasomes as a vaccine platform. Natural migrasome formation occurs at low levels and is labor intensive, however, by understanding the molecular underpinning of migrasome formation, the authors have designed a method to make engineered migrasomes from cultured, cells at higher yields utilizing a robust process. These engineered migrasomes behave like natural migrasomes. Next, the authors immunized mice with migrasomes that either expressed a model peptide or the SARS-CoV-2 spike protein. Antibodies against the spike protein were raised that could be boosted by a 2nd vaccination and these antibodies were functional as assessed by an in vitro pseudoviral assay. This new vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA that require very stringent storage conditions.

Strengths:

The authors present very robust studies detailing the biology behind migrasome formation and this fundamental understanding was used to form engineered migrasomes, which makes it possible to utilize migrasomes as a vaccine platform. The characterization of engineered migrasomes is thorough and establishes comparability with naturally occurring migrasomes. The biophysical characterization of the migrasomes is well done including thermal stability and characterization of the particle size (important characterizations for a good vaccine).

Weaknesses:

With a new vaccine platform technology, it would be nice to compare them head-to-head against a proven technology. The authors would improve the manuscript if they made some comparisons to other vaccine platforms such as a SARS-CoV-2 mRNA vaccine or even an adjuvanted recombinant spike protein. This would demonstrate a migrasome-based vaccine could elicit responses comparable to a proven vaccine technology. Additionally, understanding the integrity of the antigens expressed in their migrasomes could be useful. This could be done by looking at functional monoclonal antibodies binding to their migrasomes in a confocal microscopy experiment.

Reviewer #1 (Public Review):

Summary:

In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. Data is presented suggesting the involvement of Delta-Notch signaling in keratinocyte differentiation. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.

Strengths:

Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, perturbations that affect cytoneme number, Notch signaling, and IL-17 expression clearly lead to changes in periderm structure and gene expression.

Weaknesses:

More work is needed to determine whether the effects on keratinocyte differentiation are due to a loss of cytonemes themselves, or to broader effects of inhibiting cdc42. Moreover, more evidence is needed to support the claim that periderm cytonemes deliver Delta ligands to induce Notch signaling below. Without these aspects of the study being solidified, understanding how IL-17 affects these processes seems premature.

Reviewer #2 (Public Review):

Summary:

The aim of the study was to understand how cells of the skin communicate across dermal layers. The research group has previously demonstrated that cellular connections called airinemes contribute to this communication. The current work builds upon this knowledge by showing that differentiated keratinocytes also use cytonemes, specialized signaling filopodia, to communicate with undifferentiated keratinocytes. They show that cytonemes are the more abundant type of cellular extension used for communication between the differentiated keratinocyte layer and the undifferentiated keratinocytes. Disruption of cytoneme formation led to the expansion of the undifferentiated keratinocytes into the periderm, mimicking skin diseases like psoriasis. The authors go on to show that disruption of cytonemes results in perturbations in Notch signaling between the differentiated keratinocytes of the periderm and the underlying proliferating undifferentiated keratinocytes. Further, the authors show that Interleukin-17, also known to drive psoriasis, can restrict the formation of periderm cytonemes, possibly through the inhibition of Cdc42 expression. This work suggests that cytoneme-mediated Notch signaling plays a central role in normal epidermal regulation. The authors propose that disruption of cytoneme function may be an underlying cause of various human skin diseases.

Strengths:

The authors provide strong evidence that periderm keratinocytes cytonemes contain the notch ligand DeltaC to promote Notch activation in the underlying intermediate layer to regulate accurate epidermal maintenance.

Weaknesses:

The impact of the study would be increased if the mechanism by which Interlukin-17 and Cdc42 collaborate to regulate cytonemes was defined. Experiments measuring Cdc42 activity, rather than just measuring expression, would strengthen the conclusions.

Reviewer #3 (Public Review):

Summary:

Leveraging zebra fish as a research model, Wang et al identified "cytoneme-like structures" as a mechanism for mediating cell-cell communications among skin epidermal cells. The authors further demonstrated that the "cytoneme-like structures" can mediate Notch signaling, and the "cytoneme-like structures" are influenced by IL17 signaling.

Strengths:

Elegant zebrafish genetics, reporters, and live imaging.

Weaknesses: (minor)<br /> This paper focused on characterizing the "cytoneme-like structures" between different layers and the NOTCH signaling. However, these "cytoneme-like structures" observed in undifferentiated KC (Figure 2B), although at a slightly lower frequency, were not interpreted. In addition, it is unclear if these "cytoneme-like structures" can mediate other signaling pathways than NOTCH.

Overall, this is a solid paper with convincing data reporting the "cytoneme-like structures" in vivo, and with compelling data demonstrating the roles in NOTCH signaling and the regulation by IL17.

These findings provide a foundation for future work exploring the "cytoneme-like structures" in the mammalian system and other epithelial tissue types. This paper also suggests a potential connection between the "cytoneme-like structures" and psoriasis, which needs to be further explored in clinical samples.

Reviewer #1 (Public Review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).

In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.

Reviewer #2 (Public Review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Reviewer #3 (Public Review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.

Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.<br /> Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.

Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.

Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.

Reviewer #1 (Public Review):

Summary:

In this study the authors use an elegant set of single-molecule experiments to assess the transcriptional and post-transcriptional regulation of RecB. The question stems from a previous observation from the same lab, that RecB protein levels are low and not induced under DNA damage. The authors first show that recB transcript levels are low and have a short half-life. They further show that RecB levels are likely regulated via translational control. They provide evidence for low noise in RecB protein levels across cells and show that the translation of the mRNA increases under double-strand break conditions. Authors identify Hfq binding sites in the recBCD operon and show that Hfq regulates the levels of RecB protein without changing the mRNA levels. They suggest that RecB translation is directly controlled by Hfq binding to mRNA, as mutating one of the binding sites has a direct effect on RecB protein levels.

Strengths:

The implication of Hfq in regulation of RecB translation is important and suggests mechanisms of cellular response to DNA damage that are beyond the canonically studied mechanisms (such as transcriptional regulation by LexA). Data are clearly presented and the writing is direct and easy to follow. Overall, the study is well-designed and provides novel insights into the regulation of RecB, that is part of the complex required to process break ends.

Weaknesses:

Some key findings need additional support/ clarifications to strengthen the conclusions. These are suggested to the authors.

Reviewer #2 (Public Review):

Summary:

The authors carry out a careful and rigorous quantitative analysis of RecB transcript and protein levels at baseline and in response to DNA damage. Using single-molecule FISH and Halo-tagging in order to achieve sensitive measurements, they provide evidence that enhanced RecB protein levels in response to DNA damage are achieved through a post-transcriptional mechanism mediated by the Sm-like RNA binding protein, Hfq. In terms of biological relevance, the authors suggest that this mechanism provides a way to control the optimum level of RecB expression as both deletion and over-expression are deleterious. In addition, the proposed mechanism provides a new framework for understanding how transcriptional noise can be suppressed at the protein level.

Strengths:

Strengths of the manuscript include the rigorous approaches and orthogonal evidence to support the core conclusions, for example, the evidence that altering either Hhq or its recognition sequence on the RNA similarly enhance the protein to RNA ratio of RecB. The writing is clear and the experiments are well-controlled. The modeling approaches provide essential context to interpret the data, particularly given the small numbers of molecules per cell. The interpretations are careful and well supported.

Weaknesses:

The authors make a compelling case for the biological need to exquisitely control RecB levels, which they suggest is achieved by the pathway they have uncovered and described in this work. However, this conclusion is largely inferred as the authors only investigate the effect on cell survival in response to (high levels of) DNA damage and in response to two perturbations - genetic knock-out or over-expression, both of which are likely more dramatic than the range of expression levels observed in unstimulated and DNA damage conditions.

Reviewer #3 (Public Review):

Summary:

The work by Kalita et al. reports regulation of RecB expression by Hfq protein in E.coli cell. RecBCD is an essential complex for DNA repair and chromosome maintenance. The expression level needs to be regulated at low level under regular growth conditions but upregulated upon DNA damage. Through quantitative imaging, the authors demonstrate that recB mRNAs and proteins are expressed at low level under regular conditions. While the mRNA copy number demonstrates high noise level due to stochastic gene expression, the protein level is maintained at a lower noise level compared to expected value. Upon DNA damage, the authors claim that the recB mRNA concentration is decreased, however RecB protein level is compensated by higher translation efficiency. Through analyzing CLASH data on Hfq, they identified two Hfq binding sites on RecB polycistronic mRNA, one of which is localized at the ribosome binding site (RBS). Through measuring RecB mRNA and protein level in the ∆hfq cell, the authors conclude that binding of Hfq to the RBS region of recB mRNA suppresses translation of recB mRNA. This conclusion is further supported by the same measurement in the presence of Hfq sequestrator, the sRNA ChiX, and the deletion of the Hfq binding region on the mRNA.

Strengths:

(1) The manuscript is well-written and easy to understand.<br /> (2) While there are reported cases of Hfq regulating translation of bound mRNAs, its effect on reducing translation noise is relatively new.<br /> (3) The imaging and analysis are carefully performed with necessary controls.

Weaknesses:

The major weaknesses include a lack of mechanistic depth, and part of the conclusions are not fully supported by the data.

(1) Mechanistically, it is still unclear why upon DNA damage, translation level of recB mRNA increases, which makes the story less complete. The authors mention in the Discussion that a moderate (30%) decrease in Hfq protein was observed in previous study, which may explain the loss of translation repression on recB. However, given that this mRNA exists in very low copy number (a few per cell) and that Hfq copy number is on the order of a few hundred to a few thousand, it's unclear how 30% decrease in the protein level should resides a significant change in its regulation of recB mRNA.<br /> (2) Based on the experiment and the model, Hfq regulates translation of recB gene through binding to the RBS of the upstream ptrA gene through translation coupling. In this case, one would expect that the behavior of ptrA gene expression and its response to Hfq regulation would be quite similar to recB. Performing the same measurement on ptrA gene expression in the presence and absence of Hfq would strengthen the conclusion and model.<br /> (3) The authors agree that they cannot exclude the possibility of sRNA being involved in the translation regulation. However, this can be tested by performing the imaging experiments in the presence of Hfq proximal face mutations, which largely disrupt binding of sRNAs.<br /> (4) The data on construct with a long region of Hfq binding site on recB mRNA deleted is less convincing. There is no control to show that removing this sequence region itself has no effect on translation, and the effect is solely due to the lack of Hfq binding. A better experiment would be using a Hfq distal face mutant that is deficient in binding to the ARN motifs.<br /> (5) Ln 249-251: The authors claim that the stability of recB mRNA is not changed in ∆hfq simply based on the steady-state mRNA level. To claim so, the lifetime needs to be measured in the absence of Hfq.<br /> (6) What's the labeling efficiency of Halo-tag? If not 100% labeled, is it considered in the protein number quantification? Is the protein copy number quantification through imaging calibrated by an independent method? Does Halo tag affect the protein translation or degradation?<br /> (7) Upper panel of Fig S8a is redundant as in Fig 5B. Seems that Fig S8d is not described in the text.

Reviewer #1 (Public Review):

I thank the authors for addressing almost all my comments on the previous version of this manuscript, which studies the representation by gender and name origin of authors from Nature and Springer Nature articles in Nature News.

The representation of author identities is an important step towards equality in science, and the authors found that women are underrepresented in news quotes and mentions with respect to the proportion of women authors.

The research is rigorously conducted. It presents relevant questions and compelling answers. The documentation of the data and methods is thoroughly done, and the authors provide the code and data for reproduction.

Reviewer #2 (Public Review):

The authors have done well to address the points raised in my previous review.

The updated version of this manuscript retains the technical competence of the first, but with important changes that make the analysis more legible and results better contextualized. Specifically, the discussion is richer, the interpretation of the results is more nuanced, the terminology is more precise, and issues of clarity related to the methodology and results have been resolved.

Broad caveats remain about the nature of authorship, and who we should expect to be quoted in science journalism. Namely, who is the lead author? Ideally, the corresponding author would be included as well, or else some bibliometric definition of the most senior author on the byline. However, the authors' approach here is certainly adequate, and they did well to incorporate discussion of authorship and the scholarly division of labour in their discussion.

In sum, I find the article greatly improved and a competent analysis into the unequal use of quotations in scientific journalism.

Reviewer #1 (Public Review):

The study offers intriguing insights, yet interpretations warrant caution, as the authors themselves acknowledged in their discussion of limitations.

The observed grid-like neural activity might not signify navigating a social landscape but rather a sensory feature space. The study's design had participants associate each face with a pair of bar lengths, with the purported 'navigation' being merely a response to the morphing of bar graph images. Crucially, the task did not necessitate any social cognitive processing to estimate grid-like activity. When making social decisions in a separate task, it is unclear whether participants were actually traversing a social space mentally or simply recalling the bar graphs linked to each face to calculate decision values. Notably, during the trust game, competence and trustworthiness did not equally influence decision-making (as illustrated by Equation 1), implying the possibility that the space represented may be more perceptual than social in nature.

The existence of a universal brain representation for faces within a social context is still debatable. Participants were not required to form a cognitive map of the six faces based on social traits; they could simply remember each face's trait values. While the study suggests that reaction times correlated with the perceived social distances between faces hint at the creation of internal representations, this phenomenon could occur without a true cognitive map of the face relationships. To convincingly argue for such internal representations in the brain, additional multivariate pattern analysis would be necessary to demonstrate that these are not merely the result of perceptual differences in the bar graphs associated with each face.

Reviewer #3 (Public Review):

Liang and colleagues set out to test whether the human brain uses distance and grid-like codes in social knowledge using a design where participants had to navigate in a two-dimensional social space based on competence and warmth during an fMRI scan. They showed that participants were able to navigate the social space and found distance-based codes as well as grid-like codes in various brain regions, and the grid-like code correlated with behavior (reaction times).

On the whole, the experiment is designed appropriately for testing for distant-based and grid-like codes, and is relatively well powered for this type of study, with a large amount of behavioral training per participant. They revealed that a number of brain regions correlated positively or negatively with distance in the social space, and found grid-like codes in the frontal polar cortex and posterior medial entorhinal cortex, the latter in line with prior findings on grid-like activity in entorhinal cortex. The current paper seems quite similar conceptually and in design to previous work, most notably Park et al., 2021, Nature Neuroscience.

(1) The authors claim that this study provides evidence that humans use a spatial / grid code for abstract knowledge like social knowledge.

This data does specifically not add anything new to this argument. As with almost all studies that test for a grid code in a similar "conceptual" space (not only the current study), the problem is that, when the space is not a uniform, square/circular space, and 2-dimensional then there is no reason the code will be perfectly grid like, i.e., show six-fold symmetry. In real world scenarios of social space (as well as navigation, semantic concepts), it must be higher dimensional - or at least more than two dimensional. It is unclear if this generalizes to larger spaces where not all part of the space is relevant. Modelling work from Tim Behrens' lab (e.g., Whittington et al., 2020) and Bradley Love's lab (e.g., Mok & Love, 2019) have shown/argued this to be the case. In experimental work, like in mazes from the Mosers' labs (e.g., Derdikman et al., 2009), or trapezoid environments from the O'Keefe lab (Krupic et al., 2015), there are distortions in mEC cells, and would not pass as grid cells in terms of the six-fold symmetry criterion.

After revision, the authors now discuss some of this and the limitations and notes that future work is required to address the problem.

Reviewer #1 (Public Review):

Summary:

The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients towards COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.

In a clinical case-control study, the team found that severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.

Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.

Overall, it is a very complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates are convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms.

The authors keep the diabetic condition at the center of their study and extend on previous knowledge of glucose-induced CTSL activation and their consequences for Covid19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes. This enables novel, public awareness of the susceptibility of diabetes patients to the disease.

Reviewer #2 (Public Review):

Summary:

In this study, the authors hypothesized that individuals with diabetes have elevated blood CTSL levels, which facilitates SARS-CoV-2 infection. The authors conducted in vitro experiments, revealing that elevated glucose levels promote SARS-CoV-2 infection in wild-type cells. In contrast, CTSL knockout cells show reduced susceptibility to high glucose-promoted effects. Additionally, the authors utilized lung tissue samples obtained from both diabetic and non-diabetic patients, along with db/db diabetic and control mice. Their findings indicate that diabetic conditions lead to an elevation in CTSL activity in both human and mice.

Strengths:

The authors have effectively met their research objectives, and their conclusions are supported by the data presented. Their findings suggest that high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum to the lysosome, potentially contributing to diabetic comorbidities and complications.

Weaknesses:

(1) In Figure 1e, the authors measured plasma levels of COVID-19 related proteins, including ACE2, CTSL, and CTSB, in both diabetic and non-diabetic COVID-19 patients. Notably, only CTSL levels exhibited a significant increase in diabetic patients compared to non-diabetic patients, and these levels varied throughout the course of COVID-19. Given that the diabetes groups encompass both male and female patients, it is essential to ascertain whether the authors considered the potential impact of gender on CTSL levels. The diabetes groups comprised a higher percentage of male patients (61.3%) compared to the non-diabetes group, where males constituted only 38.7%.

(2) lines145-149: "The results showed that WT Huh7 cell cultured in high glucose medium exhibited a much higher infective rate than those in low glucose medium. However, CTSL KO Huh7 cells maintained a low infective rate of SARS-CoV-2 regardless of glucose or insulin levels (Fig. 3f-h). Therefore, hyperglycemia enhanced SARS-CoV-2 infection dependent on CTSL." However, this evidence may be insufficient to support the claim that hyperglycemia enhances SARS-CoV-2 infection dependent on CTSL. The human hepatoma cell line Huh7 might not be an ideal model to validate the authors' hypothesis regarding high blood glucose promoting SARS-CoV-2 infection through CTSL.

(3) The Abstract and Introduction sections lack effective organization.

In this revised version of the study, the authors have addressed my concerns by providing additional experiments, references and discussing further the points of controversy. I think that the authors have made improvements to the manuscript.

Reviewer #1 (Public Review):

Summary:

In this study, Liu et al. investigate the signaling pathway that triggers sliding motility in the bacterium B. subtilis in response to subinhibitory concentrations of the antibiotic chloramphenicol. The authors used a genetic approach to identify the master regulator CodY playing a regulatory role in this behavior. They used transcriptional and metabolomic profiling to delineate the spatiotemporal separation of the regulatory networks that define distinct metabolic states related to purine metabolism and pyruvate utilization, which are ultimately responsible for the induction of sliding in response to chloramphenicol. Many readers would be interested to read this work showing how extracellular signals modulate microbial physiology and metabolism.

Strengths:

This work presents numerous technical and conceptual strengths. In the opinion of this referee, the most significant conceptual strength of this work is to (once again) provide evidence that antibiotics are not merely produced by bacteria to eliminate competitors. Bacteria have evolved to respond to their presence and activate a range of physiological responses, which are poorly understood. Understanding these responses is critical to fully understand the evolutionary consequences associated with the use of antibiotics. From a technical standpoint, perhaps the most relevant aspect is the robust phenotypic assay developed by the authors to study sliding motility in the presence of chloramphenicol. This robustness enables genetic work using mutants and performing omics assays to characterize the response to chloramphenicol in detail. Additionally, two sets of results stood out and provided important value to this work. One is the comparison established between the sliding induced by chloramphenicol and the sliding generated in the ΔcodY mutant, to determine the genes and the metabolites (using transcriptomics and metabolomics) specifically associated with the response to chloramphenicol without being part of the general Cody-mediated induction of sliding. The second set of results led the authors to identify precise genes of bacterial metabolism (pdhA) responsible for the sliding phenotype in response to chloramphenicol, and conducted genetic experiments to demonstrate that the pdhA mutant does not respond to the presence of chloramphenicol.

Weaknesses:

This work has three main weaknesses, all related to transcriptomic and metabolomic analyses. Firstly, there is the challenge of understanding the essence of the omics results. This section presents an overwhelming array of genes involved in different metabolic pathways, without an obvious thread to tie these hits together. It is easy to get lost in this section. For instance, one cannot be certain if the hits from one particular metabolic pathway are significant enough to figure out to which degree is this pathway responsible for the sliding phenotype. This section contains a huge diversity of genes and pathways and needs to be streamlined. Related to this, the message of the omics experiments highlights a very close relationship between purine and pyruvate metabolism in sliding motility. However, it is unclear how these metabolic pathways may influence sliding or any other specific bacterial behavior. I do not mean to say that it is not possible, just that the connection/mechanism is missing. The third weakness concerns the omics results that sometimes are in conflict. The authors proposed that this may stem from a division of labor and the coexistence of different subpopulations with different metabolisms within the microbial community. While plausible, other possibilities are equally plausible and should be tested in a revised version of the work.

Reviewer #2 (Public Review):

Summary:

Liu and colleagues describe the transcriptional changes observed during chloramphenicol-induced surface mobility of Bacillus subtilis. Practically, they describe that numerous transcriptional regulatory pathways are influenced by the subinhibitory concentration of a translational inhibitor and some of these regulatory changes might contribute to the induction of sliding. Nevertheless, how such translational stress is translated to induction of sliding remains undetermined. The authors clearly describe their aim (line 457): "Our goal for this study was to gain insight into how B. subtilis mobilizes a colony in response to subinhibitory exposure to translation inhibitors.", this is unfortunately not solved here, only the authors characterize the transcriptional landscape differences.

Strengths:

The very thorough analysis of transcriptional changes in the wild type and codY mutant strains is appreciated, and there are definitely a plethora of changes observed related to several global transcriptional regulators in B. subtilis. I compliment the authors for this very detailed and thorough description of transcriptional changes.

Weaknesses:

While the transcriptional changes are well and carefully described, the discussion practically interprets the correlations as causations. I am not disputing that the authors are not on the correct path with their assumptions, but their conclusions are not supported by direct experimental data, especially on (1) translational stress directly inducing mobility and (2) division of labor.

Major 1:

The authors conclude that their results point towards a putative mechanism, e.g. line 460 "which suggests translation stress is a trigger for colony mobilization"; however, no experiment demonstrates this aspect. The authors do not test ppGpp-related stress (mutants in ppGpp-related genes, or mutating the functional domain of CodY), nor do they directly connect ppGpp levels dynamics with induction of subsequent pathways. Again, I understand that the authors are on the right path to connect these pathways and identify what is causing mobility induction, but no direct data is represented, solely the transcriptional changes, therefore remains slightly descriptive.

The statement in the chapter title (line 474) is not demonstrated directly and should be revised. Similarly, in line 476, the authors claim that their "data supports a model", but "support" would require direct experimental data on this aspect.

The authors even clearly indicate in lines 504-506 that they do not reveal the direct mechanism, but the rest of the discussion delivers statements that do not consider the lack of direct data.

Major 2:

Line 427: "The results are consistent with a division of metabolic labor among cells in the expanding population" - the data shows heterogeneity, but the direct division of labor is not demonstrated.

Line 442: So in this case, the proposed division of labor is disrupted in the codY mutant (no inner localisation), and hence expansion appears, suggesting a lack of a putative division of labor is not necessary for induced mobility. On the contrary, there could be heterogeneous gene expression, division of labor requires demonstration of fitness benefit from such interaction.

Division of labor assumes that a mixture of mutants would complement full sliding dynamics, and this could be easily demonstrated by fluorescent labeled cells that should be organized in a similar fashion to those observed with luciferase reporters (pucA mutant on the outer ring, while pdhA mutant interior colony part). Without such experimental demonstration, the authors can only conclude spatially heterogeneous gene expresstion without clear functional contribution to subinhibitory chrolamphenociol-induced surface mobility.

Again, the authors' statement in line 472 "reveal a regulated, spatiotemporal division of metabolism" is not demonstrated by experiment, but spatial heterogeneity is revealed here.<br /> The statement in the Discussion chapter (line 499) is also not demonstrated by experimental data: "Metabolic coordination enables surface expansion of mobilitzed B. subtilis"

Line 550: while I agree with the authors' statement that these functions work cooperatively as demonstrated by van Gestel and colleagues (2015 PloS Biol), the exploitation of these shared goods is not quantitatively equivalent, see Jautzus et al 2022 ISME J (DOI: 10.1038/s41396-022-01279-8).

In summary: the two major conclusions of the manuscript are unfortunately not demonstrated, the presented transcriptional data delivers suggestions, supported with specific mutants displaying certain phenotypes (lack of mobility induction or constitutive mobility without inducer), but it remains unclear how translational stress induces mobility and whether the transcriptional heterogeneity detected directly contributes to metabolic division of labor.

The authors should present direct evidence on the major concerns: how translational stress induces surface mobility (using ppGpp synthesis and turnover mutants and specific CdoY mutant lacking ppGpp sensing) and whether the metabolic division of labor contributes to induced surface mobility (mixing mutants and following their distribution).

Reviewer #1 (Public Review):

By identifying a loss of function mutant of IQCH in infertile patient, Ruan et al. shows that IQCH is essential for spermiogenesis by generating a knockout mouse model of IQCH. Similar to infertile patient with mutant of IQCH, Iqch knockout mice are characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. Mechanistically, IQCH regulates the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis.

Although this manuscript contains a potentially interesting piece of work that delineates a mechanism of IQCH that associates with spermatogenesis, this reviewer feels that a number of issues require clarification and re-evaluation for a better understanding of the role of IQCH in spermatogenesis.

Line 251 - 253, "To elucidate the molecular mechanism by which IQCH regulates male fertility, we performed liquid chromatography tandem mass spectrometry (LC‒MS/MS) analysis using mouse sperm lysates and detected 288 interactors of IQCH (Figure 5-source data 1)."

The reviewer had already raised significant concerns regarding the text above, noting that "LC‒MS/MS analysis using mouse sperm lysates" would not identify interactors of IQCH. However, this issue was not addressed in the revised manuscript. In the Methods section detailing LC-MS/MS, the authors stated that it was conducted on "eluates obtained from IP". However, there was no explanation provided on how IP for LC-MS/MS was performed. Additionally, it was unclear whether LC-MS or LC-MS/MS was utilized. The primary concern is that if LC‒MS/MS was conducted for the IP of IQCH, IQCH itself should have been detected in the results; however, as indicated by Figure 5-source data 1, IQCH was not listed.

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as Iqch CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in Ichq-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. Assays are done rigorously and in a quantitative manner. Overall, the claims made by the authors in this manuscript are well-supported by the data provided.

Reviewer #2 (Public Review):

The authors analysed functional MRI recordings of brain activity at rest, using state-of-the-art methods that reveal the diverse ways in which information can be integrated in the brain. In this way, they found brain areas that act as (synergistic) gateways for the 'global workspace', where conscious access to information or cognition would occur, and brain areas that serve as (redundant) broadcasters from the global workspace to the rest of the brain. The results are compelling and are consistent with the already assumed role of several networks and areas within the Global Neuronal Workspace framework. Thus, in a way, this work comes to stress the role of synergy and redundancy as complementary information processing modes, which fulfill different roles in the bigger context of information integration.

In addition, to prove that the identified high-order interactions are relevant to the phenomenon of consciousness, the same analysis was performed in subjects under anesthesia or with disorders of consciousness (DOC), showing that indeed the loss of consciousness is associated with a deficient integration of information within the gateway regions.

Reviewer #3 (Public Review):

The work proposes a model of neural information processing based on a 'synergistic global workspace,' which processes information in three principal steps: a gatekeeping step (information gathering), an information integration step, and finally, a broadcasting step. They provided an interpretation of the reduced human consciousness states in terms of the proposed model of brain information processing, which could be helpful to be implemented in other states of consciousness. The manuscript is well-organized, and the results are important and could be interesting for a broad range of literature, suggesting interesting new ideas for the field to explore.

Reviewer #1 (Public Review):

In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at the sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and a weakness of the manuscript.

Strengths:

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

Weakness:

- The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background. Pol V encroachment into heterochromatin in the h1 mutant is indirectly shown by NRPE1-dependent methylation at such ectopic sites.

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

Reviewer #2 (Public Review):

Summary:

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

Strengths:

The authors present diverse sets of genomic data comparing Arabidopsis wild-type and h1 mutant background allowing an analysis of differential recruitment of RdDM component NPRE1, which is related to changes in DNA methylation and H1 coverage. The manuscript also contains recruitment data for SUVH1 in wild-type and h1 mutant backgrounds.<br /> Furthermore, the authors make use of a line that recruits NRPE1 ectopically to show that H1 occupancy is not altered because of this recruitment. These data clearly show that there is a hierarchy in which DNA-methylation is impacted by presence of H1 while H1 distribution is independent of DNA-methylation.

Weaknesses:

The manuscript is driven by a strong and reasonable hypothesis that absence of H1 results increased access of chromatin binding factors and that this explains how the RdDM machinery is restricted from encroaching heterochromatic regions, which are particularly enriched in H1. Indeed, increased binding of NPRE1 at pericentromeric sites is observed; however, the major DNA-methylation changes at these sites are symmetric and not related to the RdDM pathway. Thus, the authors propose that many factors redistribute, which is again reasonable. The authors show redistribution of SUVH1 and relate their data to a previous report showing redistribution of the PcG machinery in H1 depletion mutants (Teano et al. in Cell reports (Volume 42, Issue 8, 29 August 2023), but the manuscript provides limited mechanistic insight as to why there is a strong increase in heterochromatin symmetric DNA-methylation.

Reviewer #1 (Public Review):

The authors design an automated 24-well Barnes maze with 2 orienting cues inside the maze, then model what strategies the mice use to reach the goal location across multiple days of learning. They consider a set of models and conclude that the animals begin with a large proportion of random choices (choices irrespective of the goal location), which over days of experience becomes a combination of spatial choices (choices targeted around the goal location) and serial choices (successive stepwise choices in a given direction). Moreover, the authors show that after the animal has many days of experience in the maze, they still often began each trial with a random choice, followed by spatial or serial choices.

This study is written concisely and the results are presented concisely. The best fit model provides valuable insight into how the animals solve this task, and therefore offers a quantitative foundation upon which tests of neural mechanisms of the components of the behavioral strategy can be performed. These tests will also benefit from the automated nature of the task.

Reviewer #2 (Public Review):

This paper uses a novel maze design to explore mouse navigation behaviour in an automated analogue of the Barnes maze. A major strength is the novel and clever experimental design which rotates the floor and intramaze cues before the start of each new trial, allowing the previous goal location to become the next starting position. The modelling sampling a Markov chain of navigation strategies is elegant, appropriate and solid, appearing to capture the behavioural data well. This work provides a valuable contribution and I'm excited to see further developments, such as neural correlates of the different strategies and switches between them.

Reviewer #3 (Public Review):

Strength:

The development of an automated Barnes maze allows for more naturalistic and uninterrupted behavior, facilitating the study of spatial learning and memory, as well as the analysis of the brain's neural networks during behavior when combined with neurophysiological techniques. The system's design has been thoughtfully considered, encompassing numerous intricate details. These details include the incorporation of flexible options for selecting start, goal, and proximal landmark positions, the inclusion of a rotating platform to prevent the accumulation of olfactory cues, and careful attention given to atomization, taking into account specific considerations such as the rotation of the maze without causing wire shortage or breakage. When combined with neurophysiological manipulations or recordings, the system provides a powerful tool for studying spatial navigation system.<br /> The behavioral experiment protocols, along with the analysis of animal behavior, are conducted with care, and the development of behavioral modeling to capture the animal's search strategy is thoughtfully executed. It is intriguing to observe how the integration of these innovative stochastic models can elucidate the evolution of mice's search strategy within a variant of the Barnes maze.

Comments on revised version:

The authors have addressed all the points I outlined in the previous round of review, resulting in significant improvements to the manuscript. However, I have one remaining comment. Given the updated inter-animal analysis (Supplementary Figure 8), it appears that male and female mice develop strategies differently across days. Male mice seem to progressively increase their employment of spatial strategy across days, at the expense of the random strategy. Conversely, female mice exhibit both spatial and serial strategies at their highest levels on day 2, with minimal changes observed on the subsequent days.<br /> These findings could alter the interpretation of Figure 5 and the corresponding text in the section "Evolution of search strategy across days".<br /> For instance, this statement on page 6 doesn't hold for female mice: "The spatial strategy was increased across days, ... largely at the expense of the random strategy."

Reviewer #1 (Public Review):

Jiang et al. demonstrated that ablating Neurexins results in alterations to glycinergic transmission and its calcium sensitivity, utilizing a robust experimental system. Specifically, the authors employed rAAV-Cre-EGFP injection around the MNTB in Nrxn1/2/3 triple conditional mice at P0, measuring Glycine receptor-dependent IPSCs from postsynaptic LSO neurons at P13-14. Notably, the authors presented a clear reduction of 60% and 30% in the amplitudes of opto- and electric stimulation-evoked IPSCs, respectively. Additionally, they observed changes in kinetics, alterations in PPR, and sensitivity to lower calcium and the calcium chelator, EGTA, indicating solid evidence for changes in presynaptic properties of glycinergic transmission.

Furthermore, the authors uncovered an unexpected increase in sIPSC frequency without altering amplitude. Although the precise mechanism remains unknown, the authors discussed this complex phenotype by considering various possibilities, including the potential scenario where the augmentation in synapses may result from Nrxn deletion rather than being a causal effect.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Jiang et al., explore the role of neurexins at glycinergic MNTB-LSO synapses. The authors utilize elegant and compelling ex vivo slice electrophysiology to assess how the genetic conditional deletion of Nrxns1-3 impacts inhibitory glycinergic synaptic transmission and found that TKO of neurexins reduced electrically and optically evoked IPSC amplitudes, slowed optically evoked IPSC kinetics and reduced presynaptic release probability. The authors use classic approaches including reduced [Ca2+] in ACSF and EGTA chelation to propose that changes in these evoked properties are likely driven by the loss of calcium channel coupling. Intriguingly, while evoked transmission was impaired, the authors reported that spontaneous IPSC frequency was increased, due to an increase in the number of synapses in LSO. Overall, this manuscript provides important insight into the role of neurexins at the glycinergic MNTB-LSO synapse and further emphasizes the need for continued study of both the non-redundant and redundant roles of neurexins.

The authors have addressed all of my previous concerns.

Reviewer #3 (Public Review):

Summary:

The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses of TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:

The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

Weaknesses:

A concern is on novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors prior work on glutamatergic synapses (Luo et al., 2020).

Reviewer #3 (Public Review):

This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method which has high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system includes field compensation coils which allows free head movement scanning with relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age are found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy to deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeter) and temporal (milliseconds) resolution, it provides a novel and powerful technology to neurodevelopment research and clinical application not limited to somatosensory areas.

The conclusions of this paper are mostly well supported by data acquired under proper method.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

The readability could be improved.

Reviewer #2 (Public Review):

Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.

(1) The conclusions of the paper are mostly supported by the data and in the revised manuscript clarification is provided concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3 and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. The revised manuscript more clearly indicates for each individual experiment which form is used. However, a discussion on the potential effects of the modifications on CCL5 in the results and discussion sections is still missing.<br /> (2) In general, authors used high concentrations of CCL5 in their experiments. In their reply to the comments they indicate that at lower CCL5 concentrations no LLPS is detected. This is important information since it may indicate the need for chemokine oligomerization for LLPS. This info should be added to the manuscript and comparison with for instance the obligate monomer CCL7 and another chemokine such as CXCL4 that easily forms oligomers may clarify whether LLPS is controlled by oligomerization.<br /> (3) Statistical analyses have been improved in the revised manuscript.

Reviewer #1 (Public Review):

The authors perform RNA-seq on FACS isolated neurons from adult worms at days 1 and 8 of adulthood to profile the gene expression changes that occur with cognitive decline. Supporting data are included indicating that by day 7 of adulthood, learning and memory are reduced, indicating that this timepoint or after represents cognitively aged worms. Neuronal identity genes are reduced in expression within the cognitively aged worms, whereas genes involved in proteostasis, transcription/chromatin, and the stress response are elevated. A number of specific examples are provided, representing markers of specific neuronal subtypes, and correlating expression changes to the erosion of particular functions (e.g. motor neurons, chemosensory neurons, aversive learning neurons, etc).

To investigate whether upregulation of genes in neurons with age is compensatory or deleterious, the authors reduced expression of a set of three significantly upregulated genes and performed behavioral assays in young adults. In each case, reduction of expression improved memory, consistent with a model in which age-associated increases impair neuronal function.

The authors then characterize learning and memory in wild type, daf-2, and daf-2/daf-16 worms with age and find that daf-2 worms have an extended ability to learn for approximately 10 days longer that wild types. This was daf-16 dependent. Memory was extended in daf-2 as well, and strikingly, daf-2;daf-16 had no short term memory even at day 1. Transcriptomic analysis of FACS-sorted neurons was performed on the three groups at day 8. The authors focus their analysis on daf-2 vs. daf-2;daf-16 and present evidence that daf-2 neurons express a stress-resistance gene program. They also find small differences between the N2 and daf-2;daf-16 neurons, which correlate with the observed behavioral differences, though these differences are modest.

The authors tested eight candidate genes that were more highly expressed in daf-2 neurons vs. daf-2;daf-16 and showed that reduction of 2 and 5 of these genes impaired learning and memory, respectively, in daf-2 worms. This finding implicates specific neuronal transcriptional targets of IIS in maintaining cognitive ability in daf-2 with age, which, importantly, are distinct from those in young wild type worms.

Overall, this is a strong study with rigorously performed experiments. The authors achieved their aim of identifying transcriptional changes in neurons that underlie loss of learning and memory in C. elegans, and how cognition is maintained in insulin/IGF-1-like signaling mutants.

Reviewer #2 (Public Review):

Weng et al. perform a comprehensive study of gene expression changes in young and old animals, in wild-type and daf-2 insulin receptor mutants, in the whole animal and specifically in the nervous system. Using this data, they identify gene families that are correlated with neuronal ageing, as well as a distinct set of genes that are upregulated in neurons of aged daf-2 mutants. This is particularly interesting as daf-2 mutants show both extended lifespan and healthier neurons in aged animals, reflected by better learning/memory in older animals compared with wild-type controls. Indeed, knockdown of several of these upregulated genes resulted in poorer learning and memory. In addition, the authors showed that several genes upregulated during ageing in wild-type neurons also contribute to learning and memory; specifically, knockdown of these genes in young animals resulted in improved memory. This indicates that (at least in this small number of cases), genes that show increased transcript levels with age in the nervous system somehow suppress memory, potentially by having damaging effects on neuronal health.

Finally, from a resource perspective, the neuronal transcriptome provided here will be very useful for C. elegans researchers as it adds to other existing datasets by providing the transcriptome of older animals (animals at day 8 of adulthood) and demonstrating the benefits of performing tissue-specific RNAseq instead of whole-animal sequencing.

The work presented here is of high quality and the authors present convincing evidence supporting their conclusions. I only have a few comments/suggestions:

(1) Do the genes identified to decrease learning/memory capacity in daf-2 animals (Figure 4d/e) also impact neuronal health? daf-2 mutant worms show delayed onset of age-related changes to neuron structure (Tank et al., 2011, J Neurosci). Does knockdown of the genes shown to affect learning also affect neuron structure during ageing, potentially one mechanism through which they modulate learning/memory?

(2) The learning and memory assay data presented in this study uses the butanone olfactory learning paradigm, which is well established by the same group. Have the authors tried other learning assays when testing for learning/memory changes after knockdown of candidate genes? Depending on the expression pattern of these genes, they may have more or less of an effect on olfactory learning versus for e.g. gustatory or mechanosensory-based learning.

(3) A comment on the 'compensatory vs dysregulatory' model as stated by the authors on page 7 - I understand that this model presents the two main options, but perhaps this is slightly too simplistic: gene expression that rises during ageing may be detrimental for memory (= dysregulatory), but at the same time may also be beneficial other physiological roles in other tissues (=compensatory).

Comments on revised version:

I am satisfied with how the authors have addressed all my comments/suggestions.

Reviewer #3 (Public Review):

Summary

In this manuscript, Weng et al. identify the neuron specific transcriptome that impacts age dependent cognitive decline. The authors design a pipeline to profile neurons from wild type and long-lived insulin receptor/IGF-1 mutants using timepoints when memory functions are declining. They discover signatures unique to neurons which validates their approach. The authors identify that genes related to neuronal identity are lost with age in wild type worms. For example, old neurons reduce the expression of genes linked to synaptic function and neuropeptide signaling and increase the expression of chromatin regulators, insulin peptides and glycoproteins. Depletion of selected genes which are upregulated in old neurons (utx-1, ins-19 and nmgp-1) leads to improved short memory function. This indicates that some genes that increase with age have detrimental effects on learning and memory. The pipeline is then used to test neuronal profiles of long-lived insulin/IGF-1 daf-2 mutants. Genes related to stress response pathways are upregulated in long lived daf-2 mutants (e.g. dod-24, F08H9.4) and those genes are required for improved neuron function.

Strengths

The manuscript is well written, and the experiments are well described. The authors take great care to explain their reasoning for performing experiments in a specific way and guide the reader through the interpretation of the results, which makes this manuscript an enjoyable and interesting read. The authors discover novel regulators of learning and memory using neuron-specific transcriptomic analysis in aged animals, which underlines the importance of cell specific deep sequencing. The timepoints of the transcriptomic profiling are elegantly chosen, as they coincide with the loss of memory and can be used to specifically reveal gene expression profiles related to neuron function. The authors discuss on the dod-24 example how powerful this approach is. In daf-2 mutants whole-body dod-24 expression differs from neuron specific profiles, which underlines the importance of precise cell specific approaches. This dataset will provide a very useful resource for the C. elegans and aging community as it complements existing datasets with additional time points and neuron specific deep profiling.

Weakness

This study nicely describes the neuron specific profiles of aged long-lived daf-2 mutants. Selected neuronal genes that were upregulated in daf-2 mutants (e.g. F08H9.4, mtl-1, dod-24, alh-2, C44B7.5) decreased learning/memory when knocked down. However, the knock down of these genes was not specific to neurons. The authors use a neuron-sensitive RNAi strain to address this concern and acknowledge this caveat in the text. While it is likely that selected candidates act only in neurons it is possible that other tissues participate as well.

Reviewer #1 (Public Review):

Summary:

The authors utiilze the model organism C. elegans to interrogate cell non-autonomous signaling between GABAergic neurons and somatic tissues. They demonstrate that RNAi of isp-1 or spg-7 in GABAergic neurons leads to lifespan extension and improved healthspan (by resistance to paraquat or heat stress), which are dependent on the transcription factor daf-16/FOXO3a.

Strengths:

The authors are clear and straightforward in their study. They examine the healthspan of C. elegans at days 3, 6, and 9 to give a wide perspective on how the phenotypes changes with aging. They use two methods to specifically knockdown isp-1 or spg-7 in GABAergic neurons: (1) a previously published rde-1 mutant that has rde-1 and sid-1 restored only in GABAergic neurons and (2) a novel model uses a sid-1 mutant that makes dsRNA of isp-1 or spg-7 in GABAergic neurons. They use multiple methods to examine healthspan. They identified daf-16/FOXO3a as the mechanism of their phenotype and ruled out other transcription factors. The authors do not use FUdR in their studies, which is known to confound experiments.

Weaknesses:

(1) Incomplete validation of GABAergic knockdown. The study relies on the specific knockdown of isp-1 or spg-7 in GABAergic neurons, but in the opinion of this reviewer, the authors do not adequately validate their models to demonstrate GABAergic specificity. For the previously published rde-1 mutant model, a simple validation of specific knockdown of GFP in GFP-labeled GABAergic neurons should be included. They should also show that GFP RNAi would not be effective in knocking down intestinal GFP, for example.

Their second model is poorly explained and not validated and this reviewer could not find similar previously published models of its kind. This model claims that dsRNA of isp-1 was made in the GABAergic neurons of a sid-1 mutant, but no evidence is shown to support this claim. The authors point to changes in phenotypes such as lifespan extension and reduced lipofuscin in the intestines as proof that knockdown is occurring in the GABAergic neurons, but this is indirect evidence. Rigorous validation of this model is needed, especially if it is the first model of its kind.

(2) Lifespan. The control lifespans using the rde-1 mutants are very short-lived and no explanation for this is provided (eg. Figure 1D, E). The authors use two RNAis in their lifespan with daf-16 and isp-1. For their controls, they should use empty vector mixed with isp-1, not only isp-1 RNAi.

(3) Cell non-autonomous effects. The claims that GABAergic mitochondrial dysfunction have effects on somatic tissues is weak. More specific tests on somatic stress resistance are warranted for their claims. Better quality images of intestinal mitochondria are needed. Examining additional tissues, such as muscle, would also strengthen their claims. For example, they could examine muscle mitochondria and determine if muscle strength is improved in their models.

(4) Dependence on daf-16/FOXO3a. The authors show that loss of daf-16 reverses the lifespan and healthspan effects in their model. Next, they show that loss of daf-16 reverses the effects of isp-1 in the intestines and in the germline. However, they only show the daf-16 mutant data and not the positive control (EV and isp-1 alone), which should be included. Furthermore, the phenotypes they examine are only a subset of somatic phenotypes, and this reviewer would be more convinced with the additional controls and with more parameters examined.

Reviewer #2 (Public Review):

Summary:

In this work, the authors show that GABAergic neurons play a role in sensing mitochondrial stress and regulating organismal aging. Thus, disrupting the mitochondrial mitochondria function in GABAergic neurons induces resistance to thermal and paraquat stresses, promotes longevity, and affects reproduction. This mechanism is regulated by the iron-sulfur subunit of complex III of the mitochondrial electron transport chain, ISP-1, and a mitochondrial quality control m-AAA protease, SPG-7, which in turn requires DAF-16/FoxO activity in GABAergic neurons.

Strengths:

A strength of this work is that the authors identify the specific site where mitochondrial stress promotes health and longevity, i.e., GABAergic neurons. In addition, the paper corroborates the findings with the appropriate experiments. How neuronal regulation of mitochondrial function impacts systemic health and aging is of interest to cell biology and neuroscience fields.

Weaknesses:

The entire paper is based on tissue-specific RNAi in GABAergic neurons, which was achieved using two different conditions of RNAi (although not for all experiments). However, multiple studies have shown deficiencies in the tissue-specific RNAi in C. elegans, especially for the rde-1(ne219) mutant used in this study. Therefore, it is necessary to repeat critical experiments by rescuing the isp-1 or spg-7 mutants in GABAergic neurons. Additionally, it is clear in the paper that perturbing mitochondrial function requires DAF-16/FoxO activity in GABAergic neurons to promote longevity, yet the downstream cellular pathways are not described.

Reviewer #3 (Public Review):

Summary:

This manuscript describes RNAi depletion of isp-1 or spg-7 in the GABAergic neurons of C. elegans leads to: lifespan extension; increased resistance to paraquat oxidative stress and heat stress; decreased brood size and mitotic germ cell numbers in the gonad and increased DNA aggregates in the oocytes; increased mitochondrial membrane potential, ATP levels, mitochondrial mass, mitochondrial DNA copies, mitochondrial DNA polymerase gamma polg-1 levels, and decreased ROS levels. The authors further show that daf-16 is necessary for GABAergic depletion of isp-1 mediated lifespan extension, stress resistance, increased mitochondrial membrane potential, mitochondrial mass and DNA copies, and decreased brood size. Unc-25 for GABA synthesis, unc-31 for neuropeptide secretion, and flp-13 neuropeptide are all in the same pathway of isp-1 RNAi in GABAergic neurons for lifespan extension and stress resistance.

Strengths:

The topic is interesting and relatively novel in terms of GABAergic mitochondrial dysfunction. The data provided support the conclusions well.

Weaknesses:

The mechanistic evidence needs to be improved substantially.

Reviewer #1 (Public Review)

Summary:

In this study, Xie and colleagues aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. The results demonstrated that Leptospira infection was able to cause intestine damage and inflammation. Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, they proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.

Strengths:

The work is well-designed and the story are interesting to me. The gut microbiota is essential for immunity and systemic health. Many life-threatening pathogens, such as SARS-CoV-2 and other gut-damaged infection, have the potential to disrupt the gut microbiota in the later stages of infection, causing some harmful gut microbiota-derived substances to enter the bloodstream. It is emphasized that in addition to exogenous pathogenic pathogens, harmful substances of intestinal origin should also be considered in critically ill patients.

Reviewer #2 (Public Review):

Severe leptospirosis in humans and some mammals often meet death in the endpoint. In this article, authors explored the role of the gut microbiota in severe leptospirosis. They found that Leptospira infection promoted a dysbiotic gut microbiota with an expansion of Proteobacteria and LPS neutralization therapy synergized with antileptospiral therapy significantly improved the survival rates in severe leptospirosis. This study is well-organized and has potentially important clinical implications not only for severe leptospirosis but also for other gut-damaged infections.

Reviewer #3 (Public Review):