Reviewer #2 (Public Review):

Summary: In the revised manuscript, the authors aim to investigate brain-wide activation patterns following administration of the anesthetics ketamine and isoflurane, and conduct comparative analysis of these patterns to understand shared and distinct mechanisms of these two anesthetics. To this end, they perform Fos immunohistochemistry in perfused brain sections to label active nuclei, use a custom pipeline to register images to the ABA framework and quantify Fos+ nuclei, and perform multiple complementary analyses to compare activation patterns across groups.

In the latest revision, the authors have made some changes in response to our previous comments on how to fix the analyses. However, the revised analyses were not changed correctly and remain flawed in several fundamental ways.

Critical problems:

(1) Before one can perform higher level analyses such as hiearchal cluster or network hub (or PC) analysis, it is fundamental to validate that you have significant differences of the raw Fos expression values in the first place. First of all, this means showing figures with the raw data (Fos expression levels) in some form in Figures 2 and 3 before showing the higher level analyses in Figures 4 and 5; this is currently switched around. Second and most importantly, when you have a large number of brain areas with large differences in mean values and variance, you need to account for this in a meaningful way. Changing to log values is a step in the right direction for mean values but does not account well for differences in variance. Indeed, considering the large variances in brain areas with high mean values and variance, it is a little difficult to believe that all brain regions, especially brain areas with low mean values, passed corrections for multiple comparisons test. We suggested Z-scores relative to control values for each brain region; this would have accounted for wide differences in mean values and variance, but this was not done. Overall, validation of anesthesia-induced differences in Fos expression levels is not yet shown.

(2) Let's assume for a moment that the raw Fos expression analyses indicate significant differences. They used hierarchal cluster analyses as a rationale for examining 53 brain areas in all subsequent analyses of Fos expression following isoflurane versus home cage or ketamine versus saline. Instead, the authors changed to 201 brain areas with no validated rationale other than effectively saying 'we wanted to look at more brain areas'. And then later, when they examined raw Fos expression values in Figures 4 and 5, they assess 43 brain areas for ketamine and 20 brain areas for isoflurane, without any rationale for why choosing these numbers of brain areas. This is a particularly big problem when they are trying to compare effects of isoflurane versus ketamine on Fos expression in these brain areas - they did not compare the same brain areas.

Less critical comments:

(3) The explanation of hierarchical level's in lines 90-95 did not make sense.

(4) I am still perplexed by why the authors consider the prelimbic and infralimbic cortex 'neuroendocrine' brain areas in the abstract. In contrast, the prelimbic and infralimbic were described better in the introduction as "associated information processing" areas.

5- It looks like overall Fos levels in the control group Home (ISO) are a magnitude (~10-fold) lower than those in the control group Saline (KET) across all regions shown. This large difference seems unlikely to be due to a biologically driven effect and seems more likely to be due to a technical issue, such as differences in staining or imaging between experiments. The authors discuss this issue but did not answer whether the Homecage-ISO experiment or at least the Fos labeling and imaging performed at the same time as for the Saline-Ketamine experiment?

Reviewer #3 (Public Review):

The present study presents a comprehensive exploration of the distinct impacts of Isoflurane and Ketamine on c-Fos expression throughout the brain. To understand the varying responses across individual brain regions to each anesthetic, the researchers employ principal component analysis (PCA) and c-Fos-based functional network analysis. The methodology employed in this research is both methodical and expansive. Notably, the utilization of a custom software package to align and analyze brain images for c-Fos positive cells stands out as an impressive addition to their approach. This innovative technique enables effective quantification of neural activity and enhances our understanding of how anesthetic drugs influence brain networks as a whole.

The primary novelty of this paper lies in the comparative analysis of two anesthetics, Ketamine and Isoflurane, and their respective impacts on brain-wide c-Fos expression. The study reveals the distinct pathways through which these anesthetics induce loss of consciousness. Ketamine primarily influences the cerebral cortex, while Isoflurane targets subcortical brain regions. This finding highlights the differing mechanisms of action employed by these two anesthetics-a top-down approach for Ketamine and a bottom-up mechanism for Isoflurane. Furthermore, this study uncovers commonly activated brain regions under both anesthetics, advancing our knowledge about the mechanisms underlying general anesthesia.

Reviewer #1 (Public Review):

Summary:<br /> The authors ran a series of experiments with separate subject populations, different stimuli, and on two different MRI scanners (one 3T, one 7T) to establish a scenes-selective region on the intraparietal gyrus that they decided to name PIGS. I think that IPA (intraparietal place area) would also have been a good choice with an allusion to a beverage rather than a domestic animal. The authors show that PIGS can be detected robustly through a series of experiments. They anatomically and functionally separate PIGS from nearby V6, which encodes optic flow. The authors determined that PIGS encodes ego-motion.

Strengths:<br /> The robust detection of PIGS in several experiments with different sets of participants and on different scanners makes these results convincing. The functional differentiation is well executed.

Weaknesses:<br /> The distinction of PIGS from nearby OPA, which has also been implied in navigation and ego-motion, is not as clear as it could be.

Impact:<br /> Overall, this is a valuable contribution to the cognitive neuroscience of the visual system. It shows that there is still room for discovering details of visual processing, given recent advances in scanning technology, statistical methods, and larger sample sizes.

Reviewer #2 (Public Review):

Summary<br /> The authors report an extensive series of neuroimaging experiments (at both 3T and 7T) to provide evidence for a scene-selective visual area in the human posterior parietal cortex (PIGS) that is distinct from the main three (parahippocampal place area, PPA; occipital place area, OPA; medial place area, MPA) typically reported in the literature. Further, they argue that in comparison with the other three, this region may specifically be involved in representing ego-motion in natural contexts. The characterization of this scene-selective region provides a useful reference point for studies of scene processing in humans.

Strengths<br /> One of the major strengths of the work is the extensive series of experiments reported, showing clear reproducibility of the main finding and providing functional insight into the region studied. The results are clearly presented and for the most part, convincing.

Weaknesses<br /> One of the major weaknesses of the work is the failure to relate the current results to other findings in the literature, making it hard to assess whether it is is a "previously undescribed scene-selective site".

First, the scene-selective region identified appears to overlap with regions that have previously been identified in terms of their retinotopic properties. In particular, it is unclear whether this region overlaps with V7/IPS0 and/or IPS1. This is particularly important since prior work has shown that OPA often overlaps with v7/IPS0 (Silson et al, 2016, Journal of Vision). The findings would be much stronger if the authors could show how the location of PIGS relates to retinotopic areas (other than V6, which they do currently consider). I wonder if the authors have retinotopic mapping data for any of the participants included in this study. If not, the authors could always show atlas-based definitions of these areas (e.g. Wang et al, 2015, Cerebral Cortex).

Second, recent studies have reported a region anterior to OPA that seems to be involved in scene memory (Steel et al, 2021, Nature Communications; Steel et al, 2023, The Journal of Neuroscience; Steel et al, 2023, biorXiv). Is this region distinct from PIGS? Based on the figures in those papers, the scene memory-related region is inferior to V7/IPS0, so characterizing the location of PIGS to V7/IPS0 as suggested above would be very helpful here as well.

If PIGS overlaps with either of V7/IPS0 or the scene memory-related area described by Steel and colleagues, then arguably it is not a newly defined region (although the characterization provided here still provides new information).

Another reason that it would be helpful to relate PIGS to this scene memory area is that this scene memory area has been shown to have activity related to the amount of visuospatial context (Steel et al, 2023, The Journal of Neuroscience). The conditions used to show the sensitivity of PIGS to ego-motion also differ in the visuospatial context that can be accessed from the stimuli. Even if PIGS appears distinct from the scene memory area, the degree of visuospatial context is an alternative account of what might be represented in PIGS.

Reviewer #3 (Public Review):

Summary:<br /> The authors report a scene-selective area in the posterior intraparietal gyrus (PIGS). This area lies outside the classical three scene-selective regions (PPA/TPA, RSC/MPA, TOS/OPA), and is selective for ego-motion.

Strengths:<br /> The authors firmly establish the location and selectivity of the new area through a series of well-crafted controlled experiments. They show that the area can be missed with too much smoothing, thus providing a case for why it has not been previously described. They show that it appears in much the same location in different subjects, with different magnetic field strengths, and with different stimulus sets. Finally, they show that it is selective for ego-motion - defined as a series of sequential photographs of an egocentric trajectory along a path. They further clarify that the area is not generically motion-selective by showing that it does not respond to biological motion without an ego-motion component to it. All statistics are standard and sound; the evidence presented is strong.

Weaknesses:<br /> There are few weaknesses in this work. If pressed, I might say that the stimuli depicting ego-motion do not, strictly speaking, depict motion, but only apparent motion between 2m apart photographs. However, this choice was made to equate frame rates and motion contrast between the 'ego-motion' and a control condition, which is a useful and valid approach to the problem. Some choices for visualization of the results might be made differently; for example, outlines of the regions might be shown in more plots for easier comparison of activation locations, but this is a minor issue.

This is a very strong paper.

Reviewer #1 (Public Review):

Summary:<br /> The present study evaluates the role of visual experience in shaping functional correlations between extrastriate visual cortex and frontal regions. The authors used fMRI to assess "resting-state" temporal correlations in three groups: sighted adults, congenitally blind adults, and neonates. Previous research has already demonstrated differences in functional correlations between visual and frontal regions in sighted compared to early blind individuals. The novel contribution of the current study lies in the inclusion of an infant dataset, which allows for an assessment of the developmental origins of these differences.

The main results of the study reveal that correlations between prefrontal and visual regions are more prominent in the blind and infant groups, with the blind group exhibiting greater lateralization. Conversely, correlations between visual and somato-motor cortices are more prominent in sighted adults. Based on these data, the authors conclude that visual experience plays an instructive role in shaping these cortical networks. This study provides valuable insights into the impact of visual experience on the development of functional connectivity in the brain.

Strengths:<br /> The dissociations in functional correlations observed among the sighted adult, congenitally blind, and neonate groups provide strong support for the study's main conclusion regarding experience-driven changes in functional connectivity profiles between visual and frontal regions.

In general, the findings in sighted adult and congenitally blind groups replicate previous studies and enhance the confidence in the reliability and robustness of the current results.

Split-half analysis provides a good measure of robustness in the infant data.

Weaknesses:<br /> There is some ambiguity in determining which aspects of these networks are shaped by experience.

This uncertainty is compounded by notable differences in data acquisition and preprocessing methods, which could result in varying signal quality across groups. Variations in signal quality may, in turn, have an impact on the observed correlation patterns.

The study's findings could benefit from being situated within a broader debate surrounding the instructive versus permissive roles of experience in the development of visual circuits.

Reviewer #2 (Public Review):

Summary:<br /> Tian et al. explore the developmental organs of cortical reorganization in blindness. Previous work has found that a set of regions in the occipital cortex show different functional responses and patterns of functional correlations in blind vs. sighted adults. In this paper, Tian et al. ask: how does this organization arise over development? Is the "starting state" more like the blind pattern, or more like the adult pattern? Their analyses reveal that the answer depends on the particular networks investigated; some functional connections in infants look more like blind than sighted adults; other functional connections look more like sighted than blind adults; and others fall somewhere in the middle, or show an altogether different pattern in infants compared with both sighted and blind adults.

Strengths:<br /> The question raised in this paper is extremely important: what is the starting state in development for visual cortical regions, and how is this organization shaped by experience? This paper is among the first to examine this question, particularly by comparing infants not only with sighted adults but also blind adults, which sheds new light on the role of visual (and cross-modal) experience. Another clear strength lies in the unequivocal nature of many results. Many results have very large effect sizes, critical interactions between regions and groups are tested and found, and infant analyses are replicated in split halves of the data.

Weaknesses:<br /> A central claim is that "infant secondary visual cortices functionally resemble those of blind more than sighted adults" (abstract, last paragraph of intro). I see two potential issues with this claim. First, a minor change: given the approaches used here, no claims should be made about the "function" of these regions, but rather their "functional correlations". Second (and more importantly), the claim that the secondary visual cortex in general resembles blind more than sighted adults is still not fully supported by the data. In fact, this claim is only true for one aspect of secondary visual area functional correlations (i.e., their connectivity to A1/M1/S1 vs. PFC). In other analyses, the infant secondary visual cortex looks more like sighted adults than blind adults (i.e., in within vs. across hemisphere correlations), or shows a different pattern from both sighted and blind adults (i.e., in occipito-frontal subregion functional connectivity). It is not clear from the manuscript why the comparison to PFC vs. non-visual sensory cortex is more theoretically important than hemispheric changes or within-PFC correlations (in fact, if anything, the within-PFC correlations strike me as the most important for understanding the development and reorganization of these secondary visual regions). It seems then that a more accurate conclusion is that the secondary visual cortex shows a mix of instructive effects of vision and reorganizing effects of blindness, albeit to a different extent than the primary visual cortex.

Relatedly, group differences in overall secondary visual cortex connectivity are particularly striking as visualized in the connectivity matrices shown in Figure S1. In the results (lines 105-112), it is noted that while the infant FC matrix is strongly correlated with both adult groups, the infant group is nonetheless more strongly correlated with the blind than sighted adults. I am concerned that these results might be at least partially explained by distance (i.e., local spread of the bold signal), since a huge portion of the variance in these FC matrices is driven by stronger correlations between regions within the same system (e.g., secondary-secondary visual cortex, frontal-frontal cortex), which are inherently closer together, relative to those between different systems (e.g., visual to frontal cortex). How do results change if only comparisons between secondary visual regions and non-visual regions are included (i.e., just the pairs of regions within the bold black rectangle on the figure), which limits the analysis to long-rang connections only? Indeed, looking at the off-diagonal comparisons, it seems that in fact there are three altogether different patterns here in the three groups. Even if the correlation between the infant pattern and blind adult pattern survives, it might be more accurate to claim that infants are different from both adult groups, suggesting both instructive effects of vision and reorganizing effects of blindness. It might help to show the correlation between each group and itself (across independent sets of subjects) to better contextualize the relative strength of correlations between the groups.

It is not clear that differences between groups should be attributed to visual experience only. For example, despite the title of the paper, the authors note elsewhere that cross-modal experience might also drive changes between groups. Another factor, which I do not see discussed, is possible ongoing experience-independent maturation. The infants scanned are extremely young, only 2 weeks old. Although no effects of age are detected, it is possible that cortex is still undergoing experience-independent maturation at this very early stage of development. For example, consider Figure 2; perhaps V1 connectivity is not established at 2 weeks, but eventually achieves the adult pattern later in infancy or childhood. Further, consider the possibility that this same developmental progression would be found in infants and children born blind. In that case, the blind adult pattern may depend on blindness-related experience only (which may or may not reflect "visual" experience per se). To deal with these issues, the authors should add a discussion of the role of maturation vs. experience and temper claims about the role of visual experience specifically (particularly in the title).

The authors measure functional correlations in three very different groups of participants and find three different patterns of functional correlations. Although these three groups differ in critical, theoretically interesting ways (i.e., in age and visual/cross-modal experience), they also differ in many uninteresting ways, including at least the following: sampling rate (TR), scan duration, multi-band acceleration, denoising procedures (CompCor vs. ICA), head motion, ROI registration accuracy, and wakefulness (I assume the infants are asleep).

Addressing all of these issues is beyond the scope of this paper, but I do feel the authors should acknowledge these confounds and discuss the extent to which they are likely (or not) to explain their results. The authors would strengthen their conclusions with analyses directly comparing data quality between groups (e.g., measures of head motion and split-half reliability would be particularly effective).

Reviewer #3 (Public Review):

Summary:<br /> This study aimed to investigate whether the differences observed in the organization of visual brain networks between blind and sighted adults result from a reorganization of an early functional architecture due to blindness, or whether the early architecture is immature at birth and requires visual experience to develop functional connections. This question was investigated through the comparison of 3 groups of subjects with resting-state functional MRI (rs-fMRI). Based on convincing analyses, the study suggests that: 1) secondary visual cortices showed higher connectivity to prefrontal cortical regions (PFC) than to non-visual sensory areas (S1/M1 and A1) in sighted infants like in blind adults, in contrast to sighted adults; 2) the V1 connectivity pattern of sighted infants lies between that of sighted adults (stronger functional connectivity with non-visual sensory areas than with PFC) and that of blind adults (stronger functional connectivity with PFC than with non-visual sensory areas); 3) the laterality of the connectivity patterns of sighted infants resembled those of sighted adults more than those of blind adults, but sighted infants showed a less differentiated fronto-occipital connectivity pattern than adults.

Strengths:<br /> - The question investigated in this article is important for understanding the mechanisms of plasticity during typical and impaired development, and the approach considered, which compares different groups of subjects including, neonates/infants and blind adults, is highly original.

- Overall, the analyses considered are solid and well-detailed. The results are quite convincing, even if the interpretation might need to be revised downwards, as factors other than visual experience may play a role in the development of functional connections with the visual system.

Weaknesses:<br /> - While it is informative to compare the "initial" state (close to birth) and the "final" states in blind and sighted adults to study the impact of post-natal and visual experience, this study does not analyze the chronology of this development and when the specialization of functional connections is completed. This would require investigating when experience-dependent mechanisms are important for the setting- establishment of multiple functional connections within the visual system. This could be achieved by analyzing different developmental periods in the same way, using open databases such as the Baby Connectome Project. Given the early, "condensed" maturation of the visual system after birth, we might expect sighted infants to show connectivity patterns similar to those of adults a few months after birth.

- The rationale for mixing full-term neonates and preterm infants (scanned at term-equivalent age) from the dHCP 3rd release is not understandable since preterms might have a very different development related to prematurity and to post-natal (including visual) experience. Although the authors show that the difference between the connectivity of visual and other sensory regions, and the one of visual and PFC regions, do not depend on age at birth, they do not show that each connectivity pattern is not influenced by prematurity. Simply not considering the preterm infants would have made the analysis much more robust, and the full-term group in itself is already quite large compared with the two adult groups. The current study setting and the analyses performed do not seem to be an adequate and sufficient model to ascertain that "a few weeks of vision after birth is ... insufficient to influence connectivity".

In a similar way, excluding the few infants with detected brain anomalies (radiological scores higher or equal to 4) would strengthen the group homogeneity by focusing on infants supposed to have a rather typical neurodevelopment. The authors quote all infants as "sighted" but this is not guaranteed as no follow-up is provided.

The post-menstrual age (PMA) at scan of the infants is also not described. The methods indicate that all were scanned at "term-equivalent age" but does this mean that there is some PMA variability between 37 and 41 weeks? Connectivity measures might be influenced by such inter-individual variability in PMA, and this could be evaluated.

- The rationale for presenting results on the connectivity of secondary visual cortices before one of the primary cortices (V1) was not clear to understand. Also, it might be relevant to better justify why only the connectivity of visual regions to non-visual sensory regions (S1-M1, A1) and prefrontal cortex (PFC) was considered in the analyses, and not the ones to other brain regions.

- In relation to the question explored, it might be informative to reposition the study in relation to what others have shown about the developmental chronology of structural and functional long-distance and short-distance connections during pregnancy and the first postnatal months.

- The authors acknowledge the methodological difficulties in defining regions of interest (ROIs) in infants in a similar way as adults. The reliability and the comparability of the ROIs positioning in infants is definitely an issue. Given that brain development is not homogeneous and synchronous across brain regions (in particular with the frontal and parietal lobes showing delayed growth), the newborn brain is not homothetic to the adult brain, which poses major problems for registration. The functional specialization of cortical regions is incomplete at birth. This raises the question of whether the findings of this study would be stable/robust if slightly larger or displaced regions had been considered, to cover with greater certainty the same areas as those considered in adults. And have other cortical parcellation approaches been considered to assess the ROIs robustness (e.g. MCRIB-S for full-terms)?

Reviewer #1 (Public Review):

Summary:

Songbirds provide a tractable system to examine neural mechanisms of sequence generation and variability. In past work, the projection from LMAN to RA (output of the anterior forebrain pathway) was shown to be critical for driving vocal variability during babbling, learning, and adulthood. LMAN is immediately adjacent to MMAN, which projects to HVC. MMAN is less well understood but, anatomically, appears to resemble LMAN in that it is the cortical output of a BG-thalamocortical loop. Because it projects to HVC, a major sequence generator for both syllable phonology and sequence, a strong prediction would be that MMAN drives sequence variability in the same way that LMAN drives phonological variability. This hypothesis predicts that MMAN lesions in a Bengalese finch would reduce sequence variability. Here, the authors test this hypothesis. They provide a surprising and important result that is well motivated and well analyzed: MMAN lesions increase sequence variability - this is exactly the opposite result from what would be predicted based on the functions of LMAN.

Strengths:

1. A very important and surprising result shows that lesions of a frontal projection from MMAN to HVC, a sequence generator for birdsong, increase syntactical variability.

2. The choice of Bengalese finches, which have complex transition structures, to examine the mechanisms of sequence generation, enabled this important discovery.

3. The idea that frontal outputs of BG-cortical loops can generate vocal variability comes from lesions/inactivations of a parallel pathway from LMAN to RA. The difference between MMAN and LMAN functions is striking and important.

Weaknesses:

1. If more attention was paid to how syllable phonology was (or was not) affected by MMAN lesions then the claims could be stronger around the specific effects on sequence.

Reviewer #2 (Public Review):

Summary:

This study investigates the neural substrates of syntax variation in Bengalese finch songs. Here, the authors tested the effects of bilateral lesions of mMAN, a brain area with inputs to HVC, a premotor area required for song production. Lesions in mMAN induce variability in syntactic elements of song specifically through increased transition entropy, variability within stereotyped song elements known as chunks, and increases in the repeat number of individual syllables. These results suggest that mMAN projections to HVC contribute to multiple aspects of song syntax in the Bengalese finch. Overall the experiments are well-designed, the analysis excellent, and the results are of high interest.

Strengths:

The study identifies a novel role for mMAN, the medial magnocellular nucleus of the anterior nidopallium, in the control of syntactic variation within adult Bengalese finch song. This is of particular interest as multiple studies previously demonstrated that mMAN lesions do not affect song structure in zebra finches. The study undertakes a thorough analysis to characterise specific aspects of variability within the song of lesioned animals. The conclusions are well supported by the data.

Weaknesses:

The study would benefit from additional mechanistic information. A more fine-grained or reversible manipulation, such as brain cooling, might allow additional insights into how mMAN influences specific aspects of syntax structure. Are repeat number increases and transition entropy resulting from shared mechanisms within mMAN, or perhaps arising from differential output to downstream pathways (i.e. projections to HVC)? Similarly, unilateral manipulations would allow the authors to further test the hypothesis that mMAN is involved in inter-hemispheric synchronization.

Reviewer #1 (Public Review):

Summary:<br /> The authors investigated causal inference in the visual domain through a set of carefully designed experiments, and sound statistical analysis. They suggest the early visual system has a crucial contribution to computations supporting causal inference.

Strengths:<br /> I believe the authors target an important problem (causal inference) with carefully chosen tools and methods. Their analysis rightly implies the specialization of visual routines for causal inference and the crucial contribution of early visual systems to perform this computation. I believe this is a novel contribution and their data and analysis are in the right direction.

Weaknesses:<br /> In my humble opinion, a few aspects deserve more attention:

1. Causal inference (or causal detection) in the brain should be quite fundamental and quite important for human cognition/perception. Thus, the underlying computation and neural substrate might not be limited to the visual system (I don't mean the authors did claim that). In fact, to the best of my knowledge, multisensory integration is one of the best-studied perceptual phenomena that has been conceptualized as a causal inference problem. Assuming the causal inference in those studies (Shams 2012; Shams and Beierholm 2022; Kording et al. 2007; Aller and Noppeney 2018; Cao et al. 2019) (and many more e.g., by Shams and colleagues), and the current study might share some attributes, one expects some findings in those domains are transferable (at least to some degree) here as well. Most importantly, underlying neural correlates that have been suggested based on animal studies and invasive recording that has been already studied, might be relevant here as well. Perhaps the most relevant one is the recent work from the Harris group on mice (Coen et al. 2021). I should emphasize, that I don't claim they are necessarily relevant, but they can be relevant given their common roots in the problem of causal inference in the brain. This is a critical topic that the authors may want to discuss in their manuscript.

2. If I understood correctly, the authors are arguing pro a mere bottom-up contribution of early sensory areas for causal inference (for instance, when they wrote "the specialization of visual routines<br /> for the perception of causality at the level of individual motion directions raises the possibility that this function is located surprisingly early in the visual system *as opposed to a higher-level visual computation*."). Certainly, as the authors suggested, early sensory areas have a crucial contribution, however, it may not be limited to that. Recent studies progressively suggest perception as an active process that also weighs in strongly, the top-down cognitive contributions. For instance, the most simple cases of perception have been conceptualized along this line (Martin, Solms, and Sterzer 2021)<br /> and even some visual illusion (Safavi and Dayan 2022), and other extensions (Kay et al. 2023). Thus, I believe it would be helpful to extend the discussion on the top-down and cognitive contributions of causal inference (of course that can also be hinted at, based on recent developments). Even adaptation, which is central in this study can be influenced by top-down factors (Keller et al. 2017). I believe, based on other work of Rolfs and colleagues, this is also aligned with their overall perspective on vision.

3. The authors rightly implicate the neural substrate of causal inference in the early sensory system. Given their study is pure psychophysics, a more elaborate discussion based on other studies that used brain measurements is needed (in my opinion) to put into perspective this conclusion. In particular, as I mentioned in the first point, the authors mainly discuss the potential neural substrate of early vision, however much has been done about the role of higher-tier cortical areas in causal inference e.g., see (Cao et al. 2019; Coen et al. 2021).

There were many areas in this manuscript that I liked: clever questions, experimental design, and statistical analysis.

Bibliography<br /> \============

Aller, Mate, and Uta Noppeney. 2018. "To Integrate or Not to Integrate: Temporal Dynamics of Bayesian Causal Inference." Biorxiv, December, 504118. .

Cao, Yinan, Christopher Summerfield, Hame Park, Bruno Lucio Giordano, and Christoph Kayser. 2019. "Causal Inference in the Multisensory Brain." Neuron 102 (5): 1076-87.e8. .

Coen, Philip, Timothy P. H. Sit, Miles J. Wells, Matteo Carandini, and Kenneth D. Harris. 2021. "The Role of Frontal Cortex in Multisensory Decisions." Biorxiv, April. Cold Spring Harbor Laboratory, 2021.04.26.441250. .

Kay, Kendrick, Kathryn Bonnen, Rachel N. Denison, Mike J. Arcaro, and David L. Barack. 2023. "Tasks and Their Role in Visual Neuroscience." Neuron 111 (11). Elsevier: 1697-1713. .

Keller, Andreas J, Rachael Houlton, Björn M Kampa, Nicholas A Lesica, Thomas D Mrsic-Flogel, Georg B Keller, and Fritjof Helmchen. 2017. "Stimulus Relevance Modulates Contrast Adaptation in Visual Cortex." Elife 6. eLife Sciences Publications, Ltd: e21589.

Kording, K. P., U. Beierholm, W. J. Ma, S. Quartz, J. B. Tenenbaum, and L. Shams. 2007. "Causal Inference in Multisensory Perception." PloS One 2: e943. .

Martin, Joshua M., Mark Solms, and Philipp Sterzer. 2021. "Useful Misrepresentation: Perception as Embodied Proactive Inference." Trends Neurosci. 44 (8): 619-28. .

Safavi, Shervin, and Peter Dayan. 2022. "Multistability, Perceptual Value, and Internal Foraging." Neuron, August. .

Shams, L. 2012. "Early Integration and Bayesian Causal Inference in Multisensory Perception." In The Neural Bases of Multisensory Processes, edited by M. M. Murray and M. T. Wallace. Frontiers in<br /> Neuroscience. Boca Raton (FL).

Shams, Ladan, and Ulrik Beierholm. 2022. "Bayesian Causal Inference: A Unifying Neuroscience Theory." Neuroscience & Biobehavioral Reviews 137 (June): 104619. .

Reviewer #2 (Public Review):

This paper seeks to determine whether the human visual system's sensitivity to causal interactions is tuned to specific parameters of a causal launching event, using visual adaptation methods. The three parameters the authors investigate in this paper are the direction of motion in the event, the speed of the objects in the event, and the surface features or identity of the objects in the event (in particular, having two objects of different colors).

The key method, visual adaptation to causal launching, has now been demonstrated by at least three separate groups and seems to be a robust phenomenon. Adaptation is a strong indicator of a visual process that is tuned to a specific feature of the environment, in this case launching interactions. Whereas other studies have focused on retinotopically-specific adaptation (i.e., whether the adaptation effect is restricted to the same test location on the retina as the adaptation stream was presented to), this one focuses on feature-specificity.

The first experiment replicates the adaptation effect for launching events as well as the lack of adaptation event for a minimally different non-causal 'slip' event. However, it also finds that the adaptation effect does not work for launching events that do not have a direction of motion more than 30 degrees from the direction of the test event. The interpretation is that the system that is being adapted is sensitive to the direction of this event, which is an interesting and somewhat puzzling result given the methods used in previous studies, which have used random directions of motion for both adaptation and test events.

The obvious interpretation would be that past studies have simply adapted to launching in every direction, but that in itself says something about the nature of this direction-specificity: it is not working through opposed detectors. For example, in something like the waterfall illusion adaptation effect, where extended exposure to downward motion leads to illusory upward motion on neutral-motion stimuli, the effect simply doesn't work if motion in two opposed directions is shown (i.e., you don't see illusory motion in both directions, you just see nothing). The fact that adaptation to launching in multiple directions doesn't seem to cancel out the adaptation effect in past work raises interesting questions about how directionality is being coded in the underlying process. In addition, one limitation of the current method is that it's not clear whether the motion-direction-specificity is also itself retinotopically-specific, that is, if one retinotopic location were adapted to launching in one direction and a different retinotopic location adapted to launching in the opposite direction, would each test location show the adaptation effect only for events in the direction presented at that location?

The second experiment tests whether the adaptation effect is similarly sensitive to differences in speed. The short answer is no; adaptation events at one speed affect test events at another. Furthermore, this is not surprising given that Kominsky & Scholl (2020) showed adaptation transfer between events with differences in speeds of the individual objects in the event (whereas all events in this experiment used symmetrical speeds). This experiment is still novel and it establishes that the speed-insensitivity of these adaptation effects is fairly general, but I would certainly have been surprised if it had turned out any other way.

The third experiment tests color (as a marker of object identity), and pits it against motion direction. The results demonstrate that adaptation to red-launching-green generates an adaptation effect for green-launching-red, provided they are moving in roughly the same direction, which provides a nice internal replication of Experiment 1 in addition to showing that the adaptation effect is not sensitive to object identity. This result forms an interesting contrast with the infant causal perception literature. Multiple papers (starting with Leslie & Keeble, 1987) have found that 6-8-month-old infants are sensitive to reversals in causal roles exactly like the ones used in this experiment. The success of adaptation transfer suggests, very clearly, that this sensitivity is not based only on perceptual processing, or at least not on the same processing that we access with this adaptation procedure. It implies that infants may be going beyond the underlying perceptual processes and inferring genuine causal content. This is also not the first time the adaptation paradigm has diverged from infant findings: Kominsky & Scholl (2020) found a divergence with the object speed differences as well, as infants categorize these events based on whether the speed ratio (agent:patient) is physically plausible (Kominsky et al., 2017), while the adaptation effect transfers from physically implausible events to physically plausible ones. This only goes to show that these adaptation effects don't exhaustively capture the mechanisms of early-emerging causal event representation.

One overarching point about the analyses to take into consideration: The authors use a Bayesian psychometric curve-fitting approach to estimate a point of subjective equality (PSE) in different blocks for each individual participant based on a model with strong priors about the shape of the function and its asymptotic endpoints, and this PSE is the primary DV across all of the studies. As discussed in Kominsky & Scholl (2020), this approach has certain limitations, notably that it can generate nonsensical PSEs when confronted with relatively extreme response patterns. The authors mentioned that this happened once in Experiment 3 and that a participant had to be replaced. An alternate approach is simply to measure the proportion of 'pass' reports overall to determine if there is an adaptation effect. I don't think this alternate analysis strategy would greatly change the results of this particular experiment, but it is robust against this kind of self-selection for effects that fit in the bounds specified by the model, and may therefore be worth including in a supplemental section or as part of the repository to better capture the individual variability in this effect.

In general, this paper adds further evidence for something like a 'launching' detector in the visual system, but beyond that, it specifies some interesting questions for future work about how exactly such a detector might function.

Kominsky, J. F., & Scholl, B. J. (2020). Retinotopic adaptation reveals distinct categories of causal perception. Cognition, 203, 104339. https://doi.org/10.1016/j.cognition.2020.104339

Kominsky, J. F., Strickland, B., Wertz, A. E., Elsner, C., Wynn, K., & Keil, F. C. (2017). Categories and Constraints in Causal Perception. Psychological Science, 28(11), 1649-1662. https://doi.org/10.1177/0956797617719930

Leslie, A. M., & Keeble, S. (1987). Do six-month-old infants perceive causality? Cognition, 25(3), 265-288. https://doi.org/10.1016/S0010-0277(87)80006-9

Reviewer #3 (Public Review):

Summary:<br /> This paper presents evidence from three behavioral experiments that causal impressions of "launching events", in which one object is perceived to cause another object to move, depending on motion direction-selective processing. Specifically, the work uses an adaptation paradigm (Rolfs et al., 2013), presenting repetitive patterns of events matching certain features to a single retinal location, then measuring subsequent perceptual reports of a test display in which the degree of overlap between two discs was varied, and participants could respond "launch" or "pass". The three experiments report results of adapting to motion direction, motion speed, and "object identity", and examine how the psychometric curves for causal reports shift in these conditions depending on the similarity of the adapter and test. While causality reports in the test display were selective for motion direction (Experiment 1), they were not selective for adapter-test speed differences (Experiment 2) nor for changes in object identity induced via color swap (Experiment 3). These results support the notion that causal perception is computed (in part) at relatively early stages of sensory processing, possibly even independently of or prior to computations of object identity.

Strengths:<br /> The setup of the research question and hypotheses is exceptional. The experiments are carefully performed (appropriate equipment, and careful control of eye movements). The slip adaptor is a really nice control condition and effectively mitigates the need to control motion direction with a drifting grating or similar. Participants were measured with sufficient precision, and a power curve analysis was conducted to determine the sample size. Data analysis and statistical quantification are appropriate. Data and analysis code are shared on publication, in keeping with open science principles. The paper is concise and well-written.

Weaknesses:<br /> The biggest uncertainty I have in interpreting the results is the relationship between the task and the assumption that the results tell us about causality impressions. The experimental logic assumes that "pass" reports are always non-causal impressions and "launch" reports are always causal impressions. This logic is inherited from Rolfs et al (2013) and Kominsky & Scholl (2020), who assert rather than measure this. However, other evidence suggests that this assumption might not be solid (Bechlivanidis et al., 2019). Specifically, "[our experiments] reveal strong causal impressions upon first encounter with collision-like sequences that the literature typically labels "non-causal"" (Bechlivanidis et al., 2019) -- including a condition that is similar to the current "pass". It is therefore possible that participants' "pass" reports could also involve causal experiences.

Furthermore, since the only report options are "launch" or "pass", it is also possible that "launch" reports are not indications of "I experienced a causal event" but rather "I did not experience a pass event". It seems possible to me that different adaptation transfer effects (e.g. selectivity to motion direction, speed, or color-swapping) change the way that participants interpret the task, or the uncertainty of their impression. For example, it could be that adaptation increases the likelihood of experiencing a "pass" event in a direction-selective manner, without changing causal impressions. Increases of "pass" impressions (or at least, uncertainty around what was experienced) would produce a leftward shift in the PSE as reported in Experiment 1, but this does not necessarily mean that experiences of causal events changed. Thus, changes in the PSEs between the conditions in the different experiments may not directly reflect changes in causal impressions. I would like the authors to clarify the extent to which these concerns call their conclusions into question.

Leaving these concerns aside, I am also left wondering about the functional significance of these specialised mechanisms. Why would direction matter but speed and object identity not? Surely object identity, in particular, should be relevant to real-world interpretations and inputs of these visual routines? Is color simply too weak an identity?

References:

Bechlivanidis, C., Schlottmann, A., & Lagnado, D. A. (2019). Causation without realism. Journal of Experimental Psychology: General, 148(5), 785-804. https://doi.org/10.1037/xge0000602

Kominsky, J. F., & Scholl, B. J. (2020). Retinotopic adaptation reveals distinct categories of causal perception. Cognition, 203, 104339.

Rolfs, M., Dambacher, M., & Cavanagh, P. (2013). Visual Adaptation of the Perception of Causality. Current Biology, 23(3), 250-254. https://doi.org/10.1016/j.cub.2012.12.017

Reviewer #1 (Public Review):

Summary and strengths. This paper starts with an exceptionally fair and balanced introduction to a topic, the mirror neuron literature, which is often debated and prone to controversies even in the choice of the terminology. In my opinion, the authors made an excellent job in this regard, and I really appreciated it. Then, they propose a novel method to look at population dynamics to compare neural selectivity and alignment between execution and observation of actions performed with different types of grip.

Weakness. Unfortunately, the goal and findings within this well-described framework are less clear to me. The authors aimed to investigate, using a novel analytic approach, whether and to what extent a match exists between population codes and neural dynamics when a monkey performs an action or observes it performed by an experimenter. This motivation stems from the fact that the general evidence in the literature is that the match between visual and motor selectivity of mirror neuron responses is essentially at a chance level. While the approach devised by the author is generally well-described and understandable, the main result obtained confirms this general finding of a lack of matching between the two contexts in 2 out of the three monkeys. Nevertheless, the authors claim that the patterns associated with execution and observation can be re-aligned with canonical correlation, indicating that these distinct neural representations show dynamical similarity that may enable the nervous system to recognize particular actions. This final conclusion is hardly acceptable to me, and constitutes my major concern, at least without a more explicit explanation: how do we know that this additional operation can be performed by the brain? Is this a computational trick to artificially align something that is naturally non-aligned, or can it capture something real and useful?<br /> Based on the accumulated evidence on space-constrained coding of others' actions by mirror neurons (e.g., Caggiano et al. 2009; Maranesi et al. 2017), recent evidence also cited by the authors (Pomper et al. 2023), and the most recent views supported even by the first author of the original discovery (i.e., Vittorio Gallese, see Bonini et al. 2022 on TICS), it seems that one of the main functions of these cells, especially in monkeys, might be to prepare actions and motor responses during social interaction rather than recognizing the actions of others - something that visual brain areas could easily do better than motor ones in most situations. In this perspective, and given the absence of causal evidence so far, the lack of visuo-motor congruence is a potentially relevant feature of the mechanism rather than something to be computationally cracked at all costs.

Specific comments on Results/Methods:<br /> I can understand, based on the authors' hypothesis, that they employed an ANOVA to preliminarily test whether and which of the recorded neurons fit their definition of "mirror neurons". However, given the emphasis on the population level, and the consolidated finding of highly different execution and observation responses, I think it could be interesting to apply the same analysis on (at least also) the whole recorded neuronal population, without any preselection-based on a single neuron statistic. Such preselection of mirror neurons could influence the results of EXE-OBS comparisons since all the neurons activated only during EXE or OBS are excluded. Related to this point, the authors could report the total number of recorded neurons per monkey/session, so that also the fraction of neurons fitting their definition of mirror neuron is explicit.<br /> Furthermore, the comparison of the dynamics of the classification accuracy in figures 4 and 5, and therefore the underlying assumption of subspaces shift in execution and observation, respectively, reveal substantial similarities between monkeys despite the different contexts, which are clearly greater than the similarities among neural subspaces shifts across task epochs: to me, this suggests that the main result is driven by the selected neural populations in different monkeys/implants rather than by an essential property of the neuronal dynamics valid across animals. Could the author comment on this issue? This could easily explain the "strange" result reported in figure 6 for monkey T.

Reviewer #2 (Public Review):

In this work, the authors set out to identify time-varying subspaces in the premotor cortical activity of monkeys as they executed/observed a reach-grasp-hold movement of 4 different objects. Then, they projected the neural activity to these subspaces and found evidence of shifting subspaces in the time course of a trial in both conditions, executing and observing. These shifting subspaces appear to be distinct in execution and observation trials. However, correlation analysis of neural dynamics reveals the similarity of dynamics in these distinct subspaces. Taken together, Zhao and Schieber speculate that the condition-dependent activity studied here provides a representation of movement that relies on the actor.<br /> This work addresses an interesting question. The authors developed a novel approach to identify instantaneous subspaces and decoded the object type from the projected neural dynamics within these subspaces. As interesting as these results might be, I have a few suggestions and questions to improve the manuscript:<br /> 1- Repeating the analyses in the paper, e.g., in Fig5, using non-MN units only or the entire population, and demonstrating that the results are specific to MNs would make the whole study much more compelling.<br /> 2- The method presented here is similar and perhaps related to principal angles (https://doi.org/10.2307/2005662). It would be interesting to confirm these results with principal angles. For instance, instead of using the decoding performance as a proxy for shifting subspaces, principal angles could directly quantify the 'shift' (similar to Gallego et al, Nat Comm, 2018). Relatedly, why the decoding of the 'object type' is used to establish the progressive shifting of the subspaces? I would be interested to see the authors' argument. The object type should be much more decodable during movement or hold, than instruction, which is probably why the chance-level decoding performance (horizontal lines) is twice the instruction segment for the movement segment.<br /> 3- Why aren't execution and observation subspaces compared together directly? Especially given that there are both types of trials in the same session with the same recorded population of neurons. Using instantaneous subspaces, or the principal angles between manifolds during exec trials vs obs trials.<br /> 4- The definition of the instantaneous subspaces is a critical point in the manuscript. I think it is slightly unclear: based on the Methods section #715-722 and the main text #173-#181, I gather that the subspaces are based on trial averaged neural activity for each of the 4 objects, separately. So for each object and per timepoint, a vector of size (1, n) -n neurons- is reduced to a vector of (1, 2 or 3 -the main text says 2, methods say 3-) which would be a single point in the low-d space. Is this description accurate? This should be clarified in the manuscript.<br /> 5- Isn't the process of projecting segments of neural dynamics and comparing the results equivalent to comparing the projection matrices in the first place? If so, that might have been a more intuitive avenue to follow.<br /> 6- Lines #385-#389: This process seems unnecessarily complicated. Also, given the number of trials available, this sometimes doesn't make sense. E.g. Monkey R exec has only 8 trials of one of the objects, so bootstrapping 20 trials 500 times would be spurious. Why not, as per Gallego et al, Nat Neurosci 2020 and Safaie et al, Nat 2023 which are cited, concatenate the trials?<br /> 7- Related to the CCA analysis, what behavioural epoch has been used here, the same as the previous analyses, i.e. 100ms? how many datapoint is that in time? Given that CCA is essentially a correlation value, too few datapoints make it rather meaningless. If that's the case, I encourage using, let's say, one window combined of I and G until movement, and one window of movement and hold, such that they are both easier to interpret. Indeed low values of exec-exec in CC2 compared to Gallego et al, Nat Neurosci, 2020 might be a sign of a methodological error.

Reviewer #3 (Public Review):

Summary:<br /> In their study, Zhao et al. investigated the population activity of mirror neurons (MNs) in the premotor cortex of monkeys either executing or observing a task consisting of reaching to, grasping, and manipulating various objects. The authors proposed an innovative method for analyzing the population activity of MNs during both execution and observation trials. This method enabled to isolate the condition-dependent variance in neural data and to study its temporal evolution over the course of single trials. The method proposed by the authors consists of building a time series of "instantaneous" subspaces with single time step resolution, rather than a single subspace spanning the entire task duration. As these subspaces are computed on an instant time basis, projecting neural activity from a given task time into them results in latent trajectories that capture condition-dependent variance while minimizing the condition-independent one. The authors then analyzed the time evolution of these instantaneous subspaces and revealed that a progressive shift is present in subspaces of both execution and observation trials, with slower shifts during the grasping and manipulating phases compared to the initial preparation phase. Finally, they compared the instantaneous subspaces between execution and observation trials and observed that neural population activity did not traverse the same subspaces in these two conditions. However, they showed that these distinct neural representations can be aligned with Canonical Correlation Analysis, indicating dynamic similarities of neural data when executing and observing the task. The authors speculated that such similarities might facilitate the nervous system's ability to recognize actions performed by oneself or another individual.

Strengths:<br /> Unlike other areas of the brain, the analysis of neural population dynamics of premotor cortex MNs is not well established. Furthermore, analyzing population activity recorded during non-trivial motor actions, distinct from the commonly used reaching tasks, serves as a valuable contribution to computational neuroscience. This study holds particular significance as it bridges both domains, shedding light on the temporal evolution of the shift in neural states when executing and observing actions. The results are moderately robust, and the proposed analytical method could potentially be used in other neuroscience contexts.

Weaknesses:<br /> While the overall clarity is satisfactory, the paper falls short in providing a clear description of the mathematical formulas for the different methods used in the study. Moreover, it was not immediately clear why the authors did not consider a (relatively) straightforward metric to quantity the progressive shift of the instantaneous subspaces, such as computing the angle between consecutive subspaces, rather than choosing a (in my opinion) more cumbersome metric based on classification of trajectory segments representing different movements.

Specific comments:<br /> In the methods, it is stated that instantaneous subspaces are found with 3 PCs. Why does it say 2 here? Another doubt on how instantaneous subspaces are computed: in the methods you state that you apply PCA on trial-averaged activity at each 50ms time step. From the next sentence, I gather that you apply PCA on an Nx4 data matrix (N being the number of neurons, and 4 being the trial-averaged activity of the four objects) every 50 ms. Is this right? It would help to explicitly specify the dimensions of the data matrix that goes into PCA computation.

It would help to include some equations in the methods section related to the LSTM decoding. Just to make sure I understood correctly: after having identified the instantaneous subspaces (every 50 ms), you projected the Instruction, Go, Movement, and Holding segments from individual trials (each containing 100 samples, since they are sampled from a 100ms window) onto each instantaneous subspace. So you have four trajectories for each subspace. In the methods, it is stated that a single LSTM classifier is trained for each subspace. Do you also have a separate classifier for each trajectory segment? What is used as input to the classifier? Each trajectory segment should be a 100x3 matrix once projected in an instantaneous subspace. Is that what (each of) the LSTMs take as input? And lastly, what is the LSTM trained to predict exactly? Just a label indicating the type of object that was manipulated in that trial? I apologize if I overlooked any detail, but I believe a clearer explanation of the LSTM, preferably with mathematical formulas, would greatly help readers understand this section.

Reviewer #1 (Public Review):

The manuscript by Hariani et al. presents experiments designed to improve our understanding of the connectivity and computational role of Unipolar Brush Cells (UBCs) within the cerebellar cortex, primarily lobes IX and X. The authors develop and cross several genetic lines of mice that express distinct fluorophores in subsets of UBCs, combined with immunocytochemistry that also distinguishes subtypes of UBCs, and they use confocal microscopy and electrophysiology to characterize the electrical and synaptic properties of subsets of so-labelled cells, and their synaptic connectivity within the cerebellar cortex. The authors then generate a computer model to test possible computational functions of such interconnected UBCs.

Using these approaches, the authors report that:<br /> 1) GRP-driven TDtomato is expressed exclusively in a subset (20%) of ON-UBCs, defined electrophysiologically (excited by mossy fiber afferent stimulation via activation of UBC AMPA and mGluR1 receptors) and immunocytochemically by their expression of mGluR1.

2) UBCs ID'd/tagged by mCitrine expression in Brainbow mouse line P079 is expressed in a similar minority subset of OFF-UBCs defined electrophysiologically (inhibited by mossy fiber afferent stimulation via activation of UBC mGluR2 receptors) and immunocytochemically by their expression of Calretinin. However, such mCitrine expression was also detected in some mGluR1 positive UBCs, which may not have shown up electrophysiologically because of the weaker fluorophore expression without antibody amplification.

3) Confocal analysis of crossed lines of mice (GRP X P079) stained with antibodies to mGluR1 and calretinin documented the existence of all possible permutations of interconnectivity between cells (ON-ON, ON-OFF, OFF-OFF, OFF-ON), but their overall abundance was low, and neither their absolute or relative abundance was quantified.

4) A computational model (NEURON ) indicated that the presence of an intermediary UBC (in a polysynaptic circuit from MF to UBC to UBC) could prolong bursts (MF-ON-ON), prolong pauses (MF-ON-OFF), cause a delayed burst (MF-OFF-OFF), cause a delayed pause (MF-OFF-ON) relative to solely MF to UBC synapses which would simply exhibit long bursts (MF-ON) or long pauses (MF-OFF).

The authors thus conclude that the pattern of interconnected UBCs provides an extended and more nuanced pattern of firing within the cerebellar cortex that could mediate longer lasting sensorimotor responses.

The cerebellum's long known role in motor skills and reflexes, and associated disorders, combined with our nascent understanding of its role in cognitive, emotional, and appetitive processing, makes understanding its circuitry and processing functions of broad interest to the neuroscience and biomedical community. The focus on UBCs, which are largely restricted to vestibular lobes of the cerebellum reduces the breadth of likely interest somewhat. The overall design of specific experiments is rigorous and the use of fluorophore expressing mouse lines is creative. The data that is presented and the writing are clear.

Reviewer #2 (Public Review):

In this paper, the authors presented a compelling rationale for investigating the role of UBCs in prolonging and diversifying signals. Based on the two types of UBCs known as ON and OFF UBC subtypes, they have highlighted the existing gaps in understanding UBCs connectivity and the need to investigate whether UBCs target UBCs of the same subtype, different subtypes, or both. The importance of this knowledge is for understanding how sensory signals are extended and diversified in the granule cell layer.

The authors designed very interesting approaches to study UBCs connectivity by utilizing transgenic mice expressing GFP and RFP in UBCs, Brainbow approach, immunohistochemical and electrophysiological analysis, and computational models to understand how the feed-forward circuits of interconnected UBCs transform their inputs.

This study provided evidence for the existence of distinct ON and OFF UBC subtypes based on their electrophysiological properties, anatomical characteristics, and expression patterns of mGluR1 and calretinin in the cerebellum. The findings support the classification of GRP UBCs as ON UBCs and P079 UBCs as OFF UBCs and suggest the presence of synaptic connections between the ON and OFF UBC subtypes. In addition, they found that GRP and P079 UBCs form parallel and convergent pathways and have different membrane capacitance and excitability. Furthermore, they showed that UBCs of the same subtype provide input to one another and modify the input to granule cells, which could provide a circuit mechanism to diversify and extend the pattern of spiking produced by mossy fiber input. Accordingly, they suggested that these transformations could provide a circuit mechanism for maintaining a sensory representation of movement for seconds.

Overall, the article is well written in a sound detailed format, very interesting with excellent discovery and suggested model.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:<br /> The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:<br /> None.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance g,K,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

My only comment relates to the statement regarding the results providing "evidence" that Kv1.4 form heteromultimers with Kv1.8 channels (see Discussion). The only data I can see from the results is that Kv1.4 channels are expressed in the membrane of type II hair cells, which is not sufficient evidence for the above claim. Is the distribution of Kv1.8 and Kv1.4 overlapping in type II hair cells? Have the authors attempted to perform some pharmacological studies on Kv1.4? For example, would gK,A be completely blocked by a Kv1.4 antagonist? Addressing at least some of these questions would strengthen your argument.

Reviewer #3 (Public Review):

Summary:<br /> This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells have unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

2. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

3. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

4. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Joint Public Review:

Zhang et. al. presents compelling results that support the identification of epigenetically mediated control for the recognition of dihydropyrimidine dehydrogenase (DPYD) gene expression that is linked with cancer treatment resistance 5-fluorouracil. The experimental approach was developed and pursued with in vitro and in vivo strategies. Combining molecular, cellular, and biochemical approaches, the authors identify a germline variant with compromised enhancer control. Several lines of evidence were presented that are consistent with increased CEBP recruitment to the DPYD regulatory domain with consequential modifications in promoter-enhancer interactions that are associated with compromised 5-fluorouracil resistance. Functional identification of promoter and enhancer elements was validated by CRISPRi and CRISPRa assays. ChIP and qPCR documented histone marks that can account for the control of DPYD gene expression were established. Consistency with data from patient-derived specimens and direct assessment of 5-fluorouracil sensitivity provides confidence in the proposed mechanisms. The model is additionally supported by genome data from a population with high "compromised allele frequency". It can be informative to directly demonstrate DPYD promoter-enhancer interactions. However, the genetic variants support the integration of regulatory activities.

Reviewer #1 (Public Review):

Summary:<br /> The authors originally investigated the function of p53 isoforms with an alternative C-terminus encoded by the Alternatively Spliced (AS) exon in place of exon 11 encoding the canonical "α" C-terminal domain. For this purpose, the authors create a mouse model with a specific deletion of the AS exon.

Strengths:<br /> Interestingly, wt or p53ΔAS/ΔAS mouse embryonic fibroblasts did not differ in cell cycle control, expression of well-known p53 target genes, proliferation under hyperoxic conditions, or the growth of tumor xenografts. However, p53-AS isoforms were shown to confer male-specific protection against lymphomagenesis in Eμ-Myc transgenic mice, prone to highly penetrant B-cell lymphomas. In fact, p53ΔAS/ΔAS Eμ-Myc mice were less protected from developing B-cell lymphomas compared to WT counterparts. The important difference that the authors find between WT and p53ΔAS/ΔAS Eμ-Myc males is a higher number of immature B cells in p53ΔAS/ΔAS vs WT mice. Higher expression of Ackr4 and lower expression of Mt2 was found in p53+/+ Eμ-Myc males compared to p53ΔAS/ΔAS counterparts, suggesting that these two transcripts are in part regulators of B-cell lymphomagenesis and enrichment for immature B cells.

Weaknesses:<br /> The manuscript is interesting but the data are not so striking and are very correlative. The authors should add functional experiments to reinforce their hypotheses and to provide, beyond potential prognostic factors, any potential mechanism at the basis of the different rates of B-cell lymphomagenesis in males vs females individuals and in WT vs p53ΔAS/ΔAS Eμ-Myc males.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript provides a detailed analysis of B-cell lymphomagenesis in mice lacking an alternative exon in the region encoding the C-terminal (regulatory) domain of the p53 protein and thus enable to assemble the so-called p53AS isoform. This isoform differs from canonical p53 by the replacement of roughly 30 c-terminal residues by about 10 residues encoded by the alternative exon. There is biochemical and biological evidence that p53AS retains strong transcriptional and somewhat enhanced suppressive activities, with mouse models expressing protein constructs similar to p53AS showing signs of increased p53 activity leading to rapid and lethal anemia. However, the precise role of the alternative p53AS variant has not been addressed so far in a mouse model aimed at demonstrating whether the lack of this particular p53 isoform (trp53ΔAS/ΔAS mice) may cause a specific pathological phenotype.

Results show that lack of AS expression does not noticeably affect p53 transcriptional activity but reveals a subtle pathogenic phenotype, with trp53ΔAS/ΔAS males, but not females, tending to develop more frequently and earlier B-cell lymphoma than WT. Next, the authors then introduced ΔAS in transgenic Eμ-Myc mice that show accelerated lymphomagenesis. They show that lack of AS caused increased lethality and larger tumor lymph nodes in p53ΔAS Eμ-Myc males compared to their p53WT Eμ-Myc male counterparts, but not in females. Comparative transcriptomics identified a small set of candidate, differentially expressed genes, including Ackr4 (atypical chemokine receptor 4), which was significantly less expressed in the spleens of ΔAS compared to WT controls. Ackr4 encodes a dummy receptor acting as an interceptor for multiple chemokines and thus may negatively regulate a chemokine/cytokine signalling axis involved in lymphomagenesis, which is down-regulated by estrogen signalling. Using in vitro cell models, the authors provide evidence that Ackr4 is a transcriptional target for p53 and that its p53-dependent activation is repressed by 17b-oestradiol. Finally, seeking evidence for a relevance for this gene in human lymphomagenesis, the authors analyse Burkitt lymphoma transcriptomic datasets and show that high ACKR4 expression correlated with better survival in males, but not in females

Strengths:<br /> A convincing demonstration of a subtle, gender-specific pathogenic phenotype associated with the lack of p53AS. The characterization of trp53ΔAS/ΔAS is well described and the data presented are convincing. This represents a significant achievement since, as mentioned, in vivo data establishing the relevance of p53AS isoform remains scarce. Based on this initial observation, the authors provide strong correlative evidence that this particular phenotype is associated by differential expression of Ackr4.

Weaknesses:<br /> The study does not demonstrate how p53AS may specifically and differentially contribute to the regulation of Ackr4, nor whether restoring Ackr4 expression may nullify the observed phenotype.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript "comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation" aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically, they look at DNA double and single-strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.

Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.

This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I find very little to critique in their experimental approaches.

Strengths:<br /> 1. Use of trans/interdisciplinary approaches ('omics, molecular biology, biochemistry, organismal biology)<br /> 2. Careful probing of TDR1 expression/enrichment<br /> 3. Identification of a completely novel protein seemingly involved in tardigrade radio-tolerance.<br /> 4. Use of multiple, diverse, tardigrade species of 'omics comparison.

Weaknesses:<br /> 1. No reverse genetics in tardigrades - all insights into TDR1 function from heterologous cell culture system.<br /> 2. Weak discussion of Dsup's role in preventing DNA damage in light of DNA damage levels measured in this manuscript.<br /> 3. Missing sequence data which is essential for making a complete review of the work.

Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read. I believe there are points still that the authors should address, but I think the editor would do well to give the authors a chance to address these points as I find this manuscript highly insightful and novel.

Reviewer #3 (Public Review):

Summary:<br /> This paper describes transcriptomes from three tardigrade species with or without treatment with ionizing radiation (IR). The authors show that IR produces numerous single-strand and double-strand breaks as expected and that these are substantially repaired within 4-8 hours. Treatment with IR induces strong upregulation of transcripts from numerous DNA repair proteins including Dsup specific to the Hypsobioidea superfamily. Transcripts from the newly described protein TDR1 with homologs in both Hypsibioidea and Macrobiotoidea supefamilies are also strongly upregulated. They show that TDR1 transcription produces newly translated TDR1 protein, which can bind DNA and co-localizes with DNA in the nucleus. At higher concentrations, TDR appears to form aggregates with DNA, which might be relevant to a possible function in DNA damage repair. When introduced into human U2OS cells treated with bleomycin, TDR1 reduces the number of double-strand breaks as detected by gamma H2A spots. This paper will be of interest to the DNA repair field and to radiobiologists.

Strengths:<br /> The paper is well-written and provides solid evidence of the upregulation of DNA repair enzymes after irradiation of tardigrades, as well as upregulation of the TRD1 protein. The reduction of gamma-H2A.X spots in U2OS cells after expression of TRD1 supports a role in DNA damage.

Weaknesses:<br /> Genetic tools are still being developed in tardigrades, so there is no mutant phenotype to support a DNA repair function for TRD1, but this may be available soon.

Reviewer #4 (Public Review):

The manuscript brings convincing results regarding genes involved in the radio-resistance of tardigrades. It is nicely written and the authors used different techniques to study these genes. There are sometimes problems with the structure of the manuscript but these could be easily solved. According to me, there are also some points which should be clarified in the result sections. The discussion section is clear but could be more detailed, although some results were actually discussed in the results section. I wish that the authors would go deeper in the comparison with other IR-resistant eucaryotes. Overall, this is a very nice study and of interest to researchers studying molecular mechanisms of ionizing radiation resistance.

I have two small suggestions regarding the content of the study itself.

1) I think the study would benefit from the analyses of a gene tree (if feasible) in order to verify if TDR1 is indeed tardigrade-specific.<br /> 2) It would be appreciated to indicate the expression level of the different genes discussed in the study, using, for example, transcript per millions (TPMs).

Reviewer #1 (Public Review):

Summary: Szathmary and colleagues explore the parabolic growth regime of replicator evolution. Parabolic growth occurs when nucleic acid strain separation is the rate-limiting step of the replication process which would have been the case for non-enzymatic replication of short oligonucleotide that could precede the emergence of ribozyme polymerases and helicases. The key result is that parabolic replication is conducive to the maintenance of genetic diversity, that is, the coexistence of numerous master sequences (the Gause principle does not apply). Another important finding is that there is no error threshold for parabolic replication except for the extreme case of zero fidelity.

Strengths:<br /> I find both the analytic and the numerical results to be quite convincing and well-described. The results of this work are potentially important because they reveal aspects of a realistic evolutionary scenario for the origin of replicators.

Weaknesses:<br /> There are no obvious technical weaknesses. It can be argued that the results represent an incremental advance because many aspects of parabolic replication have been explored previously (the relevant publications are properly cited). Obviously, the work is purely theoretical, experimental study of parabolic replication is due. In the opinion of this reviewer, though, these are understandable limitations that do not actually detract from the value of this work.

Reviewer #2 (Public Review):

Summary:

A dominant hypothesis concerning the origin of life is that, before the appearance of the first enzymes, RNA replicated non-enzymatically by templating. However, this replication was probably not very efficient, due to the propensity of single strands to bind to each other, thus inhibiting template replication. This phenomenon, known as product inhibition, has been shown to lead to parabolic growth instead of exponential growth. Previous works have shown that this situation limits competition between alternative replicators and therefore promotes RNA population diversity. The present work examines this scenario in a model of RNA replication, taking into account finite population size, mutations, and differences in GC content. The main results are (1) confirmation that parabolic growth promotes diversity, but that when the population size is small enough, sequences least efficient at replicating may nevertheless go extinct; (2) the observation that fitness is not only controlled by the replicability of sequences, but also by their GC content ; (3) the observation that parabolic growth attenuates the impact of mutations and, in particular, that the error threshold to which exponentially growing sequences are subject can be exceeded, enabling sequence identity to be maintained at higher mutation rates.

Strengths:

The analyses are sound and the observations are intriguing. Indeed, it has been noted previously that parabolic growth promotes coexistence, its role in mitigating the error threshold catastrophe - which is often presented as a major obstacle to our understanding of the origin of life - had not been examined before.

Weaknesses:

Although all the conclusions are interesting, most are not very surprising for people familiar with the literature. As the authors point out, parabolic growth is well known to promote diversity (Szathmary-Gladkih 89) and it has also been noted previously that a form of Darwinian selection can be found at small population sizes (Davis 2000). Given that under parabolic growth, no sequence is ever excluded for infinite populations, it is also not surprising to find that mutations have a less dramatic exclusionary impact.

A general weakness is the presentation of models and parameters, whose choices often appear arbitrary. Modeling choices that would deserve to be further discussed include the association of the monomers with the strands and the ensuing polymerization, which are combined into a single association/polymerization reaction (see also below), or the choice to restrict to oligomers of length L = 10. Other models, similar to the one employed here, have been proposed that do not make these assumptions, e.g. Rosenberger et al. Self-Assembly of Informational Polymers by Templated Ligation, PRX 2021. To understand how such assumptions affect the results, it would be helpful to present the model from the perspective of existing models.

The values of the (many) parameters, often very specific, also very often lack justifications. For example, why is the "predefined error factor" ε = 0.2 and not lower or higher? How would that affect the results? Similarly, in equation (11), where does the factor 0.8 come from? Why is the kinetic constant for duplex decay reaction 1.15e10−8? Are those values related to experiments, or are they chosen because specific behaviors can happen only then?

The choice of the model and parameters potentially impact the two main results, the attenuation of the error threshold and the role of GC content:

Regarding the error threshold, it is also noted (lines 379-385) that it disappears when back mutations are taken into account. This suggests that overcoming the error threshold might not be as difficult as suggested, and can be achieved in several ways, which calls into question the importance of the particular role of parabolic growth. Besides, when the concentration of replicators is low, product inhibition may be negligible, such that a "parabolic replicator" is effectively growing exponentially and an error catastrophe may occur. Do the authors think that this consideration could affect their conclusion? Can simulations be performed?

Regarding the role of the GC content, GC-rich oligomers are found to perform the worst but no rationale is provided. One may assume that it happens because GC-rich sequences are comparatively longer to release the product. However, it is also conceivable that higher GC content may help in the polymerization of the monomers as the monomers attach longer on the template (as described in Eq.(9)). This is an instance where the choice to pull into a single step the association and polymerization reactions are pulled into a single step independent of GC content may be critical. It would be important to show that the result arises from the actual physics and not from this modeling choice.

Some more specific points that would deserve to be addressed:

- Line 53: it is said that p "reflects how easily the template-reaction product complex dissociates". This statement is not correct. A reaction order p<1 reflects product inhibition, the propensity of templates to bind to each other, not slow product release. Product release can be limiting, yet a reaction order of 1 can be achieved if substrate concentrations are sufficiently high relative to oligomer concentrations (von Kiedrowski et al., 1991).

- Population size is a key parameter, and a comparison is made between small (10^3) and large (10^5) populations, but without explaining what determines the scale (small/large relative to what?).

- In the same vein, we might expect size not to be the only important parameter, but also concentration.

- Lines 543-546: if understanding correctly, the quantitative result is that the error threshold rises from 0.1 in the exponential case to 0.196 in the parabolic. Are the authors suggesting that a factor of 2 is a significant difference?

- Figure 3C: this figure shows no statistically significant effect?

- line 542: "phase transition-like species extension (Figure 4B)": such a clear threshold is not apparent.

Reviewer #1 (Public Review):

In their manuscript, Arjun et al. investigate the role of the histone acetyltransferase Gcn5 in the control of drosophila blood cell homeostasis in the larval lymph gland. They use gcn5 zygotic mutants as well as targeted knock-down and over-expression of Gcn5 in various lymph gland populations to show that these modulations impact (in a rather haphazard manner) niche cell number, blood cell progenitor maintenance, plasmatocyte differentiation, crystal cell differentiation or DNA damage accumulation. Their results suggest that Gcn5 controls autophagy and they show that decreasing the expression of the autophagy machinery increases blood cell differentiation. Using drugs to modulate the mTOR pathway, they conclude that Gcn5 levels are regulated by mTOR but that the impact of this pathway on blood cell homeostasis can override Gcn5 function.

While the authors did a lot of experiments and good quantifications of the blood cell phenotypes, many results do not make much sense or do not bring valuable information about Gcn5 mode of action. Several conclusions of the manuscripts are not backed by solid data (e.g. that Gcn5 action is mediated by TFEB and the autophagy machinery) and different aspects of the literature are not well taken into consideration. Some results (such as the validation of the knockdown and overexpression of Gcn5) seem flawed. There are some concerns about the results obtained with gcn5 zygotic mutants and an interpretation of the phenotypes observed upon manipulation of Gcn5 expression in different cell types is missing.

Important revisions are needed to improve the quality of the manuscript and confirm the authors' findings.

Reviewer #2 (Public Review):

Summary:<br /> Drosophila hematopoiesis has been shown to be governed by a number of signaling pathways such as JAK/STAT and Dpp. This important study shows the role of nutrient sensing and autophagy in determining blood cell differentiation. The authors show that General control non-derepressible 5 (Gcn5), a histone acetyltransferase affects blood cell differentiation. Gcn5 also negatively regulates autophagy through its effector TFEB which directly regulates autophagy genes. The authors also show that mTORC1 modulates Gcn5 levels and through it, TFEB activity thus acting as a fine-tuning mechanism that maintains optimal levels of autophagy.

Strengths:<br /> The main strength of the work lies in the interesting finding that cellular metabolic processes such as autophagy have a direct role in blood cell differentiation and has the potential to be of interest to those working on vertebrate haematopoiesis as well. The report has generated intriguing data, using promoters specific for sub-sections of the lymph gland, that different cellular subsets of the lymph gland contribute differently towards haematopoiesis, but this is not followed up in detail and the final conclusions are derived from a combination of whole lymph gland perturbations as well as those from specific promoters.

Weaknesses:<br /> 1. Gc5 seems to be expressed throughout the lymph gland but modulating it in the subsections does not have the same result. It is very striking that the knockdown of Gcn5 in the prohemocyte population does not have an effect on differentiation whereas overexpression does. The modulations of Gcn5 in PSC also have variable effects across hemocyte subpopulations which is not explored in the manuscript. Interestingly, also the domain deletion constructs show a differential effect on blood cell differentiation when altered solely in the prohemocytes which is not explained. While Gcn5 can be seen in all sections of the lymph gland in the first figure, under the HHLT-Gal4 and Hml-Gal4, Gcn5 looks cytoplasmic and almost completely excluded from the nucleus strikingly unlike Gcn5 expression under the Collier-Gal4 and Dome-Gal4. The rest of the experiments in the manuscript are done with multiple promoters, with autophagy flux measured by modulating Gcn5 with a pan hemocyte promoter, but the mTORC1-Gcn5 axis is explored using chemical modulators which affect the whole of the lymph gland (Fig7) or using two pro-hemocyte promoters (Fig8).

2. The knockdown of Gcn5 seems to affect the gland size (A compared to B and C). Since mTORC1 is a central regulator of cell size, it is possible that some of the effects seen in these knockdowns are potentially through mTORC1 affecting size suggesting that the signalling axis between mTORC1 and Gcn5 might not be a one-way axis as suggested in Figure 9. Also, this would mean that in experiments where absolute cell counts of crystal cells or niche cells are used to assess blood cell differentiation, further analysis to consider total cell numbers in the lymph gland would strengthen the manuscript.

3. A genetic manipulation of mTORC1 specifically in the pro hemocytes would strengthen the role of mTORC1 in the pathway rather than the chemical modulation which affects the whole of the lymph gland.

Reviewer #2 (Public Review):

Summary:<br /> This work describes the structure of Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), a lysosomal membrane protein that catalyzes the acetylation reaction of the terminal alpha-D-glucosamine group required for the degradation of heparan sulfate (HS). HS degradation takes place during the degradation of the extracellular matrix, a process required for restructuring tissue architecture, regulation of cellular function, and differentiation. During this process, HS is degraded into monosaccharides and free sulfate in lysosomes.

HGSNAT catalyzes the transfer of the acetyl group from acetyl-CoA to the terminal non-reducing amino group of alpha-D-glucosamine. The molecular mechanism by which this process occurs has not been described so far. One of the main reasons to study the mechanism of HGSNAT is that multiple mutations spanning the entire sequence of the protein, such as nonsense mutations, splice-site variants, and missense mutations lead to dysfunction that causes abnormal accumulation of HS within the lysosomes. This accumulation is a cause of mucopolysaccharidosis IIIC (MPS IIIC), an autosomal recessive neurodegenerative lysosomal storage disorder, for which there are no approved drugs or treatment strategies.

This paper provides a 3.26A structure of HGSNAT, determined by single-particle cryo-EM. The structure reveals that HGSNAT is a dimer in detergent micelles and a density assigned to acetyl-CoA. The authors speculate about the molecular mechanism of the acetylation reaction, map the mutations known to cause MPS IIIC on the structure and speculate about the nature of the HGSNAT disfunction caused by such mutations.

Strengths:<br /> The description of the architecture of HGSNAT is the highlight of the paper since this corresponds to the first description of the structure of a member of the transmembrane acyl transferase (TmAT) superfamily. The high resolution of an HGSNAT bound to acetyl-CoA is an important leap in our understanding of the HGSNAT mechanism. The density map is of high quality, except for the luminal domain. The location of the acetyl-CoA allows speculation about the mechanistic role of multiple residues surrounding this molecule. The authors thoroughly describe the architecture of HGSNAT and map the mutations leading to MPS IIIC. The description of the dimeric interphase is a novel result, and future studies are left to confirm the importance of oligomerization for function.

Weaknesses:<br /> Apart from the cryo-EM structure, the article does not provide any other experimental evidence to support or explain a molecular mechanism. Due to the complete absence of functional assays, mutagenesis analysis, or other structures such as a ternary complex or an acetylated enzyme intermediate, the mechanistic model depicted in Figure 5 should be taken with caution.

The authors discuss that H269 is an essential residue that participates in the acetylation reaction, possibly becoming acetylated during the process. However, there is no solid experimental evidence, e.g. mutagenesis analysis or structural analysis, in this or previous articles, that demonstrates this to be the case.

In the discussion part, the authors mention previous studies in which it was postulated that the catalytic reaction can be described by a random order mechanistic model or a Ping Pong Bi Bi model. However, the authors leave open the question of which of these mechanisms best describes the acetylation reaction. The structure presented here does not provide evidence that could support one mechanism or the other.

Although the authors map the mutations leading to MPS IIIC on the structure and use FoldX software to predict the impact of these mutations on folding and fold stability, there is no experimental evidence to support FoldX's predictions.

Reviewer #1 (Public Review):

This article by Navratna et al. reports the first structure of human HGSNAT in an acetyl-CoA-bound state. Through careful structural analysis, the authors propose potential reasons why certain human mutations lead to lysosomal storage disorders and outline a catalytic mechanism. The structural data are of good quality, and the manuscript is clearly written. This study represents an important step toward understanding the mechanism of HGSNAT and is valuable to the field. I have the following suggestions:

1. The authors should characterize whether the purified protein is active. Otherwise, how does one know if the detergent used maintains the protein in a biologically relevant state? The authors should at least attempt to do so. If these prove to be challenging, at the very least, the authors should try a cell-based assay to demonstrate that the GFP tag does not interfere with the function.

2. In Figure 5, the authors present a detailed schematic of the catalytic cycle, which I find to be too speculative. There is no evidence to suggest that this enzyme undergoes isomerization, similar to a transporter, between open-to-lumen and open-to-cytosol states. Could it not simply involve some movements of side chains to complete the acetyl transfer?

Reviewer #3 (Public Review):

Summary:<br /> Navratna et al. have solved the first structure of a transmembrane N-acetyltransferase (TNAT), resolving the architecture of human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in the acetyl-CoA bound state using single particle cryo-electron microscopy (cryoEM). They show that the protein is a dimer, and define the architecture of the alpha- and beta- GSNAT fragments, as well as convincingly characterizing the binding site of acetyl-CoA.

Strengths:<br /> This is the first structure of any member of the transmembrane acyl transferase superfamily, and as such it provides important insights into the architecture and acetyl-CoA binding site of this class of enzymes.

The structural data is of a high quality, with an isotropic cryoEM density map at 3.3Å facilitating the building of a high-confidence atomic model. Importantly, the density of the acetyl-CoA ligand is particularly well-defined, as are the contacting residues within the transmembrane domain.

The open-to-lumen structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional characterization of the reaction cycle of this class of enzymes.

Weaknesses:<br /> While the structural data for the open-to-lumen state presented in this work is very convincing, and clearly defines the binding site of acetyl-CoA, to get a complete picture of the enzymatic mechanism of this family, additional structures of other states will be required.

A potentially significant weakness of the study is the lack of functional validation. The enzymatic activity of the enzyme characterized was not measured, and the enzyme lacks native proteolytic processing, so it is a little unclear whether the structure represents an active enzyme.

Reviewer #1 (Public Review):

Summary:

This study describes all tangential neurons of the lobula plate (LOPs) of the fruit fly Drosophila melanogaster. Importantly, this is done in a complete manner, for the first time in any species. This means that for the first time, all neurons involved in transmitting wide-field optic flow information to the central brain are known. Exploiting known structure-function relations in these neurons (which are based on solid physiological data in different species of flies), the authors provide estimates of the physiological properties of all described neurons. Combined with transmitter predictions of these cells, this yields a full account of what information about wide-field motion is available to the central fly brain in order to derive behavioral commands from. The study goes one step further and includes anatomical descriptions and physiological property predictions for all major downstream target cells of LOPs.

Main strengths:

The paper is exceptional in three ways. First, it is the first comprehensive account of all tangential neurons of the lobula plate of an insect. This now provides the ground truth for similar studies in other insects. In particular, these results will allow neurons emerging in other species to be confidently described as novel/different from Drosophila, if they were not found in the current study. This is a major change from previously, when confidence in the non-existence of neuronal cell types in this system was impossible, as that system was not fully described.

Second, the rigorous prediction of physiological characteristics (flow-field encoding) in all anatomically described neurons provides a solid basis for system-wide modeling of optic flow encoding in Drosophila. Importantly, the presented physiological predictions include the downstream partner cells of the LOPs in the central brain, neurons for which only very few physiological descriptions exist, but which are essential for transforming optic flow input into behavioral outputs. This paper therefore opens a path towards closing the gap between sensory processing and behavior not only for a few identified and well-studied pathways, but for all wide-field motion processing that exists in a species.

Third, the connectomics work is not only based on one individual sample, but incorporates two EM volumes, analyzed with two different methods (manual tracing and auto segmentation/proofreading), using interhemispheric correspondence and inter-individual correspondence to validate the obtained neuron catalogue. Additionally, light microscopical data was used to validate the EM data. All of this provides exceptional levels of confidence in the presented results.

Main weaknesses:

While the authors compare their results with data from both larger flies and other work in Drosophila, a recent paper (Henning et al 2022) that presented novel data on the distribution of preferred motion directions in the fly lobula plate is not mentioned. This is unfortunate, as the claim of that paper is that the lobula plate contains six instead of four main tuning directions, both at the level of LOPs and T4/T5 input cells - a claim that could likely be directly confirmed or dismissed, or at least incorporated in the data presented in the current study. How would the flow-field predictions change if the data from Henning et al on T4 neurons was used as an input for the modeling rather than the classic four tuning directions?

While the authors nicely perform comparison to other fly species, a more general discussion of how the found cells relate to other insects, e.g. cells known from bees (e.g. Honkanen et al., 2023) or older work from locusts, could give the data more general relevance. While the comparison can likely not be done on a cell type level, given that the structure of the lobula complex is very different between those insects, the types of projections found and their physiologies, i.e. the overall patterns of how wide field motion is sent to the central brain, might be comparable and informative for highlighting general principles of motion processing.

Reviewer #2 (Public Review):

Summary:

In this study, Zhao, Nern, et al. investigate a population of neurons in the optic lobe of Drosophila melanogaster that process optic flow, relative motion between the insect's eyes and its environment that is generated during flight and provides useful information to the fly about its own self-motion. Although a sample of these Lobula Plate Tangential (LPT) neurons has been studied across Diptera in prior work, the full population has not been exhaustively and thoroughly cataloged in a single species, limiting our understanding of how LPT tuning properties across the population convey features of optic flow fields relevant to downstream motor regions.

Through extensive manual reconstructions in a fly electron microscopy volume, the authors of this study identify 58 LPT neurons in the fruit fly encompassing previously studied Horizontal and Vertical cells and novel cells that have not been previously characterized. Using the detailed anatomy of each cell and knowledge of upstream T4/T5 selectivity, the authors derive the predicted motion pattern map (PMPM) of each neuron. To understand how optic flow field tunings of individual LPTs align with global optic flow patterns flies are expected to encounter during flight such as translation and rotation, the authors compute the average angular difference between each PMPM and idealized rotation and translation optic flow fields. The authors also map individual LPTs to their counterparts in a second fly brain to explore LPT-LPT connectivity and downstream connectivity to central brain neuropils. They find that distinct groupings of LPTs have diverse downstream connectivity patterns and that downstream neurons align more closely to global optic flow fields that are expected during flight. This study is a valuable resource to researchers studying motion vision in the insect brain and is of interest to researchers studying sensorimotor processing by providing hypotheses for how optic flow information is integrated downstream to guide fly behavior.

Strengths:

A key strength of this study is the thoroughness with which the authors comprehensively identify individual LPT neurons in the FAFB volume. They not only conduct an impressive number of careful manual reconstructions to recover individual LPTs, but they also attempt, and often succeed, to map each individual neuron to its counterpart in light microscopy, studies across Diptera, and available auto-segmented connectome datasets such as FlyWire, FAFB-FFN1, and Hemibrain. The authors are similarly thorough when surveying individual LPT properties such as neurotransmitter identities, in some cases using multiple datasets to reconcile ambiguous neurotransmitter predictions. The care with which the complete LPT population has been identified establishes this study as a useful resource for future studies of insect motion.

In addition to providing a comprehensive catalog of individual LPTs, the authors also contextualize their findings within broader sensorimotor circuitry by considering connectivity between LPTs and from LPTs to downstream regions. Exploration of structure in downstream connectivity suggests that optic flow information is directed to various central brain neuropils through specific groups of LPTs. With some additional analyses, these results broaden the scope of this study by providing useful hypotheses for sensorimotor circuit organization.

Weaknesses:

A novel method introduced in this study is the derivation of individual LPT-predicted motion pattern maps (PMPMs) using T4 preferred directions and LPT morphology. Although this method underlies core findings in this study, such as alignment to global optic flow fields and properties of downstream integration, aspects of the methods used to derive PMPMs are not explained sufficiently well, particularly in the main text. For example, in the Methods, the authors briefly describe the process of computing a weighted sum of T4 preferred directions to obtain the PMPM for each LPT, but a detailed understanding of these preferred directions combined is missing in Figure 2 or the associated descriptions in the main text. It is also not clear how PMPMs are derived in cases where LOP layer coverages are overlapping (for example VS 13-1 in Figure 3) to yield smooth PMPMs. In addition, it is not clear how the PMPMs of bilateral LPTs such as LPT-45 and LPT-50 in Figure 4 were integrated to compute downstream target composite PMPMs. Finally, all the PMPMs were derived from the T4 preferred direction that relies on the ommatidial viewing directions ("Eyemap") introduced in Zhao et al. 2022. It is also important for the current study to give an indication of how sensitive their results are to possible inaccuracies in this map and derived T4/T5 direction selectivities.

Although the authors explore some features of connectivity from LPT to downstream partners (Figure 6), there is a lack of reconciliation of these findings with individual LPT properties explored earlier in the study, such as those presented in Figures 2-4. In that sense, there is a disconnect between the two parts of the manuscript (and a missed opportunity). For instance, an important follow-up analysis would be to use knowledge about LPT-LPT connectivity to better predict effective PMPMs of LPTs taking into account network effects. This extension would lead to a better understanding of how LPT-LPT interactions shape optic flow responses in the LOP. In addition, in Figure 6 Supplement 2 (which I recommend to move to the main figures), the authors show that LPTs can be grouped together based on similarity of output connectivity (Panel B-D) and that this structure corresponds to output synapses located in different groups of central brain neuropils. However, they do not attempt to explicitly link these groupings with individual LPT PMPMs, alignment to global optic flow patterns, LPT layer enervation, cell morphologies, and input connectivity patterns. Such an analysis would be an important step to bring the manuscript together and to get a better understanding of the organization of the whole system.

Reviewer #3 (Public Review):

Summary:

The fruit fly visual system has provided a powerful context in which to investigate fundamental questions in neural development, phototransduction, and systems neuroscience. Of recent interest is motion processing, particularly how visual motion cues are estimated locally, and then pooled to derive behaviorally meaningful signals. Many of these pooling operations have been shown to take place in the wide-field neurons in the lobula plate, cell types that have been explored using electrophysiological recordings for more than 50 years in a variety of Diptera. However, our understanding of the diversity and connectivity of these cells remains incompletely understood, and is of interest to many.

In this context, Reiser and colleagues describe the anatomy and connectivity of the complete set of Lobula Plate Tangential neurons in Drosophila, using a careful and systematic reconstruction of the FAFB dataset. Leveraging a previous study of retinal geometry, combined with their characterization of the anatomical inputs to the elementary motion detectors, T4 and T5, they then predict the motion sensitivities of each cell, their neurotransmitter identities, and map the connections of many of these cells into the central brain and contralateral optic lobe.

Strengths:

The quality of the connectomic analysis is exceptional, and the quantitative analysis that links connectivity to function is rigorous and impressive. This paper will be an important resource for the community.

Weaknesses:

Some of the findings could be better linked to previously published work in this field, and there may be a minor limitation to the predicted optimal motion axes, given one of the simplifying assumptions made.

Reviewer #1 (Public Review):

This study examined whether mitochondrial acyl-CoA thioesterase-2 (ACOT2) regulates mitochondrial matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up. When energy demand and pyruvate availability were elevated, a lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2. In mice fed a high-fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. The authors suggest that ACOT2 supports CoASH availability to facilitate β-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Based on the data provided in this study, the authors propose that ACOT2 regulates mitochondrial matrix acyl-CoA levels in white skeletal muscle to facilitate fatty acid oxidation β-oxidation. However, I do not believe the data supports this concept, since ACOT2 deletion actually increased fatty acid oxidation in the mitochondrial JO2 studies. In addition, there are some problems with the experimental data that the authors need to address. This includes the experimental conditions used to assess JO2 in the mitochondria, and not using Cre control mice.

Reviewer #2 (Public Review):

Summary:

The manuscript from Bekeova et al. entitled "Acyl-CoA thioesterase-2 facilitates P-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner" examines whether loss of acyl-CoA thioesterase-2 (ACOT2) in the mitochondrial matrix of skeletal muscle alters mitochondrial fatty acid metabolism. The authors generate data demonstrating that under normal chow conditions, loss of ACOT2 increases mitochondrial respiration of long-chain fatty acid, but also increases susceptibility to the build-up of metabolic intermediates. However, during short-term high-fat feeding (7 days), mice with knockout of skeletal muscle ACOT2 had better glucose and insulin tolerance. Interestingly, skeletal muscle ACOT2 knockout mice on chow and high-fat diet utilized more glucose during the active (dark cycle) portion of the day. These data suggest that ACOT2 may be a potential therapeutic target to improve glucose homeostasis.

Strengths:

The use of creatine kinase cre recombinase to specifically target striated muscle localizes the genetic manipulation, thus increasing the rigor of these experiments by limiting potential off-site changes in ACOT2 expression. Also, the assessment of mitochondrial respiration and response to changes in energy change via the creatine kinase clamp technique is a strength. These data provide a measurement of isolated mitochondrial respiration at physiologically relevant concentrations of ATP and ADP, while also allowing for assessment of how these mitochondria respond to changes in free energy (Fisher-Wellman et al. 2018). The indirect calorimetry data provides systemic physiological context to the striated muscle-specific genetic manipulation, while also allowing for the examination of how this change in skeletal muscle ACOT2 impacts systemic responses to different energy challenges. Finally, the extensive metabolomics, transcriptomics, and lipidomics analysis, not only provides a wealth of data but is used to further the authors' investigation of skeletal muscle ACOT2 activity in mitochondrial fatty acid oxidation and glucose homeostasis.

Weaknesses:

Several general confounding factors exist in the experimental design that could potentially impact the interpretation of the observed outcomes. First, all mice were housed at housing temperatures (22C) below the thermoneutral zone, which has been well described by many investigators to result in dramatically increased energy expenditure. Changes in total and resting energy expenditure could alter the skeletal muscle and systemic utilization of lipids, response to high-fat diet, and glucose homeostasis. Second, no dietary control was observed in these experiments. While this did not impact outcomes when the diets were not compared, once the authors began to compare normal chow to high-fat diet, numerous differences in the composition of these diets could impact the outcomes. Third, the extended food withdrawal before the glucose- and insulin tolerance tests puts the mouse in a state of extreme energy stress more akin to starvation than fasting, which can negatively impact outcomes (Ayala et al. 2010, Virtue & Vidal-Puig 2021). Fourth, the use of the Seahorse platform for the assessment of respiration of isolated mitochondria is highly debatable (Schmidt et al. 2021), particularly when the investigators also used high-resolution respirometry specifically designed for the purpose of measuring isolated mitochondrial oxygen consumption. Importantly, the use of the Seahorse platform to assess cellular respiration in this investigation is quite appropriate. Finally, while the authors present data demonstrating that ACOT2 expression is highest in Type I fibers compared to the various Type II fiber types, a large number of the experiments are performed in a muscle that is primarily composed of Type II fibers. The authors briefly acknowledge this limitation. But, is important for the reader to keep this in mind when trying to consider how these findings would translate to humans.

Impact:

The authors have generated data that implicates skeletal muscle mitochondrial coenzyme A handling as a therapeutic target in the improvement of glucose homeostasis. While the exact role of increased tissue lipid burden on insulin action, glucose uptake, and substrate metabolism is still debated, the association between increased tissue lipid and impaired tissue- and systemic glucose handling is very strong. The data herein suggest that ACOT2 represents a pharmaceutical target to improve systemic glucose homeostasis in the population with obesity.

Reviewer #3 (Public Review):

Cells can oxidize diverse substrates in the mitochondria to sustain cellular energy metabolism. However, all of these substrates require covalent thioester linkage to coenzyme A (CoA). Thus, multiple energy metabolism substrates could potentially compete for a limited pool of mitochondrial CoA. Cells encode a set of mitochondrial acyl-CoA thioesterases (ACOTs) that free CoA up by removing attached substrates. The authors hypothesized that ACOT2, a mitochondrial ACOT with a preference for long-chain acyl-CoA substrates that arise during the oxidation of lipids as a fuel source, could regulate the balance of substrates used in the mitochondria by reducing the oxidation of lipids by removing them from CoA and freeing the mitochondrial pool of CoA for use by other substrates.

To test this hypothesis, the authors generated mice with loss of ACOT2 in the skeletal muscle, where this is most expressed, and assayed the CoA composition of muscle and their glucose/fatty acid catabolism in mice that were challenged with different diets, fasting or exercise to expose the muscle to different substrates conditions. These experiments were complemented with biochemical analysis of mitochondria isolated from the muscle of control and ACOT2 animals exposed to a variety of substrates and challenged with different simulated energy demands.

On the basis of these convincing experiments, the authors argue that loss of ACOT2 both in vivo and in vitro interestingly increases glucose oxidation, while not increasing oxidation of lipids. This is particularly surprising as the CoA competition model would predict that ACOT2 loss would increase lipid oxidation while hindering glucose oxidation. The authors argue that ACOT2 facilitates lipid oxidation due to ACOT2 reversal of lipid ligation to CoA preventing feedback inhibition of the lipid oxidation pathway that occurs when lipid supply outstrips the ability of the lipid oxidation pathway to metabolize the lipids. These findings will be valuable for the field of metabolism providing insight into how ACOTs regulate substrate catabolism in cells and tissues.

Reviewer #1 (Public Review):

The authors focused on genetic variability in relation to insulin resistance. The used genetically different lines of mice and exposed them to the same diet. They found that genetic predisposition impacts the overall outcome of metabolic disturbances.

Reviewer #2 (Public Review):

Summary:<br /> In the present study, van Gerwen et al. perform deep phosphoproteomics on muscle from saline or insulin-injected mice from 5 distinct strains fed a chow or HF/HS diet. The authors follow these data by defining a variety of intriguing genetic, dietary or gene-by-diet phosphor-sites which respond to insulin accomplished through application of correlation analyses, linear mixed models and a module-based approach (WGCNA). These findings are supported by validation experiments by intersecting results with a previous profile of insulin-responsive sites (Humphrey et al, 2013) and importantly, mechanistic validation of Pfkfb3 where overexpression in L6 myotubes was sufficient to alter fatty acid-induced impairments in insulin-stimulated glucose uptake. To my knowledge, this resource provides the most comprehensive quantification of muscle phospho-proteins which occur as a result of diet in strains of mice where genetic and dietary effects can be quantifiably attributed in an accurate manner. Utilization of this resource is strongly supported by the analyses provided highlighting the complexity of insulin signaling in muscle, exemplified by contrasts to the "classically-used" C57BL6/J strain. As it stands, I view this exceptional resource as comprehensive with compelling strength of evidence behind the mechanism explored. I raised several comments in the last round of assessment but all of them have now been thoughtfully addressed.

Strengths: Generation of a novel resource to explore genetic and dietary interactions influencing the phospho-proteome in muscle. This is accompanied by elegant application of in silico tools to highlight the utility

Weaknesses: none noted

Reviewer #3 (Public Review):

Summary:<br /> The authors aimed to investigate how genetic and environmental factors influence the muscle insulin signaling network and its impact on metabolism. They utilized mass spectrometry-based phosphoproteomics to quantify phosphosites in skeletal muscle of genetically distinct mouse strains in different dietary environments, with and without insulin stimulation. The results showed that genetic background and diet both affected insulin signaling, with almost half of the insulin-regulated phosphoproteome being modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affecting insulin signaling in a strain-dependent manner.

Strengths:<br /> Study uses state-of-the-art phosphoproteomics workflow allowing quantification of a large number of phosphosites in skeletal muscle, providing a comprehensive view of the muscle insulin signaling network. The study examined five genetically distinct mouse strains in two dietary environments, allowing for the investigation of the impact of genetic and environmental factors on insulin signaling. The identification of coregulated subnetworks within the insulin signaling pathway expanded our understanding of its organization and provided insights into potential regulatory mechanisms. The study associated diverse signaling responses with insulin-stimulated glucose uptake, uncovering regulators of muscle insulin responsiveness.

Weaknesses:<br /> The limitations acknowledged by the authors, such as the need for larger cohorts and the inclusion of female mice. Moreover as acknowledged by authors, they are unable to dissect to what extent the obesity and different life span cycle for different strain affects insulin signaling. This suggest that further research is needed to validate and expand upon the findings.

Reviewer #2 (Public Review):

The authors use a high-throughput sequencing-based enrichment assay to measure how individual amino acids substitutions in the Rep proteins of AAV change the production of AAV. The key experiment involved the creation of all possible single codon mutations of the AAV2 rep gene in a barcoded format, transfection of the library into HEK293T cells for production of AAV, and sequencing to see which rep variants were enriched in the viral particles produced from the library. As the library rep variants were flanked by inverted terminal repeats for packaging into viral particles, the authors could use high-throughput sequencing of the barcodes to determine how much each rep variant supported the production of AAV. The rep gene libraries were cleverly made through a cloning process that ensured each mutant was attached to an exactly known 20nt barcode included in each mutagenic oligo (and subsequently moved to the end of the library genes by another cloning step). This allowed the authors to confidently observe nearly all rep variants in their experiments, resulting in a comprehensive map between Rep protein variants and AAV production. The overall map should act as a useful guide for AAV engineering. Not only did certain variants improve AAV production by ~2-fold and show generality across AAV capsid serotypes, the map might be used to predict greater effects through combinations of mutations, especially if augmented by natural evolutionary datasets and statistical learning.

In interpreting the results of this study, the reader should bear in mind that what has been measured and validated in high throughput is the production of intact genome-containing AAVs. The authors also successfully show transduction for selected high production variants. This is important as the efficiency by which an AAV preparations transduce cells is most relevant property for gene therapy.

Overall, this is a well-executed and well-analyzed study. The results support the conclusions and claims of the work. I see this work as a useful resource for engineering recombinant AAVs to increase their production, which should have broad impact as the use of AAVs in gene therapy grows.

Reviewer #3 (Public Review):

The study by Jain et al. on recombinant adeno-associated viruses (rAAVs) represents a valuable contribution to the fields of virus genetics and gene therapy. As non-pathogenic vectors, rAAVs have become a popular choice for delivering gene therapies. The authors have previously investigated the effects of all possible single codon substitutions, deletions, and insertions in the AAV2 cap gene on AAV production. In this study, they extend their analysis to the AAV2 rep gene and rep genes in two additional capsid serotypes, establishing a genotype-phenotype landscape that enhances our understanding of Rep protein function and offers potential strategies for improving Rep function in gene therapy applications. The experimental design is rigorous, the analyses well-executed, and the interpretations of the data are convincing. While I have a few suggestions to further refine the study, I believe it is overall an excellent piece of research.

One aspect that may warrant further consideration is the assumption, as mentioned in Figure 2's legend, that synonymous mutations are neutral and can serve as controls for normalizing the production rate. However, Figures S5-6 and Figures S11-12 suggest that synonymous mutations are not necessarily neutral, as their distribution is similar to that of nonsynonymous mutations. Thus, it may be beneficial to more thoroughly examine the potential effects of synonymous mutations on the genotype-phenotype landscape.

Additionally, previous research by Jeff Collar and others has reported that synonymous mutations can affect mRNA levels through mRNA degradation rate. It would be interesting to determine if the 20-bp barcodes located at the 3' end are positioned within the untranslated regions and could thus be employed to quantify the mRNA levels of individual variants. This information could offer insight into another potential mechanism by which single codon mutations impact the production rate of rAAV.

The authors discovered several novel mutations that enhance AAV production yet are absent in natural occurrences. This intriguing finding could benefit from further elaboration, particularly with regard to the distribution of these mutations within the protein structure and the nature of the amino acid transitions involved. It would also be informative if the authors could provide a brief discussion as to why these mutations have not been observed in nature. For instance, could it be that optimal viral fitness necessitates an intermediate production rate rather than an excessively rapid one? Expanding on these points may further enrich the paper and offer valuable insights for readers.

The authors have taken commendable steps to address the concerns I raised in my previous evaluation. They have provided comprehensive clarifications, performed necessary revisions, and expanded upon certain key points in the manuscript.

Reviewer #2 (Public Review):

Summary:

In this manuscript Nie et al investigate the effect of PARG KO and PARG inhibition (PARGi) on pADPR, DNA damage, cell viability and synthetic lethal interactions in HEK293A and Hela cells. Surprisingly, the authors report that PARG KO cells are sensitive to PARGi and show higher pADPR levels than PARG KO cells, which is abrogated upon deletion or inhibition of PARP1/PARP2. The authors explain the sensitivity of PARG KO to PARGi through incomplete PARG depletion and demonstrate complete loss of PARG activity when incomplete PARG KO cells are transfected with additional gRNAs in the presence of PARPi. Furthermore, the authors show that the sensitivity of PARG KO cells to PARGi is not caused by NAD depletion but by S-phase accumulation of pADPR on chromatin coming from unligated Okazaki fragments, which are recognized and bound by PARP1. Consistently, PARG KO or PARG inhibition show synthetic lethality with Pol beta, which is required for Okazaki fragment maturation. PARG expression levels in ovarian cancer cell lines correlate negatively with their sensitivity to PARGi.

Strengths:

The authors show that PARG is essential for removing ADP-ribosylation in S-phase.

Weaknesses:

1) This begs the question as to the relevant substrates of PARG in S-phase, which could be addressed, for example, by analysing PARylated proteins associated with replication forks in PARG-depleted cells (EdU pulldown and Af1521 enrichment followed by mass spectrometry).<br /> 2) The results showing the generation of a full PARG KO should be moved to the beginning of the Results section, right after the first Results chapter (PARG depletion leads to drastic sensitivity to PARGi), otherwise the reader is left to wonder how PARG KO cells can be sensitive to PARGi when there should be presumably no PARG present.<br /> 3) Please indicate in the first figure which isoforms were targeted with gRNAs, given that there are 5 PARG isoforms. You should also highlight that the PARG antibody only recognizes the largest isoform, which is clearly absent in your PARG KO, but other isoforms may still be produced, depending on where the cleavage sites were located.<br /> 4) FACS data need to be quantified. Scatter plots can be moved to Supplementary while quantification histograms with statistical analysis should be placed in the main figures.<br /> 5) All colony formation assays should be quantified and sensitivity plots should be shown next to example plates.<br /> 6) Please indicate how many times each experiment was performed independently and include statistical analysis.

Reviewer #3 (Public Review):

In the revised version the authors have addressed some of the reviewers' concerns, but, despite the new explanatory paragraph on page 16, the paper remains confusing because as shown in Figure 7 at the end of the Results the PARG KO 293A cells that were analyzed at the beginning of the Results are not true PARG knockouts. The authors stated that they did not rewrite the Results because they wanted to describe the experiments in the order in which they were carried out, but there is no imperative for the experiments to be described in the order in which they were done, and it would be much easier for the uninitiated reader to appreciate the significance of these studies if the true PARG KO cell data were presented at the beginning, as all three of the original reviewers proposed.

While the authors have to some extent clarified the nature of the PARG KO alleles, they have not been able to identify the source of the residual PARG activity in the PARG KO cells, in part because different commercial PARG antibodies give different and conflicting immunoblotting results. Additional sequence characterization of PARG mRNAs expressed in the PARG cKO cells, and also in-depth proteomic analysis of the different PARG bands could provide further insight into the origins and molecular identities of the various PARG proteins expressed from the different KO PARG alleles, and determine which of them might retain catalytic activity.

The authors have made no progress in identifying which are the key PARG substrates required for S phase progression, although they suggest that PARP1 itself may be an important target.

Reviewer #1 (Public Review):

Hu et al. performed sc-RNA-seq analyses of kidney cells with or without virus infection, vaccines, and vaccines+virus infections from pooled adult zebrafish. They compared within these experimental groups as well as kidney vs spleen. Their analyses identified expected populations but also revealed new hematopoietic stem/progenitor cell (HSPC), even in the spleen. Their analyses show that HSPCs in the kidney can respond to virus infection differentially and can be trained to recognize the same infection and argue that zebrafish kidney can serve as a secondary immune organ. The findings are important and interesting. The manuscript is well written and a pleasure to read. However, there are several issues with their figure presentation and figure qualities, as well as the lack of clarity in some of figure legends. Some of the data presentation can be improved for better clarity. It is also important to outline what is conserved and what is unique for fish.

Major concerns:

1. The visualization for several figure panels is very poor. Please provide high resolution images and larger font sizes for gene list or Y and X axis labels. This includes Figure 1B, Figure 1-figure supplement 2, Figure 2B-2C, 3A-3D, 4F, 5B, 6G, Figure 6-figure supplement 1B, Figure 6-figure supplement 2. Figure 7B, 8C-8E, Figure 8-figure supplement 1., 10F, 10G-10J, Figure 10-figure supplement 1.<br /> 2. What are the figures at the end of the manuscript without any figure legends?<br /> 3. It would be better to use a Table to organize the gene signatures that define each unique population of immune cells such as T, B, NK, etc.<br /> 4. What are the similarities for HSPC and immune cell populations between fish and man based on this research? It is better to form a table to compare and discuss.<br /> 5. It is highly likely that sex and age could be the biological variation for how HSPC responds to virus infections and vaccination. The author should clearly state the fish sex and age from their samples and discuss their results taking into consideration of these variations.<br /> 6. The authors claim that the spleen and kidney share HSPCs. However, their data did not demonstrate this result clearly in Figure 4A. Perhaps they should use different color to make the overlay becoming more obvious? Or include a table to show which HSPCs are shared between the kidney and spleen? Are they sure if these are just HSPCs seeding the spleen to differentiate into B cells or other immune cells?

Reviewer #2 (Public Review):

In this manuscript, the authors have meticulously constructed a comprehensive atlas delineating hematopoietic stem/progenitor cell (HSPC) and immune-cell types within the zebrafish kidney, employing single-cell transcriptome profiling analysis. Notably, these cell populations exhibited distinctive responses to viral infection. Intriguingly, the investigation revealed that HSPCs manifest positive reactivities to viral infection, indicating the effective induction of trained immunity in select HSPCs. Furthermore, the study unveiled the capacity for the generation of antigen-stimulated adaptive immunity within the kidney, suggesting a role for the zebrafish kidney as a secondary lymphoid organ. This research elucidates the distinctive features of the fish immune system and underscores the multifaceted biology of the kidney in ancient vertebrates.

Reviewer #1 (Public Review):

Summary:<br /> This useful work provides insight into agonist binding to muscle nicotinic receptors. The authors want to understand the fundamental steps in ligand binding to muscle nicotinic receptors using computational methods. The study builds on a large basis of empirical studies of the various states involved in receptor activation. However, the evidence supporting the conclusions is incomplete, because little support is available for the starting structures that are derived from ligand docking. This work is a useful starting point for more detailed work on ligand binding to this important class of receptors.

Strengths:<br /> The strengths include the number of ligands tried, and the relation to the mature analysis of the receptor function.

Weaknesses:<br /> The weaknesses are the brevity of the simulations, the concomitant lack of scope of the simulations, the lack of depth in the analysis, and the incomplete relation to other relevant work.

Reviewer #2 (Public Review):

Summary:<br /> The aim of this manuscript is to use molecular dynamics (MD) simulations to describe the conformational changes of the neurotransmitter binding site of a nicotinic receptor. The study uses a simplified model including the alpha-delta subunit interface of the extracellular domain of the channel and describes the binding of four agonists to observe conformational changes during the weak-to-strong affinity transition.

Strength:<br /> The 200 ns-long simulations of this model suggest that the agonist rotates about its centre in a 'flip' motion, while loop C 'flops' to restructure the site. The changes appear to be reproduced across simulations and different ligands and are thus a strong point of the study.

Weaknesses:<br /> After carrying out all-atom molecular dynamics, the authors revert to a model of binding using continuum Poisson-Boltzmann, surface area, and vibrational entropy. The motivations for and limitations associated with this approximate model for the thermodynamics of binding, rather than using modern atomistic MD free energy methods (that would fully incorporate configurational sampling of the protein, ligand, and solvent) could be provided. Despite this, the authors report a correlation between their free energy estimates and those inferred from the experiment. This did, however, reveal shortcomings for two of the agonists. The authors mention their trouble getting correlation to experiment for Ebt and Ebx and refer to up to 130% errors in free energy. But this is far worse than a simple proportional error, because -24 Vs -10 kcal/mol is a massive overestimation of free energy, as would be evident if the authors were to instead express results in terms of KD values (which would have an error exceeding a billion fold). The MD analysis could be improved with better measures of convergence, as well as a more careful discussion of free energy maps as a function of identified principal components, as described below. Overall, however, the study has provided useful observations and interpretations of agonist binding that will help understand pentameric ligand-gated ion channel activation.

Main points:<br /> Regarding the choice of model, some further justification of the reduced 2 subunit ECD-only model could be given. On page 5 the authors argue that, because binding free energies are independent of energy changes outside the binding pocket, they could remove the TMD and study only an ECD subunit dimer. While the assumption of distant interactions being small seems somewhat reasonable, provided conformational changes are limited and localised, how do we know the packing of TMD onto the ECD does not alter the ability of the alpha-delta interface to rearrange during weak or strong binding? They further write that "fluctuations observed at the base of the ECD were anticipated because the TMD that offers stability here was absent.". As the TMD-ECD interface is the "gating interface" that is reshaped by agonist binding, surely the TMD-ECD interface structure must affect binding. It seems a little dangerous to completely separate the agonist binding and gating infrastructure, based on some assumption of independence. Given the model was only the alpha and delta subunits and not the pentamer with TMD, I am surprised such a model was stable without some heavy restraints. The authors state that "as a further control we carried out MD simulation of a pentamer docked with ACh and found similar structural changes at the binding pocket compared to the dimer." Is this sufficient proof of the accuracy of the simplified model? How similar was the model itself with and without agonist in terms of overall RMSD and RMSD for the subunit interface and the agonist binding site, as well as the free energy of binding to each model to compare?

Although the authors repeatedly state that they have good convergence with their MD, I believe the analysis could be improved to convince us. On page 8 the authors write that the RMSD of the system converged in under 200 ns of MD. However, I note that the graph is of the entire ECD dimer, not a measure for the local binding site region. An additional RMSD of local binding site would be much more telling. You could have a structural isomerisation in the site and not even notice it in the existing graph. On page 9 the authors write that the RMSF in Figure S2 showed instability mainly in loops C and F around the pocket. Given this flexibility at the alpha-delta interface, this is why collecting those regions into one group for the calculation of RMSD convergence analysis would have been useful. They then state "the final MD configuration (with CCh) was well-aligned with the CCh-bound cryo-EM desensitized structure (7QL6)... further demonstrating that the simulation had converged." That may suggest a change occurred that is in common with the global minimum seen in cryo EM, which is good, but does not prove the MD has "converged". I would also rename Figure S3 accordingly.

The authors draw conclusions about the dominant states and pathways from their PCA component free energy projections that need clarification. It is important first to show data to demonstrate that the two PCA components chosen were dominant and accounted for most of the variance. Then when mapping free energy as a function of those two PCA components, to prove that those maps have sufficient convergence to be able to interpret them. Moreover, if the free energies themselves cannot be used to measure state stability (as seems to be the case), that the limitations are carefully explained. First, was PCA done on all MD trajectories combined to find a common PC1 & PC2, or were they done separately on each simulation? If so, how similar are they? The authors write "the first two principal components (PC-1 and PC-2) that capture the most pronounced C. displacements". How much of the total variance did these two components capture? The authors write the changes mostly concern loop C and loop F, but which data proves this? e.g. A plot of PC1 and PC2 over residue number might help.

The authors map the -kTln rho as a free energy for each simulation as a function of PC1 & PC2. It is important to reveal how well that PC1-2 space was sampled, and how those maps converged over time. The shapes of the maps and the relative depths of the wells look very different for each agonist. If the maps were sampled well and converged, the free energies themselves would tell us the stabilities of each state. Instead, the authors do not even mention this and instead talk about "variance" being the indicator of stability, stating that m3 is most stable in all cases. While I can believe 200ns could not converge a PC1-2 map and that meaningful delta G values might not be obtained from them, the issue of lack of sampling must be dealt with. On page 12 they write "Although the bottom of the well for 3 energy minima from PCA represent the most stable overall conformation of the protein, they do not convey direct information regarding agonist stability or orientation". The reasons why not must be explained; as they should do just that if the two order parameters PC1 and PC2 captured the slowest degrees of freedom for binding and sampling was sufficient. The authors write that "For all agonists and trajectories, m3 had the least variance (was most stable), again supporting convergence by 200 ns." Again the issue of actual free energy values in the maps needs to be dealt with. The probabilities expressed as -kTln rho in kcal/mol might suggest that m2 is the most stable. Instead, the authors base stability only on variance (I guess breadth of the well?), where m3 may be more localised in the chosen PC space, despite apparently having less preference during the MD (not the lowest free energy in the maps).

The motivations and justifications for the use of approximate PBSA energetics instead of atomistic MD free energies should be dealt with in the manuscript, with limitations more clearly discussed. Rather than using modern all-atom MD free energy methods for relative or absolute binding free energies, the author selects clusters from their identified states and does Poisson-Boltzmann estimates (electrostatic, vdW, surface area, vibrational entropy). I do believe the following sentence does not begin to deal with the limitations of that method: "there are limitations with regard to MM-PBSA accurately predicting absolute binding free energies (Genheden & Ryde, 2015; Hou et al., 2011) that depends on the parameterization of the ligand (Oostenbrink et al., 2004)." What are the assumptions and limitations in taking continuum electrostatics (presumably with parameters for dielectric constants and their assignments to regions after discarding solvent), surface area (with its assumptions and limitations), and of course assuming vibration of a normal mode can capture entropy. On page 30, regarding their vibrational entropy estimate, they write that the "entropy term provides insights into the disorder within the system, as well as how this disorder changes during the binding process". It is important that the extent of disorder captured by the vibrational estimate be discussed, as it is not obvious that it has captured entropy involving multiple minima on the system's true 3N-dimensional energy surface, and especially the contribution from solvent disorder in bound Vs dissociated states.

As discussed above, errors in the free energy estimates need to be more faithfully represented, as fractional errors are not meaningful. On page 21 the authors write "The match improved when free energy ratios rather than absolute values were compared." But a ratio of free energies is not a typical or expected measure of error in delta G. They also write "For ACh and CCh, there is good agreement between.Gm1 and GLA and between.Gm3 and GHA. For these agonists, in silico values overestimated experimental ones only by ~8% and ~25%. The agreement was not as good for the other 2 agonists, as calculated values overestimated experimental ones by ~45%(Ebt) and ~130% (Ebt). However, the fractional overestimation was approximately the same for GLA and GHA." See the above comment on how this may misrepresent the error. On page 21 they write, in relation to their large fractional errors, that they "do not know the origin of this factor but speculate that it could be caused by errors in ligand parameterization". However the estimates from the PBSA approach are, by design, only approximate. Both errors in parameterisation (and their likely origin) and the approximate model used, need discussion.

Reviewer #3 (Public Review):

Summary:<br /> The authors use docking and molecular dynamics (MD) simulations to investigate transient conformations that are otherwise difficult to resolve experimentally. The docking and simulations suggest an interesting series of events whereby agonists initially bind to the low-affinity site and then flip 180 degrees as the site contracts to its high-affinity conformation. This work will be of interest to the ion channel community and to biophysical studies of pentameric ligand-gated channels.

Strengths:<br /> I find the premise for the simulations to be good, starting with an antagonist-bound structure as an estimate of the low affinity binding site conformation, then docking agonists into the site and using MD to allow the site to relax to a higher affinity conformation that is similar to structures in complex with agonists. I cannot speak to the details of the simulation methods, but the predictions are interesting and provide a view into what a transient conformation that is difficult to observe experimentally might be like.

Weaknesses:<br /> Although the match in simulated vs experimental energies for two ligands was very good, the calculated energies for two other ligands were significantly different than the experiment. It is unclear to what extent the choice of method for the energy calculations influenced the results.

A control simulation, such as for an apo site, is lacking.

Reviewer #4 (Public Review):

Summary:<br /> In their manuscript "Conformational dynamics of a nicotinic receptor neurotransmitter binding site," Singh and colleagues present cogent molecular docking and dynamics simulations to explore the initial conformational changes associated with agonist binding in the muscle nicotinic acetylcholine receptor, aligned with the extensive experimental literature on this system. Their central findings are of a consistently preferred pose for agonists upon initial association with a resting channel, followed by a dramatic rotation of the ligand and contraction of a critical loop over the binding site. Principal component analysis also suggests the formation of an intermediate complex, not yet captured in structural studies. Binding free energy calculations are consistent with the evolution of a higher-affinity complex following agonist binding, with a ligand efficiency notably similar to experimental values. Snapshot comparisons provide a structural rationale for these changes on the basis of pocket volume, hydration, and rearrangement of key residues at the subunit interface.

Strengths:<br /> Docking results are clearly presented and remarkably consistent. Simulations are produced in triplicate with each of four different agonists, providing an informative basis for internal validation. They identify an intriguing transition in ligand pose, not well documented in experimental structures, and potentially applicable to mechanistic or even pharmacological modeling of this and related receptor systems. The paper seems a notable example of integrating quantitative structure-function analysis with systematic computational modeling and simulations, likely applicable to the wider journal audience.

Weaknesses:<br /> Timescales (200 ns) do not capture global rearrangements of the extracellular domain, let alone gating transitions of the channel pore, though this work may provide a launching point for more extended simulations. A more general concern is the reproducibility of the simulations, and how representative states are defined. It is not clear whether replicates were included in principal component analysis or subsequent binding energy calculations, nor how simulation intervals were associated with specific states. Structural analysis largely focuses on snapshots, with limited direct evidence of consistency across replicates or clusters. Figure legends and tables could be clarified.

Reviewer #3 (Public Review):

Summary:

In the manuscript "Ebola Virus Sequesters IRF3 in Viral Inclusion bodies to Evade Host Antiviral Immunity " by Lin Zhu et al, the authors elucidated an evasion mechanism by which EBOV evades host innate immunity.

Strengths:

Using data from immunofluorescence analysis, TEM and Western Blot, the authors conclude that Ebola virus VP35 protein evades host antiviral immunity by interacting with STING to sequester IRF3 into IBs and inhibit type-I interferon production.

Weaknesses:

Similar mechanisms have already been found in other viruses, such as SFTSV, RSV and so on. In addition, the presented results are also relatively rough, and the mechanism explained is not deep enough, so this story is not innovative

Reviewer #4 (Public Review):

The manuscript entitled "Ebola Virus Sequesters IRF3 in Viral Inclusion Bodies to Evade Host Antiviral Immunity" mainly describes that the function of IBs formed by the viral proteins VP35 and NP in evading host antiviral immunity. They proved that Ebola virus VP35 protein can interact with STING, but not IRF3, to sequester IRF3 into inclusion bodies and thereby inhibit type-I interferon production. This work will be of some interest to readers in the Ebola Virus field, however, the current data do not clearly explain the relationship of VP35 protein and IRF3.

Reviewer #1 (Public Review):

Bian et al showed that biomarker-informed PhenoAgeAccel was consistently related to an increased risk of site-specific cancer and overall cancer within and across genetic risk groups. The results showed that PhenoAgeAccel and genetic liability of a bunch of cancers serve as productive tools to facilitate the identification of cancer-susceptible individuals under an additive model. People with a high genetic risk for cancer may benefit from PhenoAgeAccel-informed interventions.

As the authors pointed out, the large sample size, the prospective design UK Biobank study, and the effective application of PhenoAgeAccel in predicting the risk of overall cancer are the major strengths of the study. Meanwhile, the CPRS seems to be a solid and comprehensive score based on incidence-weighted site-specific polygenic risk scores across 20 well-powered GWAS for cancers.

It wouldn't be very surprising to identify the association between PhenoAgeAccel and cancer risk, since the PhenoAgeAccel was constructed as a predictor for mortality which attributed a lot to cancer. Although cancer is an essential mediator for the association, sensitivity analyses using cancer-free mortality may provide an additional angle. It would be interesting to see, to what extent, PhenoAgeAccel could be reversed by environmental or lifestyle factors. G by E for PhenoAgeAccel might be worth a try.

Reviewer #2 (Public Review):

Summary:

Bian et al. calculated Phenotypic Age Acceleration (PhenoAgeAccel) via a linear model regressing Phenotypic Age on chronological age. They examined the associations between PhenoAgeAccel and cancer incidence using 374,463 individuals from the UK Biobank and found that older PhenoAge was consistently related to an increased risk of incident cancer, even among each risk group defined by genetics.

Strengths:

The study is well-designed, and uses a large sample size from the UK biobank.

Weaknesses:

Since the UK biobank has a large sample size, it should have enough power to split the dataset into discovery and validation sets. Why did the authors use 10-fold cross-validation instead of splitting the dataset?

Reviewer #1 (Public Review):

The objective of this study was to investigate the influence of the C. trachomatis effector Cdu1 on the ubiquitination of proteins in infected host cells and its correlation with the previously identified role of Cdu1 in facilitating Golgi distribution around the Chlamydia inclusion.

To achieve this, the authors created a cdu1-null mutant in C. trachomatis and employed proteomics to analyze ubiquitinated proteins in cells infected with Cdu1-producing and Cdu1-deficient chlamydiae, comparing them to mock-infected cells. The results revealed that, among the proteins specifically ubiquitinated after infection with Cdu1-deficient chlamydiae, three were other C. trachomatis effectors (InaC, IpaM, and CTL0480), members of a large family of Chlamydia effectors (Incs) that insert in the inclusion membrane.

Subsequently, the authors focused on understanding how Cdu1 shields InaC, IpaM, and CTL0480 from ubiquitination and the implications of this protection for the protein levels and functions of these Incs during infection. Data is presented showing that Cdu1 can bind to InaC, IpaM, and CTL0480, and protects these Incs and itself from ubiquitination and proteasomal degradation. This protective role of Cdu1 is dependent on its acetylation, but not on its deubiquitinating activity. Host cells infected by the cdu1 null mutant displayed defects resembling those observed in cells infected by inaC, ipaM, or ctl0480 null mutants.

Additionally, it was previously established that CTL0480 inhibits a chlamydial egress pathway involving the extrusion of the inclusion. This study now revealed that InaC and IpaM also play a role in promoting the extrusion of C. trachomatis inclusion, and the cdu1 null mutant exhibited a defect in this process. This leads to the title's conclusion that Cdu1 regulates chlamydial exit from host cells by safeguarding specific C. trachomatis effectors from degradation.

In summary, this work is excellent and impressive, both technically and conceptually, providing mechanistic insights into the action of Cdu1. The data provides convincing support for the proposed model, illustrating how the acetylation activity of Cdu1 protects itself and three Incs (InaC, IpaM, and CTL0480) from degradation. While the study indicates that the observed phenotypes in cells infected by the cdu1 null mutant are linked to reduced levels of InaC, IpaM, and CTL0480, these Incs are still detectable in cells infected by the cdu1 null mutant. Even if very unlikely, this leaves room for the possibility that Cdu1 directly promotes assembly of F-actin and Golgi repositioning around the inclusion, MYPT1 recruitment to the inclusion, and extrusion of the inclusion. Nevertheless, the major significance of this work lies in the integration of proteomics and chlamydial genetics to unveil a unique mechanism in which one effector controls the levels of other effectors, emphasizing the intricate relationships among bacterial effectors injected into host cells.

Reviewer #2 (Public Review):

Based on the corresponding author's response, the questions I raised were not addressed for various reasons. This is not necessarily a negative. The authors indicated that most of the points raised will be addressed in a separate manuscript. Specifically, the Cdu1 targeting of IkBa. They mentioned intriguing findings regarding IkBa in cells infected with a cdu1-null strain C. trachomatis in their response to reviewers. Similar to this, there appears to be a planned manuscript that will address the question of the timing of CTL0480's function in inclusion extrusion.

The lack of more direct infection-related evidence of Cdu1 interaction with various type III effectors was raised; and the authors attributed this to technical difficulties and low abundance of starting materials. It was not clear if they tried other approaches to demonstrate interaction.

Another suggestion was the quantitation of the three target effectors of Cdu1 in wild type and cdu1-null background. The authors provided western blot data and immunofluorescence images that revealed potential differences in stability/turnover kinetics. The authors might want to discuss the implications of the different kinetics of stability/turnover. For example, if all three proteins are necessary for optimal extrusion of inclusions, and concertedly act to mediate this process, all three would need to be present at the required levels. Could this be a temporal regulation strategy? Does acetylation also regulate function, interactions, etc.?

In short, the response to some of the questions is forthcoming in the form of follow-up manuscripts. New observations on the different stability profiles could be elaborated in the Discussion section, with a brief discussion on functional and/or regulatory implications.

Reviewer #3 (Public Review):

In this article by Bastidas et al. the authors examine the functions of the Chlamydia deubiquitinating enzyme 1 (Cdu1) during infections of human cells. First, a mutant lacking Cdu1 but not Cdu2 was constructed using targetron and quantitative proteomics was used to identify differences in ubiquitinated proteins (both host and bacterial) during infection. While they found minimal changes in host protein ubiquitination, they identified three Chlamydia effector proteins, IpaM, InaC and CTL0480 were all ubiquitinated in the absence of Cdu1. Microscopy and immunoprecipitations found Cdu1 directly interacts with these Chlamydia effectors and confirmed that Cdu1 mediates the stabilization of these effectors at the inclusion membrane during late infection time points. Surprisingly rather than deubiquitination driving this stabilization, the acetylation function of Cdu1 was required, and acetylation on lysine residues prevented degradative ubiquitination of Cdu1, IpaM, InaC and CTL0480. In line with this observation the authors show that loss of Cdu1 phenocopies the loss of single effector mutants of InaC, IpaM and CTL0480, including golgi stack formation and the recruitment of MYPT1 to the inclusion. The aggregation of changes to the Chlamydia inclusion does not alter growth but controls extrusion of chlamydia from cells with reduced extrusion in Cdu1 mutant Chlamydia infections. The strengths of the manuscript are the range of assays used to convincingly examine the biochemical and cellular biology underlying Cdu1 functions. The finding that acetylation of lysine residues is a mechanisms for bacterial effectors to block degradative ubiqutination is impactful and will open new investigations into this mechanism for many intracellular pathogens. The authors revisions to the manuscript have addressed my primary concerns and the authors present compelling arguments for remaining questions that are outside the scope of this study. Altogether this is an important series of findings that help to understand the mechanisms underpinning Chlamydia pathogenesis using orthologous methods and is an impactful study.

Reviewer #1 (Public Review):

In this paper, the authors developed an image analysis pipeline to automatically identify individual ‎‎neurons within a population of fluorescently tagged neurons. This application is optimized to deal with ‎‎multi-cell analysis and builds on a previous software version, developed by the same team, to resolve ‎‎individual neurons from whole-brain imaging stacks. Using advanced statistical approaches and ‎‎several heuristics tailored for C. elegans anatomy, the method successfully identifies individual ‎‎neurons with a fairly high accuracy. Thus, while specific to C. elegans, this method can ‎become ‎instrumental for a variety of research directions such as in-vivo single-cell gene expression ‎analysis ‎and calcium-based neural activity studies.‎

Reviewer #2 (Public Review):

The authors succeed in generalizing the pre-alignment procedure for their cell identification method to allow it to work effectively on data with only small subsets of cells labeled. They convincingly show that their extension accurately identifies head angle, based on finding auto florescent tissue and looking for a symmetric l/r axis. They demonstrate method works to allow the identification of a particular subset of neurons. Their approach should be a useful one for researchers wishing to identify subsets of head neurons in C. elegans, and the ideas might be useful elsewhere.

The authors also assess the relative usefulness of several atlases for making identity predictions. They attempt to give some additional general insights on what makes a good atlas, but here insights seem less clear as available data does not allow for experiments that cleanly decouple: 1. the number of examples in the atlas 2. the completeness of the atlas. and 3. the match in strain and imaging modality discussed. In the presented experiments the custom atlas, besides the strain and imaging modality mismatches discussed is also the only complete atlas with more than one example. The neuroPAL atlas, is an imperfect stand in, since a significant fraction of cells could not be identified in these data sets, making it a 60/40 mix of Openworm and a hypothetical perfect neuroPAL comparison. This waters down general insights since it is unclear if the performance is driven by strain/imaging modality or these difficulties creating a complete neuroPal atlas. The experiments do usefully explore the volume of data needed. Though generalization remains to be shown the insight is useful for future atlas building that for the specific (small) set of cells labeled in the experiments 5-10 examples is sufficient to build a accurate atlas.

Reviewer #1 (Public Review):

In this study, the authors examined the putative functions of hypothalamic groups identifiable through Foxb1 expression, namely the parvofox Foxb1 of the LHA and the PMd Foxb1, emphasizing innate defensive responses. First, they reported that chemogenetic activation of Foxb1hypothalamic cell groups led to tachypnea. The authors tend to attribute this effect to the activation of hM3Dq expressed in the parvofox Foxb1 but did not rule out the participation of the PMd Foxb1 cell group, which may as well have expressed hM3Dq, particularly considering the large volume (200 nl) of the viral construct injected. Notably, the activation of the Foxb1hypothalamic cell groups in this experiment did not alter the gross locomotor activity, such as time spent immobile state. Thus, this contrasts with the authors' finding on the optogenetic activation of the Foxb1hypothalamic fibers projecting to the dorsolateral PAG. In the second experiment, the authors applied optogenetic ChR2-mediated excitation of the Foxb1+ cell bodies' axonal endings in the dlPAG, leading to freezing and, in a few cases, bradycardia. The effective site to evoke freezing was the rostral PAGdl, and fibers positioned either ventral or caudal to this target had no response. Considering the pattern of Foxb1hypothalamic cell groups projection to the PAG, the fibers projecting to the rostral PAGdl are likely to arise from the PMd Foxb1 cell group and not from the parvofox Foxb1 of the LHA. Here, it is important to consider that activation of PMd CCK cell group, which consists of around 90% of the PMd cells, evokes escape, not freezing. According to the present findings, a specific population of PMd Foxb1 cells may be involved in producing freezing. In addition, only a few of the animals with correct fiber placement presented sudden onset of bradycardia in response to the photostimulation. Considering the authors' findings, the Foxb1+ hypothalamic groups are likely to mediate behavioral responses related to innate defensive responses, where the parvofox Foxb1 of the LHA would be involved in promoting tachypnea and the PMd Foxb1group in mediating freezing and bradycardia. These findings are exciting, and, at this point, they need to be tested in a scenario of actual exposure to a natural predator.

Reviewer #2 (Public Review):

The authors aimed to examine the role of a group of neurons expressing Foxb1 in behaviors through projections to the dlPAG. Standard chemogenetic activation or inhibition and optogentic terminal activation or inhibition at local PAG were used and results suggested that, while activation led to reduced locomotion and breathing, inhibition led to a small degree of increased locomotion.

The observed effects on breathing are evident and dramatic. However, due to the circumstance that does not permit to perform additional experiments, the conclusion is not as strong as it could be.

Reviewer #1 (Public Review):

This study investigates the underlying mechanisms of information-seeking in infancy. Eight-month-old Dutch infants were tested in a screen-based eye-tracking task in which one of two geometrical shape cues (differing in their shape and motion) either announced the location of an upcoming reward cartoon (informative) or not (non-informative). The authors measured the infants' pupil size before the cartoon appeared. Infants showed smaller pupil sizes when presented with the informative cue as compared to the noninformative cue. The decrease in pupil size in the informative condition emerged over the course of trials whereas infants' pupil size remained unchanged in the noninformative condition. The authors interpret their findings as supportive evidence of statistical learning and generalization processes organizing infants' information-seeking.

It was a pleasure to read the paper and I think the study makes a valuable contribution to our understanding of information-seeking in infancy. The manuscript is very well written and the study is cleverly designed. My following comments are based on my reading of the manuscript and the supplemental materials. It should be noted that evaluating the details of the statistical procedure the authors used lies outside my expertise. The same applies to some decisions of the authors related to pre-processing and filtering the pupil data. I very much appreciate that the authors shared all their raw data and analysis scripts openly accessible on the Open Science Framework. The study was unfortunately not preregistered, making it difficult to trace when in the study process certain decisions or assumptions were made.

My two main concerns relate to the conceptualization and definition of information-seeking and the proposed speed of the mechanisms explaining infants' behavior. I outline my general comments below before listing some more concrete issues.

1) While reading the manuscript, I was sometimes confused about what the authors refer to when talking about information-seeking - both in terms of the broader conceptualization of the phenomenon as well as when referring to their own study. What information are infants seeking? The informative value of the cue shape in terms of their motion (because it carries information about the location of a rewarding animation)? Or is the target (the rewarding video) the information being sought? From how the study is set up, I assume the authors refer mainly to the first aspect, but I think the manuscript would benefit from some clearer distinctions and definitions of terms.

More specifically, I think it could help if the authors would specify the different aspects involved in information-seeking in the introduction (e.g., seeking information "directly", seeking cues guiding them towards information, etc.). Secondly, it would help if they would sharpen their (already in some parts existing) definitions for their study and then keep consistent with their definitions throughout the methods, results, and discussion. Is the cue the information being sought or the "behavior" (motion) of the cue? Or is the target animation the information being sought and guided via the cueing?

2) Speed of the generalization process:<br /> From my understanding of the study design, the shape of the geometrical shape gains informative value over time (serving as an informative cue) and the *motion* of the shape is the actual informative or non-informative visual cue in that it either reliably highlights the actual target region (or all regions). In the generalization trials, only the shape was manipulated while the motion aspect remained consistent with the previous trials. Based on infants' behavior across learning and generalization trials, the authors make an argument about two distinct processes taking place: a slower allowing to learn where to find info and a faster generalization process. Apologies if I missed something, but given that the motion remains consistent, it's maybe not surprising that the generalization trials are "faster"? Maybe the generalization process would have been slower if not only the shape had changed but if also a novel informative motion had been introduced. Also, it would be helpful if the authors could clarify what they mean by the statistical learning process being more "data-hungry" (line 274).

3) I would find it very helpful if the authors would discuss statistical learning and information-seeking processes from other possible mechanisms such as reward learning mechanisms. For example, the authors use a "rewarding" (not informative) stimulus as the target-wouldn't it be possible that the results can be also explained by reinforcement learning processes? Relatedly, in line 396 they write that they used TD learning to predict whether "information will be delivered" and contrast this with the approach being used to predict whether a reward will be delivered. But in their study reward was being delivered, too (in the form of the target), in addition to the informative motion of the cue.

Reviewer #2 (Public Review):

Summary<br /> The study used eye tracking with a focus on pupillometry to examine how infants can learn to distinguish between informative and uninformative visual cues. Infants (n = 30, mean age = 8.2-months-old) viewed displays consisting of a sequence of stimuli: a fixation point, a central cue that predicted a subsequent informative or uninformative signal, the signal itself, and the target event (a cartoon animal, referred to as the reward). The key results are that: (1) pupil size differs depending on whether the infants anticipated an informative or uninformative signal, (2) this difference develops across trials, consistent with a slow learning process, and (3) there is rapid generalization when new shapes were introduced that shared features with the informative vs uninformative cues. The study complements a rich literature, including from this same group, showing that children are sensitive to information gains, and is interesting and important in revealing that pupil size is a physiological marker of information anticipation. We have several comments and concerns and believe that addressing them would substantially strengthen the manuscript.

Major points are related to interpretation, statistical robustness, and clarity

1. There is a tendency to overinterpret the findings.<br /> a. Throughout, the authors interpret the findings as meaning that pupil size tracks the "value" of information; however, the results do not demonstrate conclusively whether, or what kind of value information has in this task. A natural hypothesis is that infants are intrinsically motivated to predict - i.e., value the ability to predict the target event as early as possible. In a supplementary figure, the authors present evidence that infants indeed fixate on the target event sooner after seeing informative vs uninformative cues, consistent with the idea that they use the information for improving predictions. However, those results are not fully convincing, as we detail in point 2. Most importantly, the analysis is not integrated or even mentioned in the main analyses analysis. Making the link between the pupil reaction and the use of the information would greatly strengthen the paper (whether or not the supplementary findings hold up to more thorough scrutiny). Either this link should be made and discussed, or the authors should soften their conclusions about the utility of the informative cues.

b. On line 236, the text states that the evidence "...supports the growing body of evidence indicating that infants are proactive in shaping their learning environment by searching for and focusing on information-rich stimuli". The results do not show that the infants search for information, only that they have a pupil reaction that differentiates between informative and uninformative stimuli.

c. On lines 248-249, it seems a stretch to relate the changes in pupil dilation to a shift in information value onto the cue. Without some other measure (e.g., EEG), this remains speculative. While I believe the suggestion is plausible, the language should be softened to highlight this as a follow-up research question that the present research cannot directly speak to.

2. Several findings are statistically weak and several analyses are insufficiently controlled.

a. The analysis in Supplementary Figure 2, which shows that the latencies of target fixations are shorter after informative vs uninformative cues, raises several questions.<br /> i. We were unable to fully test these analyses as the OSF project seems to only contain latency data for 33 participants (including 22 of the 30 that remain in the final sample).<br /> ii. The results are described as revealing a significant difference, but the 89% confidence interval of the difference contains 0. How did the authors establish significance here?<br /> iii. How do the authors distinguish incidental fixations (which just happened to land near the target) from true predictive gaze shifts? Fixations were pooled if they occurred from 1.25 seconds before to 1 second after target onset. This is sufficient time for the eye to move in and out of the window several times. The authors should analyse the distributions of fixation durations to rule out various artifacts unrelated to target prediction.<br /> iv. Latencies to fixation were standardized, bringing the mean across each participant to 0, and yet the statistical model includes a random intercept; is there a justification for this?<br /> v. Standardizing removes information about whether fixations were proactive or reactive. It would be very interesting to see if/how information affects these two differently.<br /> vi. Since informativeness was learned across trials, it seems desirable that the model should include as random effects a trial number and an interaction between trial number and informativeness. This would allow a comparison between learning to predict and the pupil reaction. Are infants who have a stronger (or earlier) pupil reaction also more likely to show stronger learning to anticipate?

b. The main finding that pupil size differs between informative and uninformative cues is based on a 3-second analysis window. This long window most likely spans many saccades, which can affect pupil size on its own or by bringing the eye on or off visual stimuli. There is no analysis to show that the statistics of saccades or fixation locations are equivalent between the two trial types - but this is necessary to convincingly rule out a spurious artifact.

c. The second main finding that the effect of informativeness grows across trials seems statistically weak. The text (line 138) states that the interaction had a beta of 0.002, which was equal to the lower border of the 89%HDI ([0.002, 0.003]). For the second claim that pupil size decreased across informative trials, the beta is -0.002, and 89% HID is non-existent - i.e., [-0.002, -0.002]. (In general, the authors should check their numbers more carefully and make sure they are presented with a degree of precision that allows the reader to interpret them meaningfully.

d. The analyses do not indicate how well the TD model fits; we are shown only that it fits better than a linear model. On line 177 a correlation analysis is mentioned between the data and model, but the statistic cited for this test on line 179 is a mean beta coefficient, so it is impossible to know what this means. An analysis of goodness of fit or, at the very least, a figure superimposing the model and data, would be much more convincing.

3. The descriptions are very unclear in some key parts of the paper

a. The description of the TD model applied to pupil learning (starting on line 391) is very unclear. The model has to include some measure of informativeness - i.e., the match between the cued and true target location - but it is unclear how this was formalized. It is also very unclear how time within the trial is incorporated (the meaning of the TDE equation).

b. The description of the generalization analysis (Fig. 5) is also very unclear. Every single sentence in it evoked some confusion, so I will go through them one by one. "A Bayesian additive model showed that infants' pupil dilation was reduced for novel cues." Reduced relative to what? "This was specific to those novel cues that shared the features of the familiar informative cues (estimated mean difference = -0.05, 89%HDI = [-0.062, -0.038])." All the novel cues shared features with the informative cues; do the authors mean the novel cues that had the critical feature indicative of the informative cue? "The size of this effect approximated the difference between conditions that were observed for familiar stimuli (estimated mean difference = -0.067, 89% HDI = [-201 0.077, -0.057])." What is "this effect"? "Crucially, this difference was not observable at the start of the task, when the familiar stimuli were first introduced (estimated mean difference = -0.007, 89%HDI = [-0.015, 0.001])." At the start of the task, the stimuli were novel, and not familiar.

Reviewer #3 (Public Review):

Summary:<br /> The study attempts to shed light on the mechanisms underlying information-seeking in infants by investigating whether infants distinguish between informative and uninformative stimuli to resourcefully allocate their attention. The authors show that 8-month-old infants can learn whether a visual stimulus is informative or uninformative about the location of a later appearing rewarding stimulus by employing statistical regularities from the input. Specifically, infants showed decreased pupil dilation for informative over uninformative cues, which developed over the course of trials as more and more information was gathered from the input. The pattern of learning was in line with a reinforcement learning model which employed a steep learning curve in the beginning followed by a more shallow but steady learning growth over trials. After 17 trials, the authors presented novel cues that shared certain visual features with the previous stimuli and showed that pupil dilation was reduced for novel cues that shared features with the previous informative stimuli, suggesting that infants were able to generalize their acquired knowledge about the informativeness of certain features to novel stimuli. The present study adds to the existing literature about the underlying mechanisms of learning by showing that infants cannot only predict an upcoming stimulus based on statistical regularities of a preceding cue but also the informativeness of the cue itself.

Strengths:<br /> The authors use a suitable method to test the highly relevant question of whether and how infants infer the informativeness of stimuli from experience and whether they can generalize this knowledge to new stimuli. Their experiment is carefully designed and well controlled with conditions closely matched (e.g., the shape and color of objects and the structure of each trial). Their measure of interest (i.e., pupil dilation) is also examined at a time point in each trial when the conditions are the most similar, which further points to a thought-through and careful design. This empirical data is backed up with a computational approach (using a Bayesian model and training a reinforcement learning algorithm) to elucidate the learning mechanisms at play. This approach is explained concisely to readers not familiar with the models.

The results are convincing showing a clear difference between informative and uninformative condition and development over trials. Specifically, this difference is not apparent in the first trial (Fig. 2c) but develops over time which supports a learning trajectory. The data support the authors' conclusion that infants learn about the informativeness of the object cue from the input, and the employed learning algorithms give further insights into the learning trajectory of the infants. Overall, the statistical analyses seem solid and the priors for the Bayesian models are well reported.

Data and scripts are openly available fostering transparency.

Overall, the manuscript is very well and concisely written.

Weaknesses:<br /> The authors' conclusion that infants can generalize the acquired knowledge to similar but novel stimuli is weakened by methodological concerns regarding the analysis. It is not fully clear which trials the authors excluded and analyzed as they do not consistently report the trials in the manuscript (e.g., it is stated that after trial 17 the first generalization trial started, but also that trial 17 was excluded as the first trial of the generalization phase). As there are only a few novel trials and novel and familiar trials alternated, the inclusion or exclusion of trial analyses might have a significant impact on the results. Thus, this needs further clarification. The authors also mentioned that the novel stimuli shared relevant as well as irrelevant features, but it was not clear to me whether the authors could establish that only the relevant features contributed to the observed generalization effect.

Some methodological decisions were not explained and need justification, in particular, as the study is not preregistered. This includes, for example, the exclusion criteria and the choice not to analyze all generalization trials. Further, the authors did not perform model comparison (e.g., their model against a null model) and therefore do not report the strength of evidence for a difference in conditions.

Another weakness is that the sample sizes of 30 infants for the initial part and 19 infants for the generalization part of the experiment are rather small (especially with regard to the chosen weakly informative priors).

Reviewer #1 (Public Review):

Summary:<br /> This manuscript uses optical coherence tomography (OCT) to visualize tissue microstructures about 1-2 mm under the finger pad skin surface. Their geometric features are tracked and used to generate tissue strains upon skin surface indentation by a series of transparent stimuli both normal and tangential to the surface. Then movements of the stratum corneum and the upper portion of the viable epidermis are evaluated. Based upon this data, across a number of participants and ridges, around 300 in total, the findings report upon particular movements of these tissue microstructures in various loading states. A better understanding of the mechanics of the skin microstructures is important to understand how surface forces propagate toward the locations of mechanoreceptive end organs, which lie near the edge of the epidermis and dermis, from which tactile responses of at least two peripheral afferents originate. Indeed, the microstructures of the skin are likely to be important in shaping how neural afferents respond and enhance their sensitivity, receptive field characteristics, etc.

Strengths:<br /> The use of OCT in the context of analyzing the movements of skin microstructures is novel. Also novel and powerful is the use of distinct loading cases, e.g., normal, tangential, and stimulus features, e.g., edges, and curves. I am unaware of other empirical visualization studies of this sort. They are state-of-the-art in this field. Moreover, in addition to the empirical imaging observations, strain vectors in the tissues are calculated over time.

Weaknesses:<br /> The interpretation of the results and their framing relative to the overall hypotheses/questions and prior works could be articulated more clearly. In particular, the major findings of the manuscript are in newly describing a central concept regarding "ridge flanks," but such structures are neither anatomically nor mechanistically defined in a clear fashion. For example, "... it appears that the primary components of ridge deformation and, potentially, neural responses are deformations of the ridge flanks and their relative movement, rather than overall bending of the ridges themselves." From an anatomical perspective, I think what the authors mean by "ridge flanks" is a differential in strain from one lateral side of a papillary ridge to the other. But is it unclear what about the continuous layers of tissue would cause such behaviors. Perhaps a sweat duct or some other structure (not visible to OCT) would subdivide the "flanks" of a papillary ridge somehow? If not due to particular anatomy, then is the importance of the "ridge flank" due to a mechanistic phenomenon of some sort? Given that the findings of the manuscript center upon the introduction of this new concept, I think a greater effort should be made to define what exactly are the "ridge flanks." It is clear from the results, especially the sliding case, that there is something important that the manuscript is getting at with this concept.

The OCT used herein cannot visualize deep and fully into what the manuscript refers to as a "ridge" (note others have previously broken apart this concept apart into "papillary", "intermediate" and "limiting" ridges) near locations of the mechanoreceptive end organs lie at the epidermal-dermal border. Therefore, the OCT must make inferences about the movements of these deeper tissues, but cannot see them directly, and it is the movements of these deeper tissues that are likely driving the intricacies of neural firing. Note the word "ridge" is used often in the manuscript's abstract, introduction, and discussion but the definition in Fig. 1 and elsewhere differs in important ways from prior works of Cauna (expert in anatomy). Therefore, the manuscript should clarify if "ridge" refers to the papillary ridge (visible at the exterior of the skin), intermediate ridge (defined by Cauna as what the authors refer to as the primary ridge), and limiting ridge (defined by Cauna as what the authors refer to as the secondary ridge). What the authors really mean (I think) is some combination of the papillary and intermediate ridge structures, but not the full intermediate ridge. The manuscript acknowledges this in the "Limitations and future work" section, stating that these ridges cannot be resolved. This is important because the manuscript is oriented toward tracking this structure. It sets up the narrative and hypotheses to evaluate the prior works of Cauna, Gerling, Swensson, and others who all directly addressed the movement of this anatomical feature which is key to understanding ultimately how stresses at these locations might move the peripheral end organs (i.e., Merkel cells, Meissner corpuscles).

Reviewer #2 (Public Review):

Summary:<br /> The authors investigate sub-skin surface deformations to a number of different, relevant tactile stimuli, including pressure and moving stimuli. The results demonstrate and quantify the tension and compression applied from these types of touch to fingerprint ridges, where pressure flattens the ridges. Their study further revealed that on lateral movement, prominent vertical shearing occurred in ridge deformation, with somewhat inconsistent horizontal shear. This also shows how much the deeper skin layers are deformed in touch, meaning the activation of all cutaneous mechanoreceptors, as well as the possibility of other deeper non-cutaneous mechanoreceptors.

Strengths:<br /> The paper has many strengths. As well as being impactful scientifically, the methods are sound and innovative, producing interesting and detailed results. The results reveal the intricate workings of the skin layers to pressure touch, as well as sliding touch over different conditions. This makes it applicable to many touch situations and provides insights into the differential movements of the skin, and thus the encoding of touch in regards to the function of fingerprints. The work is very clearly written and presented, including how their work relates to the literature and previous hypotheses about the function of fingerprint ridges. The figures are very well-presented and show individual and group data well. The additional supplementary information is informative and the video of the skin tracking demonstrates the experiments well.

Weaknesses:<br /> There are very few weaknesses in the work, rather the authors detail well the limitations in the discussion. Therefore, this opens up lots of possibilities for future work.

Impact/significance:<br /> Overall, the work will likely have a large impact on our understanding of the mechanics of the skin. The detail shown in the study goes beyond current understanding, to add profound insights into how the skin actually deforms and moves on contact and sliding over a surface, respectively. The method could be potentially applied in many other different settings (e.g. to investigate more complex textures, and how skin deformation changes with factors like dryness and aging). This fundamental piece of work could therefore be applied to understand skin changes and how these impact touch perception. It can further be applied to understand skin mechanoreceptor function better and model these. Finally, the importance of fingertip ridges is well-detailed, demonstrating how these play a role in directly shaping our touch perception and how they can shape the interactions we have with surfaces.

Reviewer #3 (Public Review):

Summary:<br /> The publication presents unique in-vivo images of the upper layer of the epidermis of the glabrous skin when a flat object compresses or slides on the fingertip. The images are captured using OCT, and are the process of recovering the strain that fingerprints experience during the mechanical stimulation.

The most important finding is, in my opinion, that fingerprints undergo pure compression/tension without horizontal shear, hinting at the fact that the shear stress caused by the tangential load is transferred to the deeper tissues and ultimately to the mechanoreceptors (SA-I / RA-I).

Strengths:<br /> - Fascinating new insights into the mechanics of glabrous skin. To the best of my knowledge, this is the first experimental evidence of the mechanical deformation of fingerprints when subjected to dynamic mechanical stimulation. The OCT measurement allows an unprecedented measurement of the depth of the skin whereas previous works were limited to tracking the surface deformation.<br /> - The robust data analysis reveals the continuum mechanics underlying the deformation of the fingerprint ridges.

Weaknesses:<br /> I do not see any major weaknesses. The work is mainly experimental and is rigorously executed. Two points pique my curiosity, however:

1. How do the results presented in this study compare with previous finite element analysis? I am curious to know if the claim that the horizontal shear strain is transferred to the previous layer is also captured by these models. The reason is that the FEA models typically use homogeneous materials and whether or not the behavior in-silico and in-vivo matches would offer an idea of the nature of the stratum corneum.<br /> 2. Was there a specific reason why the authors chose to track only one fingerprint? From the method section, it seems that nothing would have prevented tracking a denser point cloud and reconstructing the stain on a section of the skin rather than just one ridge. With such data, the author could extend their analysis to multiple ridges interaction and get a better sense of the behavior of the entire strip of skin.

Reviewer #1 (Public Review):

This study aims to identify gene expression differences exclusively caused by cis-regulatory genetic changes by utilizing hybrid cell lines derived from human and chimpanzee. While previous attempts have focused on specific tissues, this study expands the comparison to six different tissues to investigate tissue specificity and derive insights into the evolution of gene expression.

One notable strength of this work lies in the use of composite cell lines, enabling a comparison of gene expression between human and chimpanzee within the same nucleus and shared trans factors environment. However, a potential weakness of the methodology is the use of bulk RNA-seq in diverse tissues, which limits the ability to determine cell-type-specific gene expression and chromatin accessibility regions. Their approach, using hybrid lines, naturally accounts for cell type heterogeneity avoiding the risk of false positives introduced by the otherwise confounding differences in cell type abundances between species, albeit the challenge of false negatives remains an issue. The authors now dully acknowledge this limitation in the manuscript.

Another concern is the use of two replicates derived from the same pair of individuals. While the authors produced cell lines from two pairs of individuals in a previous study (Agloglia et al., 2021). The reason for this experimental design is cost limitations. The authors now acknowledge that the use of replicates could enhance the ability to detect "more" species-specific changes in expression and chromatin accessibility. I would emphasize that replicates would increase robustness to the present findings, given that they are derived from a single pair of individuals.

Furthermore, the study offers the opportunity to relate inter-species differences to trends in molecular evolution. The authors discovered that expression variance and haploinsufficiency score do not fully account for the enrichment of divergence in cell-type-specific genes. The reviewer suggested exploring this further by incorporating external datasets that bin genes based on interindividual transcriptomics variation as a measure of extant transcriptomics constraint (e.g., GTEx reanalysis by Garcia-Perez et al., 2023 - PMID: 36777183). The authors considered this question to be out of the scope of the paper, yet in my opinion this would enhance one of the main findings of this study.

Additionally, stratifying sequence conservation on ASCA regions, which exhibit similar enrichment of cell-type-specific features, using the Zoonomia data mentioned also in the text (Andrews et al., 2023 -- PMID: 37104580) could provide valuable insights. While the author did not find Zoonomia Phastcons values available, they used PhastCons derived from a 470-way alignment of mammals. I commend the authors for their diligent efforts, which undoubtedly bolster their findings that an enrichment in ASCA is evident across all levels of sequence conservation. However, this recent analysis indicates the presence of a potential relationship between sequence conservation and ASCA. It may be advantageous to consider evaluating more quantile subdivisions of maxZ values and pPhastCons values, with the inclusion of these results in the supplementary materials. This approach would be preferable, even if the precise reasons behind the observed discrepancy are not fully elucidated.

Another potential strength of this study is the identification of specific cases of paired allele-specific expression (ASE) and allele-specific chromatin accessibility (ASCA) with biological significance. Prioritizing specific variants remains a challenge, and the authors apply a machine learning approach to identify potential causative variants that disrupt binding sites in two examples (FABP7 and GAD1 in motor neurons). However, additional work is needed to convincingly demonstrate the functionality of these selected variants. Strengthening this section with additional validation of ASE, ASCA, and the specific putative causal variants identified would enhance the overall robustness of the paper. The authors have opted to defer these validations to future studies.

Additionally, the authors support the selected ASE-ASCA pairs by examining external datasets of adult brain comparative genomics (Ma et al., 2022) and organoids (Kanton et al., 2019). While these resources are valuable for comparing observed species biases, the analysis is not systematic, even for the two selected genes. For example, it would be beneficial to investigate if FABP7 exhibits species bias in any cell type in Kanton et al.'s organoids or if GAD1 is species-biased in adult primate brains from Ma et al. Comparing these datasets with the present study, along with the Agoglia et al. reference, would provide a more comprehensive perspective. In the revised version of the manuscript the authors have evaluated the expression of GAD1 in Ma et al, and FABP7 in Sousa et al 2017. For instance, GAD1 show cell type specific species biases in the later. The authors opted for not showing this in the manuscript, However, it remains unclear why certain datasets were favored over others, or why FABP7 should not be evaluated in Kanton et al.

The use of the term "human-derived" in ASE and ASCA has now been avoided.

Finally, throughout the paper, the authors refer to "hybrid cell lines." It has been suggested to use the term "composite cell lines" instead to address potential societal concerns associated with the term "hybrid," which some may associate with reproductive relationships (Pavlovic et al., 2022 -- PMID: 35082442). The authors have presented an eloquent and persuasive explanation that I found to be highly informative.

Reviewer #3 (Public Review):

The authors utilize chimpanzee-human hybrid cell lines to assess cis-regulatory evolution. These hybrid cell lines offer a well-controlled environment, enabling clear differentiation between cis-regulatory effects and environmental or other trans effects.<br /> In their research, Wang et al. expand the range of chimpanzee-human hybrid cell lines to encompass six new developmental cell types derived from all three germ layers. This expansion allows them to discern cell type-specific cis-regulatory changes between species from more pleiotropic ones. Although the study investigates only two iPSC clones, the RNA- and ATAC-seq data produced for this paper is a valuable resource.

The authors begin their analysis by examining the relationship between allele-specific expression (ASE) as a measure of species divergence and cell type specificity. They find that cell-type-specific genes exhibit more divergent expression. By integrating this data with measures of constraint within human populations, the authors conclude that the increased divergence of tissue-specific genes is, at least in part, attributable to positive selection. A similar pattern emerges when assessing allele-specific chromatin accessibility (ASCA) as a measure of divergence of cis-regulatory elements (CREs) in the same cell lines.

By correlating these two measures, the authors identify 95 CRE-gene pairs where tissue-specific ASE aligns with tissue-specific ASCA. Among these pairs, the authors select two genes of interest for further investigation. Notably, the authors employ an intriguing machine learning approach in which they compare the inferred chromatin state of the human sequence with that of the chimpanzee sequence to pinpoint putatively causal variants.

Overall, this study delves into the examination of gene expression and chromatin accessibility within hybrid cell lines, showcasing how this data can be leveraged to identify potential causal sequence differences underlying between-species expression changes.

All in all most conclusions appear solid, with the exception of the interpretation of a cell type/state identification machine learning model to pinpoint putatively causal variants. The described variants lack any functional validation and there is no data that measure the certainty of the results.

Joint Public Review:

In this manuscript, the authors introduced an explicit ion model using the coarse-grained modelling approach to model the interactions between nucleosomes and evaluate their effects on chromatin organization. The strength of this method lies in the explicit representation of counterions, especially divalent ions, which are notoriously difficult to model. To achieve their aims and validate the accuracy of the model, the authors conducted coarse-grained molecular dynamics simulations and compared predicted values to the experimental values of the binding energies of protein-DNA complexes and the free energy profile of nucleosomal DNA unwinding and inter-nucleosome binding. Additionally, the authors employed umbrella sampling simulations to further validate their model, reproducing experimentally measured sedimentation coefficients of chromatin under varying salt concentrations of monovalent and divalent ions.

The significance of this study lies in the authors' coarse-grained model which can efficiently capture the conformational sampling of molecules while maintaining a low computational cost. The model reproduces the scale and, in some cases, the shape of the experimental free energy profile for specific molecule interactions, particularly inter-nucleosome interactions. Additionally, the authors' method resolves certain experimental discrepancies related to determining the strength of inter-nucleosomal interactions. Furthermore, the results from this study support the crucial role of intrinsic physicochemical interactions in governing chromatin organization within the nucleus.

The authors have successfully addressed the majority of my key concerns. I appreciate the clarification regarding the parameterization from Pablo's lab and the addition of comparisons of energy profiles as a function of inter-nucleosome distances.

However, the statement "The agreement is evident" may not sufficiently capture the essence of Figure S4, as there is a shortage of substantial agreement. The authors rightly acknowledge it but should delineate the nature of the observed discrepancies.

Reviewer #1 (Public Review):

Continuous attractor networks endowed with some sort of adaptation in the dynamics, whether that be through synaptic depression or firing rate adaptation, are fast becoming the leading candidate models to explain many aspects of hippocampal place cell dynamics, from hippocampal replay during immobility to theta sequences during run. Here, the authors show that a continuous attractor network endowed with spike frequency adaptation and subject to feedforward external inputs is able to account for several previously unaccounted aspects of theta sequences, including (1) sequences that move both forwards and backwards, (2) sequences that alternate between two arms of a T-maze, (3) speed modulation of place cell firing frequency, and (4) the persistence of phase information across hippocampal inactivations.

I think the main result of the paper (findings (1) and (2)) are likely to be of interest to the hippocampal community, as well as to the wider community interested in mechanisms of neural sequences. In addition, the manuscript is generally well written and the analytics are impressive. However, several issues should be addressed, which I outline below.

Major comments:

In real data, population firing rate is strongly modulated by theta (i.e., cells collectively prefer a certain phase of theta - see review paper Buzsaki, 2002) and largely oscillates at theta frequency during run. With respect to this cyclical firing rate, theta sweeps resemble "Nike" check marks, with the sweep backwards preceding the sweep forwards within each cycle before the activity is quenched at the end of the cycle. I am concerned that (1) the summed population firing rate of the model does not oscillate at theta frequency, and (2) as the authors state, the oscillatory tracking state must begin with a forward sweep. With regards to (1), can the authors show theta phase spike preference plots for the population to see if they match data? With regards to (2), can the authors show what happens if the bump is made to sweep backwards first, as it appears to do within each cycle?

I could not find the width of the external input mentioned anywhere in the text or in the table of parameters. The implication is that it is unclear to me whether, during the oscillatory tracking state, the external input is large compared to the size of the bump, so that the bump lives within a window circumscribed by the external input and so bounces off the interior walls of the input during the oscillatory tracking phase, or whether the bump is continuously pulled back and forth by the external input, in which case it could be comparable to the size of the bump. My guess based on Fig 2c is that it is the latter. Please clarify and comment.

I would argue that the "constant cycling" of theta sweeps down the arms of a T-maze was roughly predicted by Romani & Tsodyks, 2015, Figure 7. While their cycling spans several theta cycles, it nonetheless alternates by a similar mechanism, in that adaptation (in this case synaptic depression) prevents the subsequent sweep of activity from taking the same arm as the previous sweep. I believe the authors should cite this model in this context and consider the fact that both synaptic depression and spike frequency adaptation are both possible mechanisms for this phenomenon. But I certainly give the authors credit for showing how this constant cycling can occur across individual theta cycles.

The authors make an unsubstantiated claim in the paragraph beginning with line 413 that the Tsodyks and Romani (2015) model could not account for forwards and backwards sweeps. Both the firing rate adaptation and synaptic depression are symmetry breaking models that should in theory be able to push sweeps of activity in both directions, so it is far from obvious to me that both forward and backward sweeps are not possible in the Tsodyks and Romani model. The authors should either prove that this is the case (with theory or simulation) or excise this statement from the manuscript.

The section on the speed dependence of theta (starting with line 327) was very hard to understand. Can the authors show a more graphical explanation of the phenomenon? Perhaps a version of Fig 2f for slow and fast speeds, and point out that cells in the latter case fire with higher frequency than in the former?

I had a hard time understanding how the Zugaro et al., (2005) hippocampal inactivation experiment was accounted for by the model. My intuition is that while the bump position is determined partially by the location of the external input, it is also determined by the immediate history of the bump dynamics as computed via the local dynamics within the hippocampus (recurrent dynamics and spike rate adaptation). So that if the hippocampus is inactivated for an arbitrary length of time, there is nothing to keep track of where the bump should be when the activity comes back on line. Can the authors please explain more how the model accounts for this?

Can the authors comment on why the sweep lengths oscillate in the bottom panel of Fig 5b during starting at time 0.5 seconds before crossing the choice point of the T-maze? Is this oscillation in sweep length another prediction of the model? If so, it should definitely be remarked upon and included in the discussion section.

Perhaps I missed this, but I'm curious whether the authors have considered what factors might modulate the adaptation strength. In particular, might rat speed modulate adaptation strength? If so, would have interesting predictions for theta sequences at low vs high speeds.

I think the paper has a number of predictions that would be especially interesting to experimentalists but are sort of scattered throughout the manuscript. It would be beneficial to have them listed more prominently in a separate section in the discussion. This should include (1) a prediction that the bump height in the forward direction should be higher than in the backward direction, (2) predictions about bimodal and unimodal cells starting with line 366, (3) prediction of another possible kind of theta cycling, this time in the form of sweep length (see comment above), etc.

Reviewer #2 (Public Review):

In this work, the authors elaborate on an analytically tractable, continuous-attractor model to study an idealized neural network with realistic spiking phase precession/procession. The key ingredient of this analysis is the inclusion of a mechanism for slow firing-rate adaptation in addition to the otherwise fast continuous-attractor dynamics. The latter continuous-attractor dynamics classically arises from a combination of translation invariance and nonlinear rate normalization.

For strong adaptation/weak external input, the network naturally exhibits an internally generated, travelling-wave dynamics along the attractor with some characteristic speed. For small adaptation/strong external stimulus, the network recovers the classical externally driven continuous-attractor dynamics. Crucially, when both adaptation and external input are moderate, there is a competition with the internally generated and externally generated mechanisms leading to an oscillatory tracking regime. In this tracking regime, the population firing profile oscillates around the neural field tracking the position of the stimulus. The authors demonstrate by a combination of analytical and computational arguments that oscillatory tracking corresponds to realistic phase precession/procession. In particular the authors can account for the emergence of unimodal and bimodal cells, as well as some other experimental observations with respect the dependence of phase precession/procession on the animal's locomotion.

The strengths of this work are at least three-fold: 1) Given its simplicity, the proposed model has a surprisingly large explanatory power of the various experimental observations. 2) The mechanism responsible for the emergence of precession/procession can be understood as a simple yet rather illuminating competition between internally driven and externally driven dynamical trends. 3) Amazingly, and under some adequate simplifying assumptions, a great deal of analysis can be treated exactly, which allows for a detailed understanding of all parametric dependencies. This exact treatment culminates with a full characterization of the phase space of the network dynamics, as well as the computation of various quantities of interest, including characteristic speeds and oscillating frequencies.

As mentioned by the authors themselves, the main limitation of this work is that it deals with a very idealized model and it remains to see how the proposed dynamical behaviors would persists in more realistic models. For example, the model is based on a continuous attractor model that assumes perfect translation-invariance of the network connectivity pattern. Would the oscillating tracking behavior persist in the presence of connection heterogeneities? Another limitation is that the system needs to be tuned to exhibit oscillation within the theta range and that this tuning involves a priori variable parameters such as the external input strength. Is the oscillating-tracking behavior overtly sensitive to input strength variations? The author mentioned that an external pacemaker can serve to drive oscillation within the desired theta band but there is no evidence presented supporting this. A final and perhaps secondary limitation has to do with the choice of parameter, namely the time constant of neural firing which is chosen around 3ms. This seems rather short given that the fast time scale of rate models (excluding synaptic processes) is usually given by the membrane time constant, which is typically about 15ms. I suspect this latter point can easily be addressed.

Reviewer #1 (Public Review):

The study isolated extracellular vesicles (EV) from healthy controls (HCs) and Parkinson patients (PwP), using plasma from the venous blood of non-fasting people. Such EVs were characterized and validated by the presence of markers, their size, and their morphology. The main aim of the manuscript is to correlate the presence of synaptic proteins, namely SNAP-25, GAP-43, and SYNAPTOTAGMIN-1, normalized with HSP70, with the clinical progression of PwP. Changes in synaptic proteins have been documented in the CSF of Alzheimer's and Parkinson's patients. The demographics of participants are adequately presented. One important limiting, as well as puzzling aspect, is the fact that authors did not find differences between groups at the beginning of the study nor after one year, after age and sex adjustment.

Reviewer #2 (Public Review):

Hong and collaborators investigated variations in the amount of synaptic proteins in plasma extracellular vesicles (EV) in Parkinson's Disease (PD) patients on one-year follow-up. Their findings suggest that plasma EV synaptic proteins may be used as clinical biomarkers of PD progression.

It is a preliminary study using semi-quantitative analysis of synaptic proteins.

The authors have a cohort of PD patients with clinical examination and a know-how on EV purification. Regarding this latter part, they may improve their description of EV purification. EV may be broken into smaller size EV after freezing. Does it explain the relatively small size in their EV preparation? Do the authors refer to the MISEV guidelines for EV purity? Regarding synaptic protein quantification, the choice of western blotting may not be the best one. ELISA and other multiplex arrays are available. How the authors do justify their choice? Do the authors try to sort plasma EV by membrane-associated neuronal EV markers using either vesicle sorting or immunoprecipitation?

Many technical aspects may be improved. Such technical questions weakened the authors' conclusions.

Reviewer #1 (Public Review):

Summary:<br /> The authors used several zebrafish reporter lines to demonstrate the presence, regional distribution, and transcriptional profile of the immune cells in adult zebrafish brains. They identified DC-like cells distinct from microglia or other macrophages, resembling murine cDC1s. Analysis of different mutants further revealed that this DC population was dependent on Irf8, Batf3, and Csf1rb, but did not rely on Csf1ra.

Strengths:<br /> It is an elegantly designed study providing compelling evidence for further heterogeneity among brain mononuclear phagocytes in zebrafish, consisting of microglia, macrophages, and DC-like cells. This will provide a better understanding of the immune landscape in the zebrafish brain and will help to better distinguish the different cell types from microglia, and to assign specific functions.

Weaknesses:<br /> While scRNA-seq data clearly revealed different subsets of microglia, macrophages, and DCs in the brain, it remains somewhat challenging to distinguish DC-like cells from P2ry12- macrophages by immunohistochemistry or flow cytometry.

Reviewer #2 (Public Review):

The authors made an atlas of single-cell transcriptome of on a pure population of leukocytes isolated from the brain of adult Tg(cd45:DsRed) transgenic animals by flow cytometry. Seven major leukocyte populations were identified, comprising microglia, macrophages, dendritic-like cells, T cells, natural killer cells, innate lymphoid-like cells, and neutrophils. Each cluster was analyzed to characterize subclusters. Among lymphocytes, in addition to 2 subclusters expressing typical T cell markers, a group of il4+ il13+ gata3+ cells was identified as possible ILC2. This hypothesis is supported by the presence of this population in rag2KO fish, in which the frequency of lck and zap70+ cells is strongly reduced. The use of KO lines for such validations is a strength of this work (and the zebrafish model).

The subcluster analysis of mpeg1.1 + myeloid cells identified 4 groups of microglial cells, one novel group of macrophage-like cells (expressing s100a10b, sftpbb, icn, fthl27, anxa5b, f13a1b and spi1b), and several groups of DC like cells expressing the markers siglec15l, ccl19a.1, ccr7, id2a, xcr1a.1, batf3, flt3, chl1a and hepacam2. Combining these new markers and transgenic reporter fish lines, the authors then clarified the location of leukocyte subsets within the brain, showing for example that DC-like cells stand as a parenchymal population along with microglia. Reporter lines were also used to perform a detailed analysis of cell subsets, and cross with a batf3 mutant demonstrated that DC-like cells are batf3 dependent, which was similar to mouse and human cDC1. Finally, analysis of classical mononuclear phagocyte deficient zebrafish lines showed they have reduced numbers of microglia but exhibit distinct DC-like cell phenotypes. A weakness of this study is that it is mainly based on FACS sorting, which might modify the proportion of different subtypes.

This atlas of zebrafish brain leukocytes is an important new resource for scientists using the zebrafish models for neurology, immunology, and infectiology, and for those interested in the evolution of the brain and immune system.

Reviewer #3 (Public Review):

Rovira, et al., aim to characterize immune cells in the brain parenchyma and identify a novel macrophage population referred to as "dendritic-like cells". They use a combination of single-cell transcriptomics, immunohistochemistry, and genetic mutants to conclude the presence of this "dendritic-like cell" population in the brain. The strength of this manuscript is the identification of dendritic cells in the brain, which are typically found in the meningeal layers and choroid plexus. A weakness is the lack of specific reporters or labeling of this dendritic cell population using specific genes found in their single-cell dataset. Additionally, it is difficult to remove the meningeal layers from the brain samples and thus can lead to confounding conclusions. Overall, I believe this study should be accepted contingent on sufficient labeling of this population and addressing comments.

Reviewer #1 (Public Review):

Summary:<br /> This is a detailed description of the role of PKCδ in Drosophila learning and memory. The work is based on a previous study (Placais et al. 2017) that has already shown that for the establishment of long-term memory, the repetitive activity of MP1 dopaminergic neurons via the dopamine receptor DAMB is essential to increase mitochondrial energy flux in the mushroom body.

In this paper, the role of PKCδ is now introduced. PKCδ is a molecular link between the dopaminergic system and the mitochondrial pyruvate metabolism of mushroom body Kenyon cells. For this purpose, the authors establish a genetically encoded FRET-based fluorescent reporter of PKCδ-specific activity, δCKAR.

Strengths:<br /> This is a thorough study of the long-term memory of Drosophila. The work is based on the extensive, high-quality experience of the senior authors. This is particularly evident in the convincing use of behavioral assays and imaging techniques to differentiate and explore various memory phases in Drosophila. The study also establishes a new reporter to measure the activity of PKCδ - the focus of this study - in behaving animals. The authors also elucidate how recurrent spaced training sessions initiate a molecular gating mechanism, linking a dopaminergic punishment signal with the regulation of mitochondrial pyruvate metabolism. This advancement will enable a more precise molecular distinction of various memory phases and a deeper comprehension of their formation in the future.

Weaknesses:<br /> Apart from a few minor technical issues, such as the not entirely convincing visualisation of the localisation of a PKCδ reporter in the mitochondria, there are no major weaknesses. Likewise, the scientific classification of the results seems appropriate, although a somewhat more extensive discussion in relation to Drosophila would have been desirable.

Reviewer #2 (Public Review):

Summary<br /> This study deepens the former authors' investigations of the mechanisms involved in gating the long-term consolidation of an associative memory (LTM) in Drosophila melanogaster. After having previously found that LTM consolidation 1. costs energy (Plaçais and Préat, Science 2013) provided through pyruvate metabolism (Plaçais et al., Nature Comm 2017) and 2. is gated by the increased tonic activity in a type of dopaminergic neurons ('MP1 neurons') following only training protocol relevant for LTM, i.e. interspaced in time (Plaçais et al., Nature Neuro 2012), they here dig into the intra-cell signalling triggered by dopamine input and eventually responsible for the increased mitochondria activity in Kenyon Cells. They identify a particular PKC, PKCδ, as a major molecular interface in this process and describe its translocation to mitochondria to promote pyruvate metabolism, specifically after spaced training.

Methodological approach<br /> To that end, they use RNA interference against the isozyme PKCδ, in a time-controlled way and in the whole Kenyon cell populations or in the subpopulation forming the α/β lobe. This knock-down decreased the total PKCδ mRNA level in the brain by ca. 30%, and is enough to observe decreased in flies performances for LTM consolidation. Using Pyronic, a sensor for pyruvate for in vivo imaging, and pharmacological disruption of mitochondrial function, the authors then show that PKCδ knock-down prevents a high level of pyruvate from accumulating in the Kenyon cells at the time of LTM consolidation, pointing towards a role of PKCδ in promoting pyruvate metabolism. They further identify the PDH kinase PDK as a likely target for PKCδ since knocking down both PKCδ and PDK led to normal LTM performances, likely counterbalancing PKCδ knock-down alone.

To understand the timeline of PKCδ activation and to visualise its mitochondrial translocation in a subpart of Mushroom body lobes they imported in fruitfly the genetically-encoded FRET reporters of PKCδ, δCKAR, and mitochondria-δCKAR (Kajimoto et al 2010). They show that PKCδ is activated to the sensor's saturation only after spaced training, and not other types of training that are 'irrelevant' for LTM. Further, adding thermogenetic activation of dopaminergic neurons and RNA interference against Gq-coupled dopamine receptor to FRET imaging, they identify that a dopamine-triggered cascade is sufficient for the elevated PKCδ-activation.

Strengths and weaknesses<br /> The authors use a combination of new fluorescent sensors and behavioral, imaging, and pharmacological protocols they already established to successfully identify the molecular players that bridge the requirement for spaced training/dopaminergic neurons MP1 oscillatory activity and the increased metabolic activity observed during long-term memory consolidation.

The study is dense in new exciting findings and each methodological step is carefully designed. Almost all possible experiments one could think of to make this link have been done in this study, with a few exceptions that do not prevent the essential conclusions from being drawn.

The discussion is well conducted, with interesting parallels with mammals, where the possibility that this process takes place as well is yet unknown.

Impact<br /> Their findings should interest a large audience:<br /> They discover and investigate a new function for PKCδ in regulating memory processes in neurons in conjunction with other physiological functions, making this molecule a potentially valid target for neuropathological conditions. They also provide new tools in drosophila to measure PKCδ activation in cells. They identify the major players for lifting the energetic limitations preventing the formation of a long-term memory.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript reports the effects of a heterozygous mutation in the KCNT1 potassium channels on the properties of ion currents and the firing behavior of excitatory and inhibitory neurons in the cortex of mice expressing KCNT1-Y777H. In humans, this mutation as well as multiple other heterozygotic mutations produce very severe early-onset seizures and produce a major disruption of all intellectual function. In contrast, in mice, this heterozygous mutation appears to have no behavioral phenotype or any increased propensity to seizures. A relevant phenotype is, however, evident in mice with the homozygous mutation, and the authors have previously published the results of similar experiments with the homozygotes. As perhaps expected, the neuronal effects of the heterozygous mutation presented in this manuscript are generally similar but markedly smaller than the previously published findings on homozygotes. There are, however, some interesting differences, particularly on PV+ interneurons, which appear to be more excitable than wild type in the heterozygotes but more excitable in the heterozygotes. This raises the interesting question (which could be more explicitly discussed by the authors) as to whether the reported changes represent homeostatic events that suppress the seizure phenotype in the mouse heterozygotes or simply changes in excitability that do not reach the threshold for behavioral outcomes.

Strengths and Weaknesses:<br /> 1) The authors find that the heterozygous mutation in PV+ interneurons increases their excitability, a result that is opposite from their previous observation in neurons with the corresponding homozygous mutation. They propose that this results from the selective upregulation of a persistent sodium current INaP in the PV+ interneurons. While the observations are very interesting, there are three issues concerning this interpretation that should be addressed:<br /> A) The protocol for measuring the INaP current could potentially lead to results that could be (mis)interpreted in different ways in different cells. First, neither K currents nor Ca currents are blocked in these experiments. Instead, TTX is applied to the cells relatively rapidly (within 1 second) and the ramp protocol is applied immediately thereafter. It is stated that, at this time, Na currents and INaP are fully blocked but that any effects on Na-activated K currents are minimal. In theory, this would allow the pre- to post-difference current to represent a relatively uncontaminated INaP. This would, however, only work if activation of KNa currents following Na entry is very slow, taking many seconds. A good deal of literature has suggested that the kinetics of activation of KNa currents by Na influx vary substantially between cell types, such that single action potentials and single excitatory synaptic events rapidly evoke KNa currents in some cell types. This is, of course, much faster than the time of TTX application. Most importantly, the kinetics of KNa activation may be different in different neuronal types, which would lead to errors that could produce different estimates of INaP in PV+ interneurons vs other cell types.<br /> B) As the authors recognize, INaP current provides a major source of cytoplasmic sodium ions for the activation. An expected outcome of increased INaP is, therefore, further activation of KNa currents, rather than a compensatory increase in an inward current that counteracts the increase in KNa currents, as is suggested in the discussion.<br /> C) Numerical simulations, in general, provide a very useful way to evaluate the significance of experimental findings. Nevertheless, while the in-silico modeling suggests that increases in INaP can increase firing rate in models of PV+ neurons, there is as yet insufficient information on the relative locations of the INaP channels and the kinetics of sodium transfer to KNa channels to evaluate the validity of this specific model.

2) The greatest effect of TTX application would be expected to be the elimination of large transient inward sodium currents. Why are no such currents visible in the control (pre-TTX) or the difference currents (Fig. 2)? Is it possible I missed something in the methods?

3) As expected, the changes in many of the measured parameters are smaller in the present study with heterozygotes than those previously reported for the homozygous mutation. Some of the statements on the significance of some of the present findings need to be stated more clearly. For example, in the results section describing Fig. 2, it is stated that "In glutamatergic and NFS GABAergic YH-HET neurons, the overall KNa current was increased ...as measured by a significant effect of genotype ...." Later in the same paragraph it is stated that the increases in KNa current are not significant. Apparently, different tests lead to different conclusions. Both for the purpose of understanding the pathophysiological effects of changes in KNa current and for making further numerical simulations, more explicit clarifying statements should be made.

4) The effects of the KCNT1 channel blocker VU170 on potassium currents are somewhat larger and different from those of TTX, suggesting that additional sources of sodium may contribute to activating KCNT1, as suggested by the authors. Because VU170 is, however, a novel pharmacological agent, it may be appropriate to make more careful statements on this. While the original published description of this compound reported no effect on a variety of other channels, there are many that were not tested, including Na and cation channels that are known to activate KCNT1, raising the possibility of off-target effects.

5) The experiments were carried out at room temperature. Is it possible that different effects on firing patterns in heterozygotes and homozygotes would be observed at more physiological temperatures?

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Shore et al. investigate the consequent changes in excitability and synaptic efficacy of diverse neuronal populations in an animal model of juvenile epilepsy. Using electrophysiological patch-clamp recordings from dissociated neuronal cultures, the authors find diverging changes in two major populations of inhibitory cell types, namely somatostatin (SST)- and parvalbumin (PV)-positive interneurons, in mice expressing a variant of the KCNT1 potassium channel. They further suggest that the differential effects are due to a compensatory increase in the persistent sodium current in PV interneurons in pharmacological and in silico experiments.

Strengths:<br /> 1) Heterozygous KCNT1 gain of function variant was used which more accurately models the human disorder.<br /> 2) The manuscript is clearly written, and the flow is easy to follow. The authors explicitly state the similarities and differences between the current findings and the previously published results in the homozygous KCNT1 gain of function variant.<br /> 3) This study uses a variety of approaches including patch clamp recording, in silico modeling, and pharmacology that together make the claims stronger.<br /> 4) Pharmacological experiments are fraught with off-target effects and thus it bolsters the authors' claims when multiple channel blockers (TTX and VU170) are used to reconstruct the sodium-activated potassium current. Having said that, it would be helpful to see the two drug manipulations be used in the same experiment. Notably, does the more selective blocker VU170 mimic the results of TTX for NFS GABAergic cells in Figure 2? And does it unmask a genotype difference for FS GABAergic cells like the one seen in PV interneurons in Figure 5C3.

Weaknesses:<br /> 1) This study relies on recordings in dissociated cortical neurons. Although specific WT interneurons showed intrinsic membrane properties like those reported for acute brain slices, it is unclear whether the same will be true for those cells expressing KCNT1 variants. This reviewer highly recommends confirming some of the key findings using an ex vivo slice preparation. This is especially important given the discrepant result of reduced excitability of PV cells reported by Gertler et al., 2022 (cited here in the manuscript but not discussed in this context) in acute hippocampal slices for a different KCTN1 gain of function variant.<br /> 2) It is unclear how different pieces of results fit together to form a story about the disease pathophysiology. For example, hyperexcitability of PV cells would suggest more inhibition which would counter seizure propensity. However, spontaneous inhibitory postsynaptic currents show no change in pyramidal neurons. Moreover, how do the authors reconcile that the reductions in synaptic inputs onto interneurons in Figure 3B with the increases in Figure 8? This should be discussed.<br /> 3) Similarly, the results in this work are not entirely internally consistent. For example, given the good correspondence between FS and NFS GABAergic cells with PV and SST expression, why are FS GABAergic cells hyperexcitable in Figure 1? If anything, there is a tendency to show reduced excitability like the NFS GABAergic cells. Also, why do the WT I-V curves look so different between Figures 2 and 5? This reviewer suggests at least a brief explanation in the discussion.<br /> 4) Given the authors' claim that the KCNT1 activation curve is a major contributor to the observed excitability differences in specific GABA cell subtypes, it would be helpful to directly measure the activation curve in the variants experimentally as was done for WT KCNT1 in Figure 6A and use the derived kinetics in the compartmental model.

Reviewer #3 (Public Review):

Summary:<br /> The present manuscript by Shore et al. entitled Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy" describes in vitro and in silico results obtained in cortical neurons from mice carrying the KCNT1-Y777H gain-of-function (GOF) variant in the KCNT1 gene encoding for a subunit of the Na+-activated K+ (KNa) channel. This variant corresponds to the human Y796H variant found in a family with Autosomal Dominant Nocturnal Frontal lobe epilepsy. The occurrence of GOF variants in potassium channel encoding genes is well known, and among potential pathophysiological mechanisms, impaired inhibition has been documented as responsible for KCNT1-related DEEs. Therefore, building on a previous study by the same group performed in homozygous KI animals, and considering that the largest majority of pathogenic KCNT1 variants in humans occur in heterozygosis, the Authors have investigated the effects of heterozygous Kcnt1-Y777H expression on KNa currents and neuronal physiology among cortical glutamatergic and the 3 main classes of GABAergic neurons, namely those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), crossing KCNT1-Y777H mice with PV-, SST- and PV-cre mouse lines, and recording from GABAergic neurons identified by their expression of mCherry (but negative for GFP used to mark excitatory neurons).

The results obtained revealed heterogeneous effects of the variant on KNa and action potential firing rates in distinct neuronal subpopulations, ranging from no change (glutamatergic and VIP GABAergic) to decreased excitability (SST GABAergic) to increased excitability (PV GABAergic). In particular, modelling and in vitro data revealed that an increase in persistent Na current occurring in PV neurons was sufficient to overcome the effects of KCNT1 GOF and cause an overall increase in AP generation.

Strengths:<br /> The paper is very well written, the results clearly presented and interpreted, and the discussion focuses on the most relevant points.

The recordings performed in distinct neuronal subpopulations are a clear strength of the paper. The finding that the same variant can cause opposite effects and trigger specific homeostatic mechanisms in distinct neuronal populations is very relevant for the field, as it narrows the existing gap between experimental models and clinical evidence.

Weaknesses:<br /> My main concern is in the epileptic phenotype of the heterozygous mice investigated. In fact, in their previous paper the Authors state that "...Kcnt1-Y777H heterozygous mice did not exhibit any detectable epileptiform activity" (first sentence on page 4). However, in the present manuscript, they indicate twice in the discussion section that these mice exhibit "infrequent seizures". This relevant difference needs to be clarified to correctly attribute to the novel pathophysiological mechanism a role in seizure occurrence. Were such infrequent seizures clearly identified on the EEG, or were behavioral seizures? Could the authors quantify this "infrequent" value? This is crucial also to place in the proper perspective the Discussion statement regarding "... the increased INaP contribution to ... network hyperexcitability and seizures".

Also, some statistical analysis seems to be missing. For example, I could not find any for the data shown in Fig. 6. Thus, the following statement: "the model PV neurons responded to KCNT1 GOF with decreased AP firing and an increased rheobase" requires proper statistical evaluation.

Reviewer #1 (Public Review):

Summary: This paper reported interesting aberrant calcium microwaves in the hippocampus when synapsin promoter driven GCaMPs were expressed for a long period of time. These aberrant hippocampal Ca2+ micro-waves depend on the viral titre of the GECI. The microwave of Ca2+ was not observed when GECI was expressed only in a sparse set of neurons.

Strengths: These findings are important to the wide neuroscience community, especially considering a great number of investigators are using similar approaches. Results look convincing and are consistent across several laboratories.

Weaknesses: One important question is needed to further clarify the mechanisms of aberrant Ca2+ microwaves as described below.

Synapsin promoter labels both excitatory pyramidal neurons and inhibitory neurons. To avoid aberrant Ca2+ microwave, a combination of Flex virus and CaMKII-Cre or Thy-1-GCaMP6s and 6f mice were tested. However, all these approaches limit the number of infected pyramidal neurons. While the comprehensive display of these results is appreciated, a crucial question remains unanswered. To distinguish whether the microwave of Ca2+ is caused selectively via the abnormality of interneurons, or just a matter of pyramidal neuron density, testing Flex-GCaMP6 in interneuron specific mouse lines such as PV-Cre and SOM-Cre will be critical.

Reviewer #2 (Public Review):

Summary:

The authors describe and quantify a phenomenon in the CA1 and CA3 of the hippocampus that they call aberrant Ca2+ micro-waves. Micro-waves are sometimes seen during 2-photon calcium imaging of populations of neurons under certain conditions. They are spatially confined slow calcium events that start in a few cells and slowly spread to neighboring groups of cells. This phenomenon has been uttered between researchers in the field at conferences, but no one has taken the time to carefully capture and quantify micro-waves and pin down the causes. The authors show that micro-waves are dependent on the viral titre of the genetically encoded calcium indicators (GECIs), the genetic promoter (synapsin), the neuronal subtype (granule cells in the dentate gyrus do not produce micro-waves and they are not seen in the neocortex), and the density of GECI expression. The authors should be commended for their work and raising awareness to all labs doing any form of calcium imaging in populations of neurons. The authors also come up with alternative approaches to avoid artifactual micro-waves such as reducing the transduction titre (1:2 dilution of virus) and a transduction method employing sparser and cre-dependent GECI expression in principal cells using a CaMKII promoter.

Strengths:

The micro-waves reported in the paper were robustly observed across 4 laboratories and 3 different countries with various experimenters and calcium imaging set-ups. This adds significant strength to the work.

The age of mice used covered a broad range (from 6 to 43 weeks). This is a strength because is covers most ages that are used in labs that regularly do calcium imaging.

Another strength is they used different GCaMP variants (GCaMP6m, GCaMP6s, GCaMP7f), as well a red indicator: RCaMP. This shows the micro-waves are not an issue with any particular GECI, as the authors suggest.

The authors include many movies of micro-waves. This is extremely useful for researchers in the field to view them in real-time so they can identify them in their own data.

They provide a useful table with specific details of the virus injected, titre, dilution, and other information along with the incidence of micro-waves. A nice look-up table for researchers to see if their viral strategy is associated with a high or low incidence of micro-waves.

Weaknesses:

Whether micro-waves are associated with the age of mice was not quantified. This would be good to know and the authors do have this data.

The effect of mico-waves on single cell function was not analyzed. It would be useful, for example, if we knew the influence of micro-waves on place fields. Can a place cell still express a place field in a hippocampus that produces micro-waves? What effect might a microwave passing over a cell have on its place field? Mice were not trained in these experiments, so the authors do not have the data.

The CaMKII-Cre approach for flexed-syn-GCaMP expression shows no micro-waves and is convincing, but it is only from 2 animals, even though both had no micro-waves.

The authors state in their Discussion that even without observable microwaves, a syn-Ca2+-indicator transduction strategy could still be problematic. This may be true, but they do not check this in their analysis, so it remains unknown.

Reviewer #3 (Public Review):

Summary:<br /> The work by Masala and colleagues highlights a striking artifact that can result from a particular viral method for expressing genetically encoded calcium indicators (GECIs) in neurons. In a cross-institutional collaboration, the authors find that viral transduction of GECIs in the hippocampus can result in aberrant slow-traveling calcium (Ca2+) micro-waves. These Ca2+ micro-waves are distinct from previously described ictal activity but nevertheless are likely a pathological consequence of overexpression of virally transduced proteins. Ca2+ micro-waves will most likely obscure the physiology that most researchers are interested in studying with GECIs, and their presence indicates that the neural circuit is in an unintended pathological state. Interestingly this pathology was not observed using the same viral transduction methods in the visual cortex. The authors recommend several approaches that may help other experimenters avoid this confound in their own data such as reducing the titer of viral injections or using recombinase-dependent expression. The intent of this manuscript is to raise awareness of the potential unintended consequences of viral overexpression, particularly for GECIs. A rigorous investigation into the exact causes of Ca2+ micro-waves or the mech

Strengths:

The authors clearly demonstrate that Ca2+ micro-waves occur in the CA1 and CA3 regions of the hippocampus following large volume, high titer injections of adeno-associated viruses (AAV1 and AAV9) encoding GECIs. The supplementary videos provide undeniable proof of their existence.

By forming an inter-institutional collaboration, the authors demonstrate that this phenomenon is robust to changes in surgical techniques or imaging conditions.

Weaknesses:

I believe that the weaknesses of the manuscript are appropriately highlighted by the authors themselves in the discussion. I would, however, like to emphasize several additional points.

As the authors state, the exact conditions that lead to Ca2+ micro-waves are unclear from this manuscript. It is also unclear if Ca2+ micro-waves are specific to GECI expression or if high-titer viral transduction of other proteins such as genetically encoded voltage indicators, static fluorescent proteins, recombinases, etc could also cause Ca2+ micro-waves.

The authors almost exclusively tested high titer (>5x10^12 vg/mL) large volume (500-1000 nL) injections using the synapsin promoter and AAV1 serotypes. It is possible that Ca2+ micro-waves are dramatically less frequent when titers are lowered further but still kept high enough to be useful for in vivo imaging (e.g. 1x10^12 vg/mL) or smaller injection volumes are used. It is also possible that Ca2+ micro-waves occur with high titer injections using other viral promoter sequences such as EF1α or CaMKIIα. There may additionally be effects of viral serotype on micro-wave occurrence.

The number of animals in any particular condition are fairly low (Table 1) with the exception of V1 imaging and thy1-GCaMP6 imaging. This prohibits rigorous comparison of the frequency of pathological calcium activity across conditions.

Reviewer #1 (Public Review):

Summary:<br /> This study delves into the roles of dact1 and dact2 during zebrafish embryonic axis formation and craniofacial morphogenesis. The researchers seek to unravel the mechanisms by which dact1/2 influences Wnt signaling modulation throughout embryonic development and patterning. They propose distinct spatiotemporal roles for Dact1 and Dact2 proteins in zebrafish embryonic development, highlighting their involvement in modulating noncanonical Wnt signaling during convergent extension events. Their findings demonstrate that dact1 and dact2 exhibit distinct spatiotemporal expression domains during development and that dact1/2 mutation leads to convergent extension defects. Furthermore, the study attempts to establish a link between convergent extension defects resulting from dact1/2 mutation and subsequent craniofacial abnormalities during development. To investigate the connection between dact1 and dact2, compound mutants were employed since single mutants did not exhibit craniofacial phenotypes. Additionally, the research encompasses comprehensive transcriptomics and pathway analyses of differentially expressed genes in dact1/2 mutants. This analysis reveals the overexpression of a calcium-dependent cysteine protease, calpain 8. The study suggests a connection between the upregulation of calpain 8 and the observed craniofacial dysmorphology in dact1/2 mutants, implying a potential link between the altered expression of calpain 8 and the craniofacial abnormalities observed in these mutants.

Strengths:<br /> The study beautifully recapitulates previous findings on the role of dact1/2 in modulating convergent extension during zebrafish embryogenesis.

A combination of multiple approaches, including in vivo time-lapse imaging, has been employed to elucidate the etiology of the rod-like neurocranial phenotype in dact1/2 double mutant.<br /> This study utilizes and discusses several 'traditional' mutant lines and newly created ones, analyzing them through single-cell transcriptomics.

Weaknesses:<br /> 1. Enhancing Reproducibility and Robustness:<br /> To enhance the reproducibility and robustness of the findings, it would be valuable for the authors to provide specific numbers of animals used in each experiment.<br /> Explicitly stating the penetrance of the rod-like neurocranial shape in dact1/2-/- animals would provide a clearer understanding of the consistency of this phenotype.

2. Strengthening Single-Cell Data Interpretation:<br /> To further validate the single-cell data and strengthen the interpretation of the gene expression patterns, I recommend the following:<br /> -Provide a more thorough explanation of the rationale for comparing dact1/2 double mutants with gpc4 mutants.<br /> -Employ genotyping techniques after embryo collection to ensure the accuracy of animal selection based on phenotype and address the potential for contamination of wild-type "delayed" animals.<br /> -Supplement the single-cell data with secondary validation using RNA in situ or immunohistochemistry techniques.

3. Directly Investigating Non-Cell-Autonomous Effects:<br /> To directly assess the proposed non-cell-autonomous role of dact1/2, I suggest conducting transplantation experiments to examine the ability of ectodermal/neural crest cells from dact1/2 double mutants to form wild-type-like neurocranium.

4. Further Elucidating Calpain 8's Role:<br /> To strengthen the evidence supporting the critical role of Calpain 8, I recommend conducting overexpression experiments using a sensitized background to enhance the statistical significance of the findings.

Reviewer #2 (Public Review):

Summary:<br /> Non-canonical Wnt signaling plays an important role in morphogenesis, but how different components of the pathway are required to regulate different developmental events remains an open question. This paper focuses on elucidating the overlapping and distinct functions of dact1 and dact2, two Dishevelled-binding scaffold proteins, during zebrafish axis elongation and craniofacial development. By combining genetic studies, detailed phenotypic analysis, lineage tracing, and single-cell RNA-sequencing, the authors aimed to understand (1) the relative function of dact1/2 in promoting axis elongation, (2) their ability to modulate phenotypes caused by mutations in other non-canonical wnt components, and (3) pathways downstream of dact1/2.

Strong qualitative evidence was provided to support dact1/2's role in genetically modulating non-canonical wnt signaling to regulate body axis elongation and the morphology of the anterior neurocranium (ANC). However, there is currently insufficient evidence supporting the author's claim that suppression of calpain 8 by dact1/2 is important for craniofacial development and that "embryonic fields determined during gastrulation affect the CNCC ability to contribute to the craniofacial skeleton".

Strengths:<br /> (1) The generation of dact1/2 germline mutants and the use of genetic approaches to dissect their genetic interactions with wnt11f2 and gpc4 provide unambiguous and consistent results that inform the relative functions of dact1 and dact2, as well as their combined effects.

(2) Because the anterior neurocranium exhibits a spectrum of phenotypes in different genetic mutants, it is a useful system for studying how tissue morphology can be modulated by different components of the same pathway, as demonstrated in this study.

(3) The authors leveraged lineage tracing by photoconversion to dissect how dact1/2 differentially impacts the ability of different cranial neural crest populations to contribute to the anterior neurocranium. This revealed that distinct mechanisms can lead to similar phenotypes in different mutants.

Weaknesses:<br /> (1) While the qualitative data show altered morphologies in each mutant, quantifications of these phenotypes are lacking in several instances, making it difficult to gauge reproducibility and penetrance, as well as to assess the novel ANC forms described in certain mutants.

(2) Germline mutations limit the authors' ability to study a gene's spatiotemporal functional requirement. They therefore cannot concretely attribute nor separate early-stage phenotypes (during gastrulation) to/from late-stage phenotypes (ANC morphological changes).

(3) Given that dact1/2 can regulate both canonical and non-canonical wnt signaling, this study did not specifically test which of these pathways is altered in the dact1/2 mutants, and it is currently unclear whether disrupted canonical wnt signaling contributes to the craniofacial phenotypes, even though these phenotypes are typical non-canonical wnt phenotypes.

(4) The use of single-cell RNA sequencing unveiled genes and processes that are uniquely altered in the dact1/2 mutants, but not in the gpc4 mutants during gastrulation. However, how these changes lead to the manifested ANC phenotype later during craniofacial development remains unclear. The authors showed that calpain 8 is significantly upregulated in the mutant, but the fact that only 1 out of 142 calpain-overexpressing animals phenocopied dact1/2 mutants indicates the complexity of the system.

(5) Craniofacial phenotypes observed in this study are attributed to convergent extension defects but convergent extension cell movement itself was not directly examined, leaving open if changes in other cellular processes, such as cell differentiation, proliferation, or oriented division, could cause distinct phenotypes between different mutants.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors explore the roles of dact1 and dact2 during zebrafish gastrulation and craniofacial development. Previous studies used morpholino (MO) knockdowns to show that these scaffolding proteins, which interact with disheveled (Dsh), are expressed during zebrafish gastrulation and suggested that dact1 promotes canonical Wnt/B-catenin signaling, while dact2 promotes non-canonical Wnt/PCP-dependent convergent-extension (Waxman et al 2004). This study goes beyond this work by creating loss-of-function mutant alleles for each gene and unlike the MO studies finds little (dact2) to no (dact1) phenotypic defects in the homozygous mutants. Interestingly, dact1/2 double mutants have a more severe phenotype, which resembles those reported with MOs as well as homozygous wnt11/silberblick (wnt11/slb) mutants that disrupt non-canonical Wnt signaling (Heisenberg et al., 1997; 2000). Further analyses in this paper try to connect gastrulation and craniofacial defects in dact1/2 mutants with wnt11/slb and other wnt-pathway mutants. scRNAseq conducted in mutants identifies calpain 8 as a potential new target of dact1/2 and Wnt signaling.

Strengths:<br /> When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:<br /> The hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result of overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.

Major Comments:<br /> 1) One major problem throughout the paper is that the authors misrepresent the fact that wnt11f2 and gpc4 act in different cell populations at different times. Gastrulation defects in these mutants are not similar: wnt11 is required for anterior mesoderm CE during gastrulation but not during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and later craniofacial cartilage morphogenesis (LeClair et al., 2009). Overall, the non-overlapping functions of wnt11 and gpc4, both temporally and spatially, suggest that they are not part of the same pathway.

2) There are also serious problems surrounding attempts to relate single-cell data with the other data in the manuscript and many claims that lack validation. For example, in Fig 1 it is entirely unclear how the Daniocell scRNA-seq data have been used to compare dact1/2 with wnt11f2 or gpc4. With no labeling in panel 1E of this figure these comparisons are impossible to follow. Similarly, the comparisons between dact1/2 and gpc4 in scRNA-seq data in Fig. 6 as well as the choices of DEGs in dact1/2 or gpc4 mutants in Fig. 7 seem arbitrary and do not make a convincing case for any specific developmental hypothesis. Are dact1 and gpc4 or dact2 and wnt11 co-expressed in individual cells? Eyeballing similarity is not acceptable.

3) Many of the results in the paper are not novel and either confirm previous findings, particularly Waxman et al (2004), or even contradict them without good evidence. The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription or vice versa. Testing genetic interactions, including investigating the expression of wnt11f2 in dact1/2 mutants, dact1/2 expression in wnt11f2 mutants, or the ability of dact1/2 to rescue wnt11f2 loss of function would give this work a more novel, mechanistic angle.

4) The identification of calpain 8 overexpression in Dact1/2 mutants is interesting, but getting 1/142 phenotypes from mRNA injections does not meet reproducibility standards.

Reviewer #2 (Public Review):

George and colleagues present a novel open-source toolbox to model rodent locomotor patterns and electrophysiological responses of spatially modulated neurons, such as hippocampal "place cells". The present manuscript describes a comprehensive Python package ("RatInABox") with powerful capabilities to simulate a variety of environments, exploratory behaviors and concurrent responses of a variety of cell types. In addition, they provide the tools to expand these basics functions and potentially multiple different model designs, new cell types or more complex neural network architectures. The manuscript also illustrated several simple application cases. The authors have also created a comprehensive GitHub repository with more detailed explanations, tutorials and example scripts. Overall, I found both the manuscript and associated repository very clear, well written and easy the scrips easy to follow and implement, to a superior level of many commercial software packages. RatInABox fills several existing gaps in the literature and features important improvements over previous approaches; for example, the implementation of continuous 2D environments instead of tabularized state spaces. I believe this toolbox will be of great interest for many researchers in the field of spatial navigation and beyond and provide them with a remarkably powerful and flexible tool. I don't have any major issues with the manuscript. However, the manuscript can be further improved by clarifying some aspects of the toolbox, discussing its limitations and biological plausibility.

Reviewer #1 (Public Review):

In this work Wu, J., et al., highlight the importance of a previously overlooked region on kinases: the αC-β4 loop. Using PKA as a model system, the authors extensively describe the conserved regulatory elements within a kinase and how the αC-β4 loop region integrates with these important regulatory elements. Previous biochemical work on a mutation within the αC-β4 loop region, F100A showed that this region is important for the synergistic high affinity binding of ATP and the pseudo substrate inhibitor PKI. In the current manuscript, the authors assess the importance of the αC-β4 loop region using computational methods such as Local Spatial Pattern Alignment (LSP) and MD simulations. LSP analysis of the F100A mutant showed decreased values for degree centrality and betweenness centrality for several key regulatory elements within the kinase which suggests a loss in stability/connectivity in the mutant protein as compared to the WT. Additionally, based on MD simulation data, the side chain of K105, another residue within the αC-β4 loop region had altered dynamics in the F100A mutant as compared to the WT protein. While these changes in the αC-β4 loop region seem to be consistent with the previous biochemical data, the manuscript can be strengthened with additional experiments.

Comments on the revised version:

Additional experiments (both computational and experimental) assessing the role of the αC-β4 loop region (especially residues such as K105) are needed to bolster their hypothesis. My initial assessment therefore remains unchanged. While this manuscript falls short of expectations when it comes to experimental findings, it is an excellent review on the structural elements of kinases and how the newly identified αC-β4 loop region integrates with these important regions. Perhaps the experimental section (LSP analysis and MD simulation data) could be removed and this manuscript could be converted into a Review Article?

Reviewer #1 (Public Review):

Summary:

The authors try to use a gene therapy approach to cure urofacial symptoms in an HSPE2 mutant mouse model.

Strengths:

The authors have convincingly shown the expression of AAV9/HSPE2 in pelvic ganglion and liver tissues. They have also shown the defects in urethra relaxation and bladder muscle contraction in response to EFS in mutant mice, which were reversed in treated mice.

Weaknesses:

Some important and interesting data are missing. For example, whether the gene therapy can extend the life span of these mutants? The overall in vivo voiding function is missing. AAV9/HSPE2 expression in the bladder wall is not shown.

Reviewer #2 (Public Review):

In this study, Lopes and colleagues provide convincing evidence to support the potential for gene therapy to restore expression of heparanase-2 (Hpse2) in mice mutant for this gene, as occurs in urofacial syndrome. Beyond symptomatic relief for the consequences of outlet obstruction that results from Hpse2 mutation, no treatments exist. Building on prior studies describing the nature of urinary tract dysfunction in Hpse2 mutant mice, the authors applied a gene therapy approach to determine whether gene replacement could be achieved, and if so, whether restoration of HPSE2 expression could mitigate the urinary tract dysfunction and present a potential cure. Using an AAV9 viral vector encoding HPSE2, the authors performed gene replacement in neonatal wild-type or Hpse2 mutant mice and determined gene and protein expression as well as the impact on bladder outflow tract and bladder body physiology in juvenile mice. In addition to dose-dependent transduction of liver and pelvic ganglia (that innervate the bladder) with HPSE2, and demonstration of increased HPSE2 protein in Hpse2 mutant mice, the authors showed restoration of nerve-evoked outflow tract relaxation and bladder body contraction, both of which were deficient in mutant mice. They also showed that the viral vector-based approach was not deleterious to weight gain or to liver morphology. Based on these findings the authors concluded that AAV9-based HPSE2 replacement is feasible and safe, mitigates the physiological deficits in outflow tract and bladder tissue from Hpse2 mutant mice, and provides a foundation for gene replacement approaches for other genes implicated in lower urinary tract disorders.

Strengths include a rigorous experimental design, solid data in support of the conclusions, and a discussion of the limitations of the approach.

Weaknesses include a lack of discussion of the basis for differences in carbachol sensitivity in Hpse2 mutant mice, limited discussion of bladder tissue morphology in Hpse2 mutant mice, some questions over the variability of the functional data, and a need for clarification on the presentation of statistical significance of functional data

Reviewer #3 (Public Review):

Summary:

This is a really interesting study, looking at the efficacy of AAV-mediated delivery of wt HSPE2 gene into mouse mutants with the goal of rescuing lower urinary tract defects.

Strengths: Nice analysis of muscle physiology ex vivo, interesting approach.

Weaknesses: lack of rigor (see below). This is an awesome opportunity to learn much more about the disease, its affects on neurons, muscle, etc.

* Single-cell analysis of mutants versus control bladder, urethra including sphincter. This would be great also for the community.

* Detailed tables showing data from each mouse examined.

* Survival curves.

* Use of measurements that are done in vivo (spot assay for example). This sounds relatively simple.

* Assessment of viral integration in tissues besides the liver (could be done by QPCR).

* Discuss subtypes of neurons that are present and targeted in the context of mutants and controls.

Reviewer #3 (Public Review):

Summary of the findings:

The authors explore an important question concerning the underlying mechanism of representational drift, which despite intense recent interest remains obscure. The paper explores the intriguing hypothesis that drift may reflect changes in the intrinsic excitability of neurons. The authors set out to provide theoretical insight into this potential mechanism.

They construct a rate model with all-to-all recurrent connectivity, in which recurrent synapses are governed by a standard Hebbian plasticity rule. This network receives a global input, constant across all neurons, which can be varied with time. Each neuron also is driven by an "intrinsic excitability" bias term, which does vary across cells. The authors study how activity in the network evolves as this intrinsic excitability term is changed.

They find that after initial stimulation of the network, those neurons where the excitability term is set high become more strongly connected and are in turn more responsive to the input. Each day the subset of neurons with high intrinsic excitability is changed, and the network's recurrent synaptic connectivity and responsiveness gradually shift, such that the new high intrinsic excitability subset becomes both more strongly activated by the global input and also more strongly recurrently connected. These changes result in drift, reflected by a gradual decrease across time in the correlation of the neuronal population vector response to the stimulus.

The authors are able to build a classifier that decodes the "day" (i.e. which subset of neurons had high intrinsic excitability) with perfect accuracy. This is despite the fact that the excitability bias during decoding is set to 0 for all neurons, and so the decoder is really detecting those neurons with strong recurrent connectivity, and in turn strong responses to the input. The authors show that it is also possible to decode the order in which different subsets of neurons were given high intrinsic excitability on previous "days". This second result depends on the extent by which intrinsic excitability was increased: if the increase in intrinsic excitability was either too high or too low, it was not possible to read out any information about the past ordering of excitability changes.

Finally, using another Hebbian learning rule, the authors show that an output neuron, whose activity is a weighted sum of the activity of all neurons in the network, is able to read out the activity of the network. What this means specifically, is that although the set of neurons most active in the network changes, the output neuron always maintains a higher firing rate than a neuron with randomly shuffled synaptic weights, because the output neuron continuously updates its weights to sample from the highly active population at any given moment. Thus, the output neuron can read out a stable memory despite drift.

Strengths:

The authors are clear in their description of the network they construct and in their results. They convincingly show that when they change their "intrinsic excitability term", upon stimulation, the Hebbian synapses in their network gradually evolve, and the combined synaptic connectivity and altered excitability result in drifting patterns of activity in response to an unchanging input (Fig. 1, Fig. 2a). Furthermore, their classification analyses (Fig. 2) show that information is preserved in the network, and their readout neuron successfully tracks the active cells (Fig. 3). Finally, the observation that only a specific range of excitability bias values permits decoding of the temporal structure of the history of intrinsic excitability (Fig. 2f and Figure S1) is interesting, and as the authors point out, not trivial.

Weaknesses:

1) The way the network is constructed, there is no formal difference between what the authors call "input", Δ(t), and what they call "intrinsic excitability" Ɛ_i(t) (see Equation 3). These are two separate terms that are summed (Eq. 3) to define the rate dynamics of the network. The authors could have switched the names of these terms: Δ(t) could have been considered a global "intrinsic excitability term" that varied with time and Ɛ_i(t) could have been the external input received by each neuron in the network. In that case, the paper would have considered the consequence of "slow fluctuations of external input" rather than "slow fluctuations of intrinsic excitability", but the results would have been the same. The difference is therefore semantic. The consequence is that this paper is not necessarily about "intrinsic excitability", rather it considers how a Hebbian network responds to changes in excitatory drive, regardless of whether those drives are labeled "input" or "intrinsic excitability".

A revised version of the manuscript models "slope-based" excitability changes in addition to "threshold-based" changes. This serves to address the above concern that as constructed here changes in excitability threshold are not distinguishable from changes in input. However, it remains unclear what the model would do should only a subset of neurons receive a given, fixed input. In that case, are excitability changes sufficient to induce drift? This remains an important question that is not addressed by the paper in its current form.

2) Given how the learning rule that defines the input to the readout neuron is constructed, it is trivial that this unit responds to the most active neurons in the network, more so than a neuron assigned random weights. What would happen if the network included more than one "memory"? Would it be possible to construct a readout neuron that could classify two distinct patterns? Along these lines, what if there were multiple, distinct stimuli used to drive this network, rather than the global input the authors employ here? Does the system, as constructed, have the capacity to provide two distinct patterns of activity in response to two distinct inputs?

A revised version of the manuscript addresses this question, demonstrating that the network is capable of maintaining two distinct memories.

Impact:

Defining the potential role of changes in intrinsic excitability in drift is fundamental. Thus, this paper represents an important contribution. What we see here is that changes in intrinsic excitability are sufficient to induce drift. This raises the question for future work of the specific contributions of changing excitability from changing input to representational drift.

Reviewer #1 (Public Review):

Summary<br /> Here the authors have tethered a Pgp substrate to strategically place cysteine residues in the protein. Notably, the cysteine-linked substate (ANC-DNPT)- stimulate ATP hydrolyse and so are able to undergo IF to OF transitions. The authors then determined cryo-EM structures of these complexes and MD simulations of bound states. By capturing unforeseen OF conformations with substate they propose that TM1 undergoes local conformational changes that are sufficient to translocate substrates, rather than large bundle movements.

Strengths: This paper provides the first substrate (ANC-DNPT)- bound conformations of PgP and a new mechanistic model of how substrates are translocated.

Weaknesses: Although the cross-links stimulate ATP hydrolysis, it is unclear if the TM1 conformations are exactly the same under physiological conditions, since they have been covalently-trapped to the substrate.

Reviewer #3 (Public Review):

Summary: The authors used cross-linking of a known P-gp substrate in combination with single particle cyro-EM to investigate the translocation pathway of this important ABC transporter. Based on the results of this study, a new translocation mechanism is proposed that is supported by the data. While only one substrate was used, the data obtained are convincing. In addition, the proposed model will stimulate new experiments from other laboratories to proof or disproof the model.

Strengths: the combination of cross-linking and structural biology allowed novel insights in the translocation pathway of ABCB1

Weaknesses: While only one substrate was used, the data obtained are convincing. In addition, the proposed model will stimulate new experiments from other laboratories to proof or disproof the model.

Joint Public Review:

The authors previously showed that expressing formate dehydrogenase, rubisco, carbonic anhydrase, and phosphoribulokinase in Escherichia coli, followed by experimental evolution, led to the generation of strains that can metabolise CO2. Using two rounds of experimental evolution, the authors identify mutations in three genes - pgi, rpoB, and crp - that allow cells to metabolise CO2 in their engineered strain background. The authors make a strong case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle. The authors also use proteomic analysis to probe the role of the mutations in crp and rpoB. While they do not reach strong conclusions about how these mutations promote autotrophic growth, they provide some clues, leading to valuable speculation.

Comments on revised version:

The authors have thoroughly addressed the reviewers' comments. The major addition to the paper is the proteomic analysis of single and double mutants of crp and rpoB. These new data provide clues as to the role of the crp and rpoB mutations in promoting autotrophic growth, which the authors discuss. The authors acknowledge that it will require additional experiments to determine whether the speculated mechanisms are correct. Nonetheless, the new data provide valuable new insight into the role of the crp and rpoB mutations. The authors have also expanded their description of the crp and rpoB mutations, making it clearer that the effects of these mutations are likely to be distinct, albeit with potential for overlap in function.

Reviewer #1 (Public Review):

Summary and strengths<br /> This is an interesting paper that concludes that hiring more women will do more to improve the gender balance of (US) academia than improving the attrition rates of women (which are usually higher than men's). Other groups have reported similar findings but this study uses a larger than usual dataset that spans many fields and institutions, so it is a good contribution to the field.

Weaknesses<br /> The paper uses a mixture of mathematical models (basically Leslie matrices, though that term isn't mentioned here) parameterised using statistical models fitted to data. However, the description of the methods needs to be improved significantly. The author should consider citing Matrix Population Models by Caswell (Second Edition; 2006; OUP) as a general introduction to these methods, and consider citing some or all of the following as examples of similar studies performed with these models:<br /> Shaw and Stanton. 2012. Proc Roy Soc B 279:3736-3741<br /> Brower and James. 2020. PLOS One 15:e0226392<br /> James and Brower. 2022. Royal Society Open Science 9:220785<br /> Lawrence and Chen. 2015. [http://128.97.186.17/index.php/pwp/article/view/PWP-CCPR-2015-008]<br /> Danell and Hjerm. 2013. Scientometrics 94:999-1006

The analysis also runs the risk of conflating the fraction of women in a field with gender diversity! In female-dominated fields (e.g. Nursing, Education) increasing the proportion of women in the field will lead to reduced gender diversity. This does not seem to be accounted for in the analysis. It would also be helpful to state the number of men and women in each of the 111 fields in the study.

Reviewer #2 (Public Review):

Summary:<br /> This important study by LaBerge and co-authors seeks to understand the causal drivers of faculty gender demographics by quantifying the relative importance of faculty hiring and attrition across fields. They leverage historical data to describe past trends and develop models that project future scenarios that test the efficacy of targeted interventions. Overall, I found this study to be a compelling and important analysis of gendered hiring and attrition in US institutions, and one that has wide-reaching policy implications for the academy. The authors have also suggested a number of fruitful future avenues for research that will allow for additional clarity in understanding the gendered, racial, and socioeconomic disparities present in US hiring and attrition, and potential strategies for mitigating or eliminating these disparities.

Strengths:<br /> In this study, LaBerge et al use data from over 268,000 tenured and tenure-track faculty from over 100 fields at more than 12,000 PhD-granting institutions in the US. The period they examine covers 2011-2020. Their analysis provides a large-scale overview of demographics across fields, a unique strength that allows the authors to find statistically significant effects for gendered attrition and hiring across broad areas (STEM, non-STEM, and topical domains).

LaBerge et al. find gendered disparities in attrition-using both empirical data and their counterfactual model-that account for the loss of 1378 women faculty across all fields between 2011 and 2020. It is true that "this number is both a small portion of academia... and a staggering number of individual careers," as ." - as this loss of women faculty is comparable to losing more than 70 entire departments. I appreciate the authors' discussion about these losses-they note that each of these is likely unnecessary, as women often report feeling that they were pushed out of academic jobs.

LaBerge et al. also find-by developing a number of model scenarios testing the impacts of hiring, attrition, or both-that hiring has a greater impact on women's representation in the majority of academic fields in spite of higher attrition rates for women faculty relative to men at every career stage. Unlike many other studies of historical trends in gender diversity, which have often been limited to institution-specific analyses, they provide an analysis that spans over 100 fields and includes nearly all US PhD-granting institutions. They are able to project the impacts of strategies focusing on hiring or retention using models that project the impact of altering attrition risk or hiring success for women. With this approach, they show that even relatively modest annual changes in hiring accumulate over time to help improve the diversity of a given field. They also demonstrate that, across the model scenarios they employ, changes to hiring drive the largest improvement in the long-term gender diversity of a field.

Future work will hopefully - as the authors point out - include intersectional analyses to determine whether a disproportionate share of lost gender diversity is due to the loss of women of color from the professoriate. I appreciate the author's discussion of the racial demographics of women in the professoriate, and their note that "the majority of women faculty in the US are white" and thus that the patterns observed in this study are predominately driven by this demographic. I also highly appreciate their final note that "equal representation is not equivalent to equal or fair treatment," and that diversifying hiring without mitigating the underlying cause of inequity will continue to contribute to higher losses of women faculty.

Weaknesses<br /> First, and perhaps most importantly, it would be beneficial to include a distinct methods section. While the authors have woven the methods into the results section, I found that I needed to dig to find the answers to my questions about methods. I would also have appreciated additional information within the main text on the source of the data, specifics about its collection, inclusion and exclusion criteria for the present study, and other information on how the final dataset was produced. This - and additional information as the authors and editor see fit - would be helpful to readers hoping to understand some of the nuance behind the collection, curation, and analysis of this important dataset.

I would also encourage the authors to include a note about binary gender classifications in the discussion section. In particular, I encourage them to include an explicit acknowledgement that the trends assessed in the present study are focused solely on two binary genders - and do not include an analysis of nonbinary, genderqueer, or other "third gender" individuals. While this is likely because of the limitations of the dataset utilized, the focus of this study on binary genders means that it does not reflect the true diversity of gender identities represented within the professoriate.

In a similar vein, additional context on how gender was assigned on the basis of names should be added to the methods section.

I do think that some care might be warranted regarding the statement that "eliminating gendered attrition leads to only modest changes in field-level diversity" (Page 6). while I do not think that this is untrue, I do think that the model scenarios where hiring is "radical" and attrition is unchanged from present (equal representation of women and men among hires (ER) + observed attrition (OA)) shows that a sole focus on hiring dampens the gains that can otherwise be addressed via even modest interventions (see, e.g., gender-neutral attrition (GNA) + increasing representation of women among hires (IR)). I am curious as to why the authors did not include an additional scenario where hiring rates are equal and attrition is equalized (i.e., GNA + ER). The importance of including this additional model is highlighted in the discussion, where, on Page 7, the authors write: "In our forecasting analysis, we find that eliminating the gendered attrition gap, in isolation, would not substantially increase representation of women faculty in academia. Rather, progress towards gender parity depends far more heavily on increasing women's representation among new faculty hires, with the greatest change occurring if hiring is close to gender parity." I believe that this statement would be greatly strengthened if the authors can also include a comparison to a scenario where both hiring and attrition are addressed with "radical" interventions.

Reviewer #3 (Public Review):

This manuscript investigates the roles of faculty hiring and attrition in influencing gender representation in US academia. It uses a comprehensive dataset covering tenured and tenure-track faculty across various fields from 2011 to 2020. The study employs a counterfactual model to assess the impact of hypothetical gender-neutral attrition and projects future gender representation under different policy scenarios. The analysis reveals that hiring has a more significant impact on women's representation than attrition in most fields and highlights the need for sustained changes in hiring practices to achieve gender parity.

Strengths:<br /> Overall, the manuscript offers significant contributions to understanding gender diversity in academia through its rigorous data analysis and innovative methodology.

The methodology is robust, employing extensive data covering a wide range of academic fields and institutions.

Weaknesses:<br /> The primary weakness of the study lies in its focus on US academia, which may limit the generalizability of its findings to other cultural and academic contexts. Additionally, the counterfactual model's reliance on specific assumptions about gender-neutral attrition could affect the accuracy of its projections.

Additionally, the study assumes that whoever disappeared from the dataset is attrition in academia. While in reality, those attritions could be researchers who moved to another country or another institution that is not included in the AARC (Academic Analytics Research Centre) dataset.

Reviewer #1 (Public Review):

Summary:<br /> Herneisen et al characterise the Toxoplasma PDK1 orthologue SPARK and an associated protein SPARKEL in controlling important fate decisions in Toxoplasma. Over recent years this group and others have characterised the role of cAMP and cGMP signalling in negatively and positively regulating egress, motility, and invasion, respectively. This manuscript furthers this work by showing that SPARK and SPARKEL likely act upstream, or at least control the levels of the cAMP and cGMP-dependent kinases PKA and PKG, respectively, thus controlling the transition of intracellular replicating parasites into extracellular motile forms (and back again).

The authors use quantitative (phospho)proteomic techniques to elegantly demonstrate the upstream role of SPARK in controlling cAMP and cGMP pathways. They use sophisticated analysis techniques (at least for parasitology) to show the functional association between cGMP and cAMP signalling pathways. They therefore begin to unify our understanding of the complicated signalling pathways used by Toxoplasma to control key regulatory processes that control the activation and suppression of motility. The authors then use molecular and cellular assays on a range of generated transgenic lines to back up their observations made by quantitative proteomics that are clear in their design and approach.

The authors then extend their work by showing that SPARK/SPARKEL also control PKAc3 function. PKAc3 has previously been shown to negatively regulate differentiation into bradyzoite forms and this work backs up and extends this finding to show that SPARK also controls this. The authors conclude that SPARK could act as a central node of regulation of the asexual stage, keeping parasites in their lytic cell growth and preventing differentiation. Whether this is true is beyond the scope of this paper and will have to be determined at a later date.

Strengths:<br /> This is an exceptional body of work. It is elegantly performed, with state-of-the-art proteomic methodologies carefully being applied to Toxoplasma. Observations from the proteomic datasets are masterfully backed up with validation using quantitative molecular and cellular biology assays.

The paper is carefully and concisely written and is not overreaching in its conclusions. This work and its analysis set a new benchmark for the use of proteomics and molecular genetics in apicomplexan parasites.

Weaknesses:<br /> This reviewer did not identify any weaknesses.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Herneisen et al. examines the Toxoplasma SPARK kinase orthologous to mammalian PDK1 kinase. The extracellular signals trigger cascades of the second messengers and play a central role in the apicomplexan parasites' survival. In Toxoplasma, these cascades regulate active replication of the tachyzoites, which manifests as acute toxoplasmosis, or the development into drug-resilient bradyzoites characteristic of the chronic stage of the disease. This study focuses on the poorly understood signaling mechanisms acting upstream of such second messenger kinases as PKA and PKG. The authors showed that similar to PDK1, Toxoplasma SPARK appears to regulate several AGC kinases.

Strengths:<br /> The study demonstrated a strong association of the SPARK kinase with an elongin-like SPARKEL factor and an uncharacterized AGC kinase. Using a set of standard assays, the authors determined the SPARK/SPARKEL role in parasite egress and invasion. Finally, the study presented evidence of the SPARK/SPARKEL involvement in the bradyzoite differentiation.

Weaknesses:<br /> Although the study can potentially uncover essential sensing mechanisms operating in Toxoplasma, the evidence of the SPARK/SPARKEL mechanisms is weak. Specifically, due to incomplete data analysis, the SPARK/SPARKEL-dependent phosphoregulation of AGC kinases cannot be evaluated. The manuscript requires better organization and lacks guidance on the described experiments. Although the study is built on advanced genetics, at times, it is unnecessarily complicated, raising doubts rather than benefiting the study.

Reviewer #3 (Public Review):

Summary:<br /> This paper focuses on the roles of a toxoplasma protein (SPARKEL) with homology to an elongin C and the kinase SPARK that it interacts with. They demonstrate that the two proteins regulate the abundance of PKA and PKG, and that depletion of SPARKEL reduces invasion and egress (previously shown with SPARK), and that their loss also triggers spontaneous bradyzoite differentiation. The data are overall very convincing and will be of high interest to those who study Toxoplasma and related apicomplexan parasites.

Strengths:<br /> The study is very well executed with appropriate controls. The manuscript is also very well and clearly written. Overall, the work clearly demonstrates that SPARK/SPARKEL regulate invasion and egress and that their loss triggers differentiation.

Weaknesses:<br /> 1. The authors fail to discriminate between SPARK/SPARKEL acting as negative regulators of differentiation as a result of an active role in regulating stage-specific transcription/translation or as a consequence of a stress response activated when either is depleted.

2. The function of SPARKEL has not been addressed. In mammalian cells, Elongin C is part of an E3 ubiquitin ligase complex that regulates transcription and other processes. From what I can tell from the proteomic data, homologs of the Elongin B/C complex were not identified. This is an important issue as the authors find that PKG and PKA protein levels are reduced in the knockdown strains

Reviewer #1 (Public Review):

Summary:<br /> The authors previously demonstrated that species-specific variation in primate CD4 impacts its ability to serve as a functional receptor for diverse SIVs. Here, Warren and Barbachano-Guerrero et al. perform population genetics analyses and functional characterization of great ape CD4 with a particular focus on gorillas, which are natural hosts of SIVgor. They first used ancestral reconstruction to derive the ancestral hominin and hominid CD4. Using pseudotyped viruses representing a panel of envelopes from SIVcpz and HIV strains, they find that these ancestral reconstructions of CD4 are more similar to human CD4 in terms of being a broadly susceptible entry receptor (in the context of mediating entry into Cf2Th cells stably expressing human CCR5). In contrast, extant gorilla and chimpanzee CD4 are functional entry receptors for a narrower range of HIV and SIVcpz isolates. Based on these differences, authors next surveyed gorilla sequences and identified several CD4 haplotypes, specifically in the region encoding the CD4 D1 domain, which directly contacts the viral glycoprotein and thus may impact the interaction. Consistent with this possibility, the authors demonstrated that gorilla CD4 haplotypes are, on average, less capable of supporting entry than human CD4, and that some are largely unable to function as SIV entry receptors. Interestingly, individual residues found at key positions in the gorilla CD4 D1 when tested in the context of human CD4 reduce entry of some virions pseudotyped with diverse SIVcpz envelopes, suggesting that individual amino acids can in part explain the observed differences across gorilla CD4 haplotypes. Finally, the authors perform statistical tests to infer that CD4 from great apes with endemic SIV (i.e., chimpanzees and gorillas) but not non-reservoirs (i.e., orangutans, bonobos) or recent spillover hosts (i.e., humans), have been subject to selection as a result of pressure from endemic SIV.

The conclusions of this paper are mostly well supported by data.

Strengths:<br /> The functional assays are appropriate to test the stated hypothesis, and the authors use a broad diversity of envelopes from HIV and SIVcpz strains. The authors also partially characterize one potential mechanism of gorilla CD4 resistance - receptor glycosylation at the derived N15 found in 5/6 gorilla haplotypes.

Ancestral reconstruction provides a particularly interesting aspect of the study, allowing authors to infer the ancestral state of hominid CD4 relative to modern CD4 from gorillas and chimpanzees. This, coupled with evidence supporting SIV-driven selection of gorilla CD4 diversity and the characterization of functional diversity of extant haplotypes provides several interesting findings.

Weaknesses:<br /> The major inference of the work is that SIV infection of gorillas drove the observed diversity in gorilla CD4. This is supported by the majority of SNPs being localized to the CD4 D1, which directly interacts with the envelope, and the demonstrated functional consequences of that diversity for viral entry. However, SIVgor (to the best of my knowledge) only infects Western lowland gorillas (Gorilla gorilla gorilla), and one Gorilla gorilla diehli and three Gorilla beringei graueri individuals were included in the haplotype and allele frequency analyses. The presence of these haplotypes or the presence of similar allele frequencies in Eastern lowland and mountain gorillas would impact this conclusion. It would be helpful for the authors to clarify this point.

The authors appear to use a somewhat atypical approach to assess intra-population selection to compensate for relatively small numbers of NHP sequences (Fig. 6). However, they do not cite precedence for the robustness of the approach or the practice of grouping sequences from multiple species for the endemic vs other comparison. They also state in the methods that some genes encoded in the locus were removed from the analysis "because they have previously been shown to directly interact with a viral protein." This seems to undercut the analysis and prevents alternative explanations for the observed diversity in CD4 (e.g., passenger mutations from selection at a neighboring locus).

Data in Figure 5 is graphed as % infected cells instead of virus titer (TDU/mL). It's unclear why this is the case, and prevents a comparison to data in Figure 2 and Figure 4.

The lack of pseudotyping with SIVgor envelope is a surprising omission from this study, that would help to contextualize the findings. Similarly, building gorilla CD4 haplotype SNPs onto the hominin ancestor (as opposed to extant human CD4) may provide additional insights that are meaningful toward understanding the evolutionary trajectory of gorilla CD4.

Reviewer #2 (Public Review):

Lentiviral infection of primate species has been linked to the rapid mutational evolution of numerous primate genes that interact with these viruses, including genes that inhibit lentiviruses as well as genes required for viral infection. In this manuscript, Warren et al. provide further support for the diversification of CD4, the lentiviral entry receptor, to resist lentiviral infection in great ape populations. This work builds on their prior publication (Warren et al. 2019, PMCID: PMC6561292 ) and that of other groups (e.g., Russell et al. 2021, PMCID: PMC8020793; Bibollet-Ruche et al. 2019, PMCID: PMC6386711) documenting both sequence and functional diversity in CD4, specifically within (1) the CD4 domain that binds to the lentiviral envelope and (2) great ape populations with endemic lentiviruses. Thus, the paper's finding that gorilla populations exhibit diverse CD4 alleles that differ in their susceptibility to lentiviral infection is well demonstrated both here and in a prior publication.

To bolster the argument that lentiviruses are indeed the causative driver of this diversification, which seems likely from a logical perspective but is difficult to prove, Warren et al. pursue two novel lines of evidence. First, the authors reconstruct ancestral CD4 genes that predate lentiviral infection of hominid populations. They then demonstrate that resistance to lentiviral infection is a derived trait in chimpanzees and gorillas, which have been co-evolving with endemic lentiviruses, but not in humans, which only recently acquired HIV. Nevertheless, the derived resistance could be stochastic or due to drift. This argument would be strengthened by demonstrating that bonobo and orangutan CD4, which also do not have endemic lentiviruses, resemble the ancestral and human susceptibility to great-ape-infecting lentiviruses.

Second, Warren et al. provide a population genetic argument that only endemically infected primates exhibit diversifying selection, again arguing for endemic lentiviruses being the evolutionary driver. The authors compare SNP occurrence in CD4 to neighboring genes, demonstrating that non-synonymous SNP frequency is only elevated in endemically infected species. Moreover, these amino-acid-coding changes are significantly concentrated in the CD4 domain that binds the lentiviral envelope. This is a creative analysis to overcome the problem of very small sample sizes, with very few great ape individuals sequenced. The additional small number of species compared (2-3 in each group) also limits the power of the analysis; the authors could consider expanding their analysis to Old World Monkey species that do or do not have endemic lentiviruses, as well as great apes.

Overall, this manuscript lends additional support to a well-documented example of a host-virus arms race: that of lentiviruses and the viral entry receptor.

Reviewer #1 (Public Review):

Wang et al investigated the evolution, expression, and function of the X-linked miR-506 miRNA family. They showed that the miR-506 family underwent rapid evolution. They provided evidence that miR-506 appeared to have originated from the MER91C DNA transposons. Human MER91C transposon produced mature miRNAs when expressed in cultured cells. A series of mouse mutants lacking individual clusters, a combination of clusters, and the entire X-linked cluster (all 22 miRNAs) were generated and characterized. The mutant mice lacking four or more miRNA clusters showed reduced reproductive fitness (litter size reduction). They further showed that the sperm from these mutants were less competitive in polyandrous mating tests. RNA-seq revealed the impact of deletion of miR-506 on the testicular transcriptome. Bioinformatic analysis analyzed the relationship among miR-506 binding, transcriptomic changes, and target sequence conservation. The miR-506-deficient mice did not have apparent effect on sperm production, motility, and morphology. Lack of severe phenotypes is typical for miRNA mutants in other species as well. However, the miR-506-deficient males did exhibit reduced litter size, such an effect would have been quite significant in an evolutionary time scale. The number of mouse mutants and sequencing analysis represent a tour de force. This study is a comprehensive investigation of the X-linked miR-506 miRNA family. It provides important insights into the evolution and function of the miR-506 family.

The conclusions of this preprint are mostly supported by the data except being noted below. Some descriptions need to be revised for accuracy.

L219-L285: The conclusion that X-linked miR-506 family miRNAs are expanded via LINE1 retrotransposition is not supported by the data. LINE1s and SINEs are very abundant, accounting for nearly 30% of the genome. In addition, the LINE1 content of the mammalian X chromosome is twice that of the autosomes. One can easily find flanking LINE1/SINE repeat. Therefore, the analyses in Fig. 2G, Fig. 2H and Fig. S3 are not informative. In order to claim LINE1-mediated retrotransposition, it is necessary to show the hallmarks of LINE1 retrotransposition, which are only possible for new insertions. The X chromosome is known to be enriched for testis-specific multi-copy genes that are expressed in round spermatids (PMID: 18454149). The conclusion on the LINE1-mediated expansion of miR-506 family on the X chromosome is not supported by the data and does not add additional insights. I think that the LINE1 related figure panels and description (L219-L285) need to be deleted. In discussion (L557-558), "...and subsequently underwent sequence divergence via LINE1-mediated retrotransposition during evolution" should also be deleted. This section (L219-L285) needs to deal only with the origin of miR-506 from MER91C DNA transposons, which is both convincing and informative.

Fig. 3A: can you speculate/discuss why the miR-506 expression in sperm is higher than in round spermatids?

Reviewer #2 (Public Review):

In this paper, Wang and collaborators characterize the rapid evolution of the X-linked miR-506 cluster in mammals and characterize the functional reference of depleting a few or most of the miRNAs in the cluster. The authors show that the cluster originated from the MER91C DNA transposon and provide some evidence that it might have expanded through the retrotransposition of adjacent LINE1s. Although the animals depleted of most miRNAs in the cluster show normal sperm parameters, the authors observed a small but significant reduction in litter size. The authors then speculate that the depletion of most miRNAs in the cluster could impair sperm competitiveness in polyandrous mating. Using a successive mating protocol, they show that, indeed, sperm lacking most X-linked miR-506 family members is outcompeted by wild-type sperm. The authors then analyze the evolution of the miR-506 cluster and its predicted targets. They conclude that the main difference between mice and humans is the expansion of the number of target sites per transcript in humans.

The conclusions of the paper are, in most cases, supported by the data; however, a more precise and in-depth analysis would have helped build a more convincing argument in most cases.

1) In the abstracts and throughout the manuscript, the authors claim that "... these X-linked miRNA-506 family miRNA [...] have gained more targets [...] " while comparing the human miRNA-506 family to the mouse. An alternative possibility is that the mouse has lost some targets. A proper analysis would entail determining the number of targets in the mouse and human common ancestor.

2) The authors claim that the miRNA cluster expanded through L1 retrotransposition. However, the possibility of an early expansion of the cluster before the divergence of the species while the MER91C DNA transposon was active was not evaluated. Although L1 likely contributed to the diversity within mammals, the generalization may not apply to all species. For example, SINEs are closer on average than L1s to the miRNAs in the SmiR subcluster in humans and dogs, and the horse SmiR subcluster seems to have expanded by a TE-independent mechanism.

3) Some results are difficult to reconcile and would have benefited from further discussion. The miR-465 sKO has over two thousand differentially expressed transcripts and no apparent phenotype. Also, the authors show a sharp downregulation of CRISP1 at the RNA and protein level in the mouse. However, most miRNAs of the cluster increase the expression of Crisp1 on a reporter assay. The only one with a negative impact has a very mild effect. miRNAs are typically associated with target repression; however, most of the miRNAs analyzed in this study activate transcript expression.

4) More information is required to interpret the results of the differential RNA targeting by the murine and human miRNA-506 family. The materials and methods section needs to explain how the authors select their putative targets. In the text, they mention the use of four different prediction programs. Are they considering all sites predicted by any method, all sites predicted simultaneously by all methods, or something in between? Also, what are they considering as a "shared target" between mice and humans? Is it a mRNA that any miR-506 family member is targeting? Is it a mRNA targeted by the same miRNA in both species? Does the targeting need to occur in the same position determined by aligning the different 3'UTRs?

5) The authors highlight the particular evolution of the cluster derived from a transposable element. Given the tendency of transposable elements to be expressed in germ cells, the family might have originated to repress the expression of the elements while still active but then remained to control the expression of the genes where the element had been inserted. The authors did not evaluate the expression of transcripts containing the transposable element or discuss this possibility. The authors proposed an expansion of the target sites in humans. However, whether this expansion was associated with the expansion of the TE in humans was not discussed either. Clarifying whether the transposable element was still active after the divergence of the mouse and human lineages would have been informative to address this outstanding issue.

Post-transcriptional regulation is exceptionally complex in male haploid cells, and the functional relevance of many regulatory pathways remains unclear. This manuscript, together with recent findings on the role of piRNA clusters, starts to clarify the nature of the selective pressure that shapes the evolution of small RNA pathways in the male germ line.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors conducted a comprehensive study of the X-linked miR-506 family miRNAs in mice on its origin, evolution, expression, and function. They demonstrate that the X-linked miR-506 family, predominantly expressed in the testis, may be derived from MER91C DNA transposons and further expanded by retrotransposition. By genetic deletion of different combinations of 5 major clusters of this miRNA family in mice, they found these miRNAs are not required for spermatogenesis. However, by further examination, the mutant mice show mild fertility problem and inferior sperm competitiveness. The authors conclude that the X-linked miR-506 miRNAs finetune spermatogenesis to enhance sperm competition.

Strengths:

This is a comprehensive study with extensive computational and genetic dissection of the X-linked miR-506 family providing a holistic view of its evolution and function in mice. The finding that this family miRNAs could enhance sperm competition is interesting and could explain their roles in finetuning germ cell gene expression to regulate reproductive fitness.

Weaknesses:

The authors specifically addressed the function of 5 clusters of X-link miR-506 family containing 19 miRNAs. There is another small cluster containing 3 miRNAs close to the Fmr1 locus. Would this small cluster act in concert with the 5 clusters to regulate spermatogenesis? In addition, any autosomal miR-506 like miRNAs may compensate for the loss of X-linked miR-506 family. These possibilities should be discussed.

Direct molecular link to sperm competitiveness defect remains unclear but is difficult to address.

Reviewer #1 (Public Review):

Summary:<br /> The cohesin complex maintains sister chromatid cohesion from S phase to anaphase. Beyond that, DSBs trigger cohesin recruitment and post-replication cohesion at both damage sites and globally, which was originally reported in 2004. In their recent study, Ayra-Plasencia et al reported in telophase, DSBs are repaired via HR with re-coalesced sister chromatids (Ayra-Plasencia & Machín, 2019). In this study, they show that HR occurs in a Smc3-dependent way in late mitosis.

Strengths:<br /> The authors take great advantage of the yeast system, they check the DSB processing and repair of a single DSB generated by HO endonuclease, which cuts the MAT locus in chromosome III. In combination with cell synchronization, they detect the HR repair during G2/M or late mitosis. and the cohesin subunit SMC3 is critical for this repair. Beyond that, full-length Scc1 protein can be recovered upon DSBs.

Weaknesses:<br /> These new results basically support their proposal although with a very limited molecular mechanistic progression, especially compared with their recent work.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript "Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis" by Ayra-Plasencia et al. investigates regulations of HR repair in conditional cdc15 mutants, which arrests the cell cycle in late anaphase/telophase. Using a non-competitive MAT switching system of S. cerevisiae, they show that a DSB in telophase-arrested cells elicits a delayed DNA damage checkpoint response and resection. Using a degron allele of SMC3 they show that MATa-to-alpha switching requires cohesin in this context. The presence of a DSB in telophase-arrested cells leads to an increase in the kleisin subunit Scc1 and a partial rejoining of sister chromatids after they have separated in a subset of cells.

Strengths:<br /> The experiments presented are well-controlled. The induction systems are clean and well thought-out.

Weaknesses:<br /> The manuscript is very preliminary, and I have reservations about its physiological relevance. I also have reservations regarding the usage of MAT to make the point that inter-sister repair can occur in late mitosis.

Reviewer #1 (Public Review):

Summary:

This manuscript reports that a combination of two small molecules, 2C (CHIR99027 and A-485) enabled to induce the dedifferentiation of hESC-derived cardiomyocytes (CMs) into regenerative cardiac cells (RCC). These RCCs had disassembled sarcomeric structures and elevated expression of embryonic cardiogenic genes such as ISL1, which exhibited proliferative potential and were able to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Lineage tracing further suggested that RCCs originated from TNNT2+ cells, not pre-existing ISL1+ cells. Furthermore, 2C treatment increased the numbers of RCC cells in neonatal rat and adult mouse hearts and improved cardiac function post-MI in adult mice. Mechanistically, bulk RNA-seq analysis revealed that 2C led to elevated expression of embryonic cardiogenic genes while down-regulation of CM-specific genes. Single-cell RNA-seq data showed that 2C promoted cardiomyocyte transition into an intermediate state that is marked with ACTA2 and COL1A1, which subsequently transformed into RCCs. Finally, ChIP-seq analysis demonstrated that CHIR99027 enhanced H3K9Ac and H3K27Ac modifications in embryonic cardiac genes, while A-485 inhibited these modifications in cardiac-specific genes. These combined alterations effectively induced the dedifferentiation of cardiomyocytes into RCCs.

Strengths:

Overall, this work is quite comprehensive and is logically and rigorously designed. The phenotypic and functional data on 2C are strong.

Weaknesses:

The mechanistic insights of 2C are primarily derived from transcriptomic and genomic datasets without experimental verification.

Reviewer #2 (Public Review):

Summary:

The ability of cardiac cells to regenerate has been the object of intense (and sometimes controversial) research in biology. While lower organisms can robustly undergo cardiac regeneration by reactivation of the embryonic cardiogenic pathway, this ability is strongly reduced in mice, both temporally and qualitatively. Finding a way to derive precursor cells with regenerative ability from differentiated cells in mammals has been challenging.

Zhou, He, and colleagues hypothesized that ISL-1-positive cells would show regenerative capacity and developed a small molecules screen to dedifferentiate cardiomyocytes (CM) to ISL1-positive precursor cells. Using hESC-derived CM, the authors found that the combination of both, WNT activation (CHIR99021) and p300 acetyltransferase inhibition (A-485) (named 2C protocol) induces CM dedifferentiation to regenerative cardiac cells (RCCs). RCCs are proliferative and re-express embryonic cardiogenic genes while decreasing the expression of more mature cardiac genes, bringing them towards a more precursor-like state. RCCs were able to differentiate into CM, smooth muscle cells, and endothelial cells, highlighting their multipotent property. In vivo, administration of 2C in rats and mice had protective effects on myocardial infarction.

Mechanistically, the authors report that the 2C protocol drives CM-specific transcriptional and epigenetic changes.

Strengths:

The authors made a great effort to validate their data using orthogonal ways, and several hESC lines. The use of lineage tracing convincingly showed a dedifferentiation from CM. They translate their findings into an in vivo model of myocardial injury, and show functional cardiac regeneration post-injury. They also showed that 2C could surprisingly be used as a preventive treatment. Together their data may suggest a regenerative effect of 2C both in vitro and in vivo settings. If confirmed, this study might unlock therapeutic strategy for cardiac regeneration.

Weaknesses:

Several points remain puzzling to me and some aspects of this study need to be clarified and extended:

General comments:

* Experimental design & Interpretation*

1) The main hypothesis (line 50) that Isl1 cells have regenerative properties is not extremely novel (10.1172/jci.insight.80920, doi.org/10.1038/nature03215,10.1016/s1534-5807(03)00363-0).

2) Based on Table S1, concentration of A-485 used in the screen is 10uM but used throughout this study at 0.5um. Could the authors provide a rationale for this 20x reduction of concentration? It would be useful to get a titration of this compound for the effects tested.

3) It is confusing to clearly understand what proportion of CMs dedifferentiate to become RCCs. The lineage tracing data suggests only 0.6%-1.5% of cells undergo this transition. It is difficult to understand how such a small fraction can have wide effects in their different experimental settings. This is specifically true when the author quantified nuclear and cytosolic area on brightfield pictures - would the same effect on nuclear/cytosolic area be observed in Isl1 KO cells?

4) The authors totally disregard the effect of i-BET-762 that gives a very similar percentage of Isl1-positive cells when combined to 2C (Supp. 1E). What is the effect of CHIR + I-BET-762 alone?

5) It is really hard to understand the contradictory effects of A-485 on acetylation status. The authors mentioned that A-485 only has an effect on H3K27Ac and not on H3K9Ac (line 221) to later (line 226) contradict themselves by saying it also has an effect on H3K9Ac. To explain this discrepancy, the authors vaguely mentioned "further analyses" without giving any other details. It would be transparent to explain what led to this radical change in interpretation.

6) The difference in the ChIP peak height is rather minimal for the H3K9Ac data. Were the peaks normalized to the sequencing depth? What does the y-axis represent on these ChIP traces (number of counts?)

7) Would it be possible to test this 2C protocol on mESC and see if similar changes occur? How transcriptionally different would these mouse RCCs be to Isl1+ progenitors isolated from neonatal mice (P1-P5)?

Statistics & Data acquisition

1) The authors mentioned experiments were done at least 3 times and each dot plotted on a graph is an average of technical repeat for one biological repeat. My understanding would be that if I see 9 dots, it means the experiment has been done 9 times - What would be the rationale for such a high number of repeats? It is an "artificial" way to increase the power of a test and might lead to misinterpretation of the data. This becomes relevant for some figures where biological difference is minimal and they still show statistical differences (e.g. Supp 2E, Supp 3A, Supp 9C,...). This is also true for in vivo section (Fig. 4G).

It would help to have a precise clarification between technical and biological repeats in the figure legends (e.g. n=3 biological repeat (aka 3 dots on a graph) obtained from averaging XX technical repeats), as well as the specific test stated the legend in addition to the general paragraph in the methods. Providing raw numerical data so readers can re-test them independently would also be a transparent way to do it.

2) Does the author test for normality before applying a specific test? Please clarify and justify either way.

3) If each dot represents a biological repeat as stated in the method section, why do some datasets have different numbers of repeats between NC and 2C if obtained in parallel? Have repeats been excluded?

Reviewer #3 (Public Review):

Summary:

The manuscript by Zhou and colleagues describes the potential of a two-compound combination (2C), CHIR99021 and A-485, which can generate regenerative cardiac cells (RCCs) from human embryonic stem cell-derived TNNT2+ cardiomyocytes. The authors have also demonstrated this phenomenon in neonatal rats CMs in vitro. Further, the administration of 2C can generate RCCs in adult mouse hearts and significantly improve survival and cardiac function in mice subjected to myocardial infarction. Interestingly, 2C treatment induces global changes in transcription and epigenetic modifications.

Strengths of the study:

1. This study describes the potential of 2C in improving the regeneration of the heart post-MI. The findings may have a translation potential. The idea of promoting the regenerative capacity of the heart by reprogramming CMs into RCCs is interesting.

2. The authors have validated the effect of 2C independently in hESCs, rat CMs, and a model of MI.

3. The authors explored the mechanism by Single-cell RNA-seq and Chip-Seq, which points to the transcriptional and epigenetic activation of genes essential for RCC.

Weaknesses of the study:

1. The mechanism involved in the 2C-mediated generation of RCCs is still unclear. The leads found in the RNA-seq and ChIP-seq were not validated experimentally.

2. Considering the very low number of RCCs (0.6%-1.5% of cells) generated, I cannot comprehend how the heart is protected from MI. Did the author believe 2C would affect the survival or metabolism of existing CM under hypoxia? What percentage of cells were regenerated by 2C treatment post-MI?

3. I would like to know about administering 2C in mice, which could have generated RCCs- dedifferentiated CMs in the heart. Does 2C affect the cardiac functions in mice under basal conditions? Also, does 2C administration affect any physiology in mice? The cardiac structural and functional parameters are required post-2C administration.

4. It is also not tested whether 2C would affect other cell types of the heart, including fibroblasts and endothelial cells, in vitro and in vivo. Assuming the level of protection by 2C in mice, it would affect other cell types.

5. It is still being determined how the authors chose the dose of 2C for in vivo and in vitro studies, although the concentration used for screening is different. Assessing the effect of 2C in a dose-dependent manner is essential.

6. A-485 affects H3K27Ac but not H3K9Ac. However, data show that both H3K27Ac and H3K9Ac are affected. An explanation is required.

7. The authors use "regeneration" even at the screening stage. I am wondering if regeneration could be assessed by the experimental approach they adopted.

Reviewer #4 (Public Review):

Overview:

The present manuscript by Zhou and colleagues investigates the impact of a new combination of compounds termed CHIR99021 and A-485 on stimulating cardiac cell regeneration. This manuscript fits the journal and addresses an important contribution to scientific knowledge. However, the following major revisions need to be addressed as stated below.

Major comments:

-The authors should include more information that clarifies and justifies their hypothesis.<br /> -The story line is not well developed and thus not convincing since the results from different sections are not well connected.<br /> -The main text needs to be improved, and authors should explain their purpose in choosing to study ISL1-CMs. Also, to well argument why they conducted this study and its significance.<br /> -Page 3, row 57-58: Please add the references.<br /> -Page 3-4, row 67-68, authors stated "When CMs resumed contraction, they were treated with individual small molecules from a collection of over 4,000 compounds for 3 days (SI Appendix, Fig. S1C and Table S1), and then fixed and immunostained with ISL1". Please explain better, and show the results of the selected screening compounds.<br /> -Authors must make an effort to discuss their findings in a bold way in order to provide a comprehensive and articulate explanation of their results to the readers. There is much information missing from this section. This should also propose new research avenues and foresee the challenges in future investigations.<br /> -Authors must include a conclusion and future perspectives of this study.<br /> - Page 4, row 73, the authors stated that the unique compound combination 'CHIR99021 and A-485' was found to be the most efficient in promoting ISL1 expression with a healthy cell state. However, the authors should prove that by showing at least the cell viability of these compound combinations at different concentrations and timings as a supplementary figure.<br /> -There is some missing information in the methods part, for example, "Images were captured using a confocal Zeiss LSM710 and Olympus IX83 inverted microscope"; authors should include the objective used and the image size, and should include which method they used to analyze the acquired images.<br /> -Figure S3A shows that the TNNT2 mRNA expression was completely absent after 60 hours of 2C administration. Authors should explain this further.<br /> -Figure 3J, there is high variability in the graph of mCherry cells (%). Please choose a better graph, or increase the independent experiment.<br /> -Authors did not explain/discuss their results of the DNA-binding motif analysis of ISL1 in the cells treated with A-485 or 2C (Figure 7K).<br /> -Figure S1B and D: the image's labeling is not clear. In the exact same figure S1B, how can the authors explain the reduction of ISL cells? Do the authors make the treatment with the compound CHIR99021 as shown in figure S1A? If so, the authors should clarify the ISL reduction in Figure S1B.<br /> -Figure 1H: please improve the immunoblot, the level of B-actin does not match among the different conditions, or provide a relative quantification of the proteins.<br /> -Please indicate further information in the methodology part about the compounds used in this study.<br /> -Figures are not well justified and figure legends are not sufficient enough to explain the figures.<br /> -Please improve the figure legends by including more further information; for example, in Figure 2SH it is highlighted only the "DAPI (4′,6-diamidino-2- phenylindole) staining labeled nuclei as blue" but how about the other markers?<br /> -Figure 2F: the graph shows some high variations in "ns" between NC at 2C and in 60h+3d. I would recommend increasing the independent experiments. Similar observation goes also for figure 2E.<br /> -Authors should provide limitations of this study.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

Strengths:<br /> The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

Weaknesses:<br /> 1. How conserved are the residue pair of D281-E283 in other SLC6 transporters. The authors commented on the presence of these residues in SERT but it would be nice to know how widespread these residues are in other SLC6 transporters like NET, GlyT, and DAT.

2. Further, one would like to see the effect of individual mutations D281A and E283A on transport, surface expression, and EC50 of Na+ to gauge the effect on transport.

3. A clear figure of the S1 site where Na+ tends to stay prior to Na1 site interactions needs to be provided with a clear figure. Further, it is not entirely clear how access to S1 is altered if the transporter is in an outward-occluded conformation if F294 is blocking solvent access. Please comment.

4. The p-value of the EC50 differences between GAT1WT and GAT1double mutant need to be mentioned. The difference in sodium dependence EC50 seems less than twofold and it would be useful to mention how critical the role of the recruitment site is. Since the transport is not affected the site could play a transient role in attracting ions.

5. It would be very nice to know how K+ ions are attracted by this recruitment site. This could further act as a control simulation to test the preference for Na+ ions among SLC6 members.

6. Some of the important figures are not very clear. For instance, there should be a zoomed-in view of the recruitment site. The current one in Fig. 1b and 1c could be made clearer. Similarly as mentioned earlier the Na residence at the S1 site away from the Na1 and Na2 sites needs to be shown with greater clarity by putting side chain information in Fig. 6d.

7. The structural features that comprise the two principle components PC1 and PC2 should be described in greater detail.

Reviewer #2 (Public Review):

Summary:<br /> Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

Strengths:<br /> - MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

- A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

- The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

Weaknesses:<br /> - Assessing the effects of Na+ binding on the large-scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to an AF2 model that's not well packed or really a feature of the open outward conformation.

- Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

Reviewer #1 (Public Review):

Summary:<br /> Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:<br /> In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Weaknesses:<br /> I have very few critiques for this study, and my major points are barely major.

Major points<br /> 1. My sense is that the influence of Cys mutation on dimerization is going to be one of the first queries readers consider as they read the work. It would be, in my opinion, useful to bring forward the dimer section in the manuscript.

2. Relatedly, the effect of Cys mutation on the dimerization properties of preparations of recombinant protein is not very clear as it stands. Some SEC traces would be helpful; these could be included in the supplement.

3. Is there any knowledge of Cys mutants in disease for BRSK1/2?

4. In bar charts, I'd recommend plotting data points. Plus it is crucial to report in the legend what error measure is shown, the number of replicates, and the statistical method used in any tests.

5. In Figure 5b, the GAPDH loading control doesn't look quite right.

6. In Figure 7 there is no indication of what mode of detection was used for these gels.

9. Recombinant proteins - more detail should be included on how they were prepared. Was there a reducing agent present during purification? Where did they elute off SEC... consistent with a monomer of higher order species?

Reviewer #2 (Public Review):

Summary:<br /> In this study by Bendzunas et al, the authors show that the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys at an unusual CPE motif at the end of the activation segment function as repressive regulatory mechanisms in BSK1 and 2. They observed that mutation of the CPE-Cys only, contrary to the double mutation of the pair, increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family. Understanding the molecular mechanisms underlying kinase regulation by redox-active Cys residues is fundamental as it appears to be widespread in signaling proteins and provides new opportunities to develop specific covalent compounds for the targeted modulation of protein kinases.

The authors demonstrate that intramolecular cysteine disulfide bonding between conserved cysteines can function as a repressing mechanism as indicated by the effect of DTT and the consequent increase in activity by BSK-1 and -2 (WT). The cause-effect relationship of why mutation of the CPE-Cys only increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells is not clear to me. The explanation given by the authors based on molecular modeling and molecular dynamics simulations is that oxidation of the CPE-Cys (that will favor disulfide bonding) destabilizes a conserved salt bridge network critical for allosteric activation. However, no functional evidence of the impact of the salt-bridge network is provided. If you mutated the two main Cys-pairs (aE-CHRD and A-loop T+2-CPE) you lose the effect of DTT, as the disulfide pairs cannot be formed, hence no repression mechanisms take place, however when looking at individual residues I do not understand why mutating the CPE only results in the opposite effect unless it is independent of its connection with the T+2residue on the A-loop.

Strengths:<br /> This is an important and interesting study providing new knowledge in the protein kinase field with important therapeutic implications for the rationale design and development of next-generation inhibitors.

Weaknesses:<br /> There are several issues with the figures that this reviewer considers should be addressed.

Joint Public Review:

This article is a direct follow-up to the paper published last year in eLife by the same group. In the previous article, the authors discovered a zinc finger protein, Kipferl, capable of guiding the HP1 protein Rhino towards certain genomic regions enriched in GRGGN motifs and packaged in heterochromatin marked by H3K9me3. Unlike other HP1 proteins, Rhino recruitment activates the transcription of heterochromatic regions, which are then converted into piRNA source loci. The molecular mechanism by which Kipferl interacts specifically with Rhino (via its chromodomain) and not with other HP1 proteins remained enigmatic.

In this latest article, the authors go a step further by elucidating the molecular mechanisms important for the specific interaction of Rhino and not other HP1 proteins with Kipferl. A phylogenetic study carried out between the HP1 proteins of 5 Drosophila species led them to study the importance of an AA Glycine at position 31 located in the Rhino chromodomain, an AA different from the AA (aspartic acid) found at the same position in the other HP1 proteins. The authors then demonstrate, through a series of structure predictions, biochemical, and genetic experiments, that this specific AA in the Rhino-specific chromodomain explains the difference in the chromatin binding pattern between Rhino and the other Drosophila HP1 proteins. Importantly, the G31D conversion of the Rhino protein prevents interaction between Rhino and Kipferl, phenocopying a Kipfer mutant.

Strengths:

The authors' effective use of phylogenetic analyses and protein structure predictions to identify a substitution in the chromodomain that allows Rhino's specific interaction with Kipferl is very elegant. Both genetic and biochemical approaches are applied to rigorously probe the proposed explanation. They used a point mutation in the endogenous locus that replaces the Rhino-specific residue with the aspartic acid residue present in all other HP1 family members. This novel allele largely phenocopies the defects in hatch rate, chromatin organization, and piRNA production associated with kipferl mutants, and does not support Kipferl localization to clusters. The data are of high quality, the presentation is clear and concise, and the conclusions are generally well-supported.

Weaknesses:

The reviewers identified potential ways to further strengthen the manuscript.

1) The one significant omission is RNAseq on the rhino point mutant, which would allow direct comparison to cluster, transposon, and repeat expression in kipferl mutants.

2) The manuscript would benefit from adding more evolutionary comparisons. The following or similar analyses would help put the finding into a broader evolutionary perspective: i) Is Kipferl's surface interacting with Rhino also conserved in Kipferl orthologs? In other words, are the Rhino-interacting amino acids of Kipferl under any pressure to be conserved? ii) The remarkable conservation of Rhino's G31 is at odds with the arms race that is proposed to be happening between the fly's piRNA pathway proteins and transposons. Does this mean that Rhino's chromodomain is "untouchable" for such positive selection?

Reviewer #1 (Public Review):

My main concern is the use of the 700K SNP dataset. This set of SNPs suffers from a heavy ascertainment bias, which can be seen in the PCA in the supplementary material where all the aurochs cluster in the center within the variation of cattle. Given the coverage of some of the samples, multiple individuals would have less than 10K SNP covered. The majority of these are unlikely to be informative here given that they would just represent fixed positions between taurine and indicine or SNPs mostly variable in milk cattle breeds. The authors would get a much better resolution (i.e. many more SNPs to work with their very low genome coverage data) using the 1000 bull genome project VCF data set: https://www.ebi.ac.uk/ena/browser/view/PRJEB42783 which based on whole genome resequencing data from many cattle. This will certainly help with improving the resolution of qpAdm and f4 analysis, which have huge confidence intervals in most cases. Right now some individuals have huge confidence intervals ranging from 0 to 80% auroch ancestry...

I agree with the authors that qpAdm is likely to give quite a noisy estimate of ancestry here (likely explain part of the issue I mentioned above). Although qpAdm is good for model testing here for ancestry proportion the authors instead could use an explicit f4 ratio - this would allow them to specify a model which would make the result easier to interpret.

The interpretation of the different levels of allele sharing on X vs autosome being the result of sex-bias admixture is not very convincing. Could these differences simply be due to a low recombination rate on the X chromosome and/or lower effective population size, which would lead to less efficient purifying selection?

The authors suggest that 2 pop model rejection in some domestic population might be due to indicine ancestry, this seems relatively straightforward to test.

The first sentence of the paper is a bit long-winded, also dogs were domesticated before the emergence of farming societies.

It would be good to be specific about the number of genomes and coverage info in the last paragraph of the intro.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors investigated the admixture history of domestic cattle since they were introduced into Iberia, by studying genomic data from 24 ancient samples dated to ~2000-8000 years ago and comparing them to modern breeds. They aimed to (1) test for introgression from (local) wild aurochs into domestic cattle; (2) characterize the pattern of admixture (frequency, extent, sex bias, directionality) over time; (3) test for correlation between genetic ancestry and stable isotope levels (which are indicative of ecological niche); and (4) test for the hypothesized higher aurochs ancestry in a modern breed of fighting bulls.

Strengths:<br /> Overall, this study collects valuable new data that are useful for testing interesting hypotheses, such as admixture between domestic and wild populations, and correlation between genome-wide aurochs ancestry and aggressiveness.

Weaknesses:<br /> Most conclusions are partially supported by the data presented. The presence of admixed individuals in prehistorical periods supports the hypothesized introgression, although this conclusion needs to be strengthened with an analysis of potential contamination. The frequency, sex-bias, and directionality of admixture remain highly uncertain due to limitations of the data or issues with the analysis. There is considerable overlap in stable isotope values between domestic and wild groups, indicating a shared ecological niche, but variation in classification criteria for domestic vs wild groups and in skeletal elements sampled for measurements significantly weakens this claim. Lastly, the authors presented convincing evidence for relatively constant aurochs ancestry across all modern breeds, including the Lidia breed which has been bred for aggressiveness for centuries. My specific concerns are outlined below.

Contamination is a common concern for all ancient DNA studies. Contamination by modern samples is perhaps unlikely for this specific study of ancient cattle, but there is still the possibility of cross-sample contamination. The authors should estimate and report contamination estimates for each sample (based on coverage of autosomes and sex chromosomes, or heterozygosity of Y or MT DNA). Such contamination estimates are particularly important to support the presence of individuals with admixed ancestry, as a domestic sample contaminated with a wild sample (or vice versa) could appear as an admixed individual.

A major limitation of this study is uncertainty in the "population identity" for most sampled individuals (i.e., whether an individual belonged to the domesticated or wild herd when they were alive). Based on chronology, morphology, and genetic data, it is clear the Mesolithic samples from the Artusia and Mendandia sites are bona fide aurochs, but the identities of individuals from the other two sites are much less certain. Indeed, archeological and morphological evidence from El Portalon supports the presence of both domestic animals and wild aurochs, which is echoed by the inter-individual heterogeneity in genetic ancestry. Based on results shown in Fig 1C and Fig 2 it seems that individuals moo017, moo020, and possibly moo012a are likely wild aurochs that had been hunted and brought back to the site by humans. Although the presence of individuals (e.g., moo050, moo019) that can only be explained by two-source models strongly supports that interbreeding happened (if cross-contamination is ruled out), it is unclear whether these admixed individuals were raised in the domestic population or lived in the wild population and hunted.

Such uncertainty in "population identity" limits the authors' ability to make conclusions regarding the frequency, sex bias, and directionality of gene flow between domestic and wild populations. For instance, the wide range of ancestry estimates in Neolithic and Chalcolithic samples could be interpreted as evidence of (1) frequent recent gene flow or (2) mixed practices of herding and hunting and less frequent gene flow. Similarly, the statement about "bidirection introgression" (on pages 8 and 11) is not directly supported by data. As the genomic, morphological, and isotope data cannot confidently classify an individual as belonging to the domesticated or wild population, it seems impossible to conclude the direction of gene flow (if by "bidirection introgression" the authors mean something other than "bidirectional gene flow", they need to clearly explain this before reaching the conclusion.)

The f4 statistics shown in Fig 3B are insufficient to support the claim regarding sex-biased hybridization, as the f4 statistic values are not directly comparable between the X chromosome and autosomes. Because the effective population size is different for the X chromosome and autosomes (roughly 3:4 for populations with equal numbers of males and females), the expected amount of drift is different, hence the fraction of allele sharing (f4) is expected to be different. In fact, the observation that moo004 whose autosomal genome can be modeled as 100% domestic ancestry still shows a higher f4 value for the X chromosome than autosomes hints at this issue. A more robust metric to test for sex-biased admixture is the admixture proportion itself, which can be estimated by qpAdm or f4-ratio (see Patterson et al 2012). However, even with this method, criticism has been raised (e.g., Lazaridis and Reich 2017; Pfennig and Lachance, 2023). In general, detecting sex-bias admixture is a tough problem.

In general, the stable isotope analysis seems to be very underpowered, due to the issues of variation in classification criteria and skeletal sampling location discussed by the authors in supplementary material. The authors claimed a significant difference in stable nitrogen isotope between (inconsistently defined) domestic cattle and wild aurochs, but no figures or statistics are presented to support this claim. Please describe the statistical method used and the corresponding p-values. The authors can consider including a figure to better show the stable isotope results.

Reviewer #3 (Public Review):

Summary:<br /> Günther and colleagues leverage ancient DNA data to track the genomic history of one of the most important farm animals (cattle) in Iberia, a region showing peculiarities both in terms of cultural practices as well as a climatic refugium during the LGM, the latter of which could have allowed the survival of endemic lineages. They document interesting trends of hybridisation with wild aurochs over the last 8-9 millennia, including a stabilisation of auroch ancestry ~4000 years ago, at ~20%, a time coincidental with the arrival of domestic horses from the Pontic steppe. Modern breeds such as the iconic Lidia used in bullfighting or bull running retain a comparable level of auroch ancestry.

Strengths:<br /> The generation of ancient DNA data has been proven crucial to unravel the domestication history of traditional livestock, and this is challenging due to the environmental conditions of the Iberian peninsula, less favourable to DNA preservation. The authors leverage samples unearthed from key archaeological sites in Spain, including the karstic system of Atapuerca. Their results provide fresher insights into past management practices, and permit characterisation of significant shifts in hybridization with wild aurochs.

Weaknesses:<br /> - Treatment of post-mortem damage: the base quality of nucleotide transitions was recalibrated down to a quality score of 2, but for 5bp from the read termini only. In some specimens (e.g. moo022), the damage seems to extend further. Why not use dedicated tools (e.g. mapDamage), or check the robustness by conditioning on nucleotide transversions?

- Their more solid analyses are based on qpAdm, but rely on two single-sample donor populations. As the authors openly discuss, it is unclear whether CPC98 is a good proxy for Iberian aurochs despite possibly forming a monophyletic clade (the number of analysed sites is simply too low to assess this monophyly; Supplementary Table S2). Additionally, it is also unclear whether Sub1 was a fully unadmixed domestic specimen, depleted of auroch ancestry. The authors seem to suggest themselves that sex-biased introgression may have already taken place in Anatolia ("suggesting that sex-biased processes already took place prior to the arrival of cattle to Iberia").

Alternatively, I recommend using Struct-f4 as it can model the ancestry of all individuals together based on their f4 permutations, including outgroups and modern data, and without the need to define pure "right" and "left" populations such as CPC98 and Sub1. It should work with low-coverage data, and allows us to do f4-based MDS plots as well as to estimate ancestry proportions (including from ghost populations).

- In the admixture graph analyses (supplementary results), the authors use population groups based on a single sample. If these samples are pseudohaploidised (or if coverage is insufficient to estimate heterozygosity - and it is at least for moo004 and moo014), f3 values are biased, implying that the fitted graph may be wrong. The graph shown in Fig S7 is in fact hard to interpret. For example, the auroch Gyu2 from Anatolia but not the auroch CPC98 also from Anatolia received 62% of ancestry from North Africa? The Neolithic samples moo004 and moo014 also show the same shocking disparity. I would consider re-doing this analysis with more than a sample per population group.

Reviewer #1 (Public Review):

Summary:<br /> Doxorubincin has long been known to cause bone loss by increasing osteoclast and suppressing osteoblast activities. The study by Wang et al. reports a comprehensive investigation into the off-target effects of doxorubicin on bone tissues and potential mechanisms. They used a tumor-free model with wild-type mice and found that even a single dose of doxorubicin has a major influence by increasing leukopenia, DAMPs, and inflammasomes in macrophages and neutrophils, and inflammation-related cell death (pyroptosis and NETosis). The gene knockout study shows that AIM2 and NLRP3 are the major contributors to bone loss. Overall, the study confirmed previous findings regarding the impact of doxorubicin on tissue inflammation and expanded the research further into bone tissue. The presented data are consistent; however, a major question remains regarding whether doxorubicin drives inflammation and its related events. Most in vitro studies showed that the effect of doxorubincin cannot be demonstrated without LPS priming. This observation raises the question of whether doxorubincin itself could activate the inflammasome and the related events. In vivo study, on the other hand, suggested that it doesn't require LPS. The inconsistency here was not explained further. Moreover, a tumor-free mouse model was used for the study; however, immune responses in tumor-bearing models would likely be distinct from tumor-free ones. The justification for using tumor-free models is not well-established.

Strengths:<br /> The paper includes a comprehensive study that shows the effects of doxorubincin on cytokine levels in serum, the release of DAMPs and NETosis, and leukopenia using both in vivo and in vitro models. Bone marrow cells, macrophages, and neutrophils were isolated from the bone marrow, and the levels of cytokines in serum were also determined.

They employed multiple knockout models with a deficiency in Aim 2, Nlirp3, and double deficiencies to dissect the functional involvement of these two inflammasomes.

The experiments in general are well designed. The paper is also logically written, and the figures were clearly labeled.

Weaknesses:<br /> Most of the data presented are correlative, and there is not much effort to dissect the underlying molecular mechanism.

It is not entirely clear why a tumor-free model is chosen to study immune responses, as immune responses can differ significantly with or without tumor-bearing.

Immune responses in isolated macrophages, neutrophils, and bone marrow cells require priming with LPS, while such responses are not observed in vivo. There is no explanation for these differences.

The band intensities on Western blots in Fig. 4 and Fig. 5 are not quantified, and the numbers of repeats are also not provided.

Many abbreviations are used throughout the text, and some of the full names are not provided.

Fig. 5B needs a label on the X axis.

Reviewer #2 (Public Review):

Summary:<br /> Wang and collaborators have evaluated the impact of inflammation on bone loss induced by Doxorubicin, which is commonly used in chemotherapy to treat various cancers. In mice, they show that a single injection of Doxorubicin induces systemic inflammation, leukopenia, and significant bone loss associated with increased bone-resorbing osteoclast numbers. In vitro, the authors show that Doxorubicin activates the AIM2 and NLRP3 inflammasomes in macrophages and neutrophils. Importantly, they show that the full knockouts (germline deletions) of AIM2 (Aim2-/-) and NLRP3 (Nlrp3-/-) and Caspase 1 (Casp1-/-) limit (but do not completely abolish) bone loss induced 4 weeks after a single injection of Doxorubicin in mice. From these results, they conclude that Doxorubicin activates inflammasomes to cause inflammation-associated bone loss.

Strengths:<br /> While numerous studies have reported that Doxorubicin activates the inflammasome in myeloid cells and various other cell types, that Doxorubicin induces systemic inflammation, and that both the systemic inflammation and Doxorubicin treatment leads to bone loss, functional experiments demonstrating that NRLP3 and/or AIM2 loss-of-functions, and thus the systemic impairment of the inflammatory response, may prevent bone-loss induced by Doxorubicin were lacking. The strength of this manuscript is that it provides these missing data.

Weaknesses:<br /> However, one could argue that most of the conclusions drawn from the data presented here have been previously reported and that it was very much expected that reducing systemic inflammation in treated animals (in Aim2-/- and/or Nlrp3-/- mice) would preserve bone homeostasis to some extent, similarly to what has been reported in the context of cardiotoxicity induced by Doxorubicin.

Since the manuscript focuses on therapeutic considerations aiming to preserve bone homeostasis in animals treated with Doxorubicin, additional experiments evaluating and comparing various therapeutic options could improve the impact of the study. Drugs targeting the inflammasomes could be tested in addition to the genetic mouse models. Since increased osteoclast numbers (and likely bone resorption) are associated with Doxorubicin-induced bone loss, antiresorptive drugs such as Bisphosphonates or anti-RANKL antibodies could be tested and compared to anti-inflammatory drugs. Since autophagy and senescence have been shown to contribute to bone loss induced by Doxorubicin, it would be interesting to use the pharmacologic inhibitors (targeting autophagy or senescence) used in these previous studies to evaluate the relative impact of these different cellular mechanisms, on bone loss induced by Doxorubicin.

Moreover, the cellular and molecular mechanisms by which Doxorubicin induces bone loss in vivo could be further evaluated. Doxorubicin has been reported to directly affect bone-making osteoblasts and bone-resorbing osteoclasts. It would be important to determine the relative importance of the activation of the AIM2 and NLRP3 inflammasomes in these cells compared to macrophages and neutrophils. Floxed mouse lines exist for both Aim2 and Nlrp3, as well as relevant cell-specific Cre lines. Thus, cell-specific conditional knockouts could have been used in the current study, instead of using global knockout animals. Genetic tools also exist to induce the specific ablation of macrophages or neutrophils and could be used. Furthermore, it is unclear whether local inflammation is induced in the bone marrow of Doxorubicin-treated mice, and what is the relative impact of local versus systemic inflammation in bone loss in these mice. Markers of the inflammasomes, pyroptosis, and NETosis could be evaluated on bone sections, and on bone and bone marrow samples. The effect of Doxorubicin on osteoblast numbers in vivo and on bone resorption (not just osteoclast numbers) should be evaluated as well. These mechanistic aspects are important and needed to better understand the cytotoxic mechanisms triggered by Doxorubicin, and define the best therapeutic approaches to preserve bone integrity in chemotherapy.

Finally, it would be important to assess the bone mass of Doxorubicin-treated control, Aim2-/-, Nlrp3-/- and Cas1-/- mice at a later time point than 4 weeks post-injection. Nlrp3 knockout has been reported to increase the density of the cortical and trabecular bones. The bone mass of Aim2-/-, Nlrp3-/- and Cas1-/- mice at baseline may be higher than that of control mice, and it may take slightly longer for Doxorubicin to reduce bone mass to the same extent than in controls. It would be also interesting to do similar experiments using animals treated multiple times with Doxorubicin instead of using a single injection, since patients receive their chemotherapy multiple times.

Reviewer #1 (Public Review):

Summary and strengths:

This is an interesting, timely and informative article. The authors used publicly available data (made available by a funding agency) to examine some of the academic characteristics of the individuals recipients of the National Institutes of Health (NIH) k99/R00 award program during the entire history of this funding mechanism (17 years, total ~ 4 billion US dollars (annual investment of ~230 million USD)). The analysis focuses on the pedigree and the NIH funding portfolio of the institutions hosting the k99 awardees as postdoctoral researchers and the institutions hiring these individuals. The authors also analyze the data by gender, by whether the R00 portion of the awards eventually gets activated and based on whether the awardees stayed/were hired as faculty at their k99 (postdoctoral) host institution or moved elsewhere. The authors further sought to examine the rates of funding for those in systematically marginalized groups by analyzing the patterns of receiving k99 awards and hiring k99 awardees at historically black colleges and universities.

The goals and analysis are reasonable and the limitations of the data are described adequately. It is worth noting that some of the observed funding and hiring traits are in line with the Matthew effect in science (Merton, 1968: https://www.science.org/doi/10.1126/science.159.3810.56) and in science funding (Bol et al., 2018: https://www.pnas.org/doi/10.1073/pnas.1719557115). Overall, the article is a valuable addition to the research culture literature examining the academic funding and hiring traits in the United States. The findings can provide further insights for the leadership at funding and hiring institutions and science policy makers for individual and large-scale improvements that can benefit the scientific community.

Weaknesses:

The authors have addressed my recommendations in the previous review round in a satisfactory way.

Reviewer #2 (Public Review):

Summary and strengths:

Early career funding success has an immense impact on later funding success and faculty persistence, as evidenced by well-documented "rich-get-richer" or "Matthew effect" phenomena in science (e.g., Bol et al., 2018, PNAS). In this study the authors examined publicly available data on the distribution of the National Institutes of Health's K99/R00 awards - an early career postdoc-to-faculty transition funding mechanism - and showed that although 89% of K99 awardees successfully transitioned into faculty, disparities in subsequent R01 grant obtainment emerged along three characteristics: researcher mobility, gender, and institution. Men who moved to a top-25 NIH funded institution in their postdoc-to-faculty transition experienced the shortest median time to receiving a R01 award, 4.6 years, in contrast to the median 7.4 years for women working at less well-funded schools who remained at their postdoc institutions.

Amongst the three characteristics, the finding that researcher mobility has the largest effect on subsequent funding success is key and novel. Other data supplement this finding: for example, although the total number of R00 awards has increased, most of this increase is for awards to individuals moving to different institutions. In 2010, 60% of R00 awards were activated at different institutions compared to 80% in 2022. These findings enhance previous work on the relationship between mobility and ones' access to resources, collaborators, or research objects (e.g., Sugimoto and Larivière, 2023, Equity for Women in Science (Harvard University Press)).

These results empirically demonstrate that even after receiving a prestigious early career grant, researchers with less mobility belonging to disadvantaged groups at less-resourced institutions continue to experience barriers that delay them from receiving their next major grant. This result has important policy implications aimed at reducing funding disparities - mainly that interventions that focus solely on early career or early stage investigator funding alone will not achieve the desired outcome of improving faculty diversity.

The authors also highlight two incredible facts: No postdoc at a historically Black college or university (HBCU) has been awarded a K99 since the program's launch. And out of all 2,847 R00 awards given thus far, only two have been made to faculty at HBCUs. Given the track record of HBCUs for improving diversity in STEM contexts, this distribution of awards is a massive oversight that demands attention.

At no fault of the authors, the analysis is limited to only examining K99 awardees and not those who applied but did not receive the award. This limitation is solely due to the lack of data made publicly available by the NIH. If this data were available, this study would have been able to compare the trajectory of winners versus losers and therefore could potentially quantify the impact of the award itself on later funding success, much like the landmark paper by Bol et al. (PNAS; 2018) that followed the careers of an early career grant scheme in the Netherlands. Such an analysis would also provide new insights that would inform policy.

Although data on applications versus awards for the K99/R00 mechanism are limited, there exists data for applicant race and ethnicity for the 2007-2017 period, which were made available by a Freedom of Information Act request through the now defunct Rescuing Biomedical Research Initiative (https://web.archive.org/web/20180723171128/http://rescuingbiomedicalresearch.org/blog/examining-distribution-k99r00-awards-race/). These results are highly relevant given the discussion of K99 award impacts on the sociodemographic composition of U.S. biomedical faculty. During the 2007-2017 period, the K99 award rate for white applicants was 31% compared to 26.7% for Asian applicants and 16.2% for Black applicants. In terms of award totals, these funding rates amount to 1,384 awards to white applicants, 610 to Asian applicants, and 25 to Black applicants. However, the work required to include these data may be beyond the scope of the study.

The conclusions are well-supported by the data, and limitations of the data and the name-gender matching algorithm are described satisfactorily.

Reviewer #3 (Public Review):

Summary:

The researchers aim add to the literature on faculty career pathways with particular attention to how gender disparities persist in the career and funding opportunities of researchers. The researchers also examine aspects of institutional prestige that can further amplify funding and career disparities. While some factors about individuals' pathways to faculty lines are known, including the prospects of certain K award recipients, the current study provides the only known examination of the K99/R00 awardees and their pathways.

Strengths:

The authors establish a clear overview of the institutional locations of K99 and R00 awardees and the pathways for K99-to-R00 researchers and the gendered and institutional patterns of such pathways. For example, there's a clear institutional hierarchy of hiring for K99/R00 researchers that echo previous research on the rigid faculty hiring networks across fields, and a pivotal difference in the time between awards that can impact faculty careers. Moreover, there's regional clusters of hiring in certain parts of the US where multiple research universities are located. Moreover, documenting the pathways of HBCU faculty is an important extension of the study by Wapman et al. (2022: https://www.nature.com/articles/s41586-022-05222-x), and provides a more nuanced look at the pathways of faculty beyond the oft-discussed high status institutions. (However, there is a need for more refinement in this segment of the analyses). Also, the authors provide important caveats throughout the manuscript about the study's findings that show careful attention to the complexity of these patterns and attempting to limit misinterpretations of readers.

Weaknesses:

The authors have addressed my recommendations in the previous review round in a satisfactory way.

Reviewer #1 (Public Review):

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

Reviewer #2 (Public Review):

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

Reviewer #3 (Public Review):

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

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

Reviewer #1 (Public Review):

In the best genetically and biochemically understood model of eukaryotic DNA replication, the budding yeast, Saccharomyces cerevisiae, the genomic locations at which DNA replication initiates are determined by a specific sequence motif. These motifs, or ARS elements, are bound by the origin recognition complex (ORC). ORC is required for loading of the initially inactive MCM helicase during origin licensing in G1. In human cells, ORC does not have a specific sequence binding domain and origin specification is not specified by a defined motif. There have thus been great efforts over many years to try to understand the determinants of DNA replication initiation in human cells using a variety of approaches, which have gradually become more refined over time.

In this manuscript Tian et al. combine data from multiple previous studies using a range of techniques for identifying sites of replication initiation to identify conserved features of replication origins and to examine the relationship between origins and sites of ORC binding in the human genome. The authors identify a) conserved features of replication origins e.g. association with GC-rich sequences, open chromatin, promoters and CTCF binding sites. These associations have already been described in multiple earlier studies. They also examine the relationship of their determined origins and ORC binding sites and conclude that there is no relationship between sites of ORC binding and DNA replication initiation. While the conclusions concerning genomic features of origins are not novel, if true, a clear lack of colocalization of ORC and origins would be a striking finding. However, the majority of the datasets used do not report replication origins, but rather broad zones in which replication origins fire. Rather than refining the localisation of origins, the approach of combining diverse methods that monitor different objects related to DNA replication leads to a base dataset that is highly flawed and cannot support the conclusions that are drawn, as explained in more detail below.

Methods to determine sites at which DNA replication is initiated can be divided into two groups based on the genomic resolution at which they operate. Techniques such as bubble-seq, ok-seq can localise zones of replication initiation in the range ~50kb. Such zones may contain many replication origins. Conversely, techniques such as SNS-seq and ini-seq can localise replication origins down to less than 1kb. Indeed, the application of these different approaches has led to a degree of controversy in the field about whether human replication does indeed initiate at discrete sites (origins), or whether it initiates randomly in large zones with no recurrent sites being used. However, more recent work has shown that elements of both models are correct i.e. there are recurrent and efficient sites of replication initiation in the human genome, but these tend to be clustered and correspond to the demonstrated initiation zones (Guilbaud et al., 2022).

These different scales and methodologies are important when considering the approach of Tian et al. The premise that combining all available data from five techniques will increase accuracy and confidence in identifying the most important origins is flawed for two principal reasons. First, as noted above, of the different techniques combined in this manuscript, only SNS-seq can actually identify origins rather than initiation zones. It is the former that matters when comparing sites of ORC binding with replication origin sites, if a conclusion is to be drawn that the two do not co-localise.

Second, the authors give equal weight to all datasets. Certainly, in the case of SNS-seq, this is not appropriate. The technique has evolved over the years and some earlier versions have significantly different technical designs that may impact the reliability and/or resolution of the results e.g. in Foulk et al. (Foulk et al., 2015), lambda exonuclease was added to single stranded DNA from a total genomic preparation rather than purified nascent strands), which may lead to significantly different digestion patterns (ie underdigestion). Curiously, the authors do not make the best use of the largest SNS-seq dataset (Akerman et al., 2020) by ignoring these authors separation of core and stochastic origins. By blending all data together any separation of signal and noise is lost. Further, I am surprised that the authors have chosen not to use data and analysis from a recent study that provides subsets of the most highly used and efficient origins in the human genome, at high resolution (Guilbaud et al., 2022).

References

Akerman I, Kasaai B, Bazarova A, Sang PB, Peiffer I, Artufel M, Derelle R, Smith G, Rodriguez-Martinez M, Romano M, Kinet S, Tino P, Theillet C, Taylor N, Ballester B, Méchali M (2020) A predictable conserved DNA base composition signature defines human core DNA replication origins. Nat Commun, 11: 4826

Foulk MS, Urban JM, Casella C, Gerbi SA (2015) Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res, 25: 725-735

Guilbaud G, Murat P, Wilkes HS, Lerner LK, Sale JE, Krude T (2022) Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation. Nucleic Acids Res, 50: 7436-7450

Update in response to authors' comments on the original review:

While the authors have clarified their approach to some aspects of their analysis, I believe they and I are just going to have to disagree about the methodology and conclusions of this work. I do not find the authors responses sufficiently compelling to change my mind about the significance of the study or veracity of the conclusions. In my opinion, the method for identification of strong origins is not robust and of insufficient resolution. In addition, the resolution and the overlap of the MCM Chip-seq datasets is poor. While the conclusion of the paper would indeed be striking and surprising if true, I am not at all persuaded that it is based on the presented data.

Reviewer #2 (Public Review):

Tian et al. performed a meta-analysis of 113 genome-wide origin profile datasets in humans to assess the reproducibility of experimental techniques and shared genomics features of origins. Techniques to map DNA replication sites have quickly evolved over the last decade, yet little is known about how these methods fare against each other (pros and cons), nor how consistent their maps are. The authors show that high-confidence origins recapitulate several known features of origins (e.g., correspondence with open chromatin, overlap with transcriptional promoters, CTCF binding sites). However, surprisingly, they find little overlap between ORC/MCM binding sites and origin locations.

Overall, this meta-analysis provides the field with a good assessment of the current state of experimental techniques and their reproducibility, but I am worried about: (a) whether we've learned any new biology from this analysis; (b) how binding sites and origin locations can be so mismatched, in light of numerous studies that suggest otherwise; and (c) some methodological details described below.

-- I understand better the inclusion/exclusion logic for the samples. But I'm still not sure about the fragments. As the authors wrote, there is both noise and stochasticity; the former is not important but the latter is essential to include. How can these two be differentiated, and what may be the expected overlap as a function of different stochasticity rates?

-- Many of the major genomic features analyzed have already been found to be associated with origin sites. For example, the correspondence with TSS has been reported before:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320713/<br /> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547456/

-- Line 250: The most surprising finding is that there is little overlap between ORC/MCM binding sites and origin locations. The authors speculate that the overlap between ORC1 and ORC2 could be low because they come from different cell types. Equally concerning is the lack of overlap with MCM. If true, these are potentially major discoveries that butts heads with numerous other studies that have suggested otherwise.

The key missing dataset is ORC1 and ORC2 CHiP-seq from the same cell type. This shouldn't be too expensive to perform, and I hope someone performs this test soon. Without this, I remain on the fence about how much existing datasets are "junk" vs how much the prevailing hypothesis about replication needs to be revisited. Nonetheless, the authors do perform a nice analysis showing that existing techniques should be carefully used and interpreted.

Reviewer #3 (Public Review):

Summary: The authors present a thought-provoking and comprehensive re-analysis of previously published human cell genomics data that seeks to understand the relationship between the sites where the Origin Recognition Complex (ORC) binds chromatin, where the replicative helicase (Mcm2-7) is loaded, and where DNA replication actually begins (origins). The view that these should coincide is influenced by studies in yeast where ORC binds site-specifically to dedicated nucleosome-free origins where Mcm2-7 can be loaded and remains stably positioned for subsequent replication initiation. However, this is most certainly not the case in metazoans where it has already been reported that chromatin bindings sites of ORC and Mcm2-7 do not necessarily overlap, nor do they always overlap with origins. This is likely due to Mcm2-7 possessing linear mobility on DNA (i.e., it can slide) such that other chromatin-contextualized processes can displace it from the site in which it was originally loaded. Additionally, Mcm2-7 is loaded in excess and thus only a fraction of Mcm2-7 would be predicted to coincide with replication start sites. This study reaches a very similar conclusion of these previous studies: they find a high degree of discordance between ORC, Mcm2-7, and origin positions in human cells.

Strengths: The strength of this work is its comprehensive and unbiased analysis of all relevant genomics datasets. To my knowledge, this is the first attempt to integrate these observations. It also is an important cautionary tale to not confuse replication factor binding sites with the genomic loci where replication actually begins, although this point is already widely appreciated in the field.

Weaknesses: The major weakness of this paper is the lack of novel biological insight and that the comprehensive approach taken failed to provide any additional mechanistic insight regarding how and why ORC, Mcm2-7, and origin sites are selected or why they may not coincide.

Reviewer #1 (Public Review):

The mutation rate and spectrum have been found to differ between populations as well as across individuals within the same population. Hypothesizing that some of the observed variation has a genetic basis, the authors of this paper have made important contributions in the past few years in identifying genetic variants that modify mutation rate or spectrum in natural populations. This paper makes one significant step further by developing a new method for mapping genetic variants associated with the mutation spectrum, which reveals new biological insights.

Using traditional quantitative trait locus (QTL) mapping in the BXD mouse recombinant inbred lines (RILs), the authors of this paper previously identified a genetic locus associated with C>A mutation rate. However, this approach has limited power, as it suffers from multiple testing burden as well as noise in the "observed mutation spectrum phenotype" due to rarity and randomness of mutation events. To overcome these limitations, the authors developed a new method that they named "aggregate mutation spectrum distance" (AMSD), which in short measures the difference in the aggregate mutation spectrum between two groups of individuals with distinct genotypes at a specific genomic locus. With this new approach, they recover the previously reported candidate mutator locus (near Mutyh gene) and identify a new candidate locus that modifies the C>A mutation rate on only the mutator allele genetic background at the Mutyh locus. Using more rigorous statistical testing, the authors show convincingly synergistic epistatic effects between the mutator alleles at the two loci.

Overall, the analyses presented are well done and provide convincing evidence for the major findings, including the new candidate mutator locus and its epistatic interaction with the Mutyh locus. The new AMSD method introduced is innovative and outperforms traditional QTL mapping under most conditions, as demonstrated by extensive simulations. I identify no major issues with this paper and think it is very well written.

One of the major advantages of the AMSD method over QTL mapping is alleviation of the multiple testing burden, as one comparison tests for any changes in the mutation spectrum, including simultaneous, small changes in the relative abundance of multiple mutation types. The flip side of this advantage of AMSD is that, when a significant association is detected, it is not immediately clear which mutation type is driving the signal. To narrow the signal to specific candidate mutation type(s), additional analyses are needed, such as testing for differential proportions of each mutation type between individuals with or without the candidate mutator allele. However, such analysis may be less powerful when the mutator allele leads to small changes in the relative abundance of multiple mutation types. This will be an area of improvement for future studies.

Reviewer #2 (Public Review):

In this paper Sasani, Quinlan and Harris present a new method for identifying genetic factors affecting germline mutation, which is particularly applicable to genome sequence data from mutation accumulation experiments using recombinant inbred lines. These are experiments where laboratory organisms are crossed and repeatedly inbred for many generations, to build up a substantial number of identifiable germline mutations. The authors apply their method to such data from mice, and identify two genetic factors at two separate genetic loci. Clear evidence of such factors has been difficult to obtain, so this is an important finding. They further show evidence of an epistatic interaction between these factors (meaning that they do not act independently in their effects on the germline mutation process). This is exciting because such interactions are difficult to detect and few if any other examples have been studied.

The authors present a careful comparison of their method to another similar approach, quantitative trait locus (QTL) analysis, and demonstrate that in situations such as the one analysed it has greater power to detect genetic factors with a certain magnitude of effect. They also test the statistical properties of their method using simulated data and permutation tests. Overall the analysis is rigorous and well motivated, and the methods explained clearly.

The main limitation of the approach is that it is difficult to see how it might be applied beyond the context of mutation accumulation experiments using recombinant inbred lines. This is because the signal it detects, and hence its power, is based on the number of extra accumulated mutations linked to (i.e. on the same chromosome as) the mutator allele. In germline mutation studies of wild populations the number of generations involved (and hence the total number of mutations) is typically small, or else the mutator allele becomes unlinked from the mutations it has caused (due to recombination), or is lost from the population altogther (due to chance or perhaps selection against its deleterious consequences).

Nevertheless, accumulation lines are a common and well established experimental approach to studying mutation processes in many organisms, so the new method could have wide application and impact on our understanding of this fundamental biological process.

The evidence presented for an epistatic interaction is convincing, and the authors suggest some plausible potential mechanisms for how this interaction might arise, involving the DNA repair machinery and based on previous studies of the proteins implicated. However as with all such findings, given the higher degree of complexity of the proposed model it needs to be treated with greater caution, perhaps until replicated in a separate dataset or demonstrated in follow-up experiments exploring the pathway itself.

Reviewer #3 (Public Review):

Sasani et al. develop and implement a new method for mutator allele discovery in the BXD mouse population. This new method, termed "aggregate mutation spectrum distance" or AMSD, carries several notable strengths, including the ability to aggregate de novo mutations across individuals to reduce data sparsity and to combine mutation rate frequencies across multiple nucleotide contexts into a single estimate. As demonstrated by simulations, this method is better suited to mutator discovery under certain scenarios, as compared to conventional QTL or association mapping. Overall, the theoretical premise of the AMSD method is judged to be both strong and innovative, and the methodology could be extended to other species and populations to enable discovery of additional mutator alleles.

The authors then apply their method to the BXD mouse recombinant inbred mapping population. As proof-of-principle, they first successfully re-identify a known mutator locus in this population on chr4. Next, to assess possible genetic interactions involving this known mutator, Sasani et al. condition on the chr4 mutator genotype and reimplement the AMSD scan. This strategy led them to identify a second locus on chr6 that interacts epistatically with the chr4 locus; mice with "D" alleles at both loci exhibit a significantly increased burden of C>A de novo mutations, even though mice with the D allele at the chr6 locus alone show no appreciable increase in the C>A mutation fraction. This exciting discovery not only adds to the catalog of known mutator alleles, but also reveals key aspects of mutator biology and reinforces the hypothesis that segregating variants in genes associated with DNA repair influence germline mutation spectra.

Despite a high level of overall enthusiasm for this work, there are some limitations to the AMSD method. However, it is my judgement that the authors present a balanced summary of the strengths and weaknesses of their method in the revised manuscript. I also think that the authors' conclusions may actually somewhat undersell the scientific impact of their findings. As the authors note, few mutation rate modifiers have been identified in mammals. This is potentially because large- and moderate-effect modifiers are rapidly selected against due to their deleterious effects, but could also be due to pervasive epistasis wherein modifiers are only expressed on certain "permissive" genetic backgrounds, such as the chr6 locus the authors discover in this paper. The potential background dependence of mutator expression could partially shelter it from the action of selection, allowing the allele persist in populations. This discovery has significant implications for our understanding of mutation rate evolution, but only earns a cursory mention in the paper.

Reviewer #1 (Public Review):

This is an interesting study by Pinos and colleagues that examines the effect of beta carotene on atherosclerosis regression. The authors have previously shown that beta carotene reduces atherosclerosis progress and hepatic lipid metabolism, and now they seek to extend these findings by feeding mice a diet with excess beta carotene in a model of atherosclerosis regression (LDLR antisense oligo plus Western diet followed by LDLR sense oligo and chow diet). They show some metrics of lesion regression are increased upon beta carotene feeding (collagen content) while others remain equal to normal chow diet (macrophage content and lesion size). These effects are lost when beta carotene oxidase (BCO) is deleted. The study adds to the existing literature that beta carotene protects from atherosclerosis in general, and adds new information regarding regulatory T-cells. However, the study does not present significant evidence about how beta-carotene is affecting T-cells in atherosclerosis. For the most part, the conclusions are supported by the data presented, and the work is completed in multiple models, supporting its robustness. However there are a few areas that require additional information or evidence to support their conclusions and/or to align with the previously published work.

Specific additional areas of focus for the authors:<br /> The premise of the story is that b-carotene is converted into retinoic acid, which acts as a ligand of the ROR transcription factor in T-regs. The authors measure hepatic markers of retinoic acid signaling (retinyl esters, Cyp26a1 expression) but none of these are measured in the lesion, which calls into question the conclusion that Tregs in the lesion are responsible for the regression observed with b-carotene supplementation.

There does not appear to be a strong effect of Tregs on the b-carotene induced pro-regression phenotype presented in Figure 5. The only major CD25+ cell dependent b-carotene effect is on collagen content, which matches with the findings in Figure 1 +2. This mechanistically might be very interesting and novel, yet the authors do not investigate this further or add any additional detail regarding this observation. This would greatly strengthen the study and the novelty of the findings overall as it relates to b-carotene and atherosclerosis.

The title indicates that beta-carotene induces Treg 'expansion' in the lesion, but this is not measured in the study.

Revised manuscript:<br /> In the revised manuscript, the authors provide quantification of an RA-responsive gene in the plaque as evidence that RA signalling is indeed elevated upon b-carotene supplementation. It is not reduced upon blocking CD25 (Tregs) which implies that other cells in addition to Tregs are impacted by b-carotene supplementation that favourably remodels the plaque. The authors properly account for this by tempering their conclusions and recognize that Tregs are only partially responsible for the plaque phenotype upon b-carotene supplementation.

The authors chose not to further investigate why b-carotene impacted collagen production, instead including a discussion point. In this reviewer's opinion, it is a missed opportunity but hopefully something that can be investigated further by others.

Reviewer #2 (Public Review):

Pinos et al present five atherosclerosis studies in mice to investigate the impact of dietary supplementation with b-carotene on plaque remodeling during resolution. The authors use either LDLR-ko mice or WT mice injected with ASO-LDLR to establish diet-induced hyperlipidemia and promote atherogenesis during 16 weeks, and then they promote resolution by switching the mice for 3 weeks to a regular chow, either deficient or supplemented with b-carotene. Supplementation was successful, as measured by hepatic accumulation of retinyl esters. As expected, chow diet led to reduced hyperlipidemia, and plaque remodeling (both reduced CD68+ macs and increased collagen contents) without actual changes in plaque size. But, b-carotene supplementation resulted in further increased collagen contents and, importantly, a large increase in plaque regulatory T-cells (TREG). This accumulation of TREG is specific to the plaque, as it was not observed in blood or spleen. The authors propose that the anti-inflammatory properties of these TREG explain the atheroprotective effect of b-carotene, and found that treatment with anti-CD25 antibodies (to induce systemic depletion of TREG) prevents b-carotene-stimulated increase in plaque collagen and TREG.

An obvious strength is the use of two different mouse models of atherogenesis, as well as genetic and interventional approaches. The analyses of aortic root plaque size and contents are rigorous and included both male and female mice (although the data was not segregated by sex). Unfortunately, the authors did not provide data on lesions in en face preparations of the whole aorta.

Overall, the conclusion that dietary supplementation with b-carotene may be atheroprotective via induction of TREG is reasonably supported by the evidence presented. Other conclusions put forth by the authors (e.g., that vitamin A production favors TREG production or that BCO1 deficiency reduces plasma cholesterol), however, will need further experimental evidence to be substantiated.

The authors claim that b-carotene reduces blood cholesterol, but data shown herein show no differences in plasma lipids between mice fed b-carotene-deficient and -supplemented diets (Figs. 1B, 2A, and S3A). Also, the authors present no experimental data to support the idea that BCO1 activity favors plaque TREG expansion (e.g., no TREG data in Fig 3 using Bco1-ko mice).

As the authors show, the treatment with anti-CD25 resulted in only partial suppression of TREG levels. Because CD25 is also expressed in some subpopulation of effector T-cells, this could potentially cloud the interpretation of the results. Data in Fig 4H showing loss of b-carotene-stimulated increase in numbers of FoxP3+GFP+ cells in the plaque should be taken cautiously, as they come from a small number of mice. Perhaps an orthogonal approach using FoxP3-DTR mice could have produced a more robust loss of TREG and further confirmation that the loss of plaque remodeling is indeed due to loss of TREG.

Reviewer #1 (Public Review):

Summary:

The authors demonstrate that the immunosuppressive environment in pancreatic ductal adenocarcinoma (PDAC) can be mitigated by a combination of ionizing radiation (IR), CCR5 inhibition, and PD1 blockade. This combination therapy increases tissue-resident natural killer (trNK) cells that facilitate CD8 T cell activity, resulting in a reduction of E-cadherin positive tumor cells. They identify a specific "hypofunctional" NK cell population in both mouse and human PDAC that supports CD8 T cell involvement. A trNK signature is found to be associated with better survival outcomes in PDAC and other solid tumors.

Strengths:

Overall, I think this is an interesting study that combines testing of therapeutic concepts in mice with bioinformatics analysis of single-cell transcriptome data in primary tumors and exploration of clinical outcomes using signature genes in TCGA data. The key finding is that immunoregulatory properties of tumor-infiltrating/resident CD56-bright NK cells (assumed to be non-cytotoxic) are beneficial for outcome through cross-talk with DC and recruitment of CD8 T cells. The latter is specifically induced by irradiation combined with CCR5i and PD1 blockade.

"These results collectively support the notion that IR/CCR5i/αPD1 combination treatment alters immune infiltration by reducing Tregs and increasing NK and CD8 T cells, thereby resulting in greater local tumor control." I agree with this conclusion.

Weaknesses:

There are a few points to discuss and that the authors may want to address.

1) "Notably, CCR5i significantly reduced Treg infiltration but had no effect on the infiltration of other immune cells, indicating the active recruitment of CCR5+ Tregs in PDAC (Figure 2B)."<br /> CCR5i treatment seems to inhibit infiltration of CD8 T cells and NK cells to a greater extent, in relative terms, compared to Treg, albeit it is not statistically significant. If this visual inspection of the graph does not reflect reality, additional experiments may be needed to verify the selective targeting of Tregs or confirm the fact that also CD8 T cells and NK cells are affected by single agent CCR5i. The reduced recruitment of Treg, NK cells, and CD8T cells was completely reversed when combined with irradiation. In the data shown in Figure 3E it seems as if CCR5i induced infiltration of Tregs along with other immune cells. However, this said, I agree with the conclusion of the authors that this combined treatment leads to an altered immune composition and ratio between Tregs and effector cells (CD8T cells and NK cells). Could this altered composition be displayed more clearly?

2) The definition of active and hypofunctional NK cells based on solely NKG2D expression alone seems like an oversimplification. I realize it is not trivial to test tumor-infiltrating NK cells from these tumors functionally but perhaps scRNAseq of the tumors would allow for characterization of cytotoxicity scores using KEGG or GO analysis or reversed gene set enrichment in responders/non-responders. It seems as if the abstract refers to this phenotype incorrectly since the "hyporesponsive" subset is described as NKG2C-negative.

3) "The NK_C1 cluster correlates best with the hypofunction NK phenotype observed in mice as similarly displayed reduced activation (reduced NKG7, NKp80, GZMA, and PRF1) with additional expression of tissue residency markers CD103, CD49a and, surprisingly, the adaptive activating receptor NKG2C (KLRC2) (Figure 5B, C)."

There is no doubt that NK_C1 represents tumor-infiltrating NK cells with a CD56bright gene signature with a strong tissue resident score. However, the transcriptional expression of KLRC2 on these is not surprising! It is well established that KLRC2 transcripts (but not protein) are highly expressed on conventional CD56bright NK cells. There are several published sources where the authors can find such data for confirmation. Thus, this is not to be confused with adaptive NK cells having an entirely different transcriptional signature and expressing high levels of NKG2C at the cell surface. I strongly recommend re-interpreting the results based on the fact that KLRC2 is expressed at high levels in conventional CD56bright NK cells. If not, it would be important to verify that these tissue-resident NK cells express NKG2C and not NKG2A at the cell surface.

4) NCAM1 transcript alone is not sufficient to deconvolute CD56bright NK cells in TCGA data (Figure 7A). As a single marker, it likely reflects NK cell infiltration without providing further evidence on the contribution of the bright/dim components. Therefore, the use of the bright Tr NK signature described in Table 1 is very important (Figure 7B). Table 1 is not provided. Nor Supplementary Table 1. There is only one supplementary figure in the ppt attached.

Reviewer #2 (Public Review):

Summary:

This work elaborates on a combined therapeutic approach comprising ionizing radiation and CCR5i/αPD1 immunotherapy as a promising strategy in pancreatic cancer. Previous research has established that NK cell-derived CCL5 and XCL1 play a crucial role in recruiting cDC1 cells to the tumor microenvironment, contributing to tumor control. In this study, by using a murine pancreatic cancer model, the authors propose that the addition of radiation therapy to CCR5i and αPD1 immunotherapy could upregulate CD8+ T cells and a subgroup of NK cells within the tumor and result in better tumor control. They further analyzed human single-cell sequencing data from pancreatic cancer patients and identified one subgroup of NK cells (NK C1) with tissue-resident features. Subsequent cell-cell contact analysis reveals the NK-cDC1-CD8 cell axis in pancreatic cancer. By analyzing TCGA data, they found that high NK C1 signature levels were associated with better survival in pancreatic cancer patients. Thus, radiotherapy could benefit the outcome of patients bearing low NK C1 signatures. Importantly, the positive correlation between NK C1 score with survival extends beyond pancreatic cancer, showing potential applicability across various solid cancers.

Strengths:

This study could add new insight into the clinical practice by introducing such novel combined therapy and shed light on the underlying immune cell dynamics. These findings hold potential for more effective and targeted treatment in the future. Mouse experiments nicely confirmed that such combined therapy could significantly reduce tumor volume. The elegant use of single-cell sequencing analysis and human database examination enriches the narrative and strengthens the study's foundation. Additionally, the notion that NK C1 signature correlates with patient survival in various solid cancers is of high interest and relevance.

Weaknesses:

1. The role of CCR5i requires further clarification. While the authors demonstrated its capacity to reduce Treg in murine tumors, its impact on other cell populations, including NK cells and CD8+ T cells, was not observed. Nevertheless, the effect of CCR5i on tumor growth in Figure 2B should be shown. If the combination of radiotherapy and αPD1 already can achieve good outcomes as shown in Figure 3A, the necessity to include CCR5i is questioned. Overall, a more comprehensive elucidation of the roles of CCL5 and CCR5i in this context would be good.

2. In line with this, spatial plots in Figure 4 did not include the group with only radiotherapy and αPD1. This inclusion would facilitate a clearer comparison and better highlight the essential role of CCR5i.

3. NK C1 cells should be also analyzed in the mouse model. The authors suggest that NKNKG2D-ve could be the cell population. Staining of inhibitory markers should be considered, for example, TIGIT and TIM3 as presented in Figure 5B.

4. While the cell-cell contact analysis generated from single-cell sequencing data is insightful, extending this analysis to the mouse model under therapy would be highly informative. NK and CD8 cells in the tumor increased upon the combined therapy. However, cDC1 was not characterized. Analysis regarding cDC1 would provide more information on the NK/cDC1/CD8 axis.

5. Human database analysis showed a positive correlation between NK C1 score and CCL5 in pancreatic cancer. Furthermore, radiotherapy could benefit the outcome of patients bearing low NK C1 scores. It would be interesting to test if radiotherapy could also benefit patients with low CCL5 levels in this cohort.

Reviewer #3 (Public Review):

Summary:

In the submitted manuscript by Go et al, the authors evaluated the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and made a number of interesting observations, including the following: 1) CCL5 expression within the tumor microenvironment negatively correlated with clinical outcomes in human patients with PDAC; 2) there were both positive and negative correlations between CCL5 expression and the expression of specific genes (e.g. those encoding CD56 and CD16, respectively) included among gene signature lists for Treg, MDSC, TAM, and NK cells; 3) CCR5 inhibition with the inhibitor, maraviroc, reduced Treg infiltration but not that of other immune cell types in an orthotopic murine model of PDAC; 4) CCR5 inhibition augmented anti-PD1 immunotherapy when combined with ionizing radiation (IR) therapy in the murine model; 5) the above therapy resulted in increased infiltration of CD8+ cytotoxic T cells as well as of a subset of NKG2D-negative, tissue-residency (tr) marker expressing NK cells (deemed Cluster 1 NK in their data sets) that inversely correlated with the number of E-cadherin+ cells (i.e. tumor cells) and showed predicted interactions with cDC1 dendritic cells (including XCL1/XCL2 expressed by the NK and XCR1 expressed by the cDC1); 6) the authors identified a number of putative signals stemming from the trNK (e.g. IL-16, TNFSF14, FASLG, CSF, MIF) as well as incoming from cDC1s to NK (e.g. BAG6-NKp30); 7) these trNK cells positively correlated with good outcomes and with CD8+ T cell infiltrations in human PDAC as well as in many other solid tumor types; and 8) importantly, the benefit of IR therapy was specific to the subset of PDAC patients (represented in the TCGA dataset) that were predicted to have low amounts of trNK cells. The authors used murine experimental models, multiplexed imaging analyses, and a number of publicly available sequencing data sets from human tumor samples to perform their investigations. Based on their findings, the authors proposed that combining IR with CCR5 inhibition and anti-PD1 immunotherapy is a promising strategy to treat solid cancers.

Strengths:

Overall, the collective analyses and conclusions appear to be novel and could be of high and rapid impact on the field, particularly in terms of directing clinical trials to incorporate IR with CCR5 inhibition and immunotherapy. The manuscript is well written; the figures are for the most part clear; and the Discussion is very thoughtful.

Weaknesses:

There were a number of minor typographical errors, missing references, or minor issues with the figures. In general, while many of the observations provided strong suggestive evidence of relationships, phenotypes, and functions, the authors often used language to indicate that such things were confirmed, validated, or proven. In fact, there was a paucity of such functional/confirmatory experiments. This does not necessarily detract from the overall significance, excitement for, and potential impact of the study; but the language could likely be adjusted to be more in keeping with the true nature of the findings. The main title and running title are a bit different; consider making them more similar.

Reviewer #3 (Public Review):

This work by Fleck et al. and colleagues documented the auxin feeding-induced effects in adult flies, since auxin could be used in temporally control gene expression using a modified Gal4/Gal80 system. Overall, the experiments were well designed and carefully executed. The results were quantified with appropriate statistical analyses. The paper was also well written and the results were presented logically. Their findings demonstrate that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes of genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes. Researchers need to be careful when applying this strategy in their own experimental design and should perform proper controls.

Reviewer #1 (Public Review):

In recent years, Auxin treatment is frequently used for inducing targeted protein degradation in Drosophila and various other organisms. This approach provides the way to acutely alter the levels of specific proteins. In this manuscript, the authors carefully examine the impact of Auxin treatment and provide strong evidence that Auxin treatment elicits alterations in feeding activity, survival rates, lipid metabolism, and gene expression patterns. Researchers need to be aware of these effects to design experiment/controls and interpret their data.

Strengths:<br /> Regarding widespread usage of Auxin mediated gene manipulation method, it is important to address whether the application of Auxin itself causes any physiological changes. Authors provide evidence of several Auxin effects on lipid metabolism, feeding behavior and gene expression changes. Experiments are suitably designed with appropriate sample size, data analysis methods.

Weaknesses:<br /> Data shown here are limited for certain method of treatment. No time course, dose dependency information is provided, and cell-type-specific responses are unknown. Therefore, this work basically provides the cautionary note for the field for researchers who use this method suggesting the importance that they should thoroughly check the gene expression pattern for their specific tissue of interest under their normal standard or altered food conditions.

Reviewer #2 (Public Review):

In this study, Fleck and colleagues investigate the effects of auxin exposure on Drosophila melanogaster adults, focusing their analysis on feeding behavior, fatty acid metabolism, and oogenesis. The motivation for the study is that auxin-inducible transcription systems are now being used by Drosophila researchers to drive transcription using the Gal4-UAS system as a complement to Gal80ts versions of the system. I found the study to be carefully done. This study will be of interest for researchers using the Drosophila system, especially those focusing on fatty acid metabolism or physiology. The authors have adequately addressed all the minor points I raised in my review of the first submission.

Reviewer #3 (Public Review):

Summary of Work<br /> This paper conducts the largest GWAS study of A. thaliana in response to a viral infection. The paper identifies a 1.5 MB region in the chromosome associated with disease, including SNPs, structural variation, and transposon insertions. Studies further validate the association experimentally with a separate experimental infection procedure with several lines and specific T-DNA mutants. Finally, the paper presents a geographic analysis of the minor disease allele and the major association. The major take-home message of the paper is that structural variants and not only SNPs are important changes associated with disease susceptibility. The manuscript also makes a strong case for negative frequency-dependent selection maintaining a disease susceptibility locus at low frequency.

Strengths and Weaknesses<br /> A major strength of this manuscript is the large sample sizes, careful experimental design, and rigor in the follow-up experiments. For instance, mentioning non-infected controls and using methods to determine if geographic locus associations were due to chance. The strong result of a GWAS-detected locus is impressive given the complex interaction between plant genotypes and strains noted in the results. In addition to the follow-up experiments, the geographic analysis added important context and broadened the scope of the study beyond typical lab-based GWAS studies. I find very few weaknesses in this manuscript.

Support of Conclusions<br /> The support for the conclusions is exceptional. This is due to the massive amount of evidence for each statement and also due to the careful consideration of alternative explanations for the data.

Significance of Work<br /> This manuscript will be of great significance in plant disease research, both for its findings and its experimental approach. The study has very important implications for genetic associations with disease beyond plants.

Reviewer #1 (Public Review):

In this manuscript, Butkovic et al. perform a genome-wide association (GWA) study on Arabidopsis thaliana inoculated with the natural pathogen turnip mosaic virus (TuMV) in laboratory conditions, with the aim to identify genetic associations with virus infection-related parameters. For this purpose, they use a large panel of A. thaliana inbred lines and two strains of TuMV, one naïve and one pre-adapted through experimental evolution. A strong association is found between a region in chromosome 2 (1.5 Mb) and the risk of systemic necrosis upon viral infection, although the causative gene remains to be pinpointed.

This project is a remarkable tour de force, but the conclusions that can be reached from the results obtained are unfortunately underwhelming. Some aspects of the work could be clarified, and presentation modified, to help the reader.

Reviewer #2 (Public Review):

The manuscript presents a valuable investigation of genetic associations related to plant resistance against the turnip mosaic virus (TuMV) using Arabidopsis thaliana as a model. The study infects over 1,000 A. thaliana inbred lines with both ancestral and evolved TuMV and assesses four disease-related traits: infectivity, disease progress, symptom severity, and necrosis. The findings reveal that plants infected with the evolved TuMV strain generally exhibited more severe disease symptoms than those infected with the ancestral strain. However, there was considerable variation among plant lines, highlighting the complexity of plant-virus interactions.

A major genetic locus on chromosome 2 was identified, strongly associated with symptom severity and necrosis. This region contained several candidate genes involved in plant defense against viruses. The study also identified additional genetic loci associated with necrosis, some common to both viral isolates and others specific to individual isolates. Structural variations, including transposable element insertions, were observed in the genomic region linked to disease traits.

Surprisingly, the minor allele associated with increased disease symptoms was geographically widespread among the studied plant lines, contrary to typical expectations of natural selection limiting the spread of deleterious alleles. Overall, this research provides valuable insights into the genetic basis of plant responses to TuMV, highlighting the complexity of these interactions and suggesting potential avenues for improving crop resilience against viral infections.

Overall, the manuscript is well-written, and the data are generally high-quality. The study is generally well-executed and contributes to our understanding of plant-virus interactions.

Reviewer #2 (Public Review):

Summary:

The study demonstrates that deletion of a small cytoplasmic membrane protein, Tmem263, caused severe impairment of longitudinal bone growth and that the impaired bone growth was caused by suppression of expression and/or protein levels of growth hormone receptor in the liver.

Strengths:

The experimental design of the study is sound and the results are in general of supportive of the conclusions.

Weaknesses:

The study lacks mechanistic investigation into how the deletion of a gene corresponding to a small cytoplasmic membrane protein would lead to substantial reduction in the gene expression of growth hormone receptor, which takes place in the nuclei. Accordingly, the manuscript is of largely descriptive nature.

Reviewer #3 (Public Review):

Prior studies in humans and in chickens suggested that TMEM263 could play an important role in longitudinal bone growth, but a definitive assessment of the function and potential mechanism of action of this species-conserved plasma membrane protein has been lacking. Here, the authors create a TMEM263 null mouse model and convincingly show dramatic cessation of post-natal growth, which becomes apparent by day PND21. They report proportional dwarfism, highly significant bone and related phenotypes, as well as notable alterations of hepatic GH signaling to IGF1. A large body of prior work has established an essential role for GH and it's stimulation of IGF1 production in liver and other tissues in post-natal growth. On this basis, the authors conclude that the observed decrease in serum IGF1 seen in TMEM263-KO mice is causal for the growth phenotype, which seems likely. Moreover, they ascribe the low serum IGF1 to the observed decreases in hepatic GH receptor (GHR) expression and GHR/JAK2/STAT5 signaling to IGF1, which is plausible but not proven by the experiments presented.

The finding that TMEM263 is essential for normal hepatic GHR/IGF1 signaling is an important, and unexpected finding, one that is likely to stimulate further research into the underlying mechanisms of TMEM263 action, including the distinct possibility that these effects involve direct protein-protein interactions between GHR and TMEM263 on the plasma membrane of hepatocytes, and perhaps on other mouse cell types and tissues as well, where TMEM263 expression is up to 100-fold higher (Fig. 1C).

An intriguing finding of this study, which is under emphasized and should be noted in the Abstract, is the apparent feminization of liver gene expression in male TMEM263-KO mice, where many male-biased genes are downregulated, and many female-biased genes are upregulated. Further investigation of these liver gene responses by comparison to public datasets could be very useful, as it could help determine: (1) whether the TMEM263 liver phenotype is similar to that of hypophysectomized male mouse liver, where GH and GHR/STAT5/IGF1 signaling are both totally ablated; or alternatively, (2) whether the phenotype is more similar to that of a male mouse given GH as a continuous infusion, which induces widespread feminization of gene expression in the liver, and is perhaps similar to the gene responses seen in the TMEM263-KO mice. Answering this question could provide critical insight into the mechanistic basis for the hepatic effects of TMEM263 gene KO.

Comments on revised version:

The authors have addressed a majority of the concerns raised during the initial review. The evidence supporting the whole-body growth and skeletal phenotypes, as well as the disruption of GH/IGF1 signaling seen in TMEM263-KO mice, is convincing. However, there is insufficient evidence to definitively conclude that the observed alteration of hepatic GH/IGF1 signaling is causative of the body growth and skeletal phenotypes.

Reviewer #1 (Public Review):

Summary<br /> Developing vaccination capable of inducing persistent antibody responses capable of broadly neutralizing HIV strains is of high importance. However, our ability to design vaccines to achieve this is limited by our relative lack of understanding of the role of T-follicular helper (Tfh) subtypes in the responses. In this report Verma et al investigate the effects of different prime and boost vaccination strategies to induce skewed Tfh responses and its relationship to antibody levels. They initially find that live-attenuated measles vaccine, known to be effective at inducing prolonged antibody responses has a significant minority of germinal center Tfh (GC-Tfh) with a Th1 phenotype (GC-Tfh1) and then explore whether a prime and boost vaccination strategy designed to induce GC-Tfh1 is effective in the context of anti-HIV vaccination. They demonstrate that a vaccine formulation referred to as MPLA induces higher GC-Tfh1 and link this to increased antibody production.

Strengths:<br /> While there is a lot of literature on Tfh subtypes in blood, how this related to the germinal centers is not always clear. The strength of this paper is that they use a relevant model to allow some longitudinal insight into the detailed events of the germinal center Tfh (GC-Tfh) compartment across time and how this related to antibody production.

Weaknesses:<br /> The authors focus strongly on the proportion of GC-Tfh1 of GC-Tfh. There seems to be an assumption that since the MPLA vaccine has a higher number of GC-Tfh1 that this explains the higher levels of antibodies. This is not an entirely unreasonable assumption but the mechanistic link between the two is never tested.

Reviewer #2 (Public Review):

Summary:<br /> Anil Verma et al. have performed prime-boost HIV vaccination to enhance HIV-1 Env antibodies in the rhesus macaques model. The authors used two different adjuvants, a cationic liposome-based adjuvant (CAF01) and a monophosphoryl lipid A (MPLA)+QS-21 adjuvant. They demonstrated that these two adjuvants promote different transcriptomes in the GC-TFH subsets. The MPLA+QS-21 adjuvant induces abundant GC TFH1 cells expressing CXCR3 at first priming, while the CAF01 adjuvant predominantly induced GC TFH1/17 cells co-expressing CXCR3 and CCR6. Both adjuvants initiate comparable Env antibody responses. However, MPLA+QS-21 shows more significant IgG1 antibodies binding to gp140 even after 30 weeks.<br /> The enhancement of memory responses by MPLA+QS-21 consistently associates with the emergence of GC TFH1 cells that preferentially produce IFN-γ.

Strengths:<br /> The strength of this manuscript is that all experiments have been done in the rhesus macaques model with great care. This manuscript beautifully indicated that MPLA+QS-21 would be a promising adjuvant to induce the memory B cell response in the HIV vaccine.

Weaknesses:<br /> The authors did not provide clear evidence to indicate the functional relevance of GC TFH1 in IgG1 class-switch and B cell memory responses.

Reviewer #1 (Public Review):

The authors isolated a novel marine Planctomycetes bacterium with unique characteristics using a budding mode of division from the deep-sea cold seep sediment and named it Poriferisphaera heterotrophicis ZRK32. This work demonstrated that strain ZRK32 preferred nutrient-rich medium, moreover, the addition of nitrate or ammonia promoted the growth of strain ZRK32 and further caused the release of bacteriophage without killing the host. These results are interesting, well presented and documented in the revised manuscript.

Reviewer #2 (Public Review):

Summary:<br /> Planctomycetes encompass a group of bacteria with unique biological traits, the compartmentalized cells make them appear to be organisms in between prokaryotes and eukaryotes. However, only few of the Planctomycetes bacteria are cultured thus far, and hampers insight into the biological traits of this evolutionary important organisms.

This work reports the methodology details of how to isolate the deep-sea bacteria that could be recalcitrant to laboratory cultivation, and further reveals the distinct characteristics of the new species of a deep-sea Planctomycetes bacterium, such as the chronic phage release without breaking the host and promote the host and related bacteria in nitrogen utilization. Therefore, the finding of this work is of importance in extending our knowledge on bacteria.

Strengths:

Through combination of microscopic, physiological, genomics and molecular biological approaches, this reports isolation and comprehensively investigation of the first anaerobic representative of the deep-sea Planctomycetes bacterium, in particular in that of the budding division, and release phage without lysis the cells. Most of results and conclusions are supported by the experimental evidences.

Reviewer #1 (Public Review):

In this article, Vardakalis et al. propose a novel model of hippocampal oscillations whereby an external input (emulating the medial septum) can drive theta rhythms. This model displays phase-amplitude coupling of gamma oscillations, as well as theta resetting, which are known features of physiological theta that have been missing in previous models. The end goal proposed by the authors is to have a framework to explore the mechanisms of neurostimulation, which have shown promising applications in pathological conditions, but for which the underlying dynamics remain largely unknown. To reach this objective, the authors implement an existing biophysical model of the hippocampus that is able to generate gamma oscillations, and receives inputs from a set of Kuramoto oscillators to emulate theta drive originating from the medial septum.

Overall, the hypotheses and results are clearly presented and supported by high quality figures. The study is presented in a didactic way, making it easy for a broad audience to understand the significance of the results. The study does present some weaknesses that could easily be addressed by the authors. First, there are some anatomical inaccuracies: line 129 and fig1C, the authors omit medial septum projections to area CA1 (in addition to the entorhinal cortex). Moreover, in addition to CA1, CA3 also provides monosynaptic feedback projections to the medial septum CA3. Finally, an indirect projection from CA1/3 excitatory neurons to the lateral septum, which in turn sends inhibitory projections to the medial septum could be included or mentioned by the authors. This could be of particular relevance to support claims related to effects of neurostimulations, whereby minutious implementation of anatomical data could be key. If not updating their model, the authors could add this point to their limitation section, where they already do a good job of mentioning some limitations of using the EC as a sole oscillatory input to CA1. The authors test conditions of low theta inputs, which they liken to pathological states (line 112). It is not clear what pathology the authors are referring to, especially since a large amount of 'oscillopathies' in the septohippocampal system are associated with decreased gamma/PAC, but not theta oscillations (e.g. Alzheimer's disease conditions). While relevant for the clinical field, there is overall a missed opportunity to explain many experimental accounts with this novel model. Although to this day, clinical use of DBS is mostly restricted to electrical (and thus cell-type agnostic) stimulation, recent studies focusing on mechanisms of neurostimulations have manipulated specific subtypes in the medial septum and observed effects on hippocampal oscillations (e.g. see Muller & Remy, 2017 for review). Focusing stimulations in CA1 is of course relevant for clinical studies but testing mechanistic hypotheses by focusing stimulation on specific cell types could be highly informative. For instance, could the author reproduce recent optogenetic studies (e.g. Bender et al. 2015 for stimulation of fornix fibers; Etter et al., 2019 & Zutshi et al. 2018 for stimulation of septal inhibitory neurons)? Cell specific manipulations should at least be discussed by the authors.

Beyond these weaknesses, this study has a strong utility for researchers wanting to explore hypotheses in the field of neurostimulations. In particular, I see value in such models for exploring more intricate, phase specific effects of continuous, as well as close loop stimulations which are on the rise in systems neuroscience.

Reviewer #2 (Public Review):

Theta-nested gamma oscillations (TNGO) play an important role in hippocampal memory and cognitive processes and are disrupted in pathology. Deep brain stimulation has been shown to affect memory encoding. To investigate the effect of pulsed CA1 neurostimulation on hippocampal TNGO the authors coupled a physiologically realistic model of the hippocampus comprising EC, DG, CA1, and CA3 subfields with an abstract theta oscillator model of the medial septum (MS). Pathology was modeled as weakened theta input from the MS to EC simulating MS neurodegeneration known to occur in Alzheimer's disease. The authors show that if the input from the MS to EC is strong (the healthy state) the model autonomously generates TNGO in all hippocampal subfields while a single neurostimulation pulse has the effect of resetting the TNGO phase. When the MS input strength is weaker the network is quiescent but the authors find that a single CA1 neurostimulation pulse can switch it into the persistent TNGO state, provided the neurostimulation pulse is applied at the peak of the EC theta. If the MS theta oscillator model is supplemented by an additional phase-reset mechanism a single CA1 neurostimulation pulse applied at the trough of EC theta also produces the same effect. If the MS input to EC is weaker still, only a short burst of TNGO is generated by a single neurostimulation pulse. The authors investigate the physiological origin of this burst and find it results from an interplay of CAN and M currents in the CA1 excitatory cells. In this case, the authors find that TNGO can only be rescued by a theta frequency train of CA1 pulses applied at the peak of the EC theta or again at either the peak or trough if the MS oscillator model is supplemented by the phase-reset mechanism.

The main strength of this model is its use of a fairly physiologically detailed model of the hippocampus. The cells are single-compartment models but do include multiple ion channels and are spatially arranged in accordance with the hippocampal structure. This allows the understanding of how ion channels (possibly modifiable by pharmacological agents) interact with system-level oscillations and neurostimulation. The model also includes all the main hippocampal subfields. The other strength is its attention to an important topic, which may be relevant for dementia treatment or prevention, which few modeling studies have addressed.

The work has several weaknesses. First, while investigations of hippocampal neurostimulation are important there are few experimental studies from which one could judge the validity of the model findings. All its findings are therefore predictions. It would be much more convincing to first show the model is able to reproduce some measured empirical neurostimulation effect before proceeding to make predictions. Second, the model is very specific. Or if its behavior is to be considered general it has not been explained why. For example, the model shows bistability between quiescence and TNGO, however what aspect of the model underlies this, be it some particular network structure or particular ion channel, for example, is not addressed. Similarly for the various phase reset behaviors that are found. We may wonder whether a different hippocampal model of TNGO, of which there are many published (for example [1-6]) would show the same effect under neurostimulation. This seems very unlikely and indeed the quiescent state itself shown by this model seems quite artificial. Some indication that particular ion channels, CAN and M are relevant is briefly provided and the work would be much improved by examining this aspect in more detail. In summary, the work would benefit from an intuitive analysis of the basic model ingredients underlying its neurostimulation response properties. Third, while the model is fairly realistic, considerable important factors are not included and in fact, there are much more detailed hippocampal models out there (for example [5,6]). In particular, it includes only excitatory cells and a single type of inhibitory cell. This is particularly important since there are many models and experimental studies where specific cell types, for example, OLM and VIP cells, are strongly implicated in TNGO. Other missing ingredients one may think might have a strong impact on model response to neurostimulation (in particular stimulation trains) include the well-known short-term plasticity between different hippocampal cell types and active dendritic properties. Fourth the MS model seems somewhat unsupported. It is modeled as a set of coupled oscillators that synchronize. However, there is also a phase reset mechanism included. This mechanism is important because it underlies several of the phase reset behaviors shown by the full model. However, it is not derived from experimental phase response curves of septal neurons of which there is no direct measurement. The work would benefit from the use of a more biologically validated MS model.

[1] Hyafil A, Giraud AL, Fontolan L, Gutkin B. Neural cross-frequency coupling: connecting architectures, mechanisms, and functions. Trends in neurosciences. 2015 Nov 1;38(11):725-40.

[2] Tort AB, Rotstein HG, Dugladze T, Gloveli T, Kopell NJ. On the formation of gamma-coherent cell assemblies by oriens lacunosum-moleculare interneurons in the hippocampus. Proceedings of the National Academy of Sciences. 2007 Aug 14;104(33):13490-5.

[3] Neymotin SA, Lazarewicz MT, Sherif M, Contreras D, Finkel LH, Lytton WW. Ketamine disrupts theta modulation of gamma in a computer model of hippocampus. Journal of Neuroscience. 2011 Aug 10;31(32):11733-43.

[4] Ponzi A, Dura-Bernal S, Migliore M. Theta-gamma phase-amplitude coupling in a hippocampal CA1 microcircuit. PLOS Computational Biology. 2023 Mar 23;19(3):e1010942.

[5] Bezaire MJ, Raikov I, Burk K, Vyas D, Soltesz I. Interneuronal mechanisms of hippocampal theta oscillations in a full-scale model of the rodent CA1 circuit. Elife. 2016 Dec 23;5:e18566.

[6] Chatzikalymniou AP, Gumus M, Skinner FK. Linking minimal and detailed models of CA1 microcircuits reveals how theta rhythms emerge and their frequencies controlled. Hippocampus. 2021 Sep;31(9):982-1002.

Joint Public Review:

In this work, Xie et al. developed SCA-seq, which is a multiOME mapping method that can obtain chromatin accessibility, methylation, and 3D genome information at the same time. SCA-seq first uses M.CviPI DNA methyltransferase to treat chromatin, then perform proximity ligation followed by long-read sequencing. This method is highly relevant to a few previously reported long read sequencing technologies. Specifically, NanoNome, SMAC-seq, and Fiber-seq have been reported to use m6A or GpC methyltransferase accessibility to map open chromatin, or open chromatin together with CpG methylation; Pore-C and MC-3C have been reported to use long read sequencing to map multiplex chromatin interactions, or together with CpG methylation. Therefore, as a combination of NanoNome/SMAC-seq/Fiber-seq and Pore-C/MC-3C, SCA-seq is one step forward. The authors tested SCA-seq in 293T cells and performed benchmark analyses testing the performance of SCA-seq in generating each data module (open chromatin and 3D genome). The QC metrics appear to be good and I am convinced that this is a valuable addition to the toolsets of multi-OMIC long-read sequencing mapping.

Reviewer #1 (Public Review):

This research article by Watabe T and colleagues characterizes PKA waves triggered by prostaglandin E2 (PGE2). What the author discovered is that waves of PKA occur both in vitro, in MDCK epithelial monolayers, and in vivo, in the ear epidermis in mice. The PKA waves are the consequence PGE2 discharge, that in turn is triggered by Calcium bursts. Calcium level and ERK activity intensity control that mechanism by acting at different levels.<br /> This article is a technological tour de force using different biosensors and optogenetic actuators. However, what makes this article interesting is the ability of combining these tools together to dissect a complex signaling pathway at the single-cell level and with highly dynamic processes. For this reason, this paper represents the essence of modern cell biology and paves the way for the cell biology of the future.

However, we think that the paper in this stage is still partly descriptive in its nature, and more measurements are needed to increase the strength of the mechanistic insights. Here below the points that we believe that need some improvement.

1)Even though the phenomenon of PGE2 signal propagation is elegantly demonstrated and well described, the whole paper is mostly of descriptive nature - the PGE2 signal is propagated via intercellular communication and requires Ca transients as well as MAPK activity, however function of these RSPAs in dense epithelium is not taken into consideration.<br /> What is the function of these RSPAs in cellular crowding? - Does it promote cell survival or initiate apoptosis? Does it feed into epithelial reorganization during cellular crowding? Still something else? The authors discuss possible roles of this phenomenon in cell competition context, but show no experimental or statistical efforts to answer this question. I believe some additional analysis or simple experiment would help to shed some light on the functional aspect of RSPAs and increase the importance of all the elegant demonstrations and precise experimental setups that the manuscript is rich of. Monolayer experiments using some perturbations that challenge the steady state of epithelial homeostasis - drug treatments/ serum deprivation/ osmotic stress/ combined with live cell imaging and statistical methods that take into account local cell density might provide important answers to these questions. The authors could consider following some of these ideas to improve the overall value of the manuscript.

2) In the line 82-84 the authors claim: "We found that the pattern of cAMP concentration change is very similar to the activity change of PKA, indicating that a Gs protein-coupled receptor (GsPCR) mediates RSPA". In our opinion, this conclusion is not well-supported by the results. The authors should at least show that some measurement of the two patterns show correlation. Are the patterns of cAMP of the same size as the pattern of PKA? Do they have the same size depending on cell density? Do they occur at the same frequency as the PKA patterns, depending on the cell density? Do they have an all or nothing activation as PKA or their activation is shading with the distance from the source?

3) In general, the absolute radius of the waves is not a good measurement for single-cell biology studies, especially when comparing different densities or in vivo vs in vitro experiments. We suggest the authors to add the measurement of the number of the cells involved in the waves (or the radius expressed in number of cells).

4) In 6D, the authors should also show the single-cell trajectories to understand better the correlation between PKA and ERK peaks. Is the huger variability in ERK activity ratio dues to different peak time or different ERK activity levels in different cells? The authors should show both the variability in the time and intensity.

5) In lines 130-132, the authors write, "This observation indicates that the amount of PGE2 secretion is predetermined and that there is a threshold of the cytoplasmic calcium concentration for the triggered PGE2 secretion". How could the author exclude that the amount of PGE2 is not regulated in its intensity as well? For sure, there is a threshold effect regarding calcium, but this doesn't mean that PGE2 secretion can be further regulated, e.g. by further increasing calcium concentration or by other mechanisms.

6) The manuscript shows that not all calcium transients are followed by RSPAs. Does the local cell density/crowding increase the probability of overlap between calcium transients and RSPAs?

The revision of the Watabe T paper provides additional data and analyses in response to the reviewers' comments. On our side, we are satisfied by these improvements.<br /> In the answer to our first question, the authors claim that they did multiple experiments to understand the function of RSPA in MDCK cell, all providing negative results. The authors could consider publishing the negative results as well, as they can be useful for the community.

In sum, we are convinced of the value of this article, and we thank the authors for the work that has been done.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.<br /> Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway.

Strength<br /> In this study, the authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Weakness<br /> The work is hardly conclusive as to the actual biological significance of the phenomenon. It would be interesting to know more under which physiological and pathological conditions PGE2 triggers such radical PKA activity changes. It is not well explained in which tissues and organs and under what conditions this type of cell-to-cell communication could be particularly important.<br /> The authors also do not explain further why in certain cells of the cell clusters Ca2+ signals occur spontaneously and thus trigger the phenomenon. What triggers these Ca2+ changes? And why could this be linked to certain cell functions and functional changes?<br /> What explains the radius and the time span of the radial signal continuation? To what extent are these factors also related to the degradation of PGE2? The work could be stronger if such questions and their answers would be experimentally integrated and discussed.

Reviewer #1 (Public Review):

Summary:<br /> Here, Boor et al focus on the regulation of daf-7 transcription in the ASJ chemosensory neurons, which has previously shown to be sensitive to a variety of external and internal signals. Interestingly, they find that soluble (but not volatile) signals released by food activate daf-7 expression in ASJ, but that this is counteracted by signals from the ASIC channels del-3 and del-7, previously shown to detect the ingestion of food in the pharynx. Importantly, the authors find that ASJ-derived daf-7 can promote exploration, suggesting a feedback loop that influences locomotor states to promote feeding behavior. They also implicate signals known to regulate exploratory behavior (the neuropeptide receptor PDFR-1 and the neuromodulator serotonin) in the regulation of daf-7 expression in ASJ. Additionally, they identify a novel role for a pathway previously implicated in C. elegans sensory behavior, HEN-1/SCD-2, in the regulation of daf-7 in ASJ, suggesting that the SCD-2 homolog ALK may have a conserved role in feeding and metabolism.

Strengths:<br /> The studies reported here, particularly the quantitation of gene expression and the careful behavioral analysis, are rigorously done and interpreted appropriately. The results suggest that, with respect to food, DAF-7 expression encodes a state of "unmet need" - the availability of nearby food to animals that are not currently eating. This is an interesting finding that reinforces and extends our understanding of the neurobiological significance of this important signaling pathway. The identification of a role for ASJ-derived daf-7 in motor behavior is a valuable advance, as is the finding that SCD-2 acts in the AIA interneurons to influence daf-7 expression in ASJ.

Weaknesses:<br /> A limitation of the work is that some mechanistic relationships between the identified signaling pathways remains unclear, but this provides interesting opportunities for future work. There are some minor concerns about the statistical analysis in the paper, but these are unlikely to affect the authors' interpretation of their results.

Reviewer #2 (Public Review):

In this work, Boor and colleagues explored the role of microbial food cues in the regulation of neuroendocrine controlled foraging behavior. Consistent with previous reports, the authors find that C. elegans foraging behavior is regulated by the neuroendocrine TGFβ ligand encoded by daf-7. In addition to its known role in the neuroendocrine/sensory ASI neurons, Boor and colleagues show that daf-7 expression is dynamically regulated in the ASJ sensory neurons by microbial food cues - and that this regulation is important for exploration/exploitation balance during foraging. They identify at least two independent pathways by which microbial cues regulate daf-7 expression in ASJ: a gustatory pathway that promotes daf-7 expression and an opposing interoceptive pathway, also chemosensory in nature but which requires microbial ingestion to inhibit daf-7 expression via ASIC channels, encoded by del-3/del-7. In contrast, the authors show that the conserved PDF neuropeptide signaling pathway likely functions via the gustatory pathway to promote daf-7 expression. They further identify a novel role for the C. elegans ALK orthologue encoded by scd-2, which acts in interneurons to regulate daf-7 expression and foraging behavior. These results together imply that distinct cues from microbial food are used to regulate the balance between exploration and exploitation via conserved signaling pathways.

Strengths:<br /> The findings that gustatory and interoceptive inputs into foraging behavior are separable and opposing are novel and interesting, which they have shown most clearly in Figure 1 and Figure 3. These data clarify how these parallel chemosensory pathways can be integrated at the level of daf-7 expression.

It is also clear from their results that removal of the interoceptive cue (via transfer to non-digestible food) results in rapid induction of daf-7::gfp in ASJ - suggesting that this pathway is likely chemosensory and not simply nutritive in nature. They have also shown that daf-7 in ASJ plays an important role in the regulation of foraging behavior.

The role of the hen-1/scd-2 pathway in mediating the effects of ingested food is also compelling and well-interpreted, with a few small caveats, described below. This implies that important elements of this food sensing pathway may be conserved in mammals.

Weaknesses:<br /> Although not a weakness of this work per se, the roles of the 5-HT and hen-1/scd-2 pathway remain a bit unclear, likely reflecting their complex genetic contributions to foraging and daf-7 expression. Future work should clarify how these signals are integrated and whether the integration of these pathways improve exploration/exploitation balance to regulate animal fitness.

Reviewer #3 (Public Review):

Summary:<br /> In this interesting study, the authors characterize the mechanisms whereby a C. elegans TGF-beta DAF-7 responds to various forms of food cues to regulate foraging.<br /> Building on their previous findings that characterized the functional role of daf-7 in the ASJ sensory neurons in response to a bacterial pathogen and in regulating searching behaviors, the authors of this manuscript show that ingestion of E. coli OP50, a common laboratory food for the worms, suppresses ASJ expression of daf-7 and secreted water-soluble cues of OP50 increase it. They further show that the level of daf-7 expression in ASJ is positively associated with a higher level of roaming/exploration. The authors identify that the function of a C. elegans ortholog of Anaplastic Lymphoma Kinase in the interneurons AIA regulates ASJ expression of daf-7 in response to food information and the related searching behavior.

Strengths:<br /> The study addresses an important question that appeals to a wide readership. The findings are demonstrated by strong results produced from well designed experiments.

Reviewer #1 (Public Review):

The authors' aim was to test to what extent atypical organization of language is associated with a mirrored brain organization of other cognitive functions. In particular, they focused on the inferior frontal gyri (IFG) by studying the inhibitory control network. This allowed them to directly test support for the Causal hypothesis of hemispheric specialization, arguing for fast sequences of cognitive processes being better performed by a single hemisphere, versus the Statistical hypothesis of lateralization, postulating an independent lateralization of each cognitive function.

Previous studies on this topic did not focus on functions involving homotopic language regions. This limitation is bypassed in this study by assessing inhibition with a Stop-Signal Task which also engages the IFG in the contralateral site to the verb generation task. By studying a combination of structural and functional information, in addition to the activation contrasts, the authors are able to test whether atypical organization is accompanied by stronger interhemispheric connectivity. Although relying mainly on correlations and lacking important methodological information that may be critical to understand the reported effects, the results are quite straightforward. However the bilingual/monolingual status and gender of the participants is not reported which might affect the relationship between language and inhibitory control.

The conclusions of the paper are supported by the data. With their design, the authors observed that, as a group, individuals with atypical organization show a mirror organization of the whole inhibitory network to the contralateral site, supporting the Causal hypothesis at the group level. However, individual data support the Statistical hypothesis, since the segregation between language and inhibition was not observed in all individuals and a variety of configurations in bilateral and bilateral organisation of language and inhibition were also observed.

The results of this study have important implications for our understanding of the independence of different cognitive functions, which is crucial when addressing brain damage and rehabilitation. This aspect also indirectly speaks to researchers interested in evolution and in bilingualism and its relation to cognitive control. These aspects are not discussed but incorporating them would broaden the interest of the paper beyond the current implications mentioned.

Reviewer #2 (Public Review):

Language skills are traditionally associated with a network of brain regions in the left hemisphere. In this intriguing study, Esteban Villar-Rodríguez and collaborators examined whether atypical hemispheric lateralization for language determines the functional and structural organisation of the network for inhibitory control as well as its relationship with schizotypy and autistic spectrum traits. The results suggest that individuals who have atypical lateralisation of the language function have also an atypical (mirrored) lateralisation of the inhibitory control network, compared to the typical group (individuals with left-lateralised language function). Furthermore, the atypical organization of language production is associated with a greater white matter volume of the corpus callosum, and atypical lateralization of inhibitory control is related to a higher interhemispheric functional coupling of the IFC, suggesting a link between atypical functional lateralisation (language and inhibitory control) and structural and functional changes in the brain.

This study also provides interesting evidence on how atypical language lateralisation impacts some aspects of language behaviour (reading), i.e., atypical lateralization predicts worse reading accuracy. Furthermore, the results suggest an association between atypical lateralization and increased schizotypy and autistic traits.

Joint Public Review

In this study, Mitra and coworkers extend their previous analyses of the functional role of Orai in the excitability of central dopaminergic neurons in Drosophila. The authors show that a dominant-negative mutant of Orai (OraiE180A) significantly alters the gene expression profile of flight-promoting dopaminergic neurons (fpDANs), including that of Set2, E(z), and Trl, thereby shifting the level of epigenetic signatures that modulate gene expression. The Orai-Trl-Set2 pathway modulates the expression of voltage gated calcium channels, which, in turn, are involved in dopamine release. The study is generally well-done, is in-depth, and comprehensive. The finding that SOCE regulates a wide range of neuronal genes necessary for neuronal excitability and effector signaling by controlling chromatin remodeling genes is a noteworthy discovery.

The authors have adequately answered the previous concerns.

Joint Public Review:

Summary:

The existence of hox gene complexes conserved in animals with bilateral symmetry and in which the genes are arranged along the chromosome in the same order as the structures they specify along the anteroposterior axis of organisms is one of the most spectacular discoveries of recent developmental biology. In brief, homeotic mutations leads to the transformation of a given body segment of the fly into the copy of the next adjacent segment. For the sake of understanding the main observation of this work, it is important to know that in loss-of-function (LOF) alleles, a given segment develops like a copy of the segment immediately anterior to it, and in gain-of-function mutations (GOF), the affected segment develop like a copy of the immediately posterior segment. Over the last 30 years the molecular lesions associated with GOF alleles led to a model where the sequential activation of the hox genes along the chromosome result from the sequential opening of chromosomal domains. Most of these GOF alleles turned out to be deletions of boundary elements (BE) that define the extend of the segment-specific regulatory domains. The fruit fly Drosophila is a highly specialized insect with a very rapid mode of segmentation. Furthermore, the hox clusters in this lineage have split. Given these specificities it is legitimate to question whether the regulatory landscape of the BX-C we know of in D.melanogaster is the result of very high specialization in this lineage, or whether it reflects a more ancestral organization. In this article, the authors address this question by analyzing the continuous hox cluster in butterflies. They focus on the integenic region between the Antennapedia and the Ubx gene, where the split occurred in D.melanogaster. Hi-C and ATAC-seq data suggest the existence of a boundary element between 2 Topologically-Associated-Domain (TAD) which is also characterized by the presence of CTCF binding sites. Butterflies have 2 pairs of wings originating form T2 (forewing) specified by Antp and T3 specified by Ubx (hindwing). Remarkably, CRISPR mutational perturbation of this boundary leads to the hatching of butterflies with homeotic clones of cells with hindwings identities in the forewing (a posteriorly oriented homeotic transformation). In agreement with this phenotype, the authors observe ectopic expression of Ubx in these clones of cells. In other words, CRISPR mutagenesis of this BE region identified by molecular tool give rise to homeotic transformations directed towards more posterior segment as the boundary mutations that had been 1st identified on the basis of their posterior oriented homeotic transformation in Drosophila. None of the mutant clones they observed affect the hindwing, indicating that their scheme did not affect the nearby Ubx transcription unit. This is a reassuring and important 1st evidence that some of the regulatory paradigm that have been proposed in fruit flies are also at work in the common ancestor to Drosophilae and Lepideptora.

Given the large size of the Ubx transcription unit and its associated regulatory regions it is not surprising that the authors have identified ncRNA that are conserved in 4 species of Nymphalinae butterflies, some of which also present in D.melanogaster. Attempts to target the promoters by CRISPR give rise to clones of cells in both forewings and hindwings, suggesting the generation of regulatory mutations associated with both LOF and GOF transformations. The presence of clones with dual homeosis suggest the targeting of Ubx activator and repression CRMs. Unfortunately, these experiments do not allow us to make further conclusions on the role of these ncRNA or in the identification of specific regulatory elements. To the opinion of this referee, some recent papers addressing the role that these ncRNA may play into boundary function should be taken with caution, and evidences that ncRNA(s) regulate boundaries in the BX-C in a WT context are still lacking.

Strengths: the convincing GOF phenotype resulting from the targeting of the Antp-Ubx_BE

Weaknesses: the lack of comparisons with the equivalent phenotypes obtained in D.melanogaster with for example the Fub mutation

Reviewer #1 (Public Review):

Gametocytes are erythrocytic sexual stages of the malaria-causing parasite Plasmodium, and are essential for parasite transmission and reproduction in the mosquito vector. In this study, Murata et al investigated the mechanisms of gene regulation in female gametocytes in the rodent malaria model parasite Plasmodium berghei. According to current views, gene regulation in Plasmodium parasites is dominated by the family of AP2 transcription factors (TFs), such as the AP2-G TF, which drives sexual commitment. The same authors previously identified one AP2 TF, called AP2-FG, as an essential TF mediating differentiation of female gametocytes. Here, they identified a novel protein, called PFG (for partner of AP2-FG, also described as Fd2 in a recently published study), which cooperates with AP2-FG to regulate a subset of female gametocyte genes.

PFG was identified among AP2-G targets, but possesses no known DNA binding or other characterized domain. The authors show that PFG-knockout P. berghei parasites can form male and female gametocytes yet cannot transmit to mosquitoes, due to a defect in female gametocyte development. Using RNA-seq, they show that many female-specific genes are down-regulated in PFG(-)parasites. Chromatin immunoprecipitation combined with DNA sequencing (ChIP-seq) revealed that PFG colocalizes with AP2-FG on a ten-base motif that is enriched upstream of female-specific genes. Importantly, the ChIP-seq profile of PFG is unchanged in the absence of AP2-FG, suggesting that PFG binds to DNA independently of AP2-FG. Mutation of the ten-base motif in one target gene using CRISPR-Cas9 demonstrates that this motif is required for PFG localization at the gene locus. The data also show that binding of AP2-FG is affected in the absence of PFG, with disruption of AP2-FG interaction with the ten-base motif, but conservation of AP2-FG binding to distinct five-base motifs. Using a recombinant AP2 domain from AP2-FG, the authors demonstrate that the AP2 domain of AP2-FG binds to the five-base motifs. Using CRISPR they show that disruption of the five-base motifs in the genome abrogates AP2-FG binding, and using a reporter expression system they confirm that these motifs act as a cis-activating promoter element.

Through the analysis of target genes based on the presence of the ten- versus five-base motifs, the authors propose a model where AP2-FG can function in two forms, associated or not with PFG, and acting on the ten- or five-base motifs, respectively, to regulate distinct gene subsets during development of female gametocyte development.

The paper is well written, with a clear narrative, and the work is very well performed, relying on robust molecular approaches. Generally the conclusions and the model proposed by the authors are well supported by the data. Nevertheless, the study as it stands raises a number of questions. While the data convincingly show that PFG and AP2-FG cooperate to regulate the expression of a subset of female-specific genes, the paper does not show whether the two proteins actually interact with each other to form a complex. Also, how PFG binds to DNA and whether the protein has transactivating activity remains elusive, as the protein apparently possesses no known DNA-binding or activating domain. These points could be discussed in more detail in the manuscript and/or be the subject of follow up studies.

In summary, this work reveals the essential role of a Plasmodium protein with no known DNA binding or regulatory domain, illustrating that unknown facets remain to be uncovered in this fascinating pathogen.

Reviewer #2 (Public Review):

Murata et al have characterized a transcription activator previously identified in an earlier genetic screen by Russell et al (named Fd2; for female-defective 2), here named PFG. The authors show solid evidence that PFG is a partner of the previously described transcription factor AP2-FG and describe three sets of genes: genes activated by PFG or AP2-FG alone and genes activated by the complex. The authors also show differential binding to the target DNA sequences by AP2-FG to either a 10bp, if in a complex with PFG or a 5bp motif if alone. In all, this is a useful study which further elucidates the underlying regulatory network that drives development of sexual stages and ultimately transmission to mosquitoes. The data presented are clear and solid and the conclusions drawn are mostly supported by the results shown.

A few comments:

Given that the transcriptional programme is so dynamic, the timing of the ChIP-seq experiments is crucial. Could the authors clarify the timings of the different ChIP-seq experiments (AP2-FG, PFG, PFG in AP2-FG-, AP2-FG in PFG-, ...)

Fig 4c is an example of great overlap of peaks, but it would be helpful if the authors could quantify the overlaps between experiments (and describe the overlap parameters used).

It remains unclear if AP2-FG and PFG interact directly or if they bind sequentially in the transcriptional activation process. Perhaps they are part of a larger complex? Immunoprecipitation followed by mass spectrometry of the GFP-tagged version of PFG in the presence and absence of AP2-FG would be highly informative.

Reviewer #3 (Public Review):

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it but would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

Overall the authors have shown only limited willingness to comprehensively address reviewer concerns and incorporate their suggestions.

Joint Public Review:

Xie et al. propose that the asymmetric segregation of the NuRD complex is regulated in a V-ATPase-dependent manner, and plays a crucial role in determining the differential expression of the apoptosis activator egl-1 and thus critical for the life/death fate decision.

While the model is very intriguing, the reviewers raised concerns regarding the rigor of the method. One issue is with statistics (either insufficient information or inadequate use of statistics), and second is the concern that the asymmetry observed may be caused by one cell dying (resulting in protein degradation, RNA degradation etc). We recommend that the authors address these issues.

Major #1:

There are still many misleading statements/conclusions that are not rigorously tested or that are logically flawed. These issues must be thoroughly addressed for this manuscript to be solid.

1. Asymmetry detected by scRNA seq vs. imaging may not represent the same phenomenon, thus should not be discussed as two supporting pieces of evidence for the authors' model, and importantly each method has its own flaw. First, for scRNA seq, when cells become already egl-1 positive, those cells may be already dying, and thus NuRD complex's transcripts' asymmetry may not have any significance. The data presented in FigS1D, E show that there are lots of genes (6487 out of 8624) that are decreased in dying cells. Thus, it is not convincing to claim that NuRD asymmetry is regulated by differential RNA amount.

2. Regarding NuRD protein's asymmetry, there are still multiple issues. Most likely explanation of their asymmetry is purely daughter size asymmetry. Because one cell is much bigger than the other (3 times larger), NuRD components, which are not chromatin associated, would be inherited to the bigger cell 3 times more than the smaller daughter. Then, upon nuclear envelope reformation, NuRD components will enter the nucleus, and there will be 3 times more NuRD components in the bigger daughter cell. It is possible that this is actually the underling mechanism to regulate gene expression differentially, but this possibility is not properly acknowledged. Currently, the authors use chromatin associated protein (Mys-1) as 'symmetric control', but this is not necessarily a fair comparison. For NuRD asymmetry to be meaningful, an example of protein is needed that is non-chromatin associated in mitosis, distributed to daughter cells proportional to daughter cell size, and re-enter nucleus after nuclear envelope formation to show symmetric distribution. And if daughter size asymmetry is the cause of NuRD asymmetry, other lineages that do not undergo apoptosis but exhibit daughter size asymmetry would also show NuRD asymmetry. The authors should comment on this (if such examples exist, it is fine in that in those cell types, NuRD asymmetry may be used for differential gene expression, not necessarily to induce cell death, but such comparison provides the explanation for NuRD asymmetry, and puts the authors finding in a better context).

3. For the analysis of protein asymmetry between two daughters in Fig S4C, the method of calibration is unclear, making it difficult to interpret the results.

4. As for pHluorin experiments, the authors were asked to test the changes in fluorescence observed are due to changes in pH or changes in the amount of pHluorin protein. They need to add a ratio-metric method in this manuscript. A brief mention to Page 12 line 12 is insufficient to clarify this issue.

Major #2:

Some issues surrounding statistics must be resolved.

1. Fig. 1FG, 2D, 3BDEG, 5BD and 6B used either one-sample t-test or unpaired two-tailed parametric t-test for statistical comparison. These t-tests require a verification of each sample fitting to a normal distribution. The authors need to describe a statistical test used to verify a normal distribution of each sample.

2. Fig. 2D, 3D, and 3G have very small sample size (N=3-4, N=6, N=3, respectively), it is possible that a normal distribution cannot be verified. How can the authors justify the use of one-sample t-test and unpaired parametric t-test ?

3. Statistical comparison in Fig. 2D and Fig. 6B should be re-assessed. For Fig. 2D, the authors need to compare the intensity ratio of HDA-1/LIN53 between sister cells dying within 35 min and those over 400 min. For Fig. 6B, they need to compare the intensity ratio of VHA-17 between DMSO- and BafA1- treated cells at the same time point after anaphase.

Reviewer #1 (Public Review):

Huang C-K. and colleagues in this work address the understudied role of environmental conditions and external forces in cell extrusion as a fundamental part of epithelial homeostasis. They suggest that hydrostatic stress plays a significant role in counteracting cell extrusion forces through the indirect regulation of the focal adhesion kinase (FAK) - protein kinase B (AKT) survival pathway. The team nicely exploits their expertise in fabricating cell culture substrates to control hydrostatic stress on a common epithelial cell model from the kidney (i.e., MDCK). This was done by creating waving surfaces with different lengths from 50µm to 200 µm, thus creating a heterogenous distribution of monolayer forces towards the substrate. Finally, using a specific inhibitor for FAK, they suggest that the survivor pathway FAK-AKT is involved in the observed phenomenon.

In conclusion, the presented data underline the importance of considering external forces and tissue geometry in regulating epithelial homeostasis and the selective transport of water and solutes. These results may have a significant impact on understanding the basic mechanisms of epithelial physiology and pathology, such as in the kidney, intestine, or retina.

Comments on the revised version:

Overall, most of my comments were reasonably addressed. Nevertheless, one comment was not convincingly addressed ("Recommendation 5" - reviewer #1).

The authors did not show that the FAK inhibitor directly induced the reduction of AKT phosphorylation but used this experiment to conclude that FAK - AKT survivor pathway is involved in the observed phenomenon (Fig. 4). The authors mentioned that additional immunoblotting experiments are currently underway. This is a minor control for the manuscript message, but I feel it is necessary. The connection between the levels of FAK and p-AKT shown in Fig. 4E is purely correlative and can be caused by ECM adhesion-independent reasons.

Alternatively, the authors could reduce the stress on the FAK - AKT survivor pathway's involvement and conclude only on the involvement of FAK.

Reviewer #2 (Public Review):

The paper by Huan, Yong, et al. studies epithelial cell extrusion in MDCK monolayers grown on sinusoidally wavy surfaces in varying media osmolarities, finding that both curvature and osmolarity-mediated basal hydraulic stress spatially regulate extrusion events. The authors fabricated wavy substrates of varying periods and amplitude out of PDMS (and PA hydrogels) and monitored monolayer evolution and cell extrusion over time, by combining live-cell imaging with a convolutional network-based algorithm for automatic detection of extrusions.

In general, the study has been elegantly designed, starting with convincing evidence for enhanced extrusion rates in concave valleys with respect to convex hills. Next, the authors showed that hyper-osmotic medium reduced cell extrusion rate, which was demonstrated in a variety of different media compositions (e.g. with sucrose, DMSO, or NaCl), while hypo-osmotic medium increased cell extrusion rate. Additionally, the authors applied reflection interference contrast microscopy to reveal fluid spaces between the substrate and the basal side of the monolayer, which were found to grow when media composition was altered from hyper-osmotic to normal osmotic conditions. Using a 3D traction force microscopy approach, the authors demonstrated that cells on convex regions apply a downward pointing force on the substrate, opposite to cells on the concave regions. This was linked to a larger basal separation on the concave valleys as opposed to the convex hills. Finally, the authors focussed on the FAK-Akt pathway to explore the hypothesis that basal hydraulic stress interferes with focal adhesions, leading to differences in cell extrusion rates in media of different osmolarity and on convex or concave surfaces.

Comments on the revised version:

My previous comments were reasonably answered. In response to the comment that "experiments that are currently underway" for "Recommendation 5 - reviewer #1", I would also suggest the authors to either add the additional data or alter the emphasis on the FAK-AKT pathway in the manuscript accordingly if additional data is not presented.