Reviewer #2 (Public review):

Summary:

This study analyzes protein abundance data from six VAMP-seq experiments, comprising over 31,000 single amino acid substitutions, to understand how different amino acids contribute to maintaining cellular protein levels. The authors develop substitution matrices that capture the average effect of amino acid changes on protein abundance in different structural contexts (buried vs. exposed residues). Their key finding is that these simple structure-based matrices can predict mutational effects on abundance with accuracy comparable to more complex physics-based stability calculations (ΔΔG).

Major strengths:

(1) The analysis focuses on a single molecular phenotype (abundance) measured using the same experimental approach (VAMP-seq), avoiding confounding factors present when combining data from different phenotypes (e.g., mixing stability, activity, and fitness data) or different experimental methods.

(2) The demonstration that simple structural features (particularly solvent accessibility) can capture a significant portion of mutational effects on abundance.

(3) The practical utility of the matrices for analyzing protein interfaces and identifying functionally important surface residues.

Major weaknesses:

(1) The statistical rigor of the analysis could be improved. For example, when comparing exposed vs. buried classification of interface residues, or when assessing whether differences between prediction methods are significant.

(2) The mechanistic connection between stability and abundance is assumed rather than explained or investigated. For instance, destabilizing mutations might decrease abundance through protein quality control, but other mechanisms like degron exposure could also be at play.

(3) The similar performance of simple matrix-based and complex physics-based predictions calls for deeper analysis. A systematic comparison of where these approaches agree or differ could illuminate the relationship between stability and abundance. For instance, buried sites showing exposed-like behavior might indicate regions of structural plasticity, while the link between destabilization and degradation might involve partial unfolding exposing typically buried residues. The authors have all the necessary data for such analysis but don't fully exploit this opportunity.

(4) The pooling of data across proteins to construct the matrices needs better justification, given the observed differences in score distributions between proteins (for example, PTEN's distribution is shifted towards high abundance scores while ASPA and PRKN show more binary distributions).

(5) Some key methodological choices require better justification. For example, combining "to" and "from" mutation profiles for PCA despite their different behaviors, or using arbitrary thresholds (like 0.05) for residue classification.

The authors largely achieve their primary aim of showing that simple structural features can predict abundance changes. However, their secondary goal of using the matrices to identify functionally important residues would benefit from more rigorous statistical validation. While the matrices provide a useful baseline for abundance prediction, the paper could offer deeper biological insights by investigating cases where simple structure-based predictions differ from physics-based stability calculations.

This work provides a valuable resource for the protein science community in the form of easily applicable substitution matrices. The finding that such simple features can match more complex calculations is significant for the field. However, the work's impact would be enhanced by a deeper investigation of the mechanistic implications of the observed patterns, particularly in cases where abundance changes appear decoupled from stability effects.

Reviewer #3 (Public review):

"Effects of residue substitutions on the cellular abundance of proteins" by Schulze and Lindorff-Larsen revisits the classical concept of structure-aware protein substitution matrices through the scope of modern protein structure modelling approaches and comprehensive phenotypic readouts from multiplex assays of variant effects (MAVEs). The authors explore 6 unique protein MAVE datasets based on protein abundance (and thus stability) by utilizing structural information, specifically residue solvent accessibility and secondary structure type, to derive combinations of context-specific substitution matrices predicting variant abundance. They are clear to outline that the aim of the study is not to produce a new best abundance predictor but to showcase the degree of prediction afforded simply by utilizing information on residue accessibility. The performance of their matrices is robustly evaluated using a leave-one-out approach, where the abundance effects for a single protein are predicted using the remaining datasets. Using a simple classification of buried and solvent-exposed residues, and substitution matrices derived respectively for each residue group, the authors convincingly demonstrate that taking structural solvent accessibility contexts into account leads to more accurate performance than either a structure-unaware matrix, secondary structure-based matrix, or matrices combining both solvent accessibility or secondary structure. Interestingly, it is shown that the performance of the simple buried and exposed residue substitution matrices for predicting protein abundance is on par with Rosetta, an established and specialized protein variant stability predictor. More importantly, the authors finish off the paper by demonstrating the utility of the two matrices to identify surface residues that have buried-like substitution profiles, that are shown to correspond to protein interface residues, post-translational modification sites, functional residues, or putative degrons.

Strengths:

The paper makes a strong and well-supported main point, demonstrating the utility of the authors' approach through performance comparisons with alternative substitution matrices and specialized methods alike. The matrices are rigorously evaluated without introducing bias, exploring various combinations of protein datasets. Supplemental analyses are extremely comprehensive and detailed. The applicability of the substitution matrices is explored beyond abundance prediction and could have important implications in the future for identifying functionally relevant sites.

Comments:

(1) A wider discussion of the possible reasons why matrices for certain proteins seem to correlate better than others would be extremely interesting, touching upon possible points like differences or similarities in local environments, degradation pathways, post-translation modifications, and regulation. While the initial data structure differences provide a possible explanation, Figure S17A, B correlations show a more complicated picture.

(2) The performance analysis in Figure 2D seems to show that for particular proteins "less is more" when it comes to which datasets are best to derive the matrix from (CYP2C9, ASPA, PRKN). Are there any features (direct or proxy), that would allow to group proteins to maximize accuracy? Do the authors think on top of the buried vs exposed paradigm, another grouping dimension at the protein/domain level could improve performance?

(3) While the matrices and Rosetta seem to show similar degrees of correlation, do the methods both fail and succeed on the same variants? Or do they show a degree of orthogonality and could potentially be synergistic?

Overall, this work presents a valuable contribution by creatively utilizing a simple concept through cutting-edge datasets, which could be useful in various.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the cellular mechanism underlying suppression of adrenergic effects on excitatory transmission onto hypothalamic CRH neurons by stress. Experiments in ex-vivo slices show that this is a long-lasting effect that occurs through endocytosis of receptors. The authors then move into an immortalized hypothalamic cell line to enable investigation of the mechanism of changes in receptor trafficking. They use a series of immunohistochemistry, FRET, and biochemical experiments to show that application of corticosterone increases targeting of alpha1 adrenergic receptors to the late endosome and lysosome rather than the recycling endosome. Perhaps most interesting, they find that alpha1 receptors and glucocortioid receptors form a complex that is ultimately transferred to the nucleus.

Strengths:

Overall, the studies in this manuscript are rigorous and well-conducted. The data supports their conclusions, and they've shown convincingly that glucocorticoid signaling affects trafficking of alpha1 receptors in the culture system they are using. These findings are important for the field of stress research, both in understanding how two components of the stress system (norepinephrine and HPA axis) interact with each other and in neuromodulatory modulation of hypothalamic CRH neurons. Their finding that alpha1 receptors and glucocorticoid receptors form a complex is particularly interesting and maybe impactful outside of the immediate application in the hypothalamus.

Weaknesses:

The study has two primary weaknesses. First, the majority of the experiments were conducted in an immortalized hypothalamic cell line. This was necessary to conduct the type of experiments needed to test the author's hypothesis, but it remains unclear how closely these cells resemble CRH neurons, or how the same mechanism may be preserved or altered in an intact circuit. Further discussion of these points would strengthen the manuscript.<br /> Second, while experiments are generally well-designed, the authors do not show that the effects of corticosterone can be blocked with a glucocorticoid receptor antagonist. This is fairly standard pharmacology and would strengthen confidence in the findings presented in the study.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors report novel and exciting findings delineating a non-transcriptional mechanism whereby glucocorticoids desensitize CRH neurons to NE in response to somatic stress. The authors find that this desensitization induced by CORT 1. persists more than 18h, 2. reduces surface expression of AR1bR (NE receptors) by redirecting trafficking from rapid recycling to late endosomal pools and lysosomes, 3. is dependent on NE binding to the AR1bR, 4. involves cellular nitrosylation, 5. involves ubiquitination of beta-arrestin, and 5. involves interactions between glucocorticoid receptors and AR1bs, glucocorticoid receptors and ubiquitinated beta-arrestin, and AR1b and ubiquitinated beta-arrestin. While the authors do not directly provide evidence for a trimeric complex composed of these three proteins, their data that CORT causes translocation of these dimeric complexes to the cell nucleus suggests it is likely. Overall, these results are highly informative for understanding novel mechanisms mediating glucocorticoid regulation of GPCRs.

Strengths:<br /> - Good rationale for each experiment, which describes many parts of the CORT-NE desensitization mechanism<br /> - Great discussion of limitations of the approaches and the parts of the mechanism we do not fully understand yet<br /> - Appropriate approaches for questions being answered<br /> - Describes a highly novel CORT mechanism that non-transcriptionally switches GPCR trafficking dynamics, something that could have far reaching implications for other GPCRs involved in stress responses

Weaknesses:<br /> - Unclear how this mechanism would generalize to other stressor modalities. Restraint stress is a somatic stressor, but can also be considered a psychological stressor (model of depression-like behavior). A purely somatic stressor might increase the robustness of this phenomenon.<br /> - Remains unknown how nitrosylation plays into the mechanism in terms of specific proteins affected by CORT (GRK2, endophilin, clathrin possibilities)

Reviewer #3 (Public review):

Summary:

In this manuscript, Weiss et al describe a mechanism through which glucocorticoids desensitize CRH neurons in the PVN to norepinephrine. This follows on from previous work from this lab showing rapid glucocorticoid suppression of adrenergic signaling in CRH neurons specific to somatic stress activation, and modality-selective glucocorticoid negative feedback.

Specifically, their previous work shows that:<br /> (1) NE increases glutamate drive to CRH neurons<br /> (2) CORT blunts the effects of NE through a dynamin-dependent mechanism<br /> (3) This contributes to loss of NE signalling after stress (specifically when the second stressor is a physiological one)

Here they extend this line of interrogation by showing that CORT redistributes Ara1b receptors from rapid recycling endosomes to late endosomes and lysosomes. They show a time window of CORT actions and provide additional mechanistic details implicating nitric oxide-dependent nitrosylation in receptor trafficking.

Strengths:

Builds on existing work to provide additional mechanistic details.<br /> The experiments are well done and data are compelling.<br /> The link to nitrosylation is novel (but see below)

Weaknesses:

(1) The link to nitrosylation is interesting, but a bit confusing. If I understand correctly, inhibiting the production of NO or using NEM increases receptor internalization, suggesting that NO-dependent nitrosylation prevents ligand-dependent internalization. What is unclear to me is how CORT is linked to this step. I note the authors show a decrease in nitrosylation with CORT. So, does CORT decrease the activity of NOS and, thus, the production of NO? If so, then exogenously activating this system in the presence of CORT should result in a recovery of NE-dependent increase in glutamate release. Or is the GCR directly decreasing nitrosylation? Linking these elements is critical in terms of furthering our mechanistic understanding of this process.

(2) It's not clear why/how blockade of Ara1 after CORT-induced cytosolic accumulation results in a reversal of effect. Unless I misunderstood something, this requires further explanation.

Reviewer #1 (Public Review):

The authors were curious about the formation of the electrosensory lateral line, which is found in non-traditional model organisms. This issue has traditionally hampered studies because those organisms are not amenable to controlled experimental work.

The authors skillfully use CRIPR-based technologies to overcome this limitation. Together with exceptionally good whole-mount in situ hybridisation, they produced a well-supported conclusion that Bmp signalling has different roles in the development of electrosensory ampullary organs.

I would not entirely agree that Bmp signalling has "opposing" roles because the authors do not show evidence of opposition via gain-of-function experiments at different developmental times. Instead, they are simply different at different periods of organogenesis.

The study is important for understanding the development of a still-mysterious sensory system, and for its implications in evolutionary biology more generally.

Reviewer #2 (Public Review):

Campbell et al have described the dynamic pattern of two Bmps (Bmp5, Bmp4), one of their receptors (Acvr2a), putative joint inhibitors of the Bmp & Wnt pathways (sostdc1, apcdd1) and an effector of Bmp signaling phosph-Smad, in the experimentally tractable sterlet sturgeon to better understand the role of Bmp signaling in electroreceptor development. The role of Bmp signaling is poorly understood in the lateral line system. Furthermore, the development of electroreceptors in ampullary organs remains poorly understood as most recent analysis of lateral line development has focused on model organisms Xenopus and zebrafish, which the electroreceptors have been lost. They show that expression of these players is consistent with a role for Bmp signaling in electroreceptor development. Furthermore, they show that Bmp5 crispants have fewer ampullary organs. However, inhibition of Bmp signaling with the small molecule inhibitor DMH1 for 20 hours starting from stage 36 after hatching and before ampullary organ development results in supernumerary ampullary organ development. These strikingly different results lead the authors to conclude that Bmp signaling has opposing roles in ampullary organ development.

These observations are interesting and the conclusions are supported by the data presented and the study makes important contributions to our understanding of the role of Bmp signaling in electroreceptor development in lateral line development. However, the study opens and leaves unresolved a number of questions. While a definitive answer to these questions may be outside the scope of this paper, some additional experiments may help strengthen the paper.

Reviewer #1 (Public review):

Summary:

"Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This is study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

Strengths:

The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials using according to the experts' consensus recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on rigor the EEG methods is beyond my expertise as a reviewer.<br /> Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.<br /> The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.<br /> The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

Weaknesses:

Though the methods employed in the paper are generally strong, the MRS acquisition was not optimized to quantify GABA, so the findings (or lack thereof) should be interpreted with caution. Specifically, while 7T MRS affords the benefit of quantifying metabolites, such as GABA, without spectral editing, this quantification is best achieved with echo times (TE) of 68 or 80 ms in order to minimize the spectral overlap between glutamate and GABA and reduce contamination from the macromolecular signal (Finkelman et al., 2022, https://doi.org/10.1016/j.neuroimage.2021.118810). The data in the present study were acquired at TE=28 ms, and are therefore likely affected by overlapping Glu and GABA peaks at 2.3 ppm that are much more difficult to resolve at this short TE, which could directly affect the measures that are meant to characterize the Glu/GABA+ ratio/imbalance. In future research, MRS acquisition schemes should be optimized for the acquisition of Glutamate, GABA, and their relative balance.

As the authors note in the discussion, additional factors such as MRS voxel location, participant age, and participant sex could influence associations between neural noise and reading abilities and should be considered in future studies.

Appraisal:

The authors present a thorough evaluation of the neural noise hypothesis of developmental dyslexia in a sample of adolescents and young adults using multiple methods of measuring excitatory/inhibitory imbalances as an indicator of neural noise. The authors concluded that there was not support for the neural noise hypothesis of dyslexia in their study based on null significance and Bayes factors. This conclusion is justified, and further research is called for to more broadly evaluate the neural noise hypothesis in developmental dyslexia.

Impact:

This study provides an exemplar foundation for the evaluation of the neural noise hypothesis of dyslexia. Other researcher may adopt the model applied in this paper to examine neural noise in various populations with/without dyslexia, or across a continuum of reading abilities, to more thoroughly examine evidence (or lack thereof) for this hypothesis. Notably, the lack of evidence here does not rule out the possibility for a role of neural noise in dyslexia, and the authors point out that presentation with co-occurring conditions, such as ADHD, may contribute to neural noise in dyslexia. Dyslexia remains a multi-faceted and heterogenous neurodevelopmental condition, and many genetic, neurobiological and environmental factors play a role. This study demonstrates one step toward evaluating neurobiological mechanisms that may contribute to reading difficulties.

Reviewer #2 (Public review):

Summary:

This study utilized two complimentary techniques (EEG and 7T MRI/MRS) to directly test a theory of dyslexia: the neural noise hypothesis. The authors report finding no evidence to support an excitatory/inhibitory balance, as quantified by beta in EEG and Glutamate/GABA ratio in MRS. This is important work and speaks to one potential mechanism by which increased neural noise may occur in dyslexia.

Strengths:

This is a well conceived study with in depth analyses and publicly available data for independent review. The authors provide transparency with their statistics and display the raw data points along with the averages in figures for review and interpretation. The data suggest that an E/I balance issue may not underlie deficits in dyslexia and is a meaningful and needed test of a possible mechanism for increased neural noise.

Weaknesses:

The researchers did not include a visual print task in the EEG task, which limits analysis of reading specific regions such as the visual word form area, which is a commonly hypoactivated region in dyslexia. This region is a common one of interest in dyslexia, yet the researchers measured the I/E balance in only one region of interest, specific to the language network.

Reviewer #3 (Public review):

Summary:

This study by Glica and colleagues utilized EEG (i.e., Beta power, Gamma power, and aperiodic activity) and 7T MRS (i.e., MRS IE ratio, IE balance) to reevaluating the neural noise hypothesis in Dyslexia. Supported by Bayesian statistics, their results show convincing evidence of no differences in EI balance between groups, challenging the neural noise hypothesis.

Strengths:

Combining EEG and 7T MRS, this study utilized both the indirect (i.e., Beta power, Gamma power, and aperiodic activity) and direct (i.e., MRS IE ratio, IE balance) measures to reevaluating the neural noise hypothesis in Dyslexia.

Reviewer #1 (Public review):

Summary:

This manuscript addresses the question of whether spontaneous activity contributes to the clustering of retinogeniculate synapses before eye opening. The authors re-analyze a previously published dataset to answer the question. The authors conclude that synaptic clustering is eye-specific and activity dependent during the first postnatal week. While there is useful information in this manuscript, I don't see how the data meaningfully supports the claims made about clustering.<br /> In adult retinogeniculate connections, functionally specificity is supported by select pairings of retinal ganglion cells and thalamocortical cells forming dozens of synaptic connections in subcellular microcircuits called glomeruli. In this manuscript, the authors measure whether the frequency of nearby synapses is higher in the observed data than in a model where synapses are randomly distributed throughout the volume. Any real anatomical data will deviate from such a model. The interesting biological question is not whether a developmental state deviates from random. The interesting question is how much of the adult clustering occurs before eye opening. In trying to decode the analysis in this manuscript, I can't tell if the answer is 99% or 0.001%.

Strengths:

The source dataset is high resolution data showing the colocalization of multiple synaptic proteins across development. Added to this data is labeling that distinguishes axons from the right eye from axons from the left eye. The first order analysis of this data showing changes in synapse density and in the occurrence of multi-active zone synapses is useful information about the development of an important model system.

Weaknesses:

I don't think the analysis of clustering within this dataset improves our understanding of how the system works. It is possible that the result is clear to the authors based on looking at the images. As a reader trying to interpret the analysis, I ran into the following problems:

• It is not possible to estimate biologically meaningful effect sizes from the data provided. Spontaneous activity in the post natal week could be responsible for 99% or 0.001% of RGC synapse clustering.<br /> • There is no clear biological interpretation of the core measure of the publication, the normalized clustering index. The normalized clustering index starts with counting the fraction of single active zone synapses within various distances to the edge of synapses. This frequency is compared to a randomization model in which the positions of synapses are randomized throughout a volume. The authors found that the biggest deviation between the observed and randomized proximity frequency using a distance threshold of 1.5 um. They consider the deviation from the random model to be a sign of clustering. However, two RGC synapses 1.5 um apart have a good chance of coming from the same RGC axon. At this scale, real observations will, therefore, always look more clustered than a model where synapses are randomly placed in a volume. If you randomly place synapses on an axon, they will be much closer together than if you randomly place synapses within a volume. The authors normalize their clustering measure by dividing by the frequency of clustering in the normalized model. That makes the measure of clustering an ambiguous mix of synapse clustering, axon morphology, and synaptic density.<br /> • Other measures are also very derived. For instance, one argument is based on determining that the cumulative distribution of the distance of dominant-eye multi-active zone synapses with nearby single-active zone synapses from dominant-eye multi-active zone synapses is statistically different from the cumulative distribution of the distance of dominant-eye multi-active zones without nearby single-active zone synapses from dominant-eye multi-active zones. Multiple permutations of this measure are compared.<br /> • The sample size is too small for the kinds of comparisons being made. The authors point out that many STORM studies use an n of 1 while the authors have n = 3 for each of their six experimental groups. However, the critical bit is what kinds of questions you are trying to answer with a given sample size. This study depends on determining whether the differences between groups are due to age, genotype, or individual variation. This study also makes multiple comparisons of many different noisy parameters that test the same or similar hypothesis. In this context, it is unlikely that n = 3 sufficiently controls for individual variation.<br /> • There are major biological differences between groups that are difficult to control for. Between P2, P4, and P8, there are changes in cell morphology and synaptic density. There are also large differences in synapse density between wild type and KO mice. It is difficult to be confident that these differences are not responsible for the relatively subtle changes in clustering indices.<br /> • Many claims are based on complicated comparisons between groups rather than the predominating effects within the data. It is noted that: "In KO mice, dominant eye projections showed increased clustering around mAZ synapses compared to sAC synapses suggesting partial maintenance of synaptic clustering despite retinal wave defects". In contrast, I did not notice any discussion of the fact that the most striking trend in those measures is that the clustering index decreases from P2 to P8.<br /> • Statistics are improperly applied. In my first review I tried to push the authors to calculate confidence intervals for two reasons. First, I believed the reader should be able to answer questions such as whether 99% or 0.01% of RGC synaptic clustering occurred in the first postnatal week. Second, I wanted the authors to deal with the fact that n=3 is underpowered for many of the questions they were asking. While many confidence intervals can now be found leading up to a claim, it is difficult to find claims that are directly supported by the correct confidence interval. Many claims are still incorrectly based on which combinations of comparisons produced statistically significant differences and which combinations did not.

Reviewer #2 (Public review):

Summary:

This study provides a valuable data set showing changes in the spatial organization of synaptic proteins at the retinogeniculate connection during a developmental period of active axonal and synaptic remodeling. The data collected by STORM microscopy is state-of-the-art in terms of the high-resolution view of the presynaptic components of a plastic synapse. The revision has addressed many, but not all, of the initial concerns about the authors interpretation of their data. However, with the revisions, the manuscript has become very dense and difficult to follow.

Strengths:

The data presented is of good quality and provides an unprecedented view at high resolution of the presynaptic components of the retinogeniculate synapse during active developmental remodeling. This approach offers an advance to the previous mouse EM studies of this synapse because the CTB label allows identification of the eye from which the presynaptic terminal arises.

Weaknesses:

From these data the authors conclude that eye-specific increase in mAZ synapse density occur over retinogeniculate refinement, that sAZ synapses cluster close to mAZ synapses over age, and that this process depends on spontaneous activity and proximity to eye-specific mAZ synapses. While the interpretation of this data set is much more grounded in this revised submission, some of the authors' conclusions/statements still lack convincing supporting evidence.<br /> This includes:

(1) The conclusion that multi-active zone synapses are loci for synaptic clustering. This statement, or similar ones (e.g., line 407) suggest that mAZ synapses actively or through some indirect way influence the clustering of sAZ synapses. There is no evidence for this. Clustering of retinal synapses are in part due to the fact that retinal inputs synapse on the proximal dendrites. With increased synaptogenesis, there will be increased density of retinal terminals that are closely localized. And with development, perhaps sAZ synapses mature into mAZ synapses. This scenario could also explain a large part of this data set.

(2) The conclusion that, "clustering depends on spontaneous retinal activity" could be misleading to the reader given that the authors acknowledge that their data is most consistent with a failure of synaptogenesis in the mutant mice (in the rebuttal). Additionally clustering does occur in CTB+ projections around mAZ synapses.

(3). Line 403: "Since mAZ synapses are expected to have a higher release probability, they likely play an important role in driving plasticity mechanisms reliant on neurotransmission.":What evidence do the authors have that mAZ are expected to have higher release probability?

Reviewer #3 (Public review):

This study is a follow-up to a recent study of synaptic development based on a powerful data set that combines anterograde labeling, immunofluorescence labeling of synaptic proteins, and STORM imaging (Cell Reports, 2023). Specifically, they use anti-Vglut2 label to determine the size of the presynaptic structure (which they describe as the vesicle pool size), anti-Bassoon to label active zones with the resolution to count them, and anti-Homer to identify postsynaptic densities. Their previous study compared the detailed synaptic structure across the development of synapses made with contra-projecting vs. ipsi-projecting RGCs and compared this developmental profile with a mouse model with reduced retinal waves. In this study, they produce a new detailed analysis on the same data set in which they classify synapses into "multi-active zone" vs. "single-active zone" synapses and assess the number and spacing of these synapses. The authors use measurements to make conclusions about the role of retinal waves in the generation of same-eye synaptic clusters, providing key insight into how neural activity drives synapse maturation.

Strengths:

This is a fantastic data set for describing the structural details of synapse development in a part of the brain undergoing activity-dependent synaptic rearrangements. The fact that they can differentiate eye of origin is what makes this data set unique over previous structural work. The addition of example images from EM data set provides confidence in their categorization scheme.

Weaknesses:

Though the descriptions of synaptic clusters are important and represent a significant advance, the authors conclusions regarding the biological processes driving these clusters are not testable by such a small sample. This limitation is expected given the massive effort that goes into generating this data set. Of course the authors are free to speculate, but many of the conclusions of the paper are not statistically supported.

Joint Public Review:

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/β-catenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Reviewer #1 (Public review):

Summary:

In this study, Zhang et al., presented an electrophysiology method to identify the layers of macaque visual cortex with high density Neuropixels 1.0 electrode. They found several electrophysiology signal profiles for high-resolution laminar discrimination and described a set of signal metrics for fine cortical layer identification.

Strengths:

There are two major strengths. One is the use of high density electrodes. The Neuropixels 1.0 probe has 20 um spacing electrodes, which can provide high resolution for cortical laminar identification. The second strength is the analysis. They found multiple electrophysiology signal profiles which can be used for laminar discrimination. Using this new method, they could identify the most thin layer in macaque V1. The data support their conclusion.

Weaknesses:

While this electrophysiology strategy is much easier to perform even in awake animals compared to histological staining methods, it provides an indirect estimation of cortical layers. A parallel histological study can provide a direct matching between the electrode signal features and cortical laminar locations. However, there are technical challenges, for example the distortions in both electrode penetration and tissue preparation may prevent a precise matching between electrode locations and cortical layers. In this case, additional micro wires electrodes binding with Neuropixels probe can be used to inject current and mark the locations of different depths in cortical tissue after recording.

Reviewer #2 (Public review):

Summary:

This paper documents a compelling attempt to accurately determine the locations and boundaries of the anatomically and functionally defined layers in macaque primary visual cortex using voltage signals recorded from a high-density electrode array that spans the full depth of cortex with contacts at 20 um spacing. First, the authors attempt to use current source density (CSD) analysis to determine layer locations, but they report a striking failure because the results vary greatly from one electrode penetration to the next and because the spatial resolution of the underlying local field potential (LFP) signal is coarse compared to the electrical contact spacing. The authors therefore turn to examining higher frequency signals related to action potentials and provide evidence that these signals reflect changes in neuronal size and packing density, response latency and visual selectivity, which taken together can advance the state-of-the-art accuracy in making layer assignments from in vivo recordings.

Strengths:

There is a lot of nice data to look at in this paper that show interesting quantities as a function of depth in V1. Bringing all of these together offers the reader a rich data set: CSD, action potential shape, response power and coherence spectrum, and post-stimulus time response traces. Furthermore, data are displayed as a function of eye (dominant or non-dominant) and for achromatic and cone-isolating stimuli.

This paper takes a strong stand in pointing out weaknesses in the ability of CSD analysis to make consistent determinations about cortical layering in V1. Many researchers have found CSD to be problematic, and the observations here may be important to motivate other researchers to carry out rigorous comparisons and publish their results, even if they reflect negatively on the value of CSD analysis.

The paper provides a thoughtful, practical and comprehensive recipe for assigning traditional cortical layers based on easily-computed metrics from electophysiological recordings in V1, and this is likely to be useful for electrophysiologists who are now more frequently using high-density electrode arrays.

Weaknesses:

Considerable space is taken in pointing out features that are well known, for example, the latency difference associated with different retinogeniculate pathways, the activity level differences associated with input layers, and the action potential shape differences associated with white vs. gray matter. These have been used for decades as indicators of depth and location of recordings in visual cortex as electrodes were carefully advanced. High density electrodes allow this type of data to now be collected in parallel, but at discrete, regular sampling points. Perhaps more emphasis could be placed on developing a rigorous analysis of how variable vs. reproducible are quantitative metrics of these features across penetrations, as a function of distance or functional domain, and from animal to animal, but this paper certainly makes a substantial push in this direction.

Another important piece of information for assessing the ability to determine layers from spiking activity is to carry out post-mortem histological processing so that the layer determination made in vivo can be compared to anatomical layering. However, histological methods also suffer from distortion and noise, thus it remains to be seen how much can ultimately be gained by integrating histology with the physiological methods explored here.

Overall

Overall, this paper makes a compelling argument in favor of using action potentials and stimulus driven responses, instead of CSD measurements, to assign cortical layers to electrode contacts in V1. The rich presentation of data, combined with the authors' highly educated interpretation and speculation about how useful such measurements will be for layer assignment make this an important paper for many labs using high-density electrodes. It is easy to agree with much of what is postulated here and to hope that we will soon have reliable, quantitative methods to make layer assignments that will be meaningful in terms of the differentiated roles of single neurons in cortical computation. How much this will end up corresponding to the canonical layer numbering that has been used for many decades will be interesting to see.

Comments on revisions:

I found that the authors addressed my main concerns to the degree they were able. They improved the consistency of language and figures, and they added some useful quantification.

Reviewer #3 (Public review):

Summary:

Zhang et al. explored strategies for aligning electrophysiological recordings from high-density laminar electrode arrays (Neuropixels) with the pattern of lamination across cortical depth in macaque primary visual cortex (V1), with the goal of improving the spatial resolution of layer identification based on electrophysiological signals alone. The authors compare the current commonly used standard in the field - current source density (CSD) analysis - with a new set of measures largely derived from action potential (AP) frequency band signals. Individual AP band measures provide distinct cues about different landmarks or potential laminar boundaries, and together they are used to subdivide the spatial extent of array recordings into discrete layers, including the very thin layer 4A, at a level of resolution unavailable when relying on CSD analysis alone for laminar identification. The authors compare the widths of the resulting subdivisions with previously reported anatomical measurements as evidence that layers have been accurately identified. This is a bit circular, given that they also use these anatomical measurements as guidelines limiting the boundary assignments; however, the strategy is overall sensible and the electrophysiological signatures used to identify layers are generally convincing. Furthermore, by varying the pattern of visual stimulation to target chromatically sensitive inputs known to be partially segregated by layer in V1, they show localized response patterns that lend confidence to their identification of particular sublayers.

The authors compellingly demonstrate the insufficiency of CSD analysis for precisely identifying fine laminar structure, and in some cases its limited accuracy at identifying coarse structure. CSD analysis produced inconsistent results across array penetrations and across visual stimulus conditions and was not improved in spatial resolution by sampling at high density with Neuropixels probes. Instead, in order to generate a typical, informative pattern of current sources and sinks across layers, the LFP signals from the Neuropixels arrays required spatial smoothing or subsampling to approximately match the coarser (50-100 µm) spacing of other laminar arrays. Even with smoothing, the resulting CSDs in some cases predicted laminar boundaries that were inconsistent with boundaries estimated using other measures and/or unlikely given the typical sizes of individual layers in macaque V1. This point alone provides an important insight for others seeking to link their own laminar array recordings to cortical layers.

They next offer a set of measures based on analysis of AP band signals. These measures include analyses of the density, average signal spread, and spike waveforms of units identified through spike sorting, as well as analyses of AP band power spectra and local coherence profiles across recording depth. The power spectrum measures in particular yield compact peaks at particular depths, albeit with some variation across penetrations, whereas the waveform measures most convincingly identified the layer 6-white matter transition. In general, some of the new measures yield inconsistent patterns across penetrations, and some of the authors' explanations of these analyses draw intriguing but rather speculative connections to properties of anatomy and/or responsivity. However, taken as a group, the set of AP band analyses appear sufficient to determine the layer 6-white matter transition with precision and to delineate intermediate transition points likely to correspond to actual layer boundaries, and the strategy serves as a substantial advancement over consideration of CSD signals alone to match electrophysiological recordings with cortical layers.

Strengths:

The authors convincingly demonstrate the potential to resolve putative laminar boundaries using only electrophysiological recordings from Neuropixels arrays. This is particularly useful given that histological information is often unavailable for chronic recordings. They make a clear case that CSD analysis is insufficient to resolve the lamination pattern with the desired precision and offer a thoughtful set of alternative analyses, along with an order in which to consider multiple cues in order to facilitate others' adoption of the strategy. The suggested analyses can be used to reliably identify certain landmarks (the positions of layer 4c and the layer 6-white matter transition), which provide very useful constraints for specifying the remaining laminar boundaries, and consideration of average anatomical patterns makes it unlikely that the remaining laminar boundaries will be far from their true locations. Overall, the widths of the resulting layers bear a sensible resemblance to the expected widths identified by prior anatomical measurements, and at least in some cases there are satisfying signatures of chromatic visual sensitivity and latency differences across layers that are predicted by the known connectivity of the corresponding layers. Thus, the proposed analytical toolkit appears to work well for macaque V1 and has strong potential to generalize to use in other cortical regions, though area-targeted selection of stimuli may be required.

Weaknesses:

The waveform measures, in particular the unit density distribution, are likely to be sensitive to the methods and criteria used for spike sorting, which differ among experimenters/groups, and this may limit the usefulness of this particular measure for others in the community.<br /> More generally, although the sizes of identified layers comport with typical sizes identified anatomically, a more powerful confirmation would be a direct comparison with histologically identified boundaries along each penetration's trajectory. Ultimately, the absence of this type of independent confirmation limits the strength of the claim that veridical laminar boundaries can be precisely identified from electrophysiological signals alone.

Reviewer #1 (Public review):

Summary:

Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.

Strengths:

While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:

(1) mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. Instead, they suggest that ABHD2, acting as a coreceptor of mPRbeta, demonstrates phospholipase activity, indicating that downstream lipid mediators may play a dominant role when stimulated by progesterone.<br /> (2) Extensive exploration of downstream signaling pathways and the identification of several potential meiotic activity-related lipid mediators make this aspect of the study novel and potentially significant.

Weaknesses:

However, there are some weaknesses and areas that need further clarification:

(1) The mechanism governing the molecular assembly of mPRbeta and ABHD2 remains unclear. Are they constitutively associated or is their association ligand-dependent? Does P4 bind not only to mPRbeta but also to ABHD2, as indicated in Figure 6J? In the latter case, the reviewer suggests that the authors conduct a binding experiment using labeled P4 with ABHD2 to confirm this interaction and assess any potential positive or negative cooperativity with a partner receptor.

(2) The authors have diligently determined the metabolite profile using numerous egg cells. However, the interpretation of the results appears incomplete, and inconsistencies were noted between Figure 2F and Supplementary Figure 2C. Furthermore, PGE2 and D2 serve distinct roles and have different elution patterns by LC-MS/MS, thus requiring separate measurements. In addition, the extremely short half-life of PGI2 necessitates the measurement of its stable metabolite, 6-keto-PGF1a, instead. The authors also need to clarify why they measured PGF1a but not PGF2a. Unfortunately, even in the revision, authors did not adequately address the last issue (differential measurements of PGD2 and E2, 6-keto-PG!alpha be determined instead of PGI2).

(3) Although they propose PGs, LPA and S1P are important downstream mediators, the exact roles of the identified lipid mediators have not been clearly demonstrated, as receptor expression and activation were not demonstrated. While the authors showed S1PR3 expression and its importance by genetic manipulation, there was no observed change in S1P levels following P4 treatment (Supplementary Figure 2D). It is essential to identify which receptors (subtypes) are expressed and how downstream signaling pathways (PKA, Ca, MAPK, etc.) relate to oocyte phenotypes.

These clarifications and further experiments would enhance the overall impact and comprehensiveness of the study.

Comments on revisions:

Need correction and addition for differential analyses of PGD2 and PGE2, and measurement of 6-keto-PGF1alpha instead of PGI2 (Figure 2F). PGI2 is extremely unstable (T1/2, 1 min in neutral buffer) and rapidly converted nonenzymically to 6-keto-PGF1a.

Reviewer #2 (Public review):

Summary:

This interesting paper examines the earliest steps in progesterone-induced frog oocyte maturation, an example of non-genomic steroid hormone signaling that has been studied for decades but is still very incompletely understood. In fish and frog oocytes it seems clear that mPR proteins are involved, but exactly how they relay signals is less clear. In human sperm, the lipid hydrolase ABHD2 has been identified as a receptor for progesterone, and so the authors here examine whether ABHD2 might contribute to progesterone-induced oocyte maturation as well. The main results are:

(1) Knocking down ABHD2 makes oocytes less responsive to progesterone, and ectopically expressing ABHD2.S (but not the shorter ABHD2.L gene product) partially rescues responsiveness. The rescue depends upon the presence of critical residues in the protein's conserved lipid hydrolase domain, but not upon the presence of critical residues in its acyltransferase domain.

(2) Treatment of oocytes with progesterone causes a decrease in sphingolipid and glycerophospholipid content within 5 min. This is accompanied by an increase in LPA content and arachidonic acid metabolites. These species may contribute to signaling through GPCRs. Perhaps surprisingly, there was no detectable increase in sphingosine-1-phosphate, which might have been expected given the apparent substantial hydrolysis of sphingolipids. The authors speculate that S1P is formed and contributes to signaling but diffuses away.

(3) Pharmacological inhibitors of lipid-metabolizing enzymes support, for the most part, the inferences from the lipidomics studies, although there are some puzzling findings. The puzzling findings may be due to uncertainty about whether the inhbitors are working as advertised.

(4) Pharmacological inhibitors of G-protein signaling support a role for G-proteins and GPCRs in this signaling, although again there are some puzzling findings.

(5) Reticulocyte expression supports the idea that mPRβ and ABHD2 function together to generate a progesterone-regulated PLA2 activity.

(6) Knocking down or inhibiting ABHD2 inhibited progesterone-induced mPRβ internalization, and knocking down ABHD2 inhibited SNAP25∆20-induced maturation.

Strengths:<br /> All in all, this could be a very interesting paper and a nice contribution. The data add a lot to our understanding of the process, and, given how ubiquitous mPR and AdipoQ receptor signaling appear to be, something like this may be happening in many other physiological contexts.

Weaknesses:

I have several suggestions for how to make the main points more convincing.

Main criticisms:

(1) The ABHD2 knockdown and rescue, presented in Fig 1, is one of the most important findings. It can and should be presented in more detail to allow the reader to understand the experiments better. E.g.: the antisense oligos hybridize to both ABHD2.S and ABHD2.L, and they knock down both (ectopically expressed) proteins. Do they hybridize to either or both of the rescue constructs? If so, wouldn't you expect that both rescue constructs would rescue the phenotype, since they both should sequester the AS oligo? Maybe I'm missing something here.

In addition, it is critical to know whether the partial rescue (Fig 1E, I, and K) is accomplished by expressing reasonable levels of the ABHD2 protein, or only by greatly overexpressing the protein. The author's antibodies do not appear to be sensitive enough to detect the endogenous levels of ABHD2.S or .L, but they do detect the overexpressed proteins (Fig 1D). The authors could thus start by microinjecting enough of the rescue mRNAs to get detectable protein levels, and then titer down, assessing how low one can go and still get rescue. And/or compare the mRNA levels achieved with the rescue construct to the endogenous mRNAs.

Finally, please make it clear what is meant by n = 7 or n = 3 for these experiments. Does n = 7 mean 7 independently lysed oocytes from the same frog? Or 7 groups of, say, 10 oocytes from the same frog? Or different frogs on different days? I could not tell from the figure legends, the methods, or the supplementary methods. Ideally one wants to be sure that the knockdown and rescue can be demonstrated in different batches of oocytes, and that the experimental variability is substantially smaller than the effect size.

(2) The lipidomics results should be presented more clearly. First, please drop the heat map presentations (Fig 2A-C) and instead show individual time course results, like those shown in Fig 2E, which make it easy to see the magnitude of the change and the experiment-to-experiment variability. As it stands, the lipidomics data really cannot be critically assessed.

[Even as heat map data go, panels A-C are hard to understand. The labels are too small, especially on the heat map on the right side of panel B. And the 25 rows in panel C are not defined (the legend makes me think the panel is data from 10 individual oocytes, so are the 25 rows 25 metabolites? If so, are the individual oocyte data being collapsed into an average? Doesn't that defeat the purpose of assessing individual oocytes?) And those readers with red-green colorblindness (8% of men) will not be able to tell an increase from a decrease. But please don't bother improving the heat maps; they should just be replaced with more-informative bar graphs or scatter plots.]

(3) The reticulocyte lysate co-expression data are quite important, and are both intriguing and puzzling. My impression had been that to express functional membrane proteins, one needed to add some membrane source, like microsomes, to the standard kits. Yet it seems like co-expression of mPR and ABHD2 proteins in a standard kit is sufficient to yield progesterone-regulated PLA2 activity. I could be wrong here-I'm not a protein expression expert-but I was surprised by this result, and I think it is critical that the authors make absolutely certain that it is correct. Do you get much greater activities if microsomes are added? Are the specific activities of the putative mPR-ABHD2 complexes reasonable?

Comments on revisions:

The authors have satisfied my concerns with their response letter and revisions.

Reviewer #3 (Public review):

Summary:

The authors report two P4 receptors, ABHD2 and mPRβ that function as co-receptors to induce PLA2 activity and thus drive meiosis. In their experimental studies, the authors knock down ABHD2 and demonstrated inhibition of oocyte maturation and inactivation of Plk1, MAPK, and MPF, which indicated that ABHD2 is required for P4-induced oocyte maturation. Next, they showed three residues (S207, D345, H376) in the lipase domain that are crucial for ABHD2 P4-mediated oocyte maturation in functional assays. They performed global lipidomics analysis on mPRβ or ABHD2 knockdown oocytes, among which the downregulation of GPL and sphingolipid species were observed and enrichment in LPA was also detected using their metabolomics method. Furthermore, they investigated pharmacological profiles of enzymes predicted to be important for maturation based on their metabolomic analyses and ascertained the central role for PLA2 in inducing oocyte maturation downstream of P4. They showed the modulation of S1P/S1PR3 pathway on oocyte maturation and potential role for or Gαs signaling and potentially Gβγ downstream of P4.

Strengths:

The authors make a very interesting finding that ABHD2 has PLA2 catalytic activity but only in the presence of mPRβ and P4. Finally, they provided supporting data for a relationship between ABHD2/PLA2 activity and mPRβ endocytosis and further downstream signaling. Collectively, this research report defines early steps in nongenomic P4 signaling, which is of broad physiological implications.

Weaknesses:

There were concerns with the pharmacological studies presented. Many of these inhibitors are used at high (double digit micromolar) concentrations that could result in non-specific pharmacological effects and the authors have provided very little data in support of target engagement and selectivity under the multiple experimental paradigms. In addition, the use of an available ABHD2 small molecule inhibitor was lacking in these studies.

Comments on revisions:

In the revised manuscript, the authors have addressed my major concerns.

Reviewer #1 (Public review):

Summary:

This study is focused an important aspect of axon guidance at the central nervous system (CNS) midline: how neurons extend axons that either do or do not cross the CNS midline. The authors here address contradictory work in the field relating to how cell surface expression of the slit receptor Robo1 is regulated so as to generate crossed and non-crossed axon trajectories during Drosophila neural development. They use fly genetics, cell lines, and biochemical assessments to define a complex consisting of the commissureless, Nedd4 and Robo1 proteins necessary for regulating Robo1 protein expression. This work resolves certain remaining questions in the field regarding midline axon guidance, with strengths out weighing weaknesses; however, addressing some of these weaknesses would strengthen this study.

Strengths:

Strengths include:<br /> - The use of well controlled genetic gain-of-function (over expression) approaches in vivo in Drosophila to show that phosphorylation sites (there are 2, and this study allows for assessment of the contributions made by each) in the commissureless (Comm) protein are indeed required for Comm function with respect to regulating axon midline guidance via their role in directing Comm-mediated Robo1 ubiquitination and degradation in the lysosome.<br /> - The demonstration that in vitro, and in a sensitized genetic background in vivo, the Nedd4 ubiquitin ligase regulates Robo1 protein cell surface distribution and also midline axon crossing in vivo.<br /> - Important evidence here that serves to resolve many questions raised by previous studies (not from these authors) regarding how Robo1 is regulated by Comm and Nedd4 family ubiquitin ligases. Further, these results are likely to have implications for thinking about the regulation of midline guidance in more complex nervous systems.

Weaknesses:

- A weakness beyond the purview of revision but important to mention is that the authors chose not to complement their GOF experiments with gene editing approaches to generate endogenous PY mutant alleles of Comm that might have been useful in genetic interaction experiments directed toward revealing roles for endogenous Comm in the regulation of Robo1.

Comments on revised version:

In this revised manuscript the authors provide new experiments and also reasonable explanations to address concerns raised in the initial review. I am satisfied that these efforts address satisfactorily the points raised in the initial review and that this study has been strengthened. This is an interesting body of work that adds to our understanding of CNS midline guidance molecular mechanisms.

Reviewer #2 (Public review):

Summary:

Sullivan and Bashaw delve into the mechanisms that drive neural circuit assembly, and specifically, into the regulation of cell surface proteins that mediate axon pathfinding. During nervous system development, axons must traverse a molecularly and physically complex extracellular milieu to reach their synaptic targets. A fundamental, conserved repulsive signaling pathway is initiated by the Slit-Robo ligand-receptor pair. Robo, expressed on axon growth cones, binds Slit, secreted by midline cells, to prevent "pre-crossing" and "re-crossing" of axons at the midline. To control this repulsion, Robo surface levels are tightly regulated. In Drosophila, Commissureless (Comm) downregulates Robo surface levels and is required for axon crossing at the midline. Several studies suggest that PY motifs in Comm are required to localize Robo to endosomes. PY motifs have been shown to bind WW-domain containing proteins including the ubiquitin ligase Nedd4 family, so the authors propose that Comm may regulate Robo through Nedd4 interactions. Previous studies have hinted at a role for Nedd4-mediated ubiquitination of Comm in regulation of Robo localization, but there have also been conflicting data. For example, Comm mutants that are unable to be ubiquitinated mimic wild-type Comm, suggesting that ubiquitination of Comm is not required for regulation of Robo. The current study utilizes a suite of genetic analyses in Drosophila to resolve discrepancies pertaining to the mode of Comm-dependent regulation of Robo1 and proposes that Comm acts as an adapter for the Nedd4 ubiquitin ligase to recognize Robo1 as a substrate. The authors also demonstrate that Nedd4 is indeed required for midline crossing.

Strengths:

While this work is more incremental rather than field-shifting, it is nonetheless an excellent example of a rigorous, thorough analysis that culminates in enriching our mechanistic understanding of how neurons regulate cell-surface receptors in a spatiotemporal manner to control fundamental steps of circuit wiring. The experimental approach is thorough, and the manuscript is extremely well-written.

Weaknesses:

Some key experiments (eg. complex formation) were performed in cell culture in an overexpression background. However, updated experiments demonstrated complex formation using immunoprecipitation in tissues overexpression the corresponding components. Also, there was a missed opportunity to bolster the model proposed by using Comm PY mutants in several experiments.

Comments on revised version:

The revised manuscript bolsters the authors' conclusions and now provides evidence for interactions in tissue. No additional experiments are needed.

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating- prior social isolation is known to increase aggression in males by increased lunging, which is suppressed by group housing (GH). However, it is also known that single-housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., developed a modified aggression assay, to address this issue by recording aggression in Drosophila males for 2 hours, over a virgin female which is immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low-frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons promoting high-frequency lunging, similar to earlier studies, whereas Or47b neurons promote low-frequency but higher intensity tussling. Using optogenetic activation they found that three pairs of pC1 neurons- pC1SS2 increase tussling. While P1a neurons, previously implicated in promoting aggression and courtship, did not increase tussling in optogenetic activation (in the dark), they could promote aggressive tussling in thermogenetic activation carried out in the presence of visible light. It was further suggested, using a further modified aggression assay that GH males use increased tussling and are able to maintain territorial control, providing them mating advantage over SI males and this may partially overcome the effect of aging in GH males.

Strengths:

Using a series of clever neurogenetic and behavioral approaches, subsets of ORNs and pC1 neurons were implicated in promoting tussling behaviors. The authors devised a new paradigm to assay for territory control which appears better than earlier paradigms that used a food cup (Chen et al, 2002), as this new assay is relatively clutter-free, and can be eventually automated using computer vision approaches. The manuscript is generally well-written, and the claims made are largely supported by the data.

Weaknesses:

I have a few concerns regarding some of the evidence presented and claims made as well as a description of the methodology, which needs to be clarified and extended further.

(1) Typical paradigms for assaying aggression in Drosophila males last for 20-30 minutes in the presence of nutritious food/yeast paste/females or all of these (Chen et al. 2002, Nilsen et al., 2004, Dierick et al. 2007, Dankert et al., 2009, Certel & Kravitz 2012). The paradigm described in Figure 1 A, while important and more amenable for video recording and computational analysis, seems a modification of the assay from Kravitz lab (Chen et al., 2002), which involved using a female over which males fight on a food cup. The modifications include a flat surface with a central food patch and a female with its head buried in the food, (fixed female) and much longer adaptation and recording times respectively (30 minutes, 2 hours), so in that sense, this is not a 'new' paradigm but a modification of an existing paradigm and its description as new should be appropriately toned down. It would also be important to cite these earlier studies appropriately while describing the assay.

(2) Lunging is described as a 'low intensity' aggression (line 111 and associated text), however, it is considered a mid to high-intensity aggressive behavior, as compared to other lower-intensity behaviors such as wing flicks, chase, and fencing. Lunging therefore is lower in intensity 'relative' to higher intensity tussling but not in absolute terms and it should be mentioned clearly.

(3) It is often difficult to distinguish faithfully between boxing and tussling and therefore, these behaviors are often clubbed together as box, tussle by Nielsen et al., 2004 in their Markov chain analysis as well as a more detailed recent study of male aggression (Simon & Heberlein, 2020). Therefore, authors can either reconsider the description of behavior as 'box, tussle' or consider providing a video representation/computational classifier to distinguish between box and tussle behaviors.

(4) Simon & Heberlein, 2020 showed that increased boxing & tussling precede the formation of a dominance hierarchy in males, and lunges are used subsequently to maintain this dominant status. This study should be cited and discussed appropriately while introducing the paradigm.

(5) It would be helpful to provide more methodological details about the assay, for instance, a video can be helpful showing how the males are introduced in the assay chamber, are they simply dropped to the floor when the film is removed after 30 minutes (Figures 1-2)?

(6) The strain of Canton-S (CS) flies used should be mentioned as different strains of CS can have varying levels of aggression, for instance, CS from Martin Heisenberg lab shows very high levels of aggressive lunges. Are the CS lines used in this study isogenized? Are various genetic lines outcrossed into this CS background? In the methods, it is not clear how the white gene levels were controlled for various aggression experiments as it is known to affect aggression (Hoyer et al. 2008).

(7) How important it is to use a fixed female for the assay to induce tussling? Do these females remain active throughout the assay period of 2.5 hours? Is it possible to use decapitated virgin females for the assay? How will that affect male behaviors?

(8) Raster plots in Figure 2 suggest a complete lack of tussling in SH males in the first 60 minutes of the encounter, which is surprising given the longer duration of the assay as compared to earlier studies (Nielsen et al. 2004, Simon & Heberlein, 2020 and others), which are able to pick up tussling in a shorter duration of recording time. Also, the duration for tussling is much longer in this study as compared to shorter tussles shown by earlier studies. Is this due to differences in the paradigm used, strain of flies, or some other factor? While the bar plots in Figure 2D show some tussling in SH males, maybe an analysis of raster plots of various videos can be provided in the main text and included as a supplementary figure to address this.

(9) Neuronal activation experiments suggesting the involvement of pC1SS2 neurons are quite interesting. Further, the role of P1a neurons was demonstrated to be involved in increasing tussling in thermogenetic activation in the presence of light (Figure 4, Supplement 1), which is quite important as the role of vision in optogenetic activation experiments, which required to be carried out in dark, is often not mentioned. However, in the discussion (lines 309-310) it is mentioned that PC1SS2 neurons are 'necessary and sufficient' for inducing tussling. Given that P1a neurons were shown to be involved in promoting tussling, this statement should be toned down.

(10) Are Or47b neurons connected to pC1SS2 or P1a neurons?

(11) The paradigm for territory control is quite interesting and subsequent mating advantage experiments are an important addition to the eventual outcome of the aggressive strategy deployed by the males as per their prior housing conditions. It would be important to comment on the 'fitness outcome' of these encounters. For instance, is there any fitness advantage of using tussling by GH males as compared to lunging by SH males? The authors may consider analyzing the number of eggs laid and eclosed progenies from these encounters to address this.

Reviewer #2 (Public review):

Summary:

Gao et al. investigated the change of aggression strategies by the social experience and its biological significance by using Drosophila. Two modes of inter-male aggression in Drosophila are known: lunging, high-frequency but weak mode, and tussling, low-frequency but more vigorous mode. Previous studies have mainly focused on the lunging. In this paper, the authors developed a new behavioral experiment system for observing tussling behavior and found that tussling is enhanced by group rearing while lunging is suppressed. They then searched for neurons involved in the generation of tussling. Although olfactory receptors named Or67d and Or65a have previously been reported to function in the control of lunging, the authors found that these neurons do not function in the execution of tussling, and another olfactory receptor, Or47b, is required for tussling, as shown by the inhibition of neuronal activity and the gene knockdown experiments. Further optogenetic experiments identified a small number of central neurons pC1[SS2] that induce the tussling specifically. In order to further explore the ecological significance of the aggression mode change in group rearing, a new behavioral experiment was performed to examine territorial control and mating competition. Finally, the authors found that differences in the social experience (group vs. solitary rearing) are important in these biologically significant competitions. These results add a new perspective to the study of aggressive behavior in Drosophila. Furthermore, this study proposes an interesting general model in which the social experience-modified behavioral changes play a role in reproductive success.

Strengths:

A behavioral experiment system that allows stable observation of tussling, which could not be easily analyzed due to its low frequency, would be very useful. The experimental setup itself is relatively simple, just the addition of a female to the platform, so it should be applicable to future research. The finding about the relationship between the social experience and the aggression mode change is quite novel. Although the intensity of aggression changes with the social experience was already reported in several papers (Liu et al., 2011, etc), the fact that the behavioral mode itself changes significantly has rarely been addressed and is extremely interesting. The identification of sensory and central neurons required for the tussling makes appropriate use of the genetic tools and the results are clear. A major strength of the neurobiology in this study is the finding that another group of neurons (Or47b-expressing olfactory neurons and pC1[SS2] neurons), distinct from the group of neurons previously thought to be involved in low-intensity aggression (i.e. lunging), function in the tussling behavior. Further investigation of the detailed circuit analysis is expected to elucidate the neural substrate of the conflict between the two aggression modes.

Weaknesses:

The experimental systems examining the territory control and the reproductive competition in Figure 5 are novel and have advantages in exploring their biological significance. However, at this stage, the authors' claim is weak since they only show the effects of age and social experience on territorial and mating behaviors, but do not experimentally demonstrate the influence of aggression mode change itself. In the Abstract, the authors state that these findings reveal how social experience shapes fighting strategies to optimize reproductive success. This is the most important perspective of the present study, and it would be necessary to show directly that the change of aggression mode by social experience contributes to reproductive success.

In addition, a detailed description of the tussling is lacking. For example, the authors state that the tussling is less frequent but more vigorous than lunging, but while experimental data are presented on the frequency, the intensity seems to be subjective. The intensity is certainly clear from the supplementary video, but it would be necessary to evaluate the intensity itself using some index. Another problem is that there is no clear explanation of how to determine the tussling. A detailed method is required for the reproducibility of the experiment.

Reviewer #3 (Public review):

In this manuscript, Gao et al. presented a series of intriguing data that collectively suggest that tussling, a form of high-intensity fighting among male fruit flies (Drosophila melanogaster) has a unique function and is controlled by a dedicated neural circuit. Based on the results of behavioral assays, they argue that increased tussling among socially experienced males promotes access to resources. They also concluded that tussling is controlled by a class of olfactory sensory neurons and sexually dimorphic central neurons that are distinct from pathways known to control lunges, a common male-type attack behavior.

A major strength of this work is that it is the first attempt to characterize the behavioral function and neural circuit associated with Drosophila tussling. Many animal species use both low-intensity and high-intensity tactics to resolve conflicts. High-intensity tactics are mostly reserved for escalated fights, which are relatively rare. Because of this, tussling in the flies, like high-intensity fights in other animal species, has not been systematically investigated. Previous studies on fly aggressive behavior have often used socially isolated, relatively young flies within a short observation duration. Their discovery that 1) older (14-days-old) flies tend to tussle more often than younger (2-days-old) flies, 2) group-reared flies tend to tussle more often than socially isolated flies, and 3) flies tend to tussle at a later stage (mostly ~15 minutes after the onset of fighting), are the result of their creativity to look outside of conventional experimental settings. These new findings are keys for quantitatively characterizing this interesting yet under-studied behavior.

Precisely because their initial approach was creative, it is regrettable that the authors missed the opportunity to effectively integrate preceding studies in their rationale or conclusions, which sometimes led to premature claims. Also, while each experiment contains an intriguing finding, these are poorly related to each other. This obscures the central conclusion of this work. The perceived weaknesses are discussed in detail below.

Most importantly, the authors' definition of "tussling" is unclear because they did not explain how they quantified lunges and tussling, even though the central focus of the manuscript is behavior. Supplemental movies S1 and S2 appear to include "tussling" bouts in which 2 flies lunge at each other in rapid succession, and supplemental movie S3 appears to include bouts of "holding", in which one fly holds the opponent's wings and shakes vigorously. These cases raise a concern that their behavior classification is arbitrary. Specifically, lunges and tussling should be objectively distinguished because one of their conclusions is that these two actions are controlled by separate neural circuits. It is impossible to evaluate the credibility of their behavioral data without clearly describing a criterion of each behavior.

It is also confusing that the authors completely skipped the characterization of the tussling-controlling neurons they claimed to have identified. These neurons (a subset of so-called pC1 neurons labeled by previously described split-GAL4 line pC1SS2) are central to this manuscript, but the only information the authors have provided is its gross morphology in a low-resolution image (Figure 4D, E) and a statement that "only 3 pairs of pC1SS2 neurons whose function is both necessary and sufficient for inducing tussling in males" (lines 310-311). The evidence that supports this claim isn't provided. The expression pattern of pC1SS2 neurons in males has been only briefly described in reference 46. It is possible that these neurons overlap with previously characterized dsx+ and/or fru+ neurons that are important for male aggressions (measured by lunges), such as in Koganezawa et al., Curr. Biol. 2016 and Chiu et al., Cell 2020. This adds to the concern that lunge and tussling are not as clearly separated as the authors claim.

While their characterizations of tussling behaviors in wild-type males (Figures 1 and 2) are intriguing, the remaining data have little link with each other, making it difficult to understand what their main conclusion is. Figure 3 suggests that one class of olfactory sensory neurons (OSN) that express Or47b is necessary for tussling behavior. While the authors acknowledged that Or47b-expressing OSNs promote male courtship toward females presumably by detecting cuticular compounds, they provided little discussion on how a class of OSN can promote two different types of innate behavior. No evidence of a functional or circuitry relationship between the Or47b pathway and the pC1SS2 neurons was provided. It is unclear how these two components are relevant to each other. Lastly, the rationale of the experiment in Figure 5 and the interpretation of the results is confusing. The authors attributed a higher mating success rate of older, socially experienced males over younger, socially isolated males to their tendency to tussle, but tussling cannot happen when one of the two flies is not engaged. If, for instance, a socially isolated 14-day-old male does not engage in tussling as indicated in Figure 2, how can they tussle with a group-housed 14-day-old male? Because aggressive interactions in Figure 5 were not quantified, it is impossible to conclude that tussling plays a role in copulation advantage among pairs as authors argue (lines 282-288).

Despite these weaknesses, it is important to acknowledge the authors' courage to initiate an investigation into a less characterized, high-intensity fighting behavior. Tussling requires the simultaneous engagement of two flies. Even if there is confusion over the distinction between lunges and tussling, the authors' conclusion that socially experienced flies and socially isolated flies employ distinct fighting strategies is convincing. Questions that require more rigorous studies are 1) whether such differences are encoded by separate circuits, and 2) whether the different fighting strategies are causally responsible for gaining ethologically relevant resources among socially experienced flies. Enhanced transparency of behavioral data will help readers understand the impact of this study. Lastly, the manuscript often mentions previous works and results without citing relevant references. For readers to grasp the context of this work, it is important to provide information about methods, reagents, and other key resources.

Reviewer #1 (Public Review):

The paper proposes a new source reconstruction method for electroencephalography (EEG) data and claims that it can provide far superior spatial resolution than existing approaches and also superior spatial resolution to fMRI. This primarily stems from abandoning the established quasi-static approximation to Maxwell's equations.

The proposed method brings together some very interesting ideas, and the potential impact is high. However, the work does not provide the evaluations expected when validating a new source reconstruction approach. I cannot judge the success or impact of the approach based on the current set of results. This is very important to rectify, especially given that the work is challenging some long-standing and fundamental assumptions made in the field.

I also find that the clarity of the description of the methods, and how they link to what is shown in the main results hard to follow.

I am insufficiently familiar with the intricacies of Maxwell's equations to assess the validity of the assumptions and the equations being used by WETCOW. The work therefore needs assessing by someone more versed in that area. That said, how do we know that the new terms in Maxwell's equations, i.e. the time-dependent terms that are normally missing from established quasi-static-based approaches, are large enough to need to be considered? Where is the evidence for this?

I have not come across EFD, and I am not sure many in the EEG field will have. To require the reader to appreciate the contributions of WETCOW only through the lens of the unfamiliar (and far from trivial) approach of EFD is frustrating. In particular, what impact do the assumptions of WETCOW make compared to the assumptions of EFD on the overall performance of SPECTRE?

The paper needs to provide results showing the improvements obtained when WETCOW or EFD are combined with more established and familiar approaches. For example, EFD can be replaced by a first-order vector autoregressive (VAR) model, i.e. y_t = A y_{t-1} + e_t (where y_t is [num_gridpoints x 1] and A is [num_gridpoints x num_gridpoints] of autoregressive parameters).

The authors' decision not to include any comparisons with established source reconstruction approaches does not make sense to me. They attempt to justify this by saying that the spatial resolution of LORETA would need to be very low compared to the resolution being used in SPECTRE, to avoid compute problems. But how does this stop them from using a spatial resolution typically used by the field that has no compute problems, and comparing with that? This would be very informative. There are also more computationally efficient methods than LORETA that are very popular, such as beamforming or minimum norm.

In short, something like the following methods needs to be compared:

(1) Full SPECTRE (EFD plus WETCOW)<br /> (2) WETCOW + VAR or standard ("simple regression") techniques<br /> (3) Beamformer/min norm plus EFD<br /> (4) Beamformer/min norm plus VAR or standard ("simple regression") techniques

This would also allow for more illuminating and quantitative comparisons of the real data. For example, a metric of similarity between EEG maps and fMRI can be computed to compare the performance of these methods. At the moment, the fMRI-EEG analysis amounts to just showing fairly similar maps.

There are no results provided on simulated data. Simulations are needed to provide quantitative comparisons of the different methods, to show face validity, and to demonstrate unequivocally the new information that SPECTRE can _potentially_ provide on real data compared to established methods. The paper ideally needs at least 3 types of simulations, where one thing is changed at a time, e.g.:

(1) Data simulated using WETCOW plus EFD assumptions<br /> (2) Data simulated using WETCOW plus e.g. VAR assumptions<br /> (3) Data simulated using standard lead fields (based on the quasi-static Maxwell solutions) plus e.g. VAR assumptions

These should be assessed with the multiple methods specified earlier. Crucially the assessment should be quantitative showing the ability to recover the ground truth over multiple realisations of realistic noise. This type of assessment of a new source reconstruction method is the expected standard.

Reviewer #2 (Public Review):

Summary:

The manuscript claims to present a novel method for direct imaging of electric field networks from EEG data with higher spatiotemporal resolution than even fMRI. Validation of the EEG reconstructions with EEG/FMRI, EEG, and iEEG datasets are presented. Subsequently, reconstructions from a large EEG dataset of subjects performing a gambling task are presented.

Strengths:

If true and convincing, the proposed theoretical framework and reconstruction algorithm can revolutionize the use of EEG source reconstructions.

Weaknesses:

There is very little actual information in the paper about either the forward model or the novel method of reconstruction. Only citations to prior work by the authors are cited with absolutely no benchmark comparisons, making the manuscript difficult to read and interpret in isolation from their prior body of work.

Reviewer #1 (Public review):

Summary:

This study investigates the role of macrophage lipid metabolism in the intracellular growth of Mycobacterium tuberculosis. By using a CRISPR-Cas9 gene-editing approach, the authors knocked out key genes involved in fatty acid import, lipid droplet formation, and fatty acid oxidation in macrophages. Their results show that disrupting various stages of fatty acid metabolism significantly impairs the ability of Mtb to replicate inside macrophages. The mechanisms of growth restriction included increased glycolysis, oxidative stress, pro-inflammatory cytokine production, enhanced autophagy, and nutrient limitation. The study demonstrates that targeting fatty acid homeostasis at different stages of the lipid metabolic process could offer new strategies for host-directed therapies against tuberculosis.

The work is convincing and methodologically strong, combining genetic, metabolic, and transcriptomic analyses to provide deep insights into how host lipid metabolism affects bacterial survival.

Strengths:

The study uses a multifaceted approach, including CRISPR-Cas9 gene knockouts, metabolic assays, and dual RNA sequencing, to assess how various stages of macrophage lipid metabolism affect Mtb growth. The use of CRISPR-Cas9 to selectively knock out key genes involved in fatty acid metabolism enables precise investigation of how each step-lipid import, lipid droplet formation, and fatty acid oxidation affect Mtb survival. The study offers mechanistic insights into how different impairments in lipid metabolism lead to diverse antimicrobial responses, including glycolysis, oxidative stress, and autophagy. This deepens the understanding of macrophage function in immune defense.

The use of functional assays to validate findings (e.g., metabolic flux analyses, lipid droplet formation assays, and rescue experiments with fatty acid supplementation) strengthens the reliability and applicability of the results.

By highlighting potential targets for HDT that exploit macrophage lipid metabolism to restrict Mtb growth, the work has significant implications for developing new tuberculosis treatments.

Weaknesses:

The experiments were primarily conducted in vitro using CRISPR-modified macrophages. While these provide valuable insights, they may not fully replicate the complexity of the in vivo environment where multiple cell types and factors influence Mtb infection and immune responses.

Reviewer #2 (Public review):

Summary:

Host-derived lipids are an important factor during Mtb infection. In this study, using CRISPR knockouts of genes involved in fatty acid uptake and metabolism, the authors claim that a compromised uptake, storage, or metabolism of fatty acid restricts Mtb growth upon infection. Further, the authors claim that the mechanism involves increased glycolysis, autophagy, oxidative stress, pro-inflammatory cytokines, and nutrient limitation. The authors also claim that impaired lipid droplet formation restricts Mtb growth. However, promoting lipid droplet biogenesis does not reverse/promote Mtb growth.

Strengths:

The strength of the study is the use of clean HOXB8-derived primary mouse macrophage lines for generating CRISPR knockouts.

Weaknesses:

There are many weaknesses of this study, they are clubbed into four categories below

(1) Evidence and interpretations: The results shown in this study at several places do not support the interpretations made or are internally contradictory or inconsistent. There are several important observations, but none were taken forward for in-depth analysis. A<br /> a) The phenotypes of PLIN2-/-, FATP1-/-, and CPT-/- are comparable in terms of bacterial growth restriction; however, their phenotype in terms of lipid body formation, IL1B expression, etc., are not consistent. These are interesting observations and suggest additional mechanisms specific to specific target genes; however, clubbing them all as altered fatty acid uptake or catabolism-dependent phenotypes takes away this important point. b) Finding the FATP1 transcript in the HOXB8-derived FATP1-/- CRISPR KO line is a bit confusing. There is less than a two-fold decrease in relative transcript abundance in the KO line compared to the WT line, leaving concerns regarding the robustness of other experiments as well using FATP1-/- cells.<br /> c) No gene showing differential regulation in FATP-/- macrophages, which is very surprising.<br /> d) ROS measurements should be done using flow cytometry and not by microscopy to nail the actual pattern.

(2) Experimental design: For a few assays, the experimental design is inappropriate<br /> a) For autophagy flux assay, immunoblot of LC3II alone is not sufficient to make any interpretation regarding the state of autophagy. This assay must be done with BafA1 or CQ controls to assess the true state of autophagy.<br /> b) Similarly, qPCR analyses of autophagy-related gene expression do not reflect anything on the state of autophagy flux.

(3) Using correlative observations as evidence:<br /> a) Observations based on RNAseq analyses are presented as functional readouts, which is incorrect.<br /> b) Claiming that the inability to generate lipid droplets in PLIN2-/- cells led to the upregulation of several pathways in the cells is purely correlative, and the causal relationship does not exist in the data presented.

(4) Novelty: A few main observations described in this study were previously reported. That includes Mtb growth restriction in PLIN2 and FATP1 deficient cells. Similarly, the impact of Metformin and TMZ on intracellular Mtb growth is well-reported. While that validates these observations in this study, it takes away any novelty from the study.

(5) Manuscript organisation: It will be very helpful to rearrange figures and supplementary figures.

Reviewer #3 (Public review):

Summary:

This study provides significant insights into how host metabolism, specifically lipids, influences the pathogenesis of Mycobacterium tuberculosis (Mtb). It builds on existing knowledge about Mtb's reliance on host lipids and emphasizes the potential of targeting fatty acid metabolism for therapeutic intervention.

Strengths:

To generate the data, the authors use CRISPR technology to precisely disrupt the genes involved in lipid import (CD36, FATP1), lipid droplet formation (PLIN2), and fatty acid oxidation (CPT1A, CPT2) in mouse primary macrophages. The Mtb Erdman strain is used to infect the macrophage mutants. The study, revealsspecific roles of different lipid-related genes. Importantly, results challenge previous assumptions about lipid droplet formation and show that macrophage responses to lipid metabolism impairments are complex and multifaceted. The experiments are well-controlled and the data is convincing.

Overall, this well-written paper makes a meaningful contribution to the field of tuberculosis research, particularly in the context of host-directed therapies (HDTs). It suggests that manipulating macrophage metabolism could be an effective strategy to limit Mtb growth.

Weaknesses:

None noted. The manuscript provides important new knowledge that will lead mpvel to host-directed therapies to control Mtb infections.

Reviewer #1 (Public review):

Summary:

This study investigates what happens to the stimulus-driven responses of V4 neurons when an item is held in working memory. Monkeys are trained to perform memory-guided saccades: they must remember the location of a visual cue and then, after a delay, make an eye movement to the remembered location. In addition, a background stimulus (a grating) is presented that varies in contrast and orientation across trials. This stimulus serves to probe the V4 responses, is present throughout the trial, and is task-irrelevant. Using this design, the authors report memory-driven changes in the LFP power spectrum, changes in synchronization between the V4 spikes and the ongoing LFP, and no significant changes in firing rate.

Strengths:

(1) The logic of the experiment is nicely laid out.

(2) The presentation is clear and concise.

(3) The analyses are thorough, careful, and yield unambiguous results.

(4) Together, the recording and inactivation data demonstrate quite convincingly that the signal stored in FEF is communicated to V4 and that, under the current experimental conditions, the impact from FEF manifests as variations in the timing of the stimulus-evoked V4 spikes and not in the intensity of the evoked activity (i.e., firing rate).

Weaknesses:

I think there are two limitations of the study that are important for evaluating the potential functional implications of the data. If these were acknowledged and discussed, it would be easier to situate these results in the broader context of the topic, and their importance would be conveyed more fairly and transparently.

(1) While it may be true that no firing rate modulations were observed in this case, this may have been because the probe stimuli in the task were behaviorally irrelevant; if anything, they might have served as distracters to the monkey's actual task (the MGS). From this perspective, the lack of rate modulation could simply mean that the monkeys were successful in attending the relevant cue and shielding their performance from the potentially distracting effect of the background gratings. Had the visual probes been in some way behaviorally relevant and/or spatially localized (instead of full field), the data might have looked very different. With this in mind, it would be prudent to dial down the tone of the conclusions, which stretch well beyond the current experimental conditions (see recommendations).

(2) Another point worth discussing is that although the FEF delay-period activity corresponds to a remembered location, it can also be interpreted as an attended location, or as a motor plan for the upcoming eye movement. These are overlapping constructs that are difficult to disentangle, but it would be important to mention them given prior studies of attentional or saccade-related modulation in V4. The firing rate modulations reported in some of those cases provide a stark contrast with the findings here, and I again suspect that the differences may be due at least in part to the differing experimental conditions, rather than a drastically different encoding mode or functional linkage between FEF and V4.

Reviewer #2 (Public review):

Summary:

It is generally believed that higher-order areas in the prefrontal cortex guide selection during working memory and attention through signals that selectively recruit neuronal populations in sensory areas that encode the relevant feature. In this work, Parto-Dezfouli and colleagues tested how these prefrontal signals influence activity in visual area V4 using a spatial working memory task. They recorded neuronal activity from visual area V4 and found that information about visual features at the behaviorally relevant part of space during the memory period is carried in a spatially selective manner in the timing of spikes relative to a beta oscillation (phase coding) rather than in the average firing rate (rate code). The authors further tested whether there is a causal link between prefrontal input and the phase encoding of visual information during the memory period. They found that indeed inactivation of the frontal eye fields, a prefrontal area known to send spatial signals to V4, decreased beta oscillatory activity in V4 and information about the visual features. The authors went one step further to develop a neural model that replicated the experimental findings and suggested that changes in the average firing rate of individual neurons might be a result of small changes in the exact beta oscillation frequency within V4. These data provide important new insights into the possible mechanisms through which top-down signals can influence activity in hierarchically lower sensory areas and can therefore have a significant impact on the Systems, Cognitive, and Computational Neuroscience fields.

Strengths:

This is a well-written paper with a well-thought-out experimental design. The authors used a smart variation of the memory-guided saccade task to assess how information about the visual features of stimuli is encoded during the memory period. By using a grating of various contrasts and orientations as the background the authors ensured that bottom-up visual input would drive responses in visual area V4 in the delay period, something that is not commonly done in experimental settings in the same task. Moreover, one of the major strengths of the study is the use of different approaches including analysis of electrophysiological data using advanced computational methods of analysis, manipulation of activity through inactivation of the prefrontal cortex to establish causality of top-down signals on local activity signatures (beta oscillations, spike locking and information carried) as well as computational neuronal modeling. This has helped extend an observation into a possible mechanism well supported by the results.

Weaknesses:

Although the authors provide support for their conclusions from different approaches, I found that the selection of some of the analyses and statistical assessments made it harder for the reader to follow the comparison between a rate code and a phase code. Specifically, the authors wish to assess whether stimulus information is carried selectively for the relevant position through a firing rate or a phase code. Results for the rate code are shown in Figures 1B-G and for the phase code are shown in Figure 2. Whereas an F-statistic is shown over time in Figure 1F (and Figure S1) no such analysis is shown for LFP power. Similarly, following FEF inactivation there is no data on how that influences V4 firing rates and information carried by firing rates in the two conditions (for positions inside and outside the V4 RF). In the same vein, no data are shown on how the inactivation affects beta phase coding in the OUT condition.

Moreover, some of the statistical assessments could be carried out differently including all conditions to provide more insight into mechanisms. For example, a two-way ANOVA followed by post hoc tests could be employed to include comparisons across both spatial (IN, OUT) and visual feature conditions (see results in Figures 2D, S4, etc.). Figure 2D suggests that the absence of selectivity in the OUT condition (no significant difference between high and low contrast stimuli) is mainly due to an increase in slope in the OUT condition for the low contrast stimulus compared to that for the same stimulus in the IN condition. If this turns out to be true it would provide important information that the authors should address.

There are also a few conceptual gaps that leave the reader wondering whether the results and conclusion are general enough. Specifically,

(1) the authors used microstimulation in the FEF to determine RFs. It is thus possible that the FEF sites that were inactivated were largely more motor-related. Given that beta oscillations and motor preparatory activity have been found to be correlated and motor sites show increased beta oscillatory activity in the delay period, it is possible that the effect of FEF inactivation on V4 beta oscillations is due to inactivation of the main source of beta activity. Had the authors inactivated sites with a preponderance of visual neurons in the FEF would the results be different?

(2) Somewhat related to this point and given the prominence of low-frequency activity in deeper layers of the visual cortex according to some previous studies, it is not clear where the authors' V4 recordings were located. The authors report that they do have data from linear arrays, so it should be possible to address this.

(3) The authors suggest that a change in the exact frequency of oscillation underlies the increase in firing rate for different stimulus features. However, the shift in frequency is prominent for contrast but not for orientation, something that raises questions about the general applicability of this observation for different visual features.

(4) One of the major points of the study is the primacy of the phase code over the rate code during the delay period. Specifically, here it is shown that information about the visual features of a stimulus carried by the rate code is similar for relevant and irrelevant locations during the delay period. This contrasts with what several studies have shown for attention in which case information carried in firing rates about stimuli in the attended location is enhanced relative to that for stimuli in the unattended location. If we are to understand how top-down signals work in cognitive functions it is inevitable to compare working memory with attention. The possible source of this difference is not clear and is not discussed. The reader is left wondering whether perhaps a different measure or analysis (e.g. a percent explained variance analysis) might reveal differences during the delay period for different visual features across the two spatial conditions.

The use of the memory-guided saccade task has certain disadvantages in the context of this study. Although delay activity is interpreted as memory activity by the authors, it is in principle possible that it reflects preparation for the upcoming saccade, spatial attention (particularly since there is a stimulus in the RF), etc. This could potentially change the conclusion and perspective.

For the position outside the V4 RF, there is a decrease in both beta oscillations and the clustering of spikes at a specific phase. It is therefore possible that the decrease in information about the stimuli features is a byproduct of the decrease in beta power and phase locking. Decreased oscillatory activity and phase locking can result in less reliable estimates of phase, which could decrease the mutual information estimates.

The authors propose that coherent oscillations could be the mechanism through which the prefrontal cortex influences beta activity in V4. I assume they mean coherent oscillations between the prefrontal cortex and V4. Given that they do have simultaneous recordings from the two areas they could test this hypothesis on their own data, however, they do not provide any results on that.

The authors make a strong point about the relevance of changes in the oscillation frequency and how this may result in an increase in firing rate although it could also be the reverse - an increase in firing rate leading to an increase in the frequency peak. It is not clear at all how these changes in frequency could come about. A more nuanced discussion based on both experimental and modeling data is necessary to appreciate the source and role (if any) of this observation.

Reviewer #3 (Public review):

Summary:

In this report, the authors test the necessity of prefrontal cortex (specifically, FEF) activity in driving changes in oscillatory power, spike rate, and spike timing of extrastriate visual cortex neurons during a visual-spatial working memory (WM) task. The authors recorded LFP and spikes in V4 while macaques remembered a single spatial location over a delay period during which task-irrelevant background gratings were displayed on the screen with varying orientation and contrast. V4 oscillations (in the beta range) scaled with WM maintenance, and the information encoded by spike timing relative to beta band LFP about the task-irrelevant background orientation depended on remembered location. They also compared recorded signals in V4 with and without muscimol inactivation of FEF, demonstrating the importance of FEF input for WM-induced changes in oscillatory amplitude, phase coding, and information encoded about background orientations. Finally, they built a network model that can account for some of these results. Together, these results show that FEF provides meaningful input to the visual cortex that is used to alter neural activity and that these signals can impact information coding of task-irrelevant information during a WM delay.

Strengths:

(1) Elegant and robust experiment that allows for clear tests for the necessity of FEF activity in WM-induced changes in V4 activity.

(2) Comprehensive and broad analyses of interactions between LFP and spike timing provide compelling evidence for FEF-modulated phase coding of task-irrelevant stimuli at remembered location.

(3) Convincing modeling efforts.

Weaknesses:

(1) 0% contrast background data (standard memory-guided saccade task) are not reported in the manuscript. While these data cannot be used to consider information content of spike rate/time about task-irrelevant background stimuli, this condition is still informative as a 'baseline' (and a more typical example of a WM task).

(2) Throughout the manuscript, the primary measurements of neural coding pertain to task-irrelevant stimuli (the orientation/contrast of the background, which is unrelated to the animal's task to remember a spatial location). The remembered location impacts the coding of these stimulus variables, but it's unclear how this relates to WM representations themselves.

Reviewer #1 (Public review):

Summary:

In this study, Masroor Ahmad Paddar and his/her colleagues explore the noncanonical roles of ATG5 and membrane atg8ylation in regulating retromer assembly and function. They begin by examining the interactomes of ATG5 and expand the scope of these effects to include homeostatic responses to membrane stress and damage.

Strengths:

This study provides novel insights into the noncanonical function of ATG8ylation in endosomal cargo sorting process.

Weaknesses:

The direct mechanism by which ATG8ylation regulates the retromer remains unsolved.

Comments on revisions:

After revision, though the major weakness remains unsolved, other questions have been addressed experimentally or further interpreted.

Reviewer #2 (Public review):

Summary:

Padder et al. demonstrates that ATG5 mediates lysosomal repair via the recruitment of the retromer components during LLOMe-induced lysosomal damage and that mAtg8-ylation contributes to retromer-dependent cargo sorting of GLUT1. Although previous studies have suggested that during glucose withdrawal, classical autophagy contributes to retromer-dependent GLUT1 surface trafficking via interactions between LC3A and TBC1D5, the experiments here demonstrate that during basal conditions or lysosomal damage, ATGs that are not involved in mATG8ylation, such as FIP200, are not functionally required for retromer-dependent sorting of GLUT1. Overall, these studies suggest a unique role for ATG5 in the control of retromer function, and that conjugation of ATG8 to single membranes (CASM) is a partial contributors to these phenotypes.

Strengths:

(1) Overall, these studies suggest a unique non-autophagic role for ATG5 in the control of retromer function. They also demonstrate that conjugation of ATG8 to single membranes (CASM) is a partial contributors to these phenotypes. Overall, these data point to a new role for ATG5 and CASM-dependent mATG8ylation in lysosomal membrane repair and trafficking.

(2) Although the studies are overall supportive of the proposed model that the retromer is controlled by CASM-dependent mATG8-ylaytion, it is noteworthy that previous studies of GLUT1 trafficking during glucose withdrawal (Roy et al. Mol Cell, PMID: 28602638) were predominantly conducted in cells lacking ATG5 or ATG7, which would not be able to discriminate between a CASM-dependent vs. canonical autophagy-dependent pathway in the control of GLUT1 sorting. Is the lack of GLUT1 mis-sorting to lysosomes observed in FIP200 and ATG13KO cells also observed during glucose withdrawal? Notably, deficiencies in glycolysis and glucose-dependent growth have been reported in FIP200 deficient fibroblasts (Wei et al. G&D, PMID: 21764854) so there may be difference in regulation dependent on the stress imposed on a cell.

Comments on revisions:

My previous comments have been addressed.

Reviewer #3 (Public review):

In this manuscript, Padder et al. used APEX2 proximity labeling to find an interaction between ATG5 and the core components of the Retromer complex, VPS26, VPS29, and VPS35. Further studies revealed that ATG5 KO inhibited the trafficking of GLUT1 to the plasma membrane. They also found that other autophagy genes involved in membrane atg8ylation affected GLUT1 sorting. However, knocking out other essential autophagy genes such as ATG13 and FIP200 did not affect GLUT1 sorting. These findings suggest that ATG5 participates in the function of the Retromer in a noncanonical autophagy manner. Overall, the methods and techniques employed by the authors largely support their conclusions. These findings are intriguing and significant, enriching our understanding of the non-autophagic functions of autophagy proteins and the sorting of GLUT1.

Comments on revisions:

The concerns I raised have all been addressed.

Reviewer #1 (Public review):

Overall I found the approach taken by the authors to be clear and convincing. It is striking that the conclusions are similar to those obtained in a recent study using a different computational approach (finite state controllers), and lend confidence to the conclusions about the existence of an optimal memory duration. There are a few points or questions that could be addressed in greater detail in a revision:

(1) Discussion of spatial encoding

The manuscript contrasts the approach taken here (reinforcement learning in a grid world) with strategies that involve a "spatial map" such as infotaxis. The authors note that their algorithm contains "no spatial information." However, I wonder if further degrees of spatial encoding might be delineated to better facilitate comparisons with biological navigation algorithms. For example, the gridworld navigation algorithm seems to have an implicit allocentric representation, since movement can be in one of four allocentric directions (up, down, left, right). I assume this is how the agent learns to move upwind in the absence of an explicit wind direction signal. However, not all biological organisms likely have this allocentric representation. Can the agent learn the strategy without wind direction if it can only go left/right/forward/back/turn (in egocentric coordinates)? In discussing possible algorithms, and the features of this one, it might be helpful to distinguish<br /> (1) those that rely only on egocentric computations (run and tumble),<br /> (2) those that rely on a single direction cue such as wind direction,<br /> (3) those that rely on allocentric representations of direction, and<br /> (4) those that rely on a full spatial map of the environment.

(2) Recovery strategy on losing the plume

While the approach to encoding odor dynamics seems highly principled and reaches appealingly intuitive conclusions, the approach to modeling the recovery strategy seems to be more ad hoc. Early in the paper, the recovery strategy is defined to be path integration back to the point at which odor was lost, while later in the paper, the authors explore Brownian motion and a learned recovery based on multiple "void" states. Since the learned strategy works best, why not first consider learned strategies, and explore how lack of odor must be encoded or whether there is an optimal division of void states that leads to the best recovery strategies? Also, although the authors state that the learned recovery strategies resemble casting, only minimal data are shown to support this. A deeper statistical analysis of the learned recovery strategies would facilitate comparison to those observed in biology.

(3) Is there a minimal representation of odor for efficient navigation?

The authors suggest (line 280) that the number of olfactory states could potentially be reduced to reduce computational cost. This raises the question of whether there is a maximally efficient representation of odors and blanks sufficient for effective navigation. The authors choose to represent odor by 15 states that allow the agent to discriminate different spatial regimes of the stimulus, and later introduce additional void states that allow the agent to learn a recovery strategy. Can the number of states be reduced or does this lead to loss of performance? Does the optimal number of odor and void states depend on the spatial structure of the turbulence as explored in Figure 5?

Reviewer #2 (Public review):

Summary:

The authors investigate the problem of olfactory search in turbulent environments using artificial agents trained using tabular Q-learning, a simple and interpretable reinforcement learning (RL) algorithm. The agents are trained solely on odor stimuli, without access to spatial information or prior knowledge about the odor plume's shape. This approach makes the emergent control strategy more biologically plausible for animals navigating exclusively using olfactory signals. The learned strategies show parallels to observed animal behaviors, such as upwind surging and crosswind casting. The approach generalizes well to different environments and effectively handles the intermittency of turbulent odors.

Strengths:

(1) The use of numerical simulations to generate realistic turbulent fluid dynamics sets this paper apart from studies that rely on idealized or static plumes.

(2) A key innovation is the introduction of a small set of interpretable olfactory states based on moving averages of odor intensity and sparsity, coupled with an adaptive temporal memory.

(3) The paper provides a thorough analysis of different recovery strategies when an agent loses the odor trail, offering insights into the trade-offs between various approaches.

(4) The authors provide a comprehensive performance analysis of their algorithm across a range of environments and recovery strategies, demonstrating the versatility of the approach.

(5) Finally, the authors list an interesting set of real-world experiments based on their findings, that might invite interest from experimentalists across multiple species.

Weaknesses:

(1) The inclusion of Brownian motion as a recovery strategy, seems odd since it doesn't closely match natural animal behavior, where circling (e.g. flies) or zigzagging (ants' "sector search") could have been more realistic.

(2) Using tabular Q-learning is both a strength and a limitation. It's simple and interpretable, making it easier to analyze the learned strategies, but the discrete action space seems somewhat unnatural. In real-world biological systems, actions (like movement) are continuous rather than discrete. Additionally, the ground-frame actions may not map naturally to how animals navigate odor plumes (e.g. insects often navigate based on their own egocentric frame).

(3) The lack of accompanying code is a major drawback since nowadays open access to data and code is becoming a standard in computational research. Given that the turbulent fluid simulation is a key element that differentiates this paper, the absence of simulation and analysis code limits the study's reproducibility.

Reviewer #1 (Public review):

Summary:

The study by Nelson et al. is focused on formation of the Drosophila Posterior Signaling Center (PSC) which ultimately acts as a niche to support hematopoietic stem cells of the lymph gland (LG). Using a combination of genetics and live imaging, the authors show that PSC cells migrate as a tight collective and associate with multiple tissues during a trajectory that positions them at the posterior of the LG.

This is an important study that identifies Slit-Robo signaling as a regulator of PSC morphogenesis, and highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM) and cardioblasts (CBs) - in coordinated development of these three tissues during organ development. However, one point requiring clarification is the idea that PSC cells exhibit a collective cell migration; it is not clear that the cells are migrating rather than being pushed to a more dorsal position through dorsal closure and/or other similar large scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis as presented.

Strengths:

• Using expression of Hid or Grim to ablate associated tissues, they find evidence that the VM and CB of the dorsal vessel affect PSC migration/morphology whereas the alary muscles do not. Slit is expressed by both VM and CBs, and therefore Slit-Robo signaling was investigated as PSCs express Robo.

• Using a combination of approaches, the authors convincingly demonstrate that Slit expression in the CBs and VM acts to support PSC positioning. A strength is the ability to knockdown slit levels in particular tissue types using the Gal4 system and RNAi.

• Although in the analysis of robo mutants, the PSC positioning phenotype is weaker in the individual mutants (robo1 and robo2) with only the double mutant (robo1,robo2) exhibiting a phenotype comparable to the slit RNAi. The authors make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs, because PSCs show a phenotype even when CBs do not (Fig 4G).

• New insight into dorsal vessel formation by VM is presented in Fig 4A,B, as loss of the VM can affect dorsal vessel morphogenesis. This result additionally points to the VM as important.

Weaknesses:

• The authors are cautioned to temper the result that Slit-Robo signaling is intrinsic to PSC since loss of robo may affect other cell types (besides CBs and PSCs) to indirectly affect PSC migration/morphogenesis. In fact, in the robo2, robo1 mutant, the VM appears to be incorrectly positioned (Fig. 4G).

• If possible, the authors should use RNAi to knockdown Robo1 and Robo2 levels specifically in the PSCs if a Gal4 is available; might Antp.Gal4 (Fig 1K) be useful? Even if knockdown is achieved in PSCs+CBs, this would be a better/complementary experiment to support the approach outlined in Fig 4D.

• Movies are hard to interpret, as it seems unclear that the PSCs actively migrate rather than being pushed/moved indirectly due to association with VM and CBs/dorsal vessel.

Reviewer #2 (Public review):

The paper by Nelson KA, et al. explored the collective migration, coalescence and positioning of the posterior signaling center (PSC) cells in Drosophila embryo. With live imaging, the authors observed the dynamic progress of PSC migration. Throughout this process, visceral mesoderm (VM), alary muscles (Ams) and cardioblasts (CBs) are in proximity of PSC. Genetic ablation of these tissues reveals the requirement for VM and CBs, but not AMs in this process. Genetic manipulations further demonstrated that Slit-Robo signaling was critical during PSC migration and positioning. While the genetic mechanisms of positioning the PSC were explored in much detail, including using live imaging, the functional consequence of mispositioning or (partial) absence of PSC cells has not been addressed, but would much increase the relevance of their findings. A few additional issues need to be addressed as well in this otherwise well-done study.

Previous major points:

(1) The only readout in their experiments is the relative correctness of PSC positioning. Importantly, what is the functional consequence if PSC is not properly positioned? This would be particularly important with robo-sli manipulations, where the PSC is present but some cells are misplaced. What is the consequence? Are the LGs affected, like specification of their cell types, structure and function? To address this for at least the robo-slit requirement in the PSC, it may be important to manipulate them directly in the PSC with a split Gal4 system, using Antp and Odd promoters.

(2) The densely, parallel aligned fibers in the lower part of Figure 1J seemed to be visceral mesoderm, but further up (dorsally) that may be epidermis. It is possible that the PSC migrate together with the epidermis? This should be addressed.

(3) Although the authors described the standards of assessing PSC positioning as "normal" or "abnormal", it is rather subtle at times and variable in the mutant or KD/OE examples. The criteria should be more clearly delineated and analyzed double-blind, also since this is the only readout. Further examples of abnormal positioning in supplementary figures would also help.

(4) Discussion is very lengthy and should shortened.

Comments on revised version:

Although the authors have responded to my concerns as they deemed suitable, these concerns still stand for the revised version.

Reviewer #3 (Public review):

Summary:

This work is a detailed and thorough analysis of the morphogenesis of the posterior signaling center (PSC), a hematopoietic niche in the Drosophila larva. Live imaging is performed from the stage of PSC determination until the appearance of a compact lymph gland and PSC in the stage 16 embryo. This analysis is combined with genetic studies that clarify the involvement of adjacent tissue, including the visceral mesoderm, alary muscle, and cardioblasts/dorsal vessel. Lastly, the Slit/Robo signaling system is clearly implicated in the normal formation of the PSC.

Strengths:

The data are clearly presented and well documented, and fully support the conclusions drawn from the different experiments.

The authors have addressed all of my previous comments, in particular concerning the role of epidermal cell rearrangements during dorsal closure as a possible force acting on the movement of PSC cells. The authors have clarified their definition of "collective migration" as it applies to the movement of PSC. The revised paper will make an important contribution to our understanding of the mechanisms driving morphogenesis.

Joint Public Review:

Summary

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

Strengths

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

Challenges

Despite the breadth of information presented, and although many of the suggestions in the initial review were addressed well, some points related to quantification and discussion of sex differences are not fully addressed in this revision.

(1) The request for quantification of OEC bridges is not fully addressed. We note that this revision includes the following statement (page 6): "We note, however, that such bridge formation is rare following a severe spinal cord injury in adult mammals." However, the title of the paper states that olfactory ensheathing cells promote neural repair and the abstract states that "OECs transplanted near the injury site modify the inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion." Statements such as these make it more crucial to include quantification of OEC bridges, because if single images are shown of remarkable, unusual bridges, but only one sentence acknowledges the low frequency of this occurrence, then this information taken together might present the wrong takeaway to readers.

Including some sort of quantification of bridging, whether it be the number of rats exhibiting bridges, the percentage area of OECs near a lesion site, or some other meaningful analysis, would add rigor and clarity to the manuscript.

(2) The additional discussion of sex differences in OEC bridging elaborates on the choice to study female rats, citing bladder challenges in male rats, but does not note salient clinical implications of this choice. Men account for ~80% of spinal cord injuries and likely also have worsened urinary tract issues, so it would be important to acknowledge this clinical fact and consider including males in future studies.

Reviewer #1 (Public review):

Huber proposes a theory where the role of the medial temporal lobe (MTL) is memory, where properties of spatial cells in the MTL can be explained through memory function rather than spatial processing or navigation. Instantiating the theory through a computational model, the author shows that many empirical phenomena of spatial cells can be captured, and may be better accounted through a memory theory. It is an impressive computational account of MTL cells with a lot of theoretical reasoning and aims to tightly relate to various spatial cell data.

In general, the paper is well written, and has been greatly improved after revision for clarity and situating the model in the context of the literature. Below are a few responses to the author's rebuttal.

(2 & 3) In response to my previous review point 2 and 3, the author has now added "According to this model, hexagonally arranged grid cells should be the exception rather than the rule when considering more naturalistic environments." It is good to know that it captures data that show non-grid like responses in more complex and realistic environments. However, the model still focuses on explaining the spatial firing aspect of grid cells even though they are not supposed to be spatial. I noted in my previous review, "If it's not encoding a spatial attribute, it doesn't have to have a spatial field. For example, it could fire in the whole arena". The author notes inhibitory drive and habituation. Habituation happens, but then spatial cell responses are supposed (or assumed) to be still strong after many visits to that environment. More generally, I am more convinced that grid-like and spatial coding are a special case - both in navigation and memory. In a way I believe the author agrees, though the work here focuses on capturing spatial properties (which is understandable given the literature). In conclusion, though there may be theoretical disagreements, I find the points the author raises fair.

(4) The difference between mEC and lEC or PRC for encoding non-spatial vs spatial attributes is still not clear to me - though not crucial for the point of this paper.

(5) Thank you for providing a video - this makes it extremely clear how learning occurs.

Reviewer #3 (Public review):

The author presents a novel theory and computational model suggesting that grid cells do not encode space, but rather encode non-spatial attributes. Place cells in turn encode memories of where those specific attributes occurred. The theory accounts for many experimental results and generates useful predictions for future studies. The model's simplicity and potential explanatory power will interest others in the field. There are, however, a few weaknesses outlined below which undermine the theory.

Main criticisms:

(1) A crucial assumption of the model is that grid cells express grid-like firing patterns if and only if the content of experience is constant in space. It is difficult to imagine a real world example that satisfies this assumption. Odors and sounds are used as examples. While they are often more spatially diffuse than an object on the ground, odors and sounds have sources that are readily detectable and thus are not constant in space. Animals can easily navigate to a food source or to a vocalizing conspecific. This assumption is especially problematic because it predicts that all grid cells should become silent when their preferred non-spatial attribute (e.g. a specific odor) is missing. I'm not aware of any experimental data showing that grid cells become silent. On the contrary, grid cells are known to remain active across all contexts that have been tested, including across sleep/wake states. Unlike place cells, grid cells have never been shown to turn off. Since grid cells are active in all contexts, their preferred attribute must also be present in all contexts, and therefore they would not convey any information about the specific content of an experience. The author lists many attributes that could in theory be constant in a laboratory setting, but there is no data I'm aware of that shows this is true in practice. As it stands, this crucial assumption of the model remains mere speculation.

(2) The proposed novelty of this theory is that other models all assume that grid cells encode space. This is not quite true of models based on continuous attractor networks, the discussion of which is essentially absent. More specifically, attractor models focus on the importance of intrinsic dynamics within entorhinal cortex in generating the grid pattern. While this firing pattern is aligned to space during navigation and therefore can be used a representation of that space, the neural dynamics are preserved even during sleep. Similarly, it is because the grid pattern does not strictly encode physical space that grid-like signals are also observed in relation to other two-dimensional continuous variables.

(3) The use of border cells or boundary vector cells as the main (or only) source of spatial information in the hippocampus is not well supported by experimental data. Border cells in entorhinal cortex are not active in the center of an environment. Boundary-vector cells can fire farther away from the walls, but are not found in entorhinal cortex. They are located in the subiculum, a major output of the hippocampus. While the entorhinal-hippocampal circuit is a loop, the route from boundary-vector cells to place cells is much less clear than from grid cells. Moreover, both border cells and boundary-vector cells (which are conflated in this paper) comprise a small population of neurons compared to grid cells.

Minor comments:

(1) There is substantial theoretical and experimental work supporting the idea that grid cell modules instantiate continuous attractor networks, yet this class of models is largely ignored:

p. 7 "In contrast, most grid cell models (Bellmund et al., 2016; Bush et al., 2015; Castro & Aguiar, 2014; Hasselmo, 2009; Mhatre et al., 2012; Solstad et al., 2006; Sorscher et al., 2023; Stepanyuk, 2015; Widloski & Fiete, 2014) are domain specific models of spatial navigation"

The following references should be added:

McNaughton, B. L., Battaglia, F. P., Jensen, O., Moser, E. I. & Moser, M.-B. Path integration and the neural basis of the 'cognitive map'. Nat. Rev. Neurosci. 7, 663-678 (2006).

Fuhs, M. C. & Touretzky, D. S. A spin glass model of path integration in rat medial entorhinal cortex. J. Neurosci. 26, 4266-4276 (2006).

Burak, Y. & Fiete, I. R. Accurate path integration in continuous attractor network models of grid cells. PLoS Comput. Biol. 5, e1000291 (2009).

Guanella, A., Kiper, D. & Verschure, P. A model of grid cells based on a twisted torus topology. Int. J. Neural Syst. 17, 231-240 (2007).

Couey, J. J. et al. Recurrent inhibitory circuitry as a mechanism for grid formation. Nat. Neurosci. 16, 318-324 (2013).

(Note: the Bellmund et al. (2016) citation is likely a mistake and was intended to be Bellmund et al. (2018).)

(2) The author claims in two places that this model is the first to explain that grid cell population activity lies on a torus. While it may be the first explicit computational account of why grid cell activity is mapped onto a torus, these claims should be moderated for clarity, for example by adding "but see McNaughton et al. (2006) and others".

Box 1. Results Uniquely Explained by this Memory Model - the population code of grid cells lies on a torus

p.11 "In addition, this simplifying assumption allows the model to capture the finding that the population of grid cells lies on a torus (Gardner et al., 2022), although I note that the model was developed before this result was known."

(3) Lateral entorhinal cortex is largely ignored in this memory model. It should be considered that the predominance of spatial representations reported in the literature is due to historical reasons. Namely, the discovery of hippocampal place cells spurred interest in looking upstream for the source of spatial information, which was later abundantly clear in medial entorhinal cortex. Lateral entorhinal cortex is relatively understudied, but is known to encode odors, objects, and time in a way that medial entorhinal cortex does not. It is therefore confusing to assume that these attributes are instead encoded by grid cells.

Reviewer #1 (Public review):

Summary:

The manuscript of He et al. compares the roles of Hox/Gbx genes between the well-established anthozoan model, the burrowing sea anemone Nematostella, and the new scleractinian model Montipora. The authors show staggered expression of Anthox6a.1, Anthox8 and Gbx of the Montipora larva and argue that their BMP-dependent expression is responsible for the segmentation of the endomesoderm, just like they have previously demonstrated in Nematostella (despite some differences in the timing, formation of extra mesenteries, etc). The authors posit that Hox/Gbx-dependent segmentation of the endomesoderm represents an ancestral anthozoan trait. The study addresses a remarkably interesting question, but it has several important shortcomings, which the authors should try to rectify.

Strengths:

The authors introduce a new scleractinian model Montipora and present interesting data on the composition of its compact Hox cluster, its embryonic and larval development, metamorphosis, and segmentation. They also show staggered expression of Gbx, Anthox6a.1, and Anthox8, which is suggestive of their involvement in the partitioning of the gastrodermis of the polyp.

Weaknesses:

He et al. claim that Gbx and Hox genes are responsible for the segmentation of the directive axis in Montipora based on expression patterns of these genes before the onset of segmentation. In the absence of functional analyses, this claim (although likely correct) is not supported. Moreover, the authors do not show that staggered Gbx and Hox gene expression correlates with the position of the segment boundaries.

The authors use two inhibitors of BMP signaling and show that segmentation is lost in the treated animals. However, they do not provide controls, which would show that the effect of the treatment is specific to the loss of BMP function. Moreover, their transcriptomic analyses suggest that the whole BMP signaling system in Montipora is wired completely differently than in Nematostella, but they do not acknowledge and discuss this striking difference. If true, this is a very interesting result, but it requires thorough validation.

Reviewer #2 (Public review):

Building on their detailed dissection of the role of Hox-Gbx genes in endomesodermal segmentation in Nematostella, He and colleagues attempt to understand the evolutionary conservation of this process in anthozoans. In a move that should be congratulated, the authors perform this work in the coral M. capitata, a species that is not well established in the lab. The authors show convincing expression data using both RNAseq and in-situ hybridization and discover the conserved expression of Hox-Gbx genes preceding the segmentation of the enodmesoderm. The authors further attempt to understand whether BMP signalling is playing a role in this process and present data that certainly points to this being the case.

Strength:

The overall quality of the data is very high and the authors show very convincing expression data for the Hox-Gbx genes as well as putting forward a well-thought-out hypothesis for segment evolution.

Weakness:

There are a number of weaknesses in the paper which I believe can be easily addressed:

(1) The authors in many cases claim to have provided functional evidence for the role of Hox-Gbx genes in M. capitata. This is not, however, the case, and although the expression data along with their previous work in Nematostella make their claims very likely I still believe it is necessary to set a higher bar for claiming to understand function. In the abstract, for example, they claim: "These findings demonstrate the existence of a functionally conserved Hox-Gbx module....", something which is not substantiated by the data presented. At the end of the introduction, they say they "systematically interrogate the molecular functions of Hox-Gbx genes" (line 75) which again is not what is presented in the manuscript. Finally, on line 289-291 the authors state: "Taken together, our findings strongly suggest that the heterochronic deployment of a conserved Hox-Gbx module contributes to the divergent adult body plans observed between Edwardsiidae and other anthozoans." I would remove "Strongly" given the absence of functional data. There are also other examples where functional understanding is implied and I would suggest the authors tone this down throughout the manuscript.

(2) On Line 185, the authors state "To determine the function of the Hox-Gbx network in M.capitata segmentation..." when introducing their BMP experiments. I would reword this since they are looking at BMP signalling and do not look directly at Hox-Gbx function.

(3) Although the BMP inhibitor experiments are very interesting I think there is a lack of basic understanding of BMP signalling in this system. Where are the BMP components expressed and how would this match with the hypothesis derived from the data? The authors present some expression patterns in Figure S3 but do not discuss them. In addition, the authors do not show pSMAD staining etc, and do not validate that the inhibitors have an effect on this. I entirely understand the difficulties in doing such experiments in a system like this and would not suggest the authors should now do them but an acknowledgment of this in the discussion would be very welcome.

(4) In both lines 88 and 294 the authors talk about the mechanism of gastrulation. It is not clear to me how they infer this from the figure. If the authors could include some more high-resolution images that show this it would be very helpful and interesting.

(5) On line 169/170 the authors state that two Anthox6 paralogs, McAnthox6 and McAnthox6.1, were specifically expressed at the time of settlement. This is not what I see in the images. I see that McAnthox6 is expressed at 14 hpf more strongly than at the later time point. The authors should clarify this point.

(6) On lines 259-261 the authors state "How temporally and spatially coordinated gene expression can be achieved in this scenario remains an interesting and open question." This seems like a strange statement to include given that they have shown that there is no spatial and temporal collinearity in cnidarians. Surely it is not an open question to ask how it would work if there is none. I would simply remove this.

(7) The authors should cite the sources of information contained in Fig. S2 including how orthology was assigned.

Reviewer #3 (Public review):

Summary:

The authors analyze the expression of a series of genes from the Hox/Gbx family of transcription factors in the settling larva of the rice coral Montipora capitata. The first achievement of the work is developing a protocol for artificial induction of settlement in this species. In the synchronized settlers, the authors were able to follow the sequence of the subdivision of the body cavity to form individual cavities separated by mesenteries. This process has been previously studied in the starlet sea anemone, Nematostella vectensies, and this same group showed that there is a spatio-temporal sequence of expression of genes from the Hox/Gbx group, reminiscent of the sequence of Hox genes in bilaterians. The authors now repeat this analysis with orthologous genes in Montipora, and demonstrate a similar pattern. Finally, they manipulate the BMP pathway and demonstrate that in the absence of BMP signaling, the subdivision of the gastric cavity is abrogated.

Strengths:

The authors have developed a new experimental system for embryological work on cnidarians, where only a handful of systems are available. They identified orthologs of a number of homeobox genes and tested their expression. There is a detailed description of the sequence of the formation of the mesenteries, which differs from that of Namatostella, raising interesting questions about the evolution of mesentery number and the homology of mesenteries.

Weaknesses:

The in situ hybridization experiments describing the expression of the Hox/Gbx genes are not as clean and sharp as could be hoped for. This is evidently a limitation of the system. The discussion of the evolution of mesentery number does not really give new insights into the question (although just raising the discussion is interesting in its own right).

Reviewer #1 (Public review):

Summary:

A theoretical model for microbial osmoresponse was proposed. The model assumes simple phenomenological rules: (i) the change of free water volume in the cell due to osmotic imbalance based on pressure balance, (ii) Osmoregulation that assumes change of the proteome partitioning depending on the osmotic pressure that affects the osmolyte-producing protein production, (iii) The cell-wall synthesis regulation where the change of the turgor pressure to the cell-wall synthesis efficiency to go back to the target turgor pressure, (iv) Effect of Intracellular crowding assuming that the biochemical reactions slow down for more crowding and stops when the protein density (protein mass divided by free water volume) reaches a critical value. The parameter values were found in the literature or obtained by fitting to the experimental data. The authors compare the model behavior with various microorganismcs (E. coli, B. subtils, S. Cerevisiae, S. pombe), and successfully reproduced the overall trend (steady state behavior for many of them, dynamics for S. pombe). In addition, the model predicts non-trivial behavior such as the fast cell growth just after the hypoosmotic shock, which is consistent with experimental observation. The authors further make experimentally testable predictions regarding mutant behavior and transient dynamics.

Strength:

The theory assumes simple mechanistic dependence between core variables without going into specific molecular mechanisms of regulations. The simplicity allows the theory to apply to different organisms by adjusting the time scales with parameters, and the model successfully explains broad classes of observed behaviours. Mathematically, the model provides analytical expressions of the parameter dependences and an understanding of the dynamics through the phase space without being buried in the detail. This theory can serve as a base to discuss the universality and diversity of microbial osmoresponse.

Weakness:

The core part of this model is that everything is coupled with growth physiology, and, as far as I understand, the assumption (iv) (eq. 8) that imposes the global reaction rate dependence on crowding plays a crucial role. I would think this is a strong and interesting assumption. However, the abstract or discussion does not discuss the importance of this assumption. In addition, the paper does not discuss gene regulation explicitly, and some comparison with a molecular mechanism-oriented model may be beneficial to highlight the pros and cons of the current approach.

Reviewer #2 (Public review):

Summary:

In this study, Ye et al. have developed a theoretical model of osmotic pressure adaptation by osmolyte production and wall synthesis.

Strengths:

They validate their model predictions of a rapid increase in growth rate on osmotic shock experimentally using fission yeast. The study has several interesting insights which are of interest to the wider community of cell size and mechanics.

Weaknesses:

Multiple aspects of this manuscript require addressing, in terms of clarity and consistency with previous literature. The specifics are listed as major and minor comments.

Major comments:

(1) The motivation for the work is weak and needs more clarity.

(2) The link between sections is very frequently missing. The authors directly address the problem that they are trying to solve without any motivation in the results section.

(3) The parameters used in the models (symbols) need to be explained better to make the paper more readable.

(4) Throughout the paper, the authors keep switching between organisms that they are modelling. There needs to be some consistency in this aspect where they mention what organism they are trying to model, since some assumptions that they make may not be valid for both yeast as well as bacteria.

(5) The extent of universality of osmoregulation i.e the limitations are not very well highlighted.

(6) Line 198-200: It is not clear in the text what organisms the authors are writing about here. "Experiments suggested that the turgor pressure induce cell-wall synthesis, e.g., through mechanosensors on cell membrane [45, 46], by increasing the pore size of the peptidoglycan network [5], and by accelerating the moving velocity of the cell-wall synthesis machinery [31]". This however is untrue for bacteria as shown by the study (reference 22 is this paper:  E. Rojas, J. A. Theriot, and K. C. Huang, Response of escherichia coli growth rate to osmotic shock, Proceedings of the National Academy of Sciences 111, 7807 (2014).

(7) The time scale of reactions to hyperosmotic shocks does not agree with previous literature (reference 22). Therefore defining which organism you are looking at is important. Hence the statement " Because the timescale of the osmoresponse process, which is around hours (Figure 3B), is much longer than the timescale of the supergrowth phase, which is about 20 minutes, the turgor pressure at the growth rate peak can be well approximated by its immediate value after the shock." from line 447 does not seem to make sense. The authors need to address this.

Reviewer #1 (Public review):

In this important study, the authors characterized the transformation of neural representations of olfactory stimuli from the primary sensory cortex to multisensory regions in the medial temporal lobe and investigated how they were affected by non-associative learning. The authors used high-density silicon probe recordings from five different cortical regions while familiar vs. novel odors were presented to a head-restrained mouse. This is a timely study because unlike other sensory systems (e.g., vision), the progressive transformation of olfactory information is still poorly understood. The authors report that both odor identity and experience are encoded by all of these five cortical areas but nonetheless some themes emerge. Single neuron tuning of odor identity is broad in the sensory cortices but becomes narrowly tuned in hippocampal regions. Furthermore, while experience affects neuronal response magnitudes in early sensory cortices, it changes the proportion of active neurons in hippocampal regions. Thus, this study is an important step forward in the ongoing quest to understand how olfactory information is progressively transformed along the olfactory pathway.

The study is well-executed. The direct comparison of neuronal representations from five different brain regions is impressive. Conclusions are based on single neuronal level as well as population level decoding analyses. Among all the reported results, one stands out for being remarkably robust. The authors show that the anterior olfactory nucleus (AON), which receives direct input from the olfactory bulb output neurons, was far superior at decoding odor identity as well as novelty compared to all the other brain regions. This is perhaps surprising because the other primary sensory region - the piriform cortex - has been thought to be the canonical site for representing odor identity. A vast majority of studies have focused on aPCx, but direct comparisons between odor coding in the AON and aPCx are rare. The experimental design of this current study allowed the authors to do so and the AON was found to convincingly outperform aPCx. Although this result goes against the canonical model, it is consistent with a few recent studies including one that predicted this outcome based on anatomical and functional comparisons between the AON-projecting tufted cells vs. the aPCx-projecting mitral cells in the olfactory bulb (Chae, Banerjee et. al. 2022). Future experiments are needed to probe the circuit mechanisms that generate this important difference between the two primary olfactory cortices as well as their potential causal roles in odor identification.

The authors were also interested in how familiarity vs. novelty affects neuronal representation across all these brain regions. One weakness of this study is that neuronal responses were not measured during the process of habituation. Neuronal responses were measured after four days of daily exposure to a few odors (familiar) and then some other novel odors were introduced. This creates a confound because the novel vs. familiar stimuli are different odorants and that itself can lead to drastic differences in evoked neural responses. Although the authors try to rule out this confound by doing a clever decoding and Euclidian distance analysis, an alternate more straightforward strategy would have been to measure neuronal activity for each odorant during the process of habituation.

Reviewer #2 (Public review):

Summary:

This manuscript investigates how olfactory representations are transformed along the cortico-hippocampal pathway in mice during a non-associative learning paradigm involving novel and familiar odors. By recording single-unit activity in several key brain regions (AON, aPCx, LEC, CA1, and SUB), the authors aim to elucidate how stimulus identity and experience are encoded and how these representations change across the pathway.

The study addresses an important question in sensory neuroscience regarding the interplay between sensory processing and signaling novelty/familiarity. It provides insights into how the brain processes and retains sensory experiences, suggesting that the earlier stations in the olfactory pathway, the AON aPCx, play a central role in detecting novelty and encoding odor, while areas deeper into the pathway (LEC, CA1 & Sub) are more sparse and encodes odor identity but not novelty/familiarity. However, there are several concerns related to methodology, data interpretation, and the strength of the conclusions drawn.

Strengths:

The authors combine the use of modern tools to obtain high-density recordings from large populations of neurons at different stages of the olfactory system (although mostly one region at a time) with elegant data analyses to study an important and interesting question.

Weaknesses:

(1) The first and biggest problem I have with this paper is that it is very confusing, and the results seem to be all over the place. In some parts, it seems like the AON and aPCx are more sensitive to novelty; in others, it seems the other way around. I find their metrics confusing and unconvincing. For example, the example cells in Figure 1C show an AON neuron with a very low spontaneous firing rate and a CA1 with a much higher firing rate, but the opposite is true in Figure 2A. So, what are we to make of Figure 2C that shows the difference in firing rates between novel vs. familiar odors measured as a difference in spikes/sec. This seems nearly meaningless. The authors could have used a difference in Z-scored responses to normalize different baseline activity levels. (This is just one example of a problem with the methodology.)

(2) There are a lot of high-level data analyses (e.g., decoding, analyzing decoding errors, calculating mutual information, calculating distances in state space, etc.) but very little neural data (except for Figure 2C, and see my comment above about how this is flawed). So, if responses to novel vs. familiar odors are different in the AON and aPCx, how are they different? Why is decoding accuracy better for novel odors in CA1 but better for familiar odors in SUB (Figure 3A)? The authors identify a small subset of neurons that have unusually high weights in the SVM analyses that contribute to decoding novelty, but they don't tell us which neurons these are and how they are responding differently to novel vs. familiar odors.

(3) The authors call AON and aPCx "primary sensory cortices" and LEC, CA1, and Sub "multisensory areas". This is a straw man argument. For example, we now know that PCx encodes multimodal signals (Poo et al. 2021, Federman et al., 2024; Kehl et al., 2024), and LEC receives direct OB inputs, which has traditionally been the criterion for being considered a "primary olfactory cortical area". So, this terminology is outdated and wrong, and although it suits the authors' needs here in drawing distinctions, it is simplistic and not helpful moving forward.

(4) Why not simply report z-scored firing rates for all neurons as a function of trial number? (e.g., Jacobson & Friedrich, 2018). Figure 2C is not sufficient. For example, in the Discussion, they say, "novel stimuli caused larger increases in firing rates than familiar stimuli" (L. 270), but what does this mean? Odors typically increase the firing in some neurons and suppress firing in others. Where does the delta come from? Is this because novel odors more strongly activate neurons that increase their firing or because familiar odors more strongly suppress neurons?

(5) Lines 122-124 - If cells in AON and aPCx responded the same way to novel and familiar odors, then we would say that they only encode for odor and not at all for experience. So, I don't understand why the authors say these areas code for a "mixed representation of chemical identity and experience." "On the other hand," if LEC, CA1, and SUB are odor selective and only encode novel odors, then these areas, not AON and aPCx, are the jointly encoding chemical identity and experience. Also, I do not understand why, here, they say that AON and PCx respond to both while LEC, CA1, and SUB were selective for novel stimuli, but the authors then go on to argue that novelty is encoded in the AON and PCx, but not in the LEC, CA1, and SUB.

(6) Lines 132-140 - As presented in the text and the figure, this section is poorly written and confusing. Their use of the word "shuffled" is a major source of this confusion, because this typically is the control that produces outcomes at the chance level. More importantly, they did the wrong analysis here. The better and, I think, the only way to do this analysis correctly is to train on some of the odors and test on an untrained odor (i.e., what Bernardi et al., 2021 called "cross-condition generalization performance"; CCGP).

Reviewer #3 (Public review):

In this manuscript, the authors investigate how odor-evoked neural activity is modulated by experience within the olfactory-hippocampal network. The authors perform extracellular recordings in the anterior olfactory nucleus (AON), the anterior piriform (aPCx) and lateral entorhinal cortex (LEC), the hippocampus (CA1), and the subiculum (SUB), in naïve mice and in mice repeatedly exposed to the same odorants. They determine the response properties of individual neurons and use population decoding analyses to assess the effect of experience on odor information coding across these regions.

The authors' findings show that odor identity is represented in all recorded areas, but that the response magnitude and selectivity of neurons are differentially modulated by experience across the olfactory-hippocampal pathway.

Overall, this work represents a valuable multi-region data set of odor-evoked neural activity. However, limitations in the interpretability of odor experience of the behavioral paradigm, and limitations in experimental design and analysis, restrict the conclusions that can be drawn from this study.

Reviewer #1 (Public review):

Summary:

The study by Gupta et al. investigates the role of mast cells (MCs) in tuberculosis (TB) by examining their accumulation in the lungs of M. tuberculosis-infected individuals, non-human primates, and mice. The authors suggest that MCs expressing chymase and tryptase contribute to the pathology of TB and influence bacterial burden, with MC-deficient mice showing reduced lung bacterial load and pathology.

Strengths:

(1) The study addresses an important and novel topic, exploring the potential role of mast cells in TB pathology.

(2) It incorporates data from multiple models, including human, non-human primates, and mice, providing a broad perspective on MC involvement in TB.

(3) The finding that MC-deficient mice exhibit reduced lung bacterial burden is an interesting and potentially significant observation.

Weaknesses:

(1) The evidence is inconsistent across models, leading to divergent conclusions that weaken the overall impact of the study.

(2) Key claims, such as MC-mediated cytokine responses and conversion of MC subtypes in granulomas, are not well-supported by the data presented.

(3) Several figures are either contradictory or lack clarity, and important discrepancies, such as the differences between mouse and human data, are not adequately discussed.

(4) Certain data and conclusions require further clarification or supporting evidence to be fully convincing.

Reviewer #2 (Public review):

Summary:

The submitted manuscript aims to characterize the role of mast cells in TB granuloma. The manuscript reports heterogeneity in mast cell populations present within the granulomas of tuberculosis patients. With the help of previously published scRNAseq data, the authors identify transcriptional signatures associated with distinct subpopulations.

Strengths:

(1) The authors have carried out a sufficient literature review to establish the background and significance of their study.

(2) The manuscript utilizes a mast cell-deficient mouse model, which demonstrates improved lung pathology during Mtb infection, suggesting mast cells as a potential novel target for developing host-directed therapies (HDT) against tuberculosis.

Weaknesses:

(1) The manuscript requires significant improvement, particularly in the clarity of the experimental design, as well as in the interpretation and discussion of the results. Enhanced focus on these areas will provide better coherence and understanding for the readers.

(2) Throughout the manuscript, the authors have mislabelled the legends for WT B6 mice and mast cell-deficient mice. As a result, the discussion and claims made in relation to the data do not align with the corresponding graphs (Figure 1B, 3, 4, and S2). This discrepancy undermines the accuracy of the conclusions drawn from the results.

(3) The results discussed in the paper do not add a significant novel aspect to the field of tuberculosis, as the majority of the results discussed in Figure 1-2 are already known and are a re-validation of previous literature.

(4) The claims made in the manuscript are only partially supported by the presented data. Additional extensive experiments are necessary to strengthen the findings and enhance the overall scientific contribution of the work.

Reviewer #1 (Public review):

Summary:

In this manuscript, BOUTRY et al examined a cnidarian Hydra model system where spontaneous tumors manifest in laboratory settings, and lineages featuring vertically transmitted neoplastic cells (via host budding) have been sustained for over 15 years. They observed that hydras harboring long-term transmissible tumors exhibit an unexpected augmentation in tentacle count. In addition, the presence of extra tentacles, enhancing the host's foraging efficiency, correlated with an elevated budding rate, thereby promoting tumor transmission vertically. This study provided the evidence that tumors, akin to parasitic entities, can also exert control over their hosts.

Strengths:

The manuscript is well-written, and the phenotype is intriguing.

Reviewer #2 (Public review):

Background and Summary: 

This study addresses the intriguing question of whether and how tumours can develop in the freshwater polyp hydra and how they influence the fitness of the animals. Hydra is notable for its significant morphogenetic plasticity and nearly unlimited capacity for regeneration. While its growth through asexual reproduction (budding) and the associated processes of pattern formation have been extensively studied at the cellular level, the occurrence of tumours was only recently described in two strains of Hydra oligactis (Domazet-Lošo et al, 2014). Here, tumour-like tissue bulges formed within the ectodermal epithelial layer and contained increased numbers of interstitial cell-like cells which exhibited female germline markers, but none specific for somatic derivatives of interstitial stem cells (e.g., nematocytes, neurons or glandular cells). It seems likely that the cellular basis of these malformations is a misregulation of oogenesis. In wild-type polyps, interstitial-cell-related germline precursors give rise to oocytes and nurse cells, which are subsequently phagocytosed by the growing egg cell. By comparison, in the mutant strains, this uptake is disturbed, but the homeostasis between germline cells and epithelial cells must remain functional enabling further growth pattern formation in hydra. Determining whether this differentiation arrest constitutes a neoplasm also remains a challenge. 

Clonal lines of both strains have been maintained in the laboratory for years and have also been used by Boutry and colleagues. They published two further papers on the ecological and evolutionary aspects of hydra tumour formation (Boutry et al 2022, 2023), which is also the focus of this manuscript. In their paper, the authors demonstrate an increase in the number of tentacles when "tumour tissue" was transplanted to intact gastric tissue of wildtype and mutant strains. While the impact on tentacle formation is relatively modest, small, it indicates a potential influence on the cross-talk between epithelial and interstitial cells in growth control (proportion regulation). The presented data are of interest, although the underlying molecular processes remain to be demonstrated. The authors offer a different interpretation. They conclude that this growth pattern (increased number of tentacles) is correlated with "reducing the burden on the host by (over-) compensating for the reproductive costs of tumours" and claim that "transmissible tumours in hydra have evolved strategies to manipulate the phenotype of their host". 

Strength <br /> The question of whether and how tumours can develop in simple systems, here the freshwater polyp hydra, is of general interest. The authors describe transplantation experiments by using mutant strains that indicate an influence of tumour-like malformation on pattern formation. The experiments also suggest an interaction between epithelial cells and germline cells during oogenesis, interfering with the homeostatic growth control between the cell lineages. 

Weaknesses <br /> Although it is stimulating to consider a fresh perspective from other disciplines (here, ecological and evolutionary aspects), it appears that this interpretation of the data (reducing the burden on the host by (over-) compensating for the reproductive costs of tumours) is somewhat beyond what can be reasonably inferred from the evidence presented. It is essential, particularly in the context of evolutionary biology, to conduct further analysis of the underlying cell biology of these intriguing mutant hydra strains. Such cellular analysis is a relatively straightforward approach that could provide a mechanistic understanding of the phenomenon described by the authors.

Reviewer #3 (Public review):

In this study, O'Brien et al. address the need for scalable and cost-effective approaches to finding lead compounds for the treatment of the growing number of Mendelian diseases. They used state-of-the-art phenotypic screening based on an established high-dimensional phenotypic analysis pipeline in the nematode C. elegans.

First, a panel of 25 C. elegans models was created by generating CRISPR/Cas9 knock-out lines for conserved human disease genes. These mutant strains underwent behavioral analysis using the group's published methodology. Clustering analysis revealed common features for genes likely operating in similar genetic pathways or biological functions. The study also presents results from a more focused examination of ciliopathy disease models.

Subsequently, the study focuses on the NALCN channel gene family, comparing the phenotypes of mutants of nca-1, unc-77, and unc-80. This initial characterization identifies three behavioral parameters that exhibit significant differences from the wild type and could serve as indicators for pharmacological modulation.

As a proof-of-concept, O'Brien et al. present a drug repurposing screen using an FDA-approved compound library, identifying two compounds capable of rescuing the behavioral phenotype in a model with UNC80 deficiency. The relatively short time and low cost associated with creating and phenotyping these strains suggest that high-throughput worm tracking could serve as a scalable approach for drug repurposing, addressing the multitude of Mendelian diseases. Interestingly, by measuring a wide range of behavioural parameters, this strategy also simultaneously reveals deleterious side effects of tested drugs that may confound the analysis.

Considering the wealth of data generated in this study regarding important human disease genes, it is regrettable that the data is not made accessible to researchers less versed in data analysis methods. This diminishes the study's utility. It would have a far greater impact if an accessible and user-friendly online interface were established to facilitate data querying and feature extraction for specific mutants. This would empower researchers to compare their findings with the extensive dataset created here.

Another technical limitation of the study is the use of single alleles. Large deletion alleles were generated by CRISPR/Cas9 gene editing. At first glance, this seems like a good idea because it limits the risk that background mutations, present in chemically-generated alleles, will affect behavioral parameters. However, these large deletions can also remove non-coding RNAs or other regulatory genetic elements, as found, for example, in introns. Therefore, it would be prudent to validate the behavioral effects by testing additional loss-of-function alleles produced through early stop codons or targeted deletion of key functional domains.

Comments on revisions:

In this final round of revisions, the authors have improved their manuscript and provide useful information about analysis procedures and code and updated figures.

Reviewer #1 (Public review):

This paper examines the role of MLCK (myosin light chain kinase) and MLCP (myosin light chain phosphatase) in axon regeneration. Using loss-of-function approaches based on small molecule inhibitors and siRNA knockdown, the authors explore axon regeneration in cell culture and in animal models from central and peripheral nervous systems. Their evidence shows that MLCK activity facilitates axon extension/regeneration, while MLCP prevents it.

Major concerns:

(1) In the title, authors indicate that the observed effects from loss-of-function of MLCK/MLCP take place via F-actin redistribution in the growth cone. However, there are no experiments showing a causal effect between changes in axon growth mediated by MLCK/MLCP and F-actin redistribution.

(2) The author combines MLCK inhibitors with Bleb (Figure 6), trying to verify if both pairs of inhibitors act on the same target/pathway. MLCK may regulate axon growth independent of NMII activity. However, this has very important implications for the understanding not only on how NMII works and affects axon extension, but also in trying to understand what MLCP is doing. One wonders if MLCP actions, which are opposite of MLCK, also independent of NMII activity? The authors, in the discussion section, try to find an explanation for this finding, but I consider it fails since the whole rationale of the manuscript is still around how MLCK and MLCP affect NMII phosphorylation.

What follows is a discussion of the merits and limitations of different claims of the manuscript in light of the evidence presented.

(1) Using western blot and immunohistochemical analyses, authors first show that MLCK expression is increased in DRG sensory neurons following peripheral axotomy, concomitant to an increase in MLC phosphorylation, suggesting a causal effect (Figure 1). The authors claim that it is common that axon growth-promoting genes are upregulated. It would have been interesting at this point to study in this scenario the regulation of MLCP.

(2) Using DRG cultures and sciatic nerve crush in the context of MLCK inhibition (ML-7) and down-regulation, authors conclude that MLCK activity is required for mammalian peripheral axon regeneration both in vitro and in vivo (Figure 2). In parallel, the authors show that these treatments affect as expected the phosphorylation levels of MLC.

The in vitro evidence is of standard methods and convincing. However, here, as well as in all other experiments using siRNAs, no Control siRNAs were used. Authors do show that the target protein is downregulated, and they can follow transfected cells with GFP. Still, it should be noted that the standard control for these experiments has not been done.

(3) The authors then examined the role of the phosphatase MLCP in axon growth during regeneration. The authors first use a known MLCP blocker, phorbol 12,13-dibutyrate (PDBu), to show that is able to increase the levels of p-MLC, with a concomitant increase in the extent of axon regrowth of DRG neurons, both in permissive as well as non-permissive substrates. The authors repeat the experiments using the knockdown of MYPT1, a key component of the MLC-phosphatase, and again can observe a growth-promoting effect (Figure 3).

The authors further show evidence for the growth-enhancing effect in vivo, in nerve crush experiments. The evidence in vivo deserves more evidence and experimental details (see comment 2). A key weakness of the data was mentioned previously: no control siARN was used.

(4) In the next set of experiments (presented in Figure 4) authors extend the previous observations in primary cultures from the CNS. For that, they use cortical and hippocampal cultures, and pharmacological and genetic loss-of-function using the above-mentioned strategies. The expected results were obtained in both CNS neurons: inhibition or knockdown of the kinase decreases axon growth, whereas inhibition or knockdown of the phosphatase increases growth. A main weakness in this set is that drugs were used from the beginning of the experiment, and hence, they would also affect axon specification. As pointed in Materials and Method (lines 143-145) authors counted as "axons" neurites longer than twice the diameter of the cell soma, and hence would not affect the variable measured. In any case, to be sure one is only affecting axon extension in these cells, the drugs should have been used after axon specification and maturation, which occurs at least after 5 DIV.

(5) In Figure 7, the authors a local cytoskeletal action of the drug, but the evidence provided does not differentiate between a localized action of the drugs and a localized cell activity.

References:

(1) Eun-Mi Hur 1, In Hong Yang, Deok-Ho Kim, Justin Byun, Saijilafu, Wen-Lin Xu, Philip R Nicovich, Raymond Cheong, Andre Levchenko, Nitish Thakor, Feng-Quan Zhou. 2011. Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):5057-62. doi: 10.1073/pnas.1011258108.

(2) Garrido-Casado M, Asensio-Juárez G, Talayero VC, Vicente-Manzanares M. 2024. Engines of change: Nonmuscle myosin II in mechanobiology. Curr Opin Cell Biol. 2024 Apr;87:102344. doi: 10.1016/j.ceb.2024.102344.

(3) Karen A Newell-Litwa 1, Rick Horwitz 2, Marcelo L Lamers. 2015. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015 Dec;8(12):1495-515. doi: 10.1242/dmm.022103.

Reviewer #2 (Public review):

Summary:

Saijilafu et al. demonstrate that MLCK/MLCP proteins promote axonal regeneration in both the central nervous system (CNS) and peripheral nervous system (PNS) using primary cultures of adult DRG neurons, hippocampal and cortical neurons, as well as in vivo experiments involving sciatic nerve injury, spinal cord injury, and optic nerve crush. The authors show that axon regrowth is possible across different contexts through genetic and pharmacological manipulation of these proteins. Additionally, they propose that MLCK/MLCP may regulate F-actin reorganization in the growth cone, which is significant as it suggests a novel strategy for promoting axonal regeneration.

Strengths:

This manuscript presents a wide range of experimental models to address its hypothesis and biological question. Notably, the use of multiple in vivo models significantly enhances the overall validity of the study.

Weaknesses:

-The authors previously published that blocking myosin II activity stimulates axonal growth and that MLCK activates myosin II. The present work shows that inhibiting MLCK blocks axonal regeneration while blocking MLCP (the protein that dephosphorylates myosin II) produces the opposite effect. Although this contradiction is discussed, no new evidence has been added to the manuscript to clarify this mechanism or address the remaining questions. Critical unresolved questions include: what happens to myosin II expression when both MLCK and MLCP are inhibited? If MLCK/MLCP are acting through an independent mechanism, what would that mechanism be?<br /> -In the discussion, the authors mention the existence of two myosin II isoforms with opposing effects on axonal growth. Still, there is no evidence in the manuscript to support this point.<br /> -It is also unclear how MLCK/MLCP acts on the actin cytoskeleton. The authors suggest that proteins such as ADF/cofilin, Arp 2/3, Eps8, Profilin, Myosin II, and Myosin V could regulate changes in F-actin dynamics. However, this study provides no experimental evidence to determine which proteins may be involved in the mechanism.

Reviewer #1 (Public review):

Summary:

The work by Chuong et al. provides important new insights into the contribution of different molecular mechanisms in the dynamics of CNV formation. It will be of interest to anyone curious about genome architecture and evolution from yeast biologists to cancer researchers studying genome rearrangements.

Strengths:

Their results are especially striking in that the "simplest" mechanism of GAP1 amplification (non-allelic homologous recombination between the flanking Ty-LTR elements) is not the most common route taken by the cells, emphasizing the importance of experimentally testing what might seem on the surface to be obvious outcome. One of the important developments of their work is the use of their neural network simulation-based inference (nnSBI) model to derive rates of amplicon formation and their fitness effects.

Weaknesses:

The nnSBI model that derives rates of amplicon formation and fitness is still opaque to this reviewer. All of the other criticisms made in the first review have been clarified/corrected in this much-improved version of the manuscript.

Reviewer #2 (Public review):

Summary:

This study examines how local DNA features around the amino acid permease gene GAP1 influence adaptation to glutamine limited conditions through changes in GAP1 Copy Number Variation (CNV). The study is well motivated by the observation of numerous CNVs documented in many organisms, but difficulty in distinguishing the mechanisms by which they are formed, and whether or how local genomic elements influence their formation. The main finding is convincing and is that a nearby Autonomous Replicating Sequence (ARS) influences the formation of GAP1 CNVs and this is consistent with a predominate mechanism of Origin Dependent Inverted Repeat Amplification (ODIRA). These results along with finding and characterizing other mechanisms of GAP1 CNV formation will be of general interest to those studying CNVs in natural systems, experimental evolution and in tumor evolution. While the results are limited to a single CNV of interest (GAP1), the carefully controlled experimental design and quantification of CNV formation will provide a useful guide to studying other CNVs and CNVs in other organisms.

Strengths:

The study was designed to examine the effects of two flanking genomic features next to GAP1 on CNV formation and adaptation during experimental evolution. This was accomplished by removing two Long Terminal Repeats (LTRs), removing a downstream ARS, and removing both LTRs and the ARS. Although there was some heterogeneity among replicates, later shown to include the size and breakpoints of the CNV and the presence of an unmarked CNV, both marker assisted tracking of CNV formation and modeling of CNV rate and fitness effects showed that deletion of the ARS caused a clear difference compared to the control and the LTR deletion.

The consequence of deletion of local features (LTR and ARS) was quantified by genome sequencing of adaptive clones to identify the CNV size, copy number and infer the mechanism of CNV formation. This greatly added value to the study as it showed that i) ODIRA was the most common mechanism but ODIRA is enhanced by a local ARS, ii) non-allelic homologous recombination (NAHR) is also used but depends on LTRs, and iii) de novo insertion of transposable elements mediate NAHR in strains with both ARS and LTR deletions. Together, these results show how local features influence the mechanism of CNV formation, but also how alternative mechanism can substitute when primary ones are unavailable.

Weaknesses:

The CNV mutation rate and effect on fitness are hard to disentangle. The frequency of the amplified GFP provides information about mutation rate differences as well as fitness differences. The data and analysis show that each evolved population has multiple GAP1 CNV lineages within it, with some being unmarked by GFP. Thus, estimates of CNV fitness are more of a composite view of all CNV amplifications increasing in frequency during adaptation. Another unknown but potential complication is whether the local (ARS, LTR) deletions influence GAP1 expression and thus the fitness gain of GAP1 CNVs. The neural network simulation based inference does a good job at estimating both mutation rates and fitness effects, while also accounting for unmarked CNVs. However, the model does not account for population heterogeneity of CNVs and their fitness effects. Despite these limitations of distinguishing mutation rate and fitness differences, the authors' conclusions are well supported in that the LTR and ARS deletions have a clear impact on the CNV mediated evolutionary outcome and the mechanism of CNV formation.

Reviewer #4 (Public review):

Summary:

Various 'simple' models are used to mechanistically explain the formation of genomic rearrangements, often based on local sequence elements. Here the authors show these models to be lacking for the well characterised GAP1 locus as, although predicted events are observed at reasonable frequency, mutating relevant local sequence elements has surprisingly little impact on the emergence of GAP1 CNV. Rather, a similar set of mechanisms occur (at in some cases somewhat lower frequency) using different genomic elements, the outcome being that that CNV frequency is largely independent of local genomic elements, although this does of course strongly influence the actual structure of the CNVs.

Strengths:

This is a very thorough study of a very complex system.

Weaknesses:

There are limitations as previous reviews have noted, but these are well justified in the revised text and rebuttal

Reviewer #1 (Public review):

Summary:

Motivated by the existence of different behavioral strategies (e.g. model-based vs. model-free), and potentially different neural circuits that underlie them, Venditto et al. introduce a new approach for inferring which strategies animals are using from data. In particular, they extend the mixture of agents (MoA) framework to accommodate the possibility that the weighting among different strategies might change over time. These temporal dynamics are introduced via a hidden Markov model (HMM), i.e. with discrete state transitions. These state transition probabilities and initial state probabilities are fit simultaneously along with the MoA parameters, which include decay/learning rate and mixture weightings, using the EM algorithm. The authors test their model on data from Miller et al., 2017, 2022, arguing that this formulation leads to (1) better fits and (2) improved interpretability over their original model, which did not include the HMM portion. Lastly, they claim that certain aspects of OFC firing are modulated by internal state as identified by the MoA-HMM.

Strengths:

The paper is very well written and easy to follow, especially for one with a significant modeling component. Furthermore, the authors do an excellent job explaining and then disentangling many threads that are often knotted together in discussions of animal behavior and RL: model-free vs. model-based choice, outcome vs. choice-focused, exploration vs. exploitation, bias, perserveration. Each of these concepts is quantified by particular parameters of their models. Model recovery (Fig. 3) is convincing post-revision and licenses their fits to animal behavior later. While the specific claims made about behavior and neural activity are not especially surprising (e.g. the animals begin a session, in which rare vs. common transitions are not yet known, in a more exploratory mode), the MoA-HMM framework seems broadly applicable to other tasks in the field and useful for the purpose of quantification here. Overall, I believe this paper is certainly worthy of publication in a journal like eLife.

Weaknesses:

I am pleased with the authors' responses to my initial comments, and I thank them for their efforts. My main note of caution to readers is just that when it comes to applying this method to neural data, the benefits may be subtle. On one extreme, it may be possible to capture many of these effects simply by explicitly modeling time, although the authors do a good job showing that they can beat this benchmark in their case. On the other extreme, there may be multiple switches that cannot simply be a monotonic time effect, but these might be at a faster timescale than can be easily captured in this model (in Fig. 7Aii, for example, there is still lots of variance unexplained by the latent state). Quantitative justification will be required for using this model over simpler alternatives, but again, I commend the authors for providing that justification in this paper.

Reviewer #2 (Public review):

Summary:

This is an interesting and well-performed study that develops a new modeling approach (MoA-HMM) to simultaneously characterize reinforcement learning parameters of different RL agents, as well as latent behavioral states that differ in the relative contributions of those agents to the animal's choices. They performed this study in rats trained to perform the two-step task. While the major advance of the paper is developing and rigorously validating this novel technical approach, there are also a number of interesting conceptual advances. For instance, humans performing the two-step task are thought to exhibit a trade-off between model-free and model-based strategies. However, the MoA-HMM did not reveal such a trade-off in the rats, but instead suggested a trade-off between model-based exploratory vs. exploitative strategies. Additionally, the firing rates of neurons in the orbitofrontal cortex (OFC) reflected latent behavioral states estimated from the HMM, suggesting that (1) characterizing dynamic behavioral strategies might help elucidate neural dynamics supporting behavior, and (2) OFC might reflect the contributions of one or a subset of RL agents that are preferentially active or engaged in particular states identified by the HMM.

Strengths:

The claims were generally well-supported by the data. The model was validated rigorously, and was used to generate and test novel predictions about behavior and neural activity in OFC. The approach is likely to be generally useful for characterizing dynamic behavioral strategies.

Reviewer #1 (Public review):

Summary:

Wang et al. identify Hamlet, a PR-containing transcription factor, as a master regulator of reproductive development in Drosophila. Specifically, the fusion between the gonad and genital disc is necessary for the development of continuous testes and seminal vesicle tissue essential for fertility. To do this, the authors generate novel Hamlet null mutants by CRISPR/Cas9 gene editing and characterize the morphological, physiological, and gene expression changes of the mutants using immunofluorescence, RNA-seq, cut-tag, and in-situ analysis. Thus, Hamlet is discovered to regulate a unique expression program, which includes Wnt2 and Tl, that is necessary for testis development and fertility.

Strengths:

This is a rigorous and comprehensive study that identifies the Hamlet-dependent gene expression program mediating reproductive development in Drosophila. The Hamlet transcription targets are further characterized by Gal4/UAS-RNAi confirming their role in reproductive development. Finally, the study points to a role for Wnt2 and Tl as well as other Hamlet transcriptionally regulated genes in epithelial tissue fusion.

Weaknesses:

The image resolution and presentation of figures is a major issue in this study. As a non-expert, it is nearly impossible to see the morphological changes as described in the results. Quantification of all cell biological phenotypes is also lacking therefore reducing the impact of this study to those familiar with tissue fusion events in Drosophila development.

Reviewer #2 (Public review):

Strengths:

Wang and colleagues successfully uncovered an important function of the Drosophila PRDM16/PRDM3 homolog Hamlet (Ham) - a PR domain-containing transcription factor with known roles in the nervous system in Drosophila. To do so, they generated and analyzed new mutants lacking the PR domain, and also employed diverse preexisting tools. In doing so, they made a fascinating discovery: They found that PR-domain containing isoforms of ham are crucial in the intriguing development of the fly genital tract. Wang and colleagues found three distinct roles of Ham: (1) specifying the position of the testis terminal epithelium within the testis, (2) allowing normal shaping and growth of the anlagen of the seminal vesicles and paragonia and (3) enabling the crucial epithelial fusion between the seminal vesicle and the testis terminal epithelium. The mutant blocks fusion even if the parts are positioned correctly. The last finding is especially important, as there are few models allowing one to dissect the molecular underpinnings of heterotypic epithelial fusion in development. Their data suggest that they found a master regulator of this collective cell behavior. Further, they identified some of the cell biological players downstream of Ham, like for example E-Cadherin and Crumbs. In a holistic approach, they performed RNAseq and intersected them with the CUT&TAG-method, to find a comprehensive list of downstream factors directly regulated by Ham. Their function in the fusion process was validated by a tissue-specific RNAi screen. Meticulously, Wang and colleagues performed multiplexed in situ hybridization and analyzed different mutants, to gain a first understanding of the most important downstream pathways they characterized, which are Wnt2 and Toll.

This study pioneers a completely new system. It is a model for exploring a process crucial in morphogenesis across animal species, yet not well understood. Wang and colleagues not only identified a crucial regulator of heterotypic epithelial fusion but took on the considerable effort of meticulously pinning down functionally important downstream effectors by using many state-of-the-art methods. This is especially impressive, as the dissection of pupal genital discs before epithelial fusion is a time-consuming and difficult task. This promising work will be the foundation future studies build on, to further elucidate how this epithelial fusion works, for example on a cell biological and biomechanical level.

Weaknesses:

The developing testis-genital disc system has many moving parts. Myotube migration was previously shown to be crucial for testis shape. This means, that there is the potential of non-tissue autonomous defects upon knockdown of genes in the genital disc or the terminal epithelium, affecting myotube behavior which in turn affects fusion, as myotubes might create the first "bridge" bringing the epithelia together. The authors clearly showed that their driver tools do not cause expression in myoblasts/myotubes, but this does not exclude non-tissue autonomous defects in their RNAi screen. Nevertheless, this is outside the scope of this work.

However, one point that could be addressed in this study: the RNAseq and CUT&TAG experiments would profit from adding principal component analyses, elucidating similarities and differences of the diverse biological and technical replicates.

Reviewer #1 (Public review):

Summary:

The authors quantified information in gesture and speech, and investigated the neural processing of speech and gestures in pMTG and LIFG, depending on their informational content, in 8 different time-windows, and using three different methods (EEG, HD-tDCS and TMS). They found that there is a time-sensitive and staged progression of neural engagement that is correlated with the informational content of the signal (speech/gesture).

Strengths:

A strength of the paper is that the authors attempted to combine three different methods to investigate speech-gesture processing.

Weaknesses:

(1) One major issue is that there is a tight anatomical coupling between pMTG and LIFG. Stimulating one area could therefore also result in stimulation of the other area (see Silvanto and Pascual-Leone, 2008). I therefore think it is very difficult to tease apart the contribution of these areas to the speech-gesture integration process, especially considering that the authors stimulate these regions in time windows that are very close to each other in both time and space (and the disruption might last longer over time).

(2) Related to this point, it is unclear to me why the HD-TDCS/TMS is delivered in set time windows for each region. How did the authors determine this, and how do the results for TMS compare to their previous work from 2018 and 2023 (which describes a similar dataset+design)? How can they ensure they are only targeting their intended region since they are so anatomically close to each other?

(3) As the EEG signal is often not normally distributed, I was wondering whether the authors checked the assumptions for their Pearson correlations. The authors could perhaps better choose to model the different variables to see whether MI/entropy could predict the neural responses. How did they correct the many correlational analyses that they have performed?

(4) The authors use ROIs for their different analyses, but it is unclear why and on the basis of what these regions are defined. Why not consider all channels without making them part of an ROI, by using a method like the one described in my previous comment?

(5) The authors describe that they have divided their EEG data into a "lower half" and a "higher half" (lines 234-236), based on entropy scores. It is unclear why this is necessary, and I would suggest just using the entropy scores as a continuous measure.

Reviewer #2 (Public review):

Summary:

The study is an innovative and fundamental study that clarified important aspects of brain processes for integration of information from speech and iconic gesture (i.e., gesture that depicts action, movement, and shape), based on tDCS, TMS, and EEG experiments. They evaluated their speech and gesture stimuli in information-theoretic ways and calculated how informative speech is (i.e., entropy), how informative gesture is, and how much shared information speech and gesture encode. The tDCS and TMS studies found that the left IFG and pMTG, the two areas that were activated in fMRI studies on speech-gesture integration in the previous literature, are causally implicated in speech-gesture integration. The size of tDC and TMS effects are correlated with the entropy of the stimuli or mutual information, which indicates that the effects stem from the modulation of information decoding/integration processes. The EEG study showed that various ERP (event-related potential, e.g., N1-P2, N400, LPC) effects that have been observed in speech-gesture integration experiments in the previous literature, are modulated by the entropy of speech/gesture and mutual information. This makes it clear that these effects are related to information decoding processes. The authors propose a model of how the speech-gesture integration process unfolds in time, and how IFG and pMTG interact with each other in that process.

Strengths:

The key strength of this study is that the authors used information theoretic measures of their stimuli (i.e., entropy and mutual information between speech and gesture) in all of their analyses. This made it clear that the neuro-modulation (tDCS, TMS) affected information decoding/integration and ERP effects reflect information decoding/integration. This study used tDCS and TMS methods to demonstrate that left IFG and pMTG are causally involved in speech-gesture integration. The size of tDCS and TMS effects are correlated with information-theoretic measures of the stimuli, which indicate that the effects indeed stem from disruption/facilitation of the information decoding/integration process (rather than generic excitation/inhibition). The authors' results also showed a correlation between information-theoretic measures of stimuli with various ERP effects. This indicates that these ERP effects reflect the information decoding/integration process.

Weaknesses:

The "mutual information" cannot fully capture the interplay of the meaning of speech and gesture. The mutual information is calculated based on what information can be decoded from speech alone and what information can be decoded from gesture alone. However, when speech and gesture are combined, a novel meaning can emerge, which cannot be decoded from a single modality alone. When example, a person produces a gesture of writing something with a pen, while saying "He paid". The speech-gesture combination can be interpreted as "paying by signing a cheque". It is highly unlikely that this meaning is decoded when people hear speech only or see gestures only. The current study cannot address how such speech-gesture integration occurs in the brain, and what ERP effects may reflect such a process. Future studies can classify different types of speech-gesture integration and investigate neural processes that underlie each type. Another important topic for future studies is to investigate how the neural processes of speech-gesture integration change when the relative timing between the speech stimulus and the gesture stimulus changes.

Reviewer #3 (Public review):

In this useful study, Zhao et al. try to extend the evidence for their previously described two-step model of speech-gesture integration in the posterior Middle Temporal Gyrus (pMTG) and Inferior Frontal Gyrus (IFG). They repeat some of their previous experimental paradigms, but this time quantifying Information-Theoretical (IT) metrics of the stimuli in a stroop-like paradigm purported to engage speech-gesture integration. They then correlate these metrics with the disruption of what they claim to be an integration effect observable in reaction times during the tasks following brain stimulation, as well as documenting the ERP components in response to the variability in these metrics.

The integration of multiple methods, like tDCS, TMS, and ERPs to provide converging evidence renders the results solid. However, their interpretation of the results should be taken with care, as some critical confounds, like difficulty, were not accounted for, and the conceptual link between the IT metrics and what the authors claim they index is tenuous and in need of more evidence. In some cases, the difficulty making this link seems to arise from conceptual equivocation (e.g., their claims regarding 'graded' evidence), whilst in some others it might arise from the usage of unclear wording in the writing of the manuscript (e.g. the sentence 'quantitatively functional mental states defined by a specific parser unified by statistical regularities'). Having said that, the authors' aim is valuable, and addressing these issues would render the work a very useful approach to improve our understanding of integration during semantic processing, being of interest to scientists working in cognitive neuroscience and neuroimaging.

The main hurdle to achieving the aims set by the authors is the presence of the confound of difficulty in their IT metrics. Their measure of entropy, for example, being derived from the distribution of responses of the participants to the stimuli, will tend to be high for words or gestures with multiple competing candidate representations (this is what would presumptively give rise to the diversity of responses in high-entropy items). There is ample evidence implicating IFG and pMTG as key regions of the semantic control network, which is critical during difficult semantic processing when, for example, semantic processing must resolve competition between multiple candidate representations, or when there are increased selection pressures (Jackson et al., 2021). Thus, the authors' interpretation of Mutual Information (MI) as an index of integration is inextricably contaminated with difficulty arising from multiple candidate representations. This casts doubt on the claims of the role of pMTG and IFG as regions carrying out gesture-speech integration as the observed pattern of results could also be interpreted in terms of brain stimulation interrupting the semantic control network's ability to select the best candidate for a given context or respond to more demanding semantic processing.

In terms of conceptual equivocation, the use of the term 'graded' by the authors seems to be different from the usage commonly employed in the semantic cognition literature (e.g., the 'graded hub hypothesis', Rice et al., 2015). The idea of a graded hub in the controlled semantic cognition framework (i.e., the anterior temporal lobe) refers to a progressive degree of abstraction or heteromodal information as you progress through the anatomy of the region (i.e., along the dorsal-to-ventral axis). The authors, on the other hand, seem to refer to 'graded manner' in the context of a correlation of entropy or MI and the change in the difference between Reaction Times (RTs) of semantically congruent vs incongruent gesture-speech. The issue is that the discourse through parts of the introduction and discussion seems to conflate both interpretations, and the ideas in the main text do not correspond to the references they cite. This is not overall very convincing. What is it exactly the authors are arguing about the correlation between RTs and MI indexes? As stated above, their measure of entropy captures the spread of responses, which could also be a measure of item difficulty (more diverse responses imply fewer correct responses, a classic index of difficulty). Capturing the diversity of responses means that items with high entropy scores are also likely to have multiple candidate representations, leading to increased selection pressures. Regions like pMTG and IFG have been widely implicated in difficult semantic processing and increased selection pressures (Jackson et al., 2021). How is this MI correlation evidence of integration that proceeds in a 'graded manner'? The conceptual links between these concepts must be made clearer for the interpretation to be convincing.

Reviewer #1 (Public review):

Summary:

The drug Ivermectin is used to effectively treat a variety of worm parasites in the world, however resistance to Ivermectin poses a rising challenge for this treatment strategy. In this study, the authors found that loss of the E3 ubiquitin ligase UBR-1 in the worm C. elegans results in resistance to Ivermectin. In particular, the authors found that ubr-1 mutants are resistant to the effects of Ivermectin on worm viability, body size, pharyngeal pumping, and locomotion. The authors previously showed that loss of UBR-1 disrupts homeostasis of the amino acid and neurotransmitter glutamate resulting in increased levels of glutamate in C. elegans. Here, the authors found that the sensitivity of ubr-1 mutants to Ivermectin can be restored if glutamate levels are reduced using a variety of different methods. Conversely, treating worms with exogenous glutamate to increase glutamate levels also results in resistance to Ivermectin supporting the idea that increased glutamate promotes resistance to Ivermectin. The authors found that the primary known targets of Ivermectin, glutamate-gated chloride channels (GluCls), are downregulated in ubr-1 mutants providing a plausible mechanism for why ubr-1 mutants are resistant to Ivermectin. Although it is clear that loss of GluCls can lead to resistance to Ivermectin, this study suggests that one potential mechanism to decrease GluCl expression is via disruption of glutamate homeostasis that leads to increased glutamate. This study suggests that if parasitic worms become resistant to Ivermectin due to increased glutamate, their sensitivity to Ivermectin could be restored by reducing glutamate levels using drugs such as Ceftriaxone in a combination drug treatment strategy.

Strengths:

(1) The use of multiple independent assays (i.e., viability, body size, pharyngeal pumping, locomotion, and serotonin-stimulated pharyngeal muscle activity) to monitor the effects of Ivermectin

(2) The use of multiple independent approaches (got-1, eat-4, ceftriaxone drug, exogenous glutamate treatment) to alter glutamate levels to support the conclusion that increased glutamate in ubr-1 mutants contributes to Ivermectin resistance.

Weaknesses:

(1) The primary target of Ivermectin is GluCls so it is not surprising that alteration of GluCl expression or function would lead to Ivermectin resistance.

(2) It remains to be seen what percent of Ivermectin-resistant parasites in the wild have disrupted glutamate homeostasis as opposed to mutations that more directly decrease GluCl expression or function.

Reviewer #2 (Public review):

Summary:

The authors provide a very thorough investigation of the role of UBR-1 in anthelmintic resistance using the non-parasitic nematode, C. elegans. Anthelmintic resistance to macrocyclic lactones is a major problem in veterinary medicine and likely just a matter of time until resistance emerges in human parasites too. Therefore, this study providing novel insight into the mechanisms of ivermectin resistance is particularly important and significant.

Strengths:

The authors use very diverse technologies (behavior, genetics, pharmacology, genetically encoded reporters) to dissect the role of UBR-1 in ivermectin resistance. Deploying such a comprehensive suite of tools and approaches provides exceptional insight into the mechanism of how UBR-1 functions in terms of ivermectin resistance.

Weaknesses:

I do not see any major weaknesses in this study. My only concern is whether the observations made by the authors would translate to any of the important parasitic helminths in which resistance has naturally emerged in the field. This is always a concern when leveraging a non-parasitic nematode to shed light on a potential mechanism of resistance of parasitic nematodes, and I understand that it is likely beyond the scope of this paper to test some of their results in parasitic nematodes.

Reviewer #3 (Public review):

Summary:

Li et al propose to better understand the mechanisms of drug resistance in nematode parasites by studying mutants of the model roundworm C. elegans that are resistant to the deworming drug ivermectin. They provide compelling evidence that loss-of-function mutations in the E3 ubiquitin ligase encoded by the UBR-1 gene make worms resistant to the effects of ivermectin (and related compounds) on viability, body size, pharyngeal pumping rate, and locomotion and that these mutant phenotypes are rescued by a UBR-1 transgene. They propose that the mechanism is resistance is indirect, via the effects of UBR-1 on glutamate production. They show mutations (vesicular glutamate transporter eat-4, glutamate synthase got-1) and drugs (glutamate, glutamate uptake enhancer ceftriaxone) affecting glutamate metabolism/transport modulate sensitivity to ivermectin in wild-type and ubr-1 mutants. The data are generally consistent with greater glutamate tone equating to ivermectin resistance. Finally, they show that manipulations that are expected to increase glutamate tone appear to reduce expression of the targets of ivermectin, the glutamate-gated chloride channels, which is known to increase resistance.

There is a need for genetic markers of ivermectin resistance in livestock parasites that can be used to better track resistance and to tailor drug treatment. The discovery of UBR-1 as a resistance gene in C. elegans will provide a candidate marker that can be followed up in parasites. The data suggest Ceftriaxone would be a candidate compound to reverse resistance.

Strengths:

The strength of the study is the thoroughness of the analysis and the quality of the data. There can be little doubt that ubr-1 mutations do indeed confer ivermectin resistance. The use of both rescue constructs and RNAi to validate mutant phenotypes is notable. Further, the variety of manipulations they use to affect glutamate metabolism/transport makes a compelling argument for some kind of role for glutamate in resistance.

Weaknesses:

The proposed mechanism of ubr-1 resistance i.e.: UBR-1 E3 ligase regulates glutamate tone which regulates ivermectin receptor expression, is broadly consistent with the data but somewhat difficult to reconcile with the specific functions of the genes regulating glutamatergic tone. Ceftriaxone and eat-4 mutants reduce extracellular/synaptic glutamate concentrations by sequestering available glutamate in neurons, suggesting that it is extracellular glutamate that is important. But then why does rescuing ubr-1 specifically in the pharyngeal muscle have such a strong effect on ivermectin sensitivity? Is glutamate leaking out of the pharyngeal muscle into the extracellular space/synapse? Is it possible that UBR-1 acts directly on the avr-15 subunit, both of which are expressed in the muscle, perhaps as part of a glutamate sensing/homeostasis mechanism?

The use of single ivermectin dose assays can be misleading. A response change at a single dose shows that the dose-response curve has shifted, but the response is not linear with dose, so the degree of that shift may be difficult to discern and may result from a change in slope but not EC50.

Similarly, in Figure 3C, the reader is meant to understand that eat-4 mutant is epistatic to ubr-1 because the double mutant has a wild-type response to ivermectin. But eat-4 alone is more sensitive, so (eyeballing it and interpolating) the shift in EC50 caused by the ubr-1 mutant in a wild type background appears to be the same as in an eat-4 background, so arguably you are seeing an additive effect, not epistasis. For the above reasons, it would be desirable to have results for rescuing constructs in a wild-type background, in addition to the mutant background.

The added value of the pumping data in Figure 5 (using calcium imaging) over the pump counts (from video) in Figure 1G, Figure 2E, F, K, & Figure 3D, H is not clearly explained. It may have to do with the use of "dissected" pharynxes, the nature/advantage of which is not sufficiently documented in the Methods/Results.

Reviewer #1 (Public review):

Summary:

IPF is a disease lacking regressive therapies which has a poor prognosis, and so new therapies are needed. This ambitious phase 1 study builds on the authors' 2024 experience in Sci Tran Med with positive results with autologous transplantation of P63 progenitor cells in patients with COPD. The current study suggests that P63+ progenitor cell therapy is safe in patients with ILD. The authors attribute this to the acquisition of cells from a healthy upper lobe site, removed from the lung fibrosis. There are currently no cell-based therapies for ILD and in this regard the study is novel with important potential for clinical impact if validated in Phase 2 and 3 clinical trials.

Strengths:

This study addresses the need for an effective therapy for interstitial lung disease. It offers good evidence that the cells used for therapy are safe. In so doing it addresses a concern that some P63+ progenitor cells may be proinflammatory and harmful, as has been raised in the literature (articles which suggested some P63+ cells can promote honeycombing fibrosis; references 26 &35). The authors attribute the safety they observed (without proof) to the high HOPX expression of administered cells (a marker found in normal Type 1 AECs. The totality of the RNASeq suggests the cloned cells are not fibrogenic. They also offer exploratory data suggesting a relationship between clone roundness and PFT parameters (and a negative association between patient age and clone roundness).

Weaknesses:

The authors can conclude they can isolate, clone, expand, and administer P63+ progenitor cells safely; but with the small sample size and lack of a placebo group, no efficacy should be implied.

Specific points:

(1) The authors acknowledge most study weaknesses including the lack of a placebo group and the concurrent COVID-19 in half the subjects (the high-dose subjects). They indicate a phase 2 trial is underway to address these issues.

(2) The authors suggest an efficacy signal on pages 18 (improvement in 2 subjects' CT scans) and 21 (improvement in DLCO) but with such a small phase 1 study and such small increases in DLCO (+5.4%) the authors should refrain from this temptation (understandable as it is).

(3) Likewise most CT scans were unchanged and those that improved were in the mid-dose group (albeit DLCO improved in the 2 patients whose CT scans improved).

(4) The authors note an impressive 58m increase in 6MWTD in the high-dose group but again there is no placebo group, and the low-dose group has no net change in 6MWTD at 24 weeks.

(5) I also raise the question of the enrollment criteria in which 5 patients had essentially normal DLCO/VA values. In addition there is no discussion as to whether the transplanted stem cells are retained or exert benefit by a paracrine mechanism (which is the norm for cell-based therapies).

Reviewer #2 (Public review):

Summary:

This manuscript describes a first-in-human clinical trial of autologous stem cells to address IPF. The significance of this study is underscored by the limited efficacy of standard-of-care anti-fibrotic therapies and increasing knowledge of the role p63+ stem cells in lung regeneration in ARDS. While models of acute lung injury and p63+ stem cells have benefited from widespread and dynamic DAD and immune cell remodeling of damaged tissue, a key question in chronic lung disease is whether such cells could contribute to the remodeling of lung tissue that may be devoid of acute and dynamic injury. A second question is whether normal regions of the lung in an otherwise diseased organ can be identified as a source of "normal" p63+ stem cells, and how to assess these stem cells given recently identified p63+ stem cell variants emerging in chronic lung diseases including IPF. Lastly, questions of feasibility, safety, and efficacy need to be explored to set the foundation for autologous transplants to meet the huge need in chronic lung disease. The authors have addressed each of these questions to different extents in this initial study, which has yielded important if incomplete information for many of them.

Strengths:

As with a previous study from this group regarding autologous stem cell transplants for COPD (Ref. 24), they have shown that the stem cells they propagate do not form colonies in soft agar or cancers in these patients. While a full assessment of adverse events was confounded by a wave of Covid19 infections in the study participants, aside from brief fevers it appears these transplants are tolerated by these patients.

Weaknesses:

The source of stem cells for these autologous transplants is generally bronchoscopic biopsies/brushings from 5th-generation bronchi. Although stem cells have been cloned and characterized from nasal, tracheal, and distal airway biopsies, the systematic cloning and analysis of p63+ stem cells across the bronchial generations is less clear. For instance, p63+ stem cells from the nasal and tracheal mucosa appear committed to upper airway epithelia marked by 90% ciliated cells and 10% goblet cells (Kumar et al., 2011. Ref. 14). In contrast, p63+ stem cells from distal lung differentiate to epithelia replete with Club, AT2, and AT1 markers. The spectrum of p63+ stem cells in the normal bronchi of any generation is less studied. In the present study, cells are obtained by bronchoscopy from 3-5 generation bronchi and expanded by in vitro propagation. Single-cell RNAseq identifies three clusters they refer to as C1, C2, and C3, with the major C1 cluster said to have characteristics of airway basal cells and C2 possibly the same cells in states of proliferation. Perhaps the most immediate question raised by these data is the nature of the C1/C2 cells. Whereas they are clearly p63/Krt5+ cells as are other stem cells of the airways, do they display differentiation character of "upper airway" marked by ciliated/goblet cell differentiation or those of the lung marked by AT2 and AT1 fates? This could be readily determined by 3-D differentiation in so-called air-liquid interface cultures pioneered by cystic fibrosis investigators and should be done as it would directly address the validity of the sourcing protocol for autologous cells for these transplants. This would more clearly link the present study with a previous study from the same investigators (Shi et al., 2019, Ref. 9) whereby distal airway stem cells mitigated fibrosis in the murine bleomycin model. The authors should also provide methods by which the autologous cells are propagated in vitro as these could impact the quality and fate of the progenitor cells prior to transplantation.

The authors should also make a more concerted effort to compare Clusters 1, 2, and 3 with the variant stem cell identified in IPF (Wang et al., 2023, Ref. 27). While some of the markers are consistent with this variant stem cell population, others are not. A more detailed informatics analysis of normal stem cells of the airways and any variants reported could clarify whether the bronchial source of autologous stem cells is the best route to these transplants.

Other than these issues the authors should be commended for these first-in-human trials for this important condition.

Joint Public Review:

In this work, the authors develop a new computational tool, DeepTX, for studying transcriptional bursting through the analysis of single-cell RNA sequencing (scRNA-seq) data using deep learning techniques. This tool aims to describe and predict the transcriptional bursting mechanism, including key model parameters and the steady-state distribution associated with the predicted parameters. By leveraging scRNA-seq data, DeepTX provides high-resolution transcriptional information at the single-cell level, despite the presence of noise that can cause gene expression variation. The authors apply DeepTX to DNA damage experiments, revealing distinct cellular responses based on transcriptional burst kinetics. Specifically, IdU treatment in mouse stem cells increases burst size, promoting differentiation, while 5FU affects burst frequency in human cancer cells, leading to apoptosis or, depending on the dose, to survival and potential drug resistance. These findings underscore the fundamental role of transcriptional burst regulation in cellular responses to DNA damage, including cell differentiation, apoptosis, and survival. Although the insights provided by this tool are mostly well supported by the authors' methods, certain aspects would benefit from further clarification.

The strengths of this paper lie in its methodological advancements and potential broad applicability. By employing the DeepTXSolver neural network, the authors efficiently approximate stationary distributions of mRNA counts through a mixture of negative binomial distributions, establishing a simple yet accurate mapping between the kinetic parameters of the mechanistic model and the resulting steady-state distributions. This innovative use of neural networks allows for efficient inference of kinetic parameters with DeepTXInferrer, reducing computational costs significantly for complex, multi-gene models. The approach advances parameter estimation for high-dimensional datasets, leveraging the power of deep learning to overcome the computational expense typically associated with stochastic mechanistic models. Beyond its current application to DNA damage responses, the tool can be adapted to explore transcriptional changes due to various biological factors, making it valuable to the systems biology, bioinformatics, and mechanistic modelling communities. Additionally, this work contributes to the integration of mechanistic modelling and -omics data, a vital area in achieving deeper insights into biological systems at the cellular and molecular levels.

This work also presents some weaknesses, particularly concerning specific technical aspects. The tool was validated using synthetic data, and while it can predict parameters and steady-state distributions that explain gene expression behaviour across many genes, it requires substantial data for training. The authors account for measurement noise in the parameter inference process, which is commendable, yet they do not specify the exact number of samples required to achieve reliable predictions. Moreover, the tool has limitations arising from assumptions made in its design, such as assuming that gene expression counts for the same cell type follow a consistent distribution. This assumption may not hold in cases where RNA measurement timing introduces variability in expression profiles.

The authors present a deep learning pipeline to predict the steady-state distribution, model parameters, and statistical measures solely from scRNA-seq data. Results across three datasets appear robust, indicating that the tool successfully identifies genes associated with expression variability and generates consistent distributions based on its parameters. However, it remains unclear whether these results are sufficient to fully characterise the transcriptional bursting parameter space. The parameters identified by the tool pertain only to the steady-state distribution of the observed data, without ensuring that this distribution specifically originates from transcriptional bursting dynamics.

A primary concern with the TXmodel is its reliance on four independent parameters to describe gene state-switching dynamics. Although this general model can capture specific cases, such as the refractory and telegraph models, accurately estimating the parameters of the refractory model using only steady-state distributions and typical cell counts proves challenging in the absence of time-dependent data.

The claim that the GO analysis pertains specifically to DNA damage response signal transduction and cell cycle G2/M phase transition is not fully accurate. In reality, the GO analysis yielded stronger p-values for pathways related to the mitotic cell cycle checkpoint signalling. As presented, the GO analysis serves more as a preliminary starting point for further bioinformatics investigation that could substantiate these conclusions. Additionally, while GSEA analysis was performed following the GO analysis, the involvement of the cardiac muscle cell differentiation pathway remains unclear, as it was not among the GO terms identified in the initial GO analysis.

As the advancement is primarily methodological, it lacks a comprehensive comparison with traditional methods that serve similar functions. Consequently, the overall evaluation of the method, including aspects such as inference accuracy, computational efficiency, and memory cost, remains unclear. The paper would benefit from being contextualised alongside other computational tools aimed at integrating mechanistic modelling with single-cell RNA sequencing data. Additional context regarding the advantages of deep learning methods, the challenges of analysing large, high-dimensional datasets, and the complexities of parameter estimation for intricate models would strengthen the work.

Reviewer #1 (Public review):

Summary:

In this study, Hilda Tateossian et al. sought to identify the specific gene linked to hearing loss caused by otitis media effusion (OME) in individuals with Down syndrome (DS). They approached this by analyzing a series of mouse models of DS (referred to as the DpTyb lines), which include various duplications that encompass the regions of the mouse genome analogous to the human chromosome 21 (Hsa21). This allowed them to pinpoint genetic loci that may be associated with OME in DS. To control for external variables, such as genetic background and environmental influences, which could affect the development of chronic OME, all DpTyb mouse lines were maintained on a uniform C57BL/6J genetic background. The authors could show that chronic OME phenotypes were consistently reproducible across two research centers, the Francis Crick Institute and MRC Harwell Institute, supporting their conclusion while also reducing the likelihood that environmental factors could affect results.

The authors then focused on a significant locus on chromosome 16 in the Dp5Tyb mouse model that was strongly associated with OME. This locus contains only 12 genes, and it overlapped with the duplicated genomic regions in three additional mouse models (Dp1Tyb, Dp3Tyb, and Ts1Rhr), strengthening the link between this locus and OME. To identify the gene responsible within this critical interval, they conducted targeted crosses of Dp mouse lines (Dp1Tyb, Dp3Tyb, and Dp5Tyb) with gene knockout models. This strategy enabled them to normalize the copy number of specific genes within the progeny and assess the effect on OME. They found that reducing the gene dosage of Dyrk1a specifically restored a wild-type phenotype, implicating Dyrk1a as a key player in the development of OME in DS.

Given the broad biological roles of DYRK1A in various cellular pathways, the researchers also explored its effects on downstream proteins and pathways within the middle ear epithelium using immunohistochemistry and RT-qPCR. They uncovered several pathological mechanisms by which DYRK1A triplication could promote middle ear inflammation and increased vascular permeability. These mechanisms included the interaction between DYRK1A and TGFβ signaling, which affects proinflammatory cytokines IL-6 and IL-17, as well as elevated levels of VEGF in the middle ear that were accompanied by increased Hif1a expression.

At the morphological level, analyses by scanning electron microscopy further revealed a loss of cilia on the epithelial cells in the middle ears of 2-month-old Dp3Tyb and Dp5Tyb mutant mice, which likely contributes to the development of OME in DS.

Finally, to validate the relevance of their findings in humans, the researchers examined the expression of the 12 genes within the Dp5Tyb locus in samples from children with DS compared to unaffected parental controls, using qPCR. They found that among the 12 genes, DYRK1A showed the most significant fold increase in expression, further supporting its potential role in OME associated with DS.

Strengths:

(1) The manuscript is well-written and clearly presents both experimental design and results, together supporting the main conclusions.

(2) The experiments are carefully designed and executed, with data that convincingly support the identification of DYRK1A as a key gene involved in OME in DS. The use of gene knockouts to normalize Dyrk1a gene dosage within the Dp mouse lines was a thorough and successful strategy to strengthen and validate DYRK1A's causal inference in OME.

(3) The study goes beyond simple gene identification by exploring the downstream pathways and cellular effects of DYRK1A triplication. This mechanistic focus provides actionable insights into the potential molecular underpinnings of OME in DS.

(4) The study addresses a clinically important issue - OME in children with DS - and proposes DYRK1A as a practical therapeutic target. Based on data in mice and the high dose of DYRK1A in human clinical samples, the authors suggest that suppressing the activity of this gene by localized delivery of inhibitors to the middle ear cavity in DS patients can be a potential strategy for future treatment of OME.

Weaknesses:

No major weakness is identified.

The authors could discuss further the potential involvement of the other genes within the Dp5Tyb interval, and whether interactions among these genes could impact the disease or whether additional contributions to OME might be overlooked. Beyond DYRK1A expression, discussion of a more extensive analysis of the other genes within the locus in larger cohorts of individuals with DS and OME could add strength to the translational relevance of the findings.

Reviewer #2 (Public review):

This manuscript investigates the genetic basis of otitis media with effusion (OME) in children with Down syndrome (DS). Utilizing an impressive number of mouse models, the study identifies a significant locus on mouse chromosome 16 that contributes to the development of OME. Notably, the gene Dyrk1a is identified as a critical factor for OME in DS; Normalizing Dyrk1a dosage in Dp3Tyb mice restores the wild-type phenotype, highlighting its major contribution to OME in DS. The research also explores the downstream pathways affected by DYRK1A, revealing interactions with TGFβ signaling and the modulation of pro-inflammatory cytokines like IL-6 and IL-17, as well as increased VEGF levels linked to middle ear inflammation.

This work is novel in its comprehensive approach to linking specific genetic loci and genes to the development of OME in DS, and offers a refined genetic analysis, pinpointing Dyrk1a as a key gene. Additionally, the identification of some of the signaling pathways involved provides new insights into the pathophysiology of OME in DS. The findings have significant clinical implications, as they suggest that targeting Dyrk1a could be a potential therapeutic strategy for managing OME in children with DS. This could lead to improved treatment options that go beyond current surgical interventions, reducing the need for repeated tympanostomy tube placements and potentially mitigating the associated risks. Overall, this research enhances our understanding of the genetic factors underlying OME in DS, motivates future studies on the newly identified genetic loci, and opens avenues for future therapeutic developments.

Strengths:

(1) Robust methodology: The use of a comprehensive set of mouse models allows for precise localization of genetic loci associated with OME, an advancement over previous studies.

(2) Identification of Key Genes: The clear demonstration of Dyrk1a's role in OME provides a strong basis for further exploration of targeted therapies.

(3) Pathway Insights: The exploration of signaling pathways, including TGFβ and IL-6 interactions, enriches the discussion around the inflammatory mechanisms that contribute to OME in DS.

Weaknesses:

(1) Limited Human Data: While the mouse models are robust, the translation of findings to human populations could be further strengthened with comparative studies.

(2) Pathway Complexity: The study primarily focuses on Dyrk1a and its immediate inflammatory pathways, which may oversimplify the multifactorial nature of OME in DS; exploring additional genetic interactions, and further exploring the implications of the potential ciliogenesis role of DYRK1A in OME could provide a more complete view.

This study is a valuable contribution to the field of genetic research in Down syndrome, providing critical insights that could inform future therapeutic strategies for managing OME. The implications for treatment and understanding of DS phenotypes in mouse models are particularly noteworthy. The findings are well-supported and present clear avenues for further research.

Reviewer #3 (Public review):

Summary:

The authors used mouse models with nested duplications of genomic regions syntenic to human chromosome 21 to identify specific loci responsible for otitis media with effusion (OME) in people with Down syndrome. They identified two loci: one highly penetrant major locus containing the candidate gene Dyrk1a and one minor locus resulting in low penetrant OME. By normalizing the gene dosage of Dyrk1a, the authors showed it mitigated OME. Further investigation of the molecular mechanisms by which DYRK1A exerts its effect, unveiled interactions with TGFβ signaling, elevated proinflammatory cytokines (IL-6 and IL-17), and increased VEGF levels coupled with increased Hif1a activity in the middle ear.

Strengths:

(1) The manuscript is well-written and includes appropriate figures. I especially liked Figure 4, which provides an excellent graphical abstract for the genetic study.

(2) Using a panel of mouse models with nested duplications is an elegant, systematic approach to narrowing down the genetic loci linked to OME. This is a robust method for dissecting complex traits like those observed in Down syndrome.

(3) Identifying DYRK1A as a major genetic contributor to highly penetrant OME in DS could be extrapolated to individuals with isolated (nonsyndromic) OME, thus paving the way for broader exploration of its role in general OME susceptibility. This discovery also opens the door to developing genetic testing for individuals with recurrent or chronic OME, helping with diagnosis and personalized management.

(4) Identifying DYRK1A as a potential therapeutic target highlights the study's translational relevance and potential impact on treating OME in children with DS.

Weaknesses:

(1) While the mouse model findings are robust, the study lacks validation in humans. Collaborating with researchers studying OM in human cohorts to screen for DYRK1A variants and correlate these to human phenotypes could have significantly strengthened the study's translational relevance.

(2) More compelling evidence could have been provided by generating a DYRK1A overexpression knock-in mouse model in the ROSA26 locus. This approach would allow for the functional evaluation of the impact of the overexpression of this single gene. The authors could make the KI model inducible allowing for a more localized study of the gene in a subset of cells.

(3) The lack of histological findings in the cochlea does not rule out sensorineural hearing loss. The authors did not provide compelling evidence ruling out a sensorineural component. Given DYRK1A expression in various cochlear cell types (according to the gEAR resource), it is plausible that overexpression could cause dysfunction there too. Additional analysis of ABR waves, including amplitude and latency measurements, would help clarify whether the defect is exclusively middle ear-related.

(4) Although Dyrk1a is implicated as a critical gene, the study does not fully explore the potential contributions of the other 11 genes in the identified locus. These genes might also play roles in OME, whether independently or synergically.

(5) While TGFβ signaling and cytokine production are investigated, the study does not explore the full and broader pathway and network interactions. Using transcriptomics in these mice models could provide a deeper and more comprehensive understanding of the molecular mechanisms involved.

(6) The difference in wild-type phenotype restoration between double mutants: Dp3Tyb has the best rescue with no significant difference with wild type, versus Dp5Tyb failing to restore the wild-type phenotype needs further investigation. Understanding the factors accounting for these differences could identify additional modifiers within this locus.

(7) The authors stated, "We detected a one-third increase, as expected, of the number of cells positive for DYRK1A in Dp3Tyb mice (56.6%) compared to wild-type littermates (36.4%)". This measurement refers to the number of cells expressing DYRK1A rather than the actual level of DYRK1A protein expression within these cells. The number of expressing cells does not directly correlate with gene dosage, as it is likely the level of DYRK1A protein within individual cells that has a more significant impact on the phenotype. The authors should quantify the protein levels using Western blot, for example, to strengthen their findings. If the authors believe it is the number of expressing cells that is relevant, then they should provide a clear rationale for how this measure reflects gene dosage effects and its biological significance in this context.

Reviewer #1 (Public review):

Summary:

Ita Mehta and colleagues have investigated the role of putrescine in the pili-dependent surface motility of a laboratory strain of Escherichia coli. Enterobacteria, and particularly E. coli and Salmonella Typhimurium contain an enormous amount of putrescine and cadaverine compared to other bacteria. It has been estimated by Igarashi and colleagues that putrescine is present in E. coli at levels of at least 30 mM. Therefore, an investigation of the role of putrescine in E. coli is a welcome and important contribution to understanding polyamine function. The authors have used a comprehensive suite of E. coli gene deletion strains of putrescine biosynthetic, transport, and catabolic genes to understand the role of putrescine in pili-dependent surface motility.

Strengths:

Single gene deletions of arginine decarboxylase (speA) and agmatine ureohydrolase (speB), and a double gene deletion of the constitutive ornithine decarboxylase (speC) and the acid-inducible ornithine decarboxylase (speF), all of which are involved in putrescine biosynthesis, were found by the authors to be less efficient at pili-dependent surface motility. In addition, the putrescine transport genes plaP and potF are also required for efficient pili-dependent surface motility. Furthermore, the putrescine catabolic genes patA and puuA, when co-deleted, reduce pili-dependent surface motility. Transcriptomic analysis of the agmatine ureohydrolase (speB) gene deletion strain compared to the parental strain indicates a coordinated response to the speB gene deletion, including upregulation of ornithine biosynthetic genes and a downregulation of energy metabolic genes.

Weaknesses:

Because the cellular content of putrescine and other polyamines in the E. coli strains was not measured at any point in this study, and the gene deletions were not genetically complemented, it is not possible to definitively attribute physiological changes to the gene deletion strains specifically to changes in putrescine levels. Furthermore, the GT medium used for the mobility experiments contains trypsinated casein (tryptone), which may contain polyamines and most certainly contain arginine. There are two modes of putrescine biosynthesis in E. coli: one mode is the direct formation of putrescine from L-ornithine mediated by ornithine decarboxylase, and the other is the indirect pathway involving decarboxylation of arginine to form agmatine, followed by hydrolysis of agmatine to form putrescine and urea. In the absence of external arginine, putrescine is made by ornithine decarboxylase, however, in the presence of external arginine, ornithine biosynthesis is repressed and arginine decarboxylase becomes the primary biosynthetic route for putrescine biosynthesis. The GT medium used by the authors will tend to favor putrescine production from arginine. The speB gene deletion, which is used for the transcriptomic analyses, will even in the absence of external arginine, accumulate a very large amount of agmatine, greater than the level of putrescine. This will confound the interpretation of the effect of the speB gene deletion, because agmatine accumulation may be responsible for some of the effects, and the addition of external putrescine may repress agmatine accumulation. In the absence of polyamine level measurements, the relative levels of agmatine, the putrescine structural analog cadaverine, and the accumulation of decarboxylated S-adenosylmethionine, are not known. Changes to these metabolites could affect pili-dependent surface motility. Furthermore, it is not possible to conclude that the effects of gene deletions to biosynthetic, transport or catabolic genes on pili-dependent surface motility are due to changes in putrescine levels unless one takes it on faith that there must be changes to putrescine levels. Since E. coli contains such an enormous amount of putrescine, it is important to know how much putrescine must be depleted in order to exert a physiological effect.

The authors have tackled an important biomedical problem relevant to infections of the urogenital tract and also important for understanding the very unusual high level of putrescine in E. coli and related species. However, without confirmation of putrescine levels in their various strains, it would be difficult to unequivocally conclude that putrescine, or changes to its concentrations, are responsible for the physiological changes seen with the gene deletion strains.

Reviewer #2 (Public review):

Summary:

Mehta et al., in constructing E. coli strains unable to synthesize polyamines, noted that strains deficient in putrescine synthesis showed decreased movement on semisolid agar. They show that strains incapable of synthesizing putrescine have decreased expression of Type I pilin and, hence, decreased ability to perform pilin-dependent surface motility.

Strengths:

The authors characterize the specific polyamine pathways that are important for this phenomenon. RNAseq provides a detailed overview of gene expression in the strain lacking putrescine. The data suggest homeostatic control of polyamine synthesis and metabolic changes in response to putrescine.

Weaknesses:

In this version, the authors ignore phase variation of the pil operon promoter, which can be monitored via PCR. The gene expression data suggest that shifting to the pilin "off" state could help explain the phenotype.

Reviewer #3 (Public review):

Summary:

This study by Mehta et al. describes the mechanisms behind the observation that putrescine biosynthesis mutants in Escherichia coli strain W3110 are affected by surface motility. The manuscript shows that the surface motility phenotype is dependent on Type I fimbriae and that putrescine levels affect the expression level of fimbriae. The results further suggest that without putrescine, the metabolism of the cell is shifted towards the production of putrescine and away from energy metabolism.

There are two main aspects in the manuscript.

(1) The first observation is that a fimA mutant modified/decreased the motility phenotype. From this result, the authors conclude that type I fimbriae (or pili) are involved in the surface motility phenotype. Type I fimbriae are typically known to be involved in non-motile phenotypes, such as biofilm formation or adhesion. Type I fimbriae are also co-regulated with other surface structures that might impact motility. Thus, more controls are needed before concluding that the surface motility requires the type I fimbriae. For instance, the authors should have complemented the mutants and should have verified the flagella expression/motility in the fimA mutant.

(2) The second observation is that putrescine also impacts the surface motility phenotype and the expression of type I fimbriae. Although there is no genetic complementation, here the exogenous addition of putrescine to the speB mutant provides a chemical complementation method, which makes the data stronger.

In addition, testing the effect of putrescine on motility and type I fimbriae expression in additional strains of E. coli would strengthen the conclusion. This is especially important since the results are somewhat different from previous results obtained with a different strain of E. coli. The authors do note that experimental conditions are different, but testing their theory would make the conclusions stronger.

Reviewer #1 (Public review):

This manuscript investigates homeostatic structural plasticity and its interplay with synaptic scaling. It uses an integrated approach with models and experiments.<br /> First, electrophysiology and chronic imaging are used to investigate the influence of different levels of AMPA-receptor antagonist NBQX, which allows for gradual activity reduction. Low levels of NBQX lead to a decrease of activity and a homeostatic increase of synapse density, whereas high levels block neural activity and lead to a reduced number of synapses after 3 days. The authors conclude that there must be a non-linear dependency between neuronal activities and rewiring. As a mathematical model for this, a biphasic structural plasticity rule is used, which, for increasing neural activities, switches from net synapse removal to growth and back, yielding two stable states at zero activity and the homeostatic target.<br /> This rule is tested in various situations in silico, yet without attempting to reproduce the experiment. First, in network development, the biphasic rule generates a lot of unconnected silent neurons and a reasonable network structure only emerges when the neurons are additionally supported by a facilitating input current. For comparison, a linear and a simpler nonlinear homeostatic plasticity model, which had been ruled out by the experimental data, need no external drive. Second, the consequences of lasting, altered stimulation in a subgroup of neurons is explored. As expected by the design of the rule, a small increase and decrease in stimulation leads to a decrease and increase of synaptic connectivity, respectively, and stimulation silencing led to a complete disconnection of the sub-population with restoration of activity. Unlike in previous studies, an asymmetry of pre- and postsynaptic plasticity mechanisms cannot rescue this. Third, silencing only for a short time period and then overstimulating the network led to overly strong activity, which may, however, also hold without silencing. For a transiently silenced stimulation, recovery is possible, but only when there is enough recurrent excitation from the rest of the network.<br /> Following this, the second part of the manuscript explores whether synaptic scaling may adapt and up-regulate the recurrent excitation, such that activity in a normally silenced subpopulation can be restored. Indeed, fast enough synaptic scaling leads to a recovery of neuronal activity in simulations, but leads to highly synchronous activity. A systematic model analysis shows at which scaling and rewiring speeds the activity and connectivity for a silenced sub-population can be restored. In between, however, the authors analyze spine sizes and changes in their whole population AMPAR-blocking experiments that demonstrate synaptic scaling and that structural plasticity and scaling effects may be jointly regulated. This experimental "break" between a simulation and its systematic analysis makes the paper harder to read and seems unnecessary as the analyses from the experiments are not repeated for the model.

Overall, the combination of experiments and simulations is a promising approach to investigate network self-organization. Especially the gradual blocking of activity is very valuable to inform mathematical models and distinguish them from alternatives. However, it remains unclear whether the model would actually reproduce the experiment. When switching from one to the other, this entails a detour to the conceptual level which makes the narrative sometimes hard to follow.

In summary, this manuscript makes a valuable contribution to discern the mathematical shape of a homeostatic structural plasticity model and understanding the necessity of synaptic scaling in the same network. Both experimental and computational methods are solid and well described. Yet, both parts could be linked better in order to obtain conclusions with more impact and generality.

Reviewer #2 (Public review):

This manuscript by Lu et al addresses the understudied interplay between structural and functional changes underlying homeostatic plasticity. Using hippocampal organotypic slice cultures allowing chronic imaging of dendritic spines, the authors showed that a partial or complete inhibition of AMPA-type glutamate receptors differentially affects spine density, respectively leading to an increase or decrease. Based on that dataset, they built a model where activity-dependent synapse formation is regulated by a biphasic rule and tested it in stimulation- or deprivation-induced homeostatic plasticity. The model matches experimental data (from the authors and the literature) quite well, and provides a framework within which functional and structural changes coexist to regulate firing rate homeostasis.

While the correlation between changes in AMPAR numbers and in spine number/size has been well characterized during Hebbian plasticity, the situation is much less clear in homeostatic plasticity due to multiple studies yielding diverging results. This manuscript adds new experimental results to the existing data and presents a valuable effort to generate a model that can explain these divergences in a unifying framework.

The model and its successive implantation steps are well presented along a clear thread. However, the manuscript would benefit from clarifications at several key points (Hebbian vs homeostatic timeline).

First of all, it would have benefited from having an actual timeline of structural changes throughout the three days of AMPAR inhibition, especially as their experimental model allows it. This would have provided much-needed and otherwise entirely lacking information on spine dynamics (especially on transient spines) and on the respective timescale of the structural and functional changes, instead of modelling an entire timeline based solely on an experimental endpoint.

Additionally, the model would have been strengthened by an experimental dataset with homeostatic plasticity induced by higher activity (e.g. with bicuculline). To the best of my knowledge, there is currently no data on structural plasticity following scaling down, and it is also known that scaling up and down are mediated by different molecular pathways. The extension of the model from scaling up (in response to silencing) to scaling down (in response to increased activity) offers an interesting perspective, but its biological relevance is limited as there is no experimental data to support it.

Finally, the difference between weak and complete inhibition could have been more extensively characterized. The authors focus indeed on the effects of either condition on spine number, but only integrate synaptic weights following complete inhibition. This is a pity, as they show some intriguing data suggesting a differential effect on spine size by partial or complete AMPAR inhibition (although further work is required to support some of their interpretations). Since the model aims at correlating structural and functional homeostatic plasticity, the fact that it is only demonstrated for one of the two conditions tested severely undermines the claims of the authors in the discussion that the model tackles that question.

Reviewer #1 (Public review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a current anabolic therapy. The authors have achieved the aims of the study. Their conclusion that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data is useful for helping to understand it.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 10 week old, mice as a model of postmenopausal osteoporosis remains a limitation of this study, but this is now quite clearly described as a limitation,, including justifying the use of the primary spongiosa as a measurement site.

(2) Methods have been clarified. It is still necessary to properly define the micro-CT threshold in mm HA/cc^3. I think it might be at about 200mg HA/cc^3 in this study.

(3) The apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference) remains unresolved. It is still not clear whether there is a material defect in the bone, which can be partially assessed by reporting the 3 point bending test, corrected for the diameters of the bone (i.e. as stress / strain curves).

(4) It is also puzzling that both dimeric and monomeric PTH lead to a reduction in total bone area (cross sectional area?). This would suggest a reduction in bone growth. This should be discussed in the work.

Reviewer #1 (Public review):

Aging reduces tissue regeneration capacity, posing challenges for an aging population. In this study, the authors investigate impaired bone healing in aging, focusing on calvarial bones, and introduce a two-part rejuvenation strategy. Aging depletes osteoprogenitor cells and reduces their function, which hinders bone repair. Simply increasing the number of these cells does not restore their regenerative capacity in aged mice, highlighting intrinsic cellular deficits. The authors' strategy combines Wnt-mediated osteoprogenitor expansion with intermittent fasting, which remarkably restores bone healing. Intermittent fasting enhances osteoprogenitor function by targeting NAD+ pathways and gut microbiota, addressing mitochondrial dysfunction - an essential factor in aging. This approach shows promise for rejuvenating tissue repair, not only in bones but potentially across other tissues.

This study is exciting, impressive, and novel. The data presented is robust and supports the findings well.

Reviewer #2 (Public review):

Reeves et al explore a model of bone healing in the context of aging. They show that intermittent fasting can improve bone healing, even in aged animals. Their study combines a 'bone bandage' which delivers a canonical Wnt signal with intermittent fasting and shows impacts on the CD90 progenitor cell population and the healing of a critical-sized defect in the calvarium. They also explore potential regulators of this process and identify mitochondrial dysfunction in the age-related decline of stem cells. In this context, by modulating NAD+ pathways or the gut microbiota, they can also enhance healing, hinting at an effect mediated by complex impacts on multiple pathways associated with cellular metabolism.

The study shows a remarkable finding: that age-related decreases in bone healing can be restored by intermittent fasting. There is ample evidence that intermittent fasting can delay aging, but here the authors provide evidence that in an already-aged animal, intermittent fasting can restore healing to levels seen in younger animals. This is an important finding as it may hint at the potential benefits of intermittent fasting in tissue repair.

Reviewer #3 (Public review):

Summary:

This study aims to address the significant challenge of age-related decline in bone healing by developing a dual therapeutic strategy that rejuvenates osteogenic function in aged calvarial bone tissue. Specifically, the authors investigate the efficacy of combining local Wnt3a-mediated osteoprogenitor stimulation with systemic intermittent fasting (IF) to restore bone repair capacity in aged mice. The highlights are:

(1) Novel Approach with Aged Models:<br /> This pioneering study is among the first to demonstrate the rejuvenation of osteoblasts in significantly aged animals through intermitted fasting, showcasing a new avenue for regenerative therapies.

(2) Rejuvenation Potential in Aged Tissues:<br /> The findings reveal that even aged tissues retain the capacity for rejuvenation, highlighting the potential for targeted interventions to restore youthful cellular function.

(3) Enhanced Vascular Health:<br /> The study also shows that vascular structure and function can be significantly improved in aged tissues, further supporting tissue regeneration and overall health.<br /> Through this innovative approach, the authors seek to overcome intrinsic cellular deficits and environmental changes within aged osteogenic compartments, ultimately achieving bone healing levels comparable to those seen in young mice.

Strengths:

The study is a strong example of translational research, employing robust methodologies across molecular, cellular, and tissue-level analyses. The authors leverage a clinically relevant, immunocompetent mouse model and apply advanced histological, transcriptomic, and functional assays to characterise age-related changes in bone structure and function. Major strengths include the use of single-cell RNA sequencing (scRNA-seq) to profile osteoprogenitor populations within the calvarial periosteum and suture mesenchyme, as well as quantitative assessments of mitochondrial health, vascular density, and osteogenic function. Another important point is the use of very old animals (up to 88 weeks, almost 2 years) modelling the human bone aging that usually starts >65 yo. This comprehensive approach enables the authors to identify critical age-related deficits in osteoprogenitor number, function, and microenvironment, thereby justifying the combined Wnt3a and IF intervention.

[Editors' note: The manuscript was evaluated positively by all three reviewers originally. In the revised manuscript, the authors included some new data following the reviewers' suggestions, while other comments were clarified in the response to the reviewers, and by revising the manuscript text. The new data further support the major conclusions of the paper.]

Reviewer #1 (Public review):

Summary:

Nitric oxide (NO) has been implicated as a neuromodulator in the retina. Specific types of amacrine cells (ACs) produce and release NO in a light-dependent manner. NO diffuses freely through the retina and can modulate intracellular levels of cGMP, or directly modify and modulate proteins via S-nitrosylation, leading to changes in gap-junction coupling, synaptic gain, and adaptation. Although these system-wide effects have been documented, it is not well understood how the physiological function of specific neuronal types is affected by NO. This study aims to address this gap in our knowledge.

There are two major findings. 1) About a third of the retinal ganglion cells display cell-type specific adaptation to prolonged stimulus protocols. 2) Application of NO specifically affected Off-suppressed ganglion cells designated as G32 cells. The G32 cluster likely contains 3 ganglion cell types that are differentially affected.

This is the first comprehensive analysis of the functional effects of NO on ganglion cells in the retina. The cell-type specificity of the effects is surprising and provides the field with valuable new information.

Strengths:

NO was expected to produce small effects, and considerable effort was expended in validating the system to ensure that changes in the state of the preparation would not confound any effects of NO. The authors used a sequential stimulus protocol to control for changes in the sensitivity of the retina during the extended recording periods. The approach potentially increases the sensitivity of the measurements and allows more subtle effects to be observed.

Neural activity was measured by Ca-imaging. Responsive ganglion cells were grouped into 32 types using a clustering analysis. Initial control experiments demonstrated that the cell-types revealed by the analysis largely recapitulate those from their earlier landmark study using a similar approach.

Application of NO to the retina modulated responses of a single cluster of cells, labeled G32, while having little effect on the remaining 31 clusters. In separate experiments, ganglion cell spiking activity was recorded on a multi-electrode array (MEA). Together the Ca-imaging and MEA recordings provide complementary approaches and demonstrate that NO modulates the temporal but not spatial properties of affected cell-types.

Weaknesses:

The concentration of NO used in these experiments was ~0.25µM, which is 5- to 10-fold lower than the endogenous concentration previously measured in rodent retina. It is perhaps surprising that this relatively low NO concentration produced significant effects. However, the endogenous measurements were done in an eye-cup preparation, while the current experiments were performed in a bare (no choroid) preparation. Perhaps the resting NO level is lower in this preparation. It is also possible that the low concentration of NO promoted more selective effects.

Reviewer #2 (Public review):

Neuromodulators are important for circuit function, but their roles in the retinal circuitry are poorly understood. This study by Gonschorek and colleagues aims to determine the modulatory effect of nitric oxide on the response properties of retinal ganglion cells. The authors used two photon calcium imaging and multi-electrode arrays to classify and compare cell responses before and after applying a NO donor DETA-NO. The authors found that DETA-NO selectively increases activity in a subset of contrast-suppressed RGC types. In addition, the authors found cell-type specific changes in light response in the absence of pharmacological manipulation in their calcium imaging paradigm. This study focuses on an important question and the results are interesting. The limitations of the method and data interpretation are adequately discussed in the revised manuscript.

The authors have addressed my previous comments, included additional discussions on the limitations of the method, and provided a more careful interpretation of their data.

Reviewer #1 (Public review):

Summary:

This paper describes molecular dynamics simulations (MDS) of the dynamics of two T-cell receptors (TCRs) bound to the same major histocompatibility complex molecule loaded with the same peptide (pMHC). The two TCRs (A6 and B7) bind to the pMHC with similar affinity and kinetics, but employ different residue contacts. The main purpose of the study is to quantify via MDS the differences in the inter- and intra-molecular motions of these complexes, with a specific focus on what the authors describe as catch-bond behavior between the TCRs and pMHC, which could explain how T-cells can discriminate between different peptides in the presence of weak separating force.

Strengths:

The authors present extensive simulation data that indicates that, in both complexes, the number of high-occupancy inter-domain contacts initially increases with applied load, which is generally consistent with the authors' conclusion that both complexes exhibit catch-bond behavior, although to different extents. In this way, the paper somewhat expands our understanding of peptide discrimination by T-cells.

Weaknesses:

While generally well supported by data, the conclusions would nevertheless benefit from a more concise presentation of information in the figures, as well as from suggesting experimentally testable predictions.

Reviewer #2 (Public review):

In this work, Chang-Gonzalez and coworkers follow up on an earlier study on the force-dependence of peptide recognition by a T-cell receptor using all-atom molecular dynamics simulations. In this study, they compare the results of pulling on a TCR-pMHC complex between two different TCRs with the same peptide. A goal of the paper is to determine whether the newly studied B7 TCR has the same load-dependent behavior mechanism shown in the earlier study for A6 TCR. The primary result is that while the unloaded interaction strength is similar, A6 exhibits more force stabilization.

This is a detailed study, and establishing the difference between these two systems with and without applied force may establish them as a good reference setup for others who want to study mechanobiological processes if the data were made available, and could give additional molecular details for T-Cell-specialists. As written, the paper contains an overwhelming amount of details and it is difficult (for me) to ascertain which parts to focus on and which results point to the overall take-away messages they wish to convey.

Detailed comments:

(1) In Table 1 - are the values of the extension column the deviation from the average length at zero force (that is what I would term extension) or is it the distance between anchor points (which is what I would assume based on the large values. If the latter, I suggest changing the heading, and then also reporting the average extension with an asterisk indicating no extensional restraints were applied for B7-0, or just listing 0 load in the load column. Standard deviation in this value can also be reported. If it is an extension as I would define it, then I think B7-0 should indicate extension = 0+/- something. The distance between anchor points could also be labeled in Figure 1A.

(2) As in the previous paper, the authors apply "constant force" by scanning to find a particular bond distance at which a desired force is selected, rather than simply applying a constant force. I find this approach less desirable unless there is experimental evidence suggesting the pMHC and TCR were forced to be a particular distance apart when forces are applied. It is relatively trivial to apply constant forces, so in general, I would suggest this would have been a reasonable comparison. Line 243-245 speculates that there is a difference in catch bonding behavior that could be inferred because lower force occurs at larger extensions, but I do not believe this hypothesis can be fully justified and could be due to other differences in the complex.

(3) On a related note, the authors do not refer to or consider other works using MD to study force-stabilized interactions (e.g. for catch bonding systems), e.g. these cases where constant force is applied and enhanced sampling techniques are used to assess the impact of that applied force: https://www.cell.com/biophysj/fulltext/S0006-3495(23)00341-7, https://www.biorxiv.org/content/10.1101/2024.10.10.617580v1. I was also surprised not to see this paper on catch bonding in pMHC-TCR referred to, which also includes some MD simulations: https://www.nature.com/articles/s41467-023-38267-1

(4) The authors should make at least the input files for their system available in a public place (github, zenodo) so that the systems are a more useful reference system as mentioned above. The authors do not have a data availability statement, which I believe is required.

Reviewer #3 (Public review):

Summary:

The paper by Chang-Gonzalez et al. is a molecular dynamics (MD) simulation study of the dynamic recognition (load-induced catch bond) by the T cell receptor (TCR) of the complex of peptide antigen (p) and the major histocompatibility complex (pMHC) protein. The methods and simulation protocols are essentially identical to those employed in a previous study by the same group (Chang-Gonzalez et al., eLife 2024). In the current manuscript, the authors compare the binding of the same pMHC to two different TCRs, B7 and A6 which was investigated in the previous paper. While the binding is more stable for both TCRs under load (of about 10-15 pN) than in the absence of load, the main difference is that, with the current MD sampling, B7 shows a smaller amount of stable contacts with the pMHC than A6.

Strengths:

The topic is interesting because of the (potential) relevance of mechanosensing in biological processes including cellular immunology.

Weaknesses:

The study is incomplete because the claims are based on a single 1000-ns simulation at each value of the load and thus some of the results might be marred by insufficient sampling, i.e., statistical error. After the first 600 ns, the higher load of B7high than B7low is due mainly to the simulation segment from about 900 ns to 1000 ns (Figure 1D). Thus, the difference in the average value of the load is within their standard deviation (9 +/- 4 pN for B7low and 14.5 +/- 7.2 for B7high, Table 1). Even more strikingly, Figure 3E shows a lack of convergence in the time series of the distance between the V-module and pMHC, particularly for B70 (left panel, yellow) and B7low (right panel, orange). More and longer simulations are required to obtain a statistically relevant sampling of the relative position and orientation of the V-module and pMHC.

It is not clear why "a 10 A distance restraint between alphaT218 and betaA259 was applied" (section MD simulation protocol, page 9).

Reviewer #1 (Public review):

Summary:

In the manuscript entitled 'The Role of ATP Synthase Subunit e (ATP5I) in 1 Mediating the Metabolic and Antiproliferative 2 Effects of Biguanides', Lefrancois G et al. identifies ATP5I, a subunit of F1Fo-ATP synthase, as a key target of medicinal biguanides. ATP5I stabilizes F1Fo-ATP synthase dimers, essential for cristae morphology, but its role in cancer metabolism is understudied. The research shows ATP5I interacts with a biguanide analogue, and its knockout in pancreatic cancer cells mimics biguanide treatment effects, including altered mitochondria, reduced OXPHOS, and increased glycolysis. ATP5I knockout cells resist biguanide-induced antiproliferative effects, but reintroducing ATP5I restores the effects of metformin and phenformin. These findings highlight ATP5I as a promising mitochondrial target for cancer therapies. The manuscript is well written.

Strengths:

Demonstrated the experiments in systematic and well-accepted methods.

Weaknesses:

The significance of the target molecule and mechanisms may help in understanding the molecular mechanisms of metformin.

Reviewer #2 (Public review):

Summary:

The mechanism(s) by which the therapeutic drug metformin lowers blood glucose in type 2 diabetes and inhibits cell proliferation at higher concentrations remain contentious. Inhibition of complex 1 of the mitochondrial respiratory chain with consequent changes in cellular metabolites which favour allosteric activation of phosphofructokinase-1, allosteric inhibition of fructose bisphosphatase-1 and cAMP signalling and activation of AMPK which phosphorylates transcription factors are candidate mechanisms. The current manuscript proposes the e-subunit of ATP-synthase as a putative binding protein of biguanides and demonstrates that it regulates the expressivity of the Complex 1 protein NDUFB8.

Strengths:

(1) The metformin conjugate and metformin show comparable efficacy on inhibition of cell proliferation in the millimolar range.

(2) Demonstration of compromised expression of the Complex I protein NDUFB8 by the ATP5I knockout and its reversal by ATP5I expression is an important strength of the study. This shows that the decreased "sensitivity" to metformin in the ATP5I knock-out cells could be due to various proteins.

(3) Demonstration of converse effects of ATP5I KO and re-expression ATP5I on the NAD/NADH ratio.

Weaknesses:

(1) The interpretation of the cellular co-localization of the biotin-biguanide conjugate with TOMM20 (Figure 1-D) as mitochondrial "accumulation" of the conjugate is overstated because it cannot exclude binding of the conjugate to the mitochondrial membrane. It would have been more convincing if additional incubations with the biotin-biguanide conjugate in combination with metformin had shown that metformin is competitive with the biotin-conjugate.

(2) The manuscript reports the identification of 69 proteins by mass spectrometry of the pull-down assay of which 31 proteins were eluted by metformin. However, no Mass Spectrometry data is presented of the peptides identified. The methodology does not state the minimum number of peptides (1, 2?) that were used for the identification of the 31/69 proteins.

(3) The validation of ATP5I was based on the use of recombinant protein (which was 90% pure) for the SPR and the use of a single antibody to ATP5I. The validity of the immunoblotting rests on the assumption that there is no "non-specific" immunoactivity in the relevant mol wt range. Information on the validation of the antibody would be helpful.

(4) Knock-out of ATP5I markedly compromised the NAD/NADH ratio (Fig.3A) and cell proliferation (Figure 3D). These effects may be associated with decreased mitochondrial membrane potential which could explain the low efficacy of metformin (and most of the data in Figures 3-5). This possibility should be discussed. Effects of [metformin] on the NAD/NADH ratio in control cells and ATP5I-KO would have been helpful because the metformin data on cell growth is normalized as fold change relative to control, whereas the NAD/NADH ratio would represent a direct absolute measurement enabling comparison of the absolute effect in control cells with ATP5I KO.

(5) Figure-6 CRISPR/Cas9 KO at 16mM metformin in comparison with 70nM rotenone and 2 micromolar oligomycin (in serum-containing medium). The rationale for the use of such a high concentration of metformin has not been explained. In liver cells metformin concentrations above 1mM cause severe ATP depletion, whereas therapeutic (micromolar) concentrations have minimal effects on cellular ATP status. The 16mM concentration is ~2 orders of magnitude higher than therapeutic concentrations and likely linked to compromised energy status. The stronger inhibition of cell proliferation by 16mM metformin compared with rotenone or oligomycin raises the issue of whether the changes in gene expression may be linked to the greater inhibition of mitochondrial metabolism. Validation of the cellular ATP status and NAD/NADH with metformin as compared with the two inhibitors could help the interpretation of this data.

Reviewer #3 (Public review):

Most of the data are based on measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured by the Seahorse analyser in control and ATP5l KO cells. However, these measurements are conducted by a single injection of a biguanide, followed over time and presented as fold change. By doing so, the individual information on the effect of metformin and derivate on control and KO cells are lost. In addition, the usual measurement of OCR is coupled with certain inhibitors and uncouplers, such as oligomycin, FCCP, and Antimycin A/rotenone, to understand the contribution of individual complexes to respiration. Since biguanides and ATP5l KO affect protein levels of components of complex I and IV, it would be informative to measure their individual contributions/effects in the Seahorse. To further strengthen the data, it would be helpful to obtain measurements of actual ATP levels in these cells, as this would explain the activation of AMPK.

The authors report on alterations in mitochondrial morphology upon ATP5l KO, which is measured by subjective quantifications of filamentous versus puncta structures. Fiji offers great tools to quantify the mitochondrial network unbiasedly and with more accuracy using deconvolution and skeletonization of the mitochondria, providing the opportunity to measure length, shape, and number quantitatively. This will help to understand better, whether mitochondria are really fragmented upon ATP5l KO and rescued by its re-introduction.

Finally, the authors report in the last part of the paper a genetic CRISPR/Cas9 KO screen in NALM-6 cells cultured with high amounts of metformin to identify potential new mediators of metformin action. It is difficult to connect that to the rest of the paper because a) different concentrations of metformin are used and b) the metabolic effects on energy consumption are not defined. They argue about the molecular function of the obtained hits based on literature and on a comparison of the pattern of genetic alterations based on treatments with known inhibitors such as oligomycin and rotenone. However, a direct connection is not provided, thus the interpretation at the end of the results that "the OMA1-DEL1-HRI pathway mediates the antiproliferative activity of both biguanides and the F1ATPase inhibitor oligomycin" while increasing glycolysis, needs to be toned down. This is an interesting observation, but no causality is provided. In general, this part stands alone and needs to be better connected to the rest of the paper.

Reviewer #1 (Public review):

Summary:

The authors' goal was to advance the understanding of metabolic flux in the bradyzoite cyst form of the parasite T. gondii, since this is a major form of transmission of this ubiquitous parasite, but very little is understood about cyst metabolism and growth.

Nonetheless, this is an important advance in understanding and targeting bradyzoite growth.

Strengths:

The study used a newly developed technique for growing T. gondii cystic parasites in a human muscle-cell myotube format, which enables culturing and analysis of cysts. This enabled the screening of a set of anti-parasitic compounds to identify those that inhibit growth in both vegetative (tachyzoite) forms and bradyzoites (cysts). Three of these compounds were used for comparative Metabolomic profiling to demonstrate differences in metabolism between the two cellular forms.

One of the compounds yielded a pattern consistent with targeting the mitochondrial bc1 complex and suggests a role for this complex in metabolism in the bradyzoite form, an important advance in understanding this life stage.

Weaknesses:

Studies such as these provide important insights into the overall metabolic differences between different life stages, and they also underscore the challenge of interpreting individual patterns caused by metabolic inhibitors due to the systemic level of some of the targets, so that some observed effects are indirect consequences of the inhibitor action. While the authors make a compelling argument for focusing on the role of the bc1 complex, there are some inconsistencies in the patterns that underscore the complexity of metabolic systems.

Reviewer #2 (Public review):

Summary:

A particular challenge in treating infections caused by the parasite Toxoplasma gondii is to target (and ultimately clear) the tissue cysts that persist for the lifetime of an infected individual. The study by Maus and colleagues leverages the development of a powerful in vitro culture system for the cyst-forming bradyzoite stage of Toxoplasma parasites to screen a compound library for candidate inhibitors of parasite proliferation and survival. They identify numerous inhibitors capable of inhibiting both the disease-causing tachyzoite and the cyst-forming bradyzoite stages of the parasite. To characterize the potential targets of some of these inhibitors, they undertake metabolomic analyses. The metabolic signatures from these analyses lead them to identify one compound (MMV1028806) that interferes with aspects of parasite mitochondrial metabolism. The authors claim that MV1028806 targets the bc1 complex of the mitochondrial electron transport chain of the parasite, although the evidence for this is indirect and speculative. Nevertheless, the study presents an exciting approach for identifying and characterizing much-needed inhibitors for targeting tissue cysts in these parasites.

Strengths:

The study presents convincing proof-of-principle evidence that the myotube-based in vitro culture system for T. gondii bradyzoites can be used to screen compound libraries, enabling the identification of compounds that target the proliferation and/or survival of this stage of the parasite. The study also utilizes metabolomic approaches to characterize metabolic 'signatures' that provide clues to the potential targets of candidate inhibitors, although falls short of identifying the actual targets.

Weaknesses:

(1) The authors claim to have identified a compound in their screen (MMV1028806) that targets the bc1 complex of the mitochondrial electron transport chain (ETC). The evidence they present for this claim is indirect (metabolomic signatures and changes in mitochondrial membrane potential) and could be explained by the compound targeting other components of the ETC or affecting mitochondrial biology or metabolism in other ways. In order to make the conclusion that MMV1028806 targets the bc1 complex, the authors should test specifically whether MMV1028806 inhibits bc1-complex activity (i.e. in a direct enzymatic assay for bc1 complex activity). Testing the activity of MMV1028806 against other mitochondrial dehydrogenases (e.g. dihydroorotate dehydrogenase) that feed electrons into the ETC might also provide valuable insights. The experiments the authors perform also do not directly measure whether MMV1028806 impairs ETC activity, and the authors could also test whether this compound inhibits mitochondrial O2 consumption (as would be expected for a bc1 inhibitor).

(2) The authors claim that compounds targeting bradyzoites have greater lipophilicity than other compounds in the library (and imply that these compounds also have greater gastrointestinal absorbability and permeability across the blood-brain barrier). While it is an attractive idea that lipophilicity influences drug targeting against bradyzoites, the effect seems pretty small and is complicated by the fact that the comparison is being made to compounds that are not active against parasites. If the authors are correct in their assertion that lipophilicity is a major determinant of bradyzoicidal compounds compared to compounds that target tachyzoites alone, you would expect that compounds that target tachyzoites alone would have lower lipophilicity than those that target bradyzoites. It would therefore make more sense to (statistically) compare the bradyzoicidal and dual-acting compounds to those that are only active in tachyzoites (visually the differences seem small in Figure S2B). This hypothesis would be better tested through a structure-activity relationship study of select compounds (which is beyond the scope of the study). Overall, the evidence the authors present that high lipophilicity is a determinant of bradyzoite targeting is not very convincing, and the authors should present their conclusions in a more cautious manner.

(3) Page 11 and Figure 7. The authors claim that their data indicate that ATP is produced by the mitochondria of bradyzoites "independently of exogenous glucose and HDQ-target enzymes." The authors cite their previous study (Christiansen et al, 2022) as evidence that HDQ can enter bradyzoites, since HDQ causes a decrease in mitochondrial membrane potential. Membrane potential is linked to the synthesis of ATP via oxidative phosphorylation. If HDQ is really causing a depletion of membrane potential, is it surprising that the authors observe no decrease in ATP levels in these parasites? Testing the importance of HDQ-target enzymes using genetic approaches (e.g. gene knockout approaches) would provide better insights than the ATP measurements presented in the manuscript, although would require considerable extra work that may be beyond the scope of the study. Given that the authors' assay can't distinguish between ATP synthesized in the mitochondrion vs glycolysis, they may wish to interpret their data with greater caution.

Reviewer #3 (Public review):

Summary:

The authors describe an exciting 400-drug screening using a MMV pathogen box to select compounds that effectively affect the medically important Toxoplasma parasite bradyzoite stage. This work utilises a bradyzoites culture technique that was published recently by the same group. They focused on compounds that affected directly the mitochondria electron transport chain (mETC) bc1-complex and compared them with other bc1 inhibitors described in the literature such as atovaquone and HDQs. They further provide metabolomics analysis of inhibited parasites which serves to provide support for the target and to characterise the outcome of the different inhibitors.

Strengths:

This work is important as, until now, there are no effective drugs that clear cysts during T. gondii infection. So, the discovery of new inhibitors that are effective against this parasite stage in culture and thus have the potential to battle chronic infection is needed. The further metabolic characterization provides indirect target validation and highlights different metabolic outcomes for different inhibitors. The latter forms the basis for new studies in the field to understand the mode of inhibition and mechanism of bc1-complex function in detail.

The authors focused on the function of one compound, MMV1028806, that is demonstrated to have a similar metabolic outcome to burvaquone. Furthermore, the authors evaluated the importance of ATP production in tachyzoite and bradyzoites stages and under atovaquone/HDQs drugs.

Weaknesses:

Although the authors did experiments to identify the metabolomic profile of the compounds and suggested bc-1 complex as the main target of MMV1028806, they did not provide experimental validation for that.

Reviewer #1 (Public review):

Neuronal activity spatiotemporal fine-tuning of cerebral blood flow balances metabolic demands of changing neuronal activity with blood supply. Several 'feed-forward' mechanisms have been described that contribute to activity-dependent vasodilation as well as vasoconstriction leading to a reduction in perfusion. Involved messengers are ionic (K+), gaseous (NO), peptides (e.g., NPY, VIP), and other messengers (PGE2, GABA, glutamate, norepinephrine) that target endothelial cells, smooth muscle cells, or pericytes. Contributions of the respective signaling pathways likely vary across brain regions or even within specific brain regions (e.g., across the cortex) and are likely influenced by the brain's physiological state (resting, active, sleeping) or pathological departures from normal physiology.

The manuscript "Elevated pyramidal cell firing orchestrates arteriolar vasoconstriction through COX-2-derived prostaglandin E2 signaling" by B. Le Gac, et al. investigates mechanisms leading to activity-dependent arteriole constriction. Here, mainly working in brain slices from mice expressing channelrhodopsin 2 (ChR2) in all excitatory neurons (Emx1-Cre; Ai32 mice), the authors show that strong optogenetic stimulation of cortical pyramidal neurons leads to constriction that is mediated through the cyclooxygenase-2 / prostaglandin E2 / EP1 and EP3 receptor pathway with contribution of NPY-releasing interneurons and astrocytes releasing 20-HETE. Specifically, using a patch clamp, the authors show that 10-s optogenetic stimulation at 10 and 20 Hz leads to vasoconstriction (Figure 1), in line with a stimulation frequency-dependent increase in somatic calcium (Figure 2). The vascular effects were abolished in the presence of TTX and significantly reduced in the presence of glutamate receptor antagonists (Figure 3). The authors further show with RT-PCR on RNA isolated from patched cells that ~50% of analyzed cells express COX-1 or -2 and other enzymes required to produce PGE2 or PGF2a (Figure 4). Further, blockade of COX-1 and -2 (indomethacin), or COX-2 (NS-398) abolishes constriction. In animals with chronic cranial windows that were anesthetized with ketamine and medetomidine, 10-s long optogenetic stimulation at 10 Hz leads to considerable constriction, which is reduced in the presence of indomethacin. Blockade of EP1 and EP3 receptors leads to a significant reduction of the constriction in slices (Figure 5). Finally, the authors show that blockade of 20-HETE synthesis caused moderate and NPY Y1 receptor blockade a complete reduction of constriction.

The mechanistic analysis of neurovascular coupling mechanisms as exemplified here will guide further in-vivo studies and has important implications for human neuroimaging in health and disease. Most of the data in this manuscript uses brain slices as an experimental model which contrasts with neurovascular imaging studies performed in awake (headfixed) animals. However, the slice preparation allows for patch clamp as well as easy drug application and removal. Further, the authors discuss their results in view of differences between brain slices and in vivo observations experiments, including the absence of vascular tone as well as blood perfusion required for metabolite (e.g., PGE2) removal, and the presence of network effects in the intact brain. The manuscript and figures present the data clearly; regarding the presented mechanism, the data supports the authors' conclusions. Some of the data was generated in vivo in head-fixed animals under anesthesia; in this regard, the authors should revise the introduction and discussion to include the important distinction between studies performed in slices, or in acute or chronic in-vivo preparations under anesthesia (reduced network activity and reduced or blockade of neuromodulation, or in awake animals (virtually undisturbed network and neuromodulatory activity). Further, while discussed to some extent, the authors could improve their manuscript by more clearly stating if they expect the described mechanism to contribute to CBF regulation under 'resting state conditions' (i.e., in the absence of any stimulus), during short or sustained (e.g., visual, tactile) stimulation, or if this mechanism is mainly relevant under pathological conditions; especially in the context of the optogenetic stimulation paradigm being used (10-s long stimulation of many pyramidal neurons at moderate-high frequencies) and the fact that constriction leading to undersupply in response to strongly increased neuronal activity seems counterintuitive?

Reviewer #2 (Public review):

The present study by Le Gac et al. investigates the vasoconstriction of cerebral arteries during neurovascular coupling. It proposes that pyramidal neurons firing at high frequency lead to prostaglandin E2 (PGE2) release and activation of arteriolar EP1 and EP3 receptors, causing smooth muscle cell contraction. The authors further claim that interneurons and astrocytes also contribute to vasoconstriction via neuropeptide Y (NPY) and 20-hydroxyeicosatetraenoic acid (20-HETE) release, respectively. The study mainly uses brain slices and pharmacological tools in combination with Emx1-Cre; Ai32 transgenic mice expressing the H134R variant of channelrhodopsin-2 (ChR2) in the cortical glutamatergic neurons for precise photoactivation. Stimulation with 470 nm light using 10-second trains of 5-ms pulses at frequencies from 1-20 Hz revealed small constrictions at 10 Hz and robust constrictions at 20 Hz, which were abolished by TTX and partially inhibited by a cocktail of glutamate receptor antagonists. Inhibition of cyclooxygenase-1 (COX-1) or -2 (COX-2) by indomethacin blocked the constriction both ex vivo (slices) and in vivo (pial artery), and inhibition of EP1 and EP3 showed the same effect ex vivo. Single-cell RT-PCR from patched neurons confirmed the presence of the PGE2 synthesis pathway.

While the data are convincing, the overall experimental setting presents some limitations. How is the activation protocol comparable to physiological firing frequency? The delay (minutes) between the stimulation and the constriction appears contradictory to the proposed pathway, which would be expected to occur rapidly. The experiments are conducted in the absence of vascular "tone," which further questions the significance of the findings. Some of the targets investigated are expressed by multiple cell types, which makes the interpretation difficult; for example, cyclooxygenases are also expressed by endothelial cells. Finally, how is the complete inhibition of the constriction by the NPY Y1 receptor antagonist BIBP3226 consistent with a direct effect of PGE2 and 20-HETE in arterioles? Overall, the manuscript is well-written with clear data, but the interpretation and physiological relevance have some limitations. However, vasoconstriction is a rather understudied phenomenon in neurovascular coupling, and the present findings may be of significance in the context of pathological brain hypoperfusion.

Reviewer #1 (Public review):

Dwulet et al. combined experimental and modeling approaches to investigate how correlated spontaneous activity in the mouse's primary visual (V1) and primary somatosensory (S1) areas drives the development of multisensory integration in area RL. Notably, they focused on early developmental stages, before sensory experience occurs. Consistent with previous experimental findings, the authors first demonstrated that spontaneous activity becomes more sparse across development in all three areas, as measured by event amplitude, event duration, and participation ratio. Using a linear mixed model analysis to compare the maturation of this spontaneous activity, they found evidence that S1 matured the fastest. The authors then presented experimental evidence suggesting that these spontaneous events were moderately correlated both spatially and temporally.

They hypothesized that activity-dependent mechanisms use these correlations to establish connectivity across these regions. To test this hypothesis, the authors modeled a feedforward network with connections from S1 to RL and from V1 to RL, where the strength of connections depended on a Hebbian term for potentiation and a heterosynaptic term for depression. By investigating different levels of V1-S1 correlations, they found that moderate levels of correlation led to the significant development of topographically organized connectivity while maintaining a mix of bimodal and unimodal cells in RL. Additionally, when simulating a network with a more mature S1, they observed that topographical maps improved not only between S1 and RL but also between V1 and RL. Finally, the authors use linear regression to suggest that the mixture of bimodal and unimodal cells in RL is optimal for encoding the maximum amount of information from both V1 and S1.

However, there are significant gaps between the experimental data and the modeling setup, which weaken the paper's conclusions. Additionally, some key details are omitted, making it difficult to fully assess their analysis and interpret some of their figures.

(1) Some of the statistical measures and techniques in Figure 1 could benefit from clearer definitions. While the thresholds for activation (peak with at least 5% dF/F0) and events (20% of recorded cells activated simultaneously) are provided, event duration and participation rate are not clearly defined. Based on this definition of event alone, it is unclear why the minimum participation rate in Figure 1F is not 20%. Additionally, the conclusion that S1 matures earlier than RL and V1 could be strengthened by including a direct comparison between S1 and RL, as the current analysis only compares these areas to V1.

(2) The wide-field experiments in Figure 2 could be expanded to support the feedforward modeling assumptions. Currently, the spatial and temporal correlations presented leave open the possibility that these spontaneous events are traveling waves propagating from V1 to RL to S1 (or vice versa). This scenario would suggest a different connectivity scheme for the model. Clarifying this point with additional data analysis, specifically including temporal correlations involving RL, could provide stronger support for the model's assumptions.

(3) The functional correlation map in Figure 2D appears contradictory to the authors' modeling assumption that inputs are correlated spatially in V1 and S1. While V1 seed points align topographically with RL, this organization breaks down when extended into S1. In contrast, and in support of the modeling assumption, Figure 2E shows clearer topography across all three regions. A discussion of this discrepancy would be helpful, as it's a key conclusion of the figure. Additionally, it is unclear when this data was collected during development. Clarifying the developmental stage and analyzing how this map changes over time could strengthen the results.

(4) The modeling of spontaneous events with fixed amplitude and duration seems inconsistent with the experimental data in Figure 1, which shows variability in these parameters. This is particularly confusing in Figure 4, where S1 maturation is modeled as a stronger topographical alignment with RL, but the experimental data defines maturation based on amplitude, duration, and event rates. Justifying these modeling choices or adapting the model to reflect experimental variability would create a better connection between the theory and data.

(5) Several important details of the mathematical model are missing or unclear, partly due to typos. The Results section mentions the general framework of the input correlation matrix (e.g., "S1 and V1 neurons were driven by a combination of events, independent and shared in each V1 and S1" and "each independent event activated a randomly chosen, contiguous set of neurons"), but the specifics are not fully explained. Additionally, the caption of Figure 5 refers to a non-linear transfer function (a sigmoid), but these details are not provided in the Methods section, which instead suggests a linear model was used. A careful review of the main text and Methods section would help ensure that all the necessary details are included and that the story is both complete and accurate.

(6) While Figure 5 supports the paper's conclusion that a mixture of unimodal and bimodal neurons in RL optimizes information encoding, the authors missed an opportunity to strengthen the connection between the model and experimental data. Specifically, they could apply this reconstruction method to the experimental data and examine how RL's ability to reconstruct V1/S1 activity changes across development. Their model predicts that this performance would improve over time, and if this trend is observed in the experimental data, it would provide strong validation that these feedforward connections are developing in line with the model's predictions.

Reviewer #2 (Public review):

The authors aim to investigate the role of spontaneous activity in shaping the development of multisensory integration in the brain, specifically focusing on the connections between primary visual and somatosensory sensory areas (V1 and S1) and a higher-order cortical area rostro-lateral to V1 (RL). They seek to understand how spontaneous activity guides the formation of aligned topographic maps and the emergence of bimodal neurons in RL.

First, the authors found that spontaneous activity in all three areas sparsifies over time, but S1 exhibits more mature patterns earlier than V1 and RL. They claimed that correlated activity among neighboring regions of these areas during development carries topographic information. These data were used to implement a computational model that employed Hebbian rules of synaptic plasticity. The model indicated that correlated spontaneous activity can generate topographic connectivity between S1/V1 and RL and bimodal neurons in RL. The model suggested that the more mature spontaneous activity in S1 can guide map alignment between V1 and RL. In addition, the model also suggested that a mixture of bimodal and unimodal neurons in RL is optimal for decoding information from V1 and S1.

While the data presented in the manuscript is promising and provides preliminary insights into the role of spontaneous activity in multisensory integration, it would be beneficial to strengthen the experimental foundation regarding the correlation between V1, S1, and RL. Incorporating more rigorous spatio-temporal analyses of spontaneous activity could enhance the robustness of these findings.

Here are some important concerns:

(1) The analysis of how spatial topography influences activity correlations in Figure 2 has several issues.<br /> 1a. While squares in V1 and S1 covered a small area of these sensory areas, the correlated territories in RL covered the entire area of RL. The topographic map in V1 continues caudally, so where is the rest of the map in RL? Something similar applies to the relationship between S1 and RL.<br /> 1b. It is essential to know how areas were drawn. High precision is required.<br /> 1c. It is not clear if correlated activity means different events in sync or large events that cover 2 or all 3 cortical areas of interest. The figure points to the second option, which contradicts the size of events at these stages, mainly in the oldest mice analyzed here.<br /> 1d. It is fundamental to know in detail and provide examples of how the detection of events was performed. For instance, could the dispersion of light from an event in V1 close to RL cause the detection of activity in RL?

(2) For the correlations among V1, S1, and RL, it is crucial to have a consistent method to delineate the borders of cortical areas. The authors mention in one sentence that areas were drawn according to a reference map. More details are needed to convince the reader that the borders are accurate, especially because their shape and position change with age.

(3) The results from the model seem to be based on the initial bias in connectivity between neighboring cells from the different areas. Then, it seems straightforward that implementing correlated activity with Hebbian and synaptic depression rules will force the strengthening of connections between spatially close cells. Despite this apparent predisposition of the model towards a defined outcome, the flaws in the experimental data used prevent a rigorous interpretation of the computational model.

(4) In the Introduction, the authors nicely and briefly explain the role of primary and higher-order sensory cortices in information processing. They also explain how spontaneous activity during development helps to build these circuits by refining connections or establishing hierarchies. They continue explaining the relevance of aligning different topographic maps to allow multisensory integration. Then they provide some examples of sites of multisensory integration. This provides a general context for the data presented in the Results section; however, and importantly, there is no specific introduction of why they are interested in RL and its interaction with V1 and S1. The authors should introduce the RL area and explain why it is an interesting site for multisensory processing.

(5) The results shown in Figure 1 corroborate published data from Golshani et al, Rochefort et al, Murakami et al. While the reproduction of data is more than welcome, the authors should specify which part of the data is completely new and acknowledge clearly the rest as corroboration of previous data. The sentence "As described in previous experiments ..." partially acknowledges this fact but is not clear enough. In addition, the transition between this part of the manuscript and the next data is not smooth. Data seems to be used to feed the model so perhaps the organization of the manuscript leaves room for improvement.

Reviewer #3 (Public review):

Summary:

The study by Dwulet et al. explores how the development of spontaneous neural activity in primary sensory cortices influences the co-alignment of multiple sensory modalities in higher-order brain areas (HOAs). To address this question, they focus on connectivity between the primary visual (V1) and somatosensory (S1) cortices and an associative cortical area (RL) in mice. The authors combine experimental (wide-field and two-photon calcium imaging) and computational approaches to show that spontaneous activity matures at a different pace across these brain regions. Their data indicate that S1 develops more rapidly than V1, which is possibly beneficial for RL's integration of visual and somatosensory inputs through correlated spontaneous activity. Using a computational model, they demonstrate that a moderate correlation between V1 and S1 activity can optimally guide the formation of bimodal neurons in RL, which are crucial for maximizing the decodability of multisensory stimuli. This finding highlights the role of correlated spontaneous activity in primary sensory cortices in establishing co-aligned topographic multimodal sensory representations in downstream circuits.

Strengths:

The manuscript is well written and it provides strong enough evidence to support the main claim of the authors. The insights on the role of correlated activity on instructing co-aligned multisensory maps in HOAs are not trivial and are an important advancement for the field.

Weaknesses:

In the opinion of this reviewer, the study has no major weaknesses. A drawback of the work is that none of the predictions of the computational modeling have been corroborated through mechanistic experimental manipulations of early brain activity.

Reviewer #1 (Public review):

Summary:

The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix.

Strengths:

The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately.

Weaknesses:

The structures don't show how CTF18-RFC opens or loads PCNA. There are recent structures from other groups that do examine these steps in more detail, although this does not really dampen this reviewer's enthusiasm. It does mean that the authors should spend their time investigating aspects of CTF18-RFC function that were overlooked or not explored in detail in the competing papers. The paper poorly describes the interactions of CTF18-RFC with PCNA and the ATPase active sites, which are the main interest points. The nomenclature choices made by the authors make the manuscript very difficult to read.

Reviewer #2 (Public review):

Summary

Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures.

Strength & Weakness

Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. This is potentially very interesting, although some more work is needed on the quantification. Moreover, the authors argue that the Ctf18 ATP-binding domain assumes a more flexible organisation, but their visual representation could be improved.

The data are discussed accurately and relevantly, which provides an important framework for rationalising the results.

All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18-dependent clamp loading.

Reviewer #3 (Public review):

Summary:

CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader that is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit that is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex.

Relevance:

The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment, and the DNA damage response.

Strengths:

The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes.

Weaknesses:

The manuscript would have benefitted from more detailed biochemical analysis to tease apart the differences with the canonical RFC complex.

I'm not aware of using Mg depletion to trap active states of AAA ATPases. Perhaps the authors could provide a reference to successful examples of this and explain why they chose not to use the more standard practice in the field of using ATP analogues to increase the lifespan of reaction intermediates.

Overall appraisal:

Overall the work presented here is solid and important. The data is sufficient to support the stated conclusions and so I do not suggest any additional experiments.

Reviewer #1 (Public review):

This study presents Jyvaskylavirus, a new member of the Marseilleviridae family, infecting Acanthamoeba castellanii. The study provides a detailed and comprehensive genomic and structural analysis of Jyvaskylavirus. The authors identified ORF142 as the capsid penton protein and additional structural proteins that comprise the virion. Using a combination of imaging techniques the authors provide new insights into the giant virus architecture and lifecycle. The study could be improved by providing atomic coordinates and refinement statistics, comparisons with available giant virus structures could be expanded, and the novelty in terms of the first isolated example of a giant virus from Finland could be expounded upon.

The study contributes new structural and genomic diversity to the Marseilleviridae family, hinting at a broader distribution and ecological significance of giant viruses than previously thought.

Reviewer #2 (Public review):

Summary:

This paper describes the molecular characterisation of a new isolate of the giant virus Jyvaskylavirus, a member of the Marseilleviridae family infecting Acanthamoeba castellanii. The isolate comes from a boreal environment in Finland, showcasing that giant viruses can thrive in this ecological niche. The authors came up with a non-trivial isolation procedure that can be applied to characterise other members of the family and will be beneficial for the virology field. The genome shows typical Marseilleviridae features and phylogenetically belongs to their clade B. The structural characterisation was performed on the level of isolated virion morphology by negative stain EM, virions associated with cells either during the attachment or release by helium microscopy, the visualisation of the virus assembly inside cells using stained thin sections, and lastly on the protein secondary structure level by reconstructing ~6 A icosahedral map of the massive virion using cryoEM. The cryoEM density combined with gene product structure prediction enabled the identification and functional assessment of various virion proteins.

Strengths:

The detailed description of the virus isolation protocol is the largest strength of the paper and this reviewer believes it can be modified for isolating various viruses infecting small eukaryotes. The cryoEM map allows us to understand how exceptionally large virions of these viruses are stabilised by minor capsid proteins and nicely demonstrates the integration of medium-resolution cryoEM with protein structure prediction in deciphering virion protein function. The visualisation of ongoing virus assembly inside virus factories brings interesting hypotheses about the process that; however, needs to be verified in the next studies.

Weaknesses:

The conclusions from helium microscopy images are overinterpreted, as the native membrane structure cannot be preserved in a fixed and dehydrated sample. In the image, there are many other parts of the curved membrane and a lot of virions, to me it seems the specific position of the highlighted virion could arise by a random chance. The claim that the cells were imaged in the near-original state by this method should be therefore omitted. Also, no mass spectrometry data are presented that would supplement and confirm the identity of virion proteins which predicted models were fitted into the cryoEM density. For a general virology reader outside of the giant virus field, the results presented in the current state might not have enough influence and the section should be rewritten to better showcase the novelty of findings.

Reviewer #1 (Public review):

Summary:

The authors report the role of a novel gene Aff3ir-ORF2 in flow-induced atherosclerosis. They show that the gene is anti-inflammatory in nature. It inhibits the IRF5-mediated athero-progression by inhibiting the causal factor (IRF5). Furthermore, the authors show a significant connection between shear stress and Aff3ir-ORF2 and its connection to IRF5 mediated athero-progression in different established mice models which further validates the ex vivo findings.

Strengths:

(1) An adequate number of replicates were used for this study.<br /> (2) Both in vitro and in vivo validation was done.<br /> (3) The figures are well presented.<br /> (4) In vivo causality is checked with cleverly designed experiments.

Weaknesses:

(1) Inflammatory proteins must be measured with standard methods e.g ELISA as mRNA level and protein level does not always correlate.

(2) RNA seq analysis has to be done very carefully. How does the euclidean distance correlate with the differential expression of genes. Do they represent the neighborhood? If they do how does this correlation affect the conclusion of the paper?

(3) The volcano plot does not indicate the q value of the shown genes. It is advisable to calculate the q value for each of the genes which represents the FDR probability of the identified genes.

(4) GO enrichment was done against the Global gene set or a local geneset? The authors should provide more detailed information about the analysis.

(5) If the analysis was performed against a global gene set. How does that connect with this specific atherosclerotic microenvironment?

(6) What was the basal expression of genes and how did the DGE (differential gene expression) values differ?

(7) How was IRF5 picked from GO analysis? was it within the 20 most significant genes?

(8) Microscopic studies should be done more carefully? There seems to be a global expression present on the vascular wall for Aff3ir-ORF2 and the expression seems to be similar to AFF3 in Figure 1.

Reviewer #2 (Public review):

Summary:

The authors recently uncovered a novel nested gene, Aff3ir, and this work sets out to study its function in endothelial cells further. Based on differences in expression correlating with areas of altered shear stress, they investigate a role for the isoform Aff3ir-ORF2 in endothelial activation and development of atherosclerosis downstream of disturbed shear stress. Using a knockout mouse model and in vivo overexpression experiments, they demonstrate a strong potential for Aff3ir-ORF2 to alleviate atherosclerosis. They find that Aff3ir-ORF2 interacts with the pro-inflammatory transcription factor IRF5 and retains it in the cytoplasm, hence preventing upregulation of inflammation-associated genes. The data expands our knowledge of IRF5 regulation which could be relevant to researchers studying various inflammatory diseases as well as adding to our understanding of atherosclerosis development.

Strengths:

The in vivo data is solid using immunofluorescence staining to assess AFF3ir-ORF2 expression, a knockout mouse model, overexpression and knockdown studies, and rescue experiments in combination with two atherosclerotic models to demonstrate that Aff3ir-ORF2 can lessen atherosclerotic plaque formation in ApoE-/- mice.

Weaknesses:

While the in vivo data is generally convincing, a few data panels have issues and will need addressing. Also, the knockout mouse model will need to be described, since the paper referred to in the manuscript does not actually report any knockout mouse model. Hence it is unclear how Aff3ir-ORF2 is targeted, but Figure S2B shows that targeting is partial, since about 30% expression remains at the RNA level in MEFs isolated from the knockout mice.

While the effect on atherosclerosis is clear, the conclusion that this is the result of reduced endothelial cell activation is not supported by the data. The mouse model is described as a global knockout and the shRNA knockdowns (Figure 5) and overexpression data in Figure 2 are not cell type-specific. Only the overexpression construct in Figure 6 uses an ICAM-2 promoter construct, which drives expression in endothelial cells, though leaky expression of this promoter has been reported in the literature. Therefore, other cell types such as smooth muscle cells or macrophages could be responsible for the effects observed.

The weakest part of the manuscript is the in vitro experiments. While they are solidly executed, all experiments are performed in MEFs, and results are interpreted as being equivalent to endothelial cell responses. There is also an RNA-seq experiment performed on MEFs from the Aff3ir-ORF2 knockout and control mice, but the data is not disclosed other than showing some non-identifiable expression differences. The data is used to hypothesise on a role for IRF5 in the effects observed with Aff3ir-ORF2 knockout.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis, but the cell types involved and mechanisms remain unclear. The study also shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but any relevance of this mechanism for atherosclerosis or any cell types involved in the development of this disease remains largely speculative.

Reviewer #3 (Public review):

This study is to demonstrate the role of Aff3ir-ORF2 in the atheroprone flow-induced EC dysfunction and ensuing atherosclerosis in mouse models. Overall, the data quality and comprehensiveness are convincing. In silico, in vitro, and in vivo experiments and several atherosclerosis were well executed. To strengthen further, the authors can address human EC relevance.

Major comments:

(1) The tissue source in Figures 1A and 1B should be clarified, the whole aortic segments or intima? If aortic segment was used, the authors should repeat the experiments using intima, due to the focus of the current study on the endothelium.

(2) Why were MEFs used exclusively in the in vitro experiments? Can the authors repeat some of the critical experiments in mouse or human ECs?

(3) The authors should explain why AFF3ir-ORF2 overexpression did not affect the basal level expression of ICAM-1, VCAM-1, IL-1b, and IL-6 under ST conditions (Figure 2A-C).

(4) Please include data from sham controls, i.e., right carotid artery in Figure 2E.

(5) Given that the merit of the study lies in the effect of different flow patterns, the legion areas in AA and TA (Figure 3B, 3C) should be separately compared.

(6) For confirmatory purposes for the variations of IRF5 and IRF8, can the authors mine available RNA-seq or even scRNA-seq data on human or mouse atherosclerosis? This approach is important and could complement the current results that are lacking EC data.

(7) With the efficacy of using AAV-ICAM2-AFF3ir-ORF2 in atherosclerosis reduction (Figure 6), the authors are encouraged to use lung ECs isolated from the AFF3ir-ORF2-/-mice to recapitulate its regulation of IRF5.

Reviewer #1 (Public review):

Summary:

Multiple compounds that inhibit ATP-sensitive potassium (KATP) channels also chaperone channels to the surface membrane. The authors used an artificial intelligence (AI)-based virtual screening (AtomNet) to identify novel compounds that exhibit chaperoning effects on trafficking-deficient disease-causing mutant channels. One compound, which they named Aekatperone, acts as a low affinity, reversible inhibitor and effective chaperone. A cryoEM structure of KATP bound to Aekatperone showed that the molecule binds at the canonical inhibitory site.

Strengths and weaknesses:

The details of the AI screening itself are inevitably opaque but appear to differ from classical virtual screening in not involving any physical docking of test compounds into the target site. The authors mention criteria that were used to limit the number of compounds so that those with high similarity to known binders and 'sequence identity' (does this mean structural identity) were excluded. The identified molecules contain sulfonylurea-like moieties. How different are they from other sulfonylure4as?

The experimental work confirming that Aekatperone acts to traffic mutant KATP channels to the surface and acts as a low affinity, reversible, inhibitor is comprehensive and clear, with very convincing cell biological and patch-clamp data, as is the cryoEM structural analysis, for which the group are leading experts. In addition to the three positive chaperone-effective molecules, the authors identified a large number of compounds that are predicted binders but apparently have no chaperoning effect. Did any of them have an inhibitory action on channels? If so, does this give clues to separating chaperoning from inhibitory effects?

The authors suggest that the novel compound may be a promising therapeutic for the treatment of congenital hyperinsulinism due to trafficking defective KATP mutations. Because they are low-affinity, reversible, inhibitors. This is a very interesting concept, and perhaps a pulsed dosing regimen would allow trafficking without constant channel inhibition (which otherwise defeats the therapeutic purpose), although it is unclear whether the new compound will offer advantages over earlier low-affinity sulfonylurea inhibitor chaperones. These include tolbutamide which has very similar affinity and effect to Aekatperone. As the authors point out this (as well as other sulfonylureas) is currently out of favor because of potential adverse cardiovascular effects, but again, it is unclear why Aekatperone should not have the same concerns.

Reviewer #2 (Public review):

Summary:

In their study 'AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone', ElSheikh and colleagues undertake a computational screening approach to identify candidate drugs that may bind to an identified binding pocket in the SUR1 subunit of KATP channels. Other KATP channel inhibitors such as glibenclamide have been previously shown to bind in this pocket, and in addition to inhibition of KATP channel function, these inhibitors can very effectively rescue cell surface expression of trafficking deficient KATP mutations that cause excessive insulin secretion (Congenital Hyperinsulinism). However, a challenge for their utility for the treatment of hyperinsulinism has been that they are powerful inhibitors of the channels that are rescued to the channel surface. In contrast, successful therapeutic pharmacochaperones (eg. CFTR chaperones) permit the function of the channels rescued to the cell membrane. Thus, a key criterion for the authors' approach, in this case, was to identify relatively low-affinity compounds that target the glibenclamide binding site (and be washed off) - these could potentially rescue KATP surface expression but also permit KATP function.

Strengths:

The main findings of the manuscript include:

(1) Computational screening of a large virtual compound library, followed by functional screening of cell surface expression, which identified several potential candidate pharmacochaperones that target the glibenclamide binding site.

(2) Prioritization and functional characterization of Aekatperone as a low-affinity KATP inhibitor which can be readily 'washed off' in patch clamp and cell-based efflux assays. Thus the drug clearly rescues cell surface expression but can be manipulated experimentally to permit the function of rescued channels.

(3) Determination of the binding site and dynamics of this candidate drug by cryo-EM, and functional validation of several residues involved in drug sensitivity using mutagenesis and patch clamp.

The experiments are well-conceived and executed, and the study is clearly described. The results of the experiments are very straightforward and clearly support the conclusions drawn by the authors. I found the study to provide important new information about the KATP chaperone effects of certain drugs, with interesting considerations in terms of ion channel biology and human disease.

Weaknesses:

I don't have any major criticisms of the study as described, but I had some remaining questions that could be addressed in a revision.

(1) The chaperones can effectively rescue KATP trafficking mutants, but clearly not as strongly as the higher affinity inhibitor glibenclamide. Is this relationship between inhibitory potency, and efficacy of trafficking an intrinsic challenge of the approach? I suspect that it may be an intractable problem in the sense that the inhibitor-bound conformation that underlies the chaperone effect cannot be uncoupled from the inhibited gating state. But this might not be true (many partial agonist drugs with low efficacy can be strongly potent, for example). In this case, the approach is really to find a 'happy medium' of a drug that is a weak enough inhibitor to be washed away, but still strong enough to exert some satisfactory chaperone effect. Could some additional clarity be added in the discussion on whether the chaperone and gating effects can be 'uncoupled'?

(2) Based on the western blots in Figure 2B, the rescue of cell surface expression appears to require a higher concentration of AKP compared to the concentration-response of channel inhibition (~9 microM in Figure 3, perhaps even more potent in patch clamp in Figure 2C). Could the authors clarify/quantify the concentration response for trafficking rescue?

(3) A future challenge in the application of pharmacochaperones of this type in hyperinsulinism may be the manipulation of chaperone concentration in order to permit function. In experiments, it is straightforward to wash off the chaperone, but this would not be the case in an organism. I wondered if the authors had attempted to rescue channel function with diazoxide in the presence of AKP, rather than after washing off (ie. is AKP inhibition insurmountable, or can it be overcome by sufficient diazoxide).

(4) Do the authors have any information about the turnover time of KATP after the wash-off of the chaperone (how stable are the rescued channels at the cell surface)? This is a difficult question to probe when glibenclamide is used as a chaperone, but may be much simpler to address with a lower affinity chaperone like AKP.

Reviewer #1 (Public review):

Summary:

This manuscript is a focused investigation of the phosphor-regulation of a C. elegans kinesin-2 motor protein, OSM-3. In C-elegans sensory ciliary, kinesin-2 motor proteins Kinesin-II complex and OSM-3 homodimer transport IFT trains anterogradely to the ciliary tip. Kinesin-II carries OSM-3 as an inactive passenger from the ciliary base to the middle segment, where kinesin-II dissociates from IFT trains and OSM-3 gets activated and transports IFT trains to the distal segment. Therefore, activation/inactivation of OSM-3 plays an essential role in its ciliary function.

Strengths:

In this study, using mass spectrometry, the authors have shown that the NEKL-3 kinase phosphorylates a serine/threonine patch at the hinge region between coiled coils 1 and 2 of an OSM-3 dimer, referred to as the elbow region in ubiquitous kinesin-1. Phosphomimic mutants of these sites inhibit OSM-3 motility both in vitro and in vivo, suggesting that this phosphorylation is critical for the autoinhibition of the motor. Conversely, phospho-dead mutants of these sites hyperactivate OSM-3 motility in vitro and affect the localization of OSM3 in C. elegans. The authors also showed that Alanine to Tyrosine mutation of one of the phosphorylation rescues OS-3 function in live worms.

Weaknesses:

Collectively, this study presents evidence for the physiological role of OSM-3 elbow phosphorylation in its autoregulation, which affects ciliary localization and function of this motor. Overall, the work is well performed, and the results mostly support the conclusions of this manuscript. However, the work will benefit from additional experiments to further support conclusions and rule out alternative explanations, filling some logical gaps with new experimental evidence and in-text clarifications, and improving writing.

Reviewer #2 (Public review):

Summary:

The regulation of kinesin is fundamental to cellular morphogenesis. Previously, it has been shown that OSM-3, a kinesin required for intraflagellar transport (IFT), is regulated by autoinhibition. However, it remains totally elusive how the autoinhibition of OSM-3 is released. In this study, the authors have shown that NEKL-3 phosphorylates OSM-3 and releases its autoinhibition.

The authors found NEKL-3 directly phosphorylates OSM-3 (although the method is not described clearly) (Figure 1). The phophorylated residue is the "elbow" of OSM-3. The authors introduced phospho-dead (PD) and phospho-mimic (PM) mutations by genome editing and found that the OSM-3(PD) protein does not form cilia, and instead, accumulates to the axonal tips. The phenotype is similar to another constitutive active mutant of OSM-3, OSM-3(G444A) (Imanishi et al., 2006; Xie et al., 2024). osm-3(PM) has shorter cilia, which resembles with loss of function mutants of osm-3 (Figure 3). The authors did structural prediction and showed that G444E and PD mutations change the conformation of OSM-3 protein (Figure 3). In the single-molecule assays G444E and PD mutations exhibited increased landing rate (Figure 4). By unbiased genetic screening, the authors identified a suppressor mutant of osm-3(PD), in which A489T occurs. The result confirms the importance of this residue. Based on these results, the authors suggest that NEKL-3 induces phosphorylation of the elbow domain and inactivates OSM-3 motor when the motor is synthesized in the cell body. This regulation is essential for proper cilia formation.

Strengths:

The finding is interesting and gives new insight into how the IFT motor is regulated.

Weaknesses:

The methods section has not presented sufficient information to reproduce this study.

Reviewer #1 (Public review):

In this manuscript, Dillard and colleagues integrate cross-species genomic data with a systems approach to identify potential driver genes underlying human GWAS loci and establish the cell type(s) within which these genes act and potentially drive disease. Specifically, they utilize a large single-cell RNA-seq (scRNA-seq) dataset from an osteogenic cell culture model - bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) - from a genetically diverse outbred mouse population called the Diversity Outbred (DO) stock to discover network driver genes that likely underlie human bone mineral density (BMD) GWAS loci. The DO mice segregate over 40M single nucleotide variants, many of which affect gene expression levels, therefore making this an ideal population for systems genetic and co-expression analyses. The current study builds on previously published work from the same group that used co-expression analysis to identify co-expressed "modules" of genes that were enriched for BMD GWAS associations. In this study, the authors utilize a much larger scRNA-seq dataset from 80 DO BMSC-OBs, infer co-expression-based and Bayesian networks for each identified mesenchymal cell type, focused on networks with dynamic expression trajectories that are most likely driving differentiation of BMSC-OBs, and then prioritized genes ("differentiation driver genes" or DDGs) in these osteogenic differentiation networks that had known expression or splicing QTLs (eQTL/sQTLs) in any GTEx tissue that colocalized with human BMD GWAS loci. The systems analysis is impressive, the experimental methods are described in detail, and the experiments appear to be carefully done. The computational analysis of the single-cell data is comprehensive and thorough, and the evidence presented in support of the identified DDGs, including Tpx2 and Fgfrl1, is for the most part convincing. Some limitations in the data resources and methods hamper enthusiasm somewhat and are discussed below. Overall, while this study will no doubt be valuable to the BMD community, the cross-species data integration and analytical framework may be more valuable and generally applicable to the study of other diseases, especially for diseases with robust human GWAS data but for which robust human genomic data in relevant cell types is lacking.

Specific strengths of the study include the large scRNA-seq dataset on BMSC-OBs from 80 DO mice, the clustering analysis to identify specific cell types and sub-types, the comparison of cell type frequencies across the DO mice, and the CELLECT analysis to prioritize cell clusters that are enriched for BMD heritability (Figure 1). The network analysis pipeline outlined in Figure 2 is also a strength, as is the pseudotime trajectory analysis (results in Figure 3). One weakness involves the focus on genes that were previously identified as having an eQTL or sQTL in any GTEx tissue. The authors rightly point out that the GTEx database does not contain data for bone tissue, but the reason that eQTLs can be shared across many tissues - this assumption is valid for many cis-eQTLs, but it could also exclude many genes as potential DDGs with effects that are specific to bone/osteoblasts. Indeed, the authors show that important BMD driver genes have cell-type-specific eQTLs. Furthermore, the mesenchymal cell type-specific co-expression analysis by iterative WGCNA identified an average of 76 co-expression modules per cell cluster (range 26-153). Based on the limited number of genes that are detected as expressed in a given cell due to sparse per-cell read depth (400-6200 reads/cell) and dropouts, it's hard to believe that as many as 153 co-expression modules could be distinguished within any cell cluster. I would suspect some degree of model overfitting here and would expect that many/most of these identified modules have very few gene members, but the methods list a minimum module size of 20 genes. How do the numbers of modules identified in this study compare to other published scRNA-seq studies that use iterative WGCNA?

In the section "Identification of differentiation driver genes (DDGs)", the authors identified 408 significant DDGs and found that 49 (12%) were reported by the International Mouse Knockout [sic] Consortium (IMPC) as having a significant effect on whole-body BMD when knocked out in mice. Is this enrichment significant? E.g., what is the background percentage of IMPC gene knockouts that show an effect on whole-body BMD? Similarly, they found that 21 of the 408 DDGs were genes that have BMD GWAS associations that colocalize with GTEx eQTLs/sQTLs. Given that there are > 1,000 BMD GWAS associations, is this enrichment (21/408) significant? Recommend performing a hypergeometric test to provide statistical context to the reported overlaps here.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single-cell RNAseq analysis on bone marrow-derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is in parts hard to read due to the use of acronyms and there are some questions about data analysis that need to be addressed.

Reviewer #1 (Public review):

Summary:

The manuscript by Bindu et al. created an AAV-based tool (GEARAOCS) to perform in vivo genome editing of mouse astrocytes. The authors engineered a versatile AAV vector that allows for gene deletion through NHNJ, site-specific knock-in by HDR, and gene trap. By utilizing this tool, the authors deleted Sparcl1 virally in subsets of astrocytes and showed that thalamocortical synapses in cortical layer IV are indeed reduced during a critical period of ocular dominance plasticity and in adulthood, whereas there is no change in excitatory synapse number in cortical layer II/III. Furthermore, the authors made a VAMP2 gene-trap AAV vector and showed that astrocyte-derived VAMP2 is required for the maintenance of both excitatory and inhibitory synapses.

Strengths:

This AAV-based tool is versatile for astrocytic gene manipulation in vivo. The work is innovative and exciting, given the paucity of tools available to probe astrocytes in vivo.

Weaknesses:

Several important considerations need to be made for the validation and usage of this tool, including:

Major points:

(1) Efficiency and specificity of spCas9-sgRNA mediated gene knockout in astrocytes. In Figure 3, the authors utilized Sparcl1 gene deletion as the proof-of-principle experiment. The readout for Sparcl1 KO efficiency is solely the immunoreactivity using an antibody raised against Sparcl1. As the method is based on NHEJ, the indels can be diverse and can occur in one allele or two. For the tool and proof-of-principle experiment, it will be important to know the percentage of editing near the PAM site, as well as the actual sequences of indels. This can be done by single-cell PCR of edited astrocytes, similar to the published work (Ye... Chen, Nature Biotechnology 2019).

(2) Along the same line, the authors showed that GEARBOCS TagIn of Sparcl1 resulted in 12.49% efficiency based on the immunohistochemistry of mCherry tag. It is understandable that the knock-in efficiency is much reduced as compared to gene knockout. However, it remains unclear if those 12.49% knock-in cells represent sequence-correct ones, as spCas9-mediated HDR is also an error-prone process, and it may accidentally alter nucleotides near the PAM site without causing the frameshift. The author will need to consider the related evidence or make comments in the discussion.

(3) What are the efficiencies of Sparcl1 GEARBOCS GeneTrap (Figure 3V) and Vamp2 GeneTrap and HA TagIn (Figure 5)?

Minor points:

(1) Figure 3H-J. The authors only showed the representative images of Sparcl1 KO. Please consider including the control (without gRNA), given that there are still many Sparcl1+ signals in Figure 3I (likely because of its expression in other cell types?).

(2) In figure 3Q-T, it appears that some Cas9-EGFP+ astrocytes (Q) do not express Sparcl1 (R). Is Sparcl1 expressed in subsets of astrocytes? Does Cas9-EGFP or Sparcl1-TagIn alter Sparcl1 endogenous expression?

(3) On Page 8, for the explanation of the design of the GEARBOCS construct, the authors have made a self-citation (#43). That was a BioRxiv paper that is being reviewed currently.

(4) For Figures 4 and 6, the graphs seem to be made in R with the x-axis labeled as "Condition". The y-axis labels are too small to read properly, especially in print. It would be better to make the graphs clearer like Figure 2 and Figure 3.

(5) On Page 13, "Figures 3V-Y" were referred to. However, there are no Figures 3W, X, and Y.

(6) There are a few typos in the manuscript, including line 900 "immunofluorescence microscopy images of a Cas9-EGFP-positive astrocytes (green)".

Reviewer #2 (Public review):

Summary:

The present study described GEARBOCS, an adeno-associated virus tool for in vivo gene editing in astrocytes. This tool is timely and important for glial biologists who often are troubled by efficient gene targeting in astrocytes. Overall the significance of the finding is valuable, and the strength of the evidence is solid. Presumably, there will be great potential associated with GEARBOCS applications in the future.

Strengths:

As efficient tools for targeting non-neuronal cells in the brains are rather limited for astrocytes and microglia, GEARBOCS adds to the small pool of currently available tools and will provide new options for glial biologists studying these tools. As the study revealed, GEARBOCS are capable of knockout and knockin manipulations for genes of interest, also ascribed with reporter tracking and gene-trap strategy. The promising multi-functional tool will advance our understanding of astrocytes and help to further elucidate the mechanism of neuron-glia interaction.

Weaknesses:

Even though the tool seems promising and powerful. the authors failed to provide more evidence on the robustness and specificity of GEARBOCS. Also, the advantages of GEARBOCS over some of the traditional methods were not clearly stated. Some of these concerns are described below.

Reviewer #3 (Public review):

Summary:

Sivadasan Bindu et al. developed a CRISPR/Cas9-based gene-editing strategy using a single AAV vector, named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System), which enables precise genome manipulation in astrocytes. This tool was shown to effectively perform knockout, tagging, and reporter knock-in gene modifications. The utility of GEARBOCS was demonstrated in two cases: establishing astrocytes as essential for the synaptogenic factor Sparcl1 in thalamocortical synapse maintenance, and revealing that cortical astrocytes express the Vamp2 protein, which is vital for maintaining synapse numbers.

Strengths:

Astrocytes play a crucial role in brain development and function, but studying them in vivo has been challenging due to limited molecular tools for manipulation. Sivadasan Bindu et al. developed a valuable system called GEARBOCS for effective astrocyte infection via retro-orbital injection.

Weaknesses:

The manuscript provides data only from the cerebral cortex and results from P42. Additional data from other brain regions and various time points (e.g., P0-15) are needed. Results from local injection experiments would also enhance the utility of this tool for the broader glial research community.

Reviewer #1 (Public review):

Summary:

Wang et al. created a series of specific FLIM-FRET sensors to measure the activity of different Rab proteins in small cellular compartments. They apply the new sensors to monitor Rab activity in dendritic spines during induction of LTP. They find sustained (30 min) inactivation of Rab10 and transient (5 min) activation of Rab4 after glutamate uncaging in zero Mg. NMDAR function and CaMKII activation are required for these effects. Knockdown of Rab4 reduced spine volume change while knockdown of Rab10 boosted it and enhanced functional LTP (in KO mice). To test Rab effects on AMPA receptor exocytosis, the authors performed FRAP of fluorescently labeled GluA1 subunits in the plasma membrane. Within 2-3 min, new AMPARs appear on the surface via exocytosis. This process is accelerated by Rab10 knock-down and slowed by Rab4 knock-down. The authors conclude that CaMKII promotes AMPAR exocytosis by i) activating Rab4, the exocytosis driver and ii) inhibiting Rab10, possibly involved in AMPAR degradation.

Strengths:

The work is a technical tour de force, adding fundamental insights to our understanding of the crucial functions of different Rab proteins in promoting/preventing synaptic plasticity. The complexity of compartmentalized Ras signaling is poorly understood and this study makes substantial inroads. The new sensors are thoroughly characterized, seem to work very well, and will be quite useful for the neuroscience community and beyond (e.g. cancer research). The use of FLIM for read-out is compelling for precise activity measurements in rapidly expanding compartments (i.e., spines during LTP).

Weaknesses:

The interpretation of the FRAP experiments (Figure 5, Ext. Data Figure 13) is not straightforward as spine volume and surface area greatly expand during uncaging. I appreciate the correction for the added spine membrane shown in Extended Data Figure 14i, but shouldn't this be a correction factor (multiplication) derived from the volume increase instead of a subtraction?

Also, experiments were not conducted or analyzed blind, risking bias in the selection/exclusion of experiments for analysis. This reduces my confidence in the results.

Reviewer #2 (Public review):

Summary:

Wang et al. developed a set of optical sensors to monitor Rab protein activity. Their investigation into Rab activity in dendritic spines during structural long-term plasticity (sLTP) revealed sustained Rab10 inactivation (>30min) and transient Rab4 activation (~5 min). Through pharmacological and genetic manipulation to constitutively activate or inhibit Rab proteins, they found that Rab10 negatively regulates sLTP and AMPA receptor insertion, while Rab4 positively influences sLTP but only in the transient phase. The optical sensors provide new tools for studying Rab activity in cells and neurobiology. However, a full understanding of the timing of Rab activity will require a detailed characterization of sensor kinetics.

Strengths:

(1) Introduction of a series of novel sensors that can address numerous questions in Rab biology.

(2) Multiple methods to manipulate Rab proteins to reveal the roles of Rab10 and rab4 in LTP.

(3) Discovery of Rab4 activation and Rab10 inhibition with different kinetics during sLTP, correlating with their functional roles in the transient (Rab4) and both transient and sustained (Rab10) phases of sLTP.

Weaknesses:

(1) Lack of characterization of sensor kinetics, making it difficult to determine if the observed Rab kinetics during sLTP were due to sensor behavior or actual Rab activity.

(2) It is crucial to assess whether the overexpression of Rab proteins as reporters, affects Rab activity and cellular structure and physiology (e.g. spine number and size).

(3) The paper does not explain the apparently different results between NMDA receptor activation and glutamate uncaging. NMDA receptor activation increased Rab10 activity, while glutamate uncaging decreased it. NMDA receptor activation resulted in sustained Rab4 activation, whereas glutamate uncaging caused only brief activation of about 5 minutes. A potential explanation, ideally supported by data, is needed.

(4) There is a discrepancy between spine phenotype and sLTP potential with Rab10 perturbation. Rab10 perturbation affected spine density but not size, suggesting a role in spinogenesis rather than sLTP. However, glutamate uncaging affected sLTP, and spinogenesis was not examined. Explaining the discrepancy between spine size and sLTP potential is necessary. Exploring spinogenesis with glutamate uncaging would strengthen these results. Additionally, Figure 4j shows no change in synaptic transmission with Rab10 knockout, despite an increase in spine density. An explanation, ideally supported by data, is needed for the unchanged fEPSP slope despite an increase in spine density.

(5) Spine volume was imaged using acceptor fluorophores (mCherry, or mCherry/Venus) at 920nm, where the two-photon cross-section of mCherry is minimal. 920nm was also used to excite the donor fluorophore, hence the spine volume measurement based on total red channel fluorescence is the sum of minimal mCherry fluorescence from direct 920nm excitation, bleed-through from the green channel, and FRET. This confounded measurement requires correction and clarification.

Reviewer #3 (Public review):

Summary:

This study examines the roles of Rab10 and Rab4 proteins in structural long-term potentiation (sLTP) and AMPA receptor (AMPAR) trafficking in hippocampal dendritic spines using various different methods and organotypic slice cultures as the biological model.

The paper shows that Rab10 inactivation enhances AMPAR insertion and dendritic spine head volume increase during sLTP, while Rab4 supports the initial stages of these processes. The key contribution of this study is identifying Rab10 inactivation as a previously unknown facilitator of AMPAR insertion and spine growth, acting as a brake on sLTP when active. Rab4 and Rab10 seem to be playing opposing roles, suggesting a somewhat coordinated mechanism that precisely controls synaptic potentiation, with Rab4 facilitating early changes and Rab10 restricting the extent and timing of synaptic strengthening.

Strengths:

The study combines multiple techniques such as FRET/FLIM imaging, pharmacology, genetic manipulations, and electrophysiology to dissect the roles of Rab10 and Rab4 in sLTP. The authors developed highly sensitive FRET/FLIM-based sensors to monitor Rab protein activity in single dendritic spines. This allowed them to study the spatiotemporal dynamics of Rab10 and Rab4 activity during glutamate uncaging-induced sLTP. They also developed various controls to ensure the specificity of their observations. For example, they used a false acceptor sensor to verify the specificity of the Rab10 sensor response.

This study reveals previously unknown roles for Rab10 and Rab4 in synaptic plasticity, showing their opposing functions in regulating AMPAR trafficking and spine structural plasticity during LTP.

Weaknesses:

In sLTP, the initial volume of stimulated spines is an important determinant of induced plasticity. To address changes in initial volume and those induced by uncaging, the authors present Extended Data Figure 2. In my view, the methods of fitting, sample selection, or both may pose significant limitations for interpreting the overall results. While the initial spine size distribution for Rab10 experiments spans ~0.1-0.4 fL (with an unusually large single spine at the upper end), Rab4 spine distribution spans a broader range of ~0.1-0.9 fL. If the authors applied initial size-matched data selection or used polynomials rather than linear fitting, panels a, b, e, f, and g might display a different pattern. In that case, clustering analysis based on initial size may be necessary to enable a fair comparison between groups not only for this figure but also for main Figures 2 and 3.

Another limitation is the absence of in vivo validation, as the experiments were performed in organotypic hippocampal slices, which may not fully replicate the complexity of synaptic plasticity in an intact brain, where excitatory and inhibitory processes occur concurrently. High concentrations of MNI-glutamate (4 mM in this study) are known to block GABAergic responses due to its antagonistic effect on GABA-A receptors, thereby precluding the study of inhibitory network activity or connectivity [1], which is already known to be altered in organotypic slice cultures.

[1] https://www.frontiersin.org/journals/neural-circuits/articles/10.3389/neuro.04.002.2009/full

Reviewer #1 (Public review):

Summary:

This paper investigates how recurrent neural networks (RNNs) can perform context-dependent decision-making (CDM). The authors use low-rank RNN modeling and focus on a CDM task where subjects are presented with sequences of auditory pulses that vary in location and frequency, and they must determine either the prevalent location or frequency based on an external context signal. In particular, the authors focus on the problem of differentiating between two distinct selection mechanisms: input modulation, which involves altering the stimulus input representation, and selection vector modulation, which involves altering the "selection vector" of the dynamical system.

First, the authors show that rank-one networks can only implement input modulation and that higher-rank networks are required for selection vector modulation. Then, the authors use pathway-based information flow analysis to understand how information is routed to the accumulator based on context. This analysis allows the authors to introduce a novel definition of selection vector modulation that explicitly links it to changes in the effective coupling along specific pathways within the network.

The study further generates testable predictions for differentiating selection vector modulation from input modulation based on neural dynamics. In particular, the authors find that:<br /> (1) A larger proportion of selection vector modulation is expected in networks with high-dimensional connectivity.<br /> (2) Single-neuron response kernels exhibiting specific profiles (peaking between stimulus onset and choice onset) are indicative of neural dynamics in extra dimensions, supporting the presence of selection vector modulation.<br /> (3) The percentage of explained variance (PEV) of extra dynamical modes extracted from response kernels at the population level can serve as an index to quantify the amount of selection vector modulation.

Strengths:

The paper is clear and well-written, and it draws bridges between two recent important approaches in the study of CDM: circuit-level descriptions of low-rank RNNs, and differentiation across alternative mechanisms in terms of neural dynamics. The most interesting aspect of the study involves establishing a link between selection vector modulation, network dimensionality, and dimensionality of neural dynamics. The high correlation between the networks' mechanisms and their dimensionality (Figure 7d) is surprising since differentiating between selection mechanisms is generally a difficult task, and the strength of this result is further corroborated by its consistency across multiple RNN hyperparameters (Figure 7-Figure Supplement 1 and Figure 7-figure supplement 2). Interestingly, the correlation between the selection mechanism and the dimensionality of neural dynamics is also high (Figure 7g), potentially providing a promising future avenue for the study of neural recordings in this task.

Weaknesses:

The first part of the manuscript is not particularly novel, and it would be beneficial to clearly state which aspects of the analyses and derivations are different from previous literature. For example, the derivation that rank-1 RNNs cannot implement selection vector modulation is already present in the Extended Discussion of Pagan et al., 2022 (Equations 42-43). Similarly, it would be helpful to more clearly explain how the proposed pathway-based information flow analysis differs from the circuit diagram of latent dynamics in Dubreuil et al., 2022.

With regard to the results linking selection vector modulation and dimensionality, more work is required to understand the generality of these results, and how practical it would be to apply this type of analysis to neural recordings. For example, it is possible to build a network that uses input modulation and to greatly increase the dimensionality of the network simply by adding additional dimensions that do not directly contribute to the computation. Similarly, neural responses might have additional high-dimensional activity unrelated to the task. My understanding is that the currently proposed method would classify such networks incorrectly, and it is reasonable to imagine that the dimensionality of activity in high-order brain regions will be strongly dependent on activity that does not relate to this task.

Finally, a number of aspects of the analysis are not clear. The most important element to clarify is how the authors quantify the "proportion of selection vector modulation" in vanilla RNNs (Figures 7d and 7g). I could not find information about this in the Methods, yet this is a critical element of the study results. In Mante et al., 2013 and in Pagan et al., 2022 this was done by analyzing the RNN linearized dynamics around fixed points: is this the approach used also in this study? Also, how are the authors producing the trial-averaged analyses shown in Figures 2f and 3f? The methods used to produce this type of plot differ in Mante et al., 2013 and Pagan et al., 2022, and it is necessary for the authors to explain how this was computed in this case.

I am also confused by a number of analyses done to verify mathematical derivations, which seem to suggest that the results are close to identical, but not exactly identical. For example, in the histogram in Figure 6b, or the histogram in Figure 7-figure supplement 3d: what is the source of the small variability leading to some of the indices being less than 1?

Reviewer #2 (Public review):

This manuscript examines network mechanisms that allow networks of neurons to perform context-dependent decision-making.

In a recent study, Pagan and colleagues identified two distinct mechanisms by which recurrent neural networks can perform such computations. They termed these two mechanisms input-modulation and selection-vector modulation. Pagan and colleagues demonstrated that recurrent neural networks can be trained to implement combinations of these two mechanisms, and related this range of computational strategies with inter-individual variability in rats performing the same task. What type of structure in the recurrent connectivity favors one or the other mechanism however remained an open question.

The present manuscript addresses this specific question by using a class of mechanistically interpretable recurrent neural networks, low-rank RNNs.

The manuscript starts by demonstrating that unit-rank RNNs can only implement the input-modulation mechanism, but not the selection-vector modulation. The authors then build rank three networks that implement selection-vector modulation and show how the two mechanisms can be combined. Finally, they relate the amount of selection-vector modulation with the effective rank, ie the dimensionality of activity, of a trained full-rank RNN.

Strengths:

(1) The manuscript is written in a straightforward manner.<br /> (2) The analytic approach adopted in the manuscript is impressive.<br /> (3) Very clear identification of the mechanisms leading to the two types of context-dependent modulation.<br /> (4) Altogether this manuscript reports remarkable insights into a very timely question.

Weaknesses:

- The introduction could have been written in a more accessible manner for any non-expert readers.

Reviewer #1 (Public review):

Summary:

This paper advances a new understanding of plasticity in artificial neural networks. It shows that weight changes can be decomposed into two components: the first governs the magnitude (or gain) of responses in a particular layer; the second governs the relationship of those responses to the input to that layer. Then, it shows that separate control of these two factors via a surprise-based metaplasticity can avoid catastrophic forgetting as well as induce successful generalization in different conditions, through a series of simulation experiments in linear networks. The authors argue that separate control of the two factors may be at work in the brain and may underlie the ability of humans and other animals to perform successful sequential learning. The paper is hampered by confusing terminology and the precise setup of some of the simulations is unclear. The paper also focuses exclusively on the linear case, which limits confidence in the generality of the results. The paper would also benefit from the inclusion of specific predictions for neural data that would confirm the idea that the separate control of these two factors underlies successful continual learning in the brain.

Strengths:

(1) The theoretical framework developed by the paper is interesting, and could have wide applicability for both training networks and for understanding plasticity.

(2) The simulations convincingly show benefits to the coordinated eligibility model of plasticity advanced by the authors.

Weaknesses:

(1) The simulation results are limited to simple tasks in linear networks, it would be interesting to see how the intuitions developed in the linear case extend to nonlinear networks.

(2) The terminology is somewhat confusing and this can make the paper difficult to follow in some places.

(3) The details of some of the simulations are lacking.

Reviewer #2 (Public review):

Summary:

Scott and Frank propose a new method for controlling generalization and interference in neural networks that undergo continual learning. Their method called coordinated eligibility models (CEM), relies on the factorization of synaptic updates into input-driven and output-driving factors. They subsequently employ the fact that it is sufficient to orthogonalize any one of these two factors across different data points to nullify the interference during learning. They exemplify this on a number of toy tasks while comparing their result to vanilla gradient.

Strengths:

The specific mechanism proposed here is novel (while, as authors acknowledge, there is a large number of other mechanisms for the selective recruitment of synapses for the prevention of catastrophic forgetting). Furthermore, it is simple, elegant, and to a large extent biologically plausible, potentially pointing to specific and testable aspects of learning dynamics.

Weaknesses:

(1) Scope and toy nature of experiments: the model was only applied to very simple problems tailored specifically to demonstrate the strengths of the CEM method. Furthermore, single hyperparameter setting is presented for every scenario which leaves it questionable how general the numerical results are. The selection of input, output dimensionality and data set size also seems to be underexplored. Will a larger curriculum, smaller or larger dimension, compromise any of the CEM ingredients? Restriction to linear models seems arbitrary (it should be a no-time test to add non-linearity within a pytorch framework that authors used), and applicability for any non-synthetic problem is not obvious.

It is also unclear to what extent of domain knowledge is needed for surprise signals to be successfully generated. Can the authors make a stronger case about novel curriculum entries being easily recognizable by cosine distance, either in the brain or in machine learning? Can they alternatively demonstrate their method on a less toy benchmark (e.g. permuted MNIST from Kirkpatrick et al 2017 that they cite)?

Another limitation is that unlike smoother models of plasticity budgets (e.g. Kirkpatrick et al 17, Zenke et al 17), here eligibility seems to be lost forever, once surprise is applied. What happens to the model if more data from a previously visited task becomes available? Will the system be able to continue learning within the right context and how does CEM perform compared to other catastrophic-forgetting-prevention strategies?

(2) The clarity and organization must be improved. Specifically, the balance between verbal descriptions, equations, figures, and their captions needs to be improved. For example - two full-size equations are dedicated to the application of linear regression (around lines 183 and 236) while by far less obvious math such as settings for fig 7, including 'feature loadings', 'demands', etc., is presented in a hardly readable mixture figure and main text. Similarly, the surprise mechanism which is a key ingredient for the model is presented in a very non-straightforward fashion, scattered between the main text, figure, and methods. The figure legends are poorly informative in many cases as well (see minor comments for examples).

Reviewer #3 (Public review):

Summary:

This paper describes a modification of gradient descent learning, and shows in several simulations that this modification allows online learning of linear regression problems where naive gradient descent fails. The modification starts from the observation that the rank-1 weight update of online gradient learning can be written as the outer product Δw ∝ g xᵀ of a vector g and the input x. Modifying this update rule, by projecting g or x to some subspaces, i.e. Δw ∝ Pg (Qx)ᵀ, allows for preventing the typical catastrophic forgetting behavior of online gradient descent, as confirmed in the simulations. The projection matrices P and Q are updated with a "surprise"-modulation rule.

Strengths:

I find it interesting to explore the benefits of alternatives to naive online gradient learning for continual learning.

Weaknesses:

The novelty and advancement in our theoretical understanding of plasticity in neural systems are unclear. I appreciate gaining insights from simple mathematical arguments and simulations with toy models, but for this paper, I do not yet clearly see what I learned: on the mathematical/ML/simulation side it is unclear how it relates to the continual learning literature, on the neuroscience/surprise side I see only a number of papers cited but not any clear connection to data or novel insights.

More specifically:

(1) It is unclear what exactly the "coordinated eligibility theory" is. Is any update rule that satisfies Equation 4 included in the coordinated eligibility theory? If yes, what is the point: any update rule can be written in this way, including standard online gradient descent. If no, what is it? It is not Equation 5 it seems, because this is called "one of the simplest coordinated eligibility models".

(2) There is a lot of work on continual learning which is not discussed, e.g. "Orthogonal Gradient Descent for Continual Learning" (Farajtabar et al. 2019), "Continual learning in low-rank orthogonal subspaces" (Chaudhry et al. 2020), or "Keep Moving: identifying task-relevant subspaces to maximise plasticity for newly learned tasks" (Anthes et al. 2024), to name just a few. What is the novelty of this work relative to these existing works? Is the novelty in the specific projection operator? If yes, what are the benefits of this projection operator in theory and simulations? How would, for example, the approach of Farajtabar et al. 2019 perform on the tasks in Figures 3-7?

(3) There is also work on using surprise signals for multitask learning in models of biological neural networks, e.g. "Fast adaptation to rule switching using neuronal surprise" (Barry et al. 2023).

(4) What is the motivation for the projection to the unit sphere in Equation 5?

(5) What is the motivation for the surprise definition? For example, why cos(x⋅μ) = cos(|x||μ|cos(θ)) = cos(cos(θ))? (Assuming x and μ have unit length and θ is the angle between x and μ).

Reviewer #1 (Public review):

This paper presents a comprehensive study of how neural tracking of speech is affected by background noise. Using five EEG experiments and Temporal response function (TRF), it investigates how minimal background noise can enhance speech tracking even when speech intelligibility remains very high. The results suggest that this enhancement is not attention-driven but could be explained by stochastic resonance. These findings generalize across different background noise types and listening conditions, offering insights into speech processing in real-world environments.

I find this paper well-written, the experiments and results are clearly described. However, I have a few comments that may be useful to address.

(1) The behavioral accuracy and EEG results for clear speech in Experiment 4 differ from those of Experiments 1-3. Could the author provide insights into the potential reasons for this discrepancy? Might it be due to linguistic/ acoustic differences between the passages used in experiments? If so, what was the rationale behind using different passages across different experiments?

(2) Regarding peak amplitude extraction, why were the exact peak amplitudes and latencies of the TRFs for each subject not extracted, and instead, an amplitude average within a 20 ms time window based on the group-averaged TRFs used? Did the latencies significantly differ across different SNR conditions?

(3) How is neural tracking quantified in the current study? Does improved neural tracking correlate with EEG prediction accuracy or individual peak amplitudes? Given the differing trends between N1 and P2 peaks in babble and speech-matched noise in experiment 3, how is it that babble results in greater envelope tracking compared to speech-matched noise?

(4) The paper discusses how speech envelope-onset tracking varies with different background noises. Does the author expect similar trends for speech envelope tracking as well? Additionally, could you explain why envelope onsets were prioritized over envelope tracking in this analysis?

Reviewer #2 (Public review):

The author investigates the role of background noise on EEG-assessed speech tracking in a series of five experiments. In the first experiment, the influence of different degrees of background noise is investigated and enhanced speech tracking for minimal noise levels is found. The following four experiments explore different potential influences on this effect, such as attentional allocation, different noise types, and presentation mode.

The step-wise exploration of potential contributors to the effect of enhanced speech tracking for minimal background noise is compelling. The motivation and reasoning for the different studies are clear and logical and therefore easy to follow. The results are discussed in a concise and clear way. While I specifically like the conciseness, one inevitable consequence is that not all results are equally discussed in depth.

Based on the results of the five experiments, the author concludes that the enhancement of speech tracking for minimal background noise is likely due to stochastic resonance. Given broad conceptualizations of stochastic resonance as a noise benefit this is a reasonable conclusion.

This study will likely impact the field as it provides compelling support questioning the relationship between speech tracking and speech processing.

Reviewer #1 (Public review):

Summary:

Sun et al. are interested in how experience can shape the brain and specifically investigate the plasticity of the Toll-6 receptor-expressing dopaminergic neurons (DANs). To learn more about the role of Toll-6 in the DANs, the authors examine the expression of the Toll-6 receptor ligand, DNT-2. They show that DNT-2 expressing cells connect with DANs and that loss of function of DNT-2 in these cells reduces the number of PAM DANs, while overexpression causes alterations in dendrite complexity. Finally, the authors show that alterations in the levels of DNT-2 and Toll-6 can impact DAN-driven behaviors such as climbing, arena locomotion, and learning and long-term memory.

Strengths:

The authors methodically test which neurotransmitters are expressed by the 4 prominent DNT-2 expressing neurons and show that they are glutamatergic. They also use Trans-Tango and Bac-TRACE to examine the connectivity of the DNT-2 neurons to the dopaminergic circuit and show that DNT-2 neurons receive dopaminergic inputs and output to a variety of neurons including MB Kenyon cells, DAL neurons, and possibly DANS.

Weaknesses:

(1) To identify the DNT-2 neurons, the authors use CRISPR to generate a new DN2-GAL4. They note that they identified at least 12 DNT-2 plus neurons. In Supplementary Figure 1A, the DNT-2-GAL4 driver was used to express a UAS-histoneYFP nuclear marker. From these figures, it looks like DNT-2-GAL4 is labeling more than 12 neurons. Is there glial expression? This question is relevant as it is not clear how many other cell types are being manipulated with the DNT-2-GAL4 driver is used in subsequent experiments. For example, is DNT-2-GAL4--> DNT-2-RNAi is reducing DNT2 in many neurons or glia effects could be indirect.

(2) In Figure 2C the authors show that DNT-2 upregulation leads to an increase in TH levels using q-RT-PCR from whole heads. However, in Figure 3G they also show that DNT-2 overexpression also causes an increase in the number of TH neurons. It is unclear whether TH RNA increases due to expression/cell or number of TH neurons in the head.

(3)DNT-2 is also known as Spz5 and has been shown to activate Toll-6 receptors in glia (McLaughlin et al., 2019), resulting in the phagocytosis of apoptotic neurons. In addition, the knockdown of DNT-2/Spz5 throughout development causes an increase in apoptotic debris in the brain, which can lead to neurodegeneration. Indeed Figure 3H shows that an adult-specific knockdown of DNT-2 using DNT2-GAL4 causes an increase in Dcp1 signal in many neurons and not just TH neurons.

Comments on revisions:

The authors have made some changes in the text to tone down their claims. They have also provided additional images to support their work. However, requested controls are not provided, and new experiments are not added to address reviewer concerns.

Reviewer #2 (Public review):

This paper examines how structural plasticity in neural circuits, particularly in dopaminergic systems, is regulated by Drosophila neurotrophin-2 (DNT-2) and its receptors, Toll-6 and Kek-6. The authors show that these molecules are critical for modulating circuit structure, dopaminergic neuron survival, synaptogenesis, and connectivity. They demonstrate that the loss of DNT-2 or Toll-6 function leads to the loss of dopaminergic neurons, reduced dendritic arborization, and synaptic impairment, whereas overexpression of DNT-2 increases dendritic complexity and synaptogenesis. Additionally, DNT-2 and Toll-6 influence dopamine-dependent behaviors, including locomotion and long-term memory, suggesting a link between DNT-2 signaling, structural plasticity, and behavior.

A major strength of this study is the impressive cellular resolution achieved. By focusing on specific dopaminergic neurons, such as the PAM and PPL1 clusters, and using a range of molecular markers, the authors were able to clearly visualize intricate details of synapse formation, dendritic complexity, and axonal targeting within defined circuits. Given the critical role of dopaminergic pathways in learning and memory, this approach provides a valuable foundation for exploring the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. While the manuscript hints at a connection to experience-induced plasticity, the study does not establish a direct causal link between neurotrophin signaling and experience-driven changes. To support this idea, it would be necessary to observe experience-induced structural changes and demonstrate that downregulation of DNT-2 signaling prevents these changes. The closest attempt in this study was the artificial activation of DNT-2 neurons using TrpA1, which resulted in overgrowth of axonal arbors and an increase in synaptic sites in both DNT-2 and PAM neurons. However, whether the observed structural changes were dependent on DNT-2 signaling remains unclear.

In conclusion, this study demonstrates that DNT-2 and its receptors play a role in regulating the structure of dopaminergic circuits in the adult fly brain. Whether DNT-2 signaling contributes to experience-dependent structural plasticity within these circuits remains an exciting open question and warrants further investigation.

Comments on revisions:

I appreciate the authors' responses to my previous comments and have no further suggestions.

Reviewer #3 (Public review):

Summary:

The authors used the model organism Drosophila melanogaster to show that the neurotrophin Toll-6 and its ligands, DNT-2 and kek-6, play a role in maintaining the number of dopaminergic neurons and modulating their synaptic connectivity. This supports previous findings on the structural plasticity of dopaminergic neurons and suggests a molecular mechanism underlying this plasticity.

Strengths:

The experiments are overall very well designed and conclusive. Methods are in general state-of-the-art, the sample sizes are sufficient, the statistical analyses are sound, and all necessary controls are at place. The data interpretation is straight forwards, and the relevant literature is taken into consideration. Overall, the manuscript is solid and presents novel, interesting and important findings.

Weaknesses:

There are three technical weaknesses that could perhaps be improved.

First, the model of reciprocal, inhibitory feedback loops (figure 2F) is speculative. On the one hand, glutamate can act in flies as excitatory or inhibitory transmitter (line 157!), and either situation can be the case here. On the other hand, it is not clear how an increase or decrease in cAMP level translates into transmitter release. One can only conclude that two type of neurons potentially influence each other.

Second, the quantification of bouton volumes (no y-axis label in Figure 5 C and D!) and dendrite complexity are not convincingly laid out. Here, the reader expects fine-grained anatomical characterizations of the structures under investigation, and a method to precisely quantify the lengths and branching patterns of individual dendritic arborizations as well as the volume of individual axonal boutons.

Third, figure 1C shows two neurons with the goal of demonstrating between-neuron variability. It is not convincingly demonstrated that the two neurons are actually of the very same type of neuron in different flies, or two completely different neurons.

Review of the revised manuscript:

The authors have addressed some points of concern raised by the reviewers. I would like to emphasize that I find the overall research study highly interesting and important.

Reviewer #2 (Public review):

Summary:

The authors investigated the roles of IncRNA Malat1 in bone homeostasis which was initially believed to be non-functional for physiology. They found that both Malat1 KO and conditional KO in osteoblast lineage exhibit significant osteoporosis due to decreased osteoblast bone formation and increased osteoclast resorption. More interestingly, they found that deletion of Matat1 in osteoclast lineage cell does not affect osteoclast differentiation and function. Mechanistically, they found that Malat1 acts as an co-activator of b-Catenin directly regulating osteoblast activity and indirectly regulating osteoclast activity via mediating OPG, but not RANKL expression in osteoblast and chondrocyte. Their discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling.

Strengths:

The authors generated global and conditional KO mice in osteoblast and osteoclast lineage cells and carefully analyzed the role of Matat1 with both in vivo and in vitro system. The conclusion of this paper is mostly well supported by data.

Comments on revised version:

The authors have addressed all my concerns.

Reviewer #3 (Public review):

Summary:

In this manuscript, Qin and colleagues study the role of Malat1 in bone biology. This topic is interesting given the role of lncRNAs in multiple physiologic processes. A previous study (PMID 38493144) suggested a role for Malat1 in osteoclast maturation. However, the role of this lncRNA in osteoblast biology was previously not explored. Here, the authors note osteopenia with increased bone resorption in mice lacking Malat1 globally and in osteoblast lineage cells. At the mechanistic level, the authors suggest that Malat1 controls beta-catenin activity. These result advance the field regarding the role of this lncRNA in bone biology.

Strengths:

The manuscript is well-written and data are presented in a clear and easily understandable manner. The bone phenotype of osteoblast-specific Malat1 knockout mice is of high interest. The role of Malat1 in controlling beta-catenin activity and OPG expression is interesting and novel.

Weaknesses:

The lack of a bone phenotype when Malat1 is deleted with LysM-Cre is of interest given the previous report suggesting a role for this lncRNA in osteoclasts, especially in light of satisfactory deletion efficiency in this model. The data in the fracture model in Figure 8 is enhanced with quantitative data. The role of Malat1 and OPG in chondrocytes is unclear since the osteocalcin-Cre mice (which should retain normal Malat1 levels in chondrocytes) have similar bone loss as the global mutants.

Comments on revised version:

All previous comments have been addressed in a satisfactory manner.

Reviewer #1 (Public review):

Summary:

SUFU modulates Sonic hedgehog (SHH) signaling and is frequently mutated in the B-subtype of SHH driven medulloblastoma. The B-subtype occurs mostly in infants, is often metastatic, and lacks specific treatment. Yabut et al. found Fgf5 was highly expressed in the B-subtype of SHH driven medulloblastoma by examining a published microarray expression dataset. They then investigated how Fgf5 functions in the cerebellum of mice that have embryonic Sufu loss of function. This loss was induced using the hGFAP-cre transgene, which is expressed multiple cell types in the developing cerebellum, including granule neuron precursors (GNPs) derived from the rhombic lip. By measuring the area of Pax6+ cells in the external granule cell layer (EGL) of Sufu-cKO mice at postnatal day 0, they find Pax6+ cells occupy a larger area in the posterior lobe adjacent to the secondary fissure, which is poorly defined. They show that Fgf5 RNA and phosphoErk1/2 immunostaining are also higher in the same disrupted region. Some of the phosphoErk1/2+ cells are proliferative in the Sufu-cKO. Western blot analysis of Gli proteins that modulate SHH signaling found reduced expression and absence of Gli1 activity in the region of cerebellar dysgenesis in Sufu-cKO mice. This suggests the GNP expansion in this region is independent of SHH signaling. Amazingly, intraventricular injection of the FGFR1-2 antagonist AZD4547 from P0-4 and examined histologoically at P7 found the treatment restored cytoarchitecture in the cerebella of Sufu-cKO mice. This is further supported by NeuN immunostaining in the internal granule cell layer, which labels mature, non-diving neurons, and KI67 immunostaining, indicating dividing cells, and primarily found in the EGL. The mice were treated beginning at a timepoint when cerebellar cytoarchitecture was shown to be disrupted and it is indistinguishable from control following treatment. Fig.3 presents the most convincing and exciting data in this manuscript.

Sufu-cKO do not readily develop cerebellar tumors. The authors detected phosphorylated H2AX immunostaining, which labels double strand breaks, was in some cells in the EGL in regions of cerebellar dysgenesis in the Sufu-cKO, as was cleaved Caspase 3, a marker of apoptosis. P53, downstream of the double strand break pathway, protein was reduced in Sufu-cKO cerebellum. Genetically removing p53 from the Sufu-cKO cerebellum resulted in cerebellar tumors in 2 mo mice. The Sufu;p53-dKO cerebella at P0 lacked clear foliation, and the secondary fissure, even more so than the Sufu-cKO. Fgf5 RNA and signaling (pERK1/2) were also expressed ectopically.

In the revised manuscript, additional details have been added to clarify statistical analyses and to state limitations of the reported results in the absence of further experimental analyses.

Reviewer #2 (Public review):

Summary:

Mutations in SUFU are implicated in SHH medulloblastoma (MB). SUFU modulates Shh signaling in a context-dependent manner, making its role in MB pathology complex and not fully understood. This study reports that elevated FGF5 levels are associated with a specific subtype of SHH MB, particularly in pediatric cases. The authors demonstrate that Sufu deletion in a mouse model leads to abnormal proliferation of granule cell precursors (GCPs) at the secondary fissure (region B), correlating with increased Fgf5 expression. Notably, pharmacological inhibition of FGFR restores normal cerebellar development in Sufu mutant mice.

Strengths:

The identification of increased FGF5 in subsets of MB is novel and a key strength of the paper.

Weaknesses:

The study appears incomplete despite the potential significance of these findings. The current paper does not fully establish the causal relationship between Fgf5 and abnormal cerebellar development, nor does it clarify its connection to SUFU-related MB. Some conclusions seem overstated, and the central question of whether FGFR inhibition can prevent tumor formation remains untested.

Comments on revisions:

In this revised version, many of the concerns and comments raised by this and other reviewers remain unaddressed and require attention in future studies. The manuscript does not demonstrate significant improvement.

Specific Comments:

(1) In the figure provided by the authors, FGF5 appears to be highly expressed beneath the GCPs. Regarding our comment and Reviewer 1's Comment 7, it is essential to identify the cell types secreting FGF5 and clarify whether it functions in a paracrine or autocrine manner. This should be incorporated into the working model illustrated in Figure 5.<br /> (2) Contrary to the authors' claim that their results align completely with Jiwani et al. (2020), there is a discrepancy in the data. Jiwani et al. reported an increase in Gli2 levels in the Sufu mutant, whereas the current study shows the opposite result. This inconsistency needs to be addressed.

Reviewer #3 (Public review):

Summary:

The interaction between FGF signaling and SHH-mediated GNP expansion in MB, particularly in the context of Sufu LoF, has just begun to be understood. The manuscript by Yabut et al. establishes a connection between ectopic FGF5 expression and GNP over-expansion in a late stage embryonic Sufu LoF model. The data provided links region-specific interaction between aberrant FGF5 signaling with SHH subtype of medulloblastoma. New data from Yabut et al. suggest that ectopic FGF5 expression correlates with GNP expansion near the secondary fissure in Sufu LoF cerebella. Furthermore, pharmacological blockade of FGF signaling inhibits GNP proliferation. Interestingly, the data indicate that the timing of conditional Sufu deletion (E13.5 using the hGFAP-Cre line) results in different outcomes compared to later deletion (using Math1-cre line, Jiwani et al., 2020). This study provides significant insights into the molecular mechanisms driving GNP expansion in SHH subgroup MB, particularly in the context of Sufu LoF. It highlights the potential of targeting FGF5 signaling as a therapeutic strategy. Additionally, the research offers a model for better understanding MB subtypes and developing targeted treatments.

Strengths:

One notable strength of this study is the extraction and analysis of ectopic FGF5 expression from a subset of MB patient tumor samples. This translational aspect of the study enhances its relevance to human disease. By correlating findings from mouse models with patient data, the authors strengthen the validity of their conclusions and highlight the potential clinical implications of targeting FGF5 in MB therapy.

The data convincingly show that FGFR signaling activation drives GNP proliferation in Sufu conditional knockout models. This finding is supported by robust experimental evidence, including pharmacological blockade of FGF signaling, which effectively inhibits GNP proliferation. The clear demonstration of a functional link between FGFR signaling and GNP expansion underscores the potential of FGFR as a therapeutic target in SHH subgroup medulloblastoma.

Previous studies have demonstrated the inhibitory effect of FGF2 on tumor cell proliferation in certain MB types, such as the ptc mutant (Fogarty et al., 2006)(Yaguchi et al., 2009). Findings in this manuscript provide additional support suggesting multiple roles for FGF signaling in cerebellar patterning and development.

Weaknesses:

In the GEO dataset analysis, where FGF5 expression is extracted, the reporting of the P-value lacks detail on the statistical methods used, such as whether an ANOVA or t-test was employed. Providing comprehensive statistical methodologies is crucial for assessing the rigor and reproducibility of the results. The absence of this information raises concerns about the robustness of the statistical analysis.

Another concern is related to the controls used in the study. Cre recombinase induces double-strand DNA breaks within the loxP sites, and the control mice did not carry the Cre transgene (as stated in the Method section), while Sufu-cKO mice did. This discrepancy necessitates an additional control group to evaluate the effects of Cre-induced double-strand breaks on phosphorylated H2AX-DSB signaling. Including this control would strengthen the validity of the findings by ensuring that observed effects are not artifacts of Cre recombinase activity.

Although the use of the hGFAP-Cre line allows genetic access to late embryonic stage, this also targets multiple cell types, including both GNPs and cerebellar glial cells. However, the authors focus primarily on GNPs without fully addressing the potential contributions of neuron-glial interaction. This oversight could limit the understanding of the broader cellular context in which FGF signaling influences tumor development.

- Statistical analysis from the Geo expression dataset:<br /> The reviewer is satisfied with the revisions provided by the author and considers Figure 1 substantially improved.

- Generation of Sufu-cKO;Gli1-LacZ triple transgenic mice not described:<br /> >The reviewer finds that the supplementary Figure 1 revisions provided by the author do not fully address the concerns raised, and the issue remains unresolved.

- Request control group to evaluate the effects of Cre induced double-strand breaks on phosphorylated H2AX-DSB signaling:<br /> >Despite the revisions, control group (hGFAPCre;Sufu-fl/+) highlighted in the author response does not present in the revision, leaving this issue unresolved.

- hGFAP-Cre line also targets multiple celltypes, including both GNPs and cerebellar glial cells:<br /> >The author acknowledges the limitations of the study, and the reviewer concurs, noting that it enhances the contextual understanding of the findings.

Joint Public Review:

Summary:

If synaptic input is functionally clustered on dendrites, nonlinear integration could increase the computational power of neural networks. But this requires the right synapses to be located in the right places. This paper aims to address the question of whether such synaptic arrangements could arise by chance (i.e. without special rules for axon guidance or structural plasticity), and could therefore be exploited even in randomly connected networks. This is important, particularly for the dendrites and biological computation communities, where there is a pressing need to integrate decades of work at the single-neuron level with contemporary ideas about network function.

Using an abstract model where ensembles of neurons project randomly to a postsynaptic population, back-of-envelope calculations are presented that predict the probability of finding clustered synapses and spatiotemporal sequences. Using data-constrained parameters, the authors conclude that clustering and sequences are indeed likely to occur by chance (for large enough ensembles), but require strong dendritic nonlinearities and low background noise to be useful.

Strengths:

- The back-of-envelope reasoning presented can provide fast and valuable intuition. The authors have also made the effort to connect the model parameters with measured values. Even an approximate understanding of cluster probability can direct theory and experiments towards promising directions, or away from lost causes.

- I found the general approach to be refreshingly transparent and objective. Assumptions are stated clearly about the model and statistics of different circuits. Along with some positive results, many of the computed cluster probabilities are vanishingly small, and noise is found to be quite detrimental in several cases. This is important to know, and I was happy to see the authors take a balanced look at conditions that help/hinder clustering, rather than just focus on a particular regime that works.

- This paper is also a timely reminder that synaptic clusters and sequences can exist on multiple spatial and temporal scales. The authors present results pertaining to the standard `electrical' regime (~50-100 µm, <50 ms), as well as two modes of chemical signaling (~10 µm, 100-1000 ms). The senior author is indeed an authority on the latter, and the simulations in Figure 5, extending those from Bhalla (2017), are unique in this area. In my view, the role of chemical signaling in neural computation is understudied theoretically, but research will be increasingly important as experimental technologies continue to develop.

(Editors' note: the paper has been through two rounds of revisions and the authors are encouraged to finalise this as the Version of Record. The earlier reviews are here: https://elifesciences.org/reviewed-preprints/100664v2/reviews)

Reviewer #1 (Public review):

In the current manuscript, the authors use theoretical and analytical tools to examine the possibility of neural projections to engage ensembles of synaptic clusters in active dendrites. The analysis is divided into multiple models that differ in the connectivity parameters, speed of interactions and identity of the signal (electric vs. second messenger). They first show that random connectivity almost ensures the representation of presynaptic ensembles. As expected, this convergence is much more likely for small group sizes and slow processes, such as calcium dynamics. Conversely, fast signals (spikes and postsynaptic potentials) and large groups are much less likely to recruit spatially clustered inputs. Dendritic nonlinearity in the postsynaptic cells was found to play a highly important role in distinguishing these clustered activation patterns, both when activated simultaneously and in sequence. The authors tackled the difficult issue of noise, showing a beneficiary effect when noise 'happen' to fill in gaps in a sequential pattern but degraded performance at higher background activity levels. Last, the authors simulated selectivity to chemical and electrical signals. While they find that longer sequences are less perturbed by noise, in more realistic activation conditions, the signals are not well resolved in the soma.

While I think the premise of the manuscript is worth exploring, I have a number of reservations regarding the results.

(1) In the analysis, the authors made a simplifying assumption that the chemical and electrical processes are independent. However, this is not the case; excitatory inputs to spines often trigger depolarization combined with pronounced calcium influx; this mixed signaling could have dramatic implications on the analysis, particularly if the dendrites are nonlinear (see below)<br /> (2) Sequence detection in active dendrites is often simplified to investigating activation in a part of or the entirety of individual branches. However, the authors did not do that for most of their analysis. Instead, they treat the entire dendritic tree as one long branch and count how many inputs form clusters. I fail to see why the simplification is required and suspect it can lead to wrong results. For example, two inputs that are mapped to different dendrites in the 'original' morphology but then happen to fall next to each other when the branches are staggered to form the long dendrites would be counted as neighbors.<br /> (3) The simulations were poorly executed. Figures 5 and 6 show examples but no summary statistics. The authors emphasize the importance of nonlinear dendritic interactions, but they do not include them in their analysis of the ectopic signals! I find it to be wholly expected that the effects of dendritic ensembles are not pronounced when the dendrites are linear.

To provide a comprehensive analysis of dendritic integration, the authors could simulate more realistic synaptic conductances and voltage-gated channels. They would find much more complicated interactions between inputs on a single site, a sliding temporal and spatial window of nonlinear integration that depends on dendritic morphology, active and passive parameters and synaptic properties. At different activation levels, the rules of synaptic integration shift to cooperativity between different dendrites and cellular compartments, further complicated by nonlinear interactions between somatic spikes and dendritic events.

While it is tempting to extend back-of-the-napkin calculations of how many inputs can recruit nonlinear integration in active dendrites, the biological implementation is very different from this hypothetical. It is important to consider these questions, but I am not convinced that this manuscript adequately addressed the questions it set out to probe, nor does it provide information that was unknown beforehand.

Update after the first revision:

In this revision, the authors significantly improved the manuscript. They now address some of my concerns. Specifically, they show the contribution of end-effects on spreading the inputs between dendrites. This analysis reveals greater applicability of their findings to cortical cells, with long, unbranching dendrites than other neuronal types, such as Purkinje cells in the cerebellum.

They now explain better the interactions between calcium and voltage signals, which I believe improve the take-away message of their manuscript. They modified and added new figures that helped to provide more information about their simulations.<br /> However, some of my points remain valid. Figure 6 shows depolarization of ~5mV from -75. This weak depolarization would not effectively recruit nonlinear activation of NMDARs. In their paper, Branco and Hausser (2010) showed depolarizations of ~10-15mV. More importantly, the signature of NMDAR activation is the prolonged plateau potential and activation at more depolarized resting membrane potentials (their Figure 4). Thus, despite including NMDARs in the simulation, the authors do not model functional recruitment of these channels. Their simulation is thus equivalent to AMPA only drive, which can indeed summate somewhat nonlinearly.

Reviewer #2 (Public review):

Summary:

If synaptic input is functionally clustered on dendrites, nonlinear integration could increase the computational power of neural networks. But this requires the right synapses to be located in the right places. This paper aims to address the question of whether such synaptic arrangements could arise by chance (i.e. without special rules for axon guidance or structural plasticity), and could therefore be exploited even in randomly connected networks. This is important, particularly for the dendrites and biological computation communities, where there is a pressing need to integrate decades of work at the single-neuron level with contemporary ideas about network function.

Using an abstract model where ensembles of neurons project randomly to a postsynaptic population, back-of-envelope calculations are presented that predict the probability of finding clustered synapses and spatiotemporal sequences. Using data-constrained parameters, the authors conclude that clustering and sequences are indeed likely to occur by chance (for large enough ensembles), but require strong dendritic nonlinearities and low background noise to be useful.

Strengths:

- The back-of-envelope reasoning presented can provide fast and valuable intuition. The authors have also made the effort to connect the model parameters with measured values. Even an approximate understanding of cluster probability can direct theory and experiments towards promising directions, or away from lost causes.

- I found the general approach to be refreshingly transparent and objective. Assumptions are stated clearly about the model and statistics of different circuits. Along with some positive results, many of the computed cluster probabilities are vanishingly small, and noise is found to be quite detrimental in several cases. This is important to know, and I was happy to see the authors take a balanced look at conditions that help/hinder clustering, rather than just focus on a particular regime that works.

- This paper is also a timely reminder that synaptic clusters and sequences can exist on multiple spatial and temporal scales. The authors present results pertaining to the standard `electrical' regime (~50-100 µm, <50 ms), as well as two modes of chemical signaling (~10 µm, 100-1000 ms). The senior author is indeed an authority on the latter, and the simulations in Figure 5, extending those from Bhalla (2017), are unique in this area. In my view, the role of chemical signaling in neural computation is understudied theoretically, but research will be increasingly important as experimental technologies continue to develop.

Weaknesses:

- The paper is mostly let down by the presentation. In the current form, some patience is needed to grasp the main questions and results, and it is hard to keep track of the many abbreviations and definitions. A paper like this can be impactful, but the writing needs to be crisp, and the logic of the derivation accessible to non-experts. See, for instance, Stepanyants, Hof & Chklovskii (2002) for a relevant example.

It would be good to see a restructure that communicates the main points clearly and concisely, perhaps leaving other observations to an optional appendix. For the interested but time-pressed reader, I recommend starting with the last paragraph of the introduction, working through the main derivation on page 7, and writing out the full expression with key parameters exposed. Next, look at Table 1 and Figure 2J to see where different circuits and mechanisms fit in this scheme. Beyond this, the sequence derivation on page 17 and biophysical simulations in Figures 5 and 6 are also highlights.

- The analysis supporting the claim that strong nonlinearities are needed for cluster/sequence detection is unconvincing. In the analysis, different synapse distributions on a single long dendrite are convolved with a sigmoid function and then the sum is taken to reflect the somatic response. In reality, dendritic nonlinearities influence the soma in a complex and dynamic manner. It may be that the abstract approach the authors use captures some of this, but it needs to be validated with simulations to be trusted (in line with previous work, e.g. Poirazi, Brannon & Mel, (2003)).

- It is unclear whether some of the conclusions would hold in the presence of learning. In the signal-to-noise analysis, all synaptic strengths are assumed equal. But if synapses involved in salient clusters or sequences were potentiated, presumably detection would become easier? Similarly, if presynaptic tuning and/or timing was reorganized through learning, the conditions for synaptic arrangements to be useful could be relaxed. Answering these questions is beyond the scope of the study, but there is a caveat there nonetheless.

Reviewer #1 (Public review):

De Seze et al. investigated the role of guanine exchange factors (GEFs) in controlling cell protrusion and retraction. In order to causally link protein activities to the switch between the opposing cell phenotypes, they employed optogenetic versions of GEFs which can be recruited to the plasma membrane upon light exposure and activate their downstream effectors. Particularly the RhoGEF PRG could elicit both protruding and retracting phenotypes. Interestingly, the phenotype depended on the basal expression level of the optoPRG. By assessing the activity of RhoA and Cdc42, the downstream effectors of PRG, the mechanism of this switch was elucidated: at low PRG levels, RhoA is predominantly activated and leads to cell retraction, whereas at high PRG levels, both RhoA and Cdc42 are activated but PRG also sequesters the active RhoA, therefore Cdc42 dominates and triggers cell protrusion. Finally, they create a minimal model that captures the key dynamics of this protein interaction network and the switch in cell behavior.

The conclusions of this study are strongly supported by data, harnessing the power of modelling and optogenetic activation. The minimal model captures well the dynamics of RhoA and Cdc42 activation and predicts that by changing the frequency of optogenetic activation one can switch between protruding and retracting behaviour in the same cell of intermediate optoPRG level. The authors are indeed able to demonstrate this experimentally albeit with a very low number of cells. A major caveat of this study is that global changes due to PRG overexpression cannot be ruled out. Also, a quantification of absolute protein concentration, which is notoriously difficult, would be useful to put the level of overexpression here in perspective with endogenous levels. Furthermore, it remains unclear whether in cases of protein overexpression in vivo such as cancer, PRG or other GEFs can activate alternative migratory behaviours.

Previous work has implicated RhoA in both protrusion and retraction depending on the context. The mechanism uncovered here provides a convincing explanation for this conundrum. In addition to PRG, optogenetic versions of two other GEFs, LARG and GEF-H1, were used which produced either only one phenotype or less response than optoPRG, underscoring the functional diversity of RhoGEFs. The authors chose transient transfection to achieve a large range of concentration levels and, to find transfected cells at low cell density, developed a small software solution (Cell finder), which could be of interest for other researchers.

Reviewer #2 (Public review):

Summary:

This manuscript builds from the interesting observation that local recruitment of the DHPH domain of the RhoGEF PRG can induce local retraction, protrusion, or neither. The authors convincingly show that these differential responses are tied to the level of expression of the PRG transgene. This response depends on the Rho-binding activity of the recruited PH domain and is associated with and requires (co?)-activation of Cdc42. This begs the question of why this switch in response occurs. The use a computational model to predict that the timing of protein recruitment can dictate the output of the response in cells expressing intermediate levels and found that, "While the majority of cells showed mixed phenotypes irrespectively of the activation pattern, in few cells (3 out of 90) we were able to alternate the phenotype between retraction and protrusion several times at different places of the cell by changing the frequency while keeping the same total integrated intensity (Figure 6F and Supp Movie)."

Comments on the revised manuscript:

The authors have addressed the previous points and they have convincingly demonstrated that an optogenetically recruited PRG-GEF acts, as expected, as a GEF for RhoA. However, if this fragment is strongly over-expressed, it activates Cdc42, instead of RhoA. This appears to be due to sequestration of active RhoA by the overexpressed PRG-GEF.

Reviewer #2 (Public review):

Summary:

This study investigated the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on cardiovascular dynamics in subarachnoid hemorrhage (SAH) patients. The researchers conducted a randomized clinical trial with 24 SAH patients, comparing taVNS treatment to a Sham treatment group (20 minutes per day twice a day during the ICU stay). They monitored electrocardiogram (ECG) readings and vital signs to assess acute as well as middle -term changes in heart rate, heart rate variability, QT interval, and blood pressure between the two groups. The results showed that repetitive taVNS did not significantly alter heart rate, corrected QT interval, blood pressure, or intracranial pressure. However, it increased overall heart rate variability and parasympathetic activity after 5-10 days of treatment compared to the sham treatment. Acute taVNS led to an increase in heart rate, blood pressure, and peripheral perfusion index without affecting corrected QT interval, intracranial pressure, or heart rate variability. The acute post-treatment elevation in heart rate was more pronounced in patients who showed clinical improvement. In conclusion, the study found that taVNS treatment did not cause adverse cardiovascular effects, suggesting it as a safe immunomodulatory treatment for SAH patients. The mild acute increase in heart rate post-treatment could potentially serve as a biomarker for identifying SAH patients who may benefit more from taVNS therapy.

Strengths:

The paper is overall well written, and the topic is of great interest. The methods are solid and the presented data are convincing.

Comments on revisions:

The main previous weaknesses of the paper have now been fixed.

Reviewer #3 (Public review):

Summary:

The authors characterized the cardiovascular effects of acute and repetitive taVNS as an index of safety and concluded that taVNS treatment does not induce adverse cardiovascular effects such as bradycardia or QT prolongation.

Strengths:

This study contributes important information about the clinical utility of taVNS as a safe immunomodulatory treatment approach for SAH patients.

Comments on revised version:

A number of limitations were identified previously: https://elifesciences.org/reviewed-preprints/100088/reviews#peer-review-2. These major concerns were largely addressed by the authors.

Reviewer #1 (Public review):

Summary:

The authors introduced neutron crystallography coupled with room temperature X-ray crystallography to exam the redox properties of the BtFt [4Fe-4S] cluster expressed in E. coli. Neutron structure allowed the authors to exam the influence of Asp64 on the redox properties of the [4Fe-4S] cluster. The neutron structure also allowed for the identification of the hydrogen network around the [4Fe-4S] structure. This work was followed by density functional theory calculation to examine different redox states which also pointed to the role of Asp64 in affecting or dictating redox function of the [4Fe-4S] cluster. Based on the DFT work the authors examine the redox properties under oxic and anoxic conditions in wild type enzymes and in a D64N mutant again showing the role of Asp64 on the redox kinetics and redox potential of the [4Fe-4S] cluster. Lastly, the authors examined similar [4Fe-4S] ferredoxins from several organisms and with a Asp64 or Glu64 observed a similar role of Asp64 on the low potential state of the [4Fe-4S] cluster. The major conclusion of the study was to identify the role of specific amino acids, in this case Asp64, in controlling the redox state and kinetics of [4Fe-4S] clusters. The authors also demonstrate the strength of neutron crystallography when combined with classical X-ray crystallography and classical spectral/redox studies.

Strengths:

In general, the experimental design is logical and the results are convincing demonstrating the role of Asp64 on the redox properties of [4Fe-4S] clusters in ferredoxins.

Weaknesses:

The role(s) of coordinating amino acids on the redox properties of a functional group is not surprising, this reviewer believes this is a novel result in ferredoxins and does make a nice contribution to the field.

Reviewer #2 (Public review):

In this study, Wada et al. investigate the low potential ferredoxin from Bacillus thermoproteolyticus (BtFd) using a combination of neutron crystallography, x-ray crystallography, DFT and spectroscopy to determine the influence of hydrogen bonding networks on the redox potential of ferredoxin's 4Fe-4S cluster. The use of neutron diffraction allowed the authors to probe the precise location of hydrogens around the 4Fe-4S cluster, which was not possible from prior studies, even with the previously reported high-resolution (0.92 Å) structure of BtFd. This allowed the authors to revise prior models of the proposed H bonding network theorized from earlier x-ray crystallography studies ( for example, showing that there is not in fact a H bond formed between the Thr63-O𝛾1 and the [4Fe-4S]-S4 atoms). With this newly described H-bonding network established, the electronic structure of the 4Fe-4S cluster was then investigated using DFT methodology, revealing a startling role of the deprotonated surface residue Asp64, which bears substantial electronic density in the LUMO which is otherwise localized to the 4Fe-4S cluster. While aspartate is usually deprotonated at physiological pH, the authors provide compelling evidence that this aspartate has a much higher pKa than is usual, and is able to act as a protonation-dependent switch which controls the stability of the reduced state of the 4Fe-4S cluster, and thus the redox potential.

The findings of this study and the conclusions drawn from them are well supported by the data and computational work. Their findings have implications for similar control mechanisms in other, non-ferredoxin 4Fe-4S bearing electron transport proteins which have yet to be explored, providing great value to the metalloprotein community. One change that the authors may consider to enhance the clarity of the manuscript regards the nomenclature used for the varying models discussed (CM, CMNA, CMH and so forth). It would be beneficial to the reader if the nomenclature included the redox state (ox. vs red.) of the model in the model's name.

Comments on revisions:

I'm satisfied with their revisions, it looks good to me.

Reviewer #1 (Public review):

This manuscript by Bai et al concerns the expression of Scleraxis (Scx) by muscle satellite cells (SCs) and the role of that gene in regenerative myogenesis. The authors report the expression of this gene associated with tendon development in satellite cells. Genetic deletion of Scx in SCs impairs muscle regeneration, and the authors provide evidence that SCs deficient in Scx are impaired in terms of population growth and cellular differentiation. Overall, this report provides evidence of the role of this gene, unexpectedly, in SC function and adult regenerative myogenesis.

There are a few points of concern.

(1) From the data in Figure 1, it appears that all of the SCs, assessed both in vitro and in vivo, express Scx. The authors refer to a scRNA-seq dataset from their lab and one report from mdx mouse muscle that also reveal this unexpected gene expression pattern. Has this been observed in many other scRNA-seq datasets? If not, it would be important to discuss potential explanations as to why this has not been reported previously.

(2) A major point of the paper, as illustrated in Fig. 3, is that Scx-neg SCs fail to produce normal myofibers and renewed SCs following injury/regeneration. They mention in the text that there was no increased PCD by Caspase staining at 5 DPI. A failure of cell survival during the process of SC activation, proliferation, and cell fate determination (differentiation versus self-renewal) would explain most of the in vivo data. As such, this conclusion that would seem to warrant a more detailed analysis in terms of at least one or two other time points and an independent method for detecting dead/dying cells (the in vitro data in Fig. 4F is also based on assessment of activated Caspase to assess cell death). The in vitro data presented later in Fig. S4G,H do suggest an increase in cell loss during proliferative expansion of Scx-neg SCs. To what extent does cell loss (by whatever mechanism of cell death) explain both the in vivo findings of impaired regeneration and even the in vitro studies showing slower population expansion in the absence of Scx?

(3) I'm not sure I understand the description of the data or the conclusions in the section titled "Basement membrane-myofiber interaction in control and Scx cKO mice". Is there something specific to the regeneration from Scx-neg myogenic progenitors, or would these findings be expected in any experimental condition in which myogenesis was significantly delayed, with much smaller fibers in the experimental group at 5 DPI?

(4) The data presented in Fig. 4B showing differences in the purity of SC populations isolated by FACS depending on the reporter used are interesting and important for the field. The authors offer the explanation of exosomal transfer of Tdt from SCs to non-SCs. The data are consistent with this explanation, but no data are presented to support this. Are there any other explanations that the authors have considered and that could be readily tested?

(5) The Cut&Run data of Fig. 6 certainly provide evidence of direct Scx targets, especially since the authors used a novel knock-in strain for analyses. The enrichment of E-box motifs provides support for the 207 intersecting genes (scRNA-seq and Cut&Run) being direct targets. However, the rationale elaborated in the final paragraph of the Results section proposing how 4 of these genes account for the phenotypes on the Scx-neg cells and tissues is just speculation, however reasonable. These are not data, and these considerations would be more appropriate in the Discussion in the absence of any validation studies.

Comments on revisions:

The authors have adequately addressed all of the concerns I raised regarding the original submission. I have no further issues to be addressed.

Reviewer #2 (Public review):

Summary:

Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

Strengths:

Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

Weaknesses:

Scx protein expression has not been verified.

Comments on revisions:

The authors sincerely addressed all concerns, excluding the protein expression of Scx. There is convincing evidence from other experiments that indirectly indicate the protein expression of Scx. In addition, the importance of this study is solid. So, this reviewer doesn't require the authors to make more revisions.

Reviewer #1 (Public review):

Summary:

The authors present experimental and numerical results on the motility Magnetospirillum gryphiswaldense MSR-1, a magnetotactic bacterium living in sedimentary environments. The authors manufactured microfluidic chips containing three-dimensional obstacles of irregular shape, that match the statistical features of the grains observed in the sediment via micro-computer tomography. The bacteria are furthermore subject to an external magnetic field, whose intensity can be varied. The key quantity measured in the experiments is the throughput ratio, defined as the ratio between the number of bacteria that reach the end of the microfluidic channel and the number of bacteria entering it. The main result is that the throughput ratio is non-monotonic and exhibits a maximum at magnetic field strength comparable with Earth's magnetic field. The authors rationalize the throughput suppression at large magnetic fields by quantifying the number of bacteria trapped in corners between grains.

Strengths:

While magnetotactic bacteria general motility in bulk has been characterized, we know much less about their dynamics in a realistic setting, such as a disordered porous material. The micro-computer tomography of sediments and their artificial reconstruction in a microfluidic channel is a powerful method that establishes the rigorous methodology of this work. This technique can give access to further characterization of the microbial motility. The coupling of experiments and computer simulations lends considerable strength to the claims of the authors, because the model parameters (with one exception) are directly measured in the experiments.

Weaknesses:

The main weakness of the manuscript pertains to the comparison between simulations and experiments due to limitations in the tracking of bacteria in the experiments.

Impact:

Building on the present work, and refining the experimental setup may shed light on the microbial interactions in an environment such as soil which deserves further studies.

Reviewer #2 (Public review):

Summary:

The manuscript reports results of a combined experimental and simulation study of magnetotactic bacteria in microfluidic channels containing sediment-mimicking obstacles. The obstacles were produced based on micro-computer tomography reconstructions of bacteria-rich sediment samples. The swimming of bacteria through these channels is found experimentally to display the highest throughput for physiological magnetic fields. Computer simulations of active Brownian particles, parameterized based on experimental trajectories are used to quantify the swimming throughput in detail. Similar behavior as in experiments is obtained, but also considerable variability between different channel geometries. Swimming at strong field is impeded by the trapping of bacteria in corners, while at weak fields the direction of motion is almost random. The trapping effect is confirmed in the experiments, as well as the escape of bacteria with reducing field strength.

Strengths:

This is a very careful and detailed study, which draws its main strength from the fruitful combination of construction of novel microfluidic devives, their use in motility experiments, and simulations of active Brownian particles adapted to the experiment.<br /> Based on their results, the authors hypothesize that magnetotactic bacteria may have evolved to produce magnetic properties that are adapted to the geomagnetic field in order to balance movement and orientation in such crowded environments. They provide strong arguments in favor of such a hypothesis.

Weaknesses:

Some of the issues touched upon here have been studied also in other articles. It would be good to extend the list of references accordingly and discuss the relation briefly in the text.

Comments on revisions:

In their rebuttal letter, the authors have responded in detail to all points raised in my previous report. They have revised their manuscript accordingly.

Reviewer #1 (Public review):

Summary:

In this excellent manuscript by Egan et al., the authors very carefully dissect the roles of inflammasome components in restricting Salmonella Typhimurium (STm) replication in human macrophages. They show that caspase-1 is essential to mediating inflammasome responses and that caspase-4 contributes to bacterial restriction at later time points. The authors show very clear roles for the host proteins that mediate terminal lysis, gasdermin D and ninjurin-1. The unique finding in this study is that in the absence of inflammasome responses, Salmonella hypereplicates within the cytosol of macrophages. These findings suggest that caspase-1 and possibly caspase-4 play roles in restricting the replication of Salmonella in the cytosol as well as in the Salmonella containing vacuole.

Strengths:

(1) The genetic and biochemical approaches have shown for the first time in human macrophages that the caspase-1-GSDMD-NINJ1 axis is very important for restricting intracellular STm replication. In addition, they demonstrate a later role for Casp4 in control of intracellular bacterial replication.

(2) In addition, they show that in macrophages deficient in the caspase-1-GSDMD-NINJ1 axis that STm are found replicating in the cytosol, which is a novel finding. The electron microscopy is convincing that STm are in the cytosol.

(3) The authors go on to use a chloroquine resistance assay to show that inflammasome signaling also restricts STm within SCVs in human macrophages.

(4) Finally, they show that the Type 3 Secretion System encoded on Salmonella Pathogenicity Island 1 contributes to STm's cytosolic access in human macrophages.

Weaknesses:

(1) Their results with human macrophages suggest that there are differences between murine and human macrophages in inflammasome-mediated restriction of STm growth. For example, Thurston et al. showed that in murine macrophages that inflammasome activation controls the replication of mutant STm that aberrantly invades the cytosol, but only slightly limits replication of WT STm. In contrast, here the authors found that primed human macrophages rely on caspase-1, gasdermin D and ninjurin-1 to restrict WT STm. I wonder if the priming of the human macrophages in this study could account for the differences in these studies. Along those lines, do the authors see the same results presented in this study in the absence of priming the macrophages with Pam3CSK4. I think that determining whether the control of intracellular STm replication is dependent on priming is very important. Another difference with the Thurston et al. paper is the way that the STm inoculum was prepared - stationary phase bacteria that were opsonized. Could this also account for differences between the two studies rather than differences between murine and human macrophages in inflammasome-dependent control of STm?

(2) The authors show that the pore-forming proteins GSDMD and Ninj1 contribute to control of STm replication in human macrophages. Is it possible that leakage of gentamicin from the media contributes to this control?

(3) One major question that remains to be answered is whether casp-1 plays a direct role in the intracellular localization of STm. If the authors quantify the percentage of vacuolar vs. cytosolic bacteria at early time points in WT and casp-1 KO macrophages, would that be the same in the presence and absence of casp-1? If so, then this would suggest that there is a basal level of bacterial-dependent lysis of the SCV and in WT macrophages the presence of cytosolic PAMPS trigger cell death and bacteria can't replicate in the cytosol. However, in the inflammasome KO macrophages, the host cell remains alive and bacteria can replicate in the cytosol.

Comments on revisions:

The authors have addressed my previous concerns. The addition of the statements indicating the limitations of the study are an important addition.

Reviewer #2 (Public review):

Summary:

This work addresses the question of how human macrophages restrict intracellular replication of Salmonella.

Strengths:

Through a series of genetic knockouts and using specific inhibitors, Egan et al. demonstrated that the inflammasome components caspase-1, caspase-4, gasdermin D (GSDMD), and the final lytic death effector ninjurin-1 (NINJ1) are required for control of Salmonella replication in human macrophages. Interestingly, caspase-1 proved crucial in restricting Salmonella early during infection, whereas caspase-4 was essential in the later stages of infection. Furthermore, using a chloroquine resistance assay and state-of-the-art microscopy, the authors found that NAIP receptor and caspase-1 mostly regulate replication of cytosolic bacteria, with smaller, yet significant, impact on the vacuolar bacteria.

The finding that inflammasomes are critical in the restriction of replication of intracellular Salmonella in human macrophages contrasts with the published minimal role of inflammasomes in restriction of replication of intracellular Salmonella in murine macrophages. Some of these differences could be due to differences in the methodologies used in the two studies. However, the findings suggest yet another example of interspecies and intercellular differences in regulation of bacterial infections by the immune system.

Comments on revisions:

The authors may wish to comment that the measurements of released cytokines by ELISA do not discriminate between active and full-length forms of the cytokines.

Reviewer #3 (Public review):

The manuscript by Egan and coworkers investigates how Caspase-1 and Caspase-4 mediated cell death affects replication of Salmonella in human THP-1 macrophages in vitro.

Overall evaluation:

Strength of the study include the use of human cells, which exhibit notable differences (e.g., Caspase 11 vs Caspase-4/5) compared to commonly used murine models. Furthermore, the study combines inhibitors with host and bacterial genetics to elucidate mechanistic links.

Comments on revisions:

The authors have addressed my comments regarding the previous submission.

Reviewer #1 (Public review):

The authors point out that the fitness estimates obtained from different experimental assays (monoculture, pairwise competition, or bulk competition) are not generally equivalent, not even with regard to the fitness ranking of different genotypes. Using a computational model based on experimentally measured growth phenotypes for knockout strains in yeast, as well as data from Lenski's Long Term Evolution Experiment (LTEE), they derive a set of best practice rules aimed at extracting the optimal amount of information from such experiments.

The study is very complete on a technical level and I have no suggestions for further analyses. However, I feel the readability and the conceptual focus of the manuscript could be significantly improved by rearranging the material with regard to the contents of the main text vs. the Methods and the Supplement. Detailed recommendations:

(1) Regarding readability, the large number of references to material in the Methods and Supplement fragment the main text and make it difficult to follow.

(2) Conceptually, it seems to me that the current presentation obscures the reasons why we should care about fitness in the first place. In the first paragraph of Results, the authors define fitness "as any number that is sufficient to predict the genotype's relative abundance x(t) over a short-time horizon". To me, this seems like an extremely narrow and not very interesting definition. Instead, I view fitness as an intrinsic property of a genotype that allows us to predict its performance<br /> under a range of conditions, including in particular conditions that are different from the experimental setup that was used to obtain the fitness estimates. The latter viewpoint is well expressed in Supplementary Section S1, where the authors discuss the notion of fitness potential. I would recommend to move at least part of this discussion to the main text. By comparison, the arguments in favor of the logit encoding that currently opens the Results session are rather straightforward and could be shortened significantly.

(3) Similarly, the modeling strategy used in this work is quite subtle and needs to be explained more fully in the main text. The authors use growth traits (lag time, growth rate, and yield) extracted from monoculture experiments on a yeast knockout collection and feed them into a specific mathematical model to simulate pairwise and bulk competition scenarios. Since a key claim of the work is that monoculture experiments are generally poor predictors of competitive fitness, the basis for this conclusion and the assumptions on which it is based need to be described clearly in the main text. In the current version of the manuscript, this information has<br /> been largely relegated to the Methods section.

Reviewer #2 (Public review):

Summary:

The manuscript "Quantifying microbial fitness in high-throughput experiments" provides a comprehensive analysis of the various approaches to quantifying fitness in microbial evolution, focusing on three primary factors: encoding of relative abundance, time scale of measurement, and the choice of reference subpopulation. The authors systematically explore how these choices impact fitness statistics and provide recommendations aimed at standardizing practices in the field. This manuscript aims to highlight the impact of differing fitness definitions and the methodologies utilized for analysis and how that can significantly alter interpretations of mutant fitness, affecting evolutionary predictions and the overall understanding of genetic interactions in the experiments. Although this manuscript focuses on a critical issue in the quantification of fitness in high throughput experiments, it heavily relies on only one experimental dataset (Warringer et al 2003) and one organism i.e, Yeast (Saccharomyces cerevisiae) grown in a defined medium, the environmental influence is not completely captured. While the theoretical framework is strong, more experimental examples with more organisms (i.e., more datasets) in their analysis and comparison would enhance the manuscript, especially its conclusion.

Strengths:

The choices for quantifying fitness in evolution experiments are critical and highly relevant given the increasing prevalence of high-throughput experiments in evolutionary biology. The authors methodically categorize fitness statistics and their implications, providing clarity on a complex subject. This structured approach aids in understanding the nuances of fitness measurement. The manuscript effectively highlights how different choices in fitness measurement can influence fitness rankings and the understanding of epistasis, which is important for modeling evolutionary dynamics.

Weaknesses:

The theoretical framework is robust, but the manuscript could benefit from more empirical examples to illustrate how different fitness quantification methods lead to varied conclusions in experiments. The discussion on the choice of reference subpopulation could be expanded with the influence of the environment or the condition. Different types of reference groups might yield different implications for fitness calculations, and further elaboration would enhance this section. The authors overgeneralize some findings; for instance, the implications of fitness measurement choices could vary significantly across different microbes or experimental conditions. A more detailed discussion would strengthen the conclusion.

Overall, this manuscript is a significant contribution to the field of evolutionary biology, addressing a critical issue in the quantification of fitness but lacks more experimental support to make it a wider claim. By systematically exploring the factors that influence fitness measurements, the authors provide valuable insights that can guide future research - the framework is computationally thorough but needs a more detailed explanation of concepts instead of generalizing. Further work is needed, particularly to incorporate empirical examples and expand certain discussions to include environmental variation and their impact, which would improve clarity and applicability.

Reviewer #3 (Public review):

Summary:

The authors present analyses of different fitness measures derived from empirical data from yeast knock-out mutants and the long-term evolution experiment (LTEE) with Escherichia coli to explore discrepancies and identify preferred methods to estimate relative fitness in high-throughput experiments. Their work has three components. They first discuss the different "encodings" of relative abundance data and conclude that logit transformations are preferred because they transform nonlinear abundance trajectories into linear trajectories with greater predictive power. Next, they compare per-generation with per-growth cycle relative fitness estimates inferred from simulations of pairwise competitions based on published growth traits for the yeast strains and on published pairwise competition measurements for the LTEE data. Both data sets show quantitative and qualitative (i.e. rank order) discrepancies of estimates across different time scales, which are highlighted by considering possible underlying causes (i.e. trade-offs between growth traits) and consequences (i.e. epistasis among mutations affecting different growth traits). Finally, the authors compare simulated pairwise and bulk (i.e. where many mutants compete during a growth cycle in a single environment) competition assays based on the yeast knock-out mutants and demonstrate an optimal ratio of collective mutants to wild-type strains that minimizes both sampling error and overestimation of fitness estimates when compared with pairwise competitions.

Strengths:

The study deals with a highly relevant topic. Fitness is central to general evolutionary theory, but also poorly defined and implies different traits for different organisms and conditions. For microbes, which are often used in evolution experiments, high-throughput experiments may yield different measures to quantify abundance over time, from individual growth traits to bulk competition experiments. Hence, it is relevant to consider discrepancies among those measures and identify preferred measures with respect to predicting population dynamics and evolutionary processes. The present study contributes to this aim by (i) making readers aware of differences among commonly used fitness estimates, (ii) showing that simulated (yeast) and calculated (E. coli) competitive fitness may differ across time scales, and (iii) showing that bulk competitions may yield relative fitness estimates that are systematically higher than pairwise competitions. The study is rather thorough on the theory side, with extensive derivations and analyses of various fitness measures using their resource competition model in the Supplementary Information. The study ends with a few practical recommendations for preferred methods to infer relative fitness estimates, that may be useful for experimentalists and stimulate further investigations.

Weaknesses:

The study has several limitations. Perhaps the most apparent limitation is the lack of a clear answer to the question of which fitness measure is best "in the light of first principles". The authors show clear discrepancies between fitness estimates across different time scales or using different reference genotypes in bulk competition and provide useful recommendations based on practical considerations (e.g. using pairwise competitions as the "golden standard"), but it remains unclear whether these measures provide the greatest value for the questions researchers may want to answer with them (e.g. predict shifts in genotype frequencies).

A second limitation is that the authors analyse fitness differences arising solely from resource competition, whereas microbes often interact via other mechanisms, e.g. the production of anticompetitior toxins, cross-feeding of metabolites, or lack of growth to enhance their persistence in stress conditions. Without simulations of these processes, understanding discrepancies among fitness measures is necessarily limited. In addition, the analysis of trade-offs between growth traits causing these discrepancies during resource competition seems confounded by biases in measurement error or parameter estimation, at least for growth rate and lag time (Figure 2B), where the replicate estimates for the wildtype show a similar negative correlation.

Third, the study does not validate relative fitness predictions from growth traits (as is done for the yeast mutants) with measured relative fitness estimates using competition assays, while such data are available, e.g. for the LTEE. This would strengthen their inferences about preferred fitness measures.

Fourth, the analysis of epistasis between mutations affecting different growth traits (shown in Figure 3) based on the LTEE data could be better introduced and analysed more comprehensively. Now, the examples given in panels C-F seem rather idiosyncratic and readers may wonder how general these consequences of using fitness estimates based on different time scales are.

Finally, the study is generally less accessible to experimentalists due to the extensive and principled treatment of specific population dynamic models and fitness inferences. This may distract from the overarching aim to identify fitness measures that are most accurate and useful for predictions of population dynamics and evolutionary processes. In this light, the motivation for the initial discussion of the importance of how to best encode relative abundance (Figure 1) is unclear. Also, the conclusion, that logit encoding is preferred, because it linearizes logistic growth dynamics and "improves the quality of predictions", is not further motivated. Experimentalists using non-linear models to infer fitness from growth curves or competition assays may miss the relevance of this discussion.

Reviewer #1 (Public review):

Summary:

This study introduces a novel therapeutic strategy for patients with high-risk HER2-positive breast cancer and demonstrates that the incorporation of pyrotinib into adjuvant trastuzumab therapy can improve invasive disease-free survival.

Strengths:

The study features robust logic and high-quality data. Data from 141 patients across 23 centers were analyzed, thereby effectively mitigating regional biases and endowing the research findings with high applicability.

Weaknesses:

(1) Introduction and Discussion: Update the literature regarding the efficacy of pyrotinib combined with trastuzumab in treating HER2-positive advanced breast cancer.

(2) Did all the data have a normal distribution? Expand the description of statistical analysis.

(3) The novelty and innovative potential of your manuscript compared to the published literature should be described in more detail in the abstract and discussion section.

(4) Figure legend should provide a bit more detail about what readers should focus on.

(5) P-values should be clarified for the analysis.

(6) The order (A, B, and C) in Figure 3 should be labeled in the upper left corner of the Figure.

Reviewer #2 (Public review):

In this manuscript, Cao et al. evaluated the efficacy and safety of 12 months pyrotinib after trastuzumab-based adjuvant therapy in patients with high-risk, HER2-positive early or locally advanced breast cancer. Notably, the 2-year iDFS rate reached 94.59% (95% CI: 88.97-97.38) in all patients, and 94.90% (95% CI: 86.97-98.06) in patients who completed 1-year treatment of pyrotinib. This is an interesting and uplifting results, given that in ExteNET study, the 2-year iDFS rate was 93.9% (95% CI 92·4-95·2) in the 1-year neratinib group, and the 5-year iDFS survival was 90.2%, and 1-year treatment of neratinib in ExteNET study did not translate into OS benefit after 8-year follow-up. In this case, readers will be eagerly anticipating the long-term follow-up results of the current PERSIST study, as well as the results of the phase III clinical trial (NCT03980054).

I have the following comments:

(1) The introduction of the differences between pyrotinib and neratinib in terms of mechanism, efficacy, resistance, etc. is supposed to be included in the text so that authors could better highlight the clinical significance of the current trial.

(2) Please make sure that a total of 141 patients were enrolled in the study, 38 patients had a treatment duration of less than or equal to 6 months, and a total of 92 and 31 patients completed 1-year and 6-month treatment of extended adjuvant pyrotinib, respectively, which means 7 patients had a treatment duration of fewer than 6 months.

(3) The previous surgery history should be provided, and how many patients received lumpectomy, and mastectomy.

Reviewer #1 (Public review):

Summary:

This study adapts a previously published model of the cat spinal locomotor network to make predictions of how phase durations of swing and stance at different treadmill speeds in tied-belt and split-belt conditions would be altered following a lateral hemisection. The simulations make several predictions that are replicated in experimental settings.

Strengths:

(1) Despite only altering the connections in the model, the model is able to replicate very well several experimental findings. This provides strong validation for the model and highlights its utility as a tool to investigate the operations of mammalian spinal locomotor networks.

(2) The study provides insights about interactions between the left and right sides of the spinal locomotor networks, and how these interactions depend on the mode of operation, as determined by speed and state of the nervous system.

(3) The writing is logical, clear, and easy to follow.

Weaknesses:

(1) Could the authors provide a statement in the methods or results to clarify whether there were any changes in synaptic weight or other model parameters of the intact model to ensure locomotor activity in the hemisected model?

(2) The authors should remind the reader what the main differences are between state-machine, flexor-driven, and classical half-center regimes (lines 77-79).

(3) There may be changes in the wiring of spinal locomotor networks after the hemisection. Yet, without applying any sort of plasticity, the model is able to replicate many of the experimental data. Based on what was experimentally replicated or not, what does the model tell us about possible sites of plasticity after hemisection?

(4) Why are the durations on the right hemisected (fast) side similar to results in the full spinal transected model (Rybak et al. 2024)? Is it because the left is in slow mode and so there is not much drive from the left side to the right side even though the latter is still receiving supraspinal drive, as opposed to in the full transection model? (lines 202-203).

(5) There is an error with probability (line 280).

Reviewer #2 (Public review):

This is a nice article that presents interesting findings. One main concern is that I don't think the predictions from the simulation are overlaid on the animal data at any point - I understand the match is qualitative, which is fine, but even that is hard to judge without at least one figure overlaying some of the data. Second is that it's not clear how the lateral coupling strengths of the model were trained/set, so it's hard to judge how important this hemi-split-belt paradigm is. The model's predictions match the data qualitatively, which is good; but does the comparison using the hemi-split-belt paradigm not offer any corrections to the model? The discussion points to modeling plasticity after SCI, which could be good, but does that mean the fit here is so good there's no point using the data to refine?

The manuscript is well-written and interesting. The putative neural circuit mechanisms that the model uncovers are great, if they can be tested in an animal somehow.

Page 2, lines 75-6: Perhaps it belongs in the other paper on the model, but it's surprising that in the section on how the model has been revised to have different regimes of operation as speed increases, there is no reference to a lot of past literature on this idea. Just one example would be Koditschek and Full, 1999 JEB Figure 3, where they talk about exactly this idea, or similarly Holmes et al., 2006 SIAM review Figure 7, but obviously many more have put this forward over the years (Daley and Beiwener, etc). It's neat in this model to have it tied down to a detailed neural model that can be compared with the vast cat literature, but the concept of this has been talked about for at least 25+ years. Maybe a review that discusses it should be cited?

Page 2, line 88: While it makes sense to think of the sides as supraspinal vs afferent driven, respectively, what is the added insight from having them coupled laterally in this hemisection model? What does that buy you beyond complete transection (both sides no supra) compared with intact? I can see how being able to vary cycle frequencies separately of the two limbs is a good "knob" to vary when perturbing the system in order to refine the model. But there isn't a ton of context explaining how the hemi-section with split belt paradigm is important for refining the model, and therefore the science. Is it somehow importantly related to the new "regimes" of operation versus speed idea for the model?

Page 5, line 212: For the predictions from the model, a lot depends on how strong the lateral coupling of the model is, which, in turn, depends on the data the model was trained on. Were the model parameters (especially for lateral coupling of the limbs) trained on data in a context where limbs were pushed out of phase and neuronal connectivity was likely required to bring the limbs back into the same phase relationship? Because if the model had no need for lateral coupling, then it's not so surprising that the hemisected limbs behave like separate limbs, one with surpaspinal intact and one without.

Page 8, line 360: The discussion of the mechanisms (increased influence of afferents, etc) that the model reveals could be causing the changes is exciting, though I'm not sure if there is an animal model where it can be tested in vivo in a moving animal.

Page 9, line 395: There are some interesting conclusions that rely on the hemi-split-belt paradigm here.

Reviewer #1 (Public review):

Summary:

This manuscript uses the eye lens as a model to investigate basic mechanisms in the Fgf signaling pathway. Understanding Fgf signaling is of broad importance to biologists as it is involved in the regulation of various developmental processes in different tissues/organs and is often misregulated in disease states. The Fgf pathway has been studied in embryonic lens development, namely with regards to its involvement in controlling events such as tissue invagination, vesicle formation, epithelium proliferation, and cellular differentiation, thus making the lens a good system to uncover the mechanistic basis of how the modulation of this pathway drives specific outcomes. Previous work has suggested that proteins, other than the ones currently known (e.g., the adaptor protein Frs2), are likely involved in Fgfr signaling. The present study focuses on the role of Shp2 and Shc1 proteins in the recruitment of Grb2 in the events downstream of Fgfr activation.

Strengths:

The findings reveal that the juxtamembrane region of the Fgf receptor is necessary for proper control of downstream events such as facilitating key changes in transcription and cytoskeleton during tissue morphogenesis. The authors conditionally deleted all four Fgfrs in the mouse lens that resulted in molecular and morphological lens defects, most importantly, preventing the upregulation of the lens induction markers Sox2 and Foxe3 and the apical localization of F-actin, thus demonstrating the importance of Fgfrs in early lens development, i.e. during lens induction. They also examined the impact of deleting Fgfr1 and 2, on the following stage, i.e. lens vesicle development, which could be rescued by expressing constitutively active KrasG12D. By using specific mutations (e.g. Fgfr1ΔFrs lacking the Frs2 binding domain and Fgfr2LR harboring mutations that prevent binding of Frs2), it is demonstrated that the Frs2 binding site on Fgfr is necessary for specific events such as morphogenesis of lens vesicle. Further, by studying Shp2 mutations and deletions, the authors present a case for Shp2 protein to function in a context-specific manner in the role of an adaptor protein and a phosphatase enzyme. Finally, the key surprising finding from this study is that downstream of Fgfr signaling, Shc1 is an important alternative pathway - in addition to Shp2 - involved in the recruitment of Grb2 and in the subsequent activation of Ras. The methodologies, namely, mouse genetics and state-of-the-art cell/molecular/biochemical assays are appropriately used to collect the data, which are soundly interpreted to reach these important conclusions. Overall, these findings reveal the flexibility of the Fgf signaling pathway and its downstream mediators in regulating cellular events. This work is expected to be of broad interest to molecular and developmental biologists.

Weaknesses:

A weakness that needs to be discussed is that Le-Cre depends on Pax6 activation, and hence its use in specific gene deletion will not allow evaluation of the requirement of Fgfrs in the expression of Pax6 itself. But since this is the earliest Cre available for deletion in the lens, mentioning this in the discussion would make the readers aware of this issue. Referring to Jag1 among "lens-specific markers" (page 5) is debatable, suggesting changing to the lines of "the expected upregulation of Jag1 in lens vesicle". The Abstract could be modified to clearly convey the existing knowledge gap and the key findings of the present study. As it stands now, it is a bit all over the place. Some typos in the manuscript need to be fixed, e.g. "...yet its molecular mechanism remains largely resolved" - unresolved? "...in the development lens" - in the developing lens? In Figure 4 legend, "(B) Grb2 mutants Grb2 mutants displayed...", etc.

Reviewer #2 (Public review):

Summary:

I have reviewed a manuscript submitted by Wang et al., which is entitled "Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development". In this paper, the authors first examined lens phenotypes in mice with Le-Cre-mediated knockdown (KD) of all four FGFR (FGFR1-4), and found that pERK signals, Jag1, and foxe3 expression are absent or drastically reduced, indicating that FGF signaling is essential for lens induction. Next, the authors examined lens phenotypes of FGFR1/2-KD mice and found that lens fiber differentiation is compromised and that proliferative activity and cell survival are also compromised in lens epithelium. Interestingly, Kras activation rescues defects in lens growth and lens fiber differentiation in FGFR1/2-KD mice, indicating that Ras activation is a key step for lens development. Next, the authors examined the role of Frs2, Shp2, and Grb2 in FGF signaling for lens development. They confirmed that lens fiber differentiation is compromised in FGFR1/3-KD mice combined with Frs2-dysfunctional FGFR2 mutants, which is similar to lens phenotypes of Grb2-KD mice. However, lens defects are milder in mice with Shp2YF/YF and Shp2CS mutant alleles, indicating that the involvement of Shp2 is limited for the Grb2 recruitment for lens fiber differentiation. Lastly, the authors showed new evidence on the possibility that another adapter protein, Shc1, promotes Grb2 recruitment independent of Frs2/Shp2-mediated Grb2 recruitment.

Strengths:

Overall, the manuscript provides valuable data on how FGFR activation leads to Ras activation through the adapter platform of Frs2/Shp2/Grb2, which advances our understanding of complex modification of the FGF signaling pathway. The authors applied a genetic approach using mice, whose methods and results are valid to support the conclusion. The discussion also well summarizes the significance of their findings.

Weaknesses:

The authors eventually found that the new adaptor protein Shc1 is involved in Grb2 recruitments in response to FGF receptor activation. however, the main data for Shc1 are histological sections and statistical evaluation of lens size. So, my major concern is that the authors need to provide more detailed data to support the involvement of Shc1 in Grb2 recruitment of FGF signaling for lens development.

Reviewer #3 (Public review):

Summary:

The manuscript entitled "Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development" by Wang et al., investigates the molecular mechanism used by FGFR signaling to support lens development. The lens has long been known to depend on FGFR signaling for proper development. Previous investigations have demonstrated that FGFR signaling is required for embryonic lens cell survival and for lens fiber cell differentiation. The requirement of FGFR signaling for lens induction has remained more controversial as deletion of both Fgfr1 and Fgfr2 during lens placode formation does not prevent the induction of definitive lens markers such as FOXE3 or αA-crystallin. Here the authors have used the Le-Cre driver to delete all four FGFR genes from the developing lens placode demonstrating a definitive failure of lens induction in the absence of FGFR signaling. The authors focused on FGFR1 and FGFR2, the two primary FGFRs present during early lens development, and demonstrated that lens development could be significantly rescued in lenses lacking both FGFR1 and FGFR2 by expressing a constitutively active allele of KRAS. They also showed that the removal of pro-apoptotic genes Bax and Bak could also lead to a substantial rescue of lens development in lenses lacking both FGFR1 and FGFR2. In both cases, the lens rescue included both increased lens size and the expression of genes characteristic of lens cells.

Significantly the authors concentrated on the juxtamembrane domain, a portion of the FGFRs associated with FRS2. Previous investigations have demonstrated the importance of FRS2 activation for mediating a sustained level of ERK activation. FRS2 is known to associate both with GRB2 and SHP2 to activate RAS. The authors utilized a mutant allele of Fgfr1, lacking the entire juxtamembrane domain (Fgfr1ΔFrs), and an allele of Fgfr2 containing two-point mutations essential for Frs2 binding (Fgfr2LR). When combining three floxed alleles and leaving only one functional allele (Fgfr1ΔFrs or Fgfr2LR) the authors got strikingly different phenotypes. When only the Fgfr1ΔFrs allele was retained, the lens phenotype matched that of deleting both Fgfr1 and Fgfr2. However, when only the Fgfr2LR allele was retained the phenotype was significantly milder, primarily affecting lens fiber cell differentiation, suggesting that something other than FRS2 might be interacting with the juxtamembrane domain to support FGFR signaling in the lens. The authors also deleted Grb2 in the lens and showed that the phenotype was similar to that of the lenses only retaining the Fgfr2LR allele, resulting in a failure of lens fiber cell differentiation and decreased lens cell survival. However, mutating the major tyrosine phosphorylation site of GRB2 did not affect lens development. The author additionally investigated the role of SHP2 lens development by making by either deleting SHP2 or by making mutations in the SHP2 catalytic domain. The deletion of the SHP2 phosphatase activity did not affect lens development as severely as the total loss of SHP2 protein, suggesting a function for SHP2 outside of its catalytic activity. Although the loss of Shc1 alone has only a slight effect on lens size and pERK activation in the lens, the authors showed that the loss of Shc1 exacerbated the lens phenotype in lenses lacking both Frs2 and Shp2. The authors suggest that SHC1 binds to the FGFR juxtamembrane domain allowing for the recruitment of GRB2 independently of FRS2.

Strengths:

(1) The authors used a variety of genetic tools to carefully dissect the essential signals downstream of FGFR signaling during lens development.

(2) The authors made a convincing case that something other than FRS2 binding mediates FGFR signaling in the juxtamembrane domain.

(3) The authors demonstrated that despite the requirement of both the adaptor function and phosphatase activity of SHP2 are required for embryonic survival, neither of these activities is absolutely required for lens development.

(4) The authors provide more information as to why FGFR loss has a phenotype much more severe than the loss of FRS2 alone during lens development.

(5) The authors followed up their work analyzing various signaling molecules in the context of lens development with biochemical analyses of FGF-induced phosphorylation in murine embryonic fibroblasts (MEFs).

(6) In general, this manuscript represents a Herculean effort to dissect FGFR signaling in vivo with biochemical backing with cell culture experiments in vitro.

Weaknesses:

(1) The authors demonstrate that the loss of FGFR1 and FGFR2 can be compensated by a constitutive active KRAS allele in the lens and suggest that FGFRs largely support lens development only by driving ERK activation. However, the authors also saw that lens development was substantially rescued by preventing apoptosis through the deletion of BAK and BAX. To my knowledge, the deletion of BAK and BAX should not independently activate ERK. The authors do not show whether ERK activation is restored in the BAK/BAX deficient lenses. Do the authors suggest the FGFR3 and/or FGFR4 provide sufficient RAS and ERK activation for lens development when apoptosis is suppressed? Alternatively, is it the survival function of FGFR-signaling as much as a direct effect on lens differentiation?

(2) The authors make the argument that deleting all four FGFRs prevented lens induction but that the deletion of only FGFR1 and FGFR2 did not. Part of this argument is the retention of FOXE3 expression, αA-crystallin expression, and PROX1 expression in the FGFR1/2 double mutants. However, in Figure 1E, and Figure 1F, the staining of the double mutant lens tissue with FOXE3, αA-crystallin, and PROX1 is unconvincing. However, the retention of FOXE3 expression in the FGFR1/FGFR2 double mutants was previously demonstrated in Garcia et al 2011. Also, there needs to be an enlargement or inset to demonstrate the retention of pSMAD in the quadruple FGFR mutants in Figure 1D.

(3) Do the authors suggest that GRB2 is required for RAS activation and ultimately ERK activation? If so, do the authors suggest that ERK activation is not required for FGFR-signaling to mediate lens induction? This would follow considering that the GRB2 deficient lenses lack a problem with lens induction.

(4) The increase in p-Shc is only slightly higher in the Cre FGFR1f/f FGFR2r/LR than in the FGFR1f/Δfrs FGFR2f/f. Can the authors provide quantification?

(5) The authors have not shown directly that Shc1 binds to the juxtamembrane region of either Fgfr1 or Fgfr2.

(6) The authors have used the Le-Cre strain for all of their lens deletion experiments. Previous work has documented that the Le-Cre transgene can cause lens defects independent of any floxed alleles in both homozygous and hemizygous states on some genetic backgrounds (Dora et al., 2014 PLoS One 9:e109193 and Lam et al., Human Genomics 2019 13(1):10. Are the controls used in these experiments Le-Cre hemizygotes?