Reviewer #3 (Public review):

Summary:

I found the manuscript to be well-written. I have a few questions regarding the model, though the bulk of my comments are requests to provide definitions and additional clarity. There are concepts and approaches used in this manuscript that are clear boons for understanding the ecology of microbiomes but are rarely considered by researchers approaching the manuscript from a traditional biology background. The authors have clearly considered this in their writing of S1 and S2, so addressing these comments should be straightforward. The methods section is particularly informative and well-written, with sufficient explanations of each step of the derivation that should be informative to researchers in the microbial life sciences who are not well-versed with physics-inspired approaches to ecology dynamics.

Strengths:

The modeling efforts of this study primarily rely on a disordered form of the generalized Lotka-Volterra (gLV) model. This model can be appropriate for investigating certain systems, and the authors are clear about when and how more mechanistic models (i.e., consumer-resource) can lead to gLV. Phenomenological models such as this have been found to be highly useful for investigating the ecology of microbiomes, so this modeling choice seems justified, and the limitations are laid out.

Weaknesses:

The authors use metagenomic data of diseased and healthy patients that were first processed in Pasqualini et al. (2024). The use of metagenomic data leads me to a question regarding the role of sampling effort (i.e., read counts) in shaping model parameters such as $h$. This parameter is equal to the average of 1/# species across samples because the data are compositional in nature. My understanding is that $h$ was calculated using total abundances (i.e., read counts). The number of observed species is strongly influenced by sampling effort, so it would be useful if the number of reads were plotted against the number of species for healthy and diseased subjects.

However, the role of sampling effort can depend on the type of data, and my instinct about the role that sampling effort plays in species detection is primarily based on 16S data. The dependency between these two variables may be less severe for the authors' metagenomic pipeline. This potential discrepancy raises a broader issue regarding the investigation of microbial macroecological patterns and the inference of ecological parameters. Often microbial macroecology researchers rely on 16S rRNA amplicon data because that type of data is abundant and comparatively low-cost. Some in microbiology and bioinformatics are increasingly pushing researchers to choose metagenomics over 16S. Sometimes this choice is valid (discovery of new MAGs, investigate allele frequency changes within species, etc.), sometimes it is driven by the false equivalence "more data = better". The outcome, though, is that we have a body of more-or-less established microbial macroecological patterns which rest on 16S data and are now slowly incorporating results from metagenomics. To my knowledge, there has not been a systematic evaluation of the macroecological patterns that do and do not vary by one's choice in 16S vs. metagenomics. Several of the authors in this manuscript have previously compared the MAD shape for 16S and metagenomic datasets in Pasqualini et al., but moving forward, a more comprehensive study seems necessary (2024).

References

Pasqualini, Jacopo, et al. "Emergent ecological patterns and modelling of gut microbiomes in health and in disease." PLOS Computational Biology 20.9 (2024): e1012482.

Reviewer #1 (Public review):

The manuscript by Liao et al investigates the mechanisms that induce ephrin expression in spinal cord lateral motor column (LMC) neurons to facilitate axon guidance into the dorsal and ventral limb. The authors show that Sp1 and its co-activators p300 and CBP are required to induce ephrin expression to modulate the responsiveness of motor neurons to external ephrin cues. The study is well done and convincingly demonstrates the role of Sp1 in motor neuron axon guidance.

Further discussion and clarification of some results would further improve the study.

(1) The mechanism that the authors propose (Figure 7) and is also supported by their data is that Sp1 induces ephrinA5 in LMCm and ephrinB2 in LMCl to attenuate inappropriate responses to external ephrins in the limb. Therefore, deletion of Sp1 should result in mistargeting of LMCl and LMCm axons, as shown in the mouse data, but no overt changes in the number of axons in the ventral and dorsal limb. From the mouse backfills, it seems that an equal number of LMCm/LMCl project into the wrong side of the limb. However, the chick data show an increase of axons projecting into the ventral limb in the Sp1 knockout. Is this also true in the mouse? The authors state that medial and lateral LMC neurons differ in their reliance on Sp1 function but that is not supported by the mouse backfill data (27% vs 32% motor neurons mistargeted). Also, the model presented in Figure 7 does not explain how Sp1 overexpression leads to axon guidance defects.

(2) The authors do not directly show changes in ephrin expression in motor neurons, either in chick or mouse, after Sp1 knockout, which is the basis of their model. The experiment in Figure 4G seems to be Sp1 overexpression rather than knockdown (as mentioned in the results) and NSC-34 cells may not be relevant to motor neurons in vivo. NSC-34 experiments are also not described in the methods.

(3) There is no information about how the RNA-sequencing experiment was done (which neurons were isolated, how, at what age, how many replicates, etc) so it is hard to interpret the resulting data.

(4) It is unclear why the authors chose to use a Syn1-cre driver rather than a motor neuron restricted cre driver. Since this is a broad neuronal cre driver, the behavioral defects shown in Figure 7 may not be solely due to Sp1 deletion in motor neurons. Are there other relevant neuronal populations that express Sp1 that are targeted by this cre-mediated deletion?

Reviewer #2 (Public review):

Summary:

This study shows that transcription factor Sp1 is required for correct ventral vs. dorsal targeting of limb-innervating LMC motor neurons using mouse and chick as model systems. In a wild-type embryo, lateral LMC axons specifically target dorsal muscles while medial LMC axons target ventral muscles. The authors convincingly show that this specificity is lost when Sp1 is knocked down or knocked out - axons of both lateral and medial LMC motor neurons project to both dorsal and ventral muscles in mutant conditions. The authors then conduct RNA-seq and ChIP experiments to show that Sp1 loss of function disrupts Ephrin-Epha receptor signaling pathway genes. These molecules are known to provide attractive or repulsive cues to guide LMC axons to their targets. The authors show that attraction/repulsion properties of medial and lateral LMC axons to specific Ephrin/Epha molecules are in fact disrupted in Sp1 mutants using ex vivo explant studies. Finally, the authors show that behaviors like coordinated movement and grip strength are also affected in Sp1 mutant mice. This study convincingly shows that Sp1 is important for correct circuit wiring of LMC neurons, and moves the field forward by elucidating a new level of transcriptional regulation required in this process. However, the claims made by the authors that the mode of Sp1-mediated regulation is through cis-attenuation of Epha activity is not well supported. These and additional strengths and weaknesses in approach and in data interpretation are discussed below.

Strengths:

(1) The study convincingly shows that wildtype levels of Sp1 are necessary for LMC axon targeting specificity. The combination of the following approaches is a strength:<br /> a) Both loss of function and gain of function experiments are performed for Sp1 and show complementary effects on the axon targeting phenotype.<br /> b) Retrograde labeling of LMC neurons from dorsal and ventral muscles shows that Sp1 mutants clearly lose the specificity of LMC axon targeting.<br /> c) The authors also use explant experiments to show that both loss of Sp1 and gain of Sp1 show clear changes in attraction and repulsion to specific ephrin and epha receptor molecules.<br /> d) The Sp1 loss and gain of function experiments are well controlled to show that the changes in axon wiring observed are not due to cell death, cell fate switches, or due to unequal numbers of medial and lateral LMC neurons being labeled in the experiments.

(2) It is also convincing that Sp1 requires cofactors p300 and CBP for its function. In the absence of these cofactors, the gain of function phenotypes of Sp1 are subdued.

Weaknesses:

(1) The robustness of RNAseq and ChIP experiments is difficult to judge as methods are not described. For example, it is unclear if RNAseq is performed on purified motor neurons or on whole spinal cords. This is an important consideration as Sp1 is a broadly expressed protein.

(2) The authors state that expression of Ephrin A5 and Ephrin B2 is reduced based on RNAseq data, however, it is not shown that this reduction occurs specifically in LMC neurons.

(3) The authors show Sp1 ChIP peaks at Ephrin B2 promoter, but nothing is mentioned about peaks at Eprin A5 or other types of signaling molecules like Sema7a, which are also differentially expressed in Sp1 mutants. There is also no mention of the correlation between changes in gene expression seen in RNAseq data and the binding profile of Sp1 seen in ChIP data, which could help establish the robustness of these datasets.

(4) The authors conclude that Sp1 functions by activating Ephrin A5 in medial LMC and Ephrin B2 in lateral LMC. The argument, as I understand it, is that this activation leads to cis attenuation of their respective Epha receptors and therefore targeting the correct muscle. Though none of the data presented go against this hypothesis, this hypothesis is also not fully supported. Specifically:<br /> a) It would be important to know that modulation of Sp1 expression leads to changes in EphrinA5 and B2 in LMC lateral/medial neurons.<br /> b) It would also be important to show that none of the other changes caused by Sp1 are responsible for axon mistargeting by performing rescue experiments with Ephrin A5 and Ephrin B2.<br /> c) To make the most convincing case, experiments showing increased or decreased cis-binding of Ephrin molecules with Epha receptors would be necessary. This study would still be compelling without this last experiment, but the language in the abstract would need to be modulated.

(5) All behavior experiments are done in a pan-neuronal knockout of Sp1. As Sp1 is broadly expressed in neurons, a statement describing whether and why the authors think the phenotypes arise from Sp1's function in LMC motor neurons would be helpful. Experimentally, rescue experiments in which Sp1 is restored in LMC neurons or motor neurons would also make this claim more convincing.

Reviewer #3 (Public review):

Summary:

This is a compelling study on the role of Sp1 in motor axon trajectory selection, demonstrating that Sp1 is both necessary and sufficient for correct axon guidance in the limb. Sp1 regulates ephrin ligand expression to fine-tune Eph/ephrin signaling in the lateral motor column (LMC) neurons.

Strengths:

The study integrates multiple approaches. These include in ovo electroporation in chick embryos, conditional knockout mouse models, transcriptomic analyses, and functional assays such as stripe assays and behavioral testing-to provide robust evidence for Sp1's role in axon guidance mechanisms. The manuscript is well-written and scientifically rigorous, and the findings are of broad interest to the developmental neuroscience community.

Weaknesses:

Some aspects of the manuscript could be improved to enhance clarity, ensure logical flow, and strengthen the impact of the findings.

Reviewer #1 (Public review):

In this study, Li et al et al. investigated the role of miR-283 in regulating cardiac aging and its potential contribution to age-related bradyarrhythmia. Using Drosophila as a model, the authors demonstrated that systemic overexpression or knockdown of miR-283 induced age-associated bradycardia. Notably, the study found that miR-283 knockdown in ventral-lateral neurons (LNvs), rather than in the heart, was sufficient to induce bradyarrhythmia, an effect the authors linked to the upregulation of miR-283 expression in both the brain and heart. The study also explored the beneficial impact of exercise on cardiac aging, showing that endurance training mitigated bradyarrhythmia, correlating with reduced miR-283 accumulation in the brain and myocardium.

The conclusions of this paper are mostly well supported by data; however, some concerns arise from the unexpected finding that bradyarrhythmia was triggered by miR-283 knockdown in LNvs rather than in the heart, suggesting a non-cell-autonomous mechanism. A more precise mechanistic explanation linking miR-283 dysregulation in LNvs to cardiac dysfunction would strengthen the study's conclusions. While the authors propose cwo as a potential target of miR-283, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Additionally, it remains unclear whether reduced miR-283 levels in LNvs lead to accelerated aging rather than a cardiac-specific effect. Likewise, the potential influence of miR-283 on the circadian clock and its broader impact on aging warrant further investigation.

Major Comments:

(1) A significant concern arises from the unexpected outcome observed in miR-283 knockdown in LNvs, which suggests a non-cell-autonomous mechanism. Elucidating the mechanisms by which miR-283 deficiency leads to the observed phenotypes would provide a more comprehensive understanding of the study's implications.

(2) The authors propose cwo as a potential target of miR-283; however, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Similarly, direct evidence demonstrating that cwo is a bona fide target of miR-283 in LNvs should be provided.

(3) It remains unclear whether miR-283 knockdown in LNvs results in accelerated aging rather than a cardiac-specific effect. This hypothesis is supported by observations that pdf>miR-283SP animals exhibit systemic premature senescence (elevated SA-β-gal activity in both the heart and brain), cardiac dysfunction, impaired climbing ability, and reduced lifespan.

(4) The finding that reduced miR-283 levels in LNvs lead to accelerated aging raises an important, yet unexplored, question: does miR-283 influence the circadian clock, thereby broadly affecting aging?

Two aspects of this question should be addressed:<br /> (a) Is the circadian rhythm disrupted in miR-283 knockdown experiments?<br /> (b) Do circadian rhythm defects impact aging?

(5) The authors state that miR-283 knockdown in LNvs led to bradyarrhythmia, which was mainly caused by miR-283 upregulation in the whole brain and heart. However, it is unclear which experiments support this conclusion. Could the authors clarify this point?

(6) Given that miR-283 expression varies with age, could the upregulation of miR-283 in both the brain and heart be a consequence of accelerated aging rather than a specific effect of miR-283 knockdown in LNvs?

(7) While the beneficial effects of exercise on cardiac function appear clear, the claim that this effect is mediated through miR-283 function in LNvs seems premature. The data suggest that exercise-induced improvement occurs in both wild-type and miR-283-SP animals, raising the possibility that exercise acts through a miR-283-independent mechanism.

Reviewer #2 (Public review):

Summary:

The manuscript presents findings that indicate a role in controlling Drosophila heart rate for a conserved miRNA (miR-238 in flies). Further, the manuscript localizes the relevant tissue for the function of this miRNA to a subset of neurons that are heavily involved in circadian regulation, thus presenting an interesting mechanistic link between the circadian system and heart rate. Either ubiquitous knockout or ubiquitous overexpression negatively impacts several aspects of heart performance, with a pronounced effect on heart rate. Interestingly, knockdowns in the heart itself are innocuous, but knockdown in LNvS neurons recapitulates the effect on heart rate. Authors use bioinformatics to identify the clockwork orange (cwo) gene as a potential target and validate that cwo expression is reduced when miR-238 is knocked down in LNvS neurons in vivo and also validate that cwo is regulated by miR-238 in cell culture luciferase assays. Exercise shows a modest ability to restore normal cwo expression and a trend toward an effect on survival, but shows a much stronger rescue of the heart rate phenotype.

Strengths:

Evidence is strong for the effect of miR-238 in pdf-positive neurons on the control of heart rate and for cwo as a downstream effector of miR-238.

Work to identify specific targets of miR-283 is well-done and successfully identified a key downstream regulator in cwo.

The potential mechanism using miR-238 to link circadian neurons to heart rate regulation is novel and exciting.

Weaknesses:

The evidence that this is related to normal aging is rather weak, and the effect of exercise on the observed parameters is small and not necessarily working through the miR-238/cwo mechanism.

The authors seem to be conflating two hypotheses in their interpretations. Is miR-283 working through circadian mechanisms or age-related mechanisms? While it is true that aging tends to reduce heart rate, I don't think that means that any intervention that reduces heart rate is causing "senescence". Similarly, reduced survival in miR-283 knockdown flies does not prove that miR-283 promotes healthy aging per se, just that miR-283 is required for health regardless of age.

Survival reduction is quite modest which does not necessarily support the idea that the bradycardia is causing major health issues or premature senescence for the flies. The interpretation of the longevity experiments throughout the manuscript seems overstated.

The study would benefit greatly from a direct test of the author's proposed pathway for exercise to improve bradycardia.

The statement in the discussion "inducing endurance exercise of anti gravity climbing in flies with miR-283 knockdown in LNvs can improve bradyarrhythmic features by decreasing brain miR-283 expression" is not fully supported by data in the paper. There is an association there, but it cannot be said to be the full cause (or even required) without doing more experiments

The summary figure includes both data-supported mechanistic relationships and mechanisms that are inferred or assumed.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rosero and Bai examined how the well-known thermosensory neuron in C. elegans, AFD, regulates context-dependent locomotory behavior based on the tactile experience. Here they show that AFD uses discrete cGMP signalling molecules and independent of its dendritic sensory endings regulates this locomotory behavior. The authors also show here that AFD's connection to one of the hub interneurons, AIB, through gap junction/electrical synapses, is necessary and sufficient for the regulation of this context-dependent locomotion modulation.

Strengths:

This is an interesting paper showcasing how a sensory neuron in C. elegans can employ a distinct set of molecular strategies and different physical parts to regulate a completely distinct set of behaviors, which were not been shown to be regulated by AFD before. The experiments were well performed and the results are clear. However, there are some questions about the mechanism of this regulation. This reviewer thinks that the authors should address these concerns before the final published version of this manuscript.

Weaknesses:

(1) The authors argued about the role of prior exposure to different physical contexts which might be responsible for the difference in their locomotory behaviour. However, the worms in the binary chamber (with both non-uniformly sized and spaced pillars) experienced both sets of pillars for one hour prior to the assay and they were also free to move between two sets of environments during the assay. So, this is not completely a switch between two different types of tactile barriers (or not completely restricted to prior experience), but rather a difference between experiencing a more complex environment vs a simple uniform environment. They should rephrase their findings. To strictly argue about the prior experience, the authors need to somehow restrict the worms from entering the uniform assay zone during the 1hr training period.

(2) The authors here argued that the sensory endings of AFD are not required for this novel role of AFD in context-dependent locomotion modulation. However, gcy-18 has been shown to be exclusively localized to the ciliated sensory endings of AFD and even misexpression of GCY-18 in other sensory neurons also leads to localizations in sensory endings (Nguyen et. al., 2014 and Takeishi et. al., 2016). They should check whether gcy-18 or tax-2 gets mislocalized in kcc-3 or tax-1 mutants.

(3) MEC-10 was shown to be required for physical space preference through its action in FLP and not the TRNs (PMID: 28349862). Since FLP is involved in harsh touch sensation while TRNs are involved in gentle touch sensation, which are the neuron types responsible for tactile sensation in the assay arena? Does mec-10 rescue in TRNs rescue the phenotype in the current paper?

(4) The authors mention that the most direct link between TRNs and AFD is through AIB, but as far as I understand, there are no reports to suggest synapses between TRNs and AIB. However, FLP and AIB are connected through both chemical and electrical synapses, which would make more sense as per their mec-10 data. (the authors mentioned about the FLP-AIB-AFD circuit in their discussion but talked about TRNs as the sensory modality). mec-10 rescue experiment in TRNs would clarify this ambiguity.

(5) Do inx-7 or inx-10 rescue in AFD and AIB using cell-specific promoters rescue the behaviour?

(6) How Guanylyl cyclase gcy-18 function is related to the electrical synapse activity between AFD and AIB? Is AFD downstream or upstream of AIB in this context?

Reviewer #2 (Public review):

Summary:

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFD-specific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD is primarily characterized as a thermo-sensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. This represents a potentially relevant synaptic connection linking AFD to the mechanosensory-behavior circuit.

Strengths:

(1) The fact that AFD mediates tactile-context locomotion modulation is new, rather surprising, and interesting.

(2) The authors have combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with single-copy transgenes are very convincing.

(3) The study is very clearly written, and figures are nicely illustrated with diagrams that effectively convey the authors' interpretation.

Weaknesses:

(1) Whereas GCY-18 in AFD and the AFD-AIB synaptic connection clearly play a role in tactile-context locomotion modulation, whether and how they actually modulate the mechanosensory circuit and/or locomotion circuit remains unclear. The possibility of non-synaptic communication linking mechanosensory neurons and AFD (in either direction) was not explored. Thus, in the end, we have not learned much about what GCY-18 and the AFD-AIB module are doing to actuate tactile context-dependent locomotion modulation.

(2) The authors only focused on speed readout, and we don't know if the many behavioral parameters that are modulated by tactile context are also under the control of AFD-mediated modulation.

(3) The AFD-AIB gap junction reconstruction experiment was conducted in an innexin double mutant background, in which the whole nervous system's functioning might be severely impaired, and its results should be interpreted with this limitation in mind.

Reviewer #3 (Public review):

Summary:

Rosero and Bai report an unconventional role of AFD neurons in mediating tactile-dependent locomotion modulation, independent of their well-established thermosensory function. They partially elucidate the signaling mechanisms underlying this AFD-dependent behavioral modulation. The regulation does not require the sensory dendritic endings of AFD but rather the AFD neurons themselves. This process involves a distinct set of cGMP signaling proteins and CNG channel subunits separate from those involved in thermosensation or thermotaxis. Furthermore, the authors demonstrate that AIB interneurons connect AFD to mechanosensory circuits through electrical synapses. They conclude that, beyond its primary function in thermosensation, AFD contributes to context-dependent neuroplasticity and behavioral modulation via broader circuit connectivity.

While the discovery of multifunctionality in AFD is not entirely unexpected, given the limited number of neurons in C. elegans (302 in total), the molecular and cellular mechanisms underlying this AFD-dependent behavioral modulation, as revealed in this study, provide valuable insights into the field.

Strengths:

(1) The authors uncover a novel role of AFD neurons in mediating tactile-dependent locomotion modulation, distinct from their well-established thermosensory function.

(2) They provide partial insights into the signaling mechanisms underlying this AFD-dependent behavioral modulation.

(3) The neural behavior assays utilizing two types of microfluidic chambers (uniform and binary chambers) are innovative and well-designed.

(4) By comparing AFD's role in locomotion modulation to its thermosensory function throughout the study, the authors present strong evidence supporting these as two independent functions of AFD.

(5) The finding that AFD contributes to context-dependent behavioral modulation is significant, further reinforcing the growing evidence that individual neurons can serve multiple functions through broader circuit connectivity.

Weaknesses:

(1) Limited Behavioral Assays: The study relies solely on neural behavior assays conducted using two types of microfluidic chambers (uniform and binary chambers) to assess context-dependent locomotion modulation. No additional behavioral assays were performed. To strengthen the conclusions, the authors should validate their findings using an independent method, at the very least by testing AFD-ablated animals and gcy-18 mutants with a second behavioral approach.

(2) Clarity in Behavioral Assay Methodology: The methodology for conducting the behavioral assays is unclear. It appears that worms were free to move between the exploration and assay zones, with no control over the duration each worm spent in either zone. This lack of regulation may introduce variability in tactile experience across individuals, potentially affecting the reproducibility and quantitativeness of the method. The authors should clarify whether and how they accounted for this variability.

(3) Potential Developmental and Behavioral Confounds in Mutant Analysis: Several neuronal mutant strains were used in this study, yet the effects of these mutations on development and general behavior (e.g., movement ability) were not discussed. Although young adult worms were used for behavioral assays, were they at similar biological ages? To rule out confounding factors, locomotion assays assessing movement ability should be conducted (see reference PMID 25561524).

(4) Definition and Baseline Measurements for Locomotion Categories: The finding that tax-4 and kcc-3 contribute to basal locomotion but not to context-dependent locomotion modulation is intriguing. The authors argue that distinct mechanisms regulate these two processes; however, the study does not clearly define the concepts of "basal locomotion" and "context-dependent locomotion," nor does it provide baseline measurements. A clear definition and baseline data are needed to support this conclusion.

Reviewer #1 (Public review):

Summary:

In this manuscript, Campbell et al. assess how intracranial theta-burst stimulation (TBS) applied to the basolateral amygdala in 23 epilepsy patients affects neuronal spiking in the medial temporal lobe and prefrontal cortex during a visual recognition memory task.

Strengths:

This is an incredibly rare dataset; collecting single-unit spiking data from behaving humans during active intracranial stimulation is a Herculaean task, with immense potential for translational studies of how stimulation may be applied to modulate biological mechanisms of memory. The authors utilize careful, high-quality methodology throughout (e.g. task design, spike recording and sorting, statistical analysis), providing high confidence in the validity of their findings.

Weaknesses:

(1) This is an exploratory study that doesn't explore quite enough. Critically, the authors make a point of mentioning that neuronal firing properties vary across cell types, but only use baseline firing rate as a proxy metric for cell type. This leaves several important explorations on the table, not limited to the following:<br /> a) Do waveform shape features, which can also be informative of cell type, predict the effect of stimulation?<br /> b) Is the autocorrelation of spike timing, which can be informative about temporal dynamics, altered by stimulation? This is especially interesting if theta-burst stimulation either entrains theta-rhythmic spiking or is more modulatory of endogenously theta-modulated units.<br /> c) The authors reference the relevance of spike-field synchrony (30-55 Hz) in animal work, but ignore it here. Does spike-field synchrony (comparing the image presentation to post-stimulation) change in this frequency range? This does not seem beyond the scope of investigation here.<br /> d) How does multi-unit activity respond to stimulation? At this somewhat low count of neurons (total n=156 included) it would be valuable to provide input on multi-unit responses to stimulation as well.<br /> e) Several intracranial studies have implicated proximity to white matter in determining the effects of stimulation on LFPs; do the authors see an effect of white matter proximity here?

(2) It is a little confusing to interpret stimulation-induced modulation of neuronal spiking in the absence of stimulation-induced change in behavior. How do the authors findings tell us anything about the neural mechanisms of stimulation-modulated memory if memory isn't altered? In line with point #1, I would suggest a deeper dive into behavior (e.g. reaction time? Or focus on individual sessions that do change in Figure 4A?) to make a stronger statement connecting the neural results to behavioral relevance.

(3) It is not clear to me why the assessment of firing rates after image onset and after stim offset is limited to one second - this choice should be more theoretically justified, particularly for regions that spike as sparsely as these.

(4) This work coincides with another example of human intracranial stimulation investigating the effect on firing rates (doi: https://doi.org/10.1101/2024.11.28.625915). Given how incredibly rare this type of work is, I think the authors should discuss how their work converges with this work (or doesn't).

(5) What information does the pseudo-population analysis add? It's not totally clear to me.

Reviewer #2 (Public review):

Summary:

This study presents a valuable characterization of the effects of intracranial theta-burst stimulation of the basolateral amygdala on single units spiking activity in several areas in the human brain, associated with memory processing. It is written clearly and concisely, allowing readers to fully understand the analysis used.

The authors used a visual recognition memory task previously employed by their group to characterize the effects of basolateral amygdala stimulation upon memory consolidation (Inman et al, 2018). This current report is an interesting analysis to complement the results reported in the 2018 paper.

Strengths:

Rare combination of human neurophysiology and behavior -<br /> The type of experiment performed in the manuscript, which contains both neurophysiological data, behavior, and a deep brain stimulation intervention (DBS), is incredibly rare, takes many years to accomplish with tight collaboration between clinical and research teams. Our understanding of spiking dynamics of human neurons is very limited, and this report is an important piece in the puzzle that allows DBS to be used in future interventions that will benefit patients' health.

Multiple brain areas included -<br /> It's important to note that the report analyzes brain areas with which the Amygdala has extensive connections (Fig. 1A) - Hippocampus, OFC, Amygdala, ACC. It seems that neurons in all these areas were modulated by the stimulation, except the ACC, in which firing rates were so low, that only a handful of neurons were included in the analysis. This is an important demonstration that low amplitude stimulation (even when reduced to 0.5mA) can travel far and wide across the human brain.

The experiment is cleverly designed to tease apart responses due to visual stimuli (image presentation) and electrical stimulation. Authors suggest that the units modulated by stimulation are largely distinct from those responsive to image offset during trials without stimulation. The subpopulation that responds strongly also tends to have a higher baseline of firing rate. It's important to add that the chosen modulation index is more likely to be significant in neurons with higher firing rates.

Weaknesses:

Readers can benefit from understanding with more details the locations chosen for stimulation - in light of previous studies that found differences between effects based on proximity to white matter (For example - PMID 32446925, Mohan et al, Brain Stimul. 2020 and PMID 33279717 Mankin et al Brain Stimul. 2021).

Reviewer #1 (Public review):

This is an interesting manuscript aimed at improving the transcriptome characterization of 52 C. elegans neuron classes. Previous single-cell RNA seq studies already uncovered transcriptomes for these, but the data are incomplete, with a bias against genes with lower expression levels. Here, the authors use cell-specific reporter combinations to FACS purify neurons and bulk RNA sequencing to obtain better sequencing depth. This reveals more rare transcripts, as well as non-coding RNAs, pseudogenes, etc. The authors develop computational approaches to combine the bulk and scRNA transcriptome results to obtain more definitive gene lists for the neurons examined.

To ultimately understand features of any cell, from morphology to function, an understanding of the full complement of the genes it expresses is a pre-requisite. This paper gets us a step closer to this goal, assembling a current "definitive list" of genes for a large proportion of C. elegans neurons. The computational approaches used to generate the list are based on reasonable assumptions, the data appear to have been treated appropriately statistically, and the conclusions are generally warranted. I have a few issues that the authors may choose to address:

(1) As part of getting rid of cross-contamination in the bulk data, the authors model the scRNA data, extrapolate it to the bulk data and subtract out "contaminant" cell types. One wonders, however, given that low expressed genes are not represented in the scRNA data, whether the assignment of a gene to one or another cell type can really be made definitive. Indeed, it's possible that a gene is expressed at low levels in one cell, and high levels in another, and would therefore be considered a contaminant. The result would be to throw out genes that actually are expressed in a given cell type. The definitive list would therefore be a conservative estimate, and not necessarily the correct estimate.

(2) It would be quite useful to have tested some genes with lower expression levels using in vivo gene-fusion reporters to assess whether the expression assignments hold up as predicted. i.e. provide another avenue of experimentation, non-computational, to confirm that the decontamination algorithm works.

(3) In many cases, each cell class would be composed of at least 2 if not more neurons. Is it possible that differences between members of a single class would be missed by applying the cleanup algorithms? Such transcripts would be represented only in a fraction of the cells isolated by scRNAseq, and might then be considered not real.

(4) I didn't quite catch whether the precise staging of animals was matched between the bulk and scRNAseq datasets. Importantly, there are many genes whose expression is highly stage-specific or age-specific so even slight temporal differences might yield different sets of gene expression.

(5) To what extent does FACS sorting affect gene expression? Can the authors provide some controls?

Reviewer #2 (Public review):

Summary:

This study from the CenGEN consortium addresses several limitations of single-cell RNA (scRNA) and bulk RNA sequencing in C. elegans with a focus on cells in the nervous system. scRNA datasets can give very specific expression profiles, but detecting rare and non-polyA transcripts is difficult. In contrast, bulk RNA sequencing on isolated cells can be sequenced to high depth to identify rare and non-polyA transcripts but frequently suffers from RNA contamination from other cell types. In this study, the authors generate a comprehensive set of bulk RNA datasets from 53 individual neurons isolated by fluorescence-activated cell sorting (FACS). The authors combine these datasets with a previously published scRNA dataset (Taylor et al., 2021) to develop a novel method, called LittleBites, to estimate and subtract contamination from the bulk RNA data. The authors validate the method by comparing detected transcripts against gold-standard datasets on neuron-specific and non-neuronal transcripts. The authors generate an "integrated" list of protein-coding expression profiles for the 53 neuron sub-types, with fewer but higher confidence genes compared to expression profiles based only on scRNA. Also, the authors identify putative novel pan-neuronal and cell-type specific non-coding RNAs based on the bulk RNA data. LittleBites should be generally useful for extracting higher confidence data from bulk RNA-seq data in organisms where extensive scRNA datasets are available. The additional confidence in neuron-specific expression and non-coding RNA expands the already great utility of the neuronal expression reference atlas generated by the CenGEN consortium.

Strengths:

The study generates and analyzes a very comprehensive set of bulk RNA datasets from individual fluorescently tagged transgenic strains. These datasets are technically challenging to generate and significantly expand our knowledge of gene expression, particularly in cells that were poorly represented in the initial scRNA-seq datasets. Additionally, all transgenic strains are made available as a resource from the Caenorhabditis Elegans Genetics Center (CGC).

The study uses the authors' extensive experience with neuronal expression to benchmark their method for reducing contamination utilizing a set of gold-standard validated neuronal and non-neuronal genes. These gold-standard genes will be helpful for benchmarking any C. elegans gene expression study.

Weaknesses:

The bulk RNA-seq data collected by the authors has high levels of contamination and, in some cases, is based on very few cells. The methodology to remove contamination partly makes up for this shortcoming, but the high background levels of contaminating RNA in the FACS-isolated neurons limit the confidence in cell-specific transcripts.

The study does not experimentally validate any of the refined gene expression predictions, which was one of the main strengths of the initial CenGEN publication (Taylor et al, 2021). No validation experiments (e.g., fluorescence reporters or single molecule FISH) were performed for protein-coding or non-coding genes, which makes it difficult for the reader to assess how much gene predictions are improved, other than for the gold standard set, which may have specific characteristics (e.g., bias toward high expression as they were primarily identified in fluorescence reporter experiments).

The study notes that bulk RNA-seq data, in contrast to scRNA-seq data, can be used to identify which isoforms are expressed in a given cell. However, no analysis or genome browser tracks were supplied in the study to take advantage of this important information. For the community, isoform-specific expression could guide the design of cell-specific expression constructs or for predictive modeling of gene expression based on machine learning.

Reviewer #3 (Public review):

The manuscript by Barrett et al. "Integrating bulk and single cell RNA-seq refines transcriptomic profiles of individual C. elegans neurons" presents a comprehensive approach to integrating bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data to refine transcriptomic profiles of individual C. elegans neurons. The study addresses the limitations of scRNA-seq, such as the under-detection of lowly expressed and non-polyadenylated transcripts, by leveraging the sensitivity of bulk RNA-seq. The authors deploy a computational method, LittleBites, to remove non-neuronal contamination in bulk RNA-seq, that aims to enhance specificity while preserving the sensitivity advantage of bulk sequencing. Using this approach, the authors identify lowly expressed genes and non-coding RNAs (ncRNAs), many of which were previously undetected in scRNA-seq data.

Overall, the study provides high-resolution gene expression data for 53 neuron classes, covering a wide range of functional modalities and neurotransmitter usage. The integrated dataset and computational tools are made publicly available, enabling community-driven testing of the robustness and reproducibility of the study. Nevertheless, while the study represents a relevant contribution to the field, certain aspects of the work require further refinement to ensure the robustness and rigor necessary for peer-reviewed publication. Below, I outline the areas where improvements are needed to strengthen the overall impact and reliability of the findings.

(1) The study relies on thresholding to determine whether a gene is expressed or not. While this is a common practice, the choice of threshold is not thoroughly justified. In particular, the choice of two uniform cutoffs across protein-encoding RNAs and of one distinct threshold for non-coding RNAs is somewhat arbitrary and has several limitations. This reviewer recommends the authors attempt to use adaptive threshold-methods that define gene expression thresholds on a per-gene basis. Some of these methods include GiniClust2, Brennecke's variance modeling, HVG in Seurat, BASiCS, and/or MAST Hurdle model for dropout correction.

(2) Most importantly, the study lacks independent experimental validation (e.g., qPCR, smFISH, or in situ hybridization) to confirm the expression of newly detected lowly expressed genes and non-coding RNAs. This is particularly important for validating novel neuronal non-coding RNAs, which are primarily inferred from computational approaches.

(3) The novel biology is somewhat limited. One potential area of exploration would be to look at cell-type specific alternative splicing events.

(4) The integration method disproportionately benefits neuron types with limited representation in scRNA-seq, meaning well-sampled neuron types may not show significant improvement. The authors should quantify the impact of this bias on the final dataset.

(5) The authors employ a logit transformation to model single-cell proportions into count space, but they need to clarify its assumptions and potential pitfalls (e.g., how it handles rare cell types).

(6) The LittleBites approach is highly dependent on the accuracy of existing single-cell references. If the scRNA-seq dataset is incomplete or contains classification biases, this could propagate errors into the bulk RNA-seq data. The authors may want to discuss potential limitations and sensitivity to errors in the single-cell dataset, and it is critical to define minimum quality parameters (e.g. via modeling) for the scRNAseq dataset used as reference.

(7) Also very important, the LittleBites method could benefit from a more intuitive explanation and schematic to improve accessibility for non-computational readers. A supplementary step-by-step breakdown of the subtraction process would be useful.

(8) In the same vein, the ROC curves and AUROC comparisons should have clearer annotations to make results more interpretable for readers unfamiliar with these metrics.

(9) Finally, after the correlation-based decontamination of the 4,440 'unexpressed' genes, how many were ultimately discarded as non-neuronal?<br /> a) Among these non-neuronal genes, how many were actually known neuronal genes or components of neuronal pathways (e.g., genes involved in serotonin synthesis, synaptic function, or axon guidance)?<br /> b) Conversely, among the "unexpressed" genes classified as neuronal, how many were likely not neuron-specific (e.g., housekeeping genes) or even clearly non-neuronal (e.g., myosin or other muscle-specific markers)?

(10) To increase transparency and allow readers to probe false positives and false negatives, I suggest the inclusion of:<br /> a) The full list of all 4,440 'unexpressed' genes and their classification at each refinement step. In that list flag the subsets of genes potentially misclassified, including:<br /> - Neuronal genes wrongly discarded as non-neuronal.<br /> - Non-neuronal genes wrongly retained as neuronal.<br /> b) Add a certainty or likelihood ranking that quantifies confidence in each classification decision, helping readers validate neuronal vs. non-neuronal RNA assignments.<br /> This addition would enhance transparency, reproducibility, and community engagement, ensuring that key neuronal genes are not erroneously discarded while minimizing false positives from contaminant-derived transcripts.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of different specific dopaminergic neurons in the mushroom body of Drosophila larvae for learning and innate behavior. All the tested neurons are thought to be involved in punishment learning. The authors discover that artificial activation of single DANs in training leads to safety learning, but not punishment learning. Furthermore, activation of single DANs can lead to changes in locomotion behavior, which can affect light preference. The authors provide a deeper understanding of the functional diversity of single dopamine neurons; however, it is unclear how translatable these findings are to learning experiments with real punishment stimuli.

Strengths:

The authors attempt to disentangle what kind of memories are formed with the activation of different dopamine neurons - safety learning, and punishment learning, will the US be required to test for recall or not? They do indeed find differences and the results will be of interest to the learning and memory community.

Interestingly, optogenetic activation of a single DAN during training leads to safety memory, but not punishment memory. Furthermore, DAN activation also affects innate locomotion, and the authors can show that optogenetic activation of different DANs affects locomotion differently.

Weaknesses:<br /> All experiments in the manuscript use optogenetic activation of DANs, thus it is not clear what kind of memories are formed. Several stimuli can be used as punishment, such as electric shock, salt, bitter, and light - it is not clear what kind of memory the authors investigate here. The findings could be discussed in the context of what DANs respond to. Furthermore, studies in adults and larvae showed that most DANs can code for both valences - etc., aversive DANs can be activated by punishment, and inhibited by reward. Thus, safety learning might be a result of a decrease in activity in DANs during odor presentation. The authors also do not discuss possible feedback loops from MBONs to DANs across compartments. Could such connections allow for safety learning in larvae?

The authors show that artificial activation with different light intensities can form different memories and that increasing the light intensity sometimes leads to no memories. Also, using different optogenetic tools reveals different results. This again raises the question of how applicable the results will be for learning with real stimuli. Is there a natural stimulus that only induces safety learning, but no punishment learning?<br /> The authors provide a detailed behavioral analysis of locomotion behavior; however, the detailed analysis seems unnecessary for that dataset. Modulation of speed and bending rate has been described before with simpler methods (specifically for MBONs). The revealed locomotion phenotypes probably affect larval locomotion during memory recall with light activation, thus the authors should show that larvae are potentially able to move during light-on memory tests.

Reviewer #2 (Public review):

Summary:

This study provides valuable context for ongoing research on the role of dopamine in memory and locomotion. DANs have been a fascinating area of study due to their complexity, and this work dissects specific DANs, exploring their roles in different memory-related behaviors while offering some explanations. The discussions provided by the authors effectively situates the study in the broader field of learning, memory, DAN circuitry and behavioral computation in insect brains. The study achieves what it sets out to and it does so unequivocally. The experiments were elegantly designed, leaving little room for doubt in the study's claims. However, the study lacks context regarding the molecular pathways underlying these results. While it strengthens current knowledge by providing robust evidence, it does little to explore the molecular mechanisms behind these effects.

Strengths:

(1) Experiment design is one of the strengths of this study. The experiments are thorough and cover the length and breadth of the core findings of the study. Although a lot of work has already been done in studying the role of dopamine in memory and locomotion, the dissection of the functions of distinct DANs in larvae has been done meticulously with well-structured experiments.<br /> (2) This study fits quite nicely into the puzzle of memory, especially in the context of Dopamine. Previous studies in *Drosophila* adults have shown the opposing roles of DANs in locomotion depending on the context of DAN activation. This study drives that point home for larvae, providing conclusive evidence in that regard.<br /> (3) The use of clear figures and simple language is one of the strengths of this paper. The figures are comprehensive, complete and manage to narrate the story by themselves. The flow of information is smooth. The simple and effective language used maintains scientific rigor while remaining accessible to those new to the field. A pleasant read.

Weaknesses:<br /> (1) The authors have done a great job at structuring the figures. But some main figures would benefit from including the controls instead of placing them in supplementary.<br /> (2) The paper would benefit from a deeper discussion regarding molecular mechanisms underlying their results. It would be interesting to see what the authors think about different Dopamine receptors and how they relate to the findings of this paper.<br /> (3) Throughout the paper, the authors have been clear and comprehensive, but in some cases, further explanation of their choices were missing. For example, the choice to compare bending and tail velocity over other parameters within the same clusters is unclear.

Reviewer #3 (Public review):

Summary

Across species, dopamine release carries out seemingly diverse functions, like reinforcing memories and regulating locomotion and flight. However, whether distinct dopaminergic neurons (DANs) are allocated for each function is not clear. In this study, Toshima et al. have used the numerically simple organization of the Drosophila larval brain to answer this question. They use optogenetic activation to systematically stimulate a small set of DANs, individually and collectively, and study the effect on diverse functions such as memory formation, retrieval, and locomotion. They find that singly or collectively, DL1 DANs can induce punishment and/or safety memory formation and retrieval. DANs can even gate the expression of memory. Finally, the same DANs also modulate locomotion in the larvae. The authors speculate that dopaminergic neurons in other species may also share such overlapping functions. Their findings are nicely summarised in Figure 9.

Strengths

The study comprehensively activates the neurons in the DL1 cluster in a systematic manner. Individual and collective stimulation of the Dl1 DANs has been conducted to assess the induction and gating of aversive punishment memory, safety memory, and acute locomotion.

Specific adult Drosophila DANs are known to induce dual behaviors and functions. The same MP1/y1pedc DANs are recognized for gating appetitive memory expression and representing aversive teaching signals downstream of sensory stimuli such as electric shocks, bitter tastes, and heat. Neurons in the PPL1 cluster regulate adult flight and food-seeking behavior. The authors deserve credit for conducting an organized examination of dopaminergic neuron functions in larvae, which makes their findings more comparable and facilitates the proposal of a holistic model.

They have provided substantial evidence for their findings and frequently presented replicated behavioral data sets. They have been transparent about results that were difficult to explain. Additionally, they have provided an impressive body of supporting data to strengthen their main findings.

Weaknesses

The larvae exhibit directed locomotory action to express punishment or safety memory. If the larvae did not move, we would not be able to assess memory function. Hence, functional activation of DANs could result in one action, which seems like two different functions of memory expression and locomotion. It can also be argued that activation of DANs represents a teaching signal to the KCs, and then eventually, downstream of the MBONs, it results in locomotion modulation. Hence, the seeming functional diversity could be a function of different downstream neuronal pathways and not molecular context-dependent diversity inside dopaminergic neurons. The authors should address this possibility or point out the fallacy in the above argument.

The finding that activation of TH-GAL4 conveys aversive valence and R58E02-GAL4 conveys appetitive valence seems redundant (Figure 6). I understand they say this in the context of locomotion. However, they may not have mentioned similar findings in adults. In adults, artificial activation of DANs covered by the same GAL4 lines acts as aversive and appetitive teaching signals for memory formation. These references should be cited appropriately in the results and discussion if not currently included.

The evidence for the role of dopamine (Figure 7) can be bolstered by using other available RNAi lines against TH. A valium20 vector-based shRNA line is recommended. The current evidence is based mainly on non-specific pharmacological intervention with 3IY.

Reviewer #1 (Public review):

Summary:

The authors sequenced 888 individuals from the 1000 Genomes Project using the Oxford Nanopore long-read sequencing method to achieve highly sensitive, genome-wide detection of structural variants (SVs) at the population level. They conducted solid benchmarking of SV calling and systematically characterized the identified SVs. While short-read sequencing methods, including those used in the 1000 Genomes Project, have been widely applied, they exhibit high accuracy in detecting single nucleotide variants (SNVs) and small insertions and deletions but have limited sensitivity for SV detection. This study significantly enhances SV detection capabilities, establishing it as a valuable resource for human genetic research. Furthermore, the authors constructed an SV imputation panel using the generated data and imputed SVs in 488,130 individuals from the UK Biobank. They then conducted a proof-of-principle genome-wide association study (GWAS) analysis based on the imputed SVs and selected traits within the UK Biobank. Their findings demonstrate that incorporating SV-GWAS analysis provides additional insights beyond conventional GWAS frameworks focusing on SNVs, particularly in improving fine mapping.

Strengths:

The authors constructed a high-sensitivity reference panel of genome-wide SVs at the population level, addressing a critical gap in the field of human genetics. This resource is expected to significantly advance research in human genetics. They demonstrated the imputation of SVs in individuals from the UK Biobank using this panel and conducted a proof-of-concept SV-based GWAS. Their findings highlight a novel and effective strategy for integrating SVs into GWAS, which will facilitate the analysis of human genetic data from the UK Biobank and other datasets. Their conclusions are supported by comprehensive analyses.

Weaknesses:

(1) Although the authors employ state-of-the-art analytical approaches for the identification of SVs, the overall accuracy remains suboptimal, as indicated by an F1 score of 74.0%, particularly in tandem repeat regions. To enhance accuracy, it would be beneficial to explore alternative SV detection methods or develop novel approaches. Given the value of the reference panel and the fact that improved SV accuracy would lead to more precise SV imputation and GWAS results, investing effort in methodological refinement is highly encouraged.

(2) From the Methods section, it appears that the authors employed Beagle for both the "leave-one-out" imputation and the UK Biobank imputation. It would be better to explicitly clarify this in the Results section and provide a detailed description of the corresponding procedures and parameters in the Methods section for both analyses, as this represents a key aspect of the study. Additionally, Beagle is not specifically designed for SV imputation, the imputation quality of SVs is generally lower than that of SNVs. Exploring strategies to improve SV imputation, such as developing a novel method with reference panel data, may enhance performance. It is also important to assess how this reduced imputation quality may influence GWAS results. For instance, it would be useful to examine whether associated SVs exhibit higher imputation quality and whether SVs with lower quality are less likely to achieve significant association signals. In addition, the lower imputation quality observed for INV, DUP, and BND variants (Figure 3) may be due to their greater lengths (Figure 2). It is better to investigate the relationship between SV length and imputation quality.

(3) All examples presented in the manuscript focus on SVs that overlap with genes. It may also be valuable to investigate SVs that do not overlap with genes but intersect with enhancer regions. SVs can contribute to disease by altering regulatory elements, such as enhancers, which play a crucial role in gene expression. Including such analyses would further demonstrate the utility of SV-GWAS and provide deeper insights into the functional impact of SVs.

(4) The data availability link currently provides only a VCF file ("sniffles2_joint_sv_calls.vcf.gz") containing the identified SVs. It would be beneficial for the authors to make all raw sequencing data (FASTQ files) and key processed datasets (such as alignment results and merged SV and SNV files) available. Providing these resources would enable other researchers to develop improved SV detection and imputation methods or conduct further genetic analyses. Furthermore, establishing a dedicated website for data access, along with a genome browser for SV visualization, could significantly enhance the impact and accessibility of the study. Additionally, all code, particularly the SV imputation pipeline accompanied by a detailed tutorial, should be deposited in a public repository such as GitHub. This would support researchers in imputing SVs and conducting SV-GWAS on their own datasets.

Reviewer #2 (Public review):

Summary:

The authors aimed to develop a novel and efficient method for SV detection, utilizing data from the 1000 Genomes Project (1KGP) for modeling and calibration. This method was subsequently validated using UK population data and applied to identify structural variants associated with specific disease phenotypes.

Strengths:

Third-generation single-molecule sequencing data offers several advantages over traditional high-throughput sequencing methods, particularly due to its long-read lengths, which provide valuable insights into significant forms of genomic variation. The authors have developed an efficient method for detecting structural variations and optimizing the utilization of genomic data. We hope that this method will continue to be refined, enabling researchers to more effectively leverage long-read data, high-throughput data, or even a synergistic combination of both.

Weaknesses:

Although this research contributes to our ability to more effectively utilize long-length and high-throughput data, there are some key issues that need to be addressed in terms of analyzing the specific results as well as writing the article.

Reviewer #3 (Public review):

Summary:

This study successfully identified genetic loci associated with various traits by generating large-scale long-read sequencing data from a diverse set of samples. This study is significant because it not only produces large-scale long-read genome sequencing data but also demonstrates its application in actual genetics research. Given its potential utility in various fields, this study is expected to make a valuable contribution to the academic community and to this journal. However, there are several critical aspects that could be improved. Below are specific comments for consideration.

Strengths:

Producing high-quality, large-scale variant datasets and imputation datasets

Weaknesses:

(1) Data availability

Currently, it appears that only the Genomic Lens SV Panel is available on the webpage described in the Data Availability section. It is unclear whether the authors intend to release the raw sequencing data. Since the study utilized samples from the 1000 Genomes Project, there should be no restriction on making the data publicly accessible. Given this, would the authors consider making the raw sequencing reads publicly available? If so, NCBI SRA or EBI ENA would be the most appropriate repositories for data deposition. I strongly encourage the authors to consider public data release.

Additionally, accessing the Genomic Lens SV Panel data does not seem straightforward. The manuscript should provide a more detailed description of how researchers can access and utilize these data. In my opinion, the best approach would be to upload the variant data (VCF files) to a public database such as the European Variation Archive (EVA) hosted by EBI.

I strongly request that the authors publicly deposit the variant data. At a minimum:

a) The joint genotype data for all 888 samples from the 1000 Genomes Project must be publicly available.<br /> b) For the UK Biobank samples, at least allele frequency data should be disclosed.

Since eLife has a well-established data-sharing policy, compliance with these guidelines is essential for publication in this journal.

(2) Long-read sequencing data quality

While the manuscript presents N50 read length and mean or median read base quality for each sample in a table, it would be highly beneficial to visualize these data in figures as well. A violin plot or similar visualization summarizing these distributions would significantly improve data presentation.

Notably, the base quality of ONT long-read sequencing data appears lower than expected. This may be attributed to the use of pore version 9.4.1, but the unexpectedly low base quality still warrants attention. It would be helpful to include a small figure within Figure 2 to illustrate this point. A visual representation of read length distribution and base quality distribution would strengthen the manuscript.

(3) Variant detection precision, recall, and F1 score

This study focuses on insertions and deletions (indels) {greater than or equal to}50 bp, but it remains unclear how well variants <50 bp are detected. I am particularly interested in the precision, recall, and F1 score for variants between 5-49 bp.

While ONT base quality is relatively low, single-base variants are challenging to analyze, but variants {greater than or equal to}5 bp should still be detectable as their read accuracy is still approximately 90%, making analysis feasible. Given that Sniffles supports the detection of variants as small as 1 bp, I strongly encourage the authors to conduct an additional analysis.

A simple two-category classification (e.g., 5-49 bp and {greater than or equal to}50 bp) should suffice. Additionally, a comparative analysis with HiFi and short-read sequencing data would be highly valuable. If possible, I strongly recommend that all detected variants {greater than or equal to}5 bp be made publicly available as VCF files.

(4) Assembly-based methods

Given the low read accuracy and low sequencing depth in this dataset, it is understandable that genome assembly is challenging. However, the latest high-quality human genome datasets-such as those produced by the Human Pangenome Reference Consortium (HPRC)-demonstrate that assembly-based approaches provide significant advantages, particularly for resolving complex and long structural variants.

Since HPRC data also utilize 1000 Genomes Project samples, it would be highly informative to compare the accuracy of ONT sequencing in this study with HPRC's assembly-based genome data. The recent publication on 47 HPRC samples provides a valuable reference for such a comparison. Given its relevance, the authors should consider providing a comparative analysis with HPRC data.

References:

(1) A draft human pangenome reference<br /> https://www.nature.com/articles/s41586-023-05896-x

(2) The Human Pangenome Project: a global resource to map genomic diversity<br /> https://www.nature.com/articles/s41586-022-04601-8

(3) A pangenome reference of 36 Chinese populations<br /> https://www.nature.com/articles/s41586-023-06173-7

(4) Long-read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits<br /> https://www.nature.com/articles/s41588-021-00865-4

(5) Increased mutation and gene conversion within human segmental duplications<br /> https://www.nature.com/articles/s41586-023-05895-y

(6) Structural polymorphism and diversity of human segmental duplications<br /> https://www.nature.com/articles/s41588-024-02051-8

(7) Highly accurate Korean draft genomes reveal structural variation highlighting human telomere evolution<br /> https://academic.oup.com/nar/article/53/1/gkae1294/7945385

Joint Public Review:

Pannexin (Panx) hemichannels are a family of heptameric membrane proteins that form pores in the plasma membrane through which ions and relatively large organic molecules can permeate. ATP release through Panx channels during the process of apoptosis is one established biological role of these proteins in the immune system, but they are widely expressed in many cells throughout the body, including the nervous system, and likely play many interesting and important roles that are yet to be defined. Although several structures have now been solved of different Panx subtypes from different species, their biophysical mechanisms remain poorly understood, including what physiological signals control their activation. Electrophysiological measurements of ionic currents flowing in response to Panx channel activation have shown that some subtypes can be activated by strong membrane depolarization or caspase cleavage of the C-terminus. Here, Henze and colleagues set out to identify endogenous activators of Panx channels, focusing on the Panx1 and Panx2 subtypes, by fractionating mouse liver extracts and screening for activation of Panx channels expressed in mammalian cells using whole-cell patch clamp recordings. The authors present a comprehensive examination with robust methodologies and supporting data that demonstrate that lysophospholipids (LPCs) directly Panx-1 and 2 channels. These methodologies include channel mutagenesis, electrophysiology, ATP release and fluorescence assays, and molecular modelling. Mouse liver extracts were initially used to identify LPC activators, but the authors go on to individually evaluate many different types of LPCs to determine those that are more specific for Panx channel activation. Importantly, the enzymes that endogenously regulate the production of these LPCs were also assessed along with other by-products that were shown not to promote pannexin channel activation. In addition, the authors used synovial fluid from canine patients, which is enriched in LPCs, to highlight the importance of the findings in pathology. Overall, we think this is likely to be an important study because it provides strong evidence that LPCs can function as activators of Panx1 and Panx2 channels, linking two established mediators of inflammatory responses and opening an entirely new area for exploring the biological roles of Panx channels. This study provides an excellent foundation for future studies and importantly provides clinical relevance.

[Editors' note: this paper has been through two rounds of review and revisions, available here: https://sciety.org/articles/activity/10.1101/2023.10.23.563601]

Reviewer #1 (Public review):

Summary:

This study aims to understand the malaria antigen-specific cTfh profile of children and adults living in malaria holoendemic area. PBMC samples from children and adults were unstimulated or stimulated with PfSEA-1A or PfGARP in vitro for 6h and analysed by a cTfh-focused panel. Unsupervised clustering and analysis on cTfh was performed. The main conclusions are: A) the children cohort has a more diverse (cTfh1/2/17) recall responses compared to adults (mainly cTfh17) and, B) Pf-GARP stimulates better cTfh17 responses in adults, thus a promising vaccine candidate.

Strengths:

This study is, in general, well-designed and with excellent data analysis. The use of unsupervised clustering is a nice attempt to understand the heterogeneity of cTfh cells.

Weaknesses:

The authors have provided additional data in Supplementary Figures 14-16. However, I remain concerned about whether cTfh cells are truly responding to antigen stimulation. In Supplementary Figure 15A-F, the IFNg responses appear as expected, SEB elicits the strongest response, as it stimulates bulk T cells, and the staining is promising, showing a clear distinction between IFNg+ and IFNg- populations. However, in Supplementary Figure 15I-N, the IL-21 secretion assay is concerning. The FACS plots make it difficult to distinguish IL-21+ from IL-21- cells, raising concerns about the validity of this analysis. Additionally, in panel J, the responses to PfSEA-1A or PfGARP appear even greater than those to SEB stimulation. In PBMCs, only a small percentage of T cells should be specific to a particular antigen. How can the positive control (SEB) produce a weaker response than stimulation with a specific antigen? This suggests that the IL-21 secretion assay may not have worked, making the authors' interpretation unreliable.

I also have similar concerns about the IL-4 secretion in Sup Figure 16. First, the FACS plot shows that appear double-positive for IL-21 and IL-4, so it suggests the staining may be due to autofluorescence rather than true cytokine signals. Also in B-C the responses of SEB stimulation is generally weaker than stimulated by one antigen, further questioning the reliability of the IL-4 assay. In summary, I am not convinced that the in vitro antigen stimulation assay worked as intended. Consequently, the manuscript's claims regarding PfSEA-1A- and PfGARP-specific cTfh responses are not sufficiently supported by the presented data.

Reviewer #3 (Public review):

Summary:

The goal of this study was to carry out an in-depth granular and unbiased phenotyping of peripheral blood circulating Tfh specific to two malaria vaccine candidates, PfSEA-1A and PfGARP, and correlate these with age (children vs adults) and protection from malaria (antibody titers against Plasmodium antigens.) Authors further attempted to identify any specific differences of the Tfh responses to these two distinct malaria antigens.

Strengths:

The authors had access to peripheral blood samples from children and adults living in a malaria-endemic region of Kenya. The authors studied these samples using in vitro restimulation in the presence of specific malaria antigens. Authors generated a very rich data set from these valuable samples using cutting-edge spectral flow cytometry and a 21-plex panel that included a variety of surface markers, cytokines and transcription factors.

Update following first revision (R1) of the manuscript:

The authors have made a great effort to comprehensively address comments raised by the reviewers. In particular, clearly showing expression of ICOS and Bcl6 on CXCR5+ cells greatly strengthens the case for defining these cells as Tfh-like circulatory lymphocytes (cTfh).

Weaknesses:

Update following first revision (R1) of the manuscript:

Unfortunately, my main concern remains. As it stands, the study is not really on antigen-specific T cells, but rather on the overall CD4 T cell compartment plus or minus antigenic stimulation. Although authors used an in vitro restimulation strategy with malaria antigens, they do not focus on cells de-novo expressing activation markers as a result of restimulation, neither they use tetramers to detect antigen-specific T cells. Moreover, their data shows that the number of CXCR5+ CD4 T cells de-novo expressing activation markers and/or cytokines as a result of their in vitro restimulation is negligible, even when using a prototypic superantigen (SEB).

Thus, no antigen-specific CXCR5+ CD4 T cells could be analysed with the data that the authors provide in this manuscript.

Reviewer #4 (Public review):

Summary:

This manuscript is a descriptive study of circulating T follicular helper (cTfh) responses to PfSEA -1A or PfGARP (targets of new antimalaria vaccine candidates) in PBMCs from a convenience sample of children (7 yrs of age) and adults living in a malaria holo endemic Kenya using multiparameter flow cytometry and clustering analysis. This cell type promotes B cell production of long-lived antimalarial antibodies to provide protection against malaria. They find that children had a wider cTFH cytokine and TF profile cellular response in comparison to adults who responded to both antigens but had a narrower response profile.

Strengths:

Carefully done study, very detailed, nice summary model at the end of the paper. The revision provides requested clarification on a number of issues, including CD40L expression which was not differentially expressed between groups. They add additional data into the supplemental files, including IL4 and IL21 data by presenting the cytoplots.

Weaknesses:

To know the significance of these cTfh cells for long-term protection of malaria requires functional and transfer experiments in animal models which is outside the scope of this work.

Reviewer #1 (Public review):

This is a comprehensive study that sheds light on how Wag31 functions and localises in mycobacterial cells. A clear link to interactions with CL is shown using a combination of microscopy in combination with fusion fluorescent constructs, and lipid specific dyes. Furthermore, studies using mutant versions of Wag31 shed light on the functionalities of each domain in the protein. My concerns/suggestions for the manuscript are minor:

(1) Ln 130. A better clarification/discussion is required here. It is clear that both depletion and overexpression have an effect on levels of various lipids, but subsequent descriptions show that they affect different classes of lipids.<br /> (2) The pulldown assays results are interesting, but the links are tentative.<br /> (3) The authors may perhaps like to rephrase claims of effects lipid homeostasis, as my understanding is that lipid localisation rather than catabolism/breakdown is affected.

In response to the above reviews the authors have made the required changes in the revised manuscript.

Reviewer #2 (Public review):

Summary:

Kapoor et. al. investigated the role of the mycobacterial protein Wag31 in lipid and peptidoglycan synthesis and sought to delineate the role of the N- and C- terminal domains of Wag31. They demonstrated that modulating Wag31 levels influences lipid homeostasis in M. smegmatis and cardiolipin (CL) localisation in cells. Wag31 was found to preferentially bind CL-containing liposomes, and deleting the N-terminus of the protein significantly decreased this interaction. Novel interactions between Wag31 and proteins involved in lipid metabolism and cell wall synthesis were identified, suggesting that Wag31 recruits proteins to the intracellular membrane domain by direct interaction.

Strengths:

(1) The importance of Wag31 in maintaining lipid homeostasis is supported by several lines of evidence.<br /> (2) The interaction between Wag31 and cardiolipin, and the role of the N-terminus in this interaction was convincingly demonstrated.

Weakness:

(1) Interactome analysis with truncated versions of the proteins could not be performed in M. smegmatis due to protein instability.

Reviewer #3 (Public review):

Summary:

This manuscript describes the characterization of mycobacterial cytoskeleton protein Wag31, examining its role in orchestrating protein-lipid and protein-protein interactions essential for mycobacterial survival. The most significant finding is that Wag31, which directs polar elongation and maintains the intracellular membrane domain, was revealed to have membrane tethering capabilities.

Strengths:

The authors provided a detailed analysis of Wag31 domain architecture, revealing distinct functional roles: the N-terminal domain facilitates lipid binding and membrane tethering, while the C-terminal domain mediates protein-protein interactions. Overall, this study offers a robust and new understanding of Wag31 function.

Weaknesses:

The authors did not address some of the comments. The following concerns should be addressed.

• As far as I can tell, authors did not address my prior comments on Line 270, which is Line 280 in the revised manuscript: the N-terminal region is important for lipid homeostasis, but the statement in Line 270, "the maintenance of lipid homeostasis by Wag31 is a consequence of its tethering activity" requires additional proof. Please indicate the page and line numbers in the revised manuscript so that I can identify the specific changes the authors made.

• Since this pull-down assay was conducted by mixing E. coli lysate expressing Wag31 and Msm lysate expression Wag31 interactors like MurG, it is possible that the interactions are not direct. Authors acknowledge that this is a valid point, and indicated that they "will describe this caveat in the revised manuscript". I have difficulty finding where this revision was made. Please indicate the page and line numbers.

Reviewer #1 (Public review):

Summary:

In this study, the authors re-analyzed a public dataset (Rademaker et al, 2019, Nature Neuroscience) which includes fMRI and behavioral data recorded while participants held an oriented grating in visual working memory (WM) and performed a delayed recall task at the end of an extended delay period. In that experiment, participants were pre-cued on each trial as to whether there would be a distracting visual stimulus presented during the delay period (filtered noise or randomly-oriented grating). In this manuscript, the authors focused on identifying whether the neural code in retinotopic cortex for remembered orientation was 'stable' over the delay period, such that the format of the code remained the same, or whether the code was dynamic, such that information was present, but encoded in an alternative format. They identify some timepoints - especially towards the beginning/end of the delay - where the multivariate activation pattern fails to generalize to other timepoints, and interpret this as evidence for a dynamic code. Additionally, the authors compare the representational format of remembered orientation in the presence vs absence of a distracting stimulus, averaged over the delay period. This analysis suggested a 'rotation' of the representational subspace between distracting orientations and remembered orientations, which may help preserve simultaneous representations of both remembered and viewed stimuli. Intriguingly, this rotation was a bit smaller for Expt 2, in which the orientation distractor had a greater behavioral impact on the participants' behavioral working memory recall performance, suggesting that more separation between subspaces is critical for preserving intact working memory representations.

Strengths:

(1) Direct comparisons of coding subspaces/manifolds between timepoints, task conditions, and experiments is an innovative and useful approach for understanding how neural representations are transformed to support cognition

(2) Re-use of existing dataset substantially goes beyond the authors' previous findings by comparing geometry of representational spaces between conditions and timepoints, and by looking explicitly for dynamic neural representations

(3) Simulations testing whether dynamic codes can be explained purely by changes in data SNR are an important contribution, as this rules out a category of explanations for the dynamic coding results observed

Weaknesses:

(1) Primary evidence for 'dynamic coding', especially in early visual cortex, appears to be related to the transition between encoding/maintenance and maintenance/recall, but the delay period representations seem overall stable, consistent with some previous findings. However, given the simulation results, the general result that representations may change in their format appears solid, though the contribution of different trial phases remains important for considering the overall result.

(2) Converting a continuous decoding metric (angular error) to "% decoding accuracy" serves to obfuscate the units of the actual results. Decoding precision (e.g., sd of decoding error histogram) would be more interpretable and better related to both the previous study and behavioral measures of WM performance.

Reviewer #2 (Public review):

Summary:

In this work, Degutis and colleagues addressed an interesting issue related to the concurrent coding of sensory percepts and visual working memory contents in visual cortices. They used generalization analyses to test whether working memory representations change over time, diverge from sensory percepts, and vary across distraction conditions. Temporal generalization analysis demonstrated that off-diagonal decoding accuracies were lower than on-diagonal decoding accuracies, regardless of the presence of intervening distractions, implying that working memory representations can change over time. They further showed that the coding space for working memory contents showed subtle but statistically significant changes over time, potentially explaining the impaired off-diagonal decoding performance. The neural coding of sensory distractions instead remained largely stable. Generalization analyses between target and distractor codes showed overlaps but were not identical. Cross-condition decodings had lower accuracies compared to within-condition decodings. Finally, within-condition decoding revealed more reliable working memory representations in the condition with intervening random noises compared to cross-condition decoding using a trained classifier on data from the no-distraction condition, indicating a change in the VWM format between the noise distractor and no-distractor trials.

Strengths:

This paper demonstrates a clever use of generalization analysis to show changes in the neural codes of working memory contents across time and distraction conditions. It provides some insights into the differences between representations of working memory and sensory percepts, and how they can potentially coexist in overlapping brain regions.

Comments on revisions:

I appreciate the authors' efforts in addressing my previous concerns. The inclusion of additional analyses and data has strengthened the paper. I have no further concerns.

Reviewer #1 (Public review):

Summary:

The authors investigate ligand and protein-binding processes in GPCRs (including dimerization) by the multiple walker supervised molecular dynamics method. The paper is interesting and it is very well written.

Strengths:

The authors' method is a powerful tool to gain insight on the structural basis for the pharmacology of G protein-coupled receptors.

Reviewer #2 (Public review):

The study by Deganutti and co-workers is a methodological report on an adaptive sampling approach, multiple walker supervised molecular dynamics (mwSuMD), which represents an improved version of the previous SuMD.<br /> Case-studies concern complex conformational transitions in a number of G protein Coupled Receptors (GPCRs) involving long time-scale motions such as binding-unbinding and collective motions of domains or portions. GPCRs are specialized GEFs (guanine nucleotide exchange factors) of heterotrimeric Gα proteins of the Ras GTPase superfamily. They constitute the largest superfamily of membrane proteins and are of central biomedical relevance as privileged targets of currently marketed drugs.<br /> MwSuMD was exploited to address:

a) binding and unbinding of the arginine-vasopressin (AVP) cyclic peptide agonist to the V2 vasopressin receptor (V2R);<br /> b) molecular recognition of the β2-adrenergic receptor (β2-AR) and heterotrimeric GDP-bound Gs protein;<br /> c) molecular recognition of the A1-adenosine receptor (A1R) and palmotoylated and geranylgeranylated membrane-anchored heterotrimeric GDP-bound Gi protein;<br /> d) the whole process of GDP release from membrane-anchored heterotrimeric Gs following interaction with the glucagon-like peptide 1 receptor (GLP1R), converted to the active state following interaction with the orthosteric non-peptide agonist danuglipron.

The revised version has improved clarity and rigor compared to the original also thanks to the reduction in the number of complex case studies treated superficially.<br /> The mwSuMD method is solid and valuable, has wide applicability and is compatible with the most world-widely used MD engines. It may be of interest to the computational structural biology community.<br /> The huge amount of high-resolution data on GPCRs makes those systems suitable, although challenging, for method validation and development.<br /> While the approach is less energy-biased than other enhanced sampling methods, knowledge, at the atomic detail, of binding sites/interfaces and conformational states is needed to define the supervised metrics, the higher the resolution of such metrics is the more accurate the outcome is expected to be. Definition of the metrics is a user- and system-dependent process.

Reviewer #3 (Public review):

Summary:

In the present work Deganutti et al. report a structural study on GPCR functional dynamics using a computational approach called supervised molecular dynamics.

Strengths:

The study has the potential to provide novel insight into GPCR functionality. An example is the interaction between D344 and R385 identified during the Gs coupling by GLP-1R. However, validation of the findings, even computationally through for instance in silico mutagenesis study, is advisable.

Weaknesses:

No significant advance of the existing structural data on GPCR and GPCR/G protein coupling is provided. Most of the results are reproductions of the previously reported structures.

Reviewer #1 (Public review):

Summary:

Activated male Plasmodium gametocytes undergo very rapid nuclear division, while keeping the nuclear envelope intact. There is interest in how events inside the nucleus are co-ordinated with events in the parasite cytoplasm, to ensure that each nucleus is packaged into a nascent male gamete.

This manuscript by Zeeshan et al describes the organisation of a nuclear membrane bridging protein, SUN1, during nuclear division. SUN1 is expected from studies in other organisms to be a component of a bridging complex (LINC) that connects the inner nuclear membrane to the outer nuclear membrane, and from there to the cytoplasmic microtubule-organising centres, the centrosome and the basal body.

The authors show that knockout of the SUN1 in gametocytes leads to severe disruption of the mitotic spindle and failure of the basal bodies to segregate. The authors show convincingly that functional SUN1 is required for male gamete formation and subsequent oocyst development.

The authors identified several SUN1-interacting proteins, thus providing information about the nuclear membrane bridging machinery.

Strengths:

The authors have used state of the art imaging, genetic manipulation and immunoprecipitation approaches.

Weaknesses:

Technical limitations of some of the methods used make it difficult to interpret some of the micrographs.

From studies in other organisms, a protein called KASH is a critical component the bridging complex (LINC). That is, KASH links SUN1 to the outer nuclear membrane. The authors undertook a gene sequence analysis that reveals that Plasmodium lacks a KASH homologue. Thus, further work is needed to identify the functional equivalent of KASH, to understand bridging machinery in Plasmodium.

Comments on revised version:

The authors have addressed the comments and suggestions that I provided as part of a Review Commons assessment.

Reviewer #2 (Public review):

Zeeshan et al. investigate the function of the protein SUN1, a proposed nuclear envelope protein linking nuclear and cytoplasmic cytoskeleton, during the rapid male gametogenesis of the rodent malaria parasite Plasmodium berghei. They reveal that SUN1 localises to the nuclear envelope (NE) in male and female gametes and show that the male NE has unexpectedly high dynamics during the rapid process of gametogenesis. Using expansion microscopy, the authors find that SUN1 is enriched at the neck of the bipartite MTOC that links the intranuclear spindle to the basal bodies of the cytoplasmic axonemes. Upon deletion of SUN1, the basal bodies of the eight axonemes fail to segregate, no spindle is formed, and emerging gametes are anucleated, leading to a complete block in transmission. By interactomics the authors identify a divergent allantoicase-like protein, ALLAN, as a main interaction partner of SUN1 and further show that ALLAN deletion largely phenocopies the effect of SUN1.

Overall, the authors use an extensive array of fluorescence and electron microscopy techniques as well as interactomics to convincingly demonstrate that SUN1 and ALLAN play a role in maintaining the structural integrity of the bipartite MTOC during the rapid rounds of endomitosis in male gametogenesis.

Two suggestions for improvement of the work remain:

(1) Lipidomic analysis of WT and SUN1-knockout gametocytes before and after activation resulted in only minor changes in some lipid species. Without statistical analysis, it remains unclear if these changes are statistically significant and not rather due to expected biological variability. While the authors clearly toned down their conclusions in the revised manuscript, some phrasings in the results and the discussion still suggest that gametocyte activation and/or SUN1-knockout affects lipid composition. Similarly, some phrases suggest that SUN1 is responsible for the observed loops and folds in the NE and that SUN1 KO affects the NE dynamics. Currently, I do not think that the data supports these statements.

(2) It is interesting to note that ALLAN has a much more specific localisation to basal bodies than SUN1, which is located to the entire nuclear envelope. Knock out of ALLAN also exhibits a milder (but still striking) phenotype than knockout of SUN1. These observations suggest that SUN1 has additional roles in male gametogenesis besides its interaction with ALLAN, which could be discussed a bit more.

This study uses extensive microscopy and genetics to characterise an unusual SUN1-ALLAN complex, thus providing new insights into the molecular events during Plasmodium male gametogenesis, especially how the intranuclear events (spindle formation and mitosis) are linked to the cytoplasmic separation of the axonemes. The characterisation of the mutants reveals an interesting phenotype, showing that SUN1 and ALLAN are localised to and maintain the neck region of the bipartite MTOC. The authors here confirm and expand the previous knowledge about SUN1 in P. berghei, adding more detail to its localisation and dynamics, and further characterise the interaction partner ALLAN. Given the evolutionary divergence of Plasmodium, these results are interesting not only for parasitologists, but also for more general cell biologists.

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of taste cells of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi" (in terms of human taste); these kokumi stimuli appear to enhance other canonical tastes, increasing what are essentially hedonic attributes of other stimuli. The mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model, and comes to a similar conclusion, albeit with some small differences between the two rodent species.

Strengths:

The data show effects of ornithine on taste/intake in laboratory rats: In two-bottle and briefer intake tests, adding ornithine results in higher intake of most, but all not all stimuli tested. Bilateral chorda tympani (CT) nerve cuts or the addition of GPRC6A antagonists decreased or eliminated these effects. Ornithine also evoked responses by itself in the CT nerve, but mainly at higher concentrations; at lower concentrations it potentiated the response to monosodium glutamate. Finally, immunocytochemistry of taste cell expression indicated that GPRC6A was expressed predominantly in the anterior tongue, and co-localized (to a small extent) with only IP3R3, indicative of expression in a subset of type II taste receptor cells.

Weaknesses:

As the authors are aware, it is difficult to assess a complex human taste with complex attributes, such as kokumi, in an animal model. In these experiments they attempt to uncover mechanistic insights about how ornithine potentiates other stimuli by using a variety of established experimental approaches in rats. They partially succeed by finding evidence that GPRC6A may mediate effects of ornithine when it is used at lower concentrations. In the revisions they have scaled back their interpretations accordingly. A supplementary experiment measuring certain aspects of the effects of ornithine added to Miso soup in human subjects is included for the express purpose of establishing that the kokumi sensation of a complex solution is enhanced by ornithine. This (supplementary) experiment was conducted with a small sample size, and though perhaps useful, these preliminary results do not align particularly well with the animal experiments. It would be helpful to further explore human taste of ornithine in a larger and better-controlled study.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors provide compelling evidence that ornithine enhances the palatability of several chemical stimuli (i.e., IMP, MSG, MPG, Intralipos, sucrose, NaCl, quinine). Ornithine also increases CT nerve responses to MSG. Additionally, the authors provide evidence that the effects of ornithine are mediated by GPRC6A, a G-protein-coupled receptor family C group 6 subtype A, and that this receptor is expressed primarily in fungiform taste buds. Taken together, these results indicate that ornithine enhances the palatability of multiple taste stimuli in rats, and that the enhancement is mediated, at least in part, within fungiform taste buds. This finding could stand on its own. The question of whether ornithine produces these effects by eliciting kokumi-like perceptions (see below) should be presented as speculation in the Discussion section.

Weaknesses:

I am still unconvinced that the measurements in rats reflect the "kokumi" taste percept described in humans. The authors conducted long-term preference tests, 10-min avidity tests and whole chorda tympani (CT) nerve recordings. None of these procedures specifically model features of "kokumi" perception in humans, which (according to the authors) include increasing "intensity of whole complex tastes (rich flavor with complex tastes), mouthfulness (spread of taste and flavor throughout the oral cavity), and persistence of taste (lingering flavor)." While it may be possible to develop behavioral assays in rats (or mice) that effectively model kokumi taste perception in humans, the authors have not made any effort to do so. As a result, I do not think that the rat data provide support for the main conclusion of the study--that "ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor."

Why are the authors hypothesizing that the primary impacts of ornithine are on the peripheral taste system? While the CT recordings provide support for peripheral taste enhancement, they do not rule out the possibility of additional central enhancement. Indeed, based on the definition of human kokumi described above, it is likely that the effects of kokumi stimuli in humans are mediated at least in part by the central flavor system.

The authors include (in the supplemental data section) a pilot study that examined the impact of ornithine on variety of subjective measures of flavor perception in humans. The presence of this pilot study within the larger rat study does not really make sense. If the human studies are so important, as the authors state, then why did the authors relegate them to the supplemental data section? Usually one places background and negative findings in this section of a paper. Accordingly, I recommend that the human data be published in a separate article.

Reviewer #3 (Public review):

Summary:

In this study the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste. The researchers confirmed in rats their previous work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants including: inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl; salt); citric acid (sour) and quinine hydrochloride (bitter). Robust effects of ornithine were observed in the cases of IMP, MSG, MPG and sucrose; and little or no effects were observed in the cases of sodium chloride, citric acid; quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. Inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify a role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). These alternatives are appropriately discussed and, taken together, the experimental results favor the authors' interpretation that C6A mediates the Ornithine responses. The authors provide preliminary data in Suppl. 3 for the possibility of co-expression of C6A with the CaSR.

In the Discussion, the authors consider the potential effects of kokumi substances on the threshold concentrations of key tastants such as glutamate, arguing that extension of taste distribution to additional areas of the mouth (previously referred to as 'mouthfulness') and persistence of taste/flavor responses (previously referred to as 'continuity') could arise from a reduction in the threshold concentrations of umami and other substances that evoke taste responses. This concept may help to design future experiments.

Weaknesses:

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9).

The status of one of the compounds used as an inhibitor of C6A, the gallate derivative EGCG, as a potential inhibitor of the CaSR or T1R1/T1R3 is unknown. It would have been helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response.

It would have been helpful to include a positive control kokumi substance in the two bottle preference experiment (e.g., one of the known gamma glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

Reviewer #1 (Public review):

Summary:

This manuscript explores the transcriptional landscape of high-grade serous ovarian cancer (HGSOC) using consensus-independent component analysis (c-ICA) to identify transcriptional components (TCs) associated with patient outcomes. The study analyzes 678 HGSOC transcriptomes, supplemented with 447 transcriptomes from other ovarian cancer types and noncancerous tissues. By identifying 374 TCs, the authors aim to uncover subtle transcriptional patterns that could serve as novel drug targets. Notably, a transcriptional component linked to synaptic signaling was associated with shorter overall survival (OS) in patients, suggesting a potential role for neuronal interactions in the tumor microenvironment. Given notable weaknesses like lack of validation cohort or validation using other platforms (other than the 11 samples with ST), the data is considered highly descriptive and preliminary.

The study reveals significant findings by identifying a transcriptional component (TC121) associated with synaptic signaling, which is linked to shorter survival in patients with high-grade serous ovarian cancer, highlighting the potential role of neurons in the tumor microenvironment. However, the evidence could be strengthened by experimental validation to confirm the functional roles of key genes within TC121 and further exploration of its spatial aspects, including deeper analysis of neuronal and synaptic and other neuronal gene expression.

Strengths:

Innovative Methodology:<br /> The use of c-ICA to dissect bulk transcriptomes into independent components is a novel approach that allows for the identification of subtle transcriptional patterns that may be overshadowed in traditional analyses.

Comprehensive Data Integration:<br /> The study integrates a large dataset from multiple public repositories, enhancing the robustness of the findings. The inclusion of spatially resolved transcriptomes adds a valuable dimension to the analysis.

Clinical Relevance:<br /> The identification of a synaptic signaling-related TC associated with poor prognosis highlights a potential new avenue for therapeutic intervention, emphasizing the role of the tumor microenvironment in cancer progression.

Weaknesses:

Mechanistic Insights:<br /> While the study identifies TCs associated with survival, it provides limited mechanistic insights into how these components influence cancer progression. Further experimental validation is necessary to elucidate the underlying biological processes.

Generalizability:<br /> The findings are primarily based on transcriptomic data from HGSOC. It remains unclear how these results apply to other subtypes of ovarian cancer or different cancer types.

Innovative Methodology:<br /> Requires more validation using different platforms (IHC) to validate the performance of this bulk derived data. Also, the lack of control on data quality is a concern.

Clinical Application:<br /> Although the study suggests potential drug targets, the translation of these findings into clinical practice is not addressed. Probably given lack of some QA/QC procedures it'll be hard to translate these results. Future studies should focus on validating these targets in clinical settings.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data which are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns which are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC would lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (for example, Knapen et al. (2024) Commun. Med).

Strengths:

Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.<br /> The resulting annotated transcriptional components have been made available in a searchable online format.

For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers is compelling and supports the need for further mechanistic follow-up.

Further comments:

This revised version includes a suite of comparisons between the c-ICA-derived components and existing published transcriptomic/genomic-based classifications of ovarian cancers. Newly described components will require experimental validation, as acknowledged by the authors.

Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment.<br /> In this revised version, the authors additionally investigate their TC scores in single cells from a published HGSOC single-cell RNAseq dataset, highlighting examples of TC scores within and between cell types.

c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods which explicitly use a prior cell signature matrix.

Reviewer #1 (Public review):

The aim of this study was a better understanding of the reproductive life history of acoels. The acoel Hofstenia miamia, an emerging model organism, is investigated; the authors nevertheless acknowledge and address the high variability in reproductive morphology and strategies within Acoela.

The morphology of male and female reproductive organs in these hermaphroditic worms is characterised through stereo microscopy, immunohistochemistry, histology, and fluorescent in situ hybridization. The findings confirm and better detail historical descriptions. A novelty in the field is the in situ hybridization experiments, which link already published single-cell sequencing data to the worms' morphology. An interesting finding, though not further discussed by the authors, is that the known germline markers cgnl1-2 and Piwi-1 are only localized in the ovaries and not in the testes.

The work also clarifies the timing and order of appearance of reproductive organs during development and regeneration, as well as the changes upon de-growth. It shows an association of reproductive organ growth to whole body size, which will be surely taken into account and further explored in future acoel studies. This is also the first instance of non-anecdotal degrowth upon starvation in H. miamia (and to my knowledge in acoels, except recorded weight upon starvation in Convolutriloba retrogemma [1]).

Egg laying through the mouth is described in H. miamia for the first time as well as the worms' behavior in egg laying, i.e. choosing the tanks' walls rather than its floor, laying eggs in clutches, and delaying egg-laying during food deprivation. Self-fertilization is also reported for the first time.

The main strength of this study is that it expands previous knowledge on the reproductive life history traits in H. miamia and it lays the foundation for future studies on how these traits are affected by various factors, as well as for comparative studies within acoels. As highlighted above, many phenomena are addressed in a rigorous and/or quantitative way for the first time. This can be considered the start of a novel approach to reproductive studies in acoels, as the authors suggest in the conclusion. It can be also interpreted as a testimony of how an established model system can benefit the study of an understudied animal group.

The main weakness of the work is the lack of convincing explanations on the dynamics of self-fertilization, sperm storage, and movement of oocytes from the ovaries to the central cavity and subsequently to the pharynx. These questions are also raised by the authors themselves in the discussion. Another weakness (or rather missing potential strength) is the limited focus on genes. Given the presence of the single-cell sequencing atlas and established methods for in situ hybridization and even transgenesis in H. miamia, this model provides a unique opportunity to investigate germline genes in acoels and their role in development, regeneration, and degrowth. It should also be noted that employing Transmission Electron Microscopy would have enabled a more detailed comparison with other acoels, since ultrastructural studies of reproductive organs have been published for other species (cfr e.g. [2],[3],[4]). This is especially true for a better understanding of the relation between sperm axoneme and flagellum (mentioned in the Results section), as well as of sexual conflict (mentioned in the Discussion).

(1) Shannon, Thomas. 2007. 'Photosmoregulation: Evidence of Host Behavioral Photoregulation of an Algal Endosymbiont by the Acoel Convolutriloba Retrogemma as a Means of Non-Metabolic Osmoregulation'. Athens, Georgia: University of Georgia [Dissertation].<br /> (2) Zabotin, Ya. I., and A. I. Golubev. 2014. 'Ultrastructure of Oocytes and Female Copulatory Organs of Acoela'. Biology Bulletin 41 (9): 722-35.<br /> (3) Achatz, Johannes Georg, Matthew Hooge, Andreas Wallberg, Ulf Jondelius, and Seth Tyler. 2010. 'Systematic Revision of Acoels with 9+0 Sperm Ultrastructure (Convolutida) and the Influence of Sexual Conflict on Morphology'.<br /> (4) Petrov, Anatoly, Matthew Hooge, and Seth Tyler. 2006. 'Comparative Morphology of the Bursal Nozzles in Acoels (Acoela, Acoelomorpha)'. Journal of Morphology 267 (5): 634-48.

Reviewer #2 (Public review):

Summary:

While the phylogenetic position of Acoels (and Xenacoelomorpha) remains still debated, investigations of various representative species are critical to understanding their overall biology.

Hofstenia is an Acoels species that can be maintained in laboratory conditions and for which several critical techniques are available. The current manuscript provides a comprehensive and widely descriptive investigation of the productive system of Hofstenia miamia.

Strengths:

(1) Xenacoelomorpha is a wide group of animals comprising three major clades and several hundred species, yet they are widely understudied. A comprehensive state-of-the-art analysis on the reprodutive system of Hofstenia as representative is thus highly relevant.

(2) The investigations are overall very thorough, well documented, and nicely visualised in an array of figures. In some way, I particularly enjoyed seeing data displayed in a visually appealing quantitative or semi-quantitative fashion.

(3) The data provided is diverse and rich. For instance, the behavioral investigations open up new avenues for further in-depth projects.

Weaknesses:

While the analyses are extensive, they appear in some way a little uni-dimensional. For instance the two markers used were characterized in a recent scRNAseq data-set of the Srivastava lab. One might have expected slightly deeper molecular analyses. Along the same line, particularly the modes of spermatogenesis or oogenesis have not been further analysed, nor the proposed mode of sperm-storage.

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, 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) Adequate number of replicates were used for this study.<br /> (2) Both in vitro and in vivo validation was done.<br /> (3) 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.<br /> (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 neighborhood? If they do how does this correlation affect the conclusion of the paper?<br /> (3) Volcano plot does not indicate q value of the shown genes. It is advisable to calculate q value for each of the genes which represents the FDR probability of the identified genes.<br /> (4) GO enrichment was done against Global gene set or local geneset? Authors should provide more detailed information about the analysis.<br /> (5) If the analysis was performed against global gene set. How does that connect with this specific atherosclerotic microenvironment?<br /> (6) what was the basal expression of genes and how does the DGE (differential gene expression) values differ?<br /> (7) How did IRF5 picked from GO analysis? was it within 20 most significant genes?<br /> (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 like AFF3 in fig 1.

Comments on Revision:

The authors have adequately addressed my concerns.

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 understand of atherosclerosis development.

Strengths:

The in vivo data is convincing 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:

The effect on atherosclerosis is clear and there is sufficient evidence to conclude that this is the result of reduced endothelial cell activation. However, other cell types such as smooth muscle cells or macrophages could be contributing to the effects observed. The mouse model is a global knockout and the shRNA knockdowns (Fig. 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.

The in vitro experiments are solidly executed, but most experiments are performed in mouse embryonic fibroblasts (MEFs) and results extrapolated to endothelial cell responses. However, several key experiments are repeated in HUVEC, thereby making a solid case that Aff3ir-ORF2 can regulate IRF5 in both MEFs and HUVEC. It is important to note that the sequence of AFF3ir-ORF2 is not conserved in humans and lacks an initiation codon, hence the regulatory pathway is not conserved. However, the overexpression studies in HUVEC suggest that mouse AFF3ir-ORF2 can also regulate human IRF5 and hence the mechanism retains relevance for possible human health interventions.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis. The study shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but makes a solid case that this mechanism is relevant for atherosclerosis development via endothelial cell activation.

Reviewer #1 (Public review):

Summary:

Shihabeddin et al. used bioinformatic and molecular biology tools to study the unique regeneration of rod photoreceptors in a zebrafish model. The authors identified a few transcription factors that seem to play an important role in this process.

Strengths:

This manuscript is well prepared. The topic of this study is an interesting and important one. Bioinformatics clues are interesting.

Weaknesses:

Considering the importance of the mechanism, the knockdown experiments require further validation. The authors over-emphasized this study's relevance to RP disease (i.e. patients and mammals are not capable of regeneration like zebrafish). They under-explained this regeneration's relevance or difference to normal developmental process, which is pretty much conserved in evolution.

Reviewer #2 (Public review):

This is an interesting and important work from Shihabeddin et al, to identify master regulators for rod photoreceptor regenerations in a zebrafish model of Retinitis Pigmentosa. Building on their scRNA-seq data, Shihabeddin et al dissected the progenitor cell types and performed trajectory analyses to predict transcription factors that apparently drive the progenitor proliferation and differentiation into rod photoreceptors. Their analyses predicted e2f1, e2f2, and e2f3 as critical drivers of progenitor proliferation, Prdm1a as a driver of rod photoreceptor differentiation, and SP1 as a driver of rod photoreceptor maturation. Genetic experiments provide clear support for the roles of e2fs in progenitor proliferation. It's also apparent from Figure 8 that prdm1 knockdown appears to cause a decrease in rhodopsin expression. By colocalizing BrdU and Retp1, the authors inferred that the apparent "new rods" (which exhibit mixed BrdU and Retp1 signal) are decreased with prdm1, providing further support. Overall I found the work to be interesting, rigorous, and informative for the community.

I have a few suggestions for the authors to consider:

(1) Perhaps the authors can consider explaining why the Prdm1a knock-down cells would have a higher Retp1 signal per cell in Fig 9B. Is this a representative picture? This appears to contradict Figure 8's conclusion, although I could tell that the number of Retp1+ cells in the ONL appears to be lower.

(2) The authors noted "Surprisingly, the knockdown of prdm1a resulted in a significantly higher number of rhodopsin-positive cells in the INL (p=0.0293)", while it appears in Figure 9B, 9C that the difference is 2 cells vs 0 in a rightly broader field. It seems to be too strong of a statement for this effect.

(3) It appears to this reviewer that the proteomic data didn't reveal much in line with the overall hypothesis or the mechanism, and it's unclear why the authors went for proteomics rather than bulk RNA-seq or ChIP-seq for a transcription factor knock-down experiment. Overall this is a minor point.

Reviewer #3 (Public review):

Summary:

This study uses a combination of single-cell RNA-Seq to globally profile changes in gene expression in adult P23H transgenic zebrafish, which show progressive rod photoreceptor degeneration, along with age-matched controls. As expected, mitotically active retinal progenitors are identified in both conditions, the increased number of both progenitors and immature rods are observed. DrivAER-mediated gene regulatory network analysis in retinal progenitors, photoreceptor precursors, and mature rod photoreceptors respectively identified e2f1-3, prdm1a, and sp1 as top predicted transcriptional regulators of gene expression specific to these cell types. Finally, morpholino-mediated knockdown of these transcription factors led to expected defects in proliferation and rod differentiation.

Strengths:

Overall, this is a rigorous study that is convincingly executed and well-written. The data presented here will be a useful addition to existing single-cell RNA-Seq datasets obtained from regenerating zebrafish retina.

Weaknesses:

Multiple similar studies have been published and it is something of a missed opportunity in terms of identifying novel mechanisms of rod photoreceptor regeneration. Several other recent studies have used both single-cell RNA and ATAC-Seq to analyze gene regulatory networks that regulate neurogenesis in zebrafish retina following acute photoreceptor damage (Hoang, et al. 2020; Celloto, et al. 2023; Lyu, et al. 2023; Veen, et al 2023) or in other genetic models of progressive photoreceptor dystrophy such cep290 mutants (Fogerty, et al. 2022).

The gene regulatory network analysis here would also benefit from the addition of matched scATAC-Seq data, which would allow the use of more powerful tools such as Scenic+ (Bravo and de Winter, et al. 2023). It would also benefit from integration with single-cell multiome data from developing retinas (Lyu, et al. 2023). The genes selected for functional analysis here are all either robustly expressed in retinal progenitor cells (ef1-3 and aurka) or in developing rods (prdm1a), so it is not really surprising that defects are observed. Identification of factors that selectively regulate rod photoreceptor regeneration, rather than those that regulate both development and regeneration, would provide additional novelty. This would also potentially allow the use of animal mutants for candidate genes, rather than exclusively relying on morphant analysis, which may have off-target effects.

The description of the time points analyzed is vague, stating only that "fish from 6 to 12 months of age were analyzed". Since photoreceptor degeneration is progressive, it is unclear how progenitor behavior changes over time, or how the gene expression profile of other cell types such as microglia, cones, or surviving rods is altered by disease progression. Most similar studies address this by analyzing multiple time points from specific ages or times post-injury.

Reviewer #1 (Public review):

Summary:

The objective of this research is to understand how the expression of key selector transcription factors, Tal1, Gata2, Gata3, involved in GABAergic vs glutamatergic neuron fate from a single anterior hindbrain progenitor domain is transcriptionally controlled. With suitable scRNAseq, scATAC-seq, CUT&TAG, and footprinting datasets, the authors use an extensive set of computational approaches to identify putative regulatory elements and upstream transcription factors that may control selector TF expression. This data-rich study will be a valuable resource for future hypothesis testing, through perturbation approaches, of the many putative regulators identified in the study. The data are displayed in some of the main and supplemental figures in a way that makes it difficult to appreciate and understand the authors' presentation and interpretation of the data in the Results narrative. Primary images used for studying the timing and coexpression of putative upstream regulators, Insm1, E2f1, Ebf1, and Tead2 with Tal1 are difficult to interpret and do not convincingly support the authors' conclusions. There appears to be little overlap in the fluorescent labeling, and it is not clear whether the signals are located in the cell soma nucleus.

Strengths:

The main strength is that it is a data-rich compilation of putative upstream regulators of selector TFs that control GABAergic vs glutamatergic neuron fates in the brainstem. This resource now enables future perturbation-based hypothesis testing of the gene regulatory networks that help to build brain circuitry.

Weaknesses:

Some of the findings could be better displayed and discussed.

Reviewer #2 (Public review):

Summary:

In the manuscript, the authors seek to discover putative gene regulatory interactions underlying the lineage bifurcation process of neural progenitor cells in the embryonic mouse anterior brainstem into GABAergic and glutamatergic neuronal subtypes. The authors analyze single-cell RNA-seq and single-cell ATAC-seq datasets derived from the ventral rhombomere 1 of embryonic mouse brainstems to annotate cell types and make predictions or where TFs bind upstream and downstream of the effector TFs using computational methods. They add data on the genomic distributions of some of the key transcription factors and layer these onto the single-cell data to get a sense of the transcriptional dynamics.

Strengths:

The authors use a well-defined fate decision point from brainstem progenitors that can make two very different kinds of neurons. They already know the key TFs for selecting the neuronal type from genetic studies, so they focus their gene regulatory analysis squarely on the mechanisms that are immediately upstream and downstream of these key factors. The authors use a combination of single-cell and bulk sequencing data, prediction and validation, and computation.

Weaknesses:

The study generates a lot of data about transcription factor binding sites, both predicted and validated, but the data are substantially descriptive. It remains challenging to understand how the integration of all these different TFs works together to switch terminal programs on and off.

Reviewer #1 (Public review):

Summary:

The authors demonstrate impairments induced by a high cholesterol diet on GLP-1R dependent glucoregulation in vivo as well as an improvement after reduction in cholesterol synthesis with simvastatin in pancreatic islets. They also map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, and screened for key residues selected from these sites and performed detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviour, clustering, trafficking and signalling in pancreatic beta cells and primary islets, and describe an improved insulin secretion profile for the V229A mutant receptor.

These are extensive and very impressive studies indeed. I am impressed with the tireless effort exerted to understand the details of molecular mechanisms involved in the effects of cholesterol for GLP-1 activation of its receptor. In general, the study is convincing, the manuscript well written and the data well presented. Some of the changes are small and insignificant which makes one wonder how important the observations are. For instance, in Figure 2E (which is difficult to interpret anyway because the data are presented in per cent, conveniently hiding the absolute results) does not show a significant result of the cyclodextrin except for insignificant increases in basal secretion. That is not identical to impairment of GLP-1 receptor signaling!

To me the most important experiment of them all is the simvastatin experiment, but the results rest on very few numbers and there is a large variation. Apparently, in a previous study using more extensive reduction in cholesterol the opposite response was detected casting doubt on the significance of the current observation. I agree with the authors that the use of cyclodextrin may have been associated with other changes in plasma membrane structure than cholesterol depletion at the GLP-1 receptor. The entire discussion regarding the importance of cholesterol would benefit tremendously from studies of GLP-1 induced insulin secretion in people with different cholesterol levels before and after treatment with cholesterol-lowering agents. I suspect that such a study would not reveal major differences.

Comments on revisions: The authors have responded well to my criticism.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors were providing a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, by presumably decreasing the cholesterol available to interact with wt GLP-1R. The effects of this mutation are not due to differences in Ex-4:recepotor affinity. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

Weaknesses:

There are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. Future work by the authors may determine the effects of the GLP-1R V229A mutation on insulin secretion responses under diet-induced metabolic stress conditions. Furthermore, the authors may additionally investigate if V229A would have the same impact in a different cell type, especially in neurons, with implications in the regulation of satiation, gut motility, and especially nausea, which are of high translational interest.

The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is a novel direction.

Reviewer #1 (Public review):

Summary:

The authors examine CD8 T cell selective pressure in early HCV infection using. They propose that after initial CD8-T mediated loss of virus fitness, in some participants around 3 months after infection, HCV acquires compensatory mutations and improved fitness leading to virus progression.

Strengths:

Throughout the paper, the authors apply well-established approaches in studies of acute to chronic HIV infection for studies of HCV infection. This lends rigor the to the authors' work.

Reviewer #2 (Public review):

Summary:

In this work, Walker and collaborators study the evolution of hepatitis C virus (HCV) in a cohort of 14 subjects with recent HCV infections. They focus in particular on the interplay between HCV and the immune system, including the accumulation of mutations in CD8+ T cell epitopes to evade immunity. Using a computational method to estimate the fitness effects of HCV mutations, they find that intrinsic viral fitness declines as the virus mutates to escape T cell responses. In long-term infections, they found that viral fitness can rebound later in infection as HCV accumulates additional mutations.

Strengths:

This work is especially interesting for several reasons. Individuals who developed chronic infections were followed over fairly long times and, in most cases, samples of the viral population were obtained frequently. At the same time, the authors also measured CD8+ T cell and antibody responses to infection. The analysis of HCV evolution focused not only on variation within particular CD8+ T cell epitopes, but also the surrounding proteins. Overall, this work is notable for integrating information about HCV sequence evolution, host immune responses, and computational metrics of fitness and sequence variation. The evidence presented by the authors supports the main conclusions of the paper described above.

Weaknesses:

After revision, this paper has no outstanding weaknesses. Points where further investigation is needed have been clearly identified.

Reviewer #2 (Public review):

The manuscript "HNF4α-1 TET2-FBP1 axis contributes to gluconeogenesis and type 2 diabetes" from Zhang et al. presents significant and convincing findings that enhance our understanding of TET2's role in liver glucose metabolism. It highlights the epigenetic regulation of FBP1, a gluconeogenic gene, by TET2, linking this pathway to HNF4alpha which recruits TET2. The in vitro and in vivo experiments are now well-described and provide convincing evidence of TET2's impact on gluconeogenesis, particularly in fasting and HFD mice.

Comments on revisions:

The authors have thoroughly addressed all the concerns raised, and their responses adequately clarify the issues previously identified.

Minor changes:

(1) Could the authors provide some comments on why glucagon was not able to stimulate PEPCK and G6Pase mRNA levels in HepG2 cells (Fig. 3D)? Although it is not the focus of the research, it is well known that glucagon has this effect and could serve as a positive control for the quality of the preparation.

(2) Please include the sequences of the qPCR primers used for PEPCK and G6Pase in the Methods section (page 17).

Reviewer #1 (Public review):

Summary:

The crystal structure of the Sld3CBD-Cdc45 complex presented by Li et al. is a significant contribution that enhances our understanding of CMG formation during the rate-limiting step of DNA replication initiation. This structure provides crucial insights into the intermediate steps of CMG formation, and the particle analysis and model predictions compellingly describe the mechanism of Cdc45 loading. Building upon previously known Sld3 and Cdc45 structures, this study offers new perspectives on how Cdc45 is recruited to MCM DH through the Sld3-Sld7 complex. The most notable finding is the structural rearrangement of Sld3CBD upon Cdc45 binding, particularly the α8-helix conformation, which is essential for Cdc45 interaction and may also be relevant to its metazoan counterpart, Treslin. Additionally, the conformational shift in the DHHA1 domain of Cdc45 suggests a potential mechanism for its binding to Mcm2NTD. Furthermore, Sld3's ssDNA-binding experiments provide evidence of its novel functions in the DNA replication process in yeast, expanding our understanding of its role beyond Cdc45 recruitment.

Strengths:

The manuscript is generally well-written, with a precise structural analysis and a solid methodological section that will significantly advance future studies in the field. The predictions based on structural alignments are intriguing and provide a new direction for exploring CMG formation, potentially shaping the future of DNA replication research. This research also opens up several new opportunities to utilize structural biology to unravel the molecular details of the model presented in the paper.

Weaknesses:

The main weakness of the manuscript lies in the lack of detailed structural validation for the proposed Sld3-Sld7-Cdc45 model, and its CMG bound models, which could be done in the future using advanced structural biology techniques such as single particle cryo-electron microscopy. It would also be interesting to explore how Sld7 interacts with the MCM helicase, and this would help to build a detailed long-flexible model of Sld3-Sld7-Cdc45 binding to MCM DH and to show where Sld7 will lie on the structure. This will help us to understand how Sld7 functions in the complex. Also, future experiments would be needed to understand the molecular details of how Sld3 and Sld7 release from CMG is associated with ssARS1 binding.

Reviewer #2 (Public review):

Summary

The manuscript presents valuable findings, particularly in the crystal structure of the Sld3CBD-Cdc45 interaction and the identification of additional sequences involved in their binding. The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is novel, and the results provide insights into potential conformational changes that occur upon interaction. Although the single-stranded DNA binding data from Sld3 of different species is a minor weakness, the experiments support a model in which the release of Sld3 from the complex may be promoted by its binding to origin single-stranded DNA exposed by the helicase.

Strengths

• The Sld3CBD-Cdc45 structure is a novel contribution, revealing critical residues involved in the interaction.<br /> • The model structures generated from the crystal data are well presented and provide valuable insights into the interaction sequences between Sld3 and Cdc45.<br /> • The experiments testing the requirements for interaction sequences are thorough and conducted well, with clear figures supporting the conclusions.<br /> • The conformational changes observed in Sld3 and Cdc45 upon binding are interesting and enhance our understanding of the interaction.<br /> • The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is a new and valuable addition to the field.<br /> • The proposed model of Sld3 release from the complex through binding to single stranded DNA at the origin is intriguing.

Weaknesses

• The section on the binding of Sld3 complexes to origin single-stranded DNA is somewhat weakened by the use of Sld3 proteins from different species. The comparisons between Sld3-CBD, Sld3CBD-Cdc45, and Sld7-Sld3CBD-Cdc45 involve complexes from different species, limiting the comparisons' value.<br /> • Although the study reveals that Sld3 binds to different residues of Cdc45 than those previously shown to bind Mcm or GINS, the data in the paper do not shed any additional light on how GINS and Sld3 binding to Cdc45 or Mcms. would affect each other. Other previous research has suggested that the binding of GINS and Sld3 to Mcm or Cdc45 may be mutually exclusive. The authors acknowledge that a structural investigation of Sld3, Sld7, Cdc45, and MCM during the stage of GINS recruitment will be a significant goal for future research.

Reviewer #3 (Public review):

Summary:

The paper by Li et al. describes the crystal structure of a complex of Sld3-Cdc45-binding domain (CBD) with Cdc45 and a model of the dimer of an Sld3-binding protein, Sld7, with two Sld3-CBD-Cdc45 for the tethering. In addition, the authors showed the genetic analysis of the amino acid substitution of residues of Sld3 in the interface with Cdc45 and biochemical analysis of the protein interaction between Sld3 and Cdc45 as well as DNA binding activity of Sld3 to the single-strand DNAs of the ARS sequence.

Strengths:

The authors provided a nice model of an intermediate step in the assembly of an active Cdc45-MCM-GINS (CMG) double hexamers at the replication origin, which is mediated by the Sld3-Sld7 complex. The dimer of the Sld3-Sld7 complexes tethers two MCM hexamers together for the recruitment of GINS-Pol epsilon on the replication origin.

Weaknesses:

The biochemical analysis should be carefully evaluated with more quantitative ways to strengthen the authors' conclusion even in the revised version.

Reviewer #1 (Public review):

Summary:

Sandkuhler et al. re-evaluated the biological functions of TANGO2 homologs in C. elegans, yeast, and zebrafish. Compared to the previously reported role of TANGO2 homologs in transporting heme, Sandkuhler et al. expressed a different opinion on the biological functions of TANGO2 homologs. With the support of some results from their tests, they conclude that 'there is insufficient evidence to support heme transport as the primary function of TANGO2', in addition to their claims on the role of TANGO2 in modulating metabolism. While the differences are reported in this study, more work is needed to elucidate the biological function of TANGO2.

Strengths:

(1) This work revisited a set of key experiments, including the toxic heme analog GaPP survival assay, the fluorescent ZnMP accumulation assay, and the multi-organismal investigations documented by Sun et al. in Nature 2022, which is critical for comparing the two works.

(2) This work reported additional phenotypes for the C. elegans mutant of the TANGO2 homologs, including lawn avoidance, reduced pharyngeal pumping, smaller brood size, faster exhaustion under swimming test, and a shorter lifespan. These phenotypes are important for understanding the biological function of TANGO2 homologs, while they were missing from the report by Sun et al.

(3) Investigating the 'reduced GaPP consumption' as a cause of increased resistance against the toxic GaPP for the TANGO2 homologs, hrg-9 hrg-10 double null mutant provides a valuable perspective for studying the biological function of TANGO2 homologs.

(4) This work thoroughly evaluated the role of TANGO2 homologs in supporting yeast growth using multiple yeast strains and also pointed out the mitochondrial genome instability feature of the yeast strain used by Sun et al.

Weaknesses:

(1) A detailed comparison between this work and the work of Sun et al. on experimental protocols and reagents in the main text will be beneficial for readers to assess critically.

(2) The GaPP used by Sun et al. (purchased from Frontier Scientific) is more effective in killing the worm than the one used in this study (purchased from Santa Cruz). Is the different outcome due to the differences in reagents? Moreover, Sun et al. examined the lethality after 3-4 days, while this work examined the lethality after 72 hours. Would the extra 24 hours make any difference in the result?

(3) This work reported the opposite result of Sun et al. for the fluorescent ZnMP accumulation assay. However, the experimental protocols used by the two studies are massively different. Sun et al. did the ZnMP staining by incubating the L4-stage worms in an axenic mCeHR2 medium containing 40 μM ZnMP (purchased from Frontier Scientific) and 4 μM heme at 20 ℃ for 16 h, while this work placed the L4-stage worms on the OP50 E. coli seeded NGM plates treated with 40 μM ZnMP (purchased from Santa Cruz) for 16 h. The liquid axenic mCeHR2 medium is bacteria-free, heme-free, and consistent for ZnMP uptake by worms. This work has mentioned that the hrg-9 hrg-10 double null mutant has bacterial lawn avoidance and reduced pharyngeal pumping phenotypes. Therefore, the ZnMP staining protocol used in this work faces challenges in the environmental control for the wild type vs. the mutant. The authors should adopt the ZnMP staining protocol used by Sun et al. for a proper evaluation of fluorescent ZnMP accumulation.

(4) A striking difference between the two studies is that Sun et al. emphasize the biochemical function of TANGO2 homologs in heme transporting with evidence from some biochemical tests. In contrast, this work emphasizes the physiological function of TANGO2 homologs with evidence from multiple phenotypical observations. In the discussion part, the authors should address whether these observed phenotypes in this study can be due to the loss of heme transporting activities upon eliminating TANGO2 homologs. This action can improve the merit of academic debate and collaboration.

Reviewer #2 (Public review):

Summary:

This work investigates the roles of TANGO2 orthologs in different model systems and suggests bioenergetic dysfunction and oxidative stress (and not heme metabolism) as crucial pathways in TANGO2 deficiency disorders (TDD). Specifically, studies in C. elegans showed that the lack of TANGO2 ortholog activity (i) does not provide a survival benefit upon toxic heme exposure; (ii) results in a series of defects related to energy levels (reduced pharyngeal pumping, lawn avoidance, poor motility, and low brood size); (iii) reduces the fluorescence of the heme analog ZnMP in the intestine. Furthermore, upon oxidative stress, one TANGO2 ortholog, hrg-9, is upregulated compared to control conditions. Additional studies on yeast and zebrafish models failed to replicate prior findings on heme distribution and muscle integrity.

These findings have a clear therapeutic impact, as TDD currently has no cure but only symptom-managing treatments. Identifying the correct pathway to correct the disease is pivotal to finding a cure.

Although compelling, the authors' primary claim is based on indirect evidence that only hints toward it. Unfortunately, I do not see any direct and convincing evidence linking TANGO2 orthologs to bioenergetic and oxidative stress pathways.

Strengths:

(1) The study refutes and extends previous findings, highlighting new aspects of TANGO2's roles in cell physiology.

(2) The use of different model systems to address the main research questions is useful.

(3) The results suggest a broader impact than previously described, somewhat supporting the novelty of the study.

Weaknesses:

(1) The manuscript is written mainly as a criticism of a previously published paper. Although reproducibility in science is an issue that needs to be acknowledged, a manuscript should focus on the new data and the experiments that can better prove and strengthen the new claims.

(2) The current presentation of the logic of the study and its results does not help the authors deliver their message, although they possess great potential.

(3) The study is missing experiments to link hrg-9 and hrg-10 more directly to bioenergetic and oxidative stress pathways.

Reviewer #3 (Public review):

In this paper, Sandkuhler et al. reassessed the role of TANGO2 as a heme chaperone proposed by Sun et al in a recently published paper (https://doi.org/10.1038/s41586-022-05347-z) by partially repeating and failing to replicate experiments therein. Overall, Sandkuhler et al. conclude that the heme-related roles of TANGO2 had been overemphasized by Sun et al. especially because the hrg9 gene does not exclusively respond to different regimens of heme synthesis/uptake but is susceptible to a greater extent to, for example, oxidative stress.

In recent years, the discussion around the heme-related roles of TANGO2 has been tantalizing but is still far from a definitive consensus. Discrepancies between results and their interpretation are a testament to how challenging and ambitious the understanding of TANGO2 and the phenotypes associated with TANGO2 defects are. Overall, the work presented by Sandkuhler et al. in this manuscript challenges the recent developments in the field and promotes the continuous characterisation of TANGO2 in relation to heme homeostasis.

A few comments and questions:

(1) The authors stress - with evidence provided in this paper or indicated in the literature - that the primary role of TANGO2 and its homologues is unlikely to be related to heme trafficking, arguing that observed effects on heme transport are instead downstream consequences of aberrant cellular metabolism. But in light of a mounting body of evidence (referenced by the authors) connecting more or less directly TANGO2 to heme trafficking and mobilization, it is recommended that the authors comment on how they think TANGO2 could relate to and be essential for heme trafficking, albeit in a secondary, moonlighting capacity. This would highlight a seemingly common theme in emerging key players in intracellular heme trafficking, as it appears to be the case for GAPDH - with accumulating evidence of this glycolytic enzyme being critical for heme delivery to several downstream proteins.

(2) The observation - using eat-2 mutants and lawn avoidance behaviour - that survival patterns can be partially explained by reduced consumption, is fascinating. It would be interesting to quantify the two relative contributions.

(3) In the legend to Figure 1A it's a bit unclear what the differently coloured dots represent for each condition. Repeated measurements, worms, independent experiments? The authors should clarify this.

(4) It would help if the entire fluorescence images (raw and processed) for the ZnMP treatments were provided. Fluorescence images would also benefit Figure 1B.

(5) Increasingly, the understanding of heme-dependent roles relies on transient or indirect binding to unsuspected partners, not necessarily relying on a tight affinity and outdating the notion of heme as a static cofactor. Despite impressive recent advancements in the detection of these interactions (for example https://doi.org/10.1021/jacs.2c06104; cited by the authors), a full characterisation of the hemome is still elusive. Sandkuhler et al. deemed it possible but seem to question that heme binding to TANGO2 occurs. However, Sun et al. convincingly showed and characterised TANGO2 binding to heme. It is recommended that the authors comment on this.

Reviewer #1 (Public review):

Summary:

The authors aim to formalize the mathematical underpinnings of a proposed general model and discuss the relationship of this model to the ABC Score, a widely adopted heuristic for enhancer-gene predictions. While the ABC model serves as a useful binary classifier, it struggles to predict quantitative enhancer effects on gene expression. Using a graph-theoretic linear framework, the authors derive a mathematical model (the "default model") that explains how the algebraic form of the ABC Score arises under specific assumptions. They further demonstrate that the default model's predictions of enhancer additivity are inconsistent with observed non-additive enhancer effects and propose alternative assumptions to account for these discrepancies.

Strengths:

The graph-theoretic approach enables systematic exploration of enhancer interactions beyond simple additivity and enables hypothesis generation when such expectations fail. This work makes clear where assumptions are made and the consequences of those assumptions.

Weaknesses:

While the theoretical framework is elegant, I think there is always more space to demonstrate the practicality of this approach. Further guidance for how to experimentally connect this framework with typical measurements could help bolster the immediate benefits. To be clear, I do not think this is something the authors "must" do, but rather something that might help drive home the usefulness in a more accessible way.

Reviewer #2 (Public review):

Summary:

The Activity-by-Contact (ABC) model is a relatively widespread model of enhancer-gene regulation. This model leverages CRISPRi data to predict whether a gene is regulated by a given enhancer. To make this possible, this model accounts for the activity of an enhancer and its contact frequency with a target promoter in order to produce an "ABC score". However, while quantitative in its ability to predict enhancer-promoter regulation, this model is mostly phenomenological and does not commit to specific molecular mechanisms.

In this manuscript, the authors formalize the molecular and mathematical assumptions made by the ABC model. Specifically, they demonstrate a basic set of assumptions that can be made to arrive at the ABC model's mathematical structure. The resulting default model (basically, a null model) places particular emphasis on the requirement that gene activation and enhancer-gene communication must be independent and at a steady state. The authors leverage and extend a graph-based formalism they have previously spearheaded to show the generality of their conclusions with respect to different molecular realizations of the process by which enhancers interact with their promoters.

Previously published works have found that specific models of how multiple enhancers communicate with the same gene can result in additive mRNA production rates. Here, the authors demonstrate that steady-state mRNA levels are additive regardless of the specific Markovian model for how any individual enhancer communicates with the gene, as long as the model follows the basic assumptions of their default model.

By coarse-graining, both gene activation and enhancer-gene communication to simple two-state models, the authors then clearly demonstrate that the mathematical structure of the ABC model emerges. This mathematical structure implies that the ABC score summed over all the enhancers regulating a given gene must equal 1. However, experimental measurements show values ranging from 0 to 3. The authors show that, in order to explain these experimental deviations with respect to the theory, at least one of the assumptions of the default model must be broken. They demonstrate that either invoking enhancer cooperativity in mRNA production rates or breaking the assumption that individual enhancers communicate with the gene independently can explain existing experimental data.

Strengths:

By demonstrating that the mathematical structure of the ABC model emerges from a set of basic assumptions including the independence of gene activation and enhancer-gene communication, the authors succeeded in their aim to put the ABC model on a formal and molecular footing. Since some experimental results do not agree with the ABC model, the authors importantly demonstrated which assumptions of the model can be broken to explain such data. The theoretical work in this manuscript is written in a reasonably accessible manner that features how a graph theory-based approach to modeling biochemical networks can result in general statements about biological phenomena.

Weaknesses:

While the authors discuss a number of experimental techniques that can be used to test the validity of their model, a more specific discussion of proposed experiments could have strengthened the impact of the paper by providing explicit opportunities for dialogue with experimentalists.

Reviewer #1 (Public review):

This study investigates the role of site-specific DNA methylation changes during spermatogenesis and their contribution to paternal epigenetic inheritance. Using MethylCap-seq, the authors identify a transient, site-specific loss of DNA methylation at transcription start sites (TSSs) of late spermatogenesis genes during the transition from differentiating spermatogonia (KIT+) to pachytene spermatocytes (PS). This demethylation event correlates with the gain of H3K4me3, which presets nucleosome retention sites in mouse sperm. The study proposes that selective loss of DNA methylation at a subset of promoters is required for nucleosome retention and the establishment of epigenetic states that may influence embryonic gene regulation. These findings provide complementary insights to earlier work by the Peters lab, "DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos."

Overall, the study presents a valuable dataset; however, additional analyses could strengthen the conclusions and provide further mechanistic insights.

Major Comments:

(1) Prior work should be acknowledged and used for comparative analysis. A key proposal in this study is that regions undergoing DNA methylation loss retain histones, influencing the zygote's epigenetic landscape. However, previous studies (e.g., Peters et al.) have shown that regions losing methylation in DNMT3a/b knockout (KO) mice do not necessarily retain histones, suggesting additional factors are involved. Moreover, Peters et al. demonstrated that regions of low DNA methylation in sperm render paternal alleles permissive for H3K4me3 establishment in early embryos, independent of the paternal inheritance of sperm-borne H3K4me3. Comparing these findings would refine the model presented in this study.

(2) Figure 2A: The data suggest an increase in methylation peaks in PS cells. How does this align with the hypomethylation observed in Figure 1D? Reconciling these observations would improve clarity.

(3) Figure 4A: The effect size of demethylation on nucleosome retention is unclear - do all demethylated promoters retain histones or only a subset? Quantifying this would clarify whether DNA methylation loss consistently predicts nucleosome retention.

(4) Prior studies have generated bisulfite sequencing data from Tet KO sperm. Do the regions that undergo demethylation during the KIT+ to PS transition overlap with those misregulated in TET KO sperm? Integrating this comparison could provide further insight into the regulation of site-specific demethylation.

(5) The role of SCML2 enrichment in germline stem cells and its connection to H3K27me3 deposition in later germ cells is unclear. Earlier figures show that regions undergoing DNA demethylation from KIT+ to PS include genes expressed in later-stage germ cells.

Why is SCML2 enrichment occurring in germline stem cells (GSCs)? Why is H3K27me3 only acquired at later stages if SCML2 is already present? Is SCML2 preventing premature expression independent of K27ME?

Showing the dynamics of H3K27me3 and SCML2 across these stages would clarify the proposed conclusions.

Reviewer #2 (Public review):

Summary:

This study profiles the genome-wide distribution of DNA methylation using methylation capture sequencing in four stages of male germ cells: Thy1+ (undifferentiated spermatogonia), Kit+ (differentiated spermatogonia), pachytene spermatocytes, and round spermatids. These analyses revealed site-specific loss of DNA methylation in pachytene cells compared with differentiating spermatogonia. Integrated analysis using published datasets indicates that hypomethylated sites correlate with nucleosome retention sites and bivalent histone methylation sites in sperm.

Strengths:

The methyl-seq approach provides a comprehensive profile of DNA methylation in male germ cells. The concept that DNA hypomethylation in meiotic cells precedes histone modification and histone retention in sperm is interesting.

Weaknesses:

(1) In the title, the word "presets" should be changed to "precedes" or "correlates with". Preset means a causal relationship, which is not the case. This needs to be changed throughout the manuscript. For example, in the abstract, "predetermine" needs to be changed to "precede".

(2) The statement that "Based on these results, we propose that meiosis is a process of epigenetic reprogramming that sets up embryonic gene regulation" (lines 94-95) is a speculation that in the opinion of this reviewer should be removed from the text. It is too broad and not supported by the data presented.

(3) Figure 1B: details are missing. How many cells were analyzed/used? How many times was this experiment done [(The number of experiments (n)]? Were the changes statistically significant (Lines 109-111)?

(4) Figure 1A and Figure 1D: These seem to be contradictory. According to Figure 1D, leptotene/zygotene spermatocytes show bright 5mC staining. However, the diagram in 1A shows delayed recovery of DNA methylation. The authors should clarify this. It appears that 5mC was high in Kit+ spermatogonia and leptotene/zygotene spermatocytes, and then decreased in pachytene spermatocytes.

(5) L121-122: Statement: These results suggest that 5mC levels change dynamically during spermatogenesis before and after the transient reduction of DNA methylation in the premeiotic S phase. In order to make this claim about the premeiotic S phase, I suggest performing 5mC staining in premeiotic S phase cells, which can be pulse-labelled with BrdU or cite a reference if available.

Reviewer #1 (Public review):

Turner et al. present an original approach to investigate the role of Type-1 nNOS interneurons in driving neuronal network activity and in controlling vascular network dynamics in awake head-fixed mice. Selective activation or suppression of Type-1 nNOS interneurons has previously been achieved using either chemogenetic, optogenetic, or local pharmacology. Here, the authors took advantage of the fact that Type-1 nNOS interneurons are the only cortical cells that express the tachykinin receptor 1 to ablate them with a local injection of saporin conjugated to substance P (SP-SAP). SP-SAP causes cell death in 90 % of type1 nNOS interneurons without affecting microglia, astrocytes, and neurons. The authors report that the ablation has no major effects on sleep or behavior. Refining the analysis by scoring neural and hemodynamic signals with electrode recordings, calcium signal imaging, and wide-field optical imaging, the authors observe that Type-1 nNOS interneuron ablation does not change the various phases of the sleep/wake cycle. However, it does reduce low-frequency neural activity, irrespective of the classification of arousal state. Analyzing neurovascular coupling using multiple approaches, they report small changes in resting-state neural-hemodynamic correlations across arousal states, primarily mediated by changes in neural activity. Finally, they show that nNOS type 1 interneurons play a role in controlling interhemispheric coherence and vasomotion.

In conclusion, these results are interesting, use state-of-the-art methods, and are well supported by the data and their analysis. I have only a few comments on the stimulus-evoked haemodynamic responses, and these can be easily addressed.

Reviewer #2 (Public review):

Summary:

This important study by Turner et al. examines the functional role of a sparse but unique population of neurons in the cortex that express Nitric oxide synthase (Nos1). To do this, they pharmacologically ablate these neurons in the focal region of whisker-related primary somatosensory (S1) cortex using a saponin-substance P conjugate. Using widefield and 2-photon microscopy, as well as field recordings, they examine the impact of this cell-specific lesion on blood flow dynamics and neuronal population activity. Locally within the S1 cortex, they find changes in neural activity patterns, decreased delta band power, and reduced sensory-evoked changes in blood flow (specifically eliminating the sustained blood flow change after stimulation). Surprisingly, given the tiny fraction of cortical neurons removed by the lesion, they also find far-reaching effects on neural activity patterns and blood volume oscillations between the cerebral hemispheres.

Strengths:

This was a technically challenging study and the experiments were executed in an expert manner. The manuscript was well written and I appreciated the cartoon summary diagrams included in each figure. The analysis was rigorous and appropriate. Their discovery that Nos1 neurons can have far-reaching effects on blood flow dynamics and neural activity is quite novel and surprising (to me at least) and should seed many follow-up, mechanistic experiments to explain this phenomenon. The conclusions were justified by the convincing data presented.

Weaknesses:

I did not find any major flaws in the study. I have noted some potential issues with the authors' characterization of the lesion and its extent. The authors may want to re-analyse some of their data to further strengthen their conclusions. Lastly, some methodological information was missing, which should be addressed.

Reviewer #3 (Public review):

The role of type-I nNOS neurons is not fully understood. The data presented in this paper addresses this gap through optical and electrophysiological recordings in adult mice (awake and asleep).

This manuscript reports on a study on type-I nNOS neurons in the somatosensory cortex of adult mice, from 3 to 9 months of age. Most data were acquired using a combination of IOS and electrophysiological recordings in awake and asleep mice. Pharmacological ablation of the type-I nNOS populations of cells led to decreased coherence in gamma band coupling between left and right hemispheres; decreased ultra-low frequency coupling between blood volume in each hemisphere; decreased (superficial) vascular responses to sustained sensory stimulus and abolishment of the post-stimulus CBV undershoot. While the findings shed new light on the role of type-I nNOS neurons, the etiology of the discrepancies between current observations and literature observations is not clear and many potential explanations are put forth in the discussion.

Reviewer #1 (Public review):

Summary:

The present study aims to associate reproduction with age-related disease as support of the antagonistic pleiotropy hypothesis of ageing predominantly using Mendelian Randomization. The authors found evidence that early-life reproductive succes is associated with advanced ageing.

Strengths:

Large sample size. Many analyses

Weaknesses:

Still a number of doubts with regard to some of the results and their interpretation.

Reviewer #2 (Public review):

Summary:

The authors present an interesting paper where they test the antagonistic pleiotropy theory. Based on this theory they hypothesize that genetic variants associated with later onset of age at menarche and age at first birth may have a positive effect on a multitude of health outcomes later in life, such as epigenetic aging and prevalence of chronic diseases. Using a mendelian randomization and colocalization approach, the authors show that SNPs associated with later age at menarche are associated with delayed aging measurements, such as slower epigenetic aging and reduced facial aging and a lower risk of chronic diseases, such as type 2 diabetes and hypertension. Moreover, they identify 128 fertility-related SNPs that associate with age-related outcomes and they identified BMI as a mediating factor for disease risk, discussing this finding in the context of evolutionary theory.

Strengths:

The major strength of this manuscript is that it addresses the antagonistic pleiotropy theory in aging. Aging theories are not frequently empirically tested although this is highly necessary. The work is therefore relevant for the aging field as well as beyond this field, as the antagonistic pleiotropy theory addresses the link between fitness (early life health and reproduction) and aging.

The authors addressed the remarks on the previous version very well. Addressing the two points below would further increase the quality of the manuscript.

(1) In the previous version the authors mentioned that their results are also consistent with the disposable soma theory: "These results are also consistent with the disposable soma theory that suggests aging as an outcome tradeoff between an organism's investment in reproduction and somatic maintenance and repair."

Although the antagonistic pleiotropy and disposable soma theories describe different mechanisms, both provide frameworks for understanding how genes linked to fertility influence health. The antagonistic pleiotropy theory posits that genes enhancing fertility early in life may have detrimental effects later. In contrast, the disposable soma theory suggests that energy allocation involves a trade-off, where investment in fertility comes at the expense of somatic maintenance, potentially leading to poorer health in later life.

To strengthen the manuscript, a discussion section should be added to clarify the overlap and distinctions between these two evolutionary theories and suggest directions for future research in disentangling their specific mechanisms.

(2) In response to the question why the authors did not include age at menopause in addition to the already included age at first child and age at menarche the following explanation was provided: "Our manuscript focuses on the antagonistic pleiotropy theory, which posits that inherent trade-off in natural selection, where genes beneficial for early survival and reproduction (like menarche and childbirth) may have costly consequences later. So, we only included age at menarche and age at first childbirth as exposures in our research."

It remains, however, unclear why genes beneficial for early survival and reproduction would be reflected only in age at menarche and age at first childbirth, but not in age at menopause. While age at menarche marks the onset of fertility, age at menopause signifies its end. Since evolutionary selection acts directly until reproduction is no longer possible (though indirect evolutionary pressures persist beyond this point), the inclusion of additional fertility-related measures could have strengthened the analysis. A more detailed justification for focusing exclusively on age at menarche and first childbirth would enhance the clarity and rigor of the manuscript.

Reviewer #1 (Public review):

Summary:

Maladaptive decision-making is a trait commonly seen in gambling disorders. Salient cues can impact decision-making and drive gambling, though how cues affect decision-making isn't well understood. This manuscript describes the impact of cueing distinct outcomes of a validated rodent cost/benefit-making task based on the human Iowa Gambling Task. Comparing six task variants, the authors describe the effect of adding salient cues to wins (that scale with the size of win or the inverse), to every outcome regardless of loss or win, randomly to losses or wins, or to losses. Behavioral results reveal that cueing wins increased risky choices. By contrast, presenting the cues randomly or cueing the losses reduced risky choices. Risk-preferring animals of the uncued, randomly cued, and loss-cued tasks showed sensitivity to devaluation, whereas win-paired cued rats did not, suggesting cues blunt behavioral updating. Behavioral analyses were paired with computational modeling of initial acquisition which revealed that risky decision-making was related to reduced punishment learning. These data provide unique insight into how cues may bias behavior and drive gambling-related phenotypes.

Strengths:

The detailed analyses provide interesting insight into how cues impact complex decision-making. While there has been a great deal of work into the impact of cues on choice, few studies integrate multiple probabilistic outcomes. Complementing these data with computational parameters helps the reader to understand what may be driving these differences in behavior. The manuscript is well-written, clearly explaining the relevance of the results and potential future directions.

Weaknesses:

Two main questions arise from these results. The first - when do behavioral differences emerge between the task variants? Based on the results and discussion, the cues increase the salience of either the wins or the losses, biasing behavior in favor of either risky or optimal choice. If this is the case, one might expect the cues to expedite learning, particularly in the standard and loss condition. Providing an analysis of the acquisition of the tasks may provide insight into how the cues are "teaching" decision-making and might explain how biases are formed and cemented.

The second question is - does the learning period used for the modeling impact the interpretation of the behavioral results? The authors indicate that computational modeling was done on the first five sessions and used these data to predict preferences at baseline. Based on these results, punishment learning predicts choice preference. However, these animals are not naïve to the contingencies because of the forced choice training prior to the task, which may impact behavior in these early sessions. Though punishment learning may initially predict risk preference, other parameters later in training may also predict behavior at baseline. The authors also present simulated data from the models for sessions 18-20, but according to the statistical analysis section, sessions 35-40 were used for analysis (and presumably presented in Figure 1). If the simulation is carried out in sessions 35-40, do the models fit the data? Finally, though the n's are small, it would be interesting to see how the devaluation impacts computational metrics. These additional analyses may help to explain the nuanced effects of the cues in the task variants.

Reviewer #2 (Public review):

Summary:

The manuscript by Hathaway et al. describes a set of elegant behavioral experiments designed to understand which aspects of cue-reward contingencies drive risky choice behavior. The authors developed several clever variants of the well-established rodent gambling task (also developed by this group) to understand how audiovisual cues alter learning, choice behavior, and risk. Computational and sophisticated statistical approaches were used to provide evidence that: (1) audiovisual cues drive risky choice if they are paired with rewards and decrease risk if only paired with loss, (2) pairing cues with rewards reduces learning from punishment, and (3) differences in risk-taking seem to be present early on in training.

Strengths:

The paper is well-written, the experiments are well-designed, and the results are highly interesting, particularly for understanding how cues can motivate and invigorate normal and abnormal behavior.

Weaknesses:

Additional support and evidence are needed for the claims made by the authors. Some of the statements are inconsistent with the data and/or analyses or are only weakly supportive of the claims.

Reviewer #3 (Public review):

Summary:

In this work, Hathaway and colleagues aim to understand how audiovisual cues at the time of outcome promote the selection of risky choices. A real-life illustration of this effect is used in electronic gambling machines which signal a win with flashing lights and jingles, encouraging the player to keep betting. More specifically, the authors ask whether the cue has to be paired exclusively to wins, or whether it can be paired to both outcomes, or exclusively loss outcomes, or occur randomly. To tackle this question, they employ a version of the Iowa Gambling Task adapted to rats, and test the effect of different rules of cue-outcome associations on the probability of selecting the riskier options; they then test the effect of prior reward devaluation on the task; finally, the optimised computational models on the early phases of the experiment to investigate potential mechanisms underlying the behavioural differences.

Strengths:

The experimental approach is very well thought-out, in particular, the choice of the different task variants covers a wide range of different potential hypotheses. Using this approach, they find that, although rats prefer the optimal choices, there is a shift towards selecting riskier options in the variants of the task where the cue is paired to win outcomes. They analyse this population average shift by showing that there is a concurrent increase in the number of risk-taking individuals in these tasks. They also make the novel discovery that pairing cues with loss outcomes only reduces the tendency for risky decisions.

The computational strategy is appropriate and in keeping with the accepted state of the art: defining a set of candidate models, optimising them, comparing them, simulating the best ones to ensure they replicate the main experimental results, then analysing parameter estimates in the different tasks to speculate about potential mechanisms.

Weaknesses:

There is a very problematic statistical stratagem that involves categorising individuals as either risky or optimal based on their choice probabilities. As a measurement or outcome, this is fine, as previously highlighted in the results, but this label is then used as a factor in different ANOVAs to analyse the very same choice probabilities, which then constitutes a circular argument (individuals categorised as risky because they make more risky choices, make more risky choices...).

A second experiment was done to study the effect of devaluation on risky choices in the different tasks. The results, which are not very clear to understand from Figure 3, would suggest that reward devaluation affects choices in tasks where the win-cue pairing is not present. The authors interpret this result by saying that pairing wins with cues makes the individuals insensitive to reward devaluation. Counter this, if an individual is prone to making risky choices in a given task, this points to an already distorted sense of value as the most rewarding strategy is to make optimal non-risky choices.

While the overall computational approach is excellent, I believe that the choice of computational models is poor. Loss trials come at a double cost, something the authors might want to elaborate more upon, firstly the lost opportunity of not having selected a winning option which is reflected in Q-learning by the fact that r=0, and secondly a waiting period which will affect the overall reward rate. The authors choose to combine these costs by attempting to convert the time penalty into "reward currency" using three different functions that make up the three different tested models. This is a bit of a wasted opportunity as the question when comparing models is not something like "are individuals in the paired win-cue tasks more sensitive to risk? or less sensitive to time? etc" but "what is the best way of converting time into Q-value currency to fit the data?" Instead, the authors could have contrasted other models that explicitly track time as a separate variable (see for example "Impulsivity and risk-seeking as Bayesian inference under dopaminergic control" (Mikhael & Gershman 2021)) or give actions an extra risk bonus (as in "Nicotinic receptors in the VTA promote uncertainty seeking" (Naude et al 2016)). Another weakness of the computational section is the fact, that despite simulations having been made, figure 5 only shows the simulated risk scores and not the different choice probabilities which would be a much more interesting metric by which to judge model validity. In the last section, the authors ask whether the parameter estimates (obtained from optimisation on the early sessions) could be used to predict risk preference. While this is an interesting question to address, the authors give very little explanation as to how they establish any predictive relationship. A figure and more detailed explanation would have been warranted to support their claims.

Reviewer #1 (Public review):

Summary:

Here, the authors aim to investigate the potential improvements of ANNs when used to explain brain data using top-down feedback connections found in the neocortex. To do so, they use a retinotopic and tonotopic organization to model each subregion of the ventral visual (V1, V2, V4, and IT) and ventral auditory (A1, Belt, A4) regions using Convolutional Gated Recurrent Units. The top-down feedback connections are inspired by the apical tree of pyramidal neurons, modeled either with a multiplicative effect (change of gain of the activation function) or a composite effect (change of gain and threshold of the activation function).

To assess the functional impact of the top-down connections, the authors compare three architectures: a brain-like architecture derived directly from brain data analysis, a reversed architecture where all feedforward connections become feedback connections and vice versa, and a random connectivity architecture. More specifically, in the brain-like model the visual regions provide feedforward input to all auditory areas, whereas auditory areas provide feedback to visual regions.

First, the authors found that top-down feedback influences audiovisual processing and that the brain-like model exhibits a visual bias in multimodal visual and auditory tasks. Second, they discovered that in the brain-like model, the composite integration of top-down feedback, similar to that found in the neocortex, leads to an inductive bias toward visual stimuli, which is not observed in the feedforward-only model. Furthermore, the authors found that the brain-like model learns to utilize relevant stimuli more quickly while ignoring distractors. Finally, by analyzing the activations of all hidden layers (brain regions), they found that the feedforward and feedback connectivity of a region could determine its functional specializations during the given tasks.

Strengths:

The study introduces a novel methodology for designing connectivity between regions in deep learning models. The authors also employ several tasks based on audiovisual stimuli to support their conclusions. Additionally, the model utilizes backpropagation of error as a learning algorithm, making it applicable across a range of tasks, from various supervised learning scenarios to reinforcement learning agents. Conversely, the presented framework offers a valuable tool for studying top-down feedback connections in cortical models. Thus, it is a very nice study that also can give inspiration to other fields (machine learning) to start exploring new architectures.

Weaknesses:

Although the study explores some novel ideas on how to study the feedback connections of the neocortex, the data presented here are not complete in order to propose a concrete theory of the role of top-down feedback inputs in such models of the brain.

(1) The gap in the literature that the paper tries to fill in the ability of DL algorithms to predict behavior: "However, there are still significant gaps in most deep neural networks' ability to predict behavior, particularly when presented with ambiguous, challenging stimuli." and "[...] to accurately model the brain."

It is unclear to me how the presented work addresses this gap, as the only facts provided are derived from a simple categorization task that could also be solved by the feedforward-only model (see Figures 4 and 5). In my opinion, this statement is somewhat far-fetched, and there is insufficient data throughout the manuscript to support this claim.

(2) It is not clear what the advantages are between the brain-like model and a feedforward-only model in terms of performance in solving the task. Given Figures 4 and 5, it is evident that the feedforward-only model reaches almost the same performance as the brain-like model (when the latter uses the modulatory feedback with the composite function) on almost all tasks tested. The speed of learning is nearly the same: for some tested tasks the brain-like model learns faster, while for others it learns slower. Thus, it is hard to attribute a functional implication to the feedback connections given the presented figures and therefore the strong claims in the Discussion should be rephrased or toned down.

(3) The Methods section lacks sufficient detail. There is no explanation provided for the choice of hyperparameters nor for the structure of the networks (number of trainable parameters, number of nodes per layer, etc). Clarifying the rationale behind these decisions would enhance understanding. Moreover, since the authors draw conclusions based on the performance of the networks on specific tasks, it is unclear whether the comparisons are fair, particularly concerning the number of trainable parameters. Furthermore, it is not clear if the visual bias observed in the brain-like model is an emerging property of the network or has been created because of the asymmetries in the visual vs. auditory pathway (size of the layer, number of layers, etc).

Reviewer #2 (Public review):

Summary:

This work addresses the question of whether artificial deep neural network models of the brain could be improved by incorporating top-down feedback, inspired by the architecture of the neocortex.

In line with known biological features of cortical top-down feedback, the authors model such feedback connections with both, a typical driving effect and a purely modulatory effect on the activation of units in the network.

To assess the functional impact of these top-down connections, they compare different architectures of feedforward and feedback connections in a model that mimics the ventral visual and auditory pathways in the cortex on an audiovisual integration task.

Notably, one architecture is inspired by human anatomical data, where higher visual and auditory layers possess modulatory top-down connections to all lower-level layers of the same modality, and visual areas provide feedforward input to auditory layers, whereas auditory areas provide modulatory feedback to visual areas.

First, the authors find that this brain-like architecture imparts the models with a light visual bias similar to what is seen in human data, which is the opposite in a reversed architecture, where auditory areas provide a feedforward drive to the visual areas.

Second, they find that, in their model, modulatory feedback should be complemented by a driving component to enable effective audiovisual integration, similar to what is observed in neural data.

Last, they find that the brain-like architecture with modulatory feedback learns a bit faster in some audiovisual switching tasks compared to a feedforward-only model.

Overall, the study shows some possible functional implications when adding feedback connections in a deep artificial neural network that mimics some functional aspects of visual perception in humans.

Strengths:

The study contains innovative ideas, such as incorporating an anatomically inspired architecture into a deep ANN, and comparing its impact on a relevant task to alternative architectures.

Moreover, the simplicity of the model allows it to draw conclusions on how features of the architecture and functional aspects of the top-down feedback affect the performance of the network.

This could be a helpful resource for future studies of the impact of top-down connections in deep artificial neural network models of the neocortex.

Weaknesses:

Overall, the study appears to be a bit premature, as several parts need to be worked out more to support the claims of the paper and to increase its impact.

First, the functional implication of modulatory feedback is not really clear. The "only feedforward" model (is a drive-only model meant?) attains the same performance as the composite model (with modulatory feedback) on virtually all tasks tested, it just takes a bit longer to learn for some tasks, but then is also faster at others. It even reproduces the visual bias on the audiovisual switching task. Therefore, the claims "Altogether, our results demonstrate that the distinction between feedforward and feedback inputs has clear computational implications, and that ANN models of the brain should therefore consider top-down feedback as an important biological feature." and "More broadly, our work supports the conclusion that both the cellular neurophysiology and structure of feed-back inputs have critical functional implications that need to be considered by computational models of brain function" are not sufficiently supported by the results of the study. Moreover, the latter points would require showing that this model describes neural data better, e.g., by comparing representations in the model with and without top-down feedback to recorded neural activity.

Second, the analyses are not supported by supplementary material, hence it is difficult to evaluate parts of the claims. For example, it would be helpful to investigate the impact of the process time after which the output is taken for evaluation of the model. This is especially important because in recurrent and feedback models the convergence should be checked, and if the network does not converge, then it should be discussed why at which point in time the network is evaluated.

Third, the descriptions of the models in the methods are hard to understand, i.e., parameters are not described and equations are explained by referring to multiple other studies. Since the implications of the results heavily rely on the model, a more detailed description of the model seems necessary.

Lastly, the discussion and testable predictions are not very well worked out and need more details. For example, the point "This represents another testable prediction flowing from our study, which could be studied in humans by examining the optical flow (Pines et al., 2023) between auditory and visual regions during an audiovisual task" needs to be made more precise to be useful as a prediction. What did the model predict in terms of "optic flow", how can modulatory from simple driving effect be distinguished, etc.

Reviewer #3 (Public review):

Summary:

This study investigates the computational role of top-down feedback in artificial neural networks (ANNs), a feature that is prevalent in the brain but largely absent in standard ANN architectures. The authors construct hierarchical recurrent ANN models that incorporate key properties of top-down feedback in the neocortex. Using these models in an audiovisual integration task, they find that hierarchical structures introduce a mild visual bias, akin to that observed in human perception, not always compromising task performance.

Strengths:

The study investigates a relevant and current topic of considering top-down feedback in deep neural networks. In designing their brain-like model, they use neurophysiological data, such as externopyramidisation and hierarchical connectivity. Their brain-like model exhibits a visual bias that qualitatively matches human perception.

Weaknesses:

While the model is brain-inspired, it has limited bioplausibility. The model assumes a simplified and fixed hierarchy. In the brain with additional neuromodulation, the hierarchy could be more flexible and more task-dependent.

While the brain-like model showed an advantage in ignoring distracting auditory inputs, it struggled when visual information had to be ignored. This suggests that its rigid bias toward visual processing could make it less adaptive in tasks requiring flexible multimodal integration. It hence does not necessarily constitute an improvement over existing ANNs. It is unclear, whether this aspect of the model also matches human data. In general, there is no direct comparison to human data. The study does not evaluate whether the top-down feedback architecture scales well to more complex problems or larger datasets. The model is not well enough specified in the methods and some definitions are missing.

Reviewer #1 (Public review):

Summary:

Sattin, Nardin, and colleagues designed and evaluated corrective microlenses that increase the useable field of view of two long (>6mm) thin (500 um diameter) GRIN lenses used in deep-tissue two-photon imaging. This paper closely follows the thread of earlier work from the same group (esp. Antonini et al, 2020; eLife), filling out the quiver of available extended-field-of-view 2P endoscopes with these longer lenses. The lenses are made by a molding process that appears practical and easy to adopt with conventional two-photon microscopes.

Simulations are used to motivate the benefits of extended field of view, demonstrating that more cells can be recorded, with less mixing of signals in extracted traces, when recorded with higher optical resolution. In vivo tests were performed in piriform cortex, which is difficult to access, especially in chronic preparations.

The design, characterization, and simulations are clear and thorough, but they do not break new ground in optical design or biological application. However, the approach shows much promise, including for applications such as miniaturized GRIN-based microscopes. Readers will largely be interested in this work for practical reasons: to apply the authors' corrected endoscopes to their own research.

Strengths:

The text is clearly written, the ex vivo analysis is thorough and well supported, and the figures are clear. The authors achieved their aims, as evidenced by the images presented, and were able to make measurements from large numbers of cells simultaneously in vivo in a difficult preparation.

The authors did a good job of addressing issues I raised in initial review, including analyses of chromaticity and the axial field of view, descriptions of manufacturing and assembly yield, explanations in the text of differences between ex vivo and in vivo imaging conditions, and basic analysis of the in vivo recordings relative to odor presentations. They have also shortened the text, reduced repetition, and better motivated their approach in the introduction.

Reviewer #2 (Public review):

In this manuscript, the authors present an approach to correct GRIN lens aberrations, which primarily cause a decrease in signal-to-noise ratio (SNR), particularly in the lateral regions of the field-of-view (FOV), thereby limiting the usable FOV. The authors propose to mitigate these aberrations by designing and fabricating aspherical corrective lenses using ray trace simulations and two-photon lithography, respectively; the corrective lenses are then mounted on the back aperture of the GRIN lens.

This approach was previously demonstrated by the same lab for GRIN lenses shorter than 4.1 mm (Antonini et al., eLife, 2020). In the current work, the authors extend their method to a new class of GRIN lenses with lengths exceeding 6 mm, enabling access to deeper brain regions as most ventral region of the mouse brain. Specifically, they designed and characterized corrective lenses for GRIN lenses measuring 6.4 mm and 8.8 mm in length. Finally, they applied these corrected long micro-endoscopes to perform high-precision calcium signal recordings in the olfactory cortex.

Compared with alternative approaches using adaptive optics, the main strength of this method is that it does not require hardware or software modifications, nor does it limit the system's temporal resolution. The manuscript is well-written, the data are clearly presented, and the experiments convincingly demonstrate the advantages of the corrective lenses.

The implementation of these long corrected micro-endoscopes, demonstrated here for deep imaging in the mouse olfactory bulb, will also enable deep imaging in larger mammals such as rats or marmosets.

Comments on revisions:

The authors have clearly addressed all my comments.

Reviewer #3 (Public review):

Summary:

This work presents the development, characterization and use of new thin microendoscopes (500µm diameter) whose accessible field of view has been extended by the addition of a corrective optical element glued to the entrance face. Two microendoscopes of different lengths (6.4mm and 8.8mm) have been developed, allowing imaging of neuronal activity in brain regions >4mm deep. An alternative solution to increase the field of view could be to add an adaptive optics loop to the microscope to correct the aberrations of the GRIN lens. The solution presented in this paper does not require any modification of the optical microscope and can therefore be easily accessible to any neuroscience laboratory performing optical imaging of neuronal activity.

Strengths:

(1) The paper is generally clear and well written. The scientific approach is well structured, and numerous experiments and simulations are presented to evaluate the performance of corrected microendoscopes. In particular, we can highlight several consistent and convincing pieces of evidence for the improved performance of corrected microendoscopes:

- PSFs measured with corrected microendoscopes 75µm from the centre of the FOV show a significant reduction in optical aberrations compared to PSFs measured with uncorrected microendoscopes.

- Morphological imaging of fixed brain slices shows that optical resolution is maintained over a larger field of view with corrected microendoscopes compared to uncorrected ones, allowing neuronal processes to be revealed even close to the edge of the FOV.

- Using synthetic calcium data, the authors showed that the signals obtained with the corrected microendoscopes have a significantly stronger correlation with the ground truth signals than those obtained with uncorrected microendoscopes.

(2) There is a strong need for high quality microendoscopes to image deep brain regions in vivo. The solution proposed by the authors is simple, efficient and potentially easy to disseminate within the neuroscience community.

Weaknesses:

Weaknesses that were present in the first version of the paper were carefully addressed by the authors.

Reviewer #1 (Public Review):

Summary:

The study by Seo et al highlights knowledge gaps regarding the role of cerebellar complex spike (CS) activity during different phases of learning related to optokinetic reflex (OKR) in mice. The novelty of the approach is twofold: first, specifically perturbing the activity of climbing fibers (CFs) in the flocculus (as opposed to disrupting communication between the inferior olive (IO) and its cerebellar targets globally); and second, examining whether disruption of the CS activity during the putative "consolidation phase" following training affects OKR performance.

The first part of the results provides adequate evidence supporting the notion that optogenetic disruption of normal CF-Purkinje neuron (PN) signaling results in the degradation of OKR performance. As no effects are seen in OKR performance in animals subjected to optogenetic irradiation during the memory consolidation or retrieval phases, the authors conclude that CF function is not essential beyond memory acquisition. However, the manuscript does not provide a sufficiently solid demonstration that their long-term activity manipulation of CF activity is effective, thus undermining the confidence of the conclusions.

Strengths:

The main strength of the work is the aim to examine the specific involvement of the CF activity in the flocculus during distinct phases of learning. This is a challenging goal, due to the technical challenges related to the anatomical location of the flocculus as well as the IO. These obstacles are counterbalanced by the use of a well-established and easy-to-analyse behavioral model (OKR), that can lead to fundamental insights regarding the long-term cerebellar learning process.

Weaknesses:

The impact of the work is diminished by several methodological shortcomings.

Most importantly, the key finding that prolonged optogenetic inhibition of CFs (for 30 min to 6 hours after the training period) must be complemented by the demonstration that the manipulation maintains its efficacy. In its current form, the authors only show inhibition by short-term optogenetic irradiation in the context of electrical-stimulation-evoked CSs in an ex vivo preparation. As the inhibitory effect of even the eNpHR3.0 is greatly diminished during seconds-long stimulations (especially when using the yellow laser as is done in this work (see Zhang, Chuanqiang, et al. "Optimized photo-stimulation of halorhodopsin for long-term neuronal inhibition." BMC biology 17.1 (2019): 1-17. ), we remain skeptical of the extent of inhibition during the long manipulations. In short, without a demonstration of effective inhibition throughout the putative consolidation phase (for example by showing a significant decrease in CS frequency throughout the irradiation period), the main claim of the manuscript of phase-specific involvement of CF activity in OKR learning can not be considered to be based on evidence.

Second, the choice of viral targeting strategy leaves gaps in the argument for CF-specific mechanisms. CaMKII promoters are not selective for the IO neurons, and even the most precise viral injections always lead to the transfection of neurons in the surrounding brainstem, many of which project to the cerebellar cortex in the form of mossy fibers (MF). Figure 1Bii shows sparsely-labelled CFs in the flocculus, but possibly also MFs. While obtaining homogenous and strong labeling in all floccular CFs might be impossible, at the very least the authors should demonstrate that their optogenetic manipulation does not affect simple spiking in PNs.

Finally, while the paper explicitly focuses on the effects of CF-evoked complex spikes in the PNs and not, for example, on those mediated by molecular layer interneurons or via direct interaction of the CF with vestibular nuclear neurons, it would be best if these other dimensions of CF involvement in cerebellar learning were candidly discussed.

Reviewer #2 (Public Review):

Summary:

The authors aimed to explore the role of climbing fibers (CFs) in cerebellar learning, with a focus on optokinetic reflex (OKR) adaptation. Their goal was to understand how CF activity influences memory acquisition, memory consolidation, and memory retrieval by optogenetically suppressing CF inputs at various stages of the learning process.

Strengths:

The study addresses a significant question in the cerebellar field by focusing on the specific role of CFs in adaptive learning. The authors use optogenetic tools to manipulate CF activity. This provides a direct method to test the causal relationship between CF activity and learning outcomes.

Weaknesses:

Despite shedding light on the potential role of CFs in cerebellar learning, the study is hampered by significant methodological issues that question the validity of its conclusions. The absence of detailed evidence on the effectiveness of CF suppression and concerns over tissue damage from optogenetic stimulation weakens the argument that CFs are not essential for memory consolidation. These challenges make it difficult to confirm whether the study's objectives were fully met or if the findings conclusively support the authors' claims. The research commendably attempts to unravel the temporal involvement of CFs in learning but also underscores the difficulties in pinpointing specific neural mechanisms that underlie the phases of learning. Addressing these methodological issues, investigating other signals that might instruct consolidation, and understanding CFs' broader impact on various learning behaviors are crucial steps for future studies.

[Editors' note: we have included the original concerns, which the Reviewing Editor agrees with. Methodological concerns remain after revisions.]

Reviewer #1 (Public review):

Summary:

This study seeks to quantify changes in vocal behavior during development in marmosets with bilateral anterior cingulate cortex (ACC) lesions. The ACC and its role in social vocal behaviors is of great interest given previous literature on its involvement in initiation of vocalizations, processing emotional content, and its connectivity to two other critical nodes in the vocal network, the amygdala and the PAG. The authors seek to test the hypothesis that the ACC contributes to the development of mature vocal behaviors during the first few weeks of life by disrupting this process with neonatal ACC lesions. Imaging and histological analyses confirm the extent of the lesion and suggest downstream effects in connected regions. Analysis of call rates and call type proportions show no or slight differences between lesioned and controlled animals. Additional analyses on the proportion of grouped 'social' calls and certain acoustic features of a particular call, the phee, reveal more distinct differences between the groups.

Strengths:

The authors have identified that ACC lesions in early life have no or little influence on certain aspects of vocal behavior (e.g. call rate, call intervals) but larger impacts on other aspects (e.g. acoustic features of phee calls). This is difficult data to collect, especially in the difficulties of that particular time period. This data is a valuable addition to the literature on the effects of the ACC on vocal production and sparks intriguing follow-up questions on the role of different acoustic features (as related to emotional content) on vocal interactions with conspecifics over the lifespan.

The histological methods and resulting quantification of neural changes in the lesioned area and in downstream areas of interest are intriguing given the large time gap between the lesion and these analyses.

The changes to the text, figures, and additional supplemental figures to my previous review requests have made it easier to determine if conclusions are supported by the data in the manuscript. Examples include the quantification of the loss of neurons and increase in glial cells, the inclusion of changes in body weight and grip strength that could also be a result from the lesions affecting vocal behavior, and additional details on analysis methods.

Weaknesses:

The article emphasizes vocal social behavior. However, marmoset infants are recorded in isolation which allows for examining the development of vocal behavior in that particular context - reaching out to conspecifics. The text now covers the relationship between 'social' information in calls and development in this particular context. However, early-life maturation of vocal behavior is strongly influenced by social interactions with conspecifics. For example, the transition of cries and subharmonic phees which are high-entropy calls to more low-entropy mature phees is affected by social reinforcement from the parents. And this effect extends cross-context, where differences in these interaction patterns extend to vocal behavior when the marmosets are alone. Together, the results are interesting and important but may not fully capture the changes resulting from direct social interactions.

Additionally, it is an intriguing finding that the infants' phee calls have acoustic differences being 'blunted of variation, less diverse and more regular'. Though the text about how the social message conveyed by these infants was 'deficient, limited, and/or indiscriminate' is now better explained with additional text from human studies, it is still an assumption that this would directly translate to marmoset communication. Thus, experiments directed at the responses of other marmosets to these calls would still be important.

Reviewer #2 (Public review):

Summary:

Nagarajan et al. investigate the role of the anterior cingulate cortex (ACC) in vocal development of infant marmoset monkeys using lesions in this brain area. Many previous studies show that ACC plays an important role in volitional and emotion-driven vocal behavior in mammals. The experiments Nagarajan et al. performed strengthen the long-standing hypothesis that ACC influences the development of social-vocal behavior in non-human primates. Furthermore, their anatomical studies support the idea of cortical structures exerting cognitive control over subcortical networks for innate vocalization, and thus, enabling mammals to perform flexible social-vocal communication.

Strengths:

Many invasive behavioral studies in monkeys often use 2-3 animals. The authors used a sufficiently high number of animals for their experiments. This increases the power of their conclusions.

The study also investigates the impact of ACC lesions on downstream areas important for innate vocal production. This adds further evidence to the role of ACC on influencing these subcortical regions during vocal development and vocal behavior in general.

Weaknesses:

The study only provides data up to the 6th week after birth. Given the plasticity of the cortex, it would be interesting to see if these impairments in vocal behavior persist throughout adulthood or if the lesioned marmosets will recover their social-vocal behavior compared to the control animals. The authors give a reasonable explanation for why they did not provide this data.

Even though this study focuses entirely on the development of social vocalizations, providing data about altered social non-vocal behaviors that accompany ACC lesions is missing. This data can provide further insights and generate new hypothesis about the exact role of ACC in social-vocal development. For example, do these marmosets behave differently towards their conspecifics or family members and vice versa, and is this an alternate cause for the observed changes in social-vocal development? Unfortunately, the authors are unable to provide that data. Hopefully, this will be the goal of future studies.

Reviewer #3 (Public review):

Summary:

In this manuscript, Nagarajan et al. study the impact of early damage to the anterior cingulate cortex (ACC) on the vocal development of marmoset monkeys. AAC lesions were performed on neonatal marmosets and their vocal patterns and the spectrotemporal features of their calls were analyzed compared to control groups during the first six weeks of life. While the vocal repertoire was not significantly affected by ACC lesions, the authors described notable differences in the social contact call, the phee call. Marmosets with ACC damage made fewer social contact calls, and when they did, these calls were shorter, louder, and monotonic. Additionally, the study revealed that ACC damage in infancy led to permanent alterations in downstream brain areas involved in social vocalizations, such as the amygdala and periaqueductal gray.

Strengths:

This study suggests that the ACC plays a crucial role in the normal development of social vocal behavior in infant marmosets. Studying vocal behavior in marmosets can provide insights into the neural mechanisms underlying human speech and communication disorders due to their similarity in brain structure and social behavior.

The methods are robust and reliable with precise localization of the lesions with neuroimaging and histological examination.

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:

- The logic of the experiment is nicely laid out.

- The presentation is clear and concise.

- The analyses are thorough, careful, and yield unambiguous results.

- 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:

The weaknesses I noted in the first round of reviews were effectively addressed by the authors. In particular, the expanded discussion on the overlapping effects of attention, working memory, and motor planning does a great job putting the current findings against the wider context concerning the neural mechanisms of visuomotor guidance.

I think this is a well-designed and well-executed study that helps to better outline the relationship between perception and working memory given their respective neural substrates. A broad range of systems neuroscientists and experimental psychologists will find it illuminating.

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 recruiting 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 signal 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 on 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 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, a few conceptual gaps make it harder for the reader to appreciate the mechanisms that lead to the observed results and evaluate whether and how these may apply to other cases of top-down control. The fact that the visual features under study were behaviorally irrelevant make it difficult to appreciate the relevance of the finding and its relation to top-down spatial attention mechanisms that involve similar/overlapping circuits. In the same vein, 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.

Moreover, encoding of the two visual features that are manipulated in the context of the study (contrast and orientation) seems to be affected differently in certain cases, which leaves a reader wondering about the source of this variability.

Finally, although the study provides evidence in favor of a role of FEF in influencing phase coding of visual features in V4 in beta frequencies, important analysis that could have revealed the long-range mechanisms of such an effect including the analysis of intra-FEF and interareal (FEF-V4) neuronal interactions is missing from this paper

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 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:

- Elegant and robust experiment that allows for clear tests for the necessity of FEF activity in WM-induced changes in V4 activity<br /> - 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<br /> - Convincing modeling efforts

Comments on revisions:

I have no further comments for the authors. The revised manuscript appears to have adequately addressed the substantial comments raised in the previous round of review. I especially appreciate the addition of a new supplementary figure analyzing the data when no background stimulus was presented.

Reviewer #1 (Public review):

Summary:

This is an interesting study on AD(H)D. The authors combine a variety of neural and physiological metrics to study attention in a VR classroom setting. The manuscript is well written and the results are interesting, ranging from an effect of group (AD(H)D vs. control) on metrics such as envelope tracking, to multivariate regression analyses considering alpha-power, gaze, TRF, ERPs, and behaviour simultaneously. I find the first part of the results clear and strong. The multivariate analyses in Tables 1 and 2 are good ideas, but I think they would benefit from additional clarifications. Overall, I think that the methodological approach is useful in itself. The rest is interesting in that it informs us on which metrics are sensitive to group-effects and correlated with each other. I think this might be one interesting way forward. Indeed, much more work is needed to clarify how these results change with different stimuli and tasks. So, I see this as an interesting first step into more naturalistic measurement of speech attention.

Strengths:

I praise the authors for this interesting attempt to tackle a challenging topic with naturalistic experiment and metrics. I think the results broadly make sense and they contribute to a complex literature that is far from being linear and cohesive.

Weaknesses:

The authors have successfully addressed most of my concerns during the review process. Some weaknesses remain in this resubmission, but they do not make the results invalid. For example:<br /> - The EEG data was filtered twice, which is not recommended as that can introduce additional filtering artifacts. So, while I definitely do not recommend doing that, I do not expect that issue to have an impact on this specific result.<br /> - The authors did not check whether participants were somewhat familiar with the topics in the speech material. The authors agreed that this point might be beneficial for future research.<br /> - The hyperparameter tuning is consistent with previous work from the authors, and it involves selecting the optimal lambda value of the regularized regression based on the group average, thus choosing a single lambda value for all participants. In my opinion, that is not the optimal way to run those models, and I do not generally recommend using this approach. The reason is that the lambda can change depending on the magnitude of the signals and the SNR, leading to different optimal lambdas for distinct participants. On the other hand, finding those optimal lambda values for individual participants can be difficult depending on the amount of data and amount of noise, so it is sometimes necessary to apply strategies that ensure an appropriate choice of lambda. Using the group average as a metric for hyperparameter tuning produces a more stable metric and lambda value selection, which might be preferrable (even though this choice should not be taken lightly). In this specific case, I think the authors had a good reason to do so.

Comments on revisions:

The authors have done a great job at addressing my comments. I don't have any further concerns. Congratulations!

Reviewer #2 (Public review):

Summary:

While selective attention is a crucial ability of human beings, previous studies on selective attention are primarily conducted in a strictly controlled context, leaving a notable gap in underlying the complexity and dynamic nature of selective attention in a naturalistic context. This issue is particularly important for classroom learning in individuals with ADHD, as selecting the target and ignoring the distractions are pretty difficult for them but are the pre-requirement of effective learning. The authors of this study have addressed this challenge using a well-motivated study. I believe the findings of this study will be a nice addition to the fields both cognitive neuroscience and educational neuroscience.

Strengths:

To achieve the purpose of setting up a naturalistic context, the authors have based their study on a novel Virtual Reality platform. This is clever as it is usually difficult to perform such a study in the real classroom. Moreover, various techniques such as brain imaging, eye-tracking and physiological measurement are combined to collect multi-level data. They found that, different from the controls, individuals with ADHD had higher neural responses to the irrelevant rather than the target sounds, reduced speech tracking of the teacher. Additionally, the power of alpha-oscillations and frequency of gaze-shifts away from the teacher are found to be associated with the ADHD symptoms. These results provide new insights into the mechanism of selective attention among ADHD populations.

Weaknesses:

It is worth noting that nowadays there has been some studies trying to do so in the real classroom, and thus the authors should acknowledge the difference between the virtual and real classroom context and foresee the potential future changes.<br /> The approach of combining multi-level data owns advantage to obtain reliable results, but also raises significant difficult for the readers to understand the main results.

- An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

As expected, individuals with ADHD showed anomalous pattern of neural responses, and eye-tracking pattern, compared to the controls. But there are also some similarities between groups such as amount of time paying attention to teachers, etc. In general, their conclusions are supported.

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

The findings are an extension of previous efforts in understanding selective attention in the naturalistic context. The findings of this study are particularly helpful in inspiring teacher's practice and advancing the research of educational neuroscience. This study demonstrates, again, that it is important to understand the complexity of cognitive process in the naturalistic context.

Comments on revisions:

The authors have appropriately responded to my concerns. I do not have other comments. I do hope to see more data and results from the authors in future.

Reviewer #3 (Public review):

Summary:

The authors conducted a well-designed experiment, incorporating VR classroom scenes and background sound events, with both control and ADHD participants. They employed multiple neurophysiological measures, such as EEG, eye movements, and skin conductance, to investigate the mechanistic underpinnings of paying attention in class and the disruptive effects of background noise.

The results revealed that individuals with ADHD exhibited heightened sensory responses to irrelevant sounds and reduced tracking of the teacher's speech. Overall, this manuscript presented an ecologically valid paradigm for assessing neurophysiological responses in both control and ADHD groups. The analyses were comprehensive and clear, making the study potentially valuable for the application of detecting attentional deficits.

Strengths:

• The VR learning paradigm is well-designed and ecologically valid.

• The neurophysiological metrics and analyses are comprehensive, and two physiological markers are identified capable of diagnosing ADHD.

• The data shared could serve as a benchmark for future studies on attention deficits in ecologically valid scenarios.

Weaknesses:

• Several results are null results, i.e., no significant differences were found between ADHD and control populations.

Comments on revisions:

The authors have addressed all of my concerns with the original manuscript.

Reviewer #1 (Public review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings.

Comments on revisions: I have no further comments.

Reviewer #2 (Public review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population level activity in CA1.

Comments on revisions:

I have no further major requests and thank the authors for the additional data and analyses.

Reviewer #3 (Public review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head fixed mice running on a track while local field potential (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected the other side of the brain.

Strengths:

The authors use a cutting-edge technique.

Weaknesses:

Although the authors have toned down their claims, the statement in the title ("Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Theta but not Ripple Oscillations During Novel Exploration") is still unsupported.

One could write the same title while voltage imaging one mouse and recording LFP from another mouse.

To properly convey the results, the title should be modified to read "Synchronous Ensembles of Hippocampal CA1 Pyramidal Neurons Associated with Contralateral Theta but not with Contralateral Ripple Oscillations During Novel Exploration"

Without making this change, the title - and therefore the entire work - is misleading at best.

Reviewer #1 (Public review):

Summary:

The authors propose a new model of biologically realistic reinforcement learning in the direct and indirect pathway spiny projection neurons in the striatum. These pathways are widely considered to provide a neural substrate for reinforcement learning in the brain. However, we do not yet have a full understanding of mechanistic learning rules that would allow successful reinforcement learning like computations in these circuits. The authors outline some key limitations of current models and propose an interesting solution by leveraging learning with efferent inputs of selected actions. They show that the model simulations are able to recapitulate experimental findings about the activity profile in these populations in mice during spontaneous behavior. They also show how their model is able to implement off-policy reinforcement learning.

Strengths:

The manuscript has been very clearly written and the results have been presented in a readily digestible manner. The limitations of existing models, that motive the presented work, have been clearly presented and the proposed solution seems very interesting. The novel contribution in the proposed model is the idea that different patterns of activity drive current action selection and learning. Not only does this allow the model is able to implement reinforcement learning computations well, this suggestion may have interesting implications regarding why some processes selectively affect ongoing behavior and others affect learning. The model is able to recapitulate some interesting experimental findings about various activity characteristics of dSPN and iSPN pathway neuronal populations in spontaneously behaving mice. The authors also show that their proposed model can implement off-policy reinforcement learning algorithms with biologically realistic learning rules. This is interesting since off-policy learning provides some unique computational benefits and it is very likely that learning in neural circuits may, at least to some extent, implement such computations.

Weaknesses:

A weakness in this work is that it isn't clear how a key component in the model - an efferent copy of selected actions - would be accessible to these striatal populations. The authors propose several plausible candidates, but future work may clarify the feasibility of this proposal.

Reviewer #2 (Public review):

Summary:

The basal ganglia is often understood within a reinforcement learning (RL) framework, where dopamine neurons convey a reward prediction error which modulates cortico-striatal connections onto spiny projection neurons (SPNS) in the striatum. However, current models of plasticity rules are inconsistent with learning in a reinforcement learning framework.

This paper proposes a new model that describes how distinct learning rules in direct and indirect pathway striatal neurons allows them to implement reinforcement learning models. It proposes that two distinct component of striatal activity affect action selection and learning. They show that the proposed implementation allows learning in simple tasks and is consistent with experimental data from calcium imaging data in direct and indirect SPNs in freely moving mouse.

Strengths:

Despite the success of reward prediction errors at characterizing the responses of dopamine neurons as the temporal difference error within an RL framework, the implementation of RL algorithms in the rest of the basal ganglia has been unclear. A key missing aspect has been the lack of a RL implementation that is consistent with the distinction of direct- and indirect SPNs. This paper proposes a new model that is able to learn successfully in simple RL tasks and explains recent experimental results.

The author shows that their proposed model, unlike previous implementations, this model can perform well in RL tasks. The new model allows them to make experimental predictions. They test some of these predictions and show that the dynamics of dSPNs and iSPNs correspond to model predictions.

More generally, this new model can be used to understand striatal dynamics across direct and indirect SPNs in future experiments.

Weaknesses:

The authors could characterize better the reliability of their experimental predictions and the description of the parameters of some of the simulations

The authors propose some ideas about how the specificity of the striatal efferent inputs but should highlight better that this is a key feature of the model whose anatomical implementation has yet to be resolved.

Comments on revisions:

I thank the authors for their response to public and private reviews and for the clarifications and changes to the manuscript which have strengthened it. I understand the inability to implement some of the proposed additional simulation due to authors having left academia and the request for a version of record.

Reviewer #3 (Public review):

Summary:

This paper points out an inconsistency of the roles of the striatal spiny neurons projecting to the indirect pathway (iSPN) and the synaptic plasticity rule of those neurons expressing dopamine D2 receptors, and proposes a novel, intriguing mechanisms that iSPNs are activated by the efference copy of the chosen action that they are supposed to inhibit.

The proposed model was supported by simulations and analysis of the neural recording data during spontaneous behaviors.

Strengths:

Previous models suggested that the striatal neurons learn action values functions, but how the information about the chosen action is fed back to the striatum for learning was not clear. The author pointed out that this is a fundamental problem for iSPNs that are supposed to inhibit specific actions and its synaptic inputs are potentiated with dopamine dips.

The authors proposes a novel hypothesis that iSPNs are activated by efference copy of the selected action which they are supposed to inhibit during action selection. Even though intriguing and seemingly unnatural, the authors demonstrated that the model based on the hypothesis can circumvent the problem of iSPNs learning to disinhibit the actions associated with negative reward errors. They further showed by analyzing the cell-type specific neural recording data by Markowitz et al. (2018) that iSPN activities tend to be anti-correlated before and after action selection.

Weaknesses:

(1) It is not correct to call the action value learning using the externally-selected action as "off-policy." Both off-policy algorithm Q-learning and on-policy algorithm SARSA update the action value of the chosen action, which can be different from the greedy action implicated by the present action values. In standard reinforce learning terminology, on-policy or off-policy is regarding the actions in the subsequent state, whether to use the next action value of (to be) chosen action or that of greedy choice as in equation (7).<br /> It is worth noting that this paper suggested that dopamine neurons encode on-policy TD errors: Morris G, Nevet A, Arkadir D, Vaadia E, Bergman H (2006). Midbrain dopamine neurons encode decisions for future action. Nat Neurosci, 9, 1057-63. https://doi.org/10.1038/nn1743

(2) It is also confusing to contract TD learning and Q-learning, as the latter is considered as on type of TD learning. In the TD error signal by state value function (6) is dependent on the chosen action a_{t-1} implicitly in r_t and s_t based on the reward and state transition function.

(3) It is not clear why interferences of the activities for action selection and learning can be avoided, especially when actions are taken with short intervals or even temporal overlaps. How can the efference copy activation for the previous action be dissociated with the sensory cued activation for the next action selection?

(4) Although it may be difficult to single out the neural pathway that carries the efference copy signal to the striatum, it is desired to consider their requirements and difference possibilities. A major issue is that the time delay from actions to reward feedback can be highly variable.

An interesting candidate is the long-latency neurons in the CM thalamus projecting to striatal cholinergic interneurons, which are activated following low-reward actions:<br /> Minamimoto T, Hori Y, Kimura M (2005). Complementary process to response bias in the centromedian nucleus of the thalamus. Science, 308, 1798-801. https://doi.org/10.1126/science.1109154

(5) In the paragraph before Eq. (3), Eq (1) should be Eq. (2) for the iSPN.

Here are comments back to the authors' replies with the revised version:

(1) I do not agree on the use of inaccurate technical terms. On-policy does not require that the policy is greedy with respect to the actions values, as authors seem to assume here.

In fact, the policy (10) is just a standard soft-max action selection based on the action values by the difference of dSPN and iSPN outputs.

Furthermore, in the immediate reward setting tested in this paper, action values are independent of the policy, so there is no distinction between on-policy vs. off-policy. This is also apparent from the "TD" errors in (19) and (21), where there is no TD.

(2) To really compare the different forms of TD, multi-step RL tasks should be used.

(3) This fundamental limitation should be explicitly documented in the manuscript. This is not just the same as any RL algorithms. Having two action representations within each action step make temporal credit assignment more difficult.

Reviewer #1 (Public review):

Summary:

In the article titled "Polyphosphate discriminates protein conformational ensembles more efficiently than DNA promoting diverse assembly and maturation behaviors," Goyal and colleagues investigate the role of negatively charged biopolymers, i.e., polyphosphate (polyP) and DNA, play in phase separation of cytidine repressor (CytR) and fructose repressor (FruR). The authors find that both negative polymers drive the formation of metastable protein/polymer condensates. However, polyP-driven condensates form more gel- or solid-like structures over time while DNA-driven condensates tend to dissipate over time. The authors link this disparate condensate behavior to polyP-induced structures within the enzymes. Specifically, they observe the formation of polyproline II-like structures within two tested enzyme variants in the presence of polyP. Together their results provide a unique insight into the physical and structural mechanism by which two unique negatively charged polymers can induce distinct phase transitions with the same protein. This study will be a welcomed addition to the condensate field and provide new molecular insights into how binding partner-induced structural changes within a given protein can affect the mesoscale behavior of condensates. The concerns outlined below are meant to strengthen the manuscript.

Strengths:

Throughout the article, the authors used the correct techniques to probe physical changes within proteins that can be directly linked to phase transition behaviors. Their rigorous experiments create a clear picture of what occurs at the molecular level with CytR and FruR are exposed to either DNA or polyP, which are unique, highly negatively charged biopolymers found within bacteria. This work provides a new view of mechanisms by which bacteria can regulate the cytoplasmic organization upon the induction of stress. Furthermore, this is likely applicable to mammalian and plant cells and likely to numerous proteins that undergo condensation with nucleic acids and other charged biopolymers.

Weaknesses:

The biggest weakness of this study is that compares the phase behavior of enzymes driven by negatively charged polymers that have intrinsic differences in net charge and charge density. Because these properties are extremely important for controlling phase separation, any differences may result in the observed phase transitions driven by DNA and polyP. The authors should perform an additional experiment to control for these differences as best they can. The results from these experiments will provide additional insight into the importance of charge-based properties for controlling phase transitions.

Reviewer #2 (Public review):

Summary:

In this study, Goyal et al demonstrate that the assembly of proteins with polyphosphate into either condensates or aggregates can reveal information on the initial protein ensemble. They show that, unlike DNA, polyphosphate is able to effectively discriminate against initial protein ensembles with different conformational heterogeneity, structure, and compactness. The authors further show that the protein native ensemble is vital on whether polyphosphate induces phase separation or aggregation, whereas DNA induces a similar outcome regardless of the initial protein ensemble. This work provides a way to improve our mechanistic understanding of how conformational transitions of proteins may regulate or drive LLPS condensate and aggregate assemblies within biological systems.

Strengths:

This is a thoroughly conducted study that provides an alternative route for inducing phase separation that is more informative on the initial protein ensemble involved. This is particularly useful and a complementary means to investigate the role played by protein dynamics and plasticity in phase transitions. The authors use an appropriate set of techniques to investigate unique phase transitions within proteins induced by polyphosphates. An alternative protein system is used to corroborate their findings that the unique assemblies induced by polyphosphates when compared to DNA are not restricted to a single system. The work here is well-documented, easy to interpret, and of relevance for the condensate community.

Weaknesses:

The major weakness of this manuscript is that it is unclear if the information on the initial protein conformational ensemble can be determined solely from the assembly and maturation behavior and the discrimination abilities of polyphosphates. In both systems studied (CytR and FruR), polyphosphate discriminates and results in unique assemblies and maturation behaviors based on the initial protein ensemble. However, it seems the assembly and maturation behavior are not a direct result of the degree of conformational dynamics and plasticity in the initial protein. In the case of CytR, the fully-folded system forms condensates that resolubilize, while the highly disordered state immediately aggregates. Whereas, in the case of FruR, the folded state induces spontaneous aggregation, and the more dynamic, molten globular, system results in short-lived condensates. These results seem to suggest the polyphosphates' ability to discriminate between the initial protein ensemble may not be able to reveal what that initial protein ensemble is unless it is already known.

Reviewer #1 (Public review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion-the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

Weaknesses:

(1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla. Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease a new perspective with the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

(2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

(3) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.<br /> Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

(4) Referred to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Figure 2A-E but it should be replaced with 3A-E. Please do that.

Summary of the Study after revision

The revised manuscript reflects significant efforts to improve clarity, organization, and data interpretation. The refinements in anatomical descriptions, behavioral analyses, and contextual framing have strengthened the manuscript considerably. However, the study still lacks direct causal evidence linking anatomical remodeling to behavioral improvements, and the small sample size in the anatomical analyses remains a concern. The authors have addressed many points raised in the initial review, but further acknowledgement of the exploratory nature of these findings would enhance the scientific rigor of the work.

Key Improvements in the Revision

The revised manuscript demonstrates considerable progress in clarifying data presentation, refining behavioral analyses, and improving the contextualization of anatomical findings. The restructuring of the anatomical section now provides greater precision in describing motor-related pathways, integrating terminology from the Allen Brain Atlas. The addition of new figures (Figures 4 and 5) strengthens the accessibility of these findings by illustrating key connectivity patterns more effectively. Furthermore, the correlation matrices have been adjusted to improve interpretability, ensuring that the presented data contribute meaningfully to the overall narrative of the study.

The authors have also made significant improvements in their behavioral analyses, particularly in the organization and presentation of locomotor data. Figure 3 has been revised to distinctly separate results from 6-OHDA and sham animals, providing a clearer comparison of locomotor outcomes. Additional metrics, such as reaction time, locomotion bouts, and movement speed, further enhance the granularity of the analysis, making the results more informative.

The discussion surrounding anatomical connectivity has also been strengthened. The revised manuscript now places greater emphasis on motor-related pathways and refines its analysis of A13 efferents and afferents. A newly introduced figure provides a concise summary of these connections, improving the contextualization of the anatomical data within the study's broader scope. Moreover, the authors have addressed the translational relevance of their findings by acknowledging the differences between optogenetic stimulation and deep brain stimulation (DBS). Their discussion now better situates the findings within existing literature on PD-related motor circuits, providing a more balanced perspective on the potential implications of A13 stimulation.

Remaining Concerns

Despite these substantial improvements, a number of critical concerns remain. The anatomical findings, though insightful, remain largely correlative and do not establish a causal link between structural remodeling and locomotor recovery. While the authors argue that these data will serve as a reference for future investigations, their necessity for the core conclusions of the study is not entirely clear. Additionally, while the anatomical data offer an interesting perspective on A13 connectivity, their direct relevance to the study's primary goal-demonstrating the role of A13 in locomotor recovery-remains uncertain. The authors emphasize that these data will be valuable for future research, yet their integration into the study's main narrative feels somewhat supplementary. Based on this last thought of the authors it is even more relevant another key limitation lying in the small sample size used for connectivity analyses. With only two sham and three 6-OHDA animals included, the statistical confidence in the findings is inherently limited. The absence of direct statistical comparisons between ipsilesional and contralesional projections further weakens the conclusions drawn from these anatomical studies. The authors have acknowledged that obtaining the necessary samples, acquiring the data, and analyzing them is a prolonged and resource-intensive process. While this may be a valid practical limitation, it does not justify the lack of a robust statistical approach. A more rigorous statistical framework should be employed to reinforce the findings, or alternative techniques should be considered to provide additional validation. Given these constraints, it remains unclear why the authors have not opted for standard immunohistochemistry, which could provide a complementary and more statistically accessible approach to validate the anatomical findings. Employing such an approach would not only increase the robustness of the results but also strengthen the study's impact by providing an independent confirmation of the observed structural changes.

Reviewer #2 (Public review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection, thus suggesting a remodeling of the A13 connectome. Whether this remodelling contributes to pro-locomotor effects of the photostimulation of the A13 region remains unknown as causality was not addressed.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients. The study also provides a description of the A13 region connectome pertaining to motor behaviors and how it changes after a dopaminergic lesion. Although there is no causal link between anatomical and behavioral data, it raises interesting questions for further studies.

Weaknesses:

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, some uncertainty remains regarding the phenotype of neurons underlying recovery of akinesia and improvement of bradykinesia.

Figure 4 is improved, but the results from the correlation analyses remain difficult to interpret, as they may reflect changes in various impaired brain regions independently of the A13 region. While the analysis offers a snapshot of correlated changes within the connectome, it does not identify which specific cell or axonal populations are actually increasing or decreasing. Although functional MRI connectome analyses are well-established, anatomical data seem less suitable for this purpose. How can one interpret correlated changes in anatomical inputs or outputs between two distinct regions?

Figure 5 is also improved, but there is room for further enhancement. As currently presented, it is difficult to distinguish the differences between the sham and 6-OHDA groups. The first column could compare afferents, while the second column could compare efferents. Given the small sample size, it would be more appropriate to present individual data rather than the mean and standard deviation.

Appraisal and impact

Although the behavioral experiments are convincing, the low number of animals in the anatomical studies is insufficient to make any relevant statistical conclusions due to extremely low statistical power.

Reviewer #3 (Public review):

Kim, Lognon et al. present an important finding on pro-locomotor effects of optogenetic activation of the A13 region, which they identify as a dopamine-containing area of the medial zona incerta that undergoes profound remodeling in terms of afferent and efferent connectivity after administration of 6-OHDA to the MFB. The authors claim to address a model of PD-related gait dysfunction, a contentious problem that can be difficult to treat by dopaminergic medication or DBS in conventional targets. They make use of an impressive array of technologies to gain insight into the role of A13 remodeling in the 6-OHDA model of PD. The evidence provided is solid and the paper is well written, but there are several general issues that reduce the value of the paper in its current form, and a number of specific, more minor ones. Also some suggestions, that may improve the paper compared to its recent form, come to mind.

The most fundamental issue that needs to be addressed is the relation of the structural to the behavioral findings. It would be very interesting to see whether the structural heterogeneity in afferent/effects projections induced by 6-OHDA is related to the degree of symptom severity and motor improvement during A13 stimulation.

The authors provide extensive interrogation of large-scale changes in the organization of the A13 region afferent and efferent distributions. It remains unclear how many animals were included to produce Fig 4 and 5. Fig S5 suggests that only 3 animals were used, is that correct? Please provide details about the heterogeneity between animals. Please provide a table detailing how many animals were used for which experiment. Were the same animals used for several experiments?

While the authors provide evidence that photoactivation of the A13 is sufficient in driving locomotion in the OFT, this pro-locomotor effect seems to be independent of 6-OHDA induced pathophysiology. Only in the pole test do they find that there seems to be a difference between Sham vs 6-OHDA concerning effects of photoactivation of the A13. Because of these behavioral findings, optogenic activation of A13 may represent a gain of function rather than disease-specific rescue. This needs to be highlighted more explicitly in the title, abstract and conclusion.

The authors claim that A13 may be a possible target for DBS to treat gait dysfunction. However, the experimental evidence provided (in particular lack of disease-specific changes in the OFT) seem insufficient to draw such conclusions. It needs to be highlighted that optogenetic activation does not necessarily have the same effects as DBS (see the recent review from Neumann et al. in Brain: https://pubmed.ncbi.nlm.nih.gov/37450573/). This is important because ZI-DBS so far had very mixed clinical effects. The authors should provide plausible reasons for these discrepancies. Is cell-specificity, that only optogenetic interventions can achieve, necessary? Can new forms of cyclic burst DBS achieve similar specificity (Spix et al, Science 2021)? Please comment.

In a recent study, Jeon et al (Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus, 2022, Cell Reports) provided evidence on the topographically graded organization of STN afferents and McElvain et al. (Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon, 2021, Neuron) have shown similar topographical resolution for SNr efferents. Can a similar topographical organization of efferents and afferents be derived for the A13/ ZI in total?

In conclusion, this is an interesting study that can be improved taking into consideration the points mentioned above.

Reviewer #2 (Public review):

Summary:

In this extensive comparative study, Moreno-Borrallo and colleagues examine the relationships between plasma glucose levels, albumin glycation levels, diet and life-history traits across birds. Their results confirmed the expected positive relationship between plasma blood glucose level and albumin glycation rate but also provided findings that are somewhat surprising or contrast with findings of some previous studies (positive relationships between blood glucose and lifespan, or absent relationships between blood glucose and clutch mass or diet). This is the first extensive comparative analysis of glycation rates and their relationships to plasma glucose levels and life history traits in birds that is based on data collected in a single study, with blood glucose and glycation measured using unified analytical methods (except for blood glucose data for 13 species collected from a database).

Strengths:

This is an emerging topic gaining momentum in evolutionary physiology, which makes this study a timely, novel and important contribution. The study is based on a novel data set collected by the authors from 88 bird species (67 in captivity, 21 in the wild) of 22 orders, except for 13 species, for which data were collected from a database of veterinary and animal care records of zoo animals (ZIMS). This novel data set itself greatly contributes to the pool of available data on avian glycemia, as previous comparative studies either extracted data from various studies or a ZIMS database (therefore potentially containing much more noise due to different methodologies or other unstandardised factors), or only collected data from a single order, namely Passeriformes. The data further represents the first comparative avian data set on albumin glycation obtained using a unified methodology. The authors used LC-MS to determine glycation levels, which does not have problems with specificity and sensitivity that may occur with assays used in previous studies. The data analysis is thorough, and the conclusions are substantiated. Overall, this is an important study representing a substantial contribution to the emerging field evolutionary physiology focused on ecology and evolution of blood/plasma glucose levels and resistance to glycation.

Weaknesses:

Unfortunately, the authors did not record handling time (i.e., time elapsed between capture and blood sampling), which may be an important source of noise because handling-stress-induced increase in blood glucose has previously been reported. Moreover, the authors themselves demonstrate that handling stress increases variance in blood glucose levels. Both effects (elevated mean and variance) are evident in Figure ESM1.2. However, this likely makes their significant findings regarding glucose levels and their associations with lifespan or glycation rate more conservative, as highlighted by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthen the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail, and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

The authors have made some changes in the revised version. However, many of the changes were superficial, and some concerns still need to be addressed. Important details are still missing from the description of some experiments. Authors should carefully revise the manuscript to ascertain that all details that could affect interpretation of their results are presented clearly. For instance, authors still need to include details of how the metabolomics analyses were performed. Just stating that samples were "frozen for metabolomics analyses" is not enough. Was this mass-spec or NMR-based metabolomics. Assuming it was mass-spec, what kind? How was metabolite identity assigned, etc? These are important details, which need to be included. Even in cases where additional information was included, the authors did not discuss how the specific way in which certain experiments were performed could affect interpretation of their results. One example is the potential for compound carryover in their experiments. Another important one is the fact that CAPE affects bacterial growth and sporulation. Therefore, it is critical that authors acknowledge that they cannot discard the possibility that other factors besides compound interactions with the toxin are involved in their phenotypes. As stated previously, authors should also be careful when drawing conclusions from the analysis of microbiota composition data, and changes to the manuscript should be made to reflect this. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Again, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Reviewer #2 (Public review):

I appreciate the author's responses to my original review. This is a comprehensive analysis of CAPE on C. difficile activity. It seems like this compound affects all aspects of C. difficile, which could make it effective during infection but also make it difficult to understand the mechanism. Even considering the authors responses, I think it is critical for the authors to work on the conclusions regarding the infection model. There is some protection from disease by CAPE but some parameters are not substantially changed. For instance, weight loss is not significantly different in the C. difficile only group versus the C. difficile + CAPE group. Histology analysis still shows a substantial amount of pathology in the C. difficile + CAPE group. This should be discussed more thoroughly using precise language.

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI

Strengths:

Results are really good, and the CAPE shows a good and promising alternative for treating CDI.

Weaknesses:

Some references are too old or missing.

Comments on revisions:

I have read your study after comments made by all referees, and I noticed that all questions and suggestions addressed to the authors were answered and well explained. Some of the minor and major issues related to the article were also solved. I am satisfied with all the effort given by the authors to improve their manuscript.

Reviewer #3 (Public review):

Summary:

Retroviruses have been endogenized into the genome of all vertebrate animals. The envelope protein of the virus is not well conserved and acquires many mutations hence can be used to monitor viral evolution. Since they are incorporated into the host genome, they also reflect the evolution of the hosts. In this manuscript the authors have focused their analyses to the env genes of endogenous retroviruses in primates. Important observations made include the extensive recombination events between these retroviruses that were previously unknown and the discovery of HML species in genomes prior to the splitting of old and new world monkeys.

Strengths:

They explored a number of databases and made phylogenetic trees to look at the distribution of retroviral species in primates. The authors provide a strong rationale for their study design, they provide a clear description of the techniques and the bioinformatics tools used.

Weaknesses:

The manuscript is based on bioinformatics analyses only. The reference genomes do not reflect the polymorphisms in humans or other primate species. The analyses thus likely under estimate the amount of diversity in the retroviruses. Further experimental verification will be needed to confirm the observations.

Not sure which databases were used, but if not already analyzed, ERVmap.com and repeatmesker are ones that have many ERVs that are not present in the reference genomes. Also long range sequencing of the human genome has recently become available which may also be worth studying for this purpose.

Comments on revisions:

All comments have been adequately addressed.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the drift diffusion model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options between individuals with bulimia nervosa (BN) and healthy participants.

(2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has the potential to improve the understanding of pathological food choices. The article is based on secondary research data.

Weaknesses:

I have two major concerns and a major improvement point.

The major concerns deal with the reliability of the results of the DDM (first two sections of the Results, pages 6 and 7), which are central to the manuscript, and the consistency of the results with regards to the identification of mechanisms related to binge eating in BN patients (i.e. last section of the results, page 7).

(1) Ratcliff and McKoon in 2008 used tasks involving around 1000 trials per participant. The Chen et al. experiment the authors refer to involves around 400 trials per participant. On the other hand, Shevlin and colleagues ask each participant to make two sets of 42 choices with two times fewer participants than in the Chen et al. experiment. Shevlin and colleagues also fit a DDM with additional parameters (e.g. a drift rate that varies according to subjective rating of the options) as compared to the initial version of Ratcliff and McKoon. With regards to the number of parameters estimated in the DDM within each group of participants and each emotional condition, the 5- to 10-fold ratio in the number of trials between the Shevlin and colleagues' experiment and the experiments they refer to (Ratcliff and McKoon, 2008; Chen et al. 2022) raises serious concerns about a potential overfitting of the data by the DDM. This point is not highlighted in the Discussion. Robustness and sensitivity analyses are critical in this case.

The authors compare different DDMs to show that the DDM they used to report statistical results in the main text is the best according to the WAIC criterion. This may be viewed as a robustness analysis. However, the other DDM models (i.e. M0, M1, M2 in the supplementary materials) they used to make the comparison have fewer parameters to estimate than the one they used in the main text. Fits are usually expected to follow the rule that the more there are parameters to estimate in a model, the better it fits the data. Additionally, a quick plot of the data in supplementary table S12 (i.e. WAIC as a function of the number of parameters varying by food type in the model - i.e. 0 for M0, 2 for M1, 1 for M2 and 3 for M3) suggests that models M1 and potentially M2 may be also suitable: there is a break in the improvement of WAIC between model M0 and the three other models. I would thus suggest checking how the results reported in the main text differ when using models M1 and M2 instead of M3 (for the taste and health weights when comparing M3 with M1, for τS when comparing M3 with M2). If the differences are important, the results currently reported in the main text are not very reliable.

(2) The second main concern deals with the association reported between the DDM parameters and binge eating episodes (i.e. last paragraph of the results section, page 7). The authors claim that the DDM parameters "predict" binge eating episodes (in the Abstract among other places) while the binge eating frequency does not seem to have been collected prospectively. Besides this methodological issue, the interpretation of this association is exaggerated: during the task, BN patients did not make binge-related food choices in the negative emotional state. Therefore, it is impossible to draw clear conclusions about binge eating, as other explanations seem equally plausible. For example, the results the authors report with the DDM may be a marker of a strategy of the patients to cope with food tastiness in order to make restrictive-like food choices. A comparison of the authors' results with restrictive AN patients would be of interest. Moreover, correlating results of a nearly instantaneous behavior (i.e. a couple of minutes to perform the task with the 42 food choices) with an observation made over several months (i.e. binge eating frequency collected over three months) is questionable: the negative emotional state of patients varies across the day without systematically leading patients to engage in a binge eating episode in such states.

I would suggest in such an experiment to collect the binge craving elicited by each food and the overall binge craving of patients immediately before and after the task. Correlating the DDM results with these ratings would provide more compelling results. Without these data, I would suggest removing the last paragraph of the Results.

(3) My major improvement point is to tone down as much as possible any claim of a link with binge eating across the entire manuscript and to focus more on the restrictive behavior of BN patients in between binge eating episodes (see my second major concern about the methods). Additionally, since this article is a secondary research paper and since some of the authors have already used the task with AN patients, if possible I would run the same analyses with AN patients to test whether there are differences between AN (provided they were of the restrictive subtype) and BN.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

The sample size was relatively small and may have been underpowered to find differences in outcomes (i.e., food choice behaviors). Participants were all women with BN, which limits the generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. Moreover, it is unclear how long the negative affect persisted during the actual task. It is possible that any increases in negative affect would have dissipated by the time participants were engaged in the decision-making task.

Reviewer #3 (Public review):

Summary:

The study uses the food choice task, a well-established method in eating disorder research, particularly in anorexia nervosa. However, it introduces a novel analytical approach - the diffusion decision model - to deconstruct food choices and assess the influence of negative affect on how and when tastiness and healthiness are considered in decision-making among individuals with bulimia nervosa and healthy controls.

Strengths:

The introduction provides a comprehensive review of the literature, and the study design appears robust. It incorporates separate sessions for neutral and negative affect conditions and counterbalances tastiness and healthiness ratings. The statistical methods are rigorous, employing multiple testing corrections.

A key finding - that negative affect induction biases individuals with bulimia nervosa toward prioritizing tastiness over healthiness - offers an intriguing perspective on how negative affect may drive binge eating behaviors.

Weaknesses:

A notable limitation is the absence of a sample size calculation, which, combined with the relatively small sample, may have contributed to null findings. Additionally, while the affect induction method is validated, it is less effective than alternatives such as image or film-based stimuli (Dana et al., 2020), potentially influencing the results.

Another concern is the lack of clarity regarding which specific negative emotions were elicited. This is crucial, as research suggests that certain emotions, such as guilt, are more strongly linked to binge eating than others. Furthermore, recent studies indicate that negative affect can lead to both restriction and binge eating, depending on factors like negative urgency and craving (Leenaerts et al., 2023; Wonderlich et al., 2024). The study does not address this, though it could explain why, despite the observed bias toward tastiness, negative affect did not significantly impact food choices.

Reviewer #1 (Public review):

Summary:

Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains. The findings are important, given that: (1) a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking, and (2) the study offers valuable new insights into Avpr1a expression across the mouse brain. The findings are solid, and with improved data presentation and analysis, this work could serve as an important resource for the neuroscience community.

Strengths:

This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.

Weaknesses:

A few concerns remain to be addressed. The primary weakness of this manuscript lies in the robustness of its data presentation and analysis.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, limited use of photomicrographs, and low number of subjects all combine to limit the impact of the dataset. The finding of Avp-expressing cells outside of the hypothalamus and extended amygdala is poorly documented but would be novel. The Avpr1a data suggest expression in numerous brain regions. However, the data presented are difficult to interpret, with every value being an extremely small density value for a large swath of the brain. How many cells are impacted? Are puncta spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield? For a descriptive study, there is minimal statistical inference and relatively little description. The authors make a case for the novel nature of the work but do not seem, at times, to recognize a robust literature developed over the last 50 years. In conclusion, the experimental data are important and informative; however, the low number of subjects, lack of statistical power, limited description of individual brain regions, and poor quality and design of data figures reduce the overall impact.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring the function of vasopressin in the mammalian brain and developing hypotheses about cell - and circuit-level function.

Weaknesses:

(1) The style and type of data presentation, focusing on the density of individual mRNA transcript across a whole brain region, seemed incomplete in so far as the data presentation did not provide a clear visualization of the distribution of Avpr1a-expressing cells or transcript itself. However, knowing which brain regions do express transcript is itself informative.

(2) The manuscript strongly emphases on the possibility of sex differences in Avp and Avpr1a expression. However, the low number of animals used does not provide adequate statistical power to make strong inferences regarding sex differences in the data.

(3) The manuscript's methods are minimal but adequate to understand data acquisition. The description of how quantitative analyses were conducted is inadequate and would be impossible to replicate beyond identifying the program used.

Reviewer #1 (Public review):

Summary:

The authors had previously found that brief social isolation could increase the activity of these neurons, and that manipulation of these neurons could alter social behavior in a social rank-dependent fashion. This manuscript explored which of the outputs were responsible for this, identifying the central nucleus of the amygdala as the key output region. The authors identified some discrete behavior changes associated with these outputs, and found that during photostimulation of these outputs, neuronal activity appeared altered in 'social response' neurons.

Strengths:

Rigorous analysis of the anatomy. Careful examination of the heterogenous effects on cell activity due to stimulation, linking the physiology with the behavior via photostimulation during recording in vivo.

Weaknesses:

(1) There are some clear imbalances in the sample size across the different regions parsed. The CeA has a larger sample size, likely in part to the previous work suggesting differential effects depending on social rank/dominance. Given the potential variance, it may be hard to draw conclusions about the impact of stimulation across different social ranks for other groups.

(2) It is somewhat unclear why only the 'social object ratio' was used to assess the effects versus more direct measurements of social behavior.

(3) Somewhat related, while it is statistically significant, it is unclear if the change seen in face investigation of biologically significant, on average, it looks like a few-seconds difference and that was not modulated by social rank.

(4) There are several papers studying these neurons that have explored behaviors examined here, as well as the physiological connectivity that are not cited that would provide important context for this work. In particular, multiple groups have found a dopamine-mediated IPSP in the BNST, in contrast to this work. There are technical differences that may drive these differences, but not addressing them is a major weakness.

(5) The inclusion of some markers for receptors for some of these outputs is interesting, and the authors suggest that this may be important, but this is somewhat disconnected from the rest of the work performed.

Reviewer #2 (Public review):

Summary:

The authors perform a series of studies to follow up on their previous work, which established a role for dorsal raphe dopamine neurons (DRN) in the regulation of social-isolation-induced rebound in mice. In the present study, Lee et. al, use a combination of modern circuit tools to investigate putatively distinct roles of DRN dopamine transporting containing (DAT) projections to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP). Notably, they reveal that optogenetic stimulation of distinct pathways confers specific behavioral states, with DRNDAT-BLP driving aversion, DRNDAT-BNST regulating non-social exploratory behavior, and DRNDAT-CeA promoting social ability. A combination of electrophysiological studies and in situ hybridization studies reveal heterogenous dopamine and neuropeptide expression and different firing properties, providing further evidence of pathway-specific neural properties. Lastly, the authors combine optogenetics and calcium imaging to resolve social encoding properties in the DRNDAT-CeA pathway, which correlates observed social behavior to socially engaged neural ensembles.

Collectively, these studies provide an interesting way of dissecting out separable features of a complex multifaceted social-emotional state that accompanies social isolation and the perception of 'loneliness.' The main conclusions of the paper provide an important and interesting set of findings that increase our understanding of these distinct DRN projections and their role in a range of social (e.g., prosocial, dominance), non-social, and emotional behaviors. However, as noted below, the examination of these circuits within a homeostatic framework is limited given that a number of the datasets did not include an isolated condition. The DRNDAT-CeA pathway was investigated with respect to social homeostatic states in the present study for some of the datasets.

Strengths:

(1) The authors perform a comprehensive and elegant dissection of the anatomical, behavioral, molecular, and physiological properties of distinct DRN projections relevant to social, non-social, and emotional behavior, to address multifaceted and complex features of social state.

(2) This work builds on prior findings of isolation-induced changes in DRN neurons and provides a working framework for broader circuit elements that can be addressed across the social homeostatic state.

(3) This work characterizes a broader circuit implicated in social isolation and provides a number of downstream targets to explore, setting a nice foundation for future investigation.

(4) The studies account for social rank and anxiety-like behavior in several of the datasets, which are an important consideration to the interpretation of social motivation states, especially in male mice with respect to dominance behavior.

Weaknesses:

(1) The conceptual framework of the study is based on the premise of social isolation and perceived 'loneliness' under the framework of social homeostasis, analogous to hunger. In this framework, social isolation should provoke an aversive state and compensatory social contact behavior. In the authors' prior work, they demonstrate synaptic changes in DRN neurons and social rebound following acute social isolation. Thus, the prediction would be that downstream projections also would show state-dependent changes as a function of social housing conditions (e.g., grouped vs. isolated). In the current paper, a social isolation condition was not included for the majority of the studies conducted (e.g., Figures 1-6 do not include an isolated condition, Figures 7-8 do include an isolated condition). Thus, while Figure 1-6 adds a very interesting and compelling set of data that is of high value to the social behavior field with respect to social and emotional processing and general circuit characterization, these studies do not directly investigate the impacts of dynamic social homeostatic state. The main claim of the paper, including the title (e.g., separable DRN projections mediate facets of loneliness-like state), abstract, intro, and discussion presents the claim of this work under the framework of dynamic social homeostatic states, which should be interpreted with caution, as the majority of the work in the paper did not include a social isolation comparison.

(2) In Figure 1, the authors confirm co-laterals in the BNST and CeA via anatomical tracing studies. The goal of the optogenetic studies is to dissociate the functional/behavioral roles of distinct projections. However, one limitation of optogenetic projection targeting is the possibility of back-propagating action potentials (stimulation of terminals in one region may back-propagate to activate cell bodies, and then afferent projections to other regions), and/or stimulation of fibers of passage. Therefore, one limitation in the dataset for the optogenetic stimulation studies is the possibility of non-specific unintended activation of projections other than those intended (e.g., DRNDAT-CeA). This can be dealt with by administering lidocaine to prevent back-propagating action potentials.

(3) It is unclear from the test, but in the subjects' section of the methods, it appears that only male animals were included in the study, with no mention of female subjects. It should be clear to the reader that this was conducted in males only if that is the case, with consideration or discussion, about female subjects and sex as a biological variable.

(4) Averaged data are generally reported throughout the study in the form of bar graphs, across most figures. Individual data points would increase the transparency of the data.

Reviewer #3 (Public review):

Summary:

The authors investigated the role of dopaminergic neurons (dopamine transporter expressing, DAT) in the dorsal raphe nucleus (DRN) in regulating social and affective behavior through projections to the central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and the posterior subdivision of the basolateral amygdala. The largest effect observed was in the DRN-DAT projections to the CeA. Augmenting previously published results from this group (Matthews et al., 2016), the comprehensive behavioral analysis relative to social dominance, gene expression analysis, electrophysiological profiling, and in vivo imaging provides novel insights into how DRN-DAT projections to the CeA influence the engagement of social behavior in the contexts of group-housed and socially isolated mice.

Strengths:

Correlational analysis with social dominance is a nice addition to the study. The overall computational analyses performed are well-designed and rigorous.

Weaknesses:

(1) Analysis of dopamine receptor expression did not include Drd3, Drd4, or Drd5 which may provide more insights into how dopamine modulates downstream targets. This is particularly relevant to the BNST projection in which the densest innervation did not robustly co-localize with the expression of either Drd1 or Drd2. It is also possible that dopamine release from DRN-DAT neurons in any or all of these structures modulates neurotransmitter release from inputs to these regions that contain D2 receptors on their terminals.

(2) Although not the focus of this study, without pharmacological blockade of dopamine receptors, it is not possible to assess what the contribution of dopamine is to the behavioral outcomes. Given the co-release of glutamate and GABA from these neurons, it is possible that dopamine plays only a marginal role in the functional connectivity of DRN-DAT neurons. (

(3) Photostimulation parameters used during the behavioral studies (8 pulses of light delivered at 30 Hz for several minutes) could lead to confounding results limiting data interpretation. As shown in Figure 6J, 8 pulses of light delivered at 30 Hz result in a significant attenuation of the EPSC amplitude in the BLP and CeA projection. Thus, prolonged stimulation could lead to significant synaptic rundown resulting in an overall suppression of connectivity in the later stages of the behavioral analyses.

Reviewer #1 (Public review):

The authors investigated tactile spatial perception on the breast through discrimination, categorization, and direct localization tasks. They reach three main conclusions:

(1) The breast has poor tactile spatial resolution.<br /> This conclusion is based on comparing just noticeable differences, a marker of tactile spatial resolution, across four body regions, two on the breast. The data compellingly support the conclusion; the study outshines other studies on tactile spatial resolution that tend to use problematic measures of tactile resolution such as two-point-discrimination thresholds. The result will interest researchers in the field and possibly in other fields due to the intriguing tension between the finding and the sexually arousing function of touching the breast.

(2) Larger breasts are associated with lower tactile spatial resolution<br /> This conclusion is based on a strong correlation between participants' JNDs and the size of their breasts. The correlation convincingly supports the conclusion. It is of interest to the field, as it aligns with the hypothesis that nerve fibers are more sparsely distributed across larger body parts.

(3) The nipple is a landmark: perceptually a unit and an attractor for tactile percepts<br /> The data do not support these conclusions. The conclusion that the nipple is perceived as a unit is based on poor performance in tactile categorization for touches on the nipple. This categorization performance may simply mirror the breast's low tactile spatial resolution with JNDs about the size of a nipple.

The conclusion that tactile percepts are drawn towards the nipple is based on tactile localization biases towards the nipple for tactile stimuli on the breast compared to localization biases for tactile stimuli on the back. Currently, the statistical analysis of the data does not match the field, psychophysics, standards. Moreover, any bias towards the nipple could simply be another instance of regression to the mean of the stimulus distribution, given that the tested locations were centered on the nipple. This confound can only be experimentally solved by shifting the distribution of the tested locations. Finally, given that participants indicated the locations on a 3D model of the body part, further experimentation would be required to determine whether there is a perceptual bias towards the nipple or whether the authors merely find a response bias.

Further comments:

- Given that later analyses require regression models, the authors might consider using them throughout.

- The stability of the JND differences between body parts across subjects is already captured in the analysis of the JNDs; the ANOVA and the post-hoc testing would not be significant if the order were not relatively stable across participants. Thus, it is unclear why this is being evaluated again with reduced power due to improper statistics.

- The null hypothesis of an ANOVA is that at least one of the mean values is different from the others; adding participants as a factor does not provide evidence for similarity.

- The pairwise correlations between body parts seem to be exploratory in nature. Like all exploratory analyses, the question arises of how much potential extra insights outweigh the risk of false positives. It would be hard to generate data with significant differences between several conditions and not find any correlations between pairs of conditions. Thus, the a priori chance of finding a significant correlation is much higher than what a correction accounts for.

- If the JND at mid breast (measured with locations centered at the nipple) is roughly the same size as the nipple, it is not surprising that participants have difficulty with the categorical localization task on the nipple but perform better than chance on the significantly larger areola.

- To justify the conclusion that the nipple is a unit, additional data would be required. 1) One could compare psychometric curves with the nipple as the center and psychometric curves with a nearby point on the areola as the center. 2) Performance in the quadrant task could be compared for the nipple and an equally sized portion of the areola. Otherwise, the task "only" provides confirmatory evidence for a low tactile resolution in the midbreast area.

- A localization bias toward the nipple in this context does not show that the nipple is the anchor of the breast's tactile coordinate system. The result might simply be an instance of regression to the mean of the stimulus distribution (also known as experimental prior). To convincingly show localization biases towards the nipple, the tested locations should be centered at another location on the breast.

- Another problem is the visual salience of the nipple, even though Blender models were uniformly grey. With this type of direct localization, it is very difficult to distinguish perceptual from response biases even if the regression to the mean problem is solved. There are two solutions to this problem: 1) Varying the uncertainty of the tactile spatial information, for example, by using a pen that exerts lighter pressure. A perceptual bias should be stronger for more uncertain sensory information; a response bias should be the same across conditions. 2) Measure bias with a 2IFC procedure by taking advantage of the fact that sensory information is noisier if the test is presented before the standard.

- Neither signed nor absolute localization error can be compared to the results of the previous experiments. The JND should be roughly proportional to the variance of the errors.

- The statistically adequate way of testing the biases is a hierarchical regression model (LMM) with a distance of the physical location from the nipple as a predictor, and a distance of the reported location from the nipple as a dependent variable. Either variable can be unsigned or signed for greater power, for example, coding the lateral breast as negative and the medial breast as positive. The bias will show in regression coefficients smaller than 1.

- It does not matter whether distances are calculated based on skin or 3D coordinates, as Euclidean distances or based on polar coordinates. However, there should only be one consistent distance in the text across both independent and dependent variables. Calculating various versions of these measures can create issues in Frequentist Statistics. For transparency, it is good practice to report the results of other methods for calculating the distance in the supplement.

- The body part could be added as a predictor to the LMM, with differences in bias between the body parts showing a significant interaction between the two predictors. The figures suggest such an effect. However, the interpretation should take into account that 1) response biases are more likely to arise at the breast and 2) it might be harder to learn the range of locations on the back given that stimulation is not restricted to an anatomically defined region as it is the case for the breast.

Reviewer #2 (Public review):

The authors tested tactile acuity on the breast of females using several tasks and reported overall low acuity compared to the back, which is typically considered to have the worst acuity of all body parts. Moreover, there was evidence that acuity is worse the larger the breast; this finding mirrors similar findings for the hand and therefore suggests that the number of tactile sensors is fixed and must be distributed across a larger extent of skin when a body part is larger, thus resulting in comparably lower tactile acuity.

Strengths:

I find this an interesting paper with results that are relevant to the tactile community. The authors apply several tasks allowing them to link the paper with previous results. The methodology and psychophysical analysis are sound.

Weaknesses:

The analysis of localization error direction, with the result that the nipple area may be a landmark for tactile localization, is interesting and aligns the paper with some other recent papers that have suggested that such landmarks should exist. However, there are major issues with methodology and statistics, so that currently the conclusions are not supported.

In the following, line numbers refer to the re-formatted manuscript provided by the authors upon request and are mentioned for them to find the relevant passages faster.

(1) Comments on analysis of tactile acuity:

- I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

- It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

- It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

- Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

(2) Comments on analysis of "The nipple is a unit":

- Statistics in this section are not adequately described and may be partly false.

- In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant t-test is an indication of "barely above chance".

- The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

(3) Comments on analysis of "Absolute localization on the breast is anchored to the nipple"

- Again, there are things that are unclear with the statistics and description of the analysis.

- This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

- The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

- p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

- Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

(4) Comments on the Discussion:

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Additional comments:

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.

Reviewer #1 (Public review):

Summary:

The study explores the use of Transport-based morphometry (TBM) to predict hematoma expansion and growth 24 hours post-event, leveraging Non-Contrast Computed Tomography (NCCT) scans combined with clinical and location-based information. The research holds significant clinical potential, as it could enable early intervention for patients at high risk of hematoma expansion, thereby improving outcomes. The study is well-structured, with detailed methodological descriptions and a clear presentation of results. However, the practical utility of the predictive tool requires further validation, as the current findings are based on retrospective data. Additionally, the impact of this tool on clinical decision-making and patient outcomes needs to be further investigated.

Strengths

(1) Clinical Relevance: The study addresses a critical need in clinical practice by providing a tool that could enhance diagnostic accuracy and guide early interventions, potentially improving patient outcomes.

(2) Feature Visualization: The visualization and interpretation of features associated with hematoma expansion risk are highly valuable for clinicians, aiding in the understanding of model-derived insights and facilitating clinical application.

(3) Methodological Rigor: The study provides a thorough description of methods, results, and discussions, ensuring transparency and reproducibility.

Weaknesses:

(1) The limited sample size in this study raises concerns about potential model overfitting. While the reported AUCROC of 0.71 may be acceptable for clinical use, the robustness of the model could be further enhanced by employing techniques such as k-fold cross-validation. This approach, which aggregates predictive results across multiple folds, mimics the consensus of diagnoses from multiple clinicians and could improve the model's reliability for clinical application. Additionally, in clinical practice, the utility of the model may depend on specific conditions, such as achieving high specificity to identify patients at risk of hematoma expansion, thereby enabling timely interventions. Consequently, while AUC is a commonly used metric, it may not fully capture the model's clinical applicability. The authors should consider discussing alternative performance metrics, such as specificity and sensitivity, which are more aligned with clinical needs. Furthermore, evaluating the model's performance in real-world clinical scenarios would provide valuable insights into its practical utility and potential impact on patient outcomes.

(2) The authors compared the performance of TBM with clinical and location-based information, as well as other machine learning methods. While this comparison highlights the relative strengths of TBM, the study would benefit from providing concrete evidence on how this tool could enhance clinicians' ability to assess hematoma expansion in practice. For instance, it remains unclear whether integrating the model's output with a clinician's own assessment would lead to improved diagnostic accuracy or decision-making. Investigating this aspect-such as through studies evaluating the combined performance of clinician judgment and model predictions-could significantly enhance the tool's practical value.

Reviewer #2 (Public review):

Summary:

The author presents a transport-based morphometry (TBM) approach for the discovery of non-contrast computed tomography (NCCT) markers of hematoma expansion risk in spontaneous intracerebral hemorrhage (ICH) patients. The findings demonstrate that TBM can quantify hematoma morphological features and outperforms existing clinical scoring systems in predicting 24-hour hematoma expansion. In addition, the inversion model can visualize features, which makes it interpretable. In conclusion, this research has clinical potential for ICH risk stratification, improving the precision of early interventions.

Strengths:

TBM quantifies hematoma morphological changes using the Wasserstein distance, which has a well-defined physical meaning. It identifies features that are difficult to detect through conventional visual inspection (such as peripheral density distribution and density heterogeneity), which provides evidence supporting the "avalanche effect" hypothesis in hematoma expansion pathophysiology.

Weaknesses:

(1) As a methodology-focused study, the description of the methods section somewhat lacks depth and focus, which may make it challenging for readers to fully grasp the overall structure and workflow of the approach. For instance, the manuscript lacks a systematic overview of the entire process, from NCCT image input to the final prediction output. A potential improvement would be to include a workflow figure at the beginning of the manuscript, summarizing the proposed method and subsequent analytical procedures. This would help readers better understand the mechanism of the model.

(2) The description of the comparison algorithms could be more detailed. Since TBM directly utilizes NCCT images as input for prediction, while SVM and K-means are not inherently designed to process raw imaging data, it would be beneficial to clarify which specific features or input data were used for these comparison models. This would better highlight the effectiveness and advantages of the TBM method.

(3) The relatively small training and testing dataset may limit the model's performance and generalizability. Notably, while the study mentions that 1,066 patients from the ERICH dataset met the inclusion criteria, only 170 were randomly selected for the test set. Leveraging the full 1,066 ERICH cases for model training and internal validation might potentially enhance the model's robustness and performance.

(4) Some minor textual issues need to be checked and corrected, such as line 16 in the abstract "Incorporating these traits into a v achieved an AUROC of 0.71 ...".

(5) Some figures need to be reformatted (e.g., the x-axis in Figure 2 a is blocked).

Reviewer #1 (Public review):

Summary:

Govindan and Conrad use a genome-wide CRISPR screen to identify genes regulating retention of intron 4 in OGT, leveraging an intron retention reporter system previously described (PMID: 35895270). Their OGT intron 4 reporter reliably responds to O-GlcNAc levels, mirroring the endogenous splicing event. Through a genome-wide CRISPR knockout library, they uncover a range of splicing-related genes, including multiple core spliceosome components, acting as negative regulators of OGT intron 4 retention. They choose to follow up on SFSWAP, a largely understudied splicing regulator shown to undergo rapid phosphorylation in response to O-GlcNAc level changes (PMID: 32329777). RNA-sequencing reveals that SFSWAP depletion not only promotes OGT intron 4 splicing but also broadly induces exon inclusion and intron splicing, affecting decoy exon usage. While this study offers interesting insights into intron retention regulation and O-GlcNAc signaling, the RNA-Sequencing experiments lack essential controls needed to provide full confidence to the authors' conclusions.

Strengths:

(1) This study presents an elegant genetic screening approach to identify regulators of intron retention, uncovering core spliceosome genes as unexpected positive regulators of intron retention.<br /> (2) The work proposes a novel functional role for SFSWAP in splicing regulation, suggesting that it acts as a negative regulator of splicing and cassette exon inclusion, which contrasts with expected SR-related protein functions.<br /> (3) The authors suggest an intriguing model where SFSWAP, along with other spliceosome proteins, promotes intron retention by associating with decoy exons.

Weaknesses:

(1) The conclusions regarding SFSWAP's impact on alternative splicing rely on cells treated with a single pool of two siRNAs for five days. The absence of independent siRNA treatments raises concerns about potential off-target effects, which may reduce confidence in the observed SFSWAP-dependent splicing changes. Rescue experiments or using additional independent siRNA treatments would strengthen the conclusions.<br /> (2) The mechanistic role of SFSWAP in splicing would benefit from further exploration, though this may be more appropriate for future studies.

Comments on revisions:

The authors have addressed all my previous recommendations.

Reviewer #2 (Public review):

Summary:

The paper describes an effort to identify the factors responsible for intron retention and alternate exon splicing in a complex system known to be regulated by the O-GlcNAc cycling system. The CRISPR/Cas9 system was used to identify potential factors. The bioinformatic analysis is sophisticated and compelling. The conclusions are of general interest and advance the field significantly.

Strengths:

- Exhaustive analysis of potential splicing factors in an unbiased screen.<br /> - Extensive genome wide bioinformatic analysis.<br /> - Thoughtful discussion and literature survey

Weaknesses:

- No firm evidence linking SFSWA to an O-GlcNAc specific mechanism.<br /> - Resulting model leaves many unanswered questions.

Comments on revisions:

I think the authors have adequately dealt with the overall reviewer's comments.

Reviewer #3 (Public review):

Summary:

The major novel finding in this study is that SFSWAP, a splicing factor containing an RS domain but no canonical RNA binding domain, functions as a negative regulator of splicing. More specifically, it promotes retention of specific introns in a wide variety of transcripts including transcripts from the OGT gene previously studied by the Conrad lab. The balance between OGT intron retention and OGT complete splicing is an important regulator of O-GlcNAc expression levels in cells.

Strengths:

An elegant CRISPR knockout screen employed a GFP reporter, in which GFP is efficiently expressed only when the OGT retained intron is removed (so that the transcript will be exported from the nucleus to allow for translation of GFP). Factors whose CRISPR knockdown cause decreased intron retention therefore increase GFP, and these can be identified by sequencing RNA of GFP-sorted cells. SFSWAP was thus convincingly identified as a negative regulator of OGT retained intron splicing. More focused studies of OGT intron retention indicate that it may function by regulating a decoy exon previously identified in the intron, and that this may extend to other transcripts with decoy exons.

Weaknesses:<br /> The mechanism by which SFSWAP represses retained introns is unclear, although some data suggests it can operate (in OGT) at the level of a recently reported decoy exon within that intron. Interesting / appropriate speculation about possible mechanism are provided and will likely be the subject of future studies.

Overall the study is well done and carefully described.

Reviewer #1 (Public review):

Summary:

This study has preliminarily revealed the role of ACVR2A in trophoblast cell function, including its effects on migration, invasion, proliferation, and clonal formation, as well as its downstream signaling pathways.

Strengths:

The use of multiple experimental techniques, such as CRISPR/Cas9-mediated gene knockout, RNA-seq, and functional assays (e.g., Transwell, colony formation, and scratch assays), is commendable and demonstrates the authors' effort to elucidate the molecular mechanisms underlying ACVR2A's regulation of trophoblast function. The RNA-seq analysis and subsequent GSEA findings offer valuable insights into the pathways affected by ACVR2A knockout, particularly the Wnt and TCF7/c-JUN signaling pathways.

Weaknesses:

The current findings provide valuable insights into the role of ACVR2A in trophoblast cell function and its involvement in the regulation of migration, invasion, and proliferation, further validation in both in vitro and in vivo models would strengthen the conclusions. Additional techniques, such as animal models and more advanced clinical sample analyses, would help strengthen the conclusions and provide a more comprehensive understanding of the molecular pathways involved.

Reviewer #2 (Public review):

Summary:

ACVR2A is one of a handful of genes for which significant correlations between associated SNPs and the incidences of preeclampsia have been found in multiple populations. It is one of the TGFB family receptors, and multiple ligands of ACVR2A, as well as its coreceptors and related inhibitors, have been implicated in placental development, trophoblast invasion, and embryo implantation. This useful study builds on this knowledge by showing that ACVR2A knockout in trophoblast-related cell lines reduces trophoblast invasion, which could tie together many of these observations. The implication of cross-talk between the WNT and ACRV2A/SMAD2 pathways is an important contribution to the understanding of the regulation of trophoblast function.

Strengths:

(1) ACVR2A is one of very few genes implicated in preeclampsia in multiple human populations, yet its role in pathogenesis is not very well studied and this study begins to address that hole in our knowledge.

(2) ACVR2A is also indirectly implicated in trophoblast invasion and trophoblast development via its connections to many ligands, inhibitors, and coreceptors, suggesting its potential importance.

(3) The authors have used multiple cell lines to verify their most important observations.

Editors' note: Following the first round of peer review, the original reviewers were not available to review the revised manuscript. As several specific weakness detailed by the reviewers were largely addressed in the revised manuscript, they are not included here.

Reviewer #1 (Public review):

Lu et. al. proposed here a direct role of LPS in inducing hepatic fat accumulation and that metabolism of LPS therefore can mitigate fatty liver injury. With an Acyloxyacyl hydrolase whole-body KO mice, they demonstrated that Acyloxyacyl hydrolase deletion resulted in higher hepatic fat accumulation over 7 months of high glucose/high fructose diet. Previous literature has found that hepatocyte TLR4 (which is a main receptor for binding LPS) KO reduced fatty liver in MAFLD model, and this paper complement this by showing that degradation/metabolism of LPS can also reduce fatty liver. Using clodronate-liposomes to deplete KC, the authors went on to show that AOAH level decreased significantly with increased SREBP1 level, suggesting that KCs were the major source of AOAH in the liver. To explain the mechanism of LPS induced lipogenesis, the authors demostrated in vitro that LPS alone without KC can induce SREBP1 level in primary hepatocytes via mTOR activation. This result proposed a very interesting mechanism, and the translational implications of utilizing Acyloxyacyl hydrolase to decrease LPS exposure is intriguing.

The strengths of the present study include that they raised a very simplistic mechanism with LPS that is of interest in many diseases. The phenotype shown in the study is strong. The mechanism proposed by the findings are generally well supported. Manuscript significantly improved with revision. Overall, this work adds to the current understanding of the gut-liver axis and development of MAFLD, and will be of interest to many readers.

Reviewer #2 (Public review):

The authors of this article investigated the impact of the host enzyme AOAH on the progression of MASLD in mice. To achieve this, they utilized whole-body Aoah-/- mice. The authors demonstrated that AOAH reduced LPS-induced lipid accumulation in the liver, probably by decreasing the expression and activation of SREBP1. In addition, AOAH reduced hepatic inflammation and minimized tissue damage.

The authors have effectively addressed some key questions I raised. However, I still have some lingering concerns regarding the mechanisms underlying AOAH's effects.

(1) AOAH is expressed in the intestine, where it may inactivate LPS before it enters systemic circulation. In Fig. 3F, fecal LPS is significantly higher in Aoah⁻/⁻ mice compared to Aoah⁺/⁺ mice, indicating that AOAH in the intestine reduces bioactive LPS levels at the source. This implies that differences in hepatic LPS levels are already influenced by the gut environment, raising doubts about how much Kupffer cells contribute to inactivating LPS in the liver.

(2) The reliance on Kupffer cell depletion with clodronate-liposomes may overestimate the role of Kupffer cells because clodronate does not exclusively target hepatic Kupffer cells. Clodronate liposomes are taken up by macrophages systemically, potentially depleting macrophages in other organs, including the intestine and circulation. This means observed effects could also be due to loss of AOAH activity in non-hepatic macrophages.

Reviewer #3 (Public review):

Summary:

Chen et al. present a thorough statistical analysis of social interactions, more precisely, co-occupying the same chamber in the Eco-HAB measurement system. They also test the effect of manipulating the prelimbic cortex by using TIMP-1 that inhibits the MMP-9 matrix metalloproteinase. They conclude that altering neural plasticity in the prelimbic cortex does not eliminate social interactions, but it strongly impacts social information transmission.

Strengths:

The quantitative approach to analyzing social interactions is laudable and the study is interesting. It demonstrates that the Eco-HAB can be used for high throughput, standardized and automated tests of the effects of brain manipulations on social structure in large groups of mice.

Weaknesses:

A demonstration of TIMP-1 impairing neural plasticity specifically in the prelimbic cortex of the treated animals would greatly strengthen the biological conclusions. The Eco-HAB provides coarser spatial information compared to some other approaches, which may influence the conclusions.

Reviewer #1 (Public review):

Summary:

In this report, the authors made use of a murine cell line derived from a MYC-driven liver cancer to investigate the gene expression changes that accompany the switch from normoxic to hypoxia conditions during 2D growth and the switch from 2D monolayer to 3D organoid growth under normoxic conditions. They find a significant (ca. 40-50%) overlap among the genes that are dysregulated in response to hypoxia in 2D cultures and in response to spheroid formation. Unsurprisingly, hypoxia-related genes were among the most prominently deregulated under both sets of conditions. Many other pathways pertaining to metabolism, splicing, mitochondrial electron transport chain structure and function, DNA damage recognition/repair and lipid biosynthesis were also identified.

Comments on the revised manuscript:

In my original review of this manuscript, I raised 11 points that I thought needed to be addressed and/or clarified by the authors. In response, they have provided an adequate answer to only one of these (point 6), which is little more than a more thorough description of how spheroids were generated. The remaining points that I raised, which would have provided more mechanistic insight into their study were addressed by the authors with the following such comments:

- It is not the focus of this study (Points 1 and 4)

- It is worthy of further validation (Point 2)

- We apologize for not being able to validate everything (Point 3)

- This reviewer has raised an interesting question. We are investigating this hypothesis and hopefully we can give a clear answer in the future (Point 5)

- This is an excellent idea that we certainly will do it in our future experiments (Point 7)

As to responses that the authors made to the other two reviewers' comments: Most pertained to cosmetic alterations involving clarification of methods, inclusion of a new figure or rearrangement of old figures. These were generally answered. However, in response to the last point raised by Rev. 3 to compare "sgRNA abundances at the earliest harvesting time with the distribution in the library...to see whether and to what extent selection has already taken place before the three culture conditions were established", the authors responded with the comment: "This is great point. Unfortunately, we did not perform such an analysis."

I understand that it is often impossible to address all points raised by the reviewers. This can be for a variety of reasons and many times the omissions can be overlooked and accepted if the reviewer can be convinced that a good faith attempt has otherwise been made to address the other deficiencies. However, no such effort has been made here and the study remains deficient and largely descriptive as I pointed out in my original review.

Reviewer #1 (Public review):

Summary:

The authors address the role of the centromere histone core in force transduction by the kinetochore

Strengths:

They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).

Weaknesses:

The main results are shown in one figure (Fig 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that the impact is fairly limited in scope.

Comments on revisions:

The additional information provided by the authors will help the reader understand and interpret the manuscript.

Reviewer #2 (Public review):

Summary:

This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (https://doi.org/10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.

In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.

Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.

In the future, it will be interesting to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.

Strength:

- The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.

Weakness:

- How centromeric DNA exerts this effect remains unclear.

Comments on revisions:

I appreciate the authors' detailed response and their decision to list all the tested in chimeras in Table 3.

All my prior comments have been addressed.

Reviewer #1 (Public review):

The authors set out to analyse the roles of the teichoic acids of Streptococcus pneumoniae in supporting the maintenance of the periplasmic region. Previous work has proposed the periplasm to be present in Gram positive bacteria and here advanced electron microscopy approach was used. This also showed a likely role for both wall and lipo-teichoic acids in maintaining the periplasm. Next, the authors use a metabolic labelling approach to analyse the teichoic acids. This is a clear strength as this method cannot be used for most other well studied organisms. The labelling was coupled with super-resolution microscopy to be able to map the teichoic acids at the subcellular level and a series of gel separation experiments to unravel the nature of the teichoic acids and the contribution of genes previously proposed to be required for their display. The manuscript could be an important addition to the field but there are a number of technical issues which somewhat undermine the conclusions drawn at the moment. These are shown below and should be addressed. More minor points are covered in the private

Recommendations for Authors.

Weaknesses to be addressed:

(1) l. 144 Was there really only one sample that gave this resolution? Biological repeats of all experiments are required.

(2) Fig. 4A. Is the pellet recovered at "low" speeds not just some of the membrane that would sediment at this speed with or without LTA? Can a control be done using an integral membrane protein and Western Blot? Using the tacL mutant would show the behaviour of membranes alone.

(3) Fig. 4A. Using enzymatic digestion of the cell wall and then sedimentation will allow cell wall associated proteins (and other material) to become bound to the membranes and potentially effect sedimentation properties. This is what is in fact suggested by the authors (l. 1000, Fig. S6). In order to determine if the sedimentation properties observed are due to an artefact of the lysis conditions a physical breakage of the cells, using a French Press, should be carried out and then membranes purified by differential centrifugation. This is a standard, and well-established method (low-speed to remove debris and high-speed to sediment membranes) that has been used for S. pneumoniae over many years but would seem counter to the results in the current manuscript (for instance Hakenbeck, R. and Kohiyama, M. (1982), Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae. European Journal of Biochemistry, 127: 231-236).

(4) l. 303-305. The authors suggest that the observed LTA-like bands disappear in a pulse chase experiment (Fig. 6B). What is the difference between this and Fig. 5B, where the bands do not disappear? Fig. 5C is the WT and was only pulse labelled for 5 min and so would one not expect the LTA-like bands to disappear as in 6B?

(5) Fig. 6B, l. 243-269 and l. 398-410. If, as stated, most of the LTA-like bands are actually precursor then how can the quantification of LTA stand as stated in the text? The "Titration of Cellular TA" section should be re-evaluated or removed? If you compare Fig. 6C WT extract incubated at RT and 110oC it seems like a large decrease in amount of material at the higher temperature. Thus, the WT has a lot of precursors in the membrane? This needs to be quantified.

(6) L. 339-351, Fig. 6A. A single lane on a gel is not very convincing as to the role of LytR. Here, and throughout the manuscript, wherever statements concerning levels of material are made, quantification needs to be done over appropriate numbers of repeats and with densitometry data shown in SI.

(7) 14. l. 385-391. Contrary to the statement in the text, the zwitterionic TA will have associated counterions that results in net neutrality. It will just have both -ve and +ve counterions in equal amounts (dependent on their valency), which doesn't matter if it is doing the job of balancing osmolarity (rather than charge).

Comments on revisions:

The resubmitted manuscript now contains new data and changes to the text.

The authors have largely covered my previous points in both sets of reviews (Public/Recommendations).

Public Review Points:

1 & 6: I still do not see a reproducibility statement as such, with details of the number of biological repeats etc.

2 & 3. Fig S7 seems to be quite telling. As predicted after physical breakage the membrane proteins sediment at high speed (rather than low speed). This presumably also means that the LTA comes down at high and not low speed. LTA was not measured due to cost of reagents. The Microfluidizer breaks the cells using a shear force and thus is unlikely to create very small membrane fragments. Thus, the sedimentation properties of membranes containing LTA are likely dependent on the way in which the cells are lysed. It is therefore worthwhile qualifying the statements on l. 35-36, 46-47 and 212 (as Ref 8 used mechanical breakage). This will give better direction to those in the field following up the findings.

It is also a little alarming that the mutanolysin is contaminated by protease and one hopes this does not affect any of the properties of the materials being analysed.

Reviewer #2 (Public review):

The Gram-positive cell wall contains for a large part of TAs, and is essential for most bacteria. However, TA biosynthesis and regulation is highly understudied because of the difficulties in working with these molecules. This study closes some of our important knowledge gaps related to this and provides new and improved methods to study TAs. It also shows an interesting role for TAs in maintaining a 'periplasmic space' in Gram positives. Overall, this is an important piece of work. Future work will need to address the possible causal link between TAs and periplasmic space, for instance using complemented mutants and CEMOVIS. It will be interesting to see what happens with the periplasmic space in other mutants besides TA or also in strains with capsules/without capsules and in PG mutants, or in lafB (essential for production of another glycolipid) mutants. Overall, I support the publication of this revised work as it pioneers some new methods that will definitively move the field forward.

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study characterizes the role of sulfakinin and the sulfakinin receptor 1 in changes in olfactory responses associated with foraging versus mating behavior in the oriental fruit fly (Bactrocera dorsalis), a significant agricultural pest. This pathway regulates food consumption and mating receptivity in other species; here the authors use genetic disruption of sulfakinin and sulfakinin receptor 1 to provide strong evidence that changes in sulfakinin signaling modulate antennal responses to food versus pheromonal cues and alter the expression of ORs that detect relevant stimuli.

Strengths:

The authors utilize multiple complementary approaches including CRISPR/Cas9 mutagenesis, behavioral characterization, electroantennograms, RNA sequencing and heterologous expression to convincingly demonstrate the involvement of the sulfakinin pathway in the switch between foraging and mating behaviors. The use of both sulfakinin peptide and receptor mutants is a strength of the study and implicates specific signaling actors.

Weaknesses:

The authors demonstrate that SKR is expressed in olfactory neurons, however there are additional potential sites of action that may contribute to these results.

Reviewer #1 (Public review):

Summary:

This research article by Nath et al. from the Lee Lab addresses how lipolysis under starvation is achieved by a transient receptor potential channel, TRPγ, in the neuroendocrine neurons to help animals survive prolonged starvation. Through a series of genetic analyses, the authors identify that trpγ mutations specifically lead to a failure in lipolytic processes under starvation, thereby reducing animals' starvation resistance. The conclusion was confirmed through total triacylglycerol levels in the animals and lipid droplet staining in the fat bodies. This study highlights the importance of transient receptor potential (TRP) channels in the fly brain to modulate energy homeostasis and combat metabolic stress. However, the co-expression of trpγ and Dh44-R2 in the gut is not convincing, especially in the picture of the arrows pointing at the autofluorescence signals in the gut (Figure 7P). Therefore, the authors should either confirm the co-expression or acknowledge that trpγ and Dh44-R2 are not co-expressed in the gut and modify their model in Figure 8 accordingly, although clarifying their co-expression may not change the main conclusions of this study. Overall, the revised version includes the required clarifications on their important results that strengthen the interpretations of the research as well as the visibility of this study.

Strengths:

This study identifies the biological meaning of TRPγ in promoting lipolysis during starvation, advancing our knowledge about the TRPγ channel and the neural mechanisms to combat metabolic stress. Furthermore, this study demonstrates the potential of the TRPγ channel as a target to develop new therapeutic strategies for human metabolic disorders by showing that metformin and AMPK pathways are involved in its function in lipid metabolisms during starvation in Drosophila.

Reviewer #2 (Public review):

Summary

In this paper, the function of trpγ in lipid metabolism was investigated. The authors found that lipid accumulation levels were increased in trpγ mutants and remained high during starvation; the increased TAG levels in trpγ mutants were restored by the expression of active AMPK in DH44 neurons and oral administration of the anti-diabetic drug metformin. Furthermore, oral administration of lipase, TAG and free fatty acids effectively restored survival of trpγ mutants under starvation conditions. These results indicate that TRPv plays an important role in the maintenance of systemic lipid levels through the proper expression of lipase. Furthermore, authors have shown that this function is mediated by DH44R2. This study provides an interesting finding in that the neuropeptide DH44 released from the brain regulates lipid metabolism through a brain-gut axis, acting on the receptor DH44R2 expressed in gut cells.

Strengths

Using Drosophila genetics, careful analysis of which cells express trpγ regulates lipid metabolism is performed in this study. The study supports its conclusions from various angles, including not only TAG levels, but also fat droplet staining and survival rate under starved conditions, and oral administration of substances involved in lipid metabolism.

Weaknesses

The function of lipases, as well as identification of cell types, in the DH44R2-expressing cells in the gut can be investigated.

Reviewer #3 (Public review):

In this manuscript, the authors demonstrated the significance of the TRPγ channel in regulating internal TAG levels. They found high TAG levels in TRPγ mutant, which was ascribed to a deficit in the lipolysis process due to the downregulation of brummer (bmm). It was notable that the expression of TRPγ in DH44+ PI neurons, but not dILP2+ neurons, in the brain restored the internal TAG levels and that the knockdown of TRPγ in DH44+ PI neurons resulted in an increase in TAG levels. These results suggested a non-cell autonomous effect of Dh44+PI neurons. Additionally, the expression of the TRPγ channel in Dh44 R2-expressing cells restored the internal TAG levels. The authors, however, did not provide an explanation of how TRPγ might function in both presynaptic and postsynaptic cells in the non-cell autonomous manner to regulate the TAG storage. The authors further determined the effect of TRPγ mutation on the size of lipid droplets (LD) and the lifespan and found that TRPγ mutation caused an increase in the size of LD and a decrease in the lifespan, which were reverted by feeding lipase and metformin. These were creative endeavors, I thought. The finding that DH44+ PI neurons have non-cell autonomous functions in regulating bodily metabolism (mainly sugar/lipid) in addition to directing sugar nutrient sensing and consumption is likely correct, but the paper has many loose ends.

Comments on revisions:

The authors have addressed nearly all of my concerns with additional experiments and explanations.

Reviewer #1 (Public review):

This manuscript presents SAVEMONEY, a computational tool designed to enhance the utilization of Oxford Nanopore Technologies (ONT) long-read sequencing for the design and analysis of plasmid sequencing experiments. In the past few years, with the improvement in both sequencing length and accuracy, ONT sequencing is being rapidly extended to almost all omics analyses which are dominated by short-read sequencing (e.g., Illumina). However, relatively higher sequencing errors of long-read sequencing techniques including PacBio and ONT is still a major obstacle for plasmid/clone-based sequencing service that aims to achieve single base/nucleotide accuracy. This work provides a guideline for sequencing multiple plasmids together using the same ONT run without molecular barcoding, followed by data deconvolution. The whole algorithm framework is well-designed, and some real data and simulation data are utilized to support the conclusions. The tool SAVEMONEY is proposed to target users who have their own ONT sequencers and perform library preparation and sequencing by themselves, rather than relying on commercial services. As we know and discussed by the authors, in the real world, to ensure accuracy, the researchers will routinely pick up multiple colonies in the same plasmid construction and submit for Sanger sequencing. However, SAVEMONEY is not able to support the simultaneous analysis of multiple colonies in the same run, as compared to the barcoding-based approaches. This is a major limitation in the significance of this work. Encouraging computational efforts in ONT data debarcoding for mixed-plasmid or even single-cell sequencing would be more valuable in the field.

Comments on revisions:

My previous concerns have been addressed, and the revised manuscript has been significantly approved.

Reviewer #2 (Public review):

The authors developed an algorithm that allows to deconvolute plasmid sequences from a mixture of plasmids that have been sequenced by nanopore long read technology. As library preparations and barcoding of individual samples increases sequencing costs, the algorithm bypasses this need and thus decreases time on sample prep and sequencing costs. In a first step, the tool assesses which of the plasmid constructions can be mixed in a single library preparation by calculating a distance matrix between the reference plasmid and the constructions producing sequence clusters. The user is given groups of plasmids, from different clusters, to be pooled together for sequencing. After sequencing, the algorithm deconvolutes the reads by classifying them based on alignments to the reference sequence. A Bayesian analysis approach is used to obtain a consensus sequence and quality scores.

Strengths

The authors exploit one of the main advantages of long read sequencing that is to accurately resolve regions of high complexity, as regularly found in plasmids, and developed a tool that can validate plasmid constructions by reducing sequencing costs. Multiple plasmids (up to six) can be analyzed simultaneously in a single library without the need of sample barcoding, also reducing sample preparation time. Although inserts must be different, just 2 bases difference would be enough for correct assignation. Maximizes cost-efficiency for projects that require large amounts of plasmid constructions and high-throughput validation. The algorithm also allows for linear DNA analysis offering extra flexibility.

Reviewer #2 (Public review):

Summary:

This study investigates cold induced states in C. elegans, using polysome profiling and RNA seq to identify genes that are differentially regulated and concluding that cold-specific gene regulation occurs at the transcriptional level. This study also includes analysis of one gene from the differentially regulated set, lips-11 (a lipase), and finds that it is regulated in response to a specific set of ER stress factors.

Strengths:

(1) Understanding how environmental conditions are linked to stress pathways is generally interesting.<br /> (2) The study used well-established genetic tools to analyze ER stress pathways.

Weaknesses:

(1) The conclusions regarding a general transcriptional response are based on a few genes, with much of the emphasis on lips-11, which does not affect survival in response to cold.

(2) Definitive conclusions regarding transcription vs translational effects would require the use of blockers such as alpha-amanitin or cyclohexamide. Although this may be beyond the scope of the study, it does affect the breadth of the conclusions that can be made.

(3) Conclusions regarding the role of lipids are based on supplementation with oleic acid or choline, yet there is no lipid analysis of the cold animals, or after lips-1 knockdown. Although choline is important for PC production, adding choline in normal PC could have many other metabolic impacts and doesn't necessarily implicate PC without lipidomic or genetic evidence. Although they note the caveats, their evidence falls short of proving a role in PC production.

Reviewer #3 (Public review):

Summary:

The authors sought to understand the molecular mechanisms that cells use to survive cold temperatures by studying gene expression regulation in response to cold in C. elegans. They determined whether gene expression changes during cold adaptation occur primarily at the transcriptional level and identified specific pathways, such as the unfolded protein response pathway, that are activated to possibly promote survival under cold conditions.

Strengths:

Effective use of bulk RNA sequencing (RNA-seq) to measure transcript abundance and ribosome profiling (ribo-seq) to assess translation rates, providing a comprehensive view of gene expression regulation during cold adaptation. This combined approach allows for correlation between mRNA levels and their translation, thereby offering evidence for the authors' conclusion that transcriptional regulation is the primary mechanism of cold-specific gene expression changes.

Weaknesses:

Many aspects of the weakness have been addressed by the revision. Still, the weak cold sensitivity phenotype observed in ire-1 mutants suggests the ER-UPR pathway's role is likely minor, modulatory or there is an unknown compensatory mechanism responsible for surviving cold.

Reviewer #1 (Public Review):

The mechanisms that regulate establishment of the germline stem cells and germline progenitors during zebrafish reproductive development are not understood. Prior single cell analysis characterized the cell types of the early zebrafish ovary during and at stages after sexual differentiation. In this work Hsu et al. took a single approach to analyze the cell types present in the early gonad during early sex determination. As expected, they identified germline stem cells (GSCs) that express canonical GSC markers and distinct populations of progenitors. Unexpectedly, they found multiple populations of transcriptionally distinct progenitor populations that the authors termed early (those lacking the differentiation marker foxl2l), committed (those expressing fox2l2 and S-phase genes) and late (those expressing fox2l2 and meiotic genes) progenitors. Comparisons of their dataset to the published zebrafish ovary datasets confirmed the presence of these distinct progenitor populations in the ovary. Further, they convincingly validated the presence of these progenitor subtypes using fluorescent in situ hybridization. To investigate the relationship between progenitor subsets and known regulators of ovary differentiation, the authors conducted single cell analysis of gonads lacking the transcription factor, Foxl2l. As previously reported, Foxl2l absence blocks ovary differentiation and all foxl2l mutants develop testes. The single cell analysis here indicates that foxl2l is inappropriately expressed in GSCs and early progenitors and that germ cell differentiation is blocked at the committed progenitor stage since few committed progenitors and no late progenitors or meiotic transcripts were detected in the single cell analysis of foxl2l mutants. Based on the coexpression of genes that are not typically expressed together in normally developing germ cells, specifically nanos2 and foxl2l, and dmrt1 and foxl2l, the authors conclude that Foxl2l is required for the committed progenitor program and that it prevents committed progenitors from returning to the GSC state.

Overall, the data provide new insights into the cell populations of the early differentiating gonad, define distinct progenitor states, pinpoint a requirement for the ovary differentiation factor Foxl2l at a specific stage of progenitor differentiation, and generate new hypotheses to be tested. Many but not all of the conclusions are supported by compelling data, and some findings and conclusions need to be clarified in the context of the published literature.

(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation. Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.

(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.

(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination. The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated. For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.

(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.

(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.

(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.

Reviewer #2 (Public Review):

In this manuscript, Hsu et al. used scRNA-seq to profile germ cells isolated from zebrafish ovaries. They identified the transcriptional profile of germ cells representing the early stages of oogenesis, from germline stem cells to newly formed follicle stage oocytes. They identified foxl2l as a gene expressed in probable oocyte progenitor cells, one of the least understood germ cell stages in the ovary. To understand to role of Foxl2l in oogenesis, they produced loss-of-function mutations in foxl2l using CRISPR/Cas9. They found that all foxl2l mutants are males as adults, suggesting that Foxl2l is required for oogenesis. To gain more insights, they performed scRNA-seq on cells isolated from 28 dpf foxl2l mutant ovaries and found that in the absence of foxl2l, germ cells appear to arrest as early progenitors. These results argue that Foxl2l, like its medaka homolog Foxl3, is necessary for promoting oocyte vs. spermatocyte differentiation during the oocyte progenitor stage.

Reviewer #3 (Public Review):

This is the first report to show a transcriptional factor, foxl2l, is essential for the development of female germs. Without foxl2l, germ cells will be developed into sperms. The report also clearly defined the arrested stage of early germ cells in foxl2l mutants, or stages that is critical for foxl2l to play a role for the further development of female germ cells. Due to lack of cell lineage tracing, the claim of foxl2l suppression of dedifferentiate of progenitor cells to GSC based on the gene expression and cell number changes is weak. In addition, separation of early germ cell types in foxl2l mutant using marker genes from WT may not be optimal.

Reviewer #1 (Public review):

Summary:

Pradhan et al investigated the potential gustatory mechanisms that allow flies to detect cholesterol. They found that flies are indifferent to low cholesterol and avoid high cholesterol. They further showed that the ionotropic receptors Ir7g, Ir51b, and Ir56d are important for the cholesterol sensitivity in bitter neurons. The figures are clear and the behavior result is interesting. However, I have several major comments, especially on the discrepancy of the expression of these Irs with other lab published results, and the confusing finding that the same receptors (Ir7g, Ir51b) have been implicated in the detection of various seemingly unrelated compounds.

Strengths:

The results are very well presented, the figures are clear and well-made, text is easy to follow.

Weaknesses:

(1) Regarding the expression of Ir56d. The reported Ir56d expression pattern contradicts multiple previous studies (Brown et al., 2021 eLife, Figure 6a-c; Sanchez-Alcaniz et al., 2017 Nature Communications, Figure 4e-h; Koh et al., 2014 Neuron, Figure 3b). These studies, using three different driver lines, consistently showed Ir56d expression in sweet-sensing neurons and taste peg neurons. Importantly, Sanchez-Alcaniz et al. demonstrated that Ir56d is not expressed in Gr66a-expressing (bitter) neurons. This discrepancy is critical since Ir56d is identified as the key subunit for cholesterol detection in bitter neurons, and misexpression of Ir7g and Ir51b together is insufficient to confer cholesterol sensitivity (Fig.4b,d). Which Ir56d-GAL4 (and Gr66a-I-GFP) line was used in this study? Is there additional evidence (scRNA sequencing, in-situ hybridization, or immunostaining) supporting Ir56d expression in bitter neurons?

(2) Ir51b has previously been implicated in detecting nitrogenous waste (Dhakal 2021), lactic acid (Pradhan 2024), and amino acids (Aryal 2022), all by the same lab. Additionally, both Ir7g and Ir51b have been implicated in detecting cantharidin, an insect-secreted compound that flies may or may not encounter in the wild, by the same lab. Is Ir51b proposed to be a specific receptor for these chemically distinct compounds or a general multimodal receptor for aversive stimuli? Unlike other multimodal bitter receptors, the expression level of Ir51b is rather low and it's unclear which subset of GRNs express this receptor. The chemical diversity among nitrogenous waste, amino acids, lactic acid, cantharidin, and cholesterol raises questions about the specificity of these receptors and warrants further investigation and at a minimum discussion in this paper. Given the wide and seemingly unrelated sensitivity of Ir51b and Ir7g to these compounds I'm leaning towards the hypothesis that at least some of these is non-specific and ecologically irrelevant without further supporting evidence from the authors.

(3) The Benton lab Ir7g-GAL4 reporter shows no expression in adults. Additionally, two independent labellar RNA sequencing studies (Dweck, 2021 eLife; Bontonou et al., 2024 Nature Communications) failed to detect Ir7g expression in the labellum. This contradicts the authors' previous RT-PCR results (Pradhan 2024 Fig. S4, Journal of Hazardous Materials) showing Ir7g expression in the labellum. Additionally the Benton and Carlson lab Ir51b-GAL4 reporters show no expression in adults as well. Please address these inconsistencies.

(4) The premise that high cholesterol intake is harmful to flies, which makes sensory mechanisms for cholesterol avoidance necessary, is interesting but underdeveloped. Animal sensory systems typically evolve to detect ecologically relevant stimuli with dynamic ranges matching environmental conditions. Given that Drosophila primarily consume fruits and plant matter (which contain minimal cholesterol) rather than animal-derived foods (which contain higher cholesterol), the ecological relevance of cholesterol detection requires more thorough discussion. Furthermore, at high concentrations, chemicals often activate multiple receptors beyond those specifically evolved for their detection. If the cholesterol concentrations used in this study substantially exceed those encountered in the fly's natural diet, the observed responses may represent an epiphenomenon rather than an ecologically and ethologically relevant sensory mechanism. What is the cholesterol content in flies' diet and how does that compare to the concentrations used in this paper?

Reviewer #2 (Public review):

Summary:

In Cholesterol Taste Avoidance in Drosophila melanogaster, Pradhan et al. used behavioral and electrophysiological assays to demonstrate that flies can: (1) detect cholesterol through a subset of bitter-sensing gustatory receptor neurons (GRNs) and (2) avoid consuming food with high cholesterol levels. Mechanistically, they identified five members of the IR family as necessary for cholesterol detection in GRNs and for the corresponding avoidance behavior. Ectopic expression experiments further suggested that Ir7g + Ir56d or Ir51b + Ir56d may function as tuning receptors for cholesterol detection, together with the Ir25a and Ir76b co-receptors.

Strengths:

The experimental design of this study was logical and straightforward. Leveraging their expertise in the Drosophila taste system, the research team identified the molecular and cellular basis of a previously unrecognized taste category, expanding our understanding of gustation. A key strength of the study was its combination of electrophysiological recordings with behavioral genetic experiments.

Weaknesses:

My primary concern with this study is the lack of a systematic survey of the IRs of interest in the labellum GRNs. Consequently, there is no direct evidence linking the expression of putative cholesterol IRs to the B GRNs in the S6 and S7 sensilla.

Specifically, the authors need to demonstrate that the IR expression pattern explains cholesterol sensitivity in the B GRNs of S6 and S7 sensilla, but not in other sensilla. Instead of providing direct IR expression data for all candidate IRs (as shown for Ir56d in Figure 2-figure supplement 1F), the authors rely on citations from several studies (Lee, Poudel et al. 2018; Dhakal, Sang et al. 2021; Pradhan, Shrestha et al. 2024) to support their claim that Ir7g, Ir25a, Ir51b, and Ir76b are expressed in B GRNs (Lines 192-194). However, none of these studies provide GAL4 expression or in situ hybridization data to substantiate this claim.

Without a comprehensive IR expression profile for GRNs across all taste sensilla, it is difficult to interpret the ectopic expression results observed in the B GRN of the I9 sensillum or the A GRN of the L-sensillum (Figure 4). It remains equally plausible that other tuning IRs-beyond the co-receptor Ir25a and Ir76b-could interact with the ectopically expressed IRs to confer cholesterol sensitivity, rather than the proposed Ir7g + Ir56d or Ir51b + Ir56d combinations.

Reviewer #3 (Public review):

Summary:

Whether and how animals can taste cholesterol is not well understood. The study provides evidence that 1) cholesterol activates a subset of bitter-sensing gustatory receptor neurons (GRNs) in the fly labellum, but not other types of GRNs, 2) flies show aversion to high concentrations of cholesterol, and this is mediated by bitter GRNs, and 3) cholesterol avoidance depends on a specific set of ionotropic receptor (IR) subunits acting in bitter GRNs. The claims of the study are supported by electrophysiological recordings, genetic manipulations, and behavioral readouts.

Strengths:

Cholesterol taste has not been well studied, and the paper provides new insight into this question. The authors took a comprehensive and rigorous approach in several different parts of the paper, including screening the responses of all 31 labellar sensilla, screening a large panel of receptor mutants, and performing misexpression experiments with nearly every combination of the 5 IRs identified. The effects of the genetic manipulations are very clear and the results of electrophysiological and behavioral studies match nicely, for the most part. The appropriate controls are performed for all genetic manipulations.

Weaknesses:

The weaknesses of the study, described below, are relatively minor and do not detract from the main conclusions of the paper.

(1) The paper does not state what concentrations of cholesterol are present in Drosophila's natural food sources. Are the authors testing concentrations that are ethologically relevant?

(2) The paper does not state or show whether the expression of IR7g, IR51b, and IR56d is confined to bitter GRNs. Bitter-specific expression of at least some of these receptors would be necessary to explain why bitter GRNs but not sugar GRNs (or other GRN types) normally show cholesterol responses.

(3) The authors only investigated the responses of GRNs in the labellum, but GRN responses in the leg may also contribute to the avoidance of cholesterol feeding. Alternatively, leg GRNs might contribute to cholesterol attraction that is unmasked when bitter GRNs are silenced. In support of this possibility, Ahn et al. (2017) showed that Ir56d functions in sugar GRNs of the leg to promote appetitive responses to fatty acids.

(4) The authors might consider using proboscis extension as an additional readout of taste attraction or aversion, which would help them more directly link the labellar GRN responses to a behavioral readout. Using food ingestion as a readout can conflate the contribution of taste with post-ingestive effects, and the regulation of food ingestion also may involve contributions from GRNs on multiple organs, whereas organ-specific contributions can be dissociated using proboscis extension. For example, does presenting cholesterol on the proboscis lead to aversive responses in the proboscis extension assay (e.g., suppression of responses to sugar)? Does this aversion switch to attraction when bitter GRNs are silenced, as with the feeding assay?

(5) The authors claim that the cholesterol receptor is composed of IR25a, IR76b, IR56d, and either IR7g or IR51b. While the authors have shown that IR25a and IR76b are each required for cholesterol sensing, they did not show that both are required components of the same receptor complex. If the authors are relying on previous studies to make this assumption, they should state this more clearly. Otherwise, I think further misexpression experiments may be needed where only IR25a or IR76b, but not both, are expressed in GRNs.

Reviewer #1 (Public review):

The aim of this study is to test the overarching hypothesis that plasticity in BNST CRF neurons drives distinct behavioral responses to unpredictable threat in males and females. The manuscript provides solid evidence for a sex-specific role for CRF-expressing neurons in the BNST in unpredictable aversive conditioning and subsequent hypervigilance across sexes. As the authors note, this is an important question given the high prevalence of sex differences in stress-related disorders, like PTSD, and the role of hypervigilance and avoidance behaviors in these conditions. The study includes in vivo manipulation, bulk calcium imaging, and cellular resolution calcium imaging, which yield important insights into cell-type specific activity patterns. A major strength of this manuscript is the inclusion of both males and females and attention to possible behavioral and neurobiological differences between them throughout.

Reviewer #2 (Public review):

This study examined the role of CRF neurons in the BNST in both phasic and sustained fear in males and females. The authors first established a differential fear paradigm whereby shocks were consistently paired with tones (Full) or only paired with tones 50% of the time (Part), or controls who were exposed to only tones with no shocks. Recall tests established that both Full and Part conditioned male and female mice froze to the tones, with no difference between the paradigms. Additional studies using the NSF and startle test, established that neither fear paradigm produced behavioral changes in the NSF test, suggesting that these fear paradigms do not result in an increase in anxiety-like behavior. Part fear conditioning, but not Full, did enhance startle responses in males but not females, suggesting that this fear paradigm did produce sustained increases in hypervigilance in males exclusively. Photometry studies found that while undifferentiated BNST neurons all responded to shock itself, only Full conditioning in males lead to a progressive enhancement of the magnitude of this response. BNST neurons in males, but not females, were also responsive to tone onset in both fear paradigms, but only in Full fear did the magnitude of this response increase across training. Knockdown of CRF from the BNST had no effect on fear learning in males or females, nor any effect in males on fear recall in either paradigm, but in females enhanced both baseline and tone-induced freezing only in Part fear group. When looking at anxiety following fear training, it was found in males that CRF knockdown modulated anxiety in Part fear trained animals and amplified startle in Full trained males but had no effect in either test in females. Using 1P imaging, it was found that CRF neurons in the BNST generally decline in activity across both conditioning and recall trials, with some subtle sex differences emerging in the Part fear trained animals in that in females BNST CRF neurons were inhibited after both shock and omission trials but in males this only occurred after shock and not omission trials. In recall trials, CRF BNST neuron activity remained higher in Part conditioned mice relative to Full conditioned mice.

Overall, this is a very detailed and complex study that incorporates both differing fear training paradigms and males and females, as well as a suite of both state-of-the-art imaging techniques and gene knockdown approaches to isolate the role and contributions of CRF neurons in the BNST to these behavioral phenomena. The strengths of this study come from the thorough approach that the authors have taken, which in turn helped to elucidate nuanced and sex specific roles of these neurons in the BNST to differing aspects of phasic and sustained fear. More so, the methods employed provide a strong degree of cellular resolution for CRF neurons in the BNST. In general, the conclusions appropriately follow the data, although the authors do tend to minimize some of the inconsistencies across studies, although this has now been addressed to some degree. The discussion has also been improved to now address some of the inconsistencies in the data head on. Discussion of a few other points is below:

- Given the focus on CRF neurons in the BNST, it was unclear why the photometry studies were performed in undifferentiated BNST neurons as opposed to CRF neurons specifically, although the authors have now explained this in better depth making this clearer to the reader.

- The CRF KD studies are interesting, but it remains speculative as to whether these effects are mediated locally in the BNST or due to CRF signaling at downstream targets. As the literature on local pharmacological manipulation of CRF signaling within the BNST seems to be largely performed in males, the addition of pharmacological studies here would benefit this to help to resolve if these changes are indeed mediated by local impairments in CRF release within the BNST or not. While it is not essential to add these experiments, the authors have addressed this point in the discussion and highlighted studies like this as necessary in future work.

- The authors have addressed the difference between arousal and anxiety by expanding the discussion to include more focus on the behavioral measures. The CRF KD data are still somewhat confusing but better contextualized now. Overall, the manuscript has been improved by the revisions and edits the authors have made.

Reviewer #3 (Public review):

Hon et al. investigated the role of BNST CRF signaling in modulating phasic and sustained fear in male and female mice. They found that partial and full fear conditioning had similar effects in both sexes during conditioning and during recall. However, males in the partially reinforced fear conditioning group showed enhanced acoustic startle, compared to the fully reinforced fear conditioning group, an effect not seen in females. Using fiber photometry to record calcium activity in all BNST neurons, the authors show that the BNST was responsive to foot shock in both sexes and both conditioning groups. Shock response increased over the session in males in the fully conditioned fear group, an effect not observed in the partially conditioned fear group. This effect was not observed in females. Additionally, tone onset resulted in increased BNST activity in both male groups, with the tone response increasing over time in the fully conditioned fear group. This effect was less pronounced in females, with partially conditioned females exhibiting a larger BNST response. During recall in males, BNST activity was suppressed below baseline during tone presentations and was significantly greater in the partially conditioned fear group. Both female groups showed an enhanced BNST response to the tone that slowly decayed over time. Next, they knocked CRF in the BNST to examine its effect on fear conditioning, recall and anxiety-like behavior after fear. They found no effect of the knockdown in either sex or group during fear conditioning. During fear recall, BNST CRF knockdown lead to an increase in freezing in only the partially conditioned females. In the anxiety-like behavior tasks, BNST CRF knockdown lead to increased anxiolysis in the partially reinforced fear male, but not in females. Surprisingly, BNST CRF knockdown increased startle response in fully conditioned, but not partially conditioned males. An effect not observed in either female group. In a final set of experiments, the authors single photon calcium imaging to record BNST CRF cell activity during fear conditioning and recall. Approximately, 1/3 of BNST CRF cells were excited by shock in both sexes, with the rest inhibited and no differences were observed between sexes or group during fear conditioning. During recall, BNST CRF activity decreased in both sexes, an effect pronounced in male and female fully conditioned fear groups.

Overall, these data provide novel, intriguing evidence in how BNST CRF neurons may encode phasic and sustained fear differentially in males and females. The experiments were rigorous. My biggest concerns I have regard the interpretations and some conclusions from this data set, which I have stated below.

(1) It was surprising to see minimal and somewhat conflicting behavioral effects due to BNST CRF knockdown. The authors provide a representative image and address this in the conclusion. They mention the role of local vs projection CRF circuits as well as the role of GABA. I don't think those experiments are necessary for this manuscript. However, it may be worthwhile to see through in situ hybridization or IHC, to see BNST CRF levels after both full and partial conditioned fear paradigms. Additionally, it would help to see a quantification of the knockdown of the animals. The authors can add a figure showing deltaF/F changes from control.

(2) Related to the previous point, it was surprising to see an effect of the CRF deletion in the full fear group compared to the partial fear in the acoustic startle task. To strengthen the conclusion about differential recruitment of CRF during phasic and sustained fear, the experiment in my previous point could help elucidate that. Conversely, intra-BNST administration of a CRF antagonist into the BNST before the acoustic startle after both conditioning tasks could also help. Or patch from BNST CRF neurons after the conditioning tasks to measure intrinsic excitability. Not all these experiments are needed to support the conclusion, it's some examples.

(3) In Figure 5 F and K, the authors report data combined for both part and full fear conditioning. Were there any differences between the number of excited or inhibited neurons b/t the conditioning groups? Also, can the authors separate male and female traces in Fig 5 E and P?

(4) Also, regarding the calcium imaging data, what was the average length of a transient induced by shock? Were there any differences between the sexes?

Reviewer #1 (Public review):

In this study, Osiurak and colleagues investigate the neurocognitive basis of technical reasoning. They use multiple tasks from two neuroimaging studies to show that the area PF is central to technical reasoning and plays an essential role in tool-use and non-tool-use physical problem-solving, as well as both conditions of mentalizing tasks. They also demonstrate the specificity of technical reasoning, finding that area PF is not involved in the fluid-cognition task or the mentalizing network (INT+PHYS vs. PHYS-only). This work enhances our understanding of the neurocognitive basis of technical reasoning that supports advanced technologies.

Strengths:

- The topic this study focuses on is intriguing and can help us understand the neurocognitive processes involved in technical reasoning and advanced technologies.<br /> - The researchers collected fMRI data from multiple tasks. The data is rich and encompasses the mechanical problem-solving task, psychotechnical task, fluid-cognition task, and mentalizing tasks.<br /> - The article is well written.

The authors have addressed many of the reviewers' concerns in their response. They utilized both correlation analysis and coordinate analysis to tackle alternative hypotheses, namely the same-region-but-different-function interpretation and the adjacency interpretation. Additionally, ROI analysis was conducted to validate the negative results. These additional analyses have enhanced the reliability of the findings. This study offers valuable insights into the neurocognitive mechanisms underlying technical reasoning.

Weaknesses:

While the authors attempted to address the limitations of overlap analysis by correlating activation across different tasks within subjects, this issue could not be entirely resolved due to the constraints of the current experimental design. The mechanical problem-solving task was not included since the sample of subjects differed from that of other tasks. Furthermore, the fluid-cognition task was not scanned in the same run as the psychotechnical and mentalizing tasks, which may have contributed to a lack of correlation between them, thereby affecting result interpretation. Moreover, the core cognitive focus of this study, technical reasoning, may be influenced by assumptions about motion-related information. While this issue has been discussed in the discussion section, further evidence is needed to substantiate this interpretation.

Reviewer #2 (Public review):

Strengths:

The authors have done a nice job providing additional data in response to reviewer feedback. I appreciate that accuracy plots are now included, as well as a separate analysis where differences in parameter estimates are performed for participants whose accuracy data were above chance levels. I also appreciate the new figure with the sphere ROIs for each participant, as they help us appreciate anatomical variability in the peak response separately for each task.

I have four concerns related to the weaknesses of the study:

(1) Although the results still hold when removing participants whose accuracy was 50% or less, a major limitation of this study is that participants made a button press response only to the last trial in a block. This is problematic because a participant could get all trials in a block correct except for the last one, or a participant could get all trials in a block wrong, and performance would be considered equivalent-as a consequence, it is not possible for one to know if participants who are at chance are performing differently from participants who are not at chance, and it is not possible to control for variance in reaction time (a concern also raised by reviewer 3).

(2) My second concern relates to the way in which the data are interpreted based on thresholding. There is above-threshold activation in the left SMG for all tasks except the fluid cognition task. The z-scores associated with significant voxels in Figure 3 are very strong (minimum z is 6). If one were to relax the threshold of the group level maps to, e.g., p < .001, uncorrected, FDR q < .05, or FWER of .10, there will be overlapping voxels outside the SMG. The discussion of the left SMG in the manuscript is prominent and narrowly construed-the left SMG is discussed as if it were 'the' region: "This confirms that the technical-reasoning network depends upon the recruitment of the left area PF, even if additional cognitive processes involving other peripheral brain areas can be engaged depending on the task" (pp. 9). My intuition is there will be numerous other areas of overlap when using a threshold that is still highly significant (e.g., z = 3 or 4). So, for proponents of the technical reasoning hypothesis, is there a counterfactual or alternative brain area/network/system not in the left SMG?

(3) I like the new Figure 6 because it shows variability in the location of the peak coordinate at the level of single participants. And, indeed, there's considerable variability that is typical when localizing ROIs in single participants. My concern is the level at which hypothesis testing is performed. An independent SMG ROI is used to extract parameter estimates and correlate responses between tasks to show a pattern of correlation that comports with a technical reasoning model of left SMG function. This is a fine approach but it does not rule out the so-called 'same region different function' interpretation because it relies on correlation-one cannot reverse infer that the left SMG is carrying out the same function across different tasks because the response in that area is more strongly correlated between certain tasks. This finding points to that possibility and makes interesting predictions for future studies to pursue, but it cannot tell us whether common functions in the left SMG are involved in each task. E.g., one interesting prediction for future studies is to test if patients with lesions to this site are disproportionately more inaccurate in the experimental condition of the mechanical problem solving task, the psychotechnical task, the mentalizing task, but not the fluid cognition task.

(4) I appreciated the approach to testing the adjacency interpretation by showing the sphere and peak Y coordinate across the tasks. It is interesting that across the groups, there is no difference in the peak Y coordinate of the psychotechnical task and both conditions of the mentalizing task, whereas the peak Y coordinate in the fluid intelligence task is more anterior in the post-central gyrus across participants (why is that?). But why restrict the analysis to just the Y coordinate? A rigorous way to test the adjacency hypothesis is to compute Euclidean distance among X, Y, and Z coordinates between any two tasks collected in the same participant. One can then test if the Euclidean distance between, e.g., the psychotechnical task and one condition of the mentalizing task is smaller than the Euclidean distance between the psychotechnical task and the fluid cognition task. Similarly, one can test whether Euclidean distance between the INT and PHY conditions of the mentalizing task is smaller than the Euclidean distance between the INT and psychotechnical task or PHY and psychotechnical task. There is no justification to restrict this analysis to the anterior-posterior dimension only.

Reviewer #3 (Public review):

The authors have responded very thoughtfully to many of the points raised, and the revised manuscript will make a useful contribution to our understanding of some of the computations performed by area PF. In particular, the investigators' addition of analyses of peak activations, their additional clarifications that area PF is likely to be part of a larger network concerned with technical reasoning, and their responses to the reviewers' concerns about differential task difficulty have strengthened the conclusions that can be drawn from the study.

The authors' response does not completely mitigate the concern noted by all 3 reviewers that the control tasks were easier than their corresponding experimental tasks (for everything but the fluid cognition task). The specific trouble with this issue can be appreciated by looking at Figure 4A, for example, which shows that area PF was activated for many individuals in both the control task and the experimental mechanical problem-solving task, but more so for the latter. Since the experimental task was harder (and more trial time was likely spent on task trying to solve it), the concern remains that area PF was driven harder by the experimental task in part due to the more challenging nature of that task.

The revised manuscript counters that the fluid cognition task was also harder than its control condition, yet did not activate PF more than its control condition. But this response seems to sidestep the central point of the reviewers' concerns: the fundamental computations that underlie the technical reasoning tasks may also be present in the respective (non technical-reasoning-based) control tasks and drive area PF activations to greater or lesser degrees based on how much they tax those computations. The fact that the fluid cognition experimental task and control task are not differentially difficult does not mitigate this concern, it just suggests that neither of those tasks tap the same fundamental computations, whatever they may be. (As an added note, Figures 2 and 4 show that both the PHYS-only and INT+PHYS mentalizing tasks only weakly activated PF, and both of these tasks were easier than the other technical cognition tasks).

The new ROI analysis with removal of subjects who performed below 50% in the revised manuscript is somewhat helpful, but there are two remaining issues: 1) chance performance is defined by a binomial test in this case, so scores somewhat above 50% may still be at chance depending on the number of items, and thus there may have been subjects who were not removed who could not perform the tasks; 2) it would have been convincing to include accuracy as a covariate in the modeling of BOLD parameter estimates for the remaining above-chance subjects to ensure that all reported effects remain once differential task difficulty is taken into account. It also appears that the legend for Figure S2, which indicates that the figure includes just subjects who performed at or below 50%, may not be correct; does the figure instead show data from subjects who performed at or above 50%?

Despite these remaining concerns, there are many aspects of this revised study that render it a useful contribution that will likely spur further research in this very interesting area.

Reviewer #1 (Public review):

Hüppe and colleagues had already developed an apparatus and an analytical approach to capture swimming activity rhythms in krill. In a previous manuscript they explained the system, and here they employ it to show a circadian clock, supplemented by exogenous light, produces an activity pattern consistent with "twilight" diel vertical migration (DVM; a peak at sunset, a midnight sink, and a peak in the latter half of the night).

They used light:dark (LD) followed by dark:dark (DD) photoperiods at two times of the year to confirm the circadian clock, coupled with DD experiments at four times a year to show rhythmicity occurs throughout the year along with DVM in the wild population. The individual activity data show variability in the rhythmic response, which is expected. However, their results showed rhythmicity was sustained in DD throughout the year, although the amplitude decayed quickly. The interpretation of a weak clock is reasonable, and they provide a convincing justification for the adaptive nature of such a clock in a species that has a wide distributional range and experiences various photic environments. These data also show that exogenous light increases the activity response and can explain the morning activity bouts, with the circadian clock explaining the evening and late-night bouts. This acknowledgement that vertical migration can be driven by multiple proximate mechanisms is important.

The work is rigorously done, and the interpretations are sound. I see no major weaknesses in the manuscript. Because a considerable amount of processing is required to extract and interpret the rhythmic signals (see Methods and previous AMAZE paper), it is informative to have the individual activity plots of krill as a gut check on the group data.

The manuscript will be useful to the field as it provides an elegant example of looking for biological rhythms in a marine planktonic organism and disentangling the exogenous response from the endogenous one. Furthermore, as high-latitude environments change, understanding how important organisms like krill have the potential to respond will become increasingly important. This work provides a solid behavioral dataset to complement the earlier molecular data suggestive of a circadian clock in this species.

Reviewer #2 (Public review):

Summary:

This manuscript provides experimental evidence on circadian behavioural cycles in Antarctic krill. The krill were obtained directly from krill fishing vessels and the experiments were carried out on board using an advanced incubation device capable of recording activity levels over a number of days. A number of different experiments were carried out where krill were first exposed to simulated light:dark (L:D) regimes for some days followed by continuous darkness (DD). These were carried out on krill collected during late autumn and late summer. A further set of experiments was performed on krill across three different seasons (summer, autumn, winter), where incubations were all DD conditions. Activity was measured as the frequency by which an infrared beam close to the top of the incubation tube was broken over unit time. Results showed that patterns of increased and decreased activity that appeared synchronised to the LD cycle persisted during the DD period. This was interpreted as evidence of the operation of an internal (endogenous) clock. The amplitude of the behavioural cycles decreased with time in DD, which further suggests that this clock is relatively weak. The authors argued that the existence of a weak endogenous clock is an adaptation to life at high latitudes since allowing the clock to be modulated by external (exogenous) factors is an advantage when there is a high degree of seasonality. This hypothesis is further supported by seasonal DD experiments which showed that the periodicity of high and low activity levels differed between seasons.

Strengths:

Although there has been a lot of field observations of various circadian type behaviour in Antarctic krill, relatively few experimental studies have been published considering this behaviour in terms of circadian patterns of activity. Krill are not a model organism and obtaining them and incubating them in suitable conditions are both difficult undertakings. Furthermore, there is a need to consider what their natural circadian rhythms are without the overinfluence of laboratory-induced artefacts. For this reason alone, the setup of the present study is ideal to consider this aspect of krill biology. Furthermore, the equipment developed for measuring levels of activity is well-designed and likely to minimise artefacts.

Reviewer #1 (Public review):

Summary:

This manuscript by Kaya et al. studies the effect of food consumption on hippocampal sharp wave ripples (SWRs) in mice. The authors use multiple foods and forms of food delivery to show that the frequency and power of SWRs increase following food intake, and that this effect depends on the caloric content of food. The authors also studied the effects of administration of various food-intake-related hormones on SWRs during sleep, demonstrating that ghrelin negatively affects SWR rate and power, but not GLP-1, insulin, or leptin. Finally, the authors use fiber photometry to show that GABAergic neurons in the lateral hypothalamus, increase activity during a SWR event.

Strengths:

The experiments in this study seem to be well performed, and the data are well presented, visually. The data support the main conclusions of the manuscript that food intake enhances hippocampal SWRs. Taken together, this study is likely to be impactful to the study of the impact of feeding on sleep behavior, as well as the phenomena of hippocampal SWRs in metabolism.

Weaknesses:

None

Reviewer #2 (Public review):

Summary:

Kaya et al uncover an intriguing relationship between hippocampal sharp wave-ripple production and peripheral hormone exposure, food intake, and lateral hypothalamic function. These findings significantly expand our understanding of hippocampal function beyond mnemonic processes and point a direction for promising future research.

Strengths:

Some of the relationships observed in this paper are highly significant. In particular, the inverse relationship between GLP1/Leptin and Insulin/Ghrelin are particularly compelling as this aligns well with opposing hormone functions on satiety.

Reviewer #3 (Public review):

Summary:

The manuscript by Kaya et al. explores the effects of feeding on sharp wave-ripples (SWRs) in the hippocampus, which could reveal a better understanding of how metabolism is regulated by neural processes. Expanding on prior work that showed that SWRs trigger a decrease in peripheral glucose levels, the authors further tested the relationship between SWRs and meal consumption by recording LFPs from the dorsal CA1 region of the hippocampus before and after meal consumption. They found an increase in SWR magnitude during sleep after food intake, in both food-restricted and ad libitum fed conditions. Using fiber photometry to detect GABAergic neuron activity in the lateral hypothalamus, they found increased activity locked to the onset of SWRs. They conclude that the animal's satiety state modulates the amplitude and rate of SWRs, and that SWRs modulate downstream circuits involved in regulating feeding.

The authors have addressed prior requests for revision and clarification, and provide a convincing case for SWRs being modulated by satiety state. These experiments provide an important step forward in understanding how metabolism is regulated in the brain. The study will likely be of great interest in the field of learning and memory while carrying broader implications for understanding brain-body physiology.

Reviewer #2 (Public review):

This manuscript addresses an important question which has not yet been solved in the field, what is the contribution of different gamma oscillatory inputs to the development of "theta sequences" in the hippocampal CA1 region. Theta sequences have received much attention due to their proposed roles in encoding short-term behavioral predictions, mediating synaptic plasticity, and guiding flexible decision-making. Gamma oscillations in CA1 offer a readout of different inputs to this region and have been proposed to synchronize neuronal assemblies and modulate spike timing and temporal coding. However, the interactions between these two important phenomena have not been sufficiently investigated. The authors conducted place cell and local field potential (LFP) recordings in the CA1 region of rats running on a circular track. They then analyzed the phase locking of place cell spikes to slow and fast gamma rhythms, the evolution of theta sequences during behavior and the interaction between these two phenomena. They found that place cell with the strongest modulation by fast gamma oscillations were the most important contributors to the early development of theta sequences and that they also displayed a faster form of phase precession within slow gamma cycles nested with theta.

Comments on revisions:

Several important shortcomings were noted in the original manuscript. These have been addressed in this revised version with the addition of multiple new analysis, controls and clarifications. The revised manuscript has been significantly improved and its conclusions are adequately supported by the results presented.

Reviewer #1 (Public review):

Summary:

This very interesting manuscript proposes a general mechanism for how activating signaling proteins respond to species specific signals arising from a variety of stresses. In brief, the authors propose that the activating signal alters the structure by a universal allosteric mechanism.

Strengths:

The unitary mechanism proposed is appealing and testable. The propose that the allosteric module consists of crossed alpha-helical linkers with similar architecture and that their attached regulatory domains connect to phosphatases or other molecules through coiled-coli domains, such that the signal is transduced via rigidifying the alpha helices, permitting downstream enzymatic activity. The authors present genetic and structural prediction data in favor of the model for the system they are studying, and stronger structural data in other systems.

Weaknesses:

I thank the authors for making significant revisions that addressed almost all of my concerns. I hope that the authors will consider addressing my last concern, which is that the title is inappropriate. However, I do not believe that this should hold up the publication of the ms.

"A General Mechanism for Initiating the General Stress Response in Bacteria" is misleading because it suggests a broadly applicable, universal mechanism across all bacterial species, whereas the study primarily focuses on Bacillus subtilis and its RsbU phosphatase activation. While the authors propose that the mechanism may extend to other bacteria, the evidence is largely based on structural modeling rather than direct experimental validation across multiple phyla. Additionally, the phrase "General Stress Response" might imply that the paper broadly explains stress response regulation, but it specifically examines the activation of RsbU by RsbT, which is just one really small part of the broader GSR network. The redundancy in "A General Mechanism for the General Stress Response" could also create an impression of an oversimplified, universal model when stress responses are often species- and context-specific. Furthermore, the study builds upon existing knowledge of partner-switching mechanisms rather than introducing an entirely new concept, making the claim of a general mechanism overstated and misleading for the field.

Title options could be "A Conserved Activation Mechanism for the General Stress Response Phosphatase in Bacteria", "Coiled-Coil Linker-Mediated Activation of a General Stress Response Phosphatase", all of which more accurately reflect the study's scope and findings.

Reviewer #2 (Public review):

Summary:

While bacteria have the ability to induce genes in response to specific stresses, they also use the General Stress Response (GSR) to deal with growth conditions that presumably include a larger range of stresses (for instance, stationary phase growth). The activation of GSR-specific sigma factors is frequently at the heart of the induction of a GSR. Given the range of stresses that can lead to GSR induction, the regulatory inputs are frequently complex. In B. subtilis, the stressosome, a multi-protein complex, contains a set of proteins that, upon appropriate stresses, initiate partner switching cascades that free the sigma B sigma factor from an anti-sigma. The focus here is on the mode of activation of RsbU, a serine/threonine phosphatase of the PPM family, leading to sigB activation. RbsT, a component of the degradosome interacts with RsbU upon stress, activating the phosphatase activity. Once active, RsbU dephosphorylates its target (RsbV, an anti-antisigma), which in turn binds the anti-sigma. The conclusion is that flexible linker domains upstream of the phosphatase domain are the target for activation, resulting in a crossed-linker dimeric structure. The authors then use the information on RsbU to suggest that parallel approaches may be used to activate PPM phosphatases for the GSR response in other bacteria.

Strengths and Weaknesses:

(1) A strength of the work is the combination of modeling, genetics and biochemical approaches to support the idea that the flexibility of the linker of the RsbU phosphatase is critical to signalling and that this changes as a result of interactions of the signaling protein RsbT.

(2) The impact of the work, beyond better understanding of this particular signalling system, lies in the suggested parallels with other GSR system regulators in a range of bacteria. The work here provides fairly clear indications of what mutational changes would be most likely to test the model.

(3) Assuming that these predictions are shown to be correct in future work, that will leave as an intriguing question why this particular geometry has been conserved in GSR - whether they emerge from a common ancestor (found where?) and/or there is some characteristic (flexibility of modulating the response?) that is particularly important for GSR signal input. Coupled with this will be further understanding of how the linker and/or interacting proteins change in different systems.

Reviewer #3 (Public review):

Summary:

The authors present a study building on their previous work on activation of the general stress response phosphatase, RsbU, from Bacillus subtilis. Using computed structural models of the RsbU dimer the authors map previously identified activating mutations onto the structure and suggest further protein variants to test the role of the predicted linker helix and the interaction with RsbT on the activation of the phosphatase activity.

Using in vivo and in vitro activity assays, the authors demonstrate that linker variants can constitutively activate RsbU and increase the affinity of the protein for RsbT, thus showing a link between the structure of the linker region and RsbT binding.

Small angle X-ray scattering experiments on RsbU variants alone, and in complex with RsbT show structural changes consistent with a decreased flexibility of the RsbU protein, which are hypothesised to indicate an disorder-order transition in the linker when RsbT binds. This interpretation of the data is consistent with the biochemical data presented by the authors.

Further computed structure models are presented for other protein phosphates from different bacterial species and the authors propose a model for phosphatase activation by partner binding. They compare this to the activation mechanisms proposed for histidine kinase two-component systems and GGDEF proteins and suggest the individual domains could be swapped to give a toolkit of modular parts for bacterial signalling.

Strengths:

The key mutagenesis data is presented with two lines of evidence to demonstrate RsbU activation - in vivo sigma-b activation assays utilising a beta-galactosidase reporter and in vitro activity assays against the RsbV protein, which is the downstream target of RsbU. These data support the hypothesis for RsbT binding to the RsbU linker region as well as the dimerisation domain to activate the RsbU activity.

Weaknesses:

Small angle scattering curves are difficult to unambiguously interpret, but the authors present good interpretations that fit with the biochemical data presented. These interpretations should be considered as models for future testing with other methods - hydrogen/deuterium exchange mass spectrometry, would be a good additional method to use, as exchange rates in the linker region would be affected significantly by the disorder/order transition on RsbT binding.

The interpretation of the computed structure models is provided with a few caveats related to the bias in the models returned by AlphaFold2. For the full-length models of RsbU and other phosphatase proteins, the relationship of the domains to each other is likely to be the least reliable part of the models - this is apparent from the PAE plots shown in supplementary figure 8.

Comments on revisions:

The authors have addressed the review comments satisfactorily for this manuscript to stand as a version of record.

Reviewer #1 (Public review):

Summary:

Kimura et al performed a saturation mutagenesis study of CDKN2A to assess functionality of all possible missense variants and compare them to previously identified pathogenic variants. They also compared their assay result with those from in silico predictors.

Strengths:

CDKN2A is an important gene that modulate cell cycle and apoptosis, therefore it is critical to accurately assess functionality of missense variants. Overall, the paper reads well and touches upon major discoveries in a logical manner.

Weaknesses:

The paper lacks proper details for experiments and basic data, leaving the results less convincing. Analyses are superficial and does not provide variant-level resolution.

Comments on revisions

The manuscript was improved during the revision process.

Reviewer #3 (Public review):

Summary:

The authors investigate the effect of high concentrations of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in a yeast deletion strain lacking the detoxification enzyme. Transcriptomic analyses as global read out reveals that a large range of cellular functions across all compartments are affected (transcriptomic changes affect 1/3 of all genes). The authors provide additional analyses, which indicate that mitochondrial protein import is affected.

Strengths:

Global analyses (transcriptomic and functional genomics approach) to obtain an overview of changes upon yeast treatment with high doses of t-2-hex.

Weaknesses:

The use of high concentrations of t-2-hex in combination with a deletion of the detoxifying enzyme Hfd1 limits the possibility to identify physiological relevant changes. For the follow-up analysis, the authors focus on mitochondrial proteins and describe an impairment of mitochondrial protein biogenesis, but the underlying molecular modification resulting in the observed impairment is not yet known.

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

Weaknesses:

(1) Impact on Cellular Translation:

The authors demonstrate that modified saRNAs with additional components enhance transgene expression by inhibiting dsRNA-sensing pathways. However, it is unclear whether these modifications influence global cellular translation beyond the expression of GFP and mScarlet-3 (which are encoded by the saRNA itself). Conducting a polysome profiling analysis or a puromycin labeling assay would clarify whether the modified saRNAs alter overall translation efficiency. This additional data would strengthen the conclusions regarding the specificity of dsRNA-sensing inhibition.

(2) Stability and Replication Efficiency of Long saRNA Constructs:

The saRNA constructs used in this study exceed 16 kb, making them more fragile and challenging to handle. Assessing their mRNA integrity and quality would be crucial to ensure their robustness.<br /> Furthermore, the replicative capacity of the designed saRNAs should be confirmed. Since Figure 4 shows lower inflammatory cytokine production when encoding srIkBα and srIkBα-Smad7-SOCS1, it is important to determine whether this effect is due to reduced immune activation or impaired replication. Providing data on replication efficiency and expression levels of the encoded anti-inflammatory proteins would help rule out the possibility that reduced cytokine production is a consequence of lower replication.

(3) Comparative Data with Native saRNA:

Including native saRNA controls in Figures 5-7 would allow for a clearer assessment of the impact of additional genes on cytokine production. This comparison would help distinguish the effect of the encoded suppressor proteins from other potential factors.

(4) In vivo Validation and Safety Considerations:

Have the authors considered evaluating the in vivo potential of these saRNA constructs? Conducting animal studies would provide stronger evidence for their therapeutic applicability. If in vivo experiments have not been performed, discussing potential challenges - such as saRNA persistence, biodistribution, and possible secondary effects-would be valuable.

(5) Immune Response to Viral Proteins:

Since the inhibitors of dsRNA-sensing proteins (E3, NSs, and L*) are viral proteins, they would be expected to induce an immune response. Analyzing these effects in vivo would add insight into the applicability of this approach.

(6) Streamlining the Discussion Section:

The discussion is quite lengthy. To improve readability, some content - such as the rationale for gene selection-could be moved to the Results section. Additionally, the descriptions of Figure 3 should be consolidated into a single section under a broader heading for improved coherence.

Reviewer #2 (Public review):

Summary:

Lim et al. have developed a self-amplifying RNA (saRNA) design that incorporates immunomodulatory viral proteins, and show that the novel design results in enhanced protein expression in vitro in mouse primary fibroblast-like synoviocytes. They test constructs including saRNA with the vaccinia virus E3 protein and another with E3, Toscana virus NS protein and Theiler's virus L protein (E3 + NS + L), and another with srIκBα-Smad7-SOCS1. They have also tested whether ML336, an antiviral, enables control of transgene expression.

Strengths:

The experiments are generally well-designed and offer mechanistic insight into the RNA-sensing pathways that confer enhanced saRNA expression. The experiments are carried out over a long timescale, which shows the enhance effect of the saRNA E3 design compared to the control. Furthermore, the inhibitors are shown to maintain the cell number, and reduce basal activation factor-⍺ levels.

Weaknesses:

One limitation of this manuscript is that the RNA is not well characterized; some of the constructs are quite long and the RNA integrity has not been analyzed. Furthermore, for constructs with multiple proteins, it's imperative to confirm the expression of each protein to confirm that any therapeutic effect is from the effector protein (e.g. E3, NS, L). The ML336 was only tested at one concentration; it is standard in the field to do a dose-response curve. These experiments were all done in vitro in mouse cells, thus limiting the conclusion we can make about mechanisms in a human system.