Reviewer #3 (Public Review):

Summary:

Different types of retinal ganglion cell (RGC) have different temporal properties - most prominently a distinction between sustained vs. transient responses to contrast. This has been well established in multiple species, including mice. In general, RGCs with dendrites that stratify close to the ganglion cell layer (GCL) are sustained; whereas those that stratify near the middle of the inner plexiform layer (IPL) are transient. This difference in RGC spiking responses aligns with similar differences in excitatory synaptic currents as well as with differences in glutamate release in the respective layers - shown previously and here, with a glutamate sensor (iGluSnFR) expressed in the RGCs of interest. Differences in glutamate release were not explained by differences in the distinct presynaptic bipolar cells' voltage responses, which were quite similar to one another. Rather, the difference in transient vs. sustained responses seems to emerge at the bipolar cell axon terminals in the form of glutamate release. This difference in the temporal pattern of glutamate release was correlated with differences in the size of synaptic ribbons (larger in the bipolar cells with more sustained responses), which also correlated with a greater number of vesicles in the vicinity of the larger ribbons.

The main conclusion of the study relates to a correlation (because it is difficult to manipulate ribbon size or vesicle density experimentally): the bipolar cells with increased ribbon size/vesicle number would have a greater possibility of sustained release, which would be reflected in the postsynaptic RGC synaptic currents and RGC firing rates. This model proposes a mechanism for temporal channels that is independent of synaptic inhibition. Indeed, some experiments in the paper suggest that inhibition cannot explain the transient nature of glutamate release onto one of the RGC types. Still, it is surprising that such a diverse set of inhibitory interneurons in the retina would not play some role in diversifying the temporal properties of RGC responses.

Strengths:

(1) The study uses a systematic approach to evaluating temporal properties of retinal ganglion cell (RGC) spiking outputs, excitatory synaptic inputs, presynaptic voltage responses, and presynaptic glutamate release. The combination of these experiments demonstrates an important step in the conversion from voltage to glutamate release in shaping response dynamics in RGCs.

(2) The study uses a combination of electrophysiology, two-photon imaging, and scanning block-face EM to build a quantitative and coherent story about specific retinal circuits and their functional properties.

Weaknesses:

(1) There were some interesting aspects of the study that were not completely resolved, and resolving some of these issues may go beyond the current study. For example, it was interesting that different extracellular media (Ames medium vs. ACSF) generated different degrees of transient vs. sustained responses in RGCs, but it was unclear how these media might have impacted ion channels at different levels of the circuit that could explain the effects on temporal tuning.

(2) It was surprising that inhibition played such a small role in generating temporal tuning. At the same time, there were some gaps in the investigation of inhibition (e.g., IPSCs were not measured in either of the RGC types; pharmacology was used to investigate responses only in the transient RGCs).

(3) There could be additional discussion and references to the literature describing several topics, including: temporal dynamics of glutamate release at different levels of the IPL; previous evidence that release sites from a single presynaptic neuron can differ in their temporal properties depending on the postsynaptic target; previous investigations of the role of inhibition in temporal tuning within retinal circuitry.

Reviewer #1 (Public Review):

In the study "Re-focusing visual working memory during expected and unexpected memory tests" by Sisi Wang and Freek van Ede, the authors investigate the dynamics of attentional re-orienting within visual working memory (VWM). Utilizing a robust combination of behavioral measures, electroencephalography (EEG), and eye tracking, the research presents a compelling exploration of how attention is redirected within VWM under varying conditions. The research question addresses a significant gap in our understanding of cognitive processes, particularly how expected and unexpected memory tests influence the focus and re-focus of attention. The experimental design is meticulously crafted, enabling a thorough investigation of these dynamics. The figures presented are clear and effectively illustrate the findings, while the writing is concise and accessible, making the complex concepts understandable. Overall, this study provides valuable insights into the mechanisms of visual working memory and attentional re-orienting, contributing meaningfully to the field of cognitive neuroscience. Despite the strengths of the manuscript, there are several areas where improvements could be made.

Microsaccades or Saccades?

In the manuscript, the terms "microsaccades" and "saccades" are used interchangeably. For instance, "microsaccades" are mentioned in the keywords, whereas "saccades" appear in the results section. It is crucial to differentiate between these two concepts. Saccades are large, often deliberate eye movements used for scanning and shifting attention, while microsaccades are small, involuntary movements that maintain visual perception during fixation. The authors note the connection between microsaccades and attention, but it is not well-recognized that saccades are directly linked to attention. Despite the paradigm involving a fixation point, it remains unclear whether large eye movements (saccades) were removed from the analysis. The authors mention the relationship between microsaccades and attention but do not clarify whether large eye movements (saccades) were excluded from the analysis. If large eye movements were removed during data processing, this should be documented in the manuscript, including clear definitions of "microsaccades" and "saccades." If such trials were not removed, the contribution of large eye movements to the results should be shown, and an explanation provided as to why they should be considered.

Alpha Lateralization in Attentional Re-orienting

In the attentional orienting section of the results (Figure 2), the authors effectively present EEG alpha lateralization results with time-frequency plots and topographic maps. However, in the attentional re-orienting section (Figure 3), these visualizations are absent. It is important to note that the time period in attentional orienting differs from attentional re-orienting, and consequently, the time-frequency plots and topographic maps may also differ. Therefore, it may be invalid to compute alpha lateralization without a clear alpha activity difference. The authors should consider including time-frequency plots and topographic maps for the attentional re-orienting period to validate their findings.

Onset and Offset Latency of Saccade Bias

The use of the 50% peak to determine the onset and offset latency of the saccade bias is problematic. For example, if one condition has a higher peak amplitude than another, the standard for saccade bias onset would be higher, making the observed differences between the onset/offset latencies potentially driven by amplitude rather than the latencies themselves. The authors should consider a more robust method for determining saccade bias onset and offset that accounts for these amplitude differences.

Control Analysis for Trials Not Using the Initial Cue

The control analysis for trials where participants did not use the initial cue raises several questions:

(1) The authors claim that "unlike continuous alpha activity, saccades are events that can be classified on a single-trial level." However, alpha activity can also be analyzed at the single-trial level, as demonstrated by studies like "Alpha Oscillations in the Human Brain Implement Distractor Suppression Independent of Target Selection" by Wöstmann et al. (2019). If single-trial alpha activity can be used, it should be included in additional control analyses.

(2) The authors aimed to test whether the re-orienting signal observed after the test is not driven exclusively by trials where participants did not use the initial cue. They hypothesized that "in such a scenario, we should only observe attention deployment after the test stimulus in trials in which participants did not use the preceding retro cue." However, if the saccade bias is the index for attentional deployment, the authors should conduct a statistical test for significant saccade bias rather than only comparing toward-saccade after-cue trials with no-toward-saccade after-cue trials. The null results between the two conditions do not immediately suggest that there is attention deployment in both conditions.

(3) Even if attention deployment occurs in both conditions, the prolonged re-orienting effect could also be caused by trials where participants did not use the initial cue. Unexpected trials usually involve larger and longer brain activity. The authors should perform the same analysis on the time after the removal of trials without toward-saccade after the cue to address this potential confound.

Reviewer #2 (Public Review):

Summary:

This study utilized EEG-alpha activity and saccade bias to quantify the spatial allocation of attention during a working memory task. The findings indicate a second stage of internal attentional deployment following the appearance of a memory test, revealing distinct patterns between expected and unexpected test trials. The spatial bias observed during the expected test suggests a memory verification process, whereas the prolonged spatial bias during the unexpected test suggests a re-orienting response to the memory test. This work offers novel insights into the dynamics of attentional deployment, particularly in terms of orienting and re-orienting following both the cue and memory test.

Strengths:

The inclusion of both EEG-alpha activity and saccade bias yields consistent results in quantifying the attentional orienting and re-orienting processes. The data clearly delineate the dynamics of spatial attentional shifts in working memory. The findings of a second stage of attentional re-orienting may enhance our understanding of how memorized information is retrieved.

Weaknesses:

Although analyses of neural signatures and saccade bias provided clear evidence regarding the dynamics of spatial attention, the link between these signatures and behavioral performance remains unclear. Given the novelty of this study in proposing a second stage of 'verification' of memory contents, it would be more informative to present evidence demonstrating how this verification process enhances memory performance.

Reviewer #3 (Public Review):

Summary:

Wang and van Ede investigate whether and how attention re-orients within visual working memory following expected and unexpected centrally presented memory tests. Using a combination of spatial modulations in neural activity (EEG-alpha lateralization) and gaze bias quantified as time courses of microsaccade rate, the authors examined how retro cues with varying levels of reliability influence attentional deployment and subsequent memory performance. The conclusion is that attentional re-orienting occurs within visual working memory, even when tested centrally, with distinct patterns following expected and unexpected tests. The findings provide new value for the field and are likely of broad interest and impact, by highlighting working memory as an action-bound process (in)dependent on (an ambiguous) past.

Strengths:

The study uniquely integrates behavioral data (accuracy and reaction time), EEG-alpha activity, and gaze tracking to provide a comprehensive analysis of attentional re-orienting within visual working memory. As typical for this research group, the validity of the findings follows from the task design that effectively manipulates the reliability of retro cues and isolates attentional processes related to memory tests. The use of well-established markers for spatial attention (i.e. alpha lateralization) and more recently entangled dependent variable (gaze bias) is commendable. Utilizing these dependent metrics, the concise report presents a thorough analysis of the scaling effects of cue reliability on attentional deployment, both at the behavioral and neural levels. The clear demonstration of prolonged attentional deployment following unexpected memory tests is particularly noteworthy, although there are no significant time clusters per definition as time isn't a factor in a statistical sense, the jackknife approach is convincing. Overall, the evidence is compelling allowing the conclusion of a second stage of internal attentional deployment following both expected and unexpected memory tests, highlighting the importance of memory verification and re-orienting processes.

Weaknesses:

I want to stress upfront that these weaknesses are not specific to the presented work and do not affect my recommendation of the paper in its present form.

The sample size is consistent with previous studies, a larger sample could enhance the generalizability and robustness of the findings. The authors acknowledge high noise levels in EEG-alpha activity, which may affect the reliability of this marker. This is a general issue in non-invasive electrophysiology that cannot be handled by the authors but an interested reader should be aware of it. Effectively, the sensitivity of the gaze analysis appears "better" in part due to the better SNR. The latter also sets the boundaries for single-tiral analyses as the authors correctly mention. In terms of generalizability, I am convinced that the main outcome will likely generalize to different samples and stimulus types. Yet, as typical for the field future research could explore different contexts and task demands to validate and extend the findings. The authors provide here how and why (including sharing of data and code).

Reviewer #1 (Public Review):

Summary:

Mosshammer et al. studied the oxygenic photosynthetic productivity of beachrock samples containing cyanobacteria with different pigment compositions. The use of longer wavelength absorbing chlorophylls in some cyanobacteria (chlorophylls d and f) allows their photosystems to use light further in the red than canonical chlorophyll a photosystems. As such, their distribution in visible light-shaded environments, such as the beachrock studied by Mosshammer et al., allows them to perform oxygenic photosynthesis using wavelengths not capable of driving photosynthesis in most cyanobacteria, algae, or plants.

By adapting measuring systems they have previously used to study these types of beachrock samples, the authors attempt to mimic a more natural light penetration through the beachrock in order to measure oxygen production. By doing so with different wavelengths and intensities, the authors are able to show that far-red light-driven oxygen production is potentially capable of driving high levels of gross primary production.

Strengths:

The manuscript builds on previous measurement techniques used by the authors while focussing on illumination from the top of a sample rather than the specific microbial layers themselves. This provides a more environmentally realistic understanding of the beachrock community, as well as far-red light-driven photosynthesis.

The manuscript benefits from using previously defined methods to further characterize complex environmental samples.

Weaknesses:

The manuscript suffers from a lack of discussion and interpretation of the findings, and as such is more of a report.

Using the envionmental beachrock samples has inherent complications, from the variation in rock morphology, to the microbial community composition of different samples as well as within a single sample. It would benefit the authors to discuss these technical difficulties in more detail, as the light penetration through the beachrock is likely greatly limiting measurements of chlorophyll f and/or chlorophyll d-driven photosynthesis in the beachrock.

This can be seen in the different luminescence measurements (Figure 2 and supplements), that the different samples have clear differences in far-red light-driven oxygen production. While the BLACK sample produces oxygen with 740nm LED filtered with a NIR-75N filter, neither of the other two samples produce measureable oxygen under this condition. Conversely, this sample results in the lowest level of gross photosynthesis when measuring dissolved oxygen. A more detailed discussion of the variation between and within samples and measurements would benefit the overall results of the manuscript.

The PINK beachrock sample has the highest level of chlorophyll d per chlorophyll a. As FaRLiP cyanobacteria only incorporate 1 chlorophyll d per photosystem II, and none in photosytem I, is there a (relatively) high composition of Acaryochloris species in the PINK sample? If normalized to the reflectance minima can more distinct populations be identified?

For Figure 1, multiple points should be clarified. The first is that the HPLC methods are estimates of concentrations, as the extinction coefficients are not correct for the solvent solution for which the pigments elute, and are likely to be differently incorrect for each pigment. This results in quantitatively incorrect data, but qualitative comparisons between samples likely remain valid. Secondly, the pigment concentrations can also be misleading. Within the cyanobacterial cells, photosystem I harbors approximately 3 times as many chlorophylls as photosystem II. While the community numbers and photosystem stoichiometry are not necessarily relevant to the current study, the red shift in absorbance between photosystem II and photosystem I is of importance for the measurements performed. How cyanobacterial cells with differing concentrations of photosystems will absorb the red tail of the far-red LEDs, as well as impact the light penetration would be a useful discussion point.

The different samples used are from varying beachrock zonations but have the same chlorophyll f per chlorophyll a concentrations. A discussion of why this might be would be useful.

For the luminescence measurements (Figure 2 and supplements), no oxygen production is seen in the BROWN or PINK beachrock samples when the 740nm LED is filtered with a NIR-75N filter. This is likely due to multiple factors (low initial intensity compounded by penetration depth, community composition, etc.) but should be discussed. While the authors say that Chrooccidiopsis species dominate the samples, variation of absorbance between different chlorophyll f containing cyanobacteria has also been measured (see Tros et al. 2021, Chem), and the extent to which even chlorophyll f species extend into the far-red varies. Discussions about these implications would help with their characterization of the luminescence data. While the authors discuss that based on their respiration measurements the oxygen may be being consumed, resulting in an inability to measure it (lines 147-150), other explanations are clearly viable.

For the luminescence measurements, no oxygen production is discernable in the endolithic region when excited with visible light, which is at a much stronger intensity than the near-infrared light used. However, both Acaryochloris and chlorophyll f cyanobacteria are capable of driving photosynthesis with visible light. As the intensities used are much brighter than for the NIR measurements, presumably generated oxygen would be higher than what could be immediately consumed by respiration. It is important that the authors address this.

A highlighted point by the authors is the >20% of photosynthesis driven by NIR in the beachrock at comparable irradiation. However, this statement is deceiving for multiple reasons.<br /> (1) The irradiation is likely not comparable for what is reaching the cells. This is not a problem per se as illumination from above is the point, but does skew the interpretation.<br /> (2) The >20% value comes from the maximum amount of gross photosynthesis driven by NIR at ~1400 umol photons m-2s-1, whereas at other comparable illuminations the value is much, much lower (<1%). A likely interpretation of such data is that while the chlorophyll f endolithic layer is capable of producing a relatively large amount of oxygen, it is likely far less productive under most illuminations, though not zero.

The authors have the difficult task of weaving in results from laboratory, uniculture or isolated photosystem measurements with their environmental-based results. This is especially clear in lines 172-183. While the authors are correct that measurements of trapping times in chlorophyll f containing photosystems have been measured and are slower in chlorophyll f photosystem II and photosystem I relative to all chlorophyll a photosystems, the quantum yield for trapping remains high in chlorophyll f photosystem I (Tros et al. 2021, Chem). The quantum yield of trapping for chlorophyll f photosystem II is much lower for chlorophyll f than chlorophyll a complex, though improved by the attachment of phycobilisomes. However, these are intrinsic physical properties of the complexes that are not modulated in response to the environments. This could be interpreted that at low photon flux densities as measured in these experiments, the endolithic near infrared-driven oxygen production could be limited by an overall lower quantum efficiency of trapping the captured light and thus minimizing photosynthetic productivity relative to a theoretical level based on the efficiency of the chlorophyll a photosystem II. How the variations in intensity and spectral composition impact the cyanobacterial community likely involves many other factors and has not been addressed (though see Nurnberg et al. 2018, Science and Viola et al. 2022 eLife for further discussions).

Reviewer #2 (Public Review):

The authors investigate the role of near-infrared photosynthesis in primary production across three beachrock communities. This work is particularly pertinent as more cyanobacteria with far-red light acclimation capacities are discovered, underscoring the need to assess their contributions to primary production. However, the manuscript is currently very difficult to follow due to unclear correlations between the text and figures and the samples analyzed in the different experiments.. Additional explanations would also enhance clarity. For example, it would be beneficial for the authors to better define the three communities, as distinctions are not apparent. Another example is the pigment analysis, where the extinction coefficients for pigments vary in different solvents. Quantification by chromatography should use calibration curves for all pigments, not just Chl a, as is currently done. Pigments can be easily purified from cyanobacteria for this purpose.

Reviewer #3 (Public Review):

Summary:

On islands in the pacific, beachrock occurs near high tide level, composed of calcareous material. The surface of the beach rock is colonised by cyanobacteria and some eukaryotic algae. On Heron Island on the Southern Great Barrier Reef, beach rock occurs on the north and south side of the island in continuous slabs, which slope gently upwards toward the island. Thus the upper beach rock is only inundated at extreme high tides. On the south side, the major photosynthetic organism is a cyanobacterium Chroococcidiopsis, which forms tough smooth mats over all the beach rock. This cyanobacterium belongs to a newly discovered class called FaRLiP photosynthesisers, which carry out conventional photosynthesis under visible radiation using chlorophyll a (Chl a) but which deactivate most of the Chl a under near infra -red radiation (NIR) and produce chlorophyll f and chlorophyll d which can absorb NIR (700 - 760 nm). These NIR Chl molecules are repositioned in the reaction centres. In addition, an NIR-activated allophycocyanin (a phycobiliprotein) is synthesised and placed in the reaction centres. These FaRLiP cyanobacteria can carry out photosynthesis and primary production when placed under NIR. Here it is shown that in the mats of Chroococcidiopsis on the beach rock the upper layers carry out conventional photosynthesis while the lower layers carry out FaRLiP photosynthesis. It is shown that the FaRLiP-activated lower layers can produce up to 20% of the total photosynthetic primary production.

Strengths:

The authors have researched sections of beachrock obtained from the beach rock on Heron Island. The Beach Rock on Heron Island occurs on both sides of the Island lying in a semi-horizontal position slightly sloping upwards toward the Island. At normal high tide, only the upper parts are not submerged. Black crusts occur in the uppermost parts of the beachrock. Brown crusts occur in the intermediate sites and pink crusts occur at the lowest part of the beachrock.

The crusts are made up largely of cyanobacteria and the major component is a cyanobacterium of one species, tentatively identified by shape, pigmentation, and partial DNA analysis as Chroococcidiopsis.

In this investigation sections of the beach rock from different levels have been analysed using three techniques:

(1) Hyperspectral analysis to determine the layout of pigmented cells and their spectra.

(2) Bioluminescence to determine the spectra of the cells in the sections.

(3) Oxygen analysis, using luminescence lifetime imaging on special films closely applied to vertical sections of the beachrock.

(4) Oxygen production from the surface of three-dimensional blocks of beach rock, illuminated with white light or Near Infra Red (NIR) radiation, from above.

In addition, pigmentation has been analysed by High Performance Liquid Chromatography (HPLC).

These techniques allow the following conclusions:

(1) Scytonemin is a main screening compound for UV irradiation.

(2) Carotenoids also play a part in screening from UV and probably visible radiation.

(3) The cyanobacteria occur near the rock surface and contain Chl a plus some Chl f and a small amount of Chl d.

(4) HPLC pigment analysis confirms the presence of Chl a plus Chl f and a small amount of Chl d.

(5) The deeper layer with FaRLiP cyanobacteria produces oxygen under both visible light and NIR irradiation, with different P vs I curves.

(6) Using the oxygen chamber to measure oxygen exchange above the beach rock surface, it was shown that high respiration meant that only with the brown samples was significant oxygen released to the water column at lower light levels, i.e. respiration accounted for most of the primary production of oxygen except at the highest visible light intensities. And with NIR much lower levels of oxygen production only breaking compensation significantly in the brown samples.

(7) FaRLiP primary production was significant in the deeper layer.

The major new conclusion from these studies is that FaRLiP photosynthesis is a significant factor in this biofilm, and possibly other biofilms. Visible light is mostly absorbed in the upper layers and NIR reaching the lower layers induces FaRLiP photosynthesis and primary production, which can be up to 20% of the total primary production of the film.

Weaknesses:

The techniques are sufficient to justify the conclusions, especially the new result that the FaRLiP photosynthesis deeper in the films is surprisingly active with relatively high primary productivity. This is an important conclusion but it must be realised that there is some way to go to polish up the results and gain more quantitative results.

Firstly the beachrock is a heterogeneous material. So cutting a section leaves a non-homogeneous surface where various sand grains are removed, cut, or not removed. This means that when applying a luminescence film, the results are dependent on the uniformity of the surface or rather the lack of conformity. This needs to be taken into consideration in future studies.

Furthermore, previous papers have revealed that pits in the beach rock are important sites for FaRLiP cyanobacteria and the paper needs to make clear that these pits were avoided here.

Secondly, while Chroococcidiopsis is the major alga/cyanobacterium present, other algae/cyanobacteria are present and their presence needs to be factored into the results. In this regard we need more microscopic images of the surface and cross-sections of the beachrock, to reveal the nature of the bacterial and algal organisms.

Thirdly, it is not clear from this paper how far the identification of Chroococcidiopsis is firm. Presumably preliminary DNA analyses have been carried out on tell-tale genes (rRNA?). At some stage, a complete genome will be needed. Mention should be made about what has been done and what is contemplated.

Fourthly, the acclimation to FaRLiP is time-dependent. How long does it take in these beach rock sections? And has sufficient notice been taken of this time-dependent process?

Fifthly, FaRLiP is a sophisticated system as shown by Mascoli et al, 2022. It is activated in NIR by red-shifted allophycocyanin. It is also dependent on the allocation of Chl f and Chl d to special positions in the reaction centre. All this may take some time and be light-dependent. This may explain the curious increase in the slopes of light vs productivity of Fig 4 (Pink and Black) for NIR light.

The fifth point needs to be taken into account in any rewrite of the paper. The authors assume that the upwardly sloping P vs I curve is explained as follows:<br /> "This can be explained by the light attenuation due to scattering and absorption in the compacted beachrock biofilm, which prevented saturation of NIR-driven photosynthesis in the endolithic layer even at levels of incident light similar to solar irradiation on mid-day exposed beachrock."

Activation of the FaRLiP system also needs to be considered.

Reviewer #1 (Public Review):

Summary:

In this work, the authors study whether the human brain uses long term priors (acquired during our lifetime) regarding the statistics of auditory stimuli to make predictions respecting auditory stimuli. This is an important open question in the field of predictive processing.

To address this question, the authors cleverly profit from the naturally existing differences in two linguistic groups. While speakers of Spanish use phrases in which function-words (short words like, articles and prepositions) are followed by content-words (longer words like nouns, adjectives and verbs), speakers of Basque use phrases with the opposite order. Because of this, speakers of Spanish usually hear phrases in which short words are followed by longer words, and speakers of Basque experience the opposite. This difference in the order of short and longer words is hypothesized to result in a long term duration prior that is used to make predictions regarding the likely durations of incoming sounds, even if they are not linguistic in nature.

To test this, the authors used MEG to measure the mismatch responses (MMN) elicited by the omission of short and long tones that were presented in alternation. The authors report an interaction between the language background of the participants (Spanish, Basque) and the type of omission MMN (short, long), which goes in line with their predictions. They supplement these results with a source level analysis.

Strengths:

This work has many strengths. To test the main question, the authors profit from naturally occurring differences in the everyday auditory experiences of two linguistic groups, which allows to test the effect of putative auditory priors consolidated over the years. This is a direct way of testing the effect of long term priors.

The fact that the priors in question are linguistic and that the experiment was conducted using non-linguistic stimuli (i.e. simple tones), allows to test if these long term priors generalize across auditory domains.

The experimental design is elegant and the analysis pipeline appropriate. This work is very well written. In particular the introduction and discussion sections are clear and engaging. The literature review is complete.

Weaknesses:

The authors report a widespread omission response, which resembles the classical mismatch response (in MEG planar gradiometers) with strong activations in sensors over temporal regions. However the interaction reported is circumscribed to four sensors that do not overlap with the peaks of activation of the omission response.

Reviewer #2 (Public Review):

Summary:

Morucci et al. tested the influence of linguistic prosody long-term priors in forming predictions about simple acoustic rhythmic tone sequences composed of alternating tone duration, by violating context-dependent short-term priors formed during sequence listening. Spanish and Basque participants were selected due to the different rhythmic prosody of the two languages (functor-initial vs. Functor final, respectively), despite a common cultural background. The authors found that neuromagnetic responses to casual tone omissions reflected the linguistic prosody pattern of the participant's dominant language: in Spanish speakers, omission responses were larger to short tones, whereas in Basque speakers, omission responses were larger to long tones. Source localization of these responses revealed this interaction pattern in the left auditory cortex, which the authors interpret as reflecting a perceptual bias due to acoustic cues (inherent linguistic rhythms, rather than linguistic content). Importantly, this pattern was not found when the rhythmic sequence entailed pitch, rather than duration, cues. To my knowledge, this is the first study providing neural signatures of a known behavioral effect linking ambiguous rhythmic tone sequence perceptual organization to linguistic experience.

The conclusions of the study are well supported by the data. The hypotheses, albeit allowing alternative perspectives, are well justified according to the existing literature. Albeit with inconclusive results, additional analyses to test entrained oscillatory activity to the perceived rhythms have been performed, which adds explanatory power to the study.

Strengths:

(1) The choice of participants. The bilingual population of the Basque country is perfect for performing studies which need to control for cultural and socio-economic background while having profound linguistic differences. In this sense, having dominant Basque speakers as a sample equates that in Molnar et al. (2016), and thus overcomes the lack of direct behavioral evidence for a difference in rhythmic grouping across linguistic groups. Molnar et al. (2016)'s evidence on the behavioral effect is compelling, and the evidence on neural signatures provided by the present study aligns with it.

(2) The experimental paradigm. It is a well designed acoustic sequence, which considers aspects such as gap length insertion, to be able to analyze omission responses free from subsequent stimulus-driven responses, and which includes a control sequence which uses pitch instead of duration as a cue to rhythmic grouping, which provides a stronger case for the differences found between groups to be due to prosodic duration cues.

(3) Data analyses. Sound, state-of-the-art methodology in the event-related field analyses at the sensor and source levels.

Weaknesses:

(1) The main conclusion of the study reflects a known behavioral effect on rhythmic sequence perceptual organization driven by linguistic background (Molnar et al. 2016, particularly) and, thus, the novelty of the findings is restricted to neural activity evidence.

(2) Although the paradigm is well designed, there are alternative views in formulating the hypotheses. For instance, one could argue that, according to predictive coding views, omission responses should be larger when the gap occurs at the end of the pattern, as that would be where stronger expectations are placed. However, the authors provide good justification based on previous literature for the expectation of larger omission responses at the downbeat of a rhythmic pattern.

Reviewer #3 (Public Review):

Summary:

The paper investigates the effects of long-term linguistic experience on early auditory processing, a subject that has been relatively less studied compared to short-term influences. Using MEG, the study examines brain responses to auditory stimuli in speakers of Spanish and Basque, whose syntactic rules provide different degrees of exposure to durational patterns (long-short vs short-long). The findings suggest that both long-term language experience as well as short-term transitional probabilities can shape auditory predictive coding for non-linguistic sound sequences, evidenced by differences in mismatch negativity amplitudes localised to left auditory cortex.

Strengths:

The study integrates linguistics and auditory neuroscience in an interesting interdisciplinary way that may interest linguists as well as neuroscientists. The fact that long-term language experience affects early auditory predictive coding is important for understanding group and individual differences in domain-general auditory perception. It has importance for neurocognitive models of auditory perception (e.g. inclusion of long-term priors), and will be of interest to researchers in linguistics, auditory neuroscience, and the relationship between language and perception. The inclusion of a control condition based on pitch is also a strength.

Weaknesses:

The main weaknesses are the strength of the effects and generalisability. Only two languages were examined, Spanish and Basque. The sample size is also relatively small by today's standards, with N=20 in each group. Furthermore, the crucial effects are all mostly in the .01>P<.05 range, such as the crucial interaction P=.03, although I note the methods used to derive the results are sound and state-of-the-art. It would be nice to see it replicated in the future, with more participants and other languages. It would also have been nice to see behavioural data that could be correlated with neural data to better understand the real-world consequences of the effect.

Reviewer #3 (Public Review):

Summary:

This study aims to understand gene regulation of the plant bacterial pathogen Pseudomonas syringae. Although the function of some TFs has been characterized in this strain, a global picture of the gene regulatory network remains elusive. The authors conducted a large-scale ChIP-seq analysis, covering 170 out of 301 TFs of this strain, and revealed gene regulatory hierarchy with functional validation of some previously uncharacterized TFs.

Strength:

- This study provides one of the largest ChIP-seq datasets for a single bacterial strain, covering more than half of its TFs. This impressive resource enabled comprehensive systems-level analysis of the TF hierarchy.<br /> - This study identified novel gene regulation and function with validations through biochemical and genetic experiments.<br /> - The authors conducted broad analyses including comparisons between different bacterial strains, providing further insights into the diversity and conservation of gene regulatory mechanisms.

Reviewer #2 (Public Review):

Summary:

The phytopathogenic bacterium Pseudomonas syringae is comprised of many pathovars with different host plant species and has been used as a model organism to study bacterial pathogenesis in plants. Transcriptional regulation is key to plant infection and adaptation to host environments by this bacterium. However, researches have focused on limited number of transcription factors (TFs) that regulate virulence-related pathways. Thus, a comprehensive, systems-level understanding of regulatory interactions between transcription factors in P. syringae has not been achieved.

This study by Sun et al performed ChIP-seq analysis of 170 out of 301 TFs in P. syringae pv. syringae 1448A and used this unique dataset to infer transcriptional regulatory networks in this bacterium. The network analyses revealed hierarchical interactions between TFs, various network motifs, and co-regulation of target genes by TF pairs, which collectively mediate information flow. As discussed, the structure and properties of the P. syringae transcriptional regulatory networks are somewhat different from those identified in humans, yeast, and E. coli, highlighting the significance of this study. Further, the authors made use of the P. syringae transcriptional regulatory networks to find TFs of unknown functions to be involved in virulence-related pathways. For some of these TFs, their target specificity and biological functions, such as motility and biofilm formation, were experimentally validated. Of particular interest is the finding that despite conservation of TFs between P. syringae pv. syringae 1448A, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. actinidiae C48, some of the conserved TFs show different repertoires of target genes in these four P. syringae strains.

Strengths:

This study presents a systems-level analysis of transcriptional regulatory networks in relation to P. syringae virulence and metabolism, highlights differences in transcriptional regulatory landscapes of conserved TFs between different P. syringae strains, and develops a user-friendly database for mining the ChIP-seq data generated in this study. These findings and resources will be valuable to researchers in the fields of systems biology, bacteriology, and plant-microbe interactions.

Weaknesses:

No major weaknesses were found, but some of the results may need to be interpreted with caution. ChIP-seq was performed with bacterial strains overexpressing TFs. This may cause artificial binding of TFs to promoters which may not occur when TFs are expressed at physiological levels. Another caution is applied to the interpretation of the biological functions of TFs during plant infection, as biological roles of the tested TFs are mostly based on in vitro experiments.

This work advances our understanding of transcriptional regulation of virulence and metabolic pathways in plant pathogenic bacteria. Solid evidence for the claims is provided by computational analysis of newly generated data on the genome-wide binding of 170 transcription factors to their target genes, together with experimental validation of the biological functions of some of these transcription factors. The findings and resources from this study will be valuable to researchers in the fields of systems biology, bacteriology, and plant-microbe interactions.

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, expression of Sex-lethal (Sxl) protein determines sexual identity and drives female development. Functional Sxl protein is absent from males where splicing includes a termination codon-containing "poison" exon. Early during development, in the soma of female individuals, Sxl expression is initiated by an X chromosome counting mechanism that activates the Sxl establishment promoter (SxlPE) to produce an initial amount of Sxl protein. This then suppresses the inclusion of the "poison" exon, directing the constructive splicing of Sxl transcripts emerging from the Sxl maintenance promotor (SxlPM) which is activated at a later stage during development irrespective of sex. This autoregulatory loop maintains Sxl expression and commits to female development.

Sxl also determines the sexual identity of the germline. Here Sxl expression generally follows the same principles as in somatic tissues, but the way expression is initiated differs from the soma. This regulation has so far remained elusive.

In the presented manuscript, Goyal et al. show that activation of Sxl expression in the germline depends on additional regulatory DNA sequences, or sequences different from the ones driving initial Sxl expression in the soma. They further demonstrate that sisterless A (sisA), a transcription factor that is required for activation of Sxl expression in the soma, is also necessary, but not sufficient, to initiate the expression of functional Sxl protein in female germ cells. sisA expression precedes Sxl induction in the germline and its ablation by RNAi results in impaired expression of Sxl, formation of ovarian tumors, and germline loss, phenocopying the loss of Sxl. Intriguingly, this phenotype can be rescued by the forced expression of Sxl, demonstrating that the primary function of sisA in the germline is the induction of Sxl expression.

Strengths:

The clever design of probes (for RNA FISH) and reporters allowed the authors to dissect Sxl expression from different promoters to get novel insight into sex-specific gene regulation in the germline. All experiments are carefully controlled. Since Sxl regulation differs between the soma and the germline, somatic tissues provide elegant internal controls in many experiments, ensuring e.g. functionality of the reporters. Similarly, animals carrying newly generated alleles (e.g. genomic tagging of the Sxl locus) are fertile and viable, demonstrating that the genetic manipulation does not interfere with protein function. The conclusions drawn from the experimental data are sound and advance our understanding of how Sxl expression is induced in the female germline.

Weaknesses:

The assays employed by the authors provide valuable information on when Sxl promoters become active. However, since no information on the stability of the gene products (i.e. RNA and protein) is available, it remains unclear when the SxlPE promoter is switched off in the germline (conceptually it only needs to be active for a short time period to initiate production of functional Sxl protein). As correctly stated by the authors, the persisting signals observed in the germline might therefore not reflect the continuous activity of the SxlPE promoter.

Mapping of regulatory elements and their function: SxlPE with 1.5 kb of flanking upstream sequence is sufficient to recapitulate early Sxl expression in the soma. The authors now provide evidence that beyond that, additional DNA sequences flanking the SxlPE promoter are required for germline expression. However, a more precise mapping was not performed. Also, due to technical limitations, the authors could not precisely map the sisA binding sites. Since this protein is also involved in the somatic induction of Sxl, its binding sites likely reside in the region 1.5kb upstream of the SxlPE promoter, which has been reported to be sufficient for somatic regulation. The regulatory role of the sequences beyond SxlPE-1.5kb therefore remains unaddressed and it remains to be investigated which trans-acting factor(s) exert(s) its/their function(s) via this region.

The central question of how Sxl expression is initiated and controlled in the germline still remains unanswered. Since sisA is zygotically expressed in both the male and the female germline (Figure 4D), it is unlikely the factor that restricts Sxl expression to the female germline.

How does weak expression of Sxl in male tissues or expression above background after knockdown of sisA reconcile with the model that an autoregulatory feedback loop enforces constant and clonally inheritable Sxl expression once Sxl is induced? Is the current model for Sxl expression too simple or are we missing additional factors that modulate Sxl expression (such as e.g. Sister of Sex-lethal)? While I do not expect the authors to answer these questions, I would expect them to appropriately address these intriguing aspects in the discussion.

Reviewer #2 (Public Review):

Summary:

The authors wanted to determine whether cis-acting factors of Sxl - two different Sxl promoters in somatic cells - regulate Sxl in a similar way in germ cells. They also wanted to determine whether trans-acting factors known to regulate Sxl in the soma also regulate Sxl in the germline.

Regarding the cis-acting factors, they examine the Sxl "establishment promoter" (SxlPE) that is activated in female somatic cells by the presence of two X chromosomes. Slightly later in development, dosage compensation equalizes X chromosome expression in males and females and so X chromosomes can no longer be counted. The second Sxl promoter is the "maintenance promoter," (SxlPM), which is activated in both sexes. The mRNA produced from the maintenance promoter has to be alternatively splicing from early Sxl protein generated earlier in development by the PE. This leads to an autoregulatory loop that maintains Sxl expression in female somatic cells. The authors used fluorescent in situ hybridization (FISH) with oligopaints to determine the temporal activation of the PE or PM promoters. They find that - unlike the soma - the PE does not precede the PM and instead is activated contemporaneously or later than the PM - this is confusing with the later results (see below). Next, they generated transcriptional reporter constructs containing large segments of the Sxl locus, the 1.5 kb used in somatic studies, a 5.2 kb reporter, and a 10.2 kb. Interestingly the 1.5 kb reporter that was reported to recapitulate Sxl expression in soma and germline was not observed by the authors. The 5.2 kb reporter was observed in female somatic cells but not in germ cells. Only when they include an additional 5 kb downstream of the 5.2 kb reporter (here the 10.2 kb reporter) they did see expression in germ cells but this occurred at the L1 stages. Their data indicate that Sxl activity in the germ requires different cis-regulation than the soma and that the PE is activated later in germ cells than in somatic cells. The authors next use gene editing to insert epitope tags in two distinct strains in the hopes of creating an early Sxl and a later Sxl protein derived from the PE and PM, respectively. The HA-tagged protein from the PE was seen in somatic cells but never in the germline, possibly due to very low expression. The FLAG-tagged late Sxl protein is observed in L2 germ cells. Because the early HA-Sxl protein is not perceptible in germ cells, it is not possible to conclude its role in the germline. However, because late FLAG-Sxl was only observed in L2 germ cells and the PE was detected in L1, this leaves open the possibility that PE produces early HA-Sxl (which currently cannot be detected), which then alternatively splices the transcript from the PM. In other words, the soma and germline could have a similar temporal relationship between the two Sxl promoters. While I agree with the authors about this conclusion, the earlier work with the oligopaints leads to the conclusion that SE is active after PM. This is confusing.

Next, the authors wanted to turn their attention to the trans-acting factors that regulate Sxl in the soma, including Sisterless A (SisA), SisB, Runt, and the JAK/STAT ligand Unpaired. Using germline RNAi, the authors found that only knockdown of SisA causes ovarian tumors, similar to the loss of Sxl, suggesting that SisA regulates Sxl (ie the PE) in both the soma and the germline. They generated a SisA null allele using CRISPR/Cas9 and these animals had ovarian tumors and germ cell-less ovaries. FISH revealed that sisA is activated in primordial germ cells in stages 3-6 before the activation of Sxl. They used CRISPR-Cas9 to generate an endogenously-tagged SisA and found that tagged SisA was expressed in stage 3-6 PCGs, which is consistent with activating PE in the germline. They showed that sisA is upstream of Sxl as germline depletion of sisA led to a significant decrease in expression from the 10.2 kb PE reporter and in SXL protein. The authors could rescue the ovarian tumors and loss of Sxl protein upon germline depletion of sisA by supplying Sxl from another protein (the otu promoter). These data indicate that sisA is necessary for Sxl activation in the germline. However, ectopic sisA in germ cells in the testis did not lead to ectopic Sxl, suggesting that sisA is not sufficient to activate Sxl in the germline.

Strengths:

(1) The genetic and genomic approaches in this study are top-notch and they have generated reagents that will be very useful for the field.

(2) Excellent use of powerful approaches (oligo paint, reporter constructs, CRISPR-Cas9 alleles).

(3) The combination of state of art approaches and quantification of phenotypes allows the authors to make important conclusions.

Weaknesses:

(1) Confusion in line 127 (this indicates that SxlPE is not activated before SxlPM in the germline) about PE not being activated before the PM in the germline when later figures show that PE is activated in L1 and late Sxl protein is seen in L2. It would be helpful to the readers if the authors edited the text to avoid this confusion. Perhaps more explanation of the results at specific points would be helpful.

Reviewer #3 (Public Review):

Summary:

The mechanisms governing the initial female-specific activation of Sex-lethal (Sxl) in the soma, the subsequent maintenance of female-specific expression and the various functions of Sxl in somatic sex determination and dosage compensation are well documented. While Sxl is also expressed in the female germline where it plays a critical role during oogenesis, the pathway that is responsible for turning Sxl on in germ cells has been a long-standing mystery. This manuscript from Goyal et al describes studies aimed at elucidating the mechanism(s) for the sex-specific activation of the Sex-lethal (Sxl) gene in the female germline of Drosophila.

In the soma, the Sxl establishment promoter, Sxl-Pe, is regulated in pre-cellular blastoderm embryos in somatic cells by several X-linked transcription factors (sis-a, sis-b, sis-c and runt). At this stage of development, the expression of these transcription factors is proportional to gene dose, 2x females and 1x in males. The cumulative two-fold difference in the expression of these transcription factors is sufficient to turn Sxl-Pe on in female embryos. Transcripts from the Sxl-Pe promoter encode an "early" version of the female Sxl protein, and they function to activate a splicing positive autoregulatory loop by promoting the female-specific splicing of the initial pre-mRNAs derived from the Sxl maintenance promoter, Sxl-Pm (which is located upstream of Sxl-Pm). These female Sxl-Pm mRNAs encode a Sxl protein with a different N-terminus from the Sxl-Pe mRNAs, and they function to maintain female-specific splicing in the soma during the remainder of development.

In this manuscript, the authors are trying to understand how the Sxl-Pm positive autoregulatory loop is established in germ cells. If Sxl-Pe is used and its activation precedes Sxl-Pm as is true in the soma, they should be able to detect Sxl-Pe transcripts in germ cells before Sxl-Pm transcripts appear. To test this possibility, they generated RNA FISH probes complementary to the Sxl-Pe first exon (which is part of an intron sequence in the Sxl-Pm transcript) and to a "common sequence" that labels both Sxl-Pe and Sxl-Pm transcripts. Transcripts labeled by both probes were detected in germ cells beginning at stage 5 (and reaching a peak at stage 10), so either the Sxl-Pm and Sxl-Pe promoters turn on simultaneously, or Sxl-Pe is not active.

They next switched to Sxl-Pe reporters. The first Sxl-Pe:gfp reporter they used has a 1.5 kb upstream region which in other studies was found to be sufficient to drive sex-specific expression in the soma of blastoderm embryos. Also like the endogenous Sxl gene it is not expressed in germ cells at this early stage. In 2011, Hashiyama et al reported that this 1.5 kb promoter fragment was able to drive gfp expression in Vasa-positive germ cells later in development in stage 9/10 embryos. However, because of the high background of gfp in the nearby soma, their result wasn't especially convincing. Though they don't show the data, Goyal et al indicated that unlike Hashiyama et al they were unable to detect gfp expressed from this reporter in germ cells. Goyal et al extended the upstream sequences in the reporter to 5 kb, but they were still unable to detect germline expression of gfp.

Goyal et al then generated a more complicated reporter which extends 5 kb upstream of the Sxl-Pe start site and 5 kb downstream-ending at or near 4th exon of the Sxl-Pm transcript (the Sxl-Pe10 kb reporter). (The authors were not explicit as to whether the 5 kb downstream sequence extended beyond the 4th exon splice junction-in which case splicing could potentially occur with an upstream exon(s)-or terminated prior to the splice junction as seems to be indicated in their diagram.) With this reporter, they were able to detect sex-specific gfp expression in the germline beginning in L1 (first instar larva). With the caveat that gfp detection might be delayed compared to the onset of reporter activation, these findings indicated that the sequences in the reporter are able to drive sex-specific transcription in the germline at least as early as L1.

The authors next tagged the N-terminal end of the Sxl-Pe protein with HA (using Crispr/Cas9) and the N-terminal end of Sxl-Pm protein with Flag. They report that the HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis. Somatic HA-Sxl-Pe protein persists into L1, but is no longer detected in L2. However, while somatic HA-Sxl-Pe protein is detected, they were unable to detect HA-Sxl-Pe protein in germ cells. In the case of FLAG-Sxl-Pm, it could first be detected in L2 germ cells indicating that at this juncture the Sxl-positive autoregulatory loop has been activated. This contrasts with Sxl-Pm transcripts which are observed in a few germ cells at stage 5 of embryogenesis, and in most germ cells by stage 10. The authors propose (based on the expression pattern of the Sxl-Pe10kb reporter and the appearance of Flag-Sxl-Pm protein) that Sxl-Pe comes on in germ cells in L1, and that the Sxl-Pe protein activates the female splicing of Sxl-Pm transcripts, giving detectable Flag-Sxl-Pm proteins beginning in L2.

To investigate the signals that activate Sxl-Pe in germ cells, the authors tested four of the X-linked genes (sis-a, sis-b, sis-c, and runt) that function to activate Sxl-Pe in the soma in early embryos. RNAi knockdown of sis-b, sis-c, and runt had no apparent effect on oogenesis. In contrast, knockdown of sis-a resulted in tumorous ovaries, a phenotype associated with Sxl mutations. (Three different RNAi transgenes were tested-two gave this phenotype, the third did not.) Sxl-Pe10kb reporter activity in L1 female germ cells is also dependent on sis-A.

Several approaches were used to confirm a role for sis-a in a) oogenesis and b) the activation of the Sxl-Pm autoregulatory loop. They showed that sis-a germline clones (using tissue-specific Crispr/Cas9 editing) resulted in the tumorous ovary phenotype and reduced the expression of Sxl protein in these ovaries. They found that sis-a transcripts and GFP-tagged Sis-A protein are present in germ cells. Finally, they showed tumorous ovary phenotype induced by germline RNAi knockdown of sis-a can be partially rescued by expressing Sxl in the germ cells.

Critique:

While this manuscript addresses a longstanding puzzle - the mechanism activating the Sxl autoregulatory loop in female germ cells-and likely identified an important germline transcriptional activator of Sxl, sis-a, the data that they've generated doesn't make a compelling story. At every step, there are puzzle pieces that don't fit the narrative. In addition, some of their findings are inconsistent with many previous studies.

(1) The authors used RNA FISH to time the expression of Sxl-Pe and Sxl-Pm transcripts in germ cells. Transcripts complementary to Sxl-Pe and Sxl-Pm were detected at the same time in embryos beginning at stage 5. This is not a definitive experiment as it could mean a) that Sxl-Pe and Sxl-Pm turn on at the same time, b) that Sxl-Pe comes on after Sxl-Pm (as suggested by the Sxl-Pe10kb reporter) or c) Sxl-Pe never comes on.

(2) Hashiyama et al reported that they detected gfp expression in stage 9/10 germ cells from a 1.5 kb Sxl-Pe-gfp. As noted above, this result wasn't entirely convincing and thus it isn't surprising that Goyal et al were unable to reproduce it. Extending the upstream sequences to just before the 1st exon of Sxl-Pm transcripts also didn't give gfp expression in germ cells. Only when they added 5 kb downstream did they detect gfp expression. However, from this result, it isn't possible to conclude that the Sxl-Pe promoter is actually driving gfp expression in L1 germ cells. Instead, the Sxl promoter active in the germ line could be anywhere in their 10 kb reporter.

(3) At least one experiment suggests that Sxl-Pe never comes on in germ cells. The authors tagged the N-terminus of the Sxl-Pe protein with HA and the N-terminus of the Sxl-Pm protein with Flag. Though they could detect HA-Sxl-Pe protein in the soma, they didn't detect it in germ cells. On the other hand, the Flag-Sxl-Pm protein was detected in L2 germ cells (but not earlier). These results would more or less fit with those obtained for the 10 kb reporter and would support the following model: Prior to L1, Sxl-Pm transcripts are expressed and spliced in the male pattern in both male and female germ cells. During L1, Sxl protein expressed via a mechanism that depends upon a 10 kb region spanning Sxl-Pe (but not on Sxl-Pe) is produced and by L2 there are sufficient amounts of this protein to switch the splicing of Sxl-Pm transcripts from a male to a female pattern-generating Flag-tagged Sxl-Pm protein.

(4) The 10kb reporter is sex-specific, but not germline-specific. The levels of gfp in female L1 somatic cells are equal to if not greater than those in L1 female germ cells. That the Sxl-Pe10kb reporter is active in the soma complicates the conclusion that it represents a germ line-specific promoter. Germline activity is, however, sensitive to sis-A knockdowns which is plus. Presumably, somatic expression of the reporter wouldn't be sensitive to a (late) sis-A knockdown- but this wasn't shown.

(5) Their results with the HA-Sxl-Pe protein don't fit with many previous studies-assuming that the authors have explained their results properly. They report that HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis and that it then persists till L2. However, previous studies have shown that Sxl-Pe transcripts and then Sxl-Pe proteins are first detected in ~NC11-NC12 embryos. In RNase protection experiments, the Sxl-Pe exon is observed in 2-4 hr embryos, but not detected in 5-8 hr, 14-12 hr, L1, L2, L3, or pupae. Northerns give pretty much the same picture. Western blots also show that Sxl-Pe proteins are first detectable around the blastoderm stage. So it is not at all clear why HA-Sxl-Pe proteins are first observed at stage 9 which, of course, is well after the time that the Sxl-Pm autoregulatory loop is established.

Given the obvious problems with the initial timing of somatic expression described here, it is hard to know what to make of the fact that HA-tagged Sxl-Pe proteins aren't observed in germ cells.

As for the presence of HA-Sxl-Pe proteins later than expected: While RNase protection/Northern experiments showed that Sxl-Pe mRNAs are expressed in 2-4 hr embryos and disappear thereafter, one could argue from the published Western experiments that the Sxl-PE proteins expressed at the blastoderm stage persist at least until the end embryogenesis, though perhaps at somewhat lower levels than at earlier points in development. So the fact that Goyal et al were able to detect HA-Sxl-Pe proteins in stage 9 embryos and later on in L1 larva probably isn't completely unexpected. What is unexpected is that the HA-Sxl-Pe proteins weren't present earlier.

(6) The authors use RNAi and germline clones to demonstrate that sis-A is required for proper oogenesis: when sis-A activity is compromised in germ cells, i) tumorous ovary phenotypes are observed and ii) there is a reduction in the expression of Sxl-Pm protein. They are also able to rescue the phenotypic effects of sis-a knockdown by expressing a Sxl-Pm protein. While the experiments indicating sis-a is important for normal oogenesis and that at least one of its functions is to ensure that sufficient Sxl is present in the germline stem cells seem convincing, other findings would make the reader wonder whether Sis-A is actually functioning (directly) to activate Sxl transcription from promoter X.

The authors show that sis-a mRNAs and proteins are expressed in stage 3-5 germ cells (PGCs). This is not unexpected as the X-linked transcription factors that turn Sxl-Pe on are expressed prior to nuclear migration, so their protein products should be present in early PGCs. The available evidence suggests that their transcription is shut down in PGCs by the factors responsible for transcriptional quiescence (e.g., nos and pgc) in which case transcripts might be detected in only one or two PGC-which fits with their images. However, it is hard to believe that expression of Sis-A protein in pre-blastoderm embryos is relevant to the observed activation of the Sxl-Pm autoregulatory loop hours later in L2 larva.

It is also not clear how the very low level of gfp-Sis-A seen in only a small subset of migrating germ cells in stage 10 embryos (Figure S6) would be responsible for activating the Sxl-Pe10kb reporter in L1. It seems likely that the small amount of protein seen in stage 10 embryos is left over from the pre-cellular blastoderm stage. In this case, it would not be surprising to discover that the residual protein is present in both female and male stage 10 germ cells. This would raise further doubts about the relevance of the gfp-Sis-A at these early stages.

In fact, given the evidence presented implicating sis-a in activating Sxl, (the germline activation of the Sxl-Pe10kb reporter, the RNAi knockdowns, and the germ cell-specific sis-a clones) it is clear that the sis-A RNAs and proteins seen in pre-cellular blastoderm PGCs aren't relevant. The germline clone experiment (and also the RNAi knockdowns) indicates that sis-A must be transcribed in germ cells after Cas9 editing has taken place. Presumably, this would be after transcription is reactivated in the germline (~stage 10) and after the formation of the embryonic gonad (stage 14) so that the somatic gonadal cells can signal to the germ cells. With respect to the reporter, the relevant time frame for showing that sis-A is present in germ cells would be even later in L1.

(7) As noted above, the data in this manuscript do not support the idea that Sxl-Pe proteins activate the Sxl-Pm female splicing in the germline. Flybase indicates that there is at least one other Sxl promoter that could potentially generate a transcript that includes the male exon but still could encode a Sxl protein. This promoter "Sxl-Px" is located downstream of Sxl-Pm and from its position it could have been included in the authors' 10 kb reporter. The reported splicing pattern of the endogenous transcript skips exon2, and instead links an exon just downstream of Sxl-Px to the male exon. The male exon is then spliced to exon4. If the translation doesn't start and end at one of the small upstream orfs in the exons close to Sxl-Px and the male exon, a translation could begin with an AUG codon in exon4 that is in frame with the Sxl protein coding sequence. This would produce a Sxl protein that lacks aa sequences from N-terminus, but still retains some function.

Another possible explanation for how gfp is expressed from the 10 kb reporter is that the transcript includes the "z" exon described by Cline et al., 2010.

Reviewer #1 (Public Review):

In this manuscript, Liu et al. used scRNA-seq to characterize cell type-specific responses during allergic contact dermatitis (ACD) in a mouse model, specifically the hapten-induced DNFB model. Using the scRNA-seq data, they deconvolved the cell types responsible for the expression of major inflammatory cytokines such as IFNG (from CD4 and CD8 T cells), IL4/13 (from basophils), IL17A (from gd T cells), and IL1B from neutrophils and macrophages. They found the highest upregulation of a type 1 inflammatory response, centering around IFNG produced by CD4 and CD8 T cells. They further identified a subpopulation of dermal fibroblasts (pre-adipocytes found in the dermal white adipose tissue layer) that upregulate CXCL9/10 during ACD and provide functional genetic evidence in their mouse model that disrupting IFNG signaling in fibroblasts decreases CD8 T cell infiltration and overall inflammation. They identify an increase in IFNG-expressing CD8 T cells in human patient samples of ACD vs. healthy control skin and co-localization of CD8 T cells with PDGFRA+ fibroblasts, which suggests this mechanism is relevant to human ACD. This mechanism is reminiscent of recent work showing that IFNG signaling in dermal fibroblasts upregulates CXCL9/10 to recruit CD8 T cells in a mouse model of vitiligo. Overall, this is a well-presented, clear, and comprehensive manuscript. The conclusions of the study are well supported by the data, with thoughtful discussion on study limitations by the authors. One such limitation was the use of one ACD model (DNFB), which prevents an assessment of how broadly relevant this axis is. The human sample validation is limited by the multiplexing capacity of immunofluorescence markers but shows a predominance of CD8+/IFNG+ cells and PDGFRA+/CXCL10+ cells in ACD (which are virtually absent in healthy control), along with co-localization of CD8+ cells with PDGFRA+ cells. Thus, this mechanism is likely active in human ACD.

Strengths:<br /> Through deep characterization of the in vivo ACD model using scRNA-seq, the authors were able to determine which cell types were expressing the major cytokines involved in ACD inflammation, such as IFNG, IL4/13, IL17A, and IL1B. These analyses are well-presented and thoughtful, showing first that the response is IFNG-dominant, then focusing on deeper characterization of lymphocytes, myeloid cells, and fibroblasts, which are also validated and complemented by FACS experiments using canonical markers of these cell types as well as IF staining. Crosstalk analyses from the scRNA-seq data led the authors to focus on IFNG signaling fibroblasts, and in vitro experiments demonstrate that CXCL9 and CXCL10 are expressed by fibroblasts stimulated by IFNG. In vivo functional genetic evidence demonstrates an important role for IFNG signaling in fibroblasts, as KO of Ifngr1 using Pdgfra-Cre Ifngr1 fl/fl mice, showed a reduction in inflammation and CD8 T cell recruitment. Human ACD sample staining demonstrates the likely activity of the CD8 T cell IFNG-driven fibroblast response in human disease.

Weaknesses:<br /> The use of one model limits an understanding of how broad this fibroblast-T cell axis is during ACD. However, the authors chose the most commonly employed model and compared their data to work in a vitiligo model (another type 1 immune response) to demonstrate similar mechanisms at play. Human patient samples of ACD were co-stained with two markers at a time, demonstrating the presence of CD8+IFNG+ T cells, PDGFRA+CXCL10+ fibroblasts, and co-localization of PDGFRA+ fibroblasts and CD8+ T cells. However, no IF staining demonstrates co-expression of all 4 markers at once; thus, the human validation of co-localization of CD8+IFNG+ T cells and PDGFRA+CXCL10+ fibroblasts is ultimately indirect, although more likely than not to be true.

Reviewer #2 (Public Review):

Summary: The investigators apply scRNA seq and bioinformatics to identify biomarkers associated with the DNFB-induced contact dermatitis in mice. The bioinformatics component of the study appears reasonable and may provide new insights regarding TH1 driven immune reactions in ACD in mice. However, the IF data and images of tissue sections are not clear and should be improved to validate the model.

Strengths:<br /> The bioinformatics analysis.

Weaknesses:<br /> The IF data presented in 4H, 6H, 7E and 7F are not convincing and need to be correlated with routine staining on histology and different IF markers for PDGFR. Some of the IF staining data demonstrates a pattern inconsistent with its target.

Reviewer #1 (Public Review):

Review after revision

Of note the main results of this article are very similar to the results present in the previous manuscript (same Figures 1 to 9, addition of Figure 10 with no quantification).<br /> Unfortunately, the main weaknesses of the article have not been addressed:

(1) The main findings have been obtained in clones of Jurkat cells. They have not been confirmed in primary T cells. The only experiment performed in primary cells is shown in Figure S7 (primary human T lymphoblasts) for which only the distribution of FMNL1 is shown without quantification. No results presenting the effect of FMNL1 KO and expression of mutants in primary T cells are shown.

(2) Analysis in- depth of the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling) is still lacking. Only F-actin is shown, no attempt to look more precisely at actors of actin remodeling has been done.

(3) The defect in the secretion of extracellular vesicles is still very preliminary. Examples of STED images given by the authors are nice, yet no quantification is performed.

(4) Results shown in Figure S12 on the colocalization of proteins phosphorylated on Ser/Thr are still not convincing. It seems indeed that "phospho-PKC" is labeling more preferentially the CMAC positive cells (Raji) than the Jurkat T cells. It is thus particularly difficult to conclude on the co-localization and even more on the recruitment of phosphorylated-FMNL1 at the IS. Thus, these experiments are not conclusive and cannot be the basis even for their cautious conclusion: "Although all these data did not allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1bWT are specifically phosphorylated at the cIS".

The study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments.

Reviewer #2 (Public Review):

Summary

Based on i) the documented role of FMNL1 proteins in IS formation; ii) their ability to regulate F-actin dynamics; iii) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation; and iv) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified, the authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated FMNL1beta acts downstream of PKCdelta to regulate centrosome and MVB polarization to the IS and exosome release. They provide evidence that FMNL1beta accumulates at the IS where it promotes F-actin clearance from the IS center, thus allowing for MVB secretion.

Strengths

The work is based on a solid rationale, which includes previous findings by the authors establishing a link between PKCdelta, FMNL1beta phosphorylation, synaptic F-actin clearance and MVB polarization to the IS. The authors have thoroughly addressed the working hypotheses using robust tools. Among these, of particular value is an expression vector that allows for simultaneous RNAi-based knockdown of the endogenous protein of interest (here all FMNL1 isoforms) and expression of wild-type or mutated versions of the protein as YFP-tagged proteins to facilitate imaging studies. The imaging analyses, which are the core of the manuscript, have been complemented by immunoblot and immunoprecipitation studies, as well as by the measurement of exosome release (using a transfected MVB/exosome reporter to discriminate exosomes secreted by T cells).

Weaknesses

The authors have satisfactorily addressed the weaknesses pointed out in my previous review.

Reviewer #1 (Public Review):

Summary:

This paper presents valuable findings that gustation and feeding state influence the preferred environmental temperature preference in flies. Interestingly, the authors showed that by refeeding starved animals with non-nutritive sugar sucralose, they are able to tune their preference towards a higher temperature in addition to nutrient-dependent warm preference. The authors show that temperature sensing and sweet sensing gustatory neurons (SGNs) are involved in the former but not the latter. In addition, their data indicate that peptidergic signals involved in internal state and clock genes are required for taste-dependent warm preference behavior.

The authors made an analogy of their results to the cephalic phase response (CPR) in mammals where the thought, sight and taste of food prepares the animal for the consumption of food and nutrients. The authors showed that taste triggers CPR-induced temperature preference behaviors in flies. The authors also briefly covered that the combined modalities of smell and taste induced CPR responses, showing that starved orco mutant flies failed to recover temperature preference after refeeding with sucralose.

The findings of this work hold promising future research prospects, for example, whether the sight of food influences temperature preference behavior in hungry flies, or whether taste, smell and sight work together or independently in promoting CPR responses.

Futhermore, these valuable behavioral results can be further investigated in flies with the advantage of being able to dissect the neural circuitry underlying CPR and nutrient homeostasis.

Strengths:

(1) The authors convincingly showed that tasting is sufficient to drive warm temperature preference behavior in starved flies and show that it is independent of nutrient-driven warm preference.<br /> (2) By using the genetic manipulation of key internal sensors and genes controlling internal feeding and sleep state such as DH44 neurons and the per genes for eg the authors linked gustation and temperature preference behavior control to the internal state of the animal.

Weaknesses:

Most of the weaknesses of the paper have been addressed in the revision. The points mentioned below are meant to improve readability of the paper and to promote understanding of the significance of the work.<br /> (1) Supplementary fig 1 could replace Figure 1A. The purpose of Figure 1F is not clear to me as the comparison between the different food substances is not separately addressed anywhere in the text.<br /> (2) The data for the orco receptor mutant could be placed in the main figures to justify the discussion emphasising CPR-like responses.

Reviewer #2 (Public Review):

Animals constantly adjust behavior and physiology based on internal states. Hungry animals, desperate for food, exhibit physiological changes immediately upon sensing, smelling, or chewing food, known as the cephalic phase response (CPR), involving processes like increased saliva and gastrointestinal secretions. While starvation lowers body temperature, the mechanisms underlying how the sensation of food without nutrients induces behavioral responses remain unclear. Hunger stress induces changes in both behavior and physiological responses, which in flies (or at least in Drosophila melanogaster) leads to a preference for lower temperatures, analogous to the hunger-driven lower body temperature observed in mammals. In this manuscript, the authors have used Drosophila melanogaster to investigate the issue of whether taste cues can robustly trigger behavioral recovery of temperature preference in starving animals. The authors find that food detection triggers a warm preference in flies. Starved flies recover their temperature preference after food intake, with a distinction between partial and full recovery based on the duration of refeeding. Sucralose, an artificial sweetener, induces a warm preference, suggesting the importance of food-sensing cues. The paper compares the effects of sucralose and glucose refeeding, indicating that both taste cues and nutrients contribute to temperature preference recovery. The authors show that that sweet gustatory receptors (Grs) and sweet GRNs (Gustatory Receptor Neurons) play a crucial role in taste-evoked warm preference. Optogenetic experiments with CsChrimson support the idea that the excitation of sweet GRNs leads to a warm preference. The authors then examine the internal state's influence on taste-evoked warm preference, focusing on neuropeptide F (NPF) and small neuropeptide F (sNPF), analogous to mammalian neuropeptide Y. Mutations in NPF and sNPF result in a failure to exhibit taste-evoked warm preference, emphasizing their role in this process. However, these neuropeptides appear not to be critical for nutrient-induced warm preference, as indicated by increased temperature preference during glucose and fly food refeeding in mutant flies. The authors also explore the role of hunger-related factors in regulating taste-evoked warm preference. Hunger signals, including diuretic hormone (DH44) and adipokinetic hormone (AKH) neurons, are found to be essential for taste-evoked warm preference but not for nutrient-induced warm preference. Additionally, insulin-like peptide 6 (Ilp6) and Unpaired3 (Upd3), related to nutritional stress, are identified as crucial for taste-evoked warm preference. The investigation then extends into circadian rhythms, revealing that taste-evoked warm preference does not align with the feeding rhythm. While flies exhibit a rhythmic feeding pattern, taste-evoked warm preference occurs consistently, suggesting a lack of parallel coordination. Clock genes, crucial for circadian rhythms, are found to be necessary for taste-evoked warm preference but not for nutrient-induced warm preference.

Strengths:

A well-written and interesting study, investigating an intriguing issue. The claims, none of which to the best of my knowledge controversial, are backed by a substantial number of experiments.

Weakness:

The experimental setup used and the procedures for assessing the temperature preferences of flies is rather sparingly described. Additional details and data presentation would enhance the clarity and replicability of the study. I kindly request the authors to consider the following points: i) A schematic drawing or diagram illustrating the experimental setup for the temperature preference assay would greatly aid readers in understanding the spatial arrangement of the apparatus, temperature points, and the positioning of flies during the assay. The drawing should also be accompanied by specific details about the setup (dimensions, material, etc). ii) It would be beneficial to include a visual representation of the distribution of flies within the temperature gradient on the apparatus. A graphical representation, such as a heatmaps or histograms, showing the percentage of flies within each one-degree temperature bin, would offer insights into the preferences and behaviors of the flies during the assay. In addition to the detailed description of the assay and data analysis, the inclusion of actual data plots, especially for key findings or representative trials, would provide readers with a more direct visualization of the experimental outcomes. These additions will not only enhance the clarity of the presented information but also provide the reader with a more comprehensive understanding of the experimental setup and results. I appreciate the authors' attention to these points and look forward to the potential inclusion of these elements in the revised manuscript.

Update: The revised manuscript now includes heatmaps showing the distribution of the flies across the temperature bins. As well as a schematic drawing of the behavioral setup.

Reviewer #1 (Public Review):

Summary:

This paper uses a model of binge alcohol consumption in mice to examine how the behaviour and its control by a pathway between the anterior insular cortex (AIC) to the dorsolateral striatum (DLS) may differ between males and females. Photometry is used to measure the activity of AIC terminals in the DLS when animals are drinking and this activity seems to correspond to drink bouts in males but not females. The effects appear to be lateralized with inputs to the left DLS being of particular interest.

Strengths:

Increasing alcohol intake in females is of concern and the consequences for substance use disorder and brain health are not fully understood, so this is an area that needs further study. The attempt to link fine-grained drinking behaviour with neural activity has the potential to enrich our understanding of the neural basis of behaviour, beyond what can be gleaned from coarser measures of volumes consumed etc.

Weaknesses:

The introduction to the drinking in the dark (DID) paradigm is rather narrow in scope (starting line 47). This would be improved if the authors framed this in the context of other common intermittent access paradigms and gave due credit to important studies and authors that were responsible for the innovation in this area (particularly studies by Wise, 1973 and returned to popular use by Simms et al 2010 and related papers; e.g., Wise RA (1973). Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 203-210; Simms, J., Bito-Onon, J., Chatterjee, S. et al. Long-Evans Rats Acquire Operant Self-Administration of 20% Ethanol Without Sucrose Fading. Neuropsychopharmacol 35, 1453-1463 (2010).) The original drinking in the dark demonstrations should also be referenced (Rhodes et al., 2005). Line 154 Theile & Navarro 2014 is a review and not the original demonstration.

When sex differences in alcohol intake are described, more care should be taken to be clear about whether this is in terms of volume (e.g. ml) or blood alcohol levels (BAC, or at least g/kg as a proxy measure). This distinction was often lost when lick responses were being considered. If licking is similar (assuming a single lick from a male and female brings in a similar volume?), this might mean males and females consume similar volumes, but females due to their smaller size would become more intoxicated so the implications of these details need far closer consideration. What is described as identical in one measure, is not in another.

While the authors have some previous data on the AIC to DLS pathway, there are many brain regions and pathways impacted by alcohol and so the focus on this one in particular was not strongly justified. Since photometry is really an observational method, it's important to note that no causal link between activity in the pathway and drinking has been established here.

It would be helpful if the authors could further explain whether their modified lickometers actually measure individual licks. While in some systems contact with the tongue closes a circuit which is recorded, the interruption of a photobeam was used here. It's not clear to me whether the nose close to the spout would be sufficient to interrupt that beam, or whether a tongue protrusion is required. This detail is important for understanding how the photometry data is linked to behaviour. The temporal resolution of the GCaMP signal is likely not good enough to capture individual links but I think more caution or detail in the discussion of the correspondence of these events is required.

Even if the pattern of drinking differs between males and females, the use of the word "strategy" implies a cognitive process that was never described or measured.

Reviewer #2 (Public Review):

Summary:

This study looks at sex differences in alcohol drinking behaviour in a well-validated model of binge drinking. They provide a comprehensive analysis of drinking behaviour within and between sessions for males and females, as well as looking at the calcium dynamics in neurons projecting from the anterior insula cortex to the dorsolateral striatum.

Strengths:

Examining specific sex differences in drinking behaviour is important. This research question is currently a major focus for preclinical researchers looking at substance use. Although we have made a lot of progress over the last few years, there is still a lot that is not understood about sex-differences in alcohol consumption and the clinical implications of this.

Identifying the lateralisation of activity is novel, and has fundamental importance for researchers investigating functional anatomy underlying alcohol-driven behaviour (and other reward-driven behaviours).

Weaknesses:

Very small and unequal sample sizes, especially females (9 males, 5 females). This is probably ok for the calcium imaging, especially with the G-power figures provided, however, I would be cautious with the outcomes of the drinking behaviour, which can be quite variable.

For female drinking behaviour, rather than this being labelled "more efficient", could this just be that female mice (being substantially smaller than male mice) just don't need to consume as much liquid to reach the same g/kg. In which case, the interpretation might not be so much that females are more efficient, as that mice are very good at titrating their intake to achieve the desired dose of alcohol.

Reviewer #3 (Public Review):

Summary:

In this manuscript by Haggerty and Atwood, the authors use a repeated binge drinking paradigm to assess how water and ethanol intake changes in male in female mice as well as measure changes in anterior insular cortex to dorsolateral striatum terminal activity using fiber photometry. They find that overall, males and females have similar overall water and ethanol intake, but females appear to be more efficient alcohol drinkers. Using fiber photometry, they show that the anterior insular cortex (AIC) to dorsolateral striatum projections (DLS) projections have sex, fluid, and lateralization differences. The male left circuit was most robust when aligned to ethanol drinking, and water was somewhat less robust. Male right, and female and left and right, had essentially no change in photometry activity. To some degree, the changes in terminal activity appear to be related to fluid exposure over time, as well as within-session differences in trial-by-trial intake. Overall, the authors provide an exhaustive analysis of the behavioral and photometric data, thus providing the scientific community with a rich information set to continue to study this interesting circuit. However, although the analysis is impressive, there are a few inconsistencies regarding specific measures (e.g., AUC, duration of licking) that do not quite fit together across analytic domains. This does not reduce the rigor of the work, but it does somewhat limit the interpretability of the data, at least within the scope of this single manuscript.

Strengths:

- The authors use high-resolution licking data to characterize ingestive behaviors.<br /> - The authors account for a variety of important variables, such as fluid type, brain lateralization, and sex.<br /> - The authors provide a nice discussion on how this data fits with other data, both from their laboratory and others'.<br /> - The lateralization discovery is particularly novel.

Weaknesses:

- The volume of data and number of variables provided makes it difficult to find a cohesive link between data sets. This limits interpretability.<br /> - The authors describe a clear sex difference in the photometry circuit activity. However, I am curious about whether female mice that drink more similarly to males (e.g., less efficiently?) also show increased activity in the left circuit, similar to males. Oppositely, do very efficient males show weaker calcium activity in the circuit? Ultimately, I am curious about how the circuit activity maps to the behaviors described in Figures 1 and 2.<br /> - What does the change in water-drinking calcium imaging across time in males mean? Especially considering that alcohol-related signals do not seem to change much over time, I am not sure what it means to have water drinking change.

Reviewer #1 (Public Review):

Summary:

This paper introduces an efficient approach to infer properties of receptive-field subunits from the ensemble of spike-triggered stimuli. This is an important general problem in sensory coding. The results introduced in the paper make a solid contribution to both how subunits can be identified and how subunits of different types are coordinated in space.

Strengths:

A primary strength of the paper is the development of approaches that substantially speed non-negative matrix factorization and by doing so create an opportunity for a more systematic exploration of how the procedure depends on various control parameters. The improved procedure is well documented and the direct comparisons with previous procedures are helpful. The improved efficiency enabled several improvements in the procedure - notably tests of good procedures for initializing NNMF and tests of the dependence of the results on the sparsity regularization parameter.

A second strength of the paper is the exploration of the spatial relationship between different subunits. This, to my knowledge, is new and is an interesting direction. There are some concerns about this analysis (see weaknesses below), but if this analysis can be strengthened it will provide new information that will be important both functionally and developmentally.

Weaknesses:

A primary concern is that choices made about parameters for several aspects of the analysis appear to be made subjectively. Much of this centers around how much of the structure in the extracted subunits is imposed by the procedure itself, and how much reflects the underlying neural circuitry. Some specific issues related to this concern are:

- Sparsity: the use of the autocorrelation function to differentiate real vs spurious subunits should be documented and validated. For example, can the authors split data in half and show that the real subunits are stable?

- Choice of regularization: the impact of the regularization parameter on subunit properties is nicely documented. However, the choice of an appropriate regularization parameter seems somewhat arbitrary. Line 253-256 is an example of this problem: this sentence sounds circular - as if the sparsity factor was turned up until the authors obtained what they expected to obtain. Could the choice of this parameter significantly impact the properties of the extracted subunits? How sensitive are the subunit properties to that parameter? Some additional control analyses are needed to validate the parameter choice (see the crossvalidation comment below).

- Crossvalidation was not used to identify the regularization constraint value because the weight matrix from NNMF does not generalize beyond the data it was fit to. Could the authors instead hold the components matrix fixed and recompute the weight matrix, and use that approach for cross-validation (especially since it is really the components matrix that needs validating)?

The paper would benefit from a more complete comparison with known anatomy. For example, can the authors estimate the number of cones within each subunit? This is well-constrained both anatomically (at least in macaque) and, especially for midget ganglion cell subunits, functionally. In macaque, most midget bipolar cells get input from single cones, so the number of extracted subunits should be close to the number of cones. This would be a useful point of comparison for the current work.

Is the analysis of the spatial relationship between different subunit mosaics robust to the incompleteness of those mosaics? The argument on lines 496-503 should be backed up by more analysis. For example, if subunits are removed from regions where the mosaic is pretty complete, do the authors change the spatial dependence? Alternatively, could they use synthetic mosaics with properties like those measured to check the sensitivity to missing cells?

NNMF relies on accounting for each spike-triggered stimulus with a linear combination of components. Would nonlinearities - e.g. those in the bipolar cell outputs - substantially change the results?

Does the approach work for cells that receive input from multiple bipolar types? Some ganglion cells, e.g. in mice, receive input from multiple bipolar types, each accounting for a sizable percentage of the total input. There is similar anatomical work indicating that parasol cells may receive input from multiple diffuse bipolar types. It is not clear whether the current approach works in cases where the subunits of a single ganglion cell overlap. Some discussion of this would be useful.

Reviewer #2 (Public Review):

Summary:

Identifying spatial subunits within the receptive field of retinal ganglion cells can help study spatial nonlinearities and upstream computations performed by the bipolar cells. The authors significantly accelerate the implementation of the previously proposed Spike Triggered semi-non-negative Matrix Factorization (STNMF) method to identify the subunits. The authors also propose a few method improvements - better initialization; new stability-based criteria for selecting the regularization strength, and hyperparameter selection across cell types.

The authors then apply this new method to RGC populations in both the salamander retina and the macaque (marmoset) retina. The authors document the subunit sizes, numbers, and overlap across cell types. The neuroscience finding describes the anti-coordination of ON and OFF parasol receptive fields, but not for the corresponding subunits.

Overall, the authors claim that a faster and more accurate method makes scale-up to large neuronal populations feasible.

Strengths:

- The paper is well-written, easy to read and the figures are clear. The limitations are also made clear.

- The scientific findings are novel and seem to be well supported.

- The claimed speed-up of the method is potentially important for practical applications to large populations. Each innovation of the method is well-supported.

- This is a serious effort to improve the method and document the subunits in primate retina.

Weaknesses:

- The description of the method is confusing. Currently, the new method is described in the context of changes from existing methods. As someone who is not familiar with previous methods, it is very confusing to follow the details.

- I think it will help a lot with clarity to have a concise flowchart/pseudocode to summarize the algorithm and separate it from a description of the main changes from previous methods.

- Separate pseudocodes can be provided for the main method, initialization, regularization parameter selection using consensus, and identifying the regularization parameter across cell types.

- While the new method clearly shows a drastic improvement compared to the previous method on a laptop, would it be possible to get the same improvement on the previous method if it was implemented with GPU (as is standard for most AI/ML algorithms)?

- For the calculation of subunits across multiple cells, can you run multiple parallel jobs on the same computer? This may make some innovations unnecessary (like setting the same regularization strength across multiple cells).

- There are two main innovations in this paper: the fast and approximate method, and analysis of subunit mosaics for primate RGCs. It would be helpful to include an analysis of the primate RGC subunits using the older, slower, but more exact method and show that the major scientific results can be reproduced. This would validate the new method in an end-to-end manner. While this may take a while to run, it may be helpful in the supplement.

- It would be important to understand the data-efficiency of the method. The approximate method may deviate more from the exact method when the amount of data is limited.

- Would it be possible to have a few steps of the exact method at the end to ensure that the solution truly optimizes the objective function?

- Does the number of estimated subunits change with the number of observed spikes? If so, the estimates of subunit number/size must be interpreted with caution.

Reviewer #3 (Public Review):

Summary:

This work addresses the problem of determining the subunit composition of receptive fields of retinal ganglion cells (RGCs). RGCs process stimuli through non-linear transforms that largely (although not entirely) reflect the individual contributions of their input bipolar cells, which themselves process visual stimuli nonlinearly. Thus, using the correct system identification methods might correctly model the RGC cells, while revealing details of the underlying circuit, including the function of the presynaptic components. It is now well established that a model of the form of an LNLN cascade can potentially capture this bipolar-RGC circuit, although the devil is in the details. The authors present an improved method of non-negative matrix factorization (NMF) - which is one approach to this system identification problem - that can speed things up by a factor of 100, and in doing so infer plausible mosaics of the bipolar cell types supporting the identified RGC types that are recorded from.

As written, the focus of this paper seems almost entirely methodological, supporting the sped-up version of NMF, called STNMF. The >100x speedup potentially makes a lot more measurements available, since it enables much more comprehensive scans across model meta-parameters, although has its own complications that must also be methodologically addressed. The results presented are largely a demonstration and validation of the potential power of this approach using example recordings in the peripheral marmoset retina. I do not think the results themselves are meant to be evaluated as definitive, since they are often based on examples and are largely confirmatory of what is already known.

Strengths:

I have very few concerns about the paper methodologically: these methods are well laid out and demonstrated (at least up to the level of my expertise and interest), including validation with established literature.

I am also enthusiastic about some of the potential results in the retina outlined (but not fully fleshed out) in the later sections of the paper.

Weaknesses:

My main critique is to question the conceptual advance in this paper: what did we learn, and what is the targeted audience of interest? Establishing this is particularly dire for this manuscript since NMF has already been established and expounded on as a useful approach in this context (including by the author most recently in 2017) so any of the scientific results is already achievable with enough computer power using existing approaches. As currently cast, the conceptual advances here are purely methodological and relate to the utility of speeding up the approach. Also, they do not appear to generalize to other problems outside of the narrow range that it is currently applied.

Thus, two paths to improving the manuscript would be either:<br /> (1) target readers interested in the retina by fully fleshing out the current results and add more to make this into a paper about the retina rather than about the STNMF method, or<br /> (2) demonstrate that the methods might be useful outside of the very narrow set of conditions specific to identifying nonlinear bipolar cell subunits in peripheral retina under white noise stimulation.

In its current state, the Discussion addressing limitations and generality seems to suggest applicability past this narrow condition, which I do not think is the case: but would be happy to be convinced otherwise.

For fleshing out scientific results, in the current manuscript, they are currently presented to validate the approach and are largely confirmatory for what we already know about the retina (which allows for this validation). Also, much of the results are measurements based on examples, and not accumulated past a single recording in some cases. Finally, it is not clear to the extent that these results depend on the specific recordings in the peripheral marmoset retina: what about more central in the retina, or in other species?

For demonstrating the utility of the methodology: here are some of the main limitations to generalizing past this specific case:<br /> (1) the necessity of linear or near-linear processing in previous layers;<br /> (2) lack of any negative components;<br /> (3) lack of ability to account for other influences on spiking than the positive contributions of LN subunits;<br /> (4) necessity of white noise stimulation that is specifically sized for a uniform subunit size.

Together, I believe this precludes potential applications to other areas in the brain: further back in the visual system will require non-linear transforms as well as the convergence of positive and negative inputs. Other sensory systems like the auditory system are even more non-linear well before getting to even mid-level pre-cortical structures and also combine positive and negative influences. Given the importance of inhibition in the retina (including what is thought to be an important role of amacrine cells in shaping RGC responses), it is not clear how general this approach is in the retina, although the specific results shown are believable. How could this approach generalize, realistically? Could applications to other types of data be demonstrated, and/or plausibly get by these fundamental limitations? How?

Gating of Kv10 channels is unique because it involves coupling between non-domain swapped voltage sensing domains, a domain-swapped cytoplasmic ring assembly formed by the N- and C-termini, and the pore domain. Recent structural data suggests that activation of the voltage sensing domain relieves a steric hindrance to pore opening, but the contribution of the cytoplasmic domain to gating is still not well understood. This aspect is of particular importance because proteins like calmodulin interact with the cytoplasmic domain to regulate channel activity. The effects of calmodulin (CaM) in WT and mutant channels with disrupted cytoplasmic gating ring assemblies are contradictory, resulting in inhibition or activation, respectively. The underlying mechanism for these discrepancies is not understood. In the present manuscript, Reham Abdelaziz and collaborators use electrophysiology, biochemistry and mathematical modeling to describe how mutations and deletions that disrupt inter-subunit interactions at the cytoplasmic gating ring assembly affect Kv10.1 channel gating and modulation by CaM. In the revised manuscript, additional information is provided to allow readers to identify within the Kv10.1 channel structure the location of E600R, one of the key channel mutants analyzed in this study. However, the mechanistic role of the cytoplasmic domains that this study focuses on, as well as the location of the ΔPASCap deletion and other perturbations investigated in the study remain difficult to visualize without additional graphical information.

The authors focused mainly on two structural perturbations that disrupt interactions within the cytoplasmic domain, the E600R mutant and the ΔPASCap deletion. By expressing mutants in oocytes and recording currents using Two Electrode Voltage-Clamp (TEV), it is found that both ΔPASCap and E600R mutants have biphasic conductance-voltage (G-V) relations and exhibit activation and deactivation kinetics with multiple voltage-dependent components. Importantly, the mutant-specific component in the G-V relations is observed at negative voltages where WT channels remain closed. The authors argue that the biphasic behavior in the G-V relations is unlikely to result from two different populations of channels in the oocytes, because they found that the relative amplitude between the two components in the G-V relations was highly reproducible across individual oocytes that otherwise tend to show high variability in expression levels. Instead, the G-V relations for all mutant channels could be well described by an equation that considers two open states O1 and O2, and a transition between them; O1 appeared to be unaffected by any of the structural manipulations tested (i.e. E600R, ΔPASCap, and other deletions) whereas the parameters for O2 and the transition between the two open states were different between constructs. The O1 state is not observed in WT channels and is hypothesized to be associated with voltage sensor activation. O2 represents the open state that is normally observed in WT channels and is speculated to be associated with conformational changes within the cytoplasmic gating ring that follow voltage sensor activation, which could explain why the mutations and deletions disrupting cytoplasmic interactions affect primarily O2.

Severing the covalent link between the voltage sensor and pore reduced O1 occupancy in one of the deletion constructs. Although this observation is consistent with the hypothesis that voltage-sensor activation drives entry into O1, this result is not conclusive. Structural as well as functional data has established that the coupling of the voltage sensor and pore does not entirely rely on the S4-S5 covalent linker between the sensor and the pore, and thus the severed construct could still retain coupling through other mechanisms, which is consistent with the prominent voltage dependence that is observed. If both states O1 and O2 require voltage sensor activation, it is unclear why the severed construct would affect state O1 primarily, as suggested in the manuscript, as opposed to decreasing occupancy of both open states. In line with this argument, the presence of Mg2+ in the extracellular solution affected both O1 and O2. This finding suggests that entry into both O1 and O2 requires voltage-sensor activation because Mg2+ ions are known to stabilize the voltage sensor in its most deactivated conformations.

Activation towards and closure from O1 is slow, whereas channels close rapidly from O2. A rapid alternating pulse protocol was used to take advantage of the difference in activation and deactivation kinetics between the two open components in the mutants and thus drive an increasing number of channels towards state O1. Currents activated by the alternating protocol reached larger amplitudes than those elicited by a long depolarization to the same voltage. This finding is interpreted as an indication that O1 has a larger macroscopic conductance than O2. In the revised manuscript, the authors performed single-channel recordings to determine why O1 and O2 have different macroscopic conductance. The results show that at voltages where the state O1 predominates, channels exhibited longer open times and overall higher open probability, whereas at more depolarized voltages where occupancy of O2 increases, channels exhibited more flickery gating behavior and decreased open probability. These results are informative but not conclusive since single-channel amplitudes could not be resolved at strong depolarizations, limiting the extent to which the data could be analyzed. In the last revision, the authors have included one representative example showing inhibition of single channel activity by the Kv10-specific inhibitor astemizole. Group data analysis would be needed to conclusively establish that the currents that were recorded indeed correspond to Kv10 channels.

It is shown that conditioning pulses to very negative voltages result in mutant channel currents that are larger and activate more slowly than those elicited at the same voltage but starting from less negative conditioning pulses. In voltage-activated curves, O1 occupancy is shown to be favored by increasingly negative conditioning voltages. This is interpreted as indicating that O1 is primarily accessed from deeply closed states in which voltage sensors are in their most deactivated position. Consistently, a mutation that destabilizes these deactivated states is shown to largely suppress the first component in voltage-activation curves for both ΔPASCap and E600R channels.

The authors then address the role of the hidden O1 state in channel regulation by calcium-calmodulin (CaM). Stimulating calcium entry into oocytes with ionomycin and thapsigargin, assumed to enhance CaM-dependent modulation, resulted in preferential potentiation of the first component in ΔPASCap and E600R channels. This potentiation was attenuated by including an additional mutation that disfavors deeply closed states. Together, these results are interpreted as an indication that calcium-CaM preferentially stabilizes deeply closed states from which O1 can be readily accessed in mutant channels, thus favoring current activation. In WT channels lacking a conducting O1 state, CaM stabilizes deeply closed states and is therefore inhibitory. It is found that the potentiation of ΔPASCap and E600R by CaM is more strongly attenuated by mutations in the channel that are assumed to disrupt interaction with the C-terminal lobe of CaM than mutations assumed to affect interaction with the N-terminal lobe. These results are intriguing but difficult to interpret in mechanistic terms. The strong effect that calcium-CaM had on the occupancy of the O1 state in the mutants raises the possibility that O1 can be only observed in channels that are constitutively associated with CaM. To address this, a biochemical pull-down assay was carried out to establish that only a small fraction of channels are associated with CaM under baseline conditions. These CaM experiments are potentially very interesting and could have wide physiological relevance. However, the approach utilized to activate CaM is indirect and could result in additional non-specific effects on the oocytes that could affect the results.

Finally, a mathematical model is proposed consisting of two layers involving two activation steps for the voltage sensor, and one conformational change in the cytoplasmic gating ring - completion of both sets of conformational changes is required to access state O2, but accessing state O1 only requires completion of the first voltage-sensor activation step in the four subunits. The model qualitatively reproduces most major findings on the mutants. Although the model used is highly symmetric and appears simple, the mathematical form used for the rate constants in the model adds a layer of complexity to the model that makes mechanistic interpretations difficult. In addition, many transitions that from a mechanistic standpoint should not depend on voltage were assigned a voltage dependence in the model. These limitations diminish the mechanistic insight that can be reliably extracted from the model.

Reviewer #1 (Public Review):

In this manuscript, Yang et al. conduct a comprehensive investigation to demonstrate the role of adipose tissue miR-802 in obesity-associated inflammation and metabolic dysfunction. Using multiple models and techniques, they propose a mechanism where elevated levels of miR-802 in adipose tissue (both in mouse models and humans) trigger fat accumulation and inflammation, leading to increased adiposity and insulin resistance. They suggest that increased miR-802 levels in adipocytes during obesity result in the downregulation of TRAF3, a negative regulator of canonical and non-canonical NF-κB pathways. This downregulation induces inflammation through the production of cytokines/chemokines that attract and polarize macrophages. Concurrently, the NF-κB pathway induces the lipogenic transcriptional factor SREBP1, which promotes fat accumulation and further recruits pro-inflammatory macrophages. While the proposed model is supported by multiple experiments and consistent data, there are areas where the manuscript could be improved. Some improvements can be addressed in the text, while others require additional controls, experiments, or analyses.

(1) The manuscript should provide measurements of lipid droplet/adipocyte size for all models, both in vitro and in vivo. In vivo studies should also include fat weight measurements. This is crucial to determine whether miR-802, TRAF3, and SREBP1 promote adiposity/fat accumulation across all models.<br /> (2) The rationale for co-culture experiments using WAT SVF is unclear, given that miR-802 is upregulated by obesity in adipocytes, not in the stromal-vascular fraction. These experiments would be more relevant if performed using isolated adipocytes or differentiated WAT SVF.<br /> (3) Figures 1G and 1H lack a control group (time 0 or NCD). Without this control, it is impossible to determine if inflammation precedes miR-802 upregulation.<br /> (4) The statement, "The knockout of miR-802 in adipose tissue did not alter food intake, body weight, glucose level, and adiposity (data not shown)," needs more detail regarding the age and sex of the animals. These data are important and should be reported, perhaps in a supplementary figure.<br /> (5) The terms "KO" (knockout) and "KI" (knock-in) are misleading for AAV models, as they do not modify the genome. "KD" (knockdown) and "OE" (overexpression) are more accurate.<br /> (6) The statement, "miR-802 expression was unaffected in other organs (Figure S3O)," should clarify that this is except for BAT.

By addressing these points, the manuscript would present a more robust and clear demonstration of the role of miR-802 in obesity-associated inflammation and metabolic dysfunction.

Reviewer #2 (Public Review):

Yang et al. investigated the role of miR-802 in the development of adipose tissue (AT) inflammation during obesity. The authors found miR-802 levels are up-regulated in the AT of mouse models of obesity and insulin resistance as well as in the AT of humans. They further demonstrated that miR-802 regulates the intracellular levels of TRAF3 and downstream activation of the NF-kB pathway. Ultimately, controlling AT inflammation by manipulating miR-802 affected whole-body glucose homeostasis, highlighting the role of AT inflammatory status in whole-body metabolism. The study provides solid evidence on the role of adipocyte miR-802 in controlling inflammation and macrophage recruitment. However, how lipid mobilization from adipocytes and how engulfment of lipid droplets by macrophages control inflammatory phenotype in these cells could be better explored. The findings of this study will have a great impact in the field, contributing to the growing body of evidence on how microRNAs control the inflammatory microenvironment of AT and whole-body metabolism in obesity.

Reviewer #3 (Public Review):

MiR-802 appears to accumulate before macrophage numbers increase in adipose tissue in both mice and humans. The phenotype of miR-802 overexpression and deletion in vivo is sticking and novel. Deletion of miR-802 in adipose tissue after obesity onset also attenuated Adipose inflammation and improved systemic glucose homeostasis. Understanding how miR-802 affects the crosstalk between macrophage and adipocyte is a major point. For example, does miR-802 change the inflammatory of macrophages as it increases Traf3 expression in adipocytes? This is important because macrophages are the input if inflammatory mediators that will activate the TNFR receptor signaling pathway, potentially Traf3, resulting in impaired insulin stimulated Glut4 translocation and glucose uptake. Also, modulation of miR-802 levels in vivo leads to alterations in adiposity. Here, what is a direct effect of miR-802 and what is a result of simply reduced adiposity? One point that os ket is what triggers miR-802 expression, especially in obesity.

Reviewer #1 (Public Review):

In this paper, Tompary & Davachi present work looking at how memories become integrated over time in the brain, and relating those mechanisms to responses on a priming task as a behavioral measure of memory linkage. They find that remotely but not recently formed memories are behaviorally linked and that this is associated with a change in the neural representation in mPFC. They also find that the same behavioral outcomes are associated with the increased coupling of the posterior hippocampus with category-sensitive parts of the neocortex (LOC) during a post-learning rest period-again only for remotely learned information. There was also correspondence in rest connectivity (posterior hippocampus-LOC) and representational change (mPFC) such that for remote memories specifically, the initial post-learning connectivity enhancement during rest related to longer-term mPFC representational change.

This work has many strengths. The topic of this paper is very interesting, and the data provide a really nice package in terms of providing a mechanistic account of how memories become integrated over a delay. The paper is also exceptionally well-written and a pleasure to read. There are two studies, including one large behavioral study, and the findings replicate in the smaller fMRI sample. I do however have two fairly substantive concerns about the analytic approach, where more data will be required before we can know whether the interpretations are an appropriate reflection of the findings. These and other concerns are described below.

(1) One major concern relates to the lack of a pre-encoding baseline scan prior to recent learning.

a) First, I think it would be helpful if the authors could clarify why there was no pre-learning rest scan dedicated to the recent condition. Was this simply a feasibility consideration, or were there theoretical reasons why this would be less "clean"? Including this information in the paper would be helpful for context. Apologies if I missed this detail in the paper.

b) Second, I was hoping the authors could speak to what they think is reflected in the post-encoding "recent" scan. Is it possible that these data could also reflect the processing of the remote memories? I think, though am not positive, that the authors may be alluding to this in the penultimate paragraph of the discussion (p. 33) when noting the LOC-mPFC connectivity findings. Could there be the reinstatement of the old memories due to being back in the same experimental context and so forth? I wonder the extent to which the authors think the data from this scan can be reflected as strictly reflecting recent memories, particularly given it is relative to the pre-encoding baseline from before the remote memories, as well (and therefore in theory could reflect both the remote + recent). (I should also acknowledge that, if it is the case that the authors think there might be some remote memory processing during the recent learning session in general, a pre-learning rest scan might not have been "clean" either, in that it could have reflected some processing of the remote memories-i.e., perhaps a clean pre-learning scan for the recent learning session related to point 1a is simply not possible.)

c) Third, I am thinking about how both of the above issues might relate to the authors' findings, and would love to see more added to the paper to address this point. Specifically, I assume there are fluctuations in baseline connectivity profile across days within a person, such that the pre-learning connectivity on day 1 might be different from on day 2. Given that, and the lack of a pre-learning connectivity measure on day 2, it would logically follow that the measure of connectivity change from pre- to post-learning is going to be cleaner for the remote memories. In other words, could the lack of connectivity change observed for the recent scan simply be due to the lack of a within-day baseline? Given that otherwise, the post-learning rest should be the same in that it is an immediate reflection of how connectivity changes as a function of learning (depending on whether the authors think that the "recent" scan is actually reflecting "recent + remote"), it seems odd that they both don't show the same corresponding increase in connectivity-which makes me think it may be a baseline difference. I am not sure if this is what the authors are implying when they talk about how day 1 is most similar to prior investigation on p. 20, but if so it might be helpful to state that directly.

d) Fourth and very related to my point 1c, I wonder if the lack of correlations for the recent scan with behavior is interpretable, or if it might just be that this is a noisy measure due to imperfect baseline correction. Do the authors have any data or logic they might be able to provide that could speak to these points? One thing that comes to mind is seeing whether the raw post-learning connectivity values (separately for both recent and remote) show the same pattern as the different scores. However, the authors may come up with other clever ways to address this point. If not, it might be worth acknowledging this interpretive challenge in the Discussion.

(2) My second major concern is how the authors have operationalized integration and differentiation. The pattern similarity analysis uses an overall correspondence between the neural similarity and a predicted model as the main metric. In the predicted model, C items that are indirectly associated are more similar to one another than they are C items that are entirely unrelated. The authors are then looking at a change in correspondence (correlation) between the neural data and that prediction model from pre- to post-learning. However, a change in the degree of correspondence with the predicted matrix could be driven by either the unrelated items becoming less similar or the related ones becoming more similar (or both!). Since the interpretation in the paper focuses on change to indirectly related C items, it would be important to report those values directly. For instance, as evidence of differentiation, it would be important to show that there is a greater decrease in similarity for indirectly associated C items than it is for unrelated C items (or even a smaller increase) from pre to post, or that C items that are indirectly related are less similar than are unrelated C items post but not pre-learning. Performing this analysis would confirm that the pattern of results matches the authors' interpretation. This would also impact the interpretation of the subsequent analyses that involve the neural integration measures (e.g., correlation analyses like those on p. 16, which may or may not be driven by increased similarity among overlapping C pairs). I should add that given the specificity to the remote learning in mPFC versus recent in LOC and anterior hippocampus, it is clearly the case that something interesting is going on. However, I think we need more data to understand fully what that "something" is.

(3) The priming task occurred before the post-learning exposure phase and could have impacted the representations. More consideration of this in the paper would be useful. Most critically, since the priming task involves seeing the related C items back-to-back, it would be important to consider whether this experience could have conceivably impacted the neural integration indices. I believe it never would have been the case that unrelated C items were presented sequentially during the priming task, i.e., that related C items always appeared together in this task. I think again the specificity of the remote condition is key and perhaps the authors can leverage this to support their interpretation. Can the authors consider this possibility in the Discussion?

(4) For the priming task, based on the Figure 2A caption it seems as though every sequence contributes to both the control and primed conditions, but (I believe) this means that the control transition always happens first (and they are always back-to-back). Is this a concern? If RTs are changing over time (getting faster), it would be helpful to know whether the priming effects hold after controlling for trial numbers. I do not think this is a big issue because if it were, you would not expect to see the specificity of the remotely learned information. However, it would be helpful to know given the order of these conditions has to be fixed in their design.

(5) The authors should be cautious about the general conclusion that memories with overlapping temporal regularities become neurally integrated - given their findings in MPFC are more consistent with overall differentiation (though as noted above, I think we need more data on this to know for sure what is going on).

(6) It would be worth stating a few more details and perhaps providing additional logic or justification in the main text about the pre and post-exposure phases were set up and why. How many times each object was presented pre and post, and how the sequencing was determined (were any constraints put in place e.g., such that C1 and C2 did not appear close in time?). What was the cover task (I think this is important to the interpretation & so belongs in the main paper)? Were there considerations involving the fact that this is a different sequence of the same objects the participants would later be learning - e.g., interference, etc.?

Reviewer #2 (Public Review):

The manuscript by Tompary & Davachi presents results from two experiments, one behavior only and one fMRI plus behavior. They examine the important question of how to separate object memories (C1 and C2) that are never experienced together in time and become linked by shared predictive cues in a sequence (A followed by B followed by one of the C items). The authors developed an implicit priming task that provides a novel behavioral metric for such integration. They find significant C1-C2 priming for sequences that were learned 24h prior to the test, but not for recently learned sequences, suggesting that associative links between the two originally separate memories emerge over an extended period of consolidation. The fMRI study relates this behavioral integration effect to two neural metrics: pattern similarity changes in the medial prefrontal cortex (mPFC) as a measure of neural integration, and changes in hippocampal-LOC connectivity as a measure of post-learning consolidation. While fMRI patterns in mPFC overall show differentiation rather than integration (i.e., C1-C2 representational distances become larger), the authors find a robust correlation such that increasing pattern similarity in mPFC relates to stronger integration in the priming test, and this relationship is again specific to remote memories. Moreover, connectivity between the posterior hippocampus and LOC during post-learning rest is positively related to the behavioral integration effect as well as the mPFC neural similarity index, again specifically for remote memories. Overall, this is a coherent set of findings with interesting theoretical implications for consolidation theories, which will be of broad interest to the memory, learning, and predictive coding communities.

Strengths:

(1) The implicit associative priming task designed for this study provides a promising new tool for assessing the formation of mnemonic links that influence behavior without explicit retrieval demands. The authors find an interesting dissociation between this implicit measure of memory integration and more commonly used explicit inference measures: a priming effect on the implicit task only evolved after a 24h consolidation period, while the ability to explicitly link the two critical object memories is present immediately after learning. While speculative at this point, these two measures thus appear to tap into neocortical and hippocampal learning processes, respectively, and this potential dissociation will be of interest to future studies investigating time-dependent integration processes in memory.

(2) The experimental task is well designed for isolating pre- vs post-learning changes in neural similarity and connectivity, including important controls of baseline neural similarity and connectivity.

(3) The main claim of a consolidation-dependent effect is supported by a coherent set of findings that relate behavioral integration to neural changes. The specificity of the effects on remote memories makes the results particularly interesting and compelling.

(4) The authors are transparent about unexpected results, for example, the finding that overall similarity in mPFC is consistent with a differentiation rather than an integration model.

Weaknesses:

(1) The sequence learning and recognition priming tasks are cleverly designed to isolate the effects of interest while controlling for potential order effects. However, due to the complex nature of the task, it is difficult for the reader to infer all the transition probabilities between item types and how they may influence the behavioral priming results. For example, baseline items (BL) are interspersed between repeated sequences during learning, and thus presumably can only occur before an A item or after a C item. This seems to create non-random predictive relationships such that C is often followed by BL, and BL by A items. If this relationship is reversed during the recognition priming task, where the sequence is always BL-C1-C2, this violation of expectations might slow down reaction times and deflate the baseline measure. It would be helpful if the manuscript explicitly reported transition probabilities for each relevant item type in the priming task relative to the sequence learning task and discussed how a match vs mismatch may influence the observed priming effects.

(2) The choice of what regions of interest to include in the different sets of analyses could be better motivated. For example, even though briefly discussed in the intro, it remains unclear why the posterior but not the anterior hippocampus is of interest for the connectivity analyses, and why the main target is LOC, not mPFC, given past results including from this group (Tompary & Davachi, 2017). Moreover, for readers not familiar with this literature, it would help if references were provided to suggest that a predictable > unpredictable contrast is well suited for functionally defining mPFC, as done in the present study.

(3) Relatedly, multiple comparison corrections should be applied in the fMRI integration and connectivity analyses whenever the same contrast is performed on multiple regions in an exploratory manner.

Reviewer #3 (Public Review):

The authors of this manuscript sought to illuminate a link between a behavioral measure of integration and neural markers of cortical integration associated with systems consolidation (post-encoding connectivity, change in representational neural overlap). To that aim, participants incidentally encoded sequences of objects in the fMRI scanner. Unbeknownst to participants, the first two objects of the presented ABC triplet sequences overlapped for a given pair of sequences. This allowed the authors to probe the integration of unique C objects that were never directly presented in the same sequence, but which shared the same preceding A and B objects. They encoded one set of objects on Day 1 (remote condition), another set of objects 24 hours later (recent condition) and tested implicit and explicit memory for the learned sequences on Day 2. They additionally collected baseline and post-encoding resting-state scans. As their measure of behavioral integration, the authors examined reaction time during an Old/New judgement task for C objects depending on if they were preceded by a C object from an overlapping sequence (primed condition) versus a baseline object. They found faster reaction times for the primed objects compared to the control condition for remote but not recently learned objects, suggesting that the C objects from overlapping sequences became integrated over time. They then examined pattern similarity in a priori ROIs as a measure of neural integration and found that participants showing evidence of integration of C objects from overlapping sequences in the medial prefrontal cortex for remotely learned objects also showed a stronger implicit priming effect between those C objects over time. When they examined the change in connectivity between their ROIs after encoding, they also found that connectivity between the posterior hippocampus and lateral occipital cortex correlated with larger priming effects for remotely learned objects, and that lateral occipital connectivity with the medial prefrontal cortex was related to neural integration of remote objects from overlapping sequences.

The authors aim to provide evidence of a relationship between behavioral and neural measures of integration with consolidation is interesting, important, and difficult to achieve given the longitudinal nature of studies required to answer this question. Strengths of this study include a creative behavioral task, and solid modelling approaches for fMRI data with careful control for several known confounds such as bold activation on pattern analysis results, motion, and physiological noise. The authors replicate their behavioral observations across two separate experiments, one of which included a large sample size, and found similar results that speak to the reliability of the observed behavioral phenomenon. In addition, they document several correlations between neural measures and task performance, lending functional significance to their neural findings.

However, this study is not without notable weaknesses that limit the strength of the manuscript. The authors report a behavioral priming effect suggestive of integration of remote but not recent memories, leading to the interpretation that the priming effect emerges with consolidation. However, they did not observe a reliable interaction between the priming condition and learning session (recent/remote) on reaction times, meaning that the priming effect for remote memories was not reliably greater than that observed for recent. In addition, the emergence of a priming effect for remote memories does not appear to be due to faster reaction times for primed targets over time (the condition of interest), but rather, slower reaction times for control items in the remote condition compared to recent. These issues limit the strength of the claim that the priming effect observed is due to C items of interest being integrated in a consolidation-dependent manner.

Similarly, the interactions between neural variables of interest and learning session needed to strongly show a significant consolidation-related effect in the brain were sometimes tenuous. There was no reliable difference in neural representational pattern analysis fit to a model of neural integration between the short and long delays in the medial prefrontal cortex or lateral occipital cortex, nor was the posterior hippocampus-lateral occipital cortex post-encoding connectivity correlation with subsequent priming significantly different for recent and remote memories. While the relationship between integration model fit in the medial prefrontal cortex and subsequent priming (which was significantly different from that occurring for recent memories) was one of the stronger findings of the paper in favor of a consolidation-related effect on behavior, is it possible that lack of a behavioral priming effect for recent memories due to possible issues with the control condition could mask a correlation between neural and behavioral integration in the recent memory condition?

These limitations are especially notable when one considers that priming does not classically require a period of prolonged consolidation to occur, and prominent models of systems consolidation rather pertain to explicit memory. While the authors have provided evidence that neural integration in the medial prefrontal cortex, as well as post-encoding coupling between the lateral occipital cortex and posterior hippocampus, are related to faster reaction times for primed objects of overlapping sequences compared to their control condition, more work is needed to verify that the observed findings indeed reflect consolidation dependent integration as proposed.

Reviewer #1 (Public Review):

In this study, Girardello et al. use proteomics to reveal the membrane tension sensitive caveolin-1 interactome in migrating cells. The authors use EM and surface rendering to demonstrate that caveolae formed at the rear of migrating cells are complex membrane-linked multilobed structures, and they devise a robust strategy to identify caveolin-1 associated proteins using APEX2-mediated proximity biotinylation. This important dataset is further validated using proximity ligation assays to confirm key interactions, and follows up with an interrogation of a surprising relationship between caveolae and RhoGTPase signalling, where caveolin-1 recruits ROCK1 under high membrane tension conditions, and ROCK1 activity is required to reform caveolae upon reversion to isotonic solution. However, caveolin-1 recruits the RhoA inactivator ARHGAP29 when membrane tension is low and ARHGAP29 overexpression leads to disassembly of caveolae and reduced cell motility. This study builds on previous findings linking caveolae to positive feedback regulation of RhoA signalling, and provides further evidence that caveolae serve to drive rear retraction in migration but also possess an intrinsic brake to limit RhoA activation, leading the authors to suggest that cycles of caveolae assembly and disassembly could thereby be central to establish a stable cell rear for persistent cell migration

A major strength of the manuscript is the robust proteomic dataset. The experimental set up is well defined and mostly well controlled, and there is good internal validation in that the high abundance of core caveolar proteins in low membrane tension (isotonic) conditions, and absence under high membrane tension (brief hypo-osmotic shock) conditions, correlating very well with previous finding. The data could however be better presented to show where statically robust changes occur, and supplementary information should include a table of showing abundance. It's very good to see a link to PRIDE, providing a useful resource for the community.

The authors detail several known interactions and their mechanosensitivty, but also report new interactors of caveolin-1. Several mechanosensitive interactions of caveolin-1 take place at the cell rear, but others are more diffuse across the cell looking at the PLA data (e.g FLN1, CTTN, HSPB1; Figure 4A-F and Figure 4 supplement 1). It is interesting to speculate that those at the cell rear are involved in caveolae, whilst others are linked specifically to caveolin-1 (e.g. dolines). PLA or localisation analysis with Cavin1/PTRF may be able to resolve this and further specify caveolae versus non-caveolae mechanosensitive interactions.

The Cav1/ARHGAP29 influence on YAP signalling is interesting, but appear to be quite isolated from the rest of the manuscript. Does overexpression of ARHGAP29 influence YAP signalling and/or caveolar protein expression/Cav1pY14?<br /> ARHGAP29 and RhoA/ROCK1 related observations are very interesting and potentially really important. However, the link between ARHGAP29 and caveolae is not well established (other than in proteomic data). PLA or FRET could help establish this.<br /> The relationship between ARHGAP29 and RhoA signalling is not well defined. Is GAP activity important in determining the effect on migration and caveolae formation? What is the effect on RhoA activity? Alternatively, the authors could investigate YAP dependent transcriptional regulation downstream of overexpression.

Reviewer #2 (Public Review):

Girardello et al investigated the composition of the molecular machinery of caveolae governing their mechano-regulation in migrating cells. Using live cell imaging and RPE1 cells, the authors provide a spatio-temporal analysis of cavin-3 distribution during cell migration and reveal that caveolae are preferentially localized at the rear of the cell in a stable manner. They further characterize these structures using electron tomography and reveal an organization into clusters connected to the cell surface. By performing a proteomic approach, they address the interactome of caveolin-1 proteins upon mechanical stimulation by exposing RPE1 cells to hypo-osmotic shock (which aims to increase cell membrane tension) or not as a control condition. The authors identify over 300 proteins, notably proteins related to actin cytoskeleton and cell adhesion. These results were further validated in cellulo by interrogating protein-protein interactions using proximity ligation assays and hypo-osmotic shock. These experiments confirmed previous data showing that high membrane tension induces caveolae disassembly in a reversible manner. Eventually, based on literature and on the results collected by the proteomic analysis, authors investigated more deeply the molecular signaling pathway controlling caveolae assembly upon mechanical stimuli. First, they confirm the targeting of ROCK1 with Caveolin-1 and the implication of the kinase activity for caveolae formation (at the rear of the cell). Then, they show that RhoGA ARHGAP29, a factor newly identified by the proteomic analysis, is also implicated in caveolae mechano-regulation likely through YAP protein and found that overexpression of RHoGA ARHGAP29 affects cell motility. Overall, this paper interrogated the role of membrane tension in caveolae located at the rear of the cell and identified a new pathway controlling cell motility.

Strengths:

Using a proximity-based proteomic assay, the authors reveal the protein network interacting with caveolae upon mechanical stimuli. This approach is elegant and allows to identify a substantial new set of factors involved in the mechano-regulation of caveolin-1, some of which have been verified directly in the cell by PLA. This study provides a compelling set of data on the interactions between caveolae and its cortical network which was so far ill-characterized.

Weaknesses:

The methodology demonstrating an impact of membrane tension is not precise enough to directly assess a direct role on caveolae at a subcellular scale, that is between the front and the rear of the cell. First, a better characterization of the "front-rear" cellular model is encouraged. Secondly, authors frequently present osmotic shock as "high membrane tension" stimuli. While osmotic shock is widely used in the field, this study is focused only on caveolae localized at the rear of cell and it remains unclear how the level of a global mechanical stimuli triggered by an osmotic shock could mimic a local stimuli. In the present case, it remains unknown the extent to which this mechanical stress is physiologically relevant to mimic mechanical forces applied at the rear of a migrating cell.<br /> Some images are not satisfying to fully support the conclusions of the article. At this stage, the lack of an unbiased quantitative analysis of the spatio-temporal analysis of caveolae upon well-defined mechanical stimuli is also needed. Cells on images, in particular Figure 1, are difficult to see. Signal-to noise ratio in different cell area could generate a biased. Since there is inconsistency between caveolae density and localization between Figures, more solid illustrations are needed along quantitative analysis.

Reviewer #3 (Public Review):

Although the authors findings are interesting, they do little to demonstrate new scientific information or advancements in producing genetically modified livestock with improved production characteristics. While the MSTNDel273 sheep exhibited an increased number of muscle fibers, the data provided did not demonstrate a significant improvement in meat production, quality or quantity in the MSTNDel273 sheep vs WT.

The manuscript is very long, complicated and difficult to read, given the minimum amount of significant information that is provided. It reads more like a graduate student thesis than a scientific manuscript ready for publication. Given the significant findings are so minimal, the amount of text provided, figures and tables are excessive. A large number of different molecular techniques are employed to try and decipher the mechanism(s) that result in the observed phenotype = double muscling. The authors focus on the MEK-ERK-FOSL1 pathway and suggest this is the key pathway/mechanism resulting in the phenotype observed in MSTNDel273sheep. However, they provide very little "significant" evidence to support this. RNA-Seq data demonstrated that hundreds of different genes were either upregulated or down-regulated, but the authors chose to only focus on FOSL1 and associated genes. The findings do not support the idea that FOSL1 is not involved, but neither do they strongly support FOSL1 involvement. The observations made by the authors could be co-incidental and not causative in nature.

The authors indicate that sgRNA design changes in addition to changing the molar ratio of Cas9MRNA:sgRNA improved the ability to generate biallelic homozygous mutant sheep; however, the data provided to not demonstrate any significant difference. Given the small number of sheep that were actually produced and evaluated, it is extremely difficult to demonstrate anything that was analyzed to be significantly (statistically) different between MSTNDel273 sheep and WT, yet the authors seem to ignore this in much of their discussion. There is no explanation as to why the authors started with sheep that were FGF5 knockouts. The reviewer assumes that this was simply a line of sheep available from previous studies and the goal was to produce sheep with both improved hair/wool characteristics in addition to improved muscle development. However, the use of FGF5 knockout sheep complicates the ability to accurately decipher the unique aspects associated with targeting only myostatin for knock-out. At minimum, this is a variable that has to be considered in the statistical analysis. No information is provided on the methods used to produce the MSTNDel273 sheep, which seems fundamentally important. It is assumed they were produced by injecting one-cell zygotes then transferring these into surrogate females, but given the information provided, it is impossible to know. Certainly, the methods employed could have a profound effect on the outcome. There is no information provided on the sex of the animals produced and then analyzed.

Comments on revised version:

The manuscript by Chen et al. is improved and demonstrates successful gene editing in sheep embryos to obtain biallelic mutation of Mstn and FGF5. Despite the improvements in the revised manuscript, the cellular and molecular mechanism remain inadequate to conclude whether Fosl1 indeed acts downstream of myostatin. In addition, there is little that is new direction versus confirmatory for what is already well know regarding Mstn and FGF5

There are also a number of editorial mistakes e.g. the authors refer to tables S1-S4 in the materials and methods and results section, but there is no table S1-S4 provided.

Reviewer #1 (Public Review):

Summary:

The data clearly demonstrate that arpin is important for vessel barrier function, yet its genetic loss via a CRISPR strategy was not lethality, but led to viable animals in C57Blk strain at 12 weeks of age, albeit with leaky blood vessels. Pharmacological approaches were employed to demonstrate that loss of arpin led to ROCK1-dependent stress fiber formation that promoted increased permeability.

Strengths:

The results clearly demonstrate that arpin is expressed in the endothelium of blood vessels and its deficiency leads to leaky blood vessels in in vivo and in vitro models.

Weaknesses:

They conclude vessel leak was not related to enhanced Arp2/3 function through arpin deficiency, but no direct evidence of Arp2/3 activity is provided to support this conclusion. Instead, the authors concluded that ROCK1 activity was elevated in arpin knockdown cells and caused robust stress fiber formation. This idea could be strengthened by testing if ROCK1 inhibition by pharmacological block in arpin KO mice leads to less vascular leakage while pharmacological inhibition of Arp2/3 does not attenuate increased vessel permeability.

Reviewer #2 (Public Review):

Summary:

The authors have taken their previous finding that arpin is important for epithelial junctions and extended this to endothelial cells. They find that the positive effects of arpin on endothelial junctions are not dependent on Arp2/3 activity but instead on suppression of actinomyosin contractility.

Strengths:

The study uses standard approaches to test each of the components in the model. The quality of the experimental work is good and the amount of experimental evidence is sufficient to support this straightforward story.

Weaknesses:

The major weakness is that the story is a simple extension of the previous work on arpin and junctions in epithelial cells. The additional information is that the effects are not via Arp2/3 directly, but instead through an increase in actinomyosin contractility. However, the connection between arpin and increased ROCK activity is not revealed.

Reviewer #1 (Public Review):

Summary:

BMP signaling is, arguably, best known for its role in the dorsoventral patterning, but not in nematodes, where it regulates body size. In their paper, Vora et al. analyze ChIP-Seq and RNA-Seq data to identify direct transcriptional targets of SMA-3 (Smad) and SMA-9 (Schnurri) and understand the respective roles of SMA-3 and SMA-9 in the nematode model Caenorhabditis elegans. The authors use publicly available SMA-3 and SMA-9 ChIP-Seq data, own RNA-Seq data from SMA-3 and SMA-9 mutants, and bioinformatic analyses to identify the genes directly controlled by these two transcription factors (TFs) and find approximately 350 such targets for each. They show that all SMA-3-controlled targets are positively controlled by SMA-3 binding, while SMA-9-controlled targets can be either up or downregulated by SMA-9. 129 direct targets were shared by SMA-3 and SMA-9, and, curiously, the expression of 15 of them was activated by SMA-3 but repressed by SMA-9. Since genes responsible for cuticle collagen production were eminent among the SMA-3 targets, the authors focused on trying to understand the body size defect known to be elicited by the modulation of BMP signaling. Vora et al. provide compelling evidence that this defect is likely to be due to problems with the BMP signaling-dependent collagen secretion necessary for cuticle formation.

Strengths:

Vora et al. provide a valuable analysis of ChIP-Seq and RNA-Seq datasets, which will be very useful for the community. They also shed light on the mechanism of the BMP-dependent body size control by identifying SMA-3 target genes regulating cuticle collagen synthesis and by showing that downregulation of these genes affects body size in C. elegans.

Weaknesses:

(1) Although the analysis of the SMA-3 and SMA-9 ChIP-Seq and RNA-Seq data is extremely useful, the goal "to untangle the roles of Smad and Schnurri transcription factors in the developing C. elegans larva", has not been reached. While the role of SMA-3 as a transcriptional activator appears to be quite straightforward, the function of SMA-9 in the BMP signaling remains obscure. The authors write that in SMA-9 mutants, body size is affected, but they do not show any data on the mechanism of this effect.

(2) The authors clearly show that both TFs can bind independently of each other, however, by using distances between SMA-3 and SMA-9 ChIP peaks, they claim that when the peaks are close these two TFs act as complexes. In the absence of proof that SMA-3 and SMA-9 physically interact (e.g. that they co-immunoprecipitate - as they do in Drosophila), this is an unfounded claim, which should either be experimentally substantiated or toned down.

(3) The second part of the paper (the collagen story) is very loosely connected to the first part. dpy-11 encodes an enzyme important for cuticle development, and it is a differentially expressed direct target of SMA-3. dpy-11 can be bound by SMA-9, but it is not affected by this binding according to RNA-Seq. Thus, technically, this part of the paper does not require any information about SMA-9. However, this can likely be improved by addressing the function of the 15 genes, with the opposing mode of regulation by SMA-3 and SMA-9.

(4) The Discussion does not add much to the paper - it simply repeats the results in a more streamlined fashion.

Reviewer #2 (Public Review):

In the present study, Vora et al. elucidated the transcription factors downstream of the BMP pathway components Smad and Schnurri in C. elegans and their effects on body size. Using a combination of a broad range of techniques, they compiled a comprehensive list of genome-wide downstream targets of the Smads SMA-3 and SMA-9. They found that both proteins have an overlapping spectrum of transcriptional target sites they control, but also unique ones. Thereby, they also identified genes involved in one-carbon metabolism or the endoplasmic reticulum (ER) secretory pathway. In an elaborate effort, the authors set out to characterize the effects of numerous of these targets on the regulation of body size in vivo as the BMP pathway is involved in this process. Using the reporter ROL-6::wrmScarlet, they further revealed that not only collagen production, as previously shown, but also collagen secretion into the cuticle is controlled by SMA-3 and SMA-9. The data presented by Vora et al. provide in-depth insight into the means by which the BMP pathway regulates body size, thus offering a whole new set of downstream mechanisms that are potentially interesting to a broad field of researchers.

The paper is mostly well-researched, and the conclusions are comprehensive and supported by the data presented. However, certain aspects need clarification and potentially extended data.

(1) The BMP pathway is active during development and growth. Thus, it is logical that the data shown in the study by Vora et al. is based on L2 worms. However, it raises the question of if and how the pattern of transcriptional targets of SMA-3 and SMA-9 changes with age or in the male tail, where the BMP pathway also has been shown to play a role. Is there any data to shed light on this matter or are there any speculations or hypotheses?

(2) As it was shown that SMA-3 and SMA-9 potentially act in a complex to regulate the transcription of several genes, it would be interesting to know whether the two interact with each other or if the cooperation is more indirect.

(3) It would help the understanding of the data even more if the authors could specifically state if there were collagens among the genes regulated by SMA-3 and SMA-9 and which.

(4) The data on the role of SMA-3 and SMA-9 in the regulation of the secretion of collagens from the hypodermis is highly intriguing. The authors use ROL-6 as a reporter for the secretion of collagens. Is ROL-6 a target of SMA-9 or SMA-3? Even if this is not the case, the data would gain even more strength if a comparable quantification of the cuticular levels of ROL-6 were shown in Figure 6, and potentially a ratio of cuticular versus hypodermal levels. By that, the levels of secretion versus production can be better appreciated.

(5) It is known that the BMP pathway controls several processes besides body size. The discussion would benefit from a broader overview of how the identified genes could contribute to body size. The focus of the study is on collagen production and secretion, but it would be interesting to have some insights into whether and how other identified proteins could play a role or whether they are likely to not be involved here (such as the ones normally associated with lipid metabolism, etc.).

Reviewer #1 (Public Review):

Summary:

This manuscript by Aybar-Torres et al investigated the effect of common human STING1 variants on STING-mediated T cell phenotypes in mice. The authors previously made knock-in mice expressing human STING1 alleles HAQ or AQ, and here they established a new knock-in line Q293. The authors stimulated cells isolated from these mice with STING agonists and found that all three human mutant alleles resist cell death, leading to the conclusion that R293 residue is essential for STING-mediated cell death (there are several caveats with this conclusion, more below). The authors also bred HAQ and AQ alleles to the mouse Sting1-N153S SAVI mouse and observed varying levels of rescue of disease phenotypes with the AQ allele showing more complete rescue than the HAQ allele. The Q293 allele was not tested in the SAVI model. They conclude that the human common variants such as HAQ and AQ have a dominant negative effect over the gain-of-function SAVI mutants.

Strengths:

The authors and Dr. Jin's group previously made important observations of common human STING1 variants, and these knock-in mouse models are essential for understanding the physiological function of these alleles.

Weaknesses:

However, although some of the observations reported here are interesting, the data collectively does not support a unified model. The authors seem to be drawing two sets of conclusions from in vitro and in vivo experiments, and neither mechanism is clear. Several experiments need better controls, and these knock-in mice need more comprehensive functional characterization.

Reviewer #2 (Public Review):

Aybar-Torres and colleagues utilize common human STING alleles to dissect the mechanism of SAVI inflammatory disease. The authors demonstrate that these common alleles alleviate SAVI pathology in mice, and perhaps more importantly use the differing functionality of these alleles to provide insight into requirements of SAVI disease induction. Their findings suggest that it is residue A230 and/or Q293 that are required for SAVI induction, while the ability to induce an interferon-dependent inflammatory response is not. This is nicely exemplified by the AQ/SAVI mice that have an intact inflammatory response to STING activation, yet minimal disease progression. As both mutants seem to be resistant STING-dependent cell death, this manuscript also alludes to the importance of STING-dependent cell death, rather than STING-dependent inflammation, in the progression of SAVI pathology. I believe this manuscript makes some important connections between STING pathology mouse models and human genetics that would contribute to the field.

Summary:

This paper described the dynamics of the nuclear substructure called PML Nucleolar Association (PNA) in response to DNA damage on ribosomal DNA (rDNA) repeats. The authors showed that the PNA with rDNA repeats is induced by the inhibition of topoisomerases and RNA polymerase I and that the PNA formation is modulated by RAD51, thus homologous recombination. Artificially induced DNA double-strand breaks (DSBs) in rDNA repeats stimulate the formation of PNA with DSB markers. This DSB-triggered PNA formation is regulated by DSB repair pathways.

Strengths:

This paper illustrates a unique DNA damage-induced sub-nuclear structure containing the PML body, which is specifically associated with the nucleolus. Moreover, the dynamics of this PML Nucleolar Association (PNA) require topoisomerases and RNA polymerase I and are modulated by RAD51-mediated homologous recombination and non-homologous end-joining. This study provides a unique regulation of DSB repair at rDNA repeats associated with the unique-membrane-less subnuclear structure.

Weaknesses:

Although the PNA formation on rDNA repeat is nicely shown by cytological analysis, the biological significance of PNA in DSB repair is not fully addressed.

Reviewer #1 (Public Review):

Summary:<br /> In this work, the authors examine the activity and function of D1 and D2 MSNs in dorsomedial striatum (DMS) during an interval timing task. In this task, animals must first nose poke into a cued port on the left or right; if not rewarded after 6 seconds, they must switch to the other port. Thus, this task requires animals to estimate if at least 6 seconds have passed after the first nose poke. After verifying that animals estimate the passage of 6 seconds, the authors examine striatal activity during this interval. They report that D1-MSNs tend to decrease activity, while D2-MSNs increase activity, throughout this interval. They suggest that this activity follows a drift-diffusion model, in which activity increases (or decreases) to a threshold after which a decision is made. The authors next report that optogenetically inhibiting D1 or D2 MSNs, or pharmacologically blocking D1 and D2 receptors, increased the average wait time. This suggests that both D1 and D2 neurons contribute to the estimate of time, with a decrease in their activity corresponding to a decrease in the rate of 'drift' in their drift-diffusion model. Lastly, the authors examine MSN activity while pharmacologically inhibiting D1 or D2 receptors. The authors observe most recorded MSNs neurons decrease their activity over the interval, with the rate decreasing with D1/D2 receptor inhibition.

Major strengths:<br /> The study employs a wide range of techniques - including animal behavioral training, electrophysiology, optogenetic manipulation, pharmacological manipulations, and computational modeling. The question posed by the authors - how striatal activity contributes to interval timing - is of importance to the field and has been the focus of many studies and labs. This paper contributes to that line of work by investigating whether D1 and D2 neurons have similar activity patterns during the timed interval, as might be expected based on prior work based on striatal manipulations. However, the authors find that D1 and D2 neurons have distinct activity patterns. They then provide a decision-making model that is consistent with all results. The data within the paper is presented very clearly, and the authors have done a nice job presenting the data in a transparent manner (e.g., showing individual cells and animals). Overall, the manuscript is relatively easy to read and clear, with sufficient detail given in most places regarding the experimental paradigm or analyses used.

Major weaknesses:<br /> One weakness to me is the impact of identifying whether D1 and D2 had similar or different activity patterns. Does observing increasing/decreasing activity in D2 versus D1, or different activity patterns in D1 and D2, support one model of interval timing over another, or does it further support a more specific idea of how DMS contributes to interval timing?

I found the results presented in Figures 2 and 3 to be a little confusing or misleading. In Figure 2, the authors appear to claim that D1 neurons decrease their activity over the time interval while D2 neurons increase activity. The authors use this result to suggest that D1/D2 activity patterns are different. In Figure 3, a different analysis is done, and this time D2 neurons do not significantly increase their activity with time, conflicting with Figure 2. While in both figures, there is a significant difference between the mean slopes across the population, the secondary effect of positive/negative slope for D2/D1 neurons changes. I find this especially confusing as the authors refer back to the positive/negative slope for D2/D1 neurons result throughout the rest of the text.

It is a bit unclear to me how the authors chose the parameters for the model, and how well the model explains behavior is quantified. It seems that the authors didn't perform cross-validation across trials (i.e., they chose parameters that explained behavior across all trials combined, rather than choosing parameters from a subset of trials and determining whether those parameters are robust enough to explain behavior on held-out trials). I think this would increase the robustness of the result.

In addition, it remains a bit unclear to me how the authors changed the specific parameters they did to model the optogenetic manipulation. It seems these parameters were chosen because they fit the manipulation data. This makes me wonder if this model is flexible enough that there is almost always a set of parameters that would explain any experimental result; in other words, I'm not sure this model has high explanatory power.

Lastly, the results are based on a relatively small dataset (tens of cells).

Impact:<br /> The task and data presented by the authors are very intriguing, and there are many groups interested in how striatal activity contributes to the neural perception of time. The authors perform a wide variety of experiments and analysis to examine how DMS activity influences time perception during an interval-timing task, allowing for insight into this process. However, the significance of the key finding -- that D1 and D2 activity is distinct across time -- remains somewhat ambiguous to me.

Reviewer #2 (Public Review):

(1) Regarding the results in Figure 2 and Figure 5: for the heatmaps in Fig.2F and Fig.2E, the overall activity pattern of D1 and D2 MSNs looks very similar, both D1 and D2 MSNs contains neurons showing decreasing or increasing activity during interval timing. And the optogenetic and pharmacologic inhibition of either D1 or D2 MSNs resulted in similar behavior outcomes. To me, the D1 and D2 MSN activities were more complementary than opposing. If the authors want to emphasize the opposing side of D1 and D2 MSNs, then the manipulation experiments need to be re-designed, since the average activity of D2 MSNs increased, while D1 MSNs decreased during interval timing, instead of using inhibitory manipulations in both pathways, the authors should use inhibitory manipulation in D2-MSNs, while using optogenetic or pharmacology to activate D1-MSNs. In this way, the authors can demonstrate the opposing role of D1 and D2 MSNs and the functions of increased activity in D2-MSNs and decreased activity in D1-MSNs.

(2) Regarding the results in Figure 3 C and D, Figure 6 H and Figure 7 D, what is the sample size? From the single data points in the figures, it seems that the authors were using the number of cells to do statistical tests and plot the figures. For example, Figure 3 C, if the authors use n= 32 D2 MSNs and n= 41D1 MSNs to do the statistical test, it could make a small difference to be statistically significant. The authors should use the number of mice to do the statistical tests.

(3) Regarding the results in Figure 5, what is the reason for the increase in the response times? The authors should plot the position track during intervals (0-6 s) with or without optogenetic or pharmacologic inhibition. The authors can check Figures 3, 5, and 6 in the paper https://doi.org/10.1016/j.cell.2016.06.032 for reference to analyze the data.

Reviewer #3 (Public Review):

Summary:<br /> The cognitive striatum, also known as the dorsomedial striatum, receives input from brain regions involved in high-level cognition and plays a crucial role in processing cognitive information. However, despite its importance, the extent to which different projection pathways of the striatum contribute to this information processing remains unclear. In this paper, Bruce et al. conducted a study using various causal and correlational techniques to investigate how these pathways collectively contribute to interval timing in mice. Their results were consistent with previous research, showing that the direct and indirect striatal pathways perform opposing roles in processing elapsed time. Based on their findings, the authors proposed a revised computational model in which two separate accumulators track evidence for elapsed time in opposing directions. These results have significant implications for understanding the neural mechanisms underlying cognitive impairment in neurological and psychiatric disorders, as disruptions in the balance between direct and indirect pathway activity are commonly observed in such conditions.

Strengths:<br /> The authors employed a well-established approach to study interval timing and employed optogenetic tagging to observe the behavior of specific cell types in the striatum. Additionally, the authors utilized two complementary techniques to assess the impact of manipulating the activity of these pathways on behavior. Finally, the authors utilized their experimental findings to enhance the theoretical comprehension of interval timing using a computational model.

Weaknesses:<br /> The behavioral task used in this study is best suited for investigating elapsed time perception, rather than interval timing. Timing bisection tasks are often employed to study interval timing in humans and animals. In the optogenetic experiment, the laser was kept on for too long (18 seconds) at high power (12 mW). This has been shown to cause adverse effects on population activity (for example, through heating the tissue) that are not necessarily related to their function during the task epochs. Given the systemic delivery of pharmacological interventions, it is difficult to conclude that the effects are specific to the dorsomedial striatum. Future studies should use the local infusion of drugs into the dorsomedial striatum.

Comments on revised version:

Thank you for the comprehensive revisions. Most of my (addressable) concerns were addressed. The current version of your manuscript appears significantly improved.

Reviewer #1 (Public Review):

Summary:

This review evaluates the SCellBOW framework, which applies phenotype algebra to obtain vectors from cancer subclusters or user-defined subclusters.

Strengths:

SCellBOW employs an innovative application of NLP-inspired techniques to analyze scRNA-seq data, facilitating the identification and visualization of phenotypically divergent cell subpopulations.

The framework demonstrates robustness in accurately representing various cell types across multiple datasets, highlighting its versatility and utility in different biological contexts.

By simulating the impact of specific malignant subpopulations on disease prognosis, SCellBOW provides valuable insights into the relative risk and aggressiveness of cancer subpopulations, which is crucial for personalized therapeutic strategies.

The identification of a previously unknown and aggressive AR−/NElow subpopulation in metastatic prostate cancer underscores the potential of SCellBOW in uncovering clinically significant findings.

Weaknesses:

The reliance on bulk RNA-seq data as a reference raises concerns about potentially misleading results due to the presence of RNA expression from immune cells in the TME. It is unclear if SCellBOW adequately addresses this issue, which could affect the accuracy of the cancer subcluster vectors.

The method of extracting vectors in phenotype algebra appears to be a straightforward subtraction operation. This simplicity might limit its efficiency in excluding associations with phenotypes from specific subpopulations, potentially leading to inaccurate interpretations of the data.

The review would benefit from additional validation studies to assess the effectiveness of SCellBOW in distinguishing between cancerous and non-cancerous signals, particularly in heterogeneous tumor environments.

Further clarification on how SCellBOW handles mixed-cell populations within bulk RNA-seq data would strengthen the evaluation of its applicability and reliability in diverse research settings.

Reviewer #2 (Public Review):

Summary:

The authors developed a novel tool, SCellBOW, to perform cell clustering and infer survival risks on individual cancer cell clusters from the single-cell RNA seq dataset. The key ideas/techniques used in the tool include transfer learning, bag of words (BOW), and phenotype algebra which is similar to word algebra from natural language processing (NLP). Comparisons with existing methods demonstrated that SCellBOW provides superior clustering results and exhibits robust performance across a wide range of datasets. Importantly, a distinguishing feature of SCellBOW compared to other tools is its ability to assign risk scores to specific cancer cell clusters. Using SCellBOW, the authors identified a new group of prostate cancer cells characterized by a highly aggressive and dedifferentiated phenotype.

Strengths:

The application of natural language processing (NLP) to single-cell RNA sequencing (scRNA-seq) datasets is both smart and insightful. Encoding gene expression levels as word frequencies is a creative way to apply text analysis techniques to biological data. When combined with transfer learning, this approach enhances our ability to describe the heterogeneity of different cells, offering a novel method for understanding the biological behavior of individual cells and surpassing the capabilities of existing cell clustering methods. Moreover, the ability of the package to predict risk, particularly within cancer datasets, significantly expands the potential applications.

Weaknesses:

Given the promising nature of this tool, it would be beneficial for the authors to test the risk-stratification functionality on other types of tumors with high heterogeneity, such as liver and pancreatic cancers, which currently lack clinically relevant and well-recognized stratification methods. Additionally, it would be worthwhile to investigate how the tool could be applied to spatial transcriptomics by analyzing cell embeddings from different layers within these tissues.

Reviewer #1 (Public Review):

This study unveils a novel role for ferritin in Drosophila larval brain development. Furthermore, it pinpoints that the observed defects in larval brain development resulting from ferritin knockdown are attributed to impaired Fe-S cluster activity and ATP production. Overall this is a well-conducted and novel study.

The author have adequately addressed the concerns.

Reviewer #2 (Public Review):

Summary:

Zhixin and collaborators have investigated if the molecular pathways present in glia play a role in the proliferation, maintenance and differentiation of Neural Stem Cells. In this case, Drosophila Neuroblasts are used as models. Authors find that neuronal iron metabolism modulated by glial ferritin is an essential element for Neuroblast proliferation and differentiation. They show that loss of glial ferritin is sufficient to impact the number of neuroblasts. Remarkably, authors have identified that ferritin produced in the glia is secreted to be used as an iron source by the neurons. Therefore iron defects in glia have serious consequences in neuroblasts and likely vice versa. Interestingly, preventing iron absorption in the intestine is sufficient to reduce NB number. Furthermore, they have identified Zip13 as another regulator of the process. Evidence presented strongly indicates that the loss of neuroblasts is due to premature differentiation rather than cell death.

Strengths:

- Comprehensive analysis of the impact of glial iron metabolism in neuroblast behaviour by genetic and drug-based approaches as well as using a second model (mouse) for some validations.

- Using cutting edge methods such as RNAseq as well as very elegant and clean approaches such as RNAi-resistant lines or temperature-sensitive tools

- Goes beyond the state of the art highlighting iron as a key element in neuroblast formation as well as as a target in tumor treatments.

Comments on latest version:

The authors have successfully and convincingly addressed all comments from this reviewer. The modifications, changes and additions have increased the robustness of the results and clearly increased the readability of the manuscript.

This reviewer also appreciates all the efforts and extra work conducted by the authors to finish in a reasonable time all the experiments suggested by all reviewers.

In this study, the authors confirm that one of the genes classified as essential in a Tn-mutagenesis study in A. baumannii, Aeg1, is, in fact, an essential gene. The strength of the work is that it discovered that the depletion of Aeg1 leads to cell filamentation and that activation mutations in various cell division genes can suppress the requirement for Aeg1. These results suggest that Aeg1 plays an important role in cell division. The work's weakness is that it lacks convincing evidence to define Aeg1's place or role in the divisome assembly pathway. It is unclear whether proteins are at the division site under the wildtype condition and when Aeg1 is depleted, and whether Aeg1 is indeed required for a set of division proteins to the division site.

Reviewer comments:

The revised manuscript partially addressed two of the three major concerns from the previous assessment: (1) the functionality test of fluorescent fusion proteins using a spotting assay, and (2) membrane protein topology in the bacterial two-hybrid assays by constructing a C-terminal T25 fusion.

(1) In the spotting assay, all fluorescent fusion proteins rescued the growth of the corresponding deletion strain, which suggests these fusion proteins are functional. However, fluorescent images of these fusion proteins were diffusive, and only a few cells showed the expected midcell/membrane localization pattern for cell division proteins. This observation raised the concern that these fusion proteins may be cleaved in the middle, leading to the separation of the untagged fusion partner and diffusive fluorescent protein in the cytoplasm, which would explain the positive spotting rescue results. This phenomenon is commonly observed in other bacterial species. A western blot using an antibody targeting either the fluorescent protein or the fusion partner is widely used to examine whether the fusion protein is expressed at its full length.

(2) The authors constructed a C-terminal fusion of Aeg1 and showed that it still interacted with ZipA and FtsN. This result supports the authors' suggestion that the N-terminus of Aeg1 may not be the predicated membrane-targeting domain. Along the same line, the membrane topology of ZipA should also be considered. ZipA's N terminus is in the membrane facing the periplasm, and its C terminal domain is in the cytoplasm. Therefore, the PUT18C fusion will place the T18 domain of ZipA in the periplasm. All other division proteins' N termini are in the cytoplasm.

(3) Colocalization images did not show significant midcell localizations for each fluorescent protein; most cells showed diffusive cytoplasmic fluorescence. In all other species, midcell localization of cell division proteins is prominent in dividing cells, especially for early division proteins such as ZipA (at least 40-50% of cells show midcell bands). In A. baumannii, divisome localization timing may differ from other species, but this possibility needs to be established before the colocalization pattern is examined. Compounding this issue is that in Aeg1 depletion strains, some cells expressing ZipA, FtsB, FtsL, and FtsN fusions showed roughly regularly spaced puncta in long filamentous cells. It is hard to explain why this was observed if, under the WT condition, these fusions do not localize to the midcell. These results again raised concerns that these fusion proteins may not be functional and the observations are protein aggregates.

Besides these major issues, experimental observations did not support some claims in the main text. For example: (1) In the two-hybrid assay, only ZipA and FtsN showed significant interactions with Aeg1, as judged by the darkness of the blue spots. FtsL and FtsB showed pale spots. The quantified values accompanying this figure did not appear to agree with the image. (2) The spotting rescue assay showed that only FtsB-E56A and FtsA-E202K was able to bypass Aeg1 depletion (full dilution set comparable to that of Aeg1 complementation), but the main text claimed that FtsA-D124A and V144L, and FtsW-M254I and S274G also rescued the growth. These claims could be misleading.

Reviewer #1 (Public Review):

Summary:

Understanding the mechanisms of how organisms respond to environmental stresses is a key goal of biological research. Assessment of transcriptional responses to stress can provide some insights into those underlying mechanisms. The researchers quantified traits, fitness, and gene expression (transcriptional) response to salinity stress (control vs stress treatments) for 130 accessions of rice (three replicates for each accession), which were grown in the field in the Philippines. This experimental design allowed for many different types of downstream analyses to better understand the biology of the system. These analyses included estimating the strength of selection imposed on transcription in each environment, evaluating possible trade-offs in gene expression, testing whether salinity induces transcriptional decoherence, and conducting various eQTL-type analyses.

Strengths:

The study provides an extensive analysis of gene expression responses to stress in rice and offers some insights into underlying mechanisms of salinity responses in this important crop system. The fact that the study was conducted under field conditions is a major plus, as the gene expression responses to soil salinity are more realistic than if the study was conducted in a greenhouse or growth chamber. The preprint is generally well-written and the methods and results are mostly well-described.

Weaknesses:

While the study makes good use of analyzing the dataset, it is not clear how the current work advances our understanding of gene regulatory evolution or plant responses to soil salinity generally. Overall, the results are consistent with other prior studies of gene expression and studies of selection across environmental conditions. Some of the framing of the paper suggests that there is more novelty to this study than there is in reality. That said, the results will certainly be useful for those working in rice and should be interesting to scientists interested in how gene expression responses to stress occur under field conditions. I detail other concerns I had about the preprint below:

The abstract on lines 33-35 illustrates some of my concerns about the overstatement of the novelty of the current study. For example, is it really true that the role of gene expression in mediating stress response and adaptation is largely unexplored? There have been numerous studies that have evaluated gene expression responses to stresses in a wide range of organisms. Perhaps, I am missing something critically different about this study. If so, I would recommend that the authors reword this sentence to clarify what gap is being filled by this study. Further, is it really the case that none of them have evaluated how the correlational structure of gene expression changes in response to stresses in plants, as implied in lines 263-265? Don't the various modules and PC analyses of gene expression get at this question?

There were some places in the methods of the preprint that required more information to properly evaluate. For example, more information should be provided on lines 664-668 about how G, E, and GxE effects were established, especially since this is so central to this study. What programs/software (R? SAS? Other?) were used for these analyses? If R, how were the ANOVAs/models fit? What type of ANOVA was used? How exactly was significance determined for each term? Which effects were considered fixed and which were random? If the goal was to fit mixed models, why not use an approach like voom-limma (Law et al. 2014 Genome Biology)? More details should also be added to lines 688-709 about these analyses, including what software/programs were used for these analyses.

One thing that I found a bit confusing throughout was the intermixing of different terms and types of selection. In particular, there seemed to be some inconsistencies with the usage of quantitative genetics terms for selection (e.g. directional, stabilizing) vs molecular evolution terms for selection (e.g. positive, purifying). I would encourage the authors to think carefully about what they mean by each of these terms and make sure that those definitions are consistently applied here.

It would be useful to clarify the reasons for the inherent bias in the detection of conditional neutrality (CN) and antagonistic pleiotropy (AP; Lines 187-196). It is also not clear to me what the authors did to deal with the bias in terms of adjusting P-value thresholds for CN and AP the way it is currently written. Further, I found the discussion of antagonistic pleiotropy and conditional neutrality to be a bit confusing for a couple of reasons, especially around lines 489-491. First of all, does it really make sense to contrast gene expression versus local adaptation, when lots of local adaptation likely involves changes in gene expression? Second, the implication that antagonistic pleiotropy is more common for local adaptation than the results found in this study seems questionable. Conditional neutrality appears to be more common for local adaptation as well: see Table 2 of Wadgymar et al. 2017 Methods in Ecology and Evolution. That all said, it is always difficult to conclude that there are no trade-offs (antagonistic pleiotropy) for a particular locus, as the detecting trade-offs may only manifest in some years and not others and can require large sample sizes if they are subtle in effect.

Reviewer #2 (Public Review):

The authors investigate the gene expression variation in a rice diversity panel under normal and saline growth conditions to gain insight into the underlying molecular adaptive response to salinity. They present a convincing case to demonstrate that environmental stress can induce selective pressure on gene expression, which is in agreement to their earlier study (Groen et al, 2020). The data seems to be a good fit for their study and overall the analytic approach is robust.

(1) The work started by investigating the effect of genotype and their interaction at each transcript level using 3'-end-biased mRNA sequencing, and detecting a wide-spread GXE effect. Later, using the total filled grain number as a proxy of fitness, they estimated the strength of selection on each transcript and reported stronger selective pressure in a saline environment. However, this current framework relies on precise estimation of fitness and, therefore can be sensitive to the choice of fitness proxy.

(2) Furthermore, the authors decomposed the genetic architecture of expression variation into cis- and trans-eQTL in each environment separately and reported more unique environment-specific trans-eQTLs than cis-. The relative contribution of cis- and trans-eQTL depends on both the abundance and effect size. I wonder why the latter was not reported while comparing these two different genetic architectures. If the authors were to compare the variation explained by these two categories of eQTL instead of their frequency, would the inference that trans-eQTLs are primarily associated with expression variation still hold?

(3) Next, the authors investigated the relationship between cis- and trans-eQTLs at the transcript level and revealed an excess of reinforcement over the compensation pattern. Here, I struggle to understand the motivation for testing the relationship by comparing the effect of cis-QTL with the mean effect of all trans-eQTLs of a given transcript. My concern is that taking the mean can diminish the effect of small trans-eQTLs potentially biasing the relationship towards the large-effect eQTLs.

Reviewer #3 (Public Review):

In this work, the authors conducted a large-scale field trial of 130 indica accessions in normal vs. moderate salt stress conditions. The experiment consists of 3 replicates for each accession in each treatment, making it 780 plants in total. Leaf transcriptome, plant traits, and final yield were collected. Starting from a quantitative genetics framework, the authors first dissected the heritability and selection forces acting on gene expression. After summarizing the selection force acting on gene expression (or plant traits) in each environment, the authors described the difference in gene expression correlation between environments. The final part consists of eQTL investigation and categorizing cis- and trans-effects acting on gene expression.

Building on the group's previous study and using a similar methodology (Groen et al. 2020, 2021), the unique aspect of this study is in incorporating large-scale empirical field works and combining gene expression data with plant traits. Unlike many systems biology studies, this study strongly emphasizes the quantitative genetics perspective and investigates the empirical fitness effects of gene expression data. The large amounts of RNAseq data (one sample for each plant individual) also allow heritability calculation. This study also utilizes the population genetics perspective to test for traces of selection around eQTL. As there are too many genes to fit in multiple regression (for selection analysis) and to construct the G-matrix (for breeder's equation), grouping genes into PCs is a very good idea.

Building on large amounts of data, this study conducted many analyses and described some patterns, but a central message or hypothesis would still be necessary. Currently, the selection analysis, transcript correlation structure change, and eQTL parts seem to be independent. The manuscript currently looks like a combination of several parallel works, and this is reflected in the Results, where each part has its own short introduction (e.g., 185-187, 261-266, 349-353). It would be great to discuss how these patterns observed could be translated to larger biological insights. On a related note, since this and the previous studies (focusing on dry-wet environments) use a similar methodology, one would also wonder what the conclusions from these studies would be. How do they agree or disagree with each other?

Many analyses were done separately for each environment, and results from these two environments are listed together for comparison. Especially for the eQTL part, no specific comparison was discussed between the two environments. It would be interesting to consider whether one could fit the data in more coherent models specifically modeling the X-by-environment effects, where X might be transcripts, PCs, traits, transcript-transcript correlation, or eQTLs.

As stated, grouping genes into PCs is a good idea, but although in theory, the PCs are orthogonal, each gene still has some loadings on each PC (ie. each PC is not controlled by a completely different set of genes). Another possibility is to use any gene grouping method, such as WGCNA, to group genes into modules and use the PC1 of each module. There, each module would consist of completely different sets of genes, and one would be more likely to separate the biological functions of each module. I wonder whether the authors could discuss the pros and cons of these methods.

Reviewer #4 (Public Review):

The manuscript examines how patterns of selection on gene expression differ between a normal field environment and a field environment with elevated salinity based on transcript abundances obtained from leaves of a diverse panel of rice germplasm. In addition, the manuscript also maps expression QTL (eQTL) that explains variation in each environment. One highlight from the mapping is that a small group of trans-mapping regulators explains some gene expression variation for large sets of transcripts in each environment. The overall scope of the datasets is impressive, combining large field studies that capture information about fecundity, gene expression, and trait variation at multiple sites. The finding related to patterns indicating increased LD among eQTLs that have cis-trans compensatory or reinforcing effects is interesting in the context of other recent work finding patterns of epistatic selection. However, other analyses in the manuscript are less compelling or do not make the most of the value of collected data. Revisions are also warranted to improve the precision with which field-specific terminology is applied and the language chosen when interpreting analytical findings.

Selection of gene expression:<br /> One strength of the dataset is that gene expression and fecundity were measured for the same genotypes in multiple environments. However, the selection analyses are largely conducted within environments. The addition of phenotypic selection analyses that jointly analyze gene expression across environments and or selection on reaction norms would be worthwhile.

Gene expression trade-offs:<br /> The terminology and possibly methods involved in the section on gene expression trade-offs need amendment. I specifically recommend discontinuing reference to the analysis presented as an analysis of antagonistic pleiotropy (rather than more general trade-offs) because pleiotropy is defined as a property of a genotype, not a phenotype. Gene expression levels are a molecular phenotype, influenced by both genotype and the environment. By conducting analyses of selection within environments as reported, the analysis does not account for the fact that the distribution of phenotypic values, the fitness surface, or both may differ across environments. Thus, this presents a very different situation than asking whether the genotypic effect of a QTL on fitness differs across environments, which is the context in which the contrasting terms antagonistic pleiotropy and conditional neutrality have been traditionally applied. A more interesting analysis would be to examine whether the covariance of phenotype with fitness has truly changed between environments or whether the phenotypic distribution has just shifted to a different area of a static fitness surface.

Biological processes under selection / Decoherence: PCs are likely not the most ideal way to cluster genes to generate consolidated metrics for a selection gradient analysis. Because individual genes will contribute to multiple PCs, the current fractional majority-rule method applied to determine whether a PC is under direct or indirect selection for increased or decreased expression comes across as arbitrary and with the potential for double-counting genes. A gene co-expression network analysis could be more appropriate, as genes only belong to one module and one can examine how selection is acting on the eigengene of a co-expression module. Building gene co-expression modules would also provide a complementary and more concrete framework for evaluating whether salinity stress induces "decoherence" and which functional groups of genes are most impacted.

Selection of traits:<br /> Having paired organismal and molecular trait data is a strength of the manuscript, but the organismal trait data are underutilized. The manuscript as written only makes weak indirect inferences based on GO categories or assumed gene functions to connect selection at the organismal and molecular levels. Stronger connections could be made for instance by showing a selection of co-expression module eigengene values that are also correlated with traits that show similar patterns of selection, or by demonstrating that GWAS hits for trait variation co-localize to cis-mapping eQTL.

Genetic architecture of gene expression variation:<br /> The descriptive statistics of the eQTL analysis summarize counts of eQTLs observed in each environment, but these numbers are not broken down to the molecular trait level (e.g., what are the median and range of cis- and trans-eQTLs per gene). In addition, genetic architecture is a combination of the numbers and relative effect sizes of the QTLs. It would be useful to provide information about the relative distributions of phenotypic variance explained by the cis- vs. trans- eQTLs and whether those distributions vary by environment. The motivation for examining patterns of cis-trans compensation specifically for the results obtained under high salinity conditions is unclear to me. If the lines sampled have predominantly evolved under low salinity conditions and the hypothesis being evaluated relates to historical experience of stabilizing selection, then my intuition is that evaluating the eQTL patterns under normal conditions provides the more relevant test of the hypothesis.

Reviewer #1 (Public Review):

The study starts with the notion that in an AD-like disease model, ILC2s in the Rag1 knock-out were expanded and contained relatively more IL-5+ and IL-13+ ILC2s. This was confirmed in the Rag2 knock-out mouse model.

By using a chimeric mouse model in which wild-type knock-out splenocytes were injected into irradiated Rag1 knock-out mice, it was shown that even though the adaptive lymphocyte compartment was restored, there were increased AD-like symptoms and increased ILC2 expansion and activity. Moreover, in the reverse chimeric model, i.e. injecting a mix of wild-type and Rag1 knock-out splenocytes into irradiated wild-type animals, it was shown that the Rag1 knock-out ILC2s expanded more and were more active. Therefore, the authors could conclude that the RAG1 mediated effects were ILC2 cell-intrinsic.

Subsequent fate-mapping experiments using the Rag1Cre;reporter mouse model showed that there were indeed RAGnaïve and RAGexp ILC2 populations within naïve mice. Lastly, the authors performed multi-omic profiling, using single-cell RNA sequencing and ATAC-sequencing, in which a specific gene expression profile was associated with ILC2. These included well-known genes but the authors notably also found expression of Ccl1 and Ccr8 within the ILC2. The authors confirmed their earlier observations that in the RAGexp ILC2 population, the Th2 regulome was more suppressed, i.e. more closed, compared to the RAGnaïve population, indicative of the suppressive function of RAG on ILC2 activity. I do agree with the authors' notion that the main weakness was that this study lacks the mechanism by which RAG regulates these changes in ILC2s.

The manuscript is very well written and easy to follow, and the compelling conclusions are well supported by the data. The experiments are meticulously designed and presented. I wish to commend the authors for the study's quality.

Even though the study is compelling and well supported by the presented data, some additional context could increase the significance:

(1) The presence of the RAGnaïve and RAGexp ILC2 populations raises some questions on the (different?) origin of these populations. It is known that there are different waves of ILC2 origin (most notably shown in the Schneider et al Immunity 2019 publication, PMID 31128962). I believe it would be very interesting to further discuss or possibly show if there are different origins for these two ILC populations.

Several publications describe the presence and origin of ILC2s in/from the thymus (PMIDs 33432227 24155745). Could the authors discuss whether there might be a common origin for the RAGexp ILC2 and Th2 cells from a thymic lineage? If true that the two populations would be derived from different populations, e.g. being the embryonic (possibly RAGnaïve) vs. adult bone marrow/thymus (possibly RAGexp), this would show a unique functional difference between the embryonic derived ILC2 vs. adult ILC2.

(2) On line 104 & Figures 1C/G etc. the authors describe that in the RAG knock-out ILC2 are relatively more abundant in the lineage negative fraction. On line 108 they further briefly mentioned that this observation is an indication of enhanced ILC2 expansion. Since the study includes an extensive multi-omics analysis, could the authors discuss whether they have seen a correlation of RAG expression in ILC2 with regulation of genes associated with proliferation, which could explain this phenomenon?

Reviewer #2 (Public Review):

Summary:

The study by Ver Heul et al., investigates the consequences of RAG expression for type 2 innate lymphoid cell (ILC2) function. RAG expression is essential for the generation of the receptors expressed by B and T cells and their subsequent development. Innate lymphocytes, which arise from the same initial progenitor populations, are in part defined by their ability to develop in the absence of RAG expression. However, it has been described in multiple studies that a significant proportion of innate lymphocytes show a history of Rag expression. In compelling studies several years ago, members of this research team revealed that early Rag expression during the development of Natural Killer cells (Karo et al., Cell 2014), the first described innate lymphocyte, had functional consequences.

Here, the authors revisit this topic, a worthwhile endeavour given the broad history of Rag expression within all ILCs and the common use of RAG-deficient mice to specifically assess ILC function. Focusing on ILC2s and utilising state-of-the-art approaches, the authors sought to understand whether early expression of Rag during ILC2 development had consequences for activity, fitness, or function. Having identified cell-intrinsic effects in vivo, the authors investigated the causes of this, identifying epigenetic changes associated with the accessibility genes associated with core ILC2 functions.

The manuscript is well written and does an excellent job of supporting the reader through reasonably complex transcriptional and epigenetic analyses, with considerate use of explanatory diagrams. Overall I think that the conclusions are fair, the topic is thought-provoking, and the research is likely of broad immunological interest. I think that the extent of functional data and mechanistic insight is appropriate.

Strengths:

- The logical and stepwise use of mouse models to first demonstrate the impact on ILC2 function in vivo and a cell-intrinsic role. Initial analyses show enhanced cytokine production by ILC2 from RAG-deficient mice. Then through two different chimeric mice (including BM chimeras), the authors convincingly show this is cell intrinsic and not simply as a result of lymphopenia. This is important given other studies implicating enhanced ILC function in RAG-/- mice reflect altered competition for resources (e.g. cytokines).

- Use of Rag expression fate mapping to support analyses of how cells were impacted - this enables a robust platform supporting subsequent analyses of the consequences of Rag expression for ILC2.

- Use of snRNA-seq supports gene expression and chromatin accessibility studies - these reveal clear differences in the data sets consistent with altered ILC2 function.

- Convincing evidence of epigenetic changes associated with loci strongly linked to ILC2 function. This forms a detailed analysis that potentially helps explain some of the altered ILC2 functions observed in ex vivo stimulation assays.

- Provision of a wealth of expression data and bioinformatics analyses that can serve as valuable resources to the field.

Weaknesses:

- Lack of insight into precisely how early RAG expression mediates its effects, although I think this is beyond the scale of this current manuscript. Really this is the fundamental next question from the data provided here.

- The epigenetic analyses provide evidence of differences in the state of chromatin, but there is no data on what may be interacting or binding at these sites, impeding understanding of what this means mechanistically.

- Focus on ILC2 from skin-draining lymph nodes rather than the principal site of ILC2 activity itself (the skin). This may well reflect the ease at which cells can be isolated from different tissues.

- Comparison with ILC2 from other sites would have helped to substantiate findings and compensate for the reliance on data on ILC2 from skin-draining lymph nodes, which are not usually assessed amongst ILC2 populations.

- The studies of how ILC2 are impacted are a little limited, focused exclusively on IL-13 and IL-5 cytokine expression.

Reviewer #3 (Public Review):

In this study, Ver Heul et al. investigate the role of RAG expression in ILC2 functions. While RAG genes are not required for the development of ILCs, previous studies have reported a history of expression in these cells. The authors aim to determine the potential consequences of this expression in mature cells. They demonstrate that ILC2s from RAG1 or RAG2 deficient mice exhibit increased expression of IL-5 and IL-13 and suggest that these cells are expanded in the absence of RAG expression. However, it is unclear whether this effect is due to a direct impact of RAG genes or a consequence of the lack of T and B cells in this condition. This ambiguity represents a key issue with this study: distinguishing the direct effects of RAG genes from the indirect consequences of a lymphopenic environment.

The authors focus their study on ILC2s found in the skin-draining lymph nodes, omitting analysis of tissues where ILC2s are more enriched, such as the gut, lungs, and fat tissue. This approach is surprising given the goal of evaluating the role of RAG genes in ILC2s across different tissues. The study shows that ILC2s derived from RAG-/- mice are more activated than those from WT mice, and RAG-deficient mice show increased inflammation in an atopic dermatitis (AD)-like disease model. The authors use an elegant model to distinguish ILC2s with a history of RAG expression from those that never expressed RAG genes. However, this model is currently limited to transcriptional and epigenomic analyses, which suggest that RAG genes suppress the type 2 regulome at the Th2 locus in ILC2s.

The authors report a higher frequency of ILC2s in RAG-/- mice in skin-draining lymph nodes, which is expected as these mice lack T and B cells, leading to ILC expansion. Previous studies have reported hyper-activation of ILCs in RAG-deficient mice, suggesting that this is not necessarily an intrinsic phenomenon. For example, RAG-/- mice exhibit hyperphosphorylation of STAT3 in the gut, leading to hyperactivation of ILC3s. This study does not currently provide conclusive evidence of an intrinsic role of RAG genes in the hyperactivation of ILC2s. The splenocyte chimera model is artificial and does not reflect a normal environment in tissues other than the spleen. Similarly, the mixed BM model does not demonstrate an intrinsic role of RAG genes, as RAG1-/- BM cells cannot contribute to the B and T cell pool, leading to an expected expansion of ILC2s. As the data are currently presented it is expected that a proportion of IL-5-producing cells will come from the RAG1-/- BM.

Overall, the level of analysis could be improved. Total cell numbers are not presented, the response of other immune cells to IL-5 and IL-13 (except the eosinophils in the splenocyte chimera mice) is not analyzed, and the analysis is limited to skin-draining lymph nodes.

The authors have a promising model in which they can track ILC2s that have expressed RAG or not. They need to perform a comprehensive characterization of ILC2s in these mice, which develop in a normal environment with T and B cells. Approximately 50% of the ILC2s have a history of RAG expression. It would be valuable to know whether these cells differ from ILC2s that never expressed RAG, in terms of proliferation and expression of IL-5 and IL-13. These analyses should be conducted in different tissues, as ILC2s adapt their phenotype and transcriptional landscape to their environment. Additionally, the authors should perform their AD-like disease model in these mice.

The authors provide a valuable dataset of single-nuclei RNA sequencing (snRNA-seq) and ATAC sequencing (snATAC-seq) from RAGexp (RAG fate map-positive) and RAGnaïve (RAG fate map-negative) ILC2s. This elegant approach demonstrates that ILC2s with a history of RAG expression are epigenomically suppressed. However, key genes such as IL-5 and IL-13 do not appear to be differentially regulated between RAGexp and RAGnaïve ILC2s according to Table S5. Although the authors show that the regulome activity of IL-5 and IL-13 is decreased in RAGexp ILC2s, how do the authors explain that these genes are not differentially expressed between the RAGexp and RAGnaïve ILC2? I think that it is important to validate this in vivo.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors experimentally demonstrated the heterogeneous behavior of sarcomeres in cardiomyocytes and that a stochastic component exists in their contractile activity, which cancels out at the level of myofibrils.

Strengths:

The experiments and data analysis are robust and valid. With very good statistics and unbiased methods, they show cellular activity at the individual level and highlight the heterogeneity between biological networks. The similarity of the results to the study cited in [24] demonstrates the validity of the in vitro setup for answering these questions and the feasibility of such in-vitro systems to extend our knowledge of physiology.

Weaknesses:

Compared to the current literature ([24]), the study does not show a high degree of innovation. It mainly confirms what has been established in the past. The authors complemented the published experiments by developing an in vitro setup with stem cells and by changing the stiffness of the substrate to simulate pathological conditions. However, the experiments they performed do not allow them to explain more than the study in [24], and the conclusions of their study are based on interpretation and speculation about the possible mechanism underlying the observations.

Reviewer #2 (Public Review):

Summary:

Sarcomeres, the contractile units of skeletal and cardiac muscle, contract in a concerted fashion to power myofibril and thus muscle fiber contraction.

Muscle fiber contraction depends on the stiffness of the elastic substrate of the cell, yet it is not known how this dependence emerges from the collective dynamics of sarcomeres. Here, the authors analyze the contraction time series of individual sarcomeres using live imaging of fluorescently labeled cardiomyocytes cultured on elastic substrates of different stiffness. They find that reduced collective contractility of muscle fibers on unphysiologically stiff substrates is partially explained by a lack of synchronization in the contraction of individual sarcomeres.

This lack of synchronization is at least partially stochastic, consistent with the notion of a tug-of-war between sarcomeres on stiff sarcomeres. A particular irregularity of sarcomere contraction cycles is 'popping', the extension of sarcomeres beyond their rest length. The statistics of 'popping' suggest that this is a purely random process.

Strengths:

This study thus marks an important shift of perspective from whole-cell analysis towards an understanding of the collective dynamics of coupled, stochastic sarcomeres.

Weaknesses:

Further insight into mechanisms could be provided by additional analyses and/or comparisons to mathematical models.

Reviewer #3 (Public Review):

Summary:

The manuscript of Haertter and coworkers studied the variation of length of a single sarcomere and the response of microfibrils made by sarcomeres of cardiomyocytes on soft gel substrates of varying stiffnesses.

The measurements at the level of a single sarcomere are an important new result of this manuscript. They are done by combining the labeling of the sarcomeres z line using genetic manipulation and a sophisticated tracking program using machine learning. This single sarcomere analysis shows strong heterogeneities of the sarcomeres that can show fast oscillations not synchronized with the average behavior of the cell<br /> and what the authors call popping events which are large amplitude oscillations. Another important result is the fact that cardiomyocyte contractility decreases with the substrate stiffness although the properties of single sarcomeres do not seem to depend on substrate stiffness.

The authors suggest that the cardiomyocyte cell behavior is dominated by sarcomere heterogeneity. They show that the heterogeneity between sarcomeres is stochastic and that the contribution of static heterogeneity (such as composition differences between sarcomeres)<br /> is small.

Strengths:

All the results are to my knowledge new and original and deserve attention.

Weaknesses:

However, I find the manuscript a bit frustrating because the authors only give very qualitative explanations of the phenomena that they observe. They mention that popping could be explained by a nonlinear force-velocity relation of the sarcomere leading to a rapid detachment of all motors. However, they do not explicitly provide a theoretical description. How would the popping depend on the parameters and in particular on the substrate stiffness? Would the popping statistics be affected by the stiffness? It is also not clear to me how the dependence on the soft gel stiffness of the cardiomyocyte cell can be explained by the stochasticity of the sarcomere properties. Can any of the results found by the authors be explained by existing theories of cardiomyocytes? The only one I know is that of Safran and coworkers.

I also found the paper very difficult to read. The authors should perhaps reorganize the structure of the presentation in order to highlight what the new and important results are.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Yang et al report a novel regulatory role of SIRT4 in the progression of kidney fibrosis. The authors showed that in the fibrotic kidney, SIRT4 exhibited an increased nuclear localization. Deletion of Sirt4 in renal tubule epithelium attenuated the extent of kidney fibrosis following injury, while overexpression of SIRT4 aggravates kidney fibrosis. Employing a battery of in vitro and in vivo experiments, the authors demonstrated that SIRT4 interacts with U2AF2 in the nucleus upon TGF-β1 stimulation or kidney injury and deacetylates U2AF2 at K413, resulting in elevated CCN2 expression through alternative splicing of Ccn2 gene to promote kidney fibrosis. The authors further showed that the translocation of SIRT4 is through the BAX/BAK pore complex and is dependent on the ERK1/2-mediated phosphorylation of SIRT4 at S36, and consequently the binding of SIRT4 to importin α1. This fundamental work substantially advances our understanding of the progression of kidney fibrosis and uncovers a novel SIRT4-U2AF2-CCN2 axis as a potential therapeutic target for kidney fibrosis.

Strengths:

Overall, this is an extensive, well-performed study. The results are convincing, and the conclusions are mostly well supported by the data. The message is interesting to a wider community working on kidney fibrosis, protein acetylation, and SIRT4 biology.

Weaknesses:

The manuscript could be further strengthened if the authors could address a few points listed below:

(1) In the results part 3.9, an in vitro deacetylation assay employing recombinant SIRT4 and U2AF2 should be included to support the conclusion that SIRT4 is a deacetylase of U2AF2. Similarly, an in vitro binding assay can be included to confirm whether SIRT4 and U2AF2 are directly interacted.

(2) In Figure 6D, the Western Blot data using U2AF2-K453Q is confusing and is quite disconnected from the rest of the data and not explained. This data can be removed or explained why U2AF2-K453Q is employed here.

(3) Although ERK inhibitor U0126 blocked the nuclear translocation of SIRT4 in vivo, have the authors checked whether treatment with U0126 could affect the expression of kidney fibrosis markers in UUO mice?

(4) The format of gene and protein abbreviations in the manuscript should be standardized.

(5) There are a few grammar issues throughout the manuscript. The English/grammar could be stronger, thus improving the overall accessibility of the science to readers.

Reviewer #2 (Public Review):

Summary:

This manuscript presents a novel and significant investigation into the role of SIRT4 For CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing and its impact on the development of kidney fibrosis.

Strengths:

The authors' main conclusion is that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via pre-mRNA splicing. Additionally, the study reveals that SIRT4 translocates from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, further promoting its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Overall, the findings are fully convincing. The current study, to some extent, shows potential importance in this field. 

Weaknesses:

(1) Exosomes containing anti-SIRT4 antibodies were found to effectively mitigate UUO-induced kidney fibrosis in mice. While the protein loading capacity and loading methods were not mentioned.

(2) The method section is incomplete, and many methods like cell culture, cell transfection, gene expression profiling analysis, and splicing analysis, were not introduced in detail.

(3) The authors should compare their results with previous studies and mention clearly how their work is important in comparison to what has already been reported in the Discussion section.

Reviewer #3 (Public Review):

Summary:

Yang et al reported in this paper that TGF-beta induces SIRT4 activation, TGF-beta activated SIRT4 then modulates U2AF2 alternative splicing, U2AF2 in turn causes CCN2 for expression. The mechanism is described as this: mitochondrial SIRT4 transport into the cytoplasm in response to TGF-β stimulation, phosphorylated by ERK in the cytoplasm, and pathway and then undergo nuclear translocation by forming the complex with importin α1. In the nucleus, SIRT4 can then deacetylate U2AF2 at K413 to facilitate the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Moreover, they used exosomes to deliver Sirt4 antibodies to mitigate renal fibrosis in a mouse model. TGF-beta has been widely reported for its role in fibrosis induction.

Strengths:

TGF-beta induction of SIRT4 translocation from mitochondria to nuclei for epigenetics or gene regulation remains largely unknown. The findings presented here that SIRT4 is involved in U2AF2 deacetylation and CCN2 expression are interesting.

Weaknesses:

SIRT4 plays a critical role in mitochondria involved in respiratory chain reaction. This role of SIRT4 is critically involved in many cell functions. It is hard to rule out such a mitochondrial activity of SIRT4 in renal fibrosis. Moreover, the major concern is what kind of message mitochondrial SIRT4 proteins receive from TGF-beta. Although nuclear SIRT4 is increased in response to TNF treatment, it is likely de novo synthesized SIRT4 proteins can also undergo nuclear translocation upon cytokine stimulation. TGF-beta-induced mitochondrial calcium uptake and acetyl-CoA should be evaluated for calcium and acetyl-CoA may contribute to the gene expression regulation in nuclei.

Reviewer #1 (Public Review):

Summary:

Zhao et al. used the human forebrain organoid model, transgenic mice model, and embryonic neural progenitor cells to investigate the mutation previously identified in Williams Syndrome. They found abnormal proliferation and differentiation induced by this mutation, as well as altered expression profiles corresponding with aberrant cell clusters. This is regulated through the binding of GTF2IRD1 to transthyretin (TTR) promoter regions and tested on three models mentioned above on neurodevelopmental deficits.

Strengths:

Authors have applied both cell culture, organoid culture and in vivo model to test the previously reported mutation found in Williams Syndrome. They investigated cell behavior including proliferation and differentiation, while using the NGS technique to identify potential signaling pathways that are highly involved and can serve as a candidate to save the phenotype.

Reviewer #2 (Public Review):

Summary:

The study by Xingsen Zhao et al on "A human forebrain organoid model reveals the essential function of GTF2IRD1-TTR-ERK axis for the neurodevelopmental deficits of Williams Syndrome" presents a forebrain organoid model for WS and has identified defects in neurogenesis. The authors have performed scRNAseq from these patients' derived forebrain organoids showing upregulation expression in genes related to cell proliferation while genes involved in neuronal differentiation were downregulated. The major findings presented in this study are an increase in the size of SOX2+ ventricular zone in WS forebrain organoids with an altered developmental trajectory and aberrant excitatory neurogenesis. The study also presents evidence that transthyretin (TTR) has a reduced expression in WS organoids, and its expression is regulated by the transcription factor -GTF2IRD1. The authors then go on identity mechanistic details of TTR function on MAPK/ERK pathway which has been known to be involved in brain development. Overall, this is a well-constructed study revealing the function of one of the key genes that is deleted in WS and provides novel insights into mechanisms underlying the abnormal neurogenesis in WS brain.

Strengths:

WS patients have neurocognitive disorders which most likely stem from defects in early neurodevelopment. This study has investigated a WS forebrain organoid model with scRNAseq and identified differences in cell proliferation and differentiation. This study has presented some new evidence regarding the function and regulation of TTR and its regulator GTF2IRD1 during brain development.

Weaknesses:

Though the evidence presented for the mechanism of action of TTR on the MAPK pathway is unclear and lacks depth. It would require identifying downstream targets of TTR and how it interacts with the MAPK pathway.

Reviewer #1 (Public Review):

Summary:

Young (2.5 mo [adolescent]) rats were tasked to either press one lever for immediate reward or another for delayed reward. The task had a complex structure in which (1) the number of pellets provided on the immediate reward lever changed as a function of the decisions made, (2) rats were prevented from pressing the same lever three times in a row. Importantly, this task is very different from most intertemporal choice tasks which adjust delay (to the delayed lever), whereas this task held the delay constant and adjusted the number of 20 mg sucrose pellets provided on the immediate value lever.

Analyses are based on separating sessions into groups, but group membership includes arbitrary requirements and many sessions have been dropped from the analyses. Computational modeling is based on an overly simple reinforcement learning model, as evidenced by fit parameters pegging to the extremes. The neural analysis is overly complex and does not contain the necessary statistics to assess the validity of their claims.

Strengthes:

The task is interesting.

Weaknesses:

Behavior:

The basic behavioral results from this task are not presented. For example, "each recording session consisted of 40 choice trials or 45 minutes". What was the distribution of choices over sessions? Did that change between rats? Did that change between delays? Were there any sequence effects? (I recommend looking at reaction times.) Were there any effects of pressing a lever twice vs after a forced trial? This task has a very complicated sequential structure that I think I would be hard pressed to follow if I were performing this task. Before diving into the complex analyses assuming reinforcement learning paradigms or cognitive control, I would have liked to have understood the basic behaviors the rats were taking. For example, what was the typical rate of lever pressing? If the rats are pressing 40 times in 45 minutes, does waiting 8s make a large difference?

For that matter, the reaction time from lever appearance to lever pressing would be very interesting (and important). Are they making a choice as soon as the levers appear? Are they leaning towards the delay side, but then give in and choose the immediate lever? What are the reaction time hazard distributions?

It is not clear that the animals on this task were actually using cognitive control strategies on this task. One cannot assume from the task that cognitive control is key. The authors only consider a very limited number of potential behaviors (an overly simple RL model). On this task, there are a lot of potential behavioral strategies: "win-stay/lose-shift", "perseveration", "alternation", even "random choices" should be considered.

The delay lever was assigned to the "non-preferred side". How did side bias affect the decisions made?

The analyses based on "group" are unjustified. The authors compare the proportion of delayed to immediate lever press choices on the non-forced trials and then did k-means clustering on this distribution. But the distribution itself was not shown, so it is unclear whether the "groups" were actually different. They used k=3, but do not describe how this arbitrary number was chosen. (Is 3 the optimal number of clusters to describe this distribution?) Moreover, they removed three group 1 sessions with an 8s delay and two group 2 sessions with a 4s delay, making all the group 1 sessions 4s delay sessions and all group 2 sessions 8s delay sessions. They then ignore group 3 completely. These analyses seem arbitrary and unnecessarily complex. I think they need to analyze the data by delay. (How do rats handle 4s delay sessions? How do rats handle 6s delay sessions? How do rats handle 8s delay sessions?). If they decide to analyze the data by strategy, then they should identify specific strategies, model those strategies, and do model comparison to identify the best explanatory strategy. Importantly, the groups were session-based, not rat based, suggesting that rats used different strategies based on the delay to the delayed lever.

The reinforcement learning model used was overly simple. In particular, the RL model assumes that the subjects understand the task structure, but we know that even humans have trouble following complex task structures. Moreover, we know that rodent decision-making depends on much more complex strategies (model-based decisions, multi-state decisions, rate-based decisions, etc). There are lots of other ways to encode these decision variables, such as softmax with an inverse temperature rather than epsilon-greedy. The RL model was stated as a given and not justified. As one critical example, the RL model fit to the data assumed a constant exponential discounting function, but it is well-established that all animals, including rodents, use hyperbolic discounting in intertemporal choice tasks. Presumably this changes dramatically the effect of 4s and 8s. As evidence that the RL model is incomplete, the parameters found for the two groups were extreme. (Alpha=1 implies no history and only reacting to the most recent event. Epsilon=0.4 in an epsilon-greedy algorithm is a 40% chance of responding randomly.)

The authors do add a "dbias" (which is a preference for the delayed lever) term to the RL model, but note that it has to be maximal in the 4s condition to reproduce group 2 behavior, which means they are not doing reinforcement learning anymore, just choosing the delayed lever.

Neurophysiology:

The neurophysiology figures are unclear and mostly uninterpretable; they do not show variability, statistics or conclusive results.

As with the behavior, I would have liked to have seen more traditional neurophysiological analyses first. What do the cells respond to? How do the manifolds change aligned to the lever presses? Are those different between lever presses? Are there changes in cellular information (both at the individual and ensemble level) over time in the session? How do cellular responses differ during that delay while both levers are out, but the rats are not choosing the immediate lever?

Figure 3, for example, claims that some of the principal components tracked the number of pellets on the immediate lever ("ival"), but they are just two curves. No statistics, controls, or justification for this is shown. BTW, on Figure 3, what is the event at 200s?

I'm confused. On Figure 4, the number of trials seems to go up to 50, but in the methods, they say that rats received 40 trials or 45 minutes of experience.

At the end of page 14, the authors state that the strength of the correlation did not differ by group and that this was "predicted" by the RL modeling, but this statement is nonsensical, given that the RL modeling did not fit the data well, depended on extreme values. Moreover, this claim is dependent on "not statistically detectable", which is, of course, not interpretable as "not different".

There is an interesting result on page 16 that the increases in theta power were observed before a delayed lever press but not an immediate lever press, and then that the theta power declined after an immediate lever press. These data are separated by session group (again group 1 is a subset of the 4s sessions, group 2 is a subset of the 8s sessions, and group 3 is ignored). I would much rather see these data analyzed by delay itself or by some sort of strategy fit across delays. That being said, I don't see how this description shows up in Figure 6. What does Figure 6 look like if you just separate the sessions by delay?

Discussion:

Finally, it is unclear to what extent this task actually gets at the questions originally laid out in the goals and returned to in the discussion. The idea of cognitive effort is interesting, but there is no data presented that this task is cognitive at all. The idea of a resourced cognitive effort and a resistance cognitive effort is interesting, but presumably the way one overcomes resistance is through resource-limited components, so it is unclear that these two cognitive effort strategies are different.

The authors state that "ival-tracking" (neurons and ensembles that presumably track the number of pellets being delivered on the immediate lever - a fancy name for "expectations") "taps into a resourced-based form of cognitive effort", but no evidence is actually provided that keeping track of the expectation of reward on the immediate lever depends on attention or mnemonic resources. They also state that a "dLP-biased strategy" (waiting out the delay) is a "resistance-based form of cognitive effort" but no evidence is made that going to the delayed side takes effort.

The authors talk about theta synchrony, but never actually measure theta synchrony, particularly across structures such as amygdala or ventral hippocampus. The authors try to connect this to "the unpleasantness of the delay", but provide no measures of pleasantness or unpleasantness. They have no evidence that waiting out an 8s delay is unpleasant.

The authors hypothesize that the "ival-tracking signal" (the expectation of number of pellets on the immediate lever) "could simply reflect the emotional or autonomic response". Aside from the fact that no evidence for this is provided, if this were to be true, then, in what sense would any of these signals be related to cognitive control?

Reviewer #2 (Public Review):

Summary:

This manuscript explores the neuronal signals that underlie resistance vs resource-based models of cognitive effort. The authors use a delayed discounting task and computational models to explore these ideas. The authors find that the ACC strongly tracks value and time, which is consistent with prior work. Novel contributions include quantification of a resource-based control signal among ACC ensembles, and linking ACC theta oscillations to a resistance-based strategy.

Strengths:

The experiments and analyses are well done and have the potential to generate an elegant explanatory framework for ACC neuronal activity. The inclusion of local-field potential / spike-field analyses is particularly important because these can be measured in humans.

Weaknesses:

I had questions that might help me understand the task and details of neuronal analyses.

(1) The abstract, discussion, and introduction set up an opposition between resource and resistance-based forms of cognitive effort. It's clear that the authors find evidence for each (ACC ensembles = resource, theta=resistance?) but I'm not sure where the data fall on this dichotomy.<br /> a. An overall very simple schematic early in the paper (prior to the MCML model? or even the behavior) may help illustrate the main point.<br /> b. In the intro, results, and discussion, it may help to relate each point to this dichotomy.<br /> c. What would resource-based signals look like? What would resistance based signals look like? Is the main point that resistance-based strategies dominate when delays are short, but resource-based strategies dominate when delays are long?<br /> d. I wonder if these strategies can be illustrated? Could these two measures (dLP vs ival tracking) be plotted on separate axes or extremes, and behavior, neuronal data, LFP, and spectral relationships be shown on these axes? I think Figure 2 is working towards this. Could these be shown for each delay length? This way, as the evidence from behavior, model, single neurons, ensembles, and theta is presented, it can be related to this framework, and the reader can organize the findings.

(2) The task is not clear to me.<br /> a. I wonder if a task schematic and a flow chart of training would help readers.<br /> b. This task appears to be relatively new. Has it been used before in rats (Oberlin and Grahame is a mouse study)? Some history / context might help orient readers.<br /> c. How many total sessions were completed with ascending delays? Was there criteria for surgeries? How many total recording sessions per animal (of the 54?)<br /> d. How many trials completed per session (40 trials OR 45 minutes)? Where are there errors? These details are important for interpreting Figure 1.

(3) Figure 1 is unclear to me.<br /> a. Delayed vs immediate lever presses are being plotted - but I am not sure what is red, and what is blue. I might suggest plotting each animal.<br /> b. How many animals and sessions go into each data point?<br /> c. Table 1 (which might be better referenced in the paper) refers to rats by session. Is it true that some rats (2 and 8) were not analyzed for the bulk of the paper? Some rats appear to switch strategies, and some stay in one strategy. How many neurons come from each rat?<br /> d. Task basics - RT, choice, accuracy, video stills - might help readers understand what is going into these plots<br /> e. Does the animal move differently (i.e., RTs) in G1 vs. G2?

(4) I wasn't sure how clustered G1 vs. G2 vs G3 are. To make this argument, the raw data (or some axis of it) might help.<br /> a. This is particularly important because G3 appears to be a mix of G1 and G2, although upon inspection, I'm not sure how different they really are<br /> b. Was there some objective clustering criteria that defined the clusters?<br /> c. Why discuss G3 at all? Can these sessions be removed from analysis?

(5) The same applies to neuronal analyses in Fig 3 and 4<br /> a. What does a single neuron peri-event raster look like? I would include several of these.<br /> b. What does PC1, 2 and 3 look like for G1, G2, and G3?<br /> c. Certain PCs are selected, but I'm not sure how they were selected - was there a criteria used? How was the correlation between PCA and ival selected? What about PCs that don't correlate with ival?<br /> d. If the authors are using PCA, then scree plots and PETHs might be useful, as well as comparisons to PCs from time-shuffled / randomized data.

(6) I had questions about the spectral analysis<br /> a. Theta has many definitions - why did the authors use 6-12 Hz? Does it come from the hippocampal literature, and is this the best definition of theta?. What about other bands (delta - 1-4 Hz), theta (4-7 Hz); and beta - 13- 30 Hz? These bands are of particular importance because they have been associated with errors, dopamine, and are abnormal in schizophrenia and Parkinson's disease.<br /> b. Power spectra and time-frequency analyses may justify the authors focus. I would show these (y-axis - frequency, x-axis - time, z-axis, power).

(7) PC3 as an autocorrelation doesn't seem the to be right way to infer theta entrainment or spike-field relationships, as PCA can be vulnerable to phantom oscillations, and coherence can be transient. It is also difficult to compare to traditional measures of phase-locking. Why not simply use spike-field coherence? This is particularly important with reference to the human literature, which the authors invoke.

Reviewer #3 (Public Review):

Summary:

The study investigated decision making in rats choosing between small immediate rewards and larger delayed rewards, in a task design where the size of the immediate rewards decreased when this option was chosen and increased when it was not chosen. The authors conceptualise this task as involving two different types of cognitive effort; 'resistance-based' effort putatively needed to resist the smaller immediate reward, and 'resource-based' effort needed to track the changing value of the immediate reward option. They argue based on analyses of the behaviour, and computational modelling, that rats use different strategies in different sessions, with one strategy in which they consistently choose the delayed reward option irrespective of the current immediate reward size, and another strategy in which they preferentially choose the immediate reward option when the immediate reward size is large, and the delayed reward option when the immediate reward size is small. The authors recorded neural activity in anterior cingulate cortex (ACC) and argue that ACC neurons track the value of the immediate reward option irrespective of the strategy the rats are using. They further argue that the strategy the rats are using modulates their estimated value of the immediate reward option, and that oscillatory activity in the 6-12Hz theta band occurs when subjects use the 'resistance-based' strategy of choosing the delayed option irrespective of the current value of the immediate reward option. If solid, these findings will be of interest to researchers working on cognitive control and ACCs involvement in decision making. However, there are some issues with the experiment design, reporting, modelling and analysis which currently preclude high confidence in the validity of the conclusions.

Strengths:

The behavioural task used is interesting and the recording methods should enable the collection of good quality single unit and LFP electrophysiology data. The authors recorded from a sizable sample of subjects for this type of study. The approach of splitting the data into sessions where subjects used different strategies and then examining the neural correlates of each is in principle interesting, though I have some reservations about the strength of evidence for the existence of multiple strategies.

Weaknesses:

The dataset is very unbalanced in terms of both the number of sessions contributed by each subject, and their distribution across the different putative behavioural strategies (see table 1), with some subjects contributing 9 or 10 sessions and others only one session, and it is not clear from the text why this is the case. Further, only 3 subjects contribute any sessions to one of the behavioural strategies, while 7 contribute data to the other such that apparent differences in brain activity between the two strategies could in fact reflect differences between subjects, which could arise due to e.g. differences in electrode placement. To firm up the conclusion that neural activity is different in sessions where different strategies are thought to be employed, it would be important to account for potential cross-subject variation in the data. The current statistical methods don't do this as they all assume fixed effects (e.g. using trials or neurons as the experimental unit and ignoring which subject the neuron/trial came from).

It is not obvious that the differences in behaviour between the sessions characterised as using the 'G1' and 'G2' strategies actually imply the use of different strategies, because the behavioural task was different in these sessions, with a shorter wait (4 seconds vs 8 seconds) for the delayed reward in the G1 strategy sessions where the subjects consistently preferred the delayed reward irrespective of the current immediate reward size. Therefore the differences in behaviour could be driven by difference in the task (i.e. external world) rather than a difference in strategy (internal to the subject). It seems plausible that the higher value of the delayed reward option when the delay is shorter could account for the high probability of choosing this option irrespective of the current value of the immediate reward option, without appealing to the subjects using a different strategy.

Further, even if the differences in behaviour do reflect different behavioural strategies, it is not obvious that these correspond to allocation of different types of cognitive effort. For example, subjects' failure to modify their choice probabilities to track the changing value of the immediate reward option might be due simply to valuing the delayed reward option higher, rather than not allocating cognitive effort to tracking immediate option value (indeed this is suggested by the neural data). Conversely, if the rats assign higher value to the delayed reward option in the G1 sessions, it is not obvious that choosing it requires overcoming 'resistance' through cognitive effort.

The RL modelling used to characterise the subject's behavioural strategies made some unusual and arguably implausible assumptions:

i) The goal of the agent was to maximise the value of the immediate reward option (ival), rather than the standard assumption in RL modelling that the goal is to maximise long-run (e.g. temporally discounted) reward. It is not obvious why the rats should be expected to care about maximising the value of only one of their two choice options rather than distributing their choices to try and maximise long run reward.

ii) The modelling assumed that the subject's choice could occur in 7 different states, defined by the history of their recent choices, such that every successive choice was made in a different state from the previous choice. This is a highly unusual assumption (most modelling of 2AFC tasks assumes all choices occur in the same state), as it causes learning on one trial not to generalise to the next trial, but only to other future trials where the recent choice history is the same.

iii) The value update was non-standard in that rather than using the trial outcome (i.e. the amount of reward obtained) as the update target, it instead appeared to use some function of the value of the immediate reward option (it was not clear to me from the methods exactly how the fival and fqmax terms in the equation are calculated) irrespective of whether the immediate reward option was actually chosen.

iv) The model used an e-greedy decision rule such that the probability of choosing the highest value option did not depend on the magnitude of the value difference between the two options. Typically, behavioural modelling uses a softmax decision rule to capture a graded relationship between choice probability and value difference.

v) Unlike typical RL modelling where the learned value differences drive changes in subjects' choice preferences from trial to trial, to capture sensitivity to the value of the immediately rewarding option the authors had to add in a bias term which depended directly on this value (not mediated by any trial-to-trial learning). It is not clear how the rat is supposed to know the current trial ival if not by learning over previous trials, nor what purpose the learning component of the model serves if not to track the value of the immediate reward option.

Given the task design, a more standard modelling approach would be to treat each choice as occurring in the same state, with the (temporally discounted) value of the outcomes obtained on each trial updating the value of the chosen option, and choice probabilities driven in a graded way (e.g. softmax) by the estimated value difference between the options. It would be useful to explicitly perform model comparison (e.g. using cross-validated log-likelihood with fitted parameters) of the authors proposed model against more standard modelling approaches to test whether their assumptions are justified. It would also be useful to use logistic regression to evaluate how the history of choices and outcomes on recent trials affects the current trial choice, and compare these granular aspects of the choice data with simulated data from the model.

There were also some issues with the analyses of neural data which preclude strong confidence in their conclusions:

Figure 4I makes the striking claim that ACC neurons track the value of the immediately rewarding option equally accurately in sessions where two putative behavioural strategies were used, despite the behaviour being insensitive to this variable in the G1 strategy sessions. The analysis quantifies the strength of correlation between a component of the activity extracted using a decoding analysis and the value of the immediate reward option. However, as far as I could see this analysis was not done in a cross-validated manner (i.e. evaluating the correlation strength on test data that was not used for either training the MCML model or selecting which component to use for the correlation). As such, the chance level correlation will certainly be greater than 0, and it is not clear whether the observed correlations are greater than expected by chance.

An additional caveat with the claim that ACC is tracking the value of the immediate reward option is that this value likely correlates with other behavioural variables, notably the current choice and recent choice history, that may be encoded in ACC. Encoding analyses (e.g. using linear regression to predict neural activity from behavioural variables) could allow quantification of the variance in ACC activity uniquely explained by option values after controlling for possible influence of other variables such as choice history (e.g. using a coefficient of partial determination).

Figure 5 argues that there are systematic differences in how ACC neurons represent the value of the immediate option (ival) in the G1 and G2 strategy sessions. This is interesting if true, but it appears possible that the effect is an artefact of the different distribution of option values between the two session types. Specifically, due to the way that ival is updated based on the subjects' choices, in G1 sessions where the subjects are mostly choosing the delayed option, ival will on average be higher than in G2 sessions where they are choosing the immediate option more often. The relative number of high, medium and low ival trials in the G1 and G2 sessions will therefore be different, which could drive systematic differences in the regression fit in the absence of real differences in the activity-value relationship. I have created an ipython notebook illustrating this, available at: https://notebooksharing.space/view/a3c4504aebe7ad3f075aafaabaf93102f2a28f8c189ab9176d4807cf1565f4e3. To verify that this is not driving the effect it would be important to balance the number of trials at each ival level across sessions (e.g. by subsampling trials) before running the regression.

Joint Public Review:

Summary:

This study retrospectively analyzed clinical data to develop a risk prediction model for pulmonary hypertension in high-altitude populations. This finding holds clinical significance as it can be used for intuitive and individualized prediction of pulmonary hypertension risk in these populations. The strength of evidence is high, utilizing a large cohort of 6,603 patients and employing statistical methods such as LASSO regression. The model demonstrates satisfactory performance metrics, including AUC values and calibration curves, enhancing its clinical applicability.

Strengths:

(1) Large Sample Size: The study utilizes a substantial cohort of 6,603 subjects, enhancing the reliability and generalizability of the findings.

(2) Robust Methodology: The use of advanced statistical techniques, including least absolute shrinkage and selection operator (LASSO) regression and multivariate logistic regression, ensures the selection of optimal predictive features.

(3) Clinical Utility: The developed nomograms are user-friendly and can be easily implemented in clinical settings, particularly in resource-limited high-altitude regions.

(4) Performance Metrics: The models demonstrate satisfactory performance, with strong AUC values and well-calibrated curves, indicating accurate predictions.

Weaknesses:

(1) Lack of External Validation: The models were validated internally, but external validation with cohorts from other high-altitude regions is necessary to confirm their generalizability.

(2) Simplistic Predictors: The reliance on ECG and basic demographic data may overlook other potential predictors that could improve the models' accuracy and predictive power.

(3) Regional Specificity: The study's cohort is limited to Tibet, and the findings may not be directly applicable to other high-altitude populations without further validation.

Reviewer #5 (Public Review):

After reading the manuscript and the concerns raised by reviewer 2 I see both sides of the argument - the relative location of trigeminal nucleus versus the inferior olive is quite different in elephants (and different from previous studies in elephants), but when there is a large disproportionate magnification of a behaviorally relevant body part at most levels of the nervous system (certainly in the cortex and thalamus), you can get major shifting in the location of different structures. In the case of the elephant, it looks like there may be a lot of shifting. Something that is compelling is that the number of modules separated but the myelin bands correspond to the number of trunk folds which is different in the different elephants. This sort of modular division based on body parts is a general principle of mammalian brain organization (demonstrated beautifully for the cuneate and gracile nucleus in primates, VP in most of species, S1 in a variety of mammals such as the star nosed mole and duck-billed platypus). I don't think these relative changes in the brainstem would require major genetic programming - although some surely exist. Rodents and elephants have been independently evolving for over 60 million years so there is a substantial amount of time for changes in each l lineage to occur.

I agree that the authors have identified the trigeminal nucleus correctly, although comparisons with more out-groups would be needed to confirm this (although I'm not suggesting that the authors do this). I also think the new figure (which shows previous divisions of the brainstem versus their own) allows the reader to consider these issues for themselves. When reviewing this paper, I actually took the time to go through atlases of other species and even look at some of my own data from highly derived species. Establishing homology across groups based only on relative location is tough especially when there appears to be large shifts in the relative location of structures. My thoughts are that the authors did an extraordinary amount of work on obtaining, processing and analyzing this extremely valuable tissue. They document their work with images of the tissue and their arguments for their divisions are solid. I feel that they have earned the right to speculate - with qualifications - which they provide.

Reviewer #1 (Public Review):

This manuscript remains an intriguing investigation of the elephant brainstem, with particular attention drawn to possible sensory and motor representation of the renowned trunk of African and Asian elephants. As the authors note, this area has traditionally been identified as part of the superior olivary complex and associated with the fine motor control of the trunk; however, notable patterns within myelin stripes suggest that its parcellation may relate to specific regions/folds found along the long axis of the trunk, including elaborated regions for the trunk "finger" distal end.

In this iteration of the manuscript, the researchers have provided peripherin antibody staining within the regions they have identified as the trigeminal nucleus and the superior olive. These data, with abundant peripherin expression within climbing fibers of the presumed superior olive and relatively lower expression within the trigeminal nucleus, bolster their interpretation of having comprehensively identified the trigeminal nucleus and trunk representation via a battery of neuroanatomical methods.

All other conclusions remain the same, and these data have provoked intriguing and animated discussion on classification of neuroanatomical structure, particularly in species with relatively limited access to specimens. Most significantly, these discussions have underscored the fundamental nature of comparative methods (from protein to cellular to anatomical levels), including interpreting homologous structures among species of varying levels of relatedness.

Reviewer #2 (Public Review):

Here I submit my previous review and a great deal of additional information following on from the initial review and the response by the authors.

* Initial Review *

Assessment:

This manuscript is based upon the unprecedented identification of an apparently highly unusual trigeminal nuclear organization within the elephant brainstem, related to a large trigeminal nerve in these animals. The apparently highly specialized elephant trigeminal nuclear complex identified in the current study has been classified as the inferior olivary nuclear complex in four previous studies of the elephant brainstem. The entire study is predicated upon the correct identification of the trigeminal sensory nuclear complex and the inferior olivary nuclear complex in the elephant, and if this is incorrect, then the remainder of the manuscript is merely unsupported speculation. There are many reasons indicating that the trigeminal nuclear complex is misidentified in the current study, rendering the entire study, and associated speculation, inadequate at best, and damaging in terms of understanding elephant brains and behaviour at worst.

Original Public Review:

The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.<br /> The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, but the current study differs.

Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others appears to be just "lateral nuclei" with no suggestion of what might be there instead.

Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occur. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400,000 and 500,000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

But let's look more specifically at the justification outlined in the current study to support their identification of the unusually located trigeminal sensory nuclei of the brainstem.

(1) Intense cytochrome oxidase reactivity<br /> (2) Large size of the putative trunk module<br /> (3) Elongation of the putative trunk module<br /> (4) Arrangement of these putative modules correspond to elephant head anatomy<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds<br /> (6) Location apparently matches other mammals<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals

Let's examine these justifications more closely.

(1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. In order to obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. It is likely that the histochemical staining observed is background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.<br /> Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

(4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

(6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species, the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

(7) The dual to quadruple repetition of rostro-caudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. But, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

(8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to detail to support the major changes indicated by the authors.

So what are these "bumps" in the elephant brainstem?

Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship to the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

* Review of Revised Manuscript *

Assessment:

There is a clear dichotomy between the authors and this reviewer regarding the identification of specific structures, namely the inferior olivary nuclear complex and the trigeminal nuclear complex, in the brainstem of the elephant. The authors maintain the position that in the elephant alone, irrespective of all the published data on other mammals and previously published data on the elephant brainstem, these two nuclear complexes are switched in location. The authors maintain that their interpretation is correct, but this reviewer maintains that this interpretation is erroneous. The authors expressed concern that the remainder of the paper was not addressed by the reviewer, but the reviewer maintains that these sequelae to the misidentification of nuclear complexes in the elephant brainstem render any of these speculations irrelevant as the critical structures are incorrectly identified. It is this reviewer's opinion that this paper is incorrect. I provide a lot of detail below in order to provide support to the opinion I express.

Public Review of Current Submission:

As indicated in my previous review of this manuscript (see above), it is my opinion that the authors have misidentified, and indeed switched, the inferior olivary nuclear complex (IO) and the trigeminal nuclear complex (Vsens). It is this specific point only that I will address in this second review, as this is the crucial aspect of this paper - if the identification of these nuclear complexes in the elephant brainstem by the authors is incorrect, the remainder of the paper does not have any scientific validity.

The authors, in their response to my initial review, claim that I "bend" the comparative evidence against them. They further claim that as all other mammalian species exhibit a "serrated" appearance of the inferior olive, and as the elephant does not exhibit this appearance, what was previously identified as the inferior olive is actually the trigeminal nucleus and vice versa.

For convenience, I will refer to IOM and VsensM as the identification of these structures according to Maseko et al (2013) and other authors and will use IOR and VsensR to refer to the identification forwarded in the study under review.<br /> The IOM/VsensR certainly does not have a serrated appearance in elephants. Indeed, from the plates supplied by the authors in response (Referee Fig. 2), the cytochrome oxidase image supplied and the image from Maseko et al (2013) shows a very similar appearance. There is no doubt that the authors are identifying structures that closely correspond to those provided by Maseko et al (2013). It is solely a contrast in what these nuclear complexes are called and the functional sequelae of the identification of these complexes (are they related to the trunk sensation or movement controlled by the cerebellum?) that is under debate.

Elephants are part of the Afrotheria, thus the most relevant comparative data to resolve this issue will be the identification of these nuclei in other Afrotherian species. Below I provide images of these nuclear complexes, labelled in the standard nomenclature, across several Afrotherian species.

(A) Lesser hedgehog tenrec (Echinops telfairi)

Tenrecs brains are the most intensively studied of the Afrotherian brains, these extensive neuroanatomical studies were undertaken primarily by Heinz Künzle. Below I append images (coronal sections stained with cresol violet) of the IO and Vsens (labelled in the standard mammalian manner) in the lesser hedgehog tenrec. It should be clear that the inferior olive is located in the ventral midline of the rostral medulla oblongata (just like the rat) and that this nucleus is not distinctly serrated. The Vsens is located in the lateral aspect of the medulla skirted laterally by the spinal trigeminal tract (Sp5). These images and the labels indicating structures correlate precisely with that provided by Künzle (1997, 10.1016/S0168- 0102(97)00034-5), see his Figure 1K,L. Thus, in the first case of a related species, there is no serrated appearance of the inferior olive, the location of the inferior olive is confirmed through connectivity with the superior colliculus (a standard connection in mammals) by Künzle (1997), and the location of Vsens is what is considered to be typical for mammals. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Peer Review Image 1.

(B) Giant otter shrew (Potomogale velox)

The otter shrews are close relatives of the Tenrecs. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see hints of the serration of the IO as defined by the authors, but we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Peer Response Image 2.

(C) Four-toed sengi (Petrodromus tetradactylus)

The sengis are close relatives of the Tenrecs and otter shrews, these three groups being part of the Afroinsectiphilia, a distinct branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see vague hints of the serration of the IO (as defined by the authors), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Peer Response Image 3.

(D) Rock hyrax (Procavia capensis)

The hyraxes, along with the sirens and elephants form the Paenungulata branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per the standard mammalian anatomy. Here we see hints of the serration of the IO (as defined by the authors), but we also see evidence of a more "bulbous" appearance of subnuclei of the IO (particularly the principal nucleus), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Peer Review Image 4.

(E) West Indian manatee (Trichechus manatus)

The sirens are the closest extant relatives of the elephants in the Afrotheria. Below I append images of cresyl violet (top) and myelin (bottom) stained coronal sections (taken from the University of Wisconsin-Madison Brain Collection, https://brainmuseum.org, and while quite low in magnification they do reveal the structures under debate) through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see the serration of the IO (as defined by the authors). Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Peer Review Image 5.

These comparisons and the structural identification, with which the authors agree as they only distinguish the elephants from the other Afrotheria, demonstrate that the appearance of the IO can be quite variable across mammalian species, including those with a close phylogenetic affinity to the elephants. Not all mammal species possess a "serrated" appearance of the IO. Thus, it is more than just theoretically possible that the IO of the elephant appears as described prior to this study.

So what about elephants? Below I append a series of images from coronal sections through the African elephant brainstem stained for Nissl, myelin, and immunostained for calretinin. These sections are labelled according to standard mammalian nomenclature. In these complete sections of the elephant brainstem, we do not see a serrated appearance of the IOM (as described previously and in the current study by the authors). Rather the principal nucleus of the IOM appears to be bulbous in nature. In the current study, no image of myelin staining in the IOM/VsensR is provided by the authors. However, in the images I provide, we do see the reported myelin stripes in all stains - agreement between the authors and reviewer on this point. The higher magnification image to the bottom left of the plate shows one of the IOM/VsensR myelin stripes immunostained for calretinin, and within the myelin stripes axons immunopositive for calretinin are seen (labelled with an arrow). The climbing fibres of the elephant cerebellar cortex are similarly calretinin immunopositive (10.1159/000345565). In contrast, although not shown at high magnification, the fibres forming the Sp5 in the elephant (in the Maseko description, unnamed in the description of the authors) show no immunoreactivity to calretinin.

Peer Review Image 6.

Peripherin Immunostaining

In their revised manuscript the authors present immunostaining of peripherin in the elephant brainstem. This is an important addition (although it does replace the only staining of myelin provided by the authors which is unusual as the word myelin is in the title of the paper) as peripherin is known to specifically label peripheral nerves. In addition, as pointed out by the authors, peripherin also immunostains climbing fibres (Errante et al., 1998). The understanding of this staining is important in determining the identification of the IO and Vsens in the elephant, although it is not ideal for this task as there is some ambiguity. Errante and colleagues (1998; Fig. 1) show that climbing fibres are peripherin-immunopositive in the rat. But what the authors do not evaluate is the extensive peripherin staining in the rat Sp5 in the same paper (Errante et al, 1998, Fig. 2). The image provided by the authors of their peripherin immunostaining (their new Figure 2) shows what I would call the Sp5 of the elephant to be strongly peripherin immunoreactive, just like the rat shown in Errant et al (1998), and moreover in the precise position of the rat Sp5! This makes sense as this is where the axons subserving the "extraordinary" tactile sensitivity of the elephant trunk would be found (in the standard model of mammalian brainstem anatomy). Interestingly, the peripherin immunostaining in the elephant is clearly lamellated...this coincides precisely with the description of the trigeminal sensory nuclei in the elephant by Maskeo et al (2013) as pointed out by the authors in their rebuttal. Errante et al (1998) also point out peripherin immunostaining in the inferior olive, but according to the authors this is only "weakly present" in the elephant IOM/VsensR. This latter point is crucial. Surely if the elephant has an extraordinary sensory innervation from the trunk, with 400,000 axons entering the brain, the VsensR/IOM should be highly peripherin-immunopositive, including the myelinated axon bundles?! In this sense, the authors argue against their own interpretation - either the elephant trunk is not a highly sensitive tactile organ, or the VsensR is not the trigeminal nuclei it is supposed to be.

Summary:

(1) Comparative data of species closely related to elephants (Afrotherians) demonstrates that not all mammals exhibit the "serrated" appearance of the principal nucleus of the inferior olive.

(2) The location of the IO and Vsens as reported in the current study (IOR and VsensR) would require a significant, and unprecedented, rearrangement of the brainstem in the elephants independently. I argue that the underlying molecular and genetic changes required to achieve this would be so extreme that it would lead to lethal phenotypes. Arguing that the "switcheroo" of the IO and Vsens does occur in the elephant (and no other mammals) and thus doesn't lead to lethal phenotypes is a circular argument that cannot be substantiated.

(3) Myelin stripes in the subnuclei of the inferior olivary nuclear complex are seen across all related mammals as shown above. Thus, the observation made in the elephant by the authors in what they call the VsensR, is similar to that seen in the IO of related mammals, especially when the IO takes on a more bulbous appearance. These myelin stripes are the origin of the olivocerebellar pathway and are indeed calretinin immunopositive in the elephant as I show.

(4) What the authors see aligns perfectly with what has been described previously, the only difference being the names that nuclear complexes are being called. But identifying these nuclei is important, as any functional sequelae, as extensively discussed by the authors, is entirely dependent upon accurately identifying these nuclei.

(4) The peripherin immunostaining scores an own goal - if peripherin is marking peripheral nerves (as the authors and I believe it is), then why is the VsensR/IOM only "weakly positive" for this stain? This either means that the "extraordinary" tactile sensitivity of the elephant trunk is non-existent, or that the authors have misinterpreted this staining. That there is extensive staining in the fibre pathway dorsal and lateral to the IOR (which I call the spinal trigeminal tract), supports the idea that the authors have misinterpreted their peripherin immunostaining.

(5) Evolutionary expediency. The authors argue that what they report is an expedient way in which to modify the organisation of the brainstem in the elephant to accommodate the "extraordinary" tactile sensitivity. I disagree. As pointed out in my first review, the elephant cerebellum is very large and comprised of huge numbers of morphologically complex neurons. The inferior olivary nuclei in all mammals studied in detail to date, give rise to the climbing fibres that terminate on the Purkinje cells of the cerebellar cortex. It is more parsimonious to argue that, in alignment with the expansion of the elephant cerebellum (for motor control of the trunk), the inferior olivary nuclei (specifically the principal nucleus) have had additional neurons added to accommodate this cerebellar expansion. Such an addition of neurons to the principal nucleus of the inferior olive could readily lead to the loss of the serrated appearance of the principal nucleus of the inferior olive and would require far less modifications in the developmental genetic program that forms these nuclei. This type of quantitative change appears to be the primary way in which structures are altered in the mammalian brainstem.

Reviewer #3 (Public Review):

Summary:

The study claims to investigate trunk representations in elephant trigeminal nuclei located in the brainstem. The researchers identify large protrusions visible from the ventral surface of the brainstem, which they examined using a range of histological methods. However, this ventral location is usually where the inferior olivary complex is found, which challenges the author's assertions about the nucleus under analysis. They find that this brainstem nucleus of elephants contains repeating modules, with a focus on the anterior and largest unit which they define as the putative nucleus principalis trunk module of the trigeminal. The nucleus exhibits low neuron density, with glia outnumbering neurons significantly. The study also utilizes synchrotron X-ray phase contrast tomography to suggest that myelin-stripe-axons traverse this module. The analysis maps myelin-rich stripes in several specimens and concludes that based on their number and patterning they likely correspond with trunk folds; however this conclusion is not well supported if the nucleus has been misidentified.

Strengths:

The strength of this research lies in its comprehensive use of various anatomical methods, including Nissl staining, myelin staining, Golgi staining, cytochrome oxidase labeling, and synchrotron X-ray phase contrast tomography. The inclusion of quantitative data on cell numbers and sizes, dendritic orientation and morphology, and blood vessel density across the nucleus adds a quantitative dimension. Furthermore, the research is commendable for its high-quality and abundant images and figures, effectively illustrating the anatomy under investigation.

Weaknesses:

While the research provides potentially valuable insights if revised to focus on the structure that appears to be an inferior olivary nucleus, there are certain additional weaknesses that warrant further consideration. First, the suggestion that myelin stripes solely serve to separate sensory or motor modules rather than functioning as an "axonal supply system" lacks substantial support due to the absence of information about the neuronal origins and the termination targets of the axons. Postmortem fixed brain tissue limits the ability to trace full axon projections. While the study acknowledges these limitations, it is important to exercise caution in drawing conclusions about the precise role of myelin stripes without a more comprehensive understanding of their neural connections.

Second, the quantification presented in the study lacks comparison to other species or other relevant variables within the elephant specimens (i.e., whole brain or brainstem volume). The absence of comparative data to different species limits the ability to fully evaluate the significance of the findings. Comparative analyses could provide a broader context for understanding whether the observed features are unique to elephants or more common across species. This limitation in comparative data hinders a more comprehensive assessment of the implications of the research within the broader field of neuroanatomy. Furthermore, the quantitative comparisons between African and Asian elephant specimens should include some measure of overall brain size as a covariate in the analyses. Addressing these weaknesses would enable a richer interpretation of the study's findings.

Reviewer #4 (Public Review):

Summary:

The authors report a novel isomorphism in which the folds of the elephant trunk are recognizably mapped onto the principal sensory trigeminal nucleus in the brainstem. Further, they identify the enlarged nucleus as being situated in this species in an unusual ventral midline position.

Strengths:

The identity of the purported trigeminal nucleus and the isomorphic mapping with the trunk folds is supported by multiple lines of evidence: enhanced staining for cytochrome oxidase, an enzyme associated with high metabolic activity; dense vascularization, consistent with high metabolic activity; prominent myelinated bundles that partition the nucleus in a 1:1 mapping of the cutaneous folds in the trunk periphery; near absence of labeling for the anti-peripherin antibody, specific for climbing fibers, which can be seen as expected in the inferior olive; and a high density of glia.

Weaknesses:

Despite the supporting evidence listed above, the identification of the gross anatomical bumps, conspicuous in the ventral midline, is problematic. This would be the standard location of the inferior olive, with the principal trigeminal nucleus occupying a more dorsal position. This presents an apparent contradiction which at a minimum needs further discussion. Major species-specific specializations and positional shifts are well-documented for cortical areas, but nuclear layouts in the brainstem have been considered as less malleable.

Reviewer #2 (Public Review):

BTB domains are protein-protein interaction domains found in diverse eukaryotic proteins, including transcription factors. It was previously known that many of the Drosophila transcription factor BTB domains are of the TTK-type - these are defined as having a highly-conserved motif, FxLRWN, at their N-terminus, and they thereby differ from the mammalian BTB domains. Whereas the well-characterised mammalian BTB domains are dimeric, several Drosophila TTK-BTB domains notably form multimers and function as chromosome architectural proteins. The aims of this work were (i) to determine the structural basis of multimerisation of the Drosophila TTK-BTB domains, (ii) to determine how different Drosophila TTK-BTB domains interact with each other, and (iii) to investigate the evolution of this subtype of BTB domain.

The work significantly advances our understanding of the biology of BTB domains. The conclusions of the paper are mostly well-supported, although some aspects need clarification:

Hexameric organisation of the TTK-type BTB domains:<br /> Using cryo-EM, the authors showed that the CG6765 TTK-type BTB domain forms a hexameric assembly in which three "classic" BTB dimers interact via a beta-sheet interface involving the B3 strand. This is particularly interesting, as this region of the BTB domain has recently been implicated in protein-protein interactions in a mammalian BTB-transcription factor, MIZ1. SEC-MALS analysis indicated that the LOLA TTK-type BTB domain is also hexameric, and SAXS data was consistent with a hexameric assembly of the CG6765- and LOLA BTB domains.

The data regarding the hexameric organisation is convincing. However, interpreting the role of specific regions of the BTB domain is difficult because the description of the molecular contacts lacks depth.

Heteromeric interactions between TTK-type BTB domains:<br /> The authors use yeast two-hybrid assays to study heteromeric interactions between various Drosophila TTK-type BTB domains. Such assays are notorious for producing false positives, and this needs to be mentioned. Although the authors suggest that the heteromeric interactions are mediated via the newly-identify B3 interaction interface, there is no evidence to support this, since mutation of B3 yielded insoluble proteins.

Evolution of the TTK-type BTB domains:<br /> The authors carried out a bioinformatics analysis of BTB proteins and showed that most of the Drosophila BTB transcription factors (24 out of 28) are of the TTK-type. They investigated how the TTK-type BTB domains emerged during evolution, and showed that these are only found in Arthropoda, and underwent lineage-specific expansion in the modern phylogenetic groups of insects. These findings are well-supported by the evidence.

Reviewer #1 (Public Review):

Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.

I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some additional points for consideration for the same.

Major points.<br /> (1) It would be valuable to discuss alternative multimer assembly interfaces, considering the diverse ways POZs can multimerize. For instance, the Potassium channel POZ domains form tetramers. A comparison of their inter-subunit interface with that of TTK and non-TTK POZs could provide insightful contrasts.

(2) The so-called TTK motif, despite its unique sequence signature, essentially corresponds to the N-terminal extension observed in other "non-TTK" proteins such as Miz-1. Given Miz-1's structure, it becomes evident that the utilization of the N-terminal extension for dimerization is shared with the TTK family, suggesting a common evolutionary origin in metazoan transcription factors. Early phylogenetic trees (e.g. in PMID: 9917379) support the grouping of the TTK-like POZs with other animal Transcription factors containing POZ domains such as those with Kelch repeats further suggesting that the extension might be ancestral. Structural investigations by modeling prominent examples or comparing known structures of similar POZ domains, could support this inference. Control comparisons with POZ domains from fungi, plants and amoebozoans like Dictyostelium could offer additional insights.

(3) Exploring the ancestral presence of the aforementioned extension in metazoan transcription factors could serve as a foundation for understanding the evolutionary pathway of hexamerization. This analysis could shed light on exposed structural regions that had the potential to interact post-dimerization with the N-terminal extension and also might provide insights into the evolution of multimer interfaces, as observed in the Potassium channel.

(4) Considering the role of conserved residues in the multimer interface is crucial. Reference to conserved residues involved in multimer formation, such as discussed in PMID: 9917379, would enrich the discussion.

Reviewer #1 (Public Review):

The authors describe a comprehensive analysis of sex-biased expression across multiple tissues and species of mouse. Their results are broadly consistent with previous work, and their methods are robust, as the large volume of work in this area has converged toward a standardized approach.

I have a few quibbles with the findings, and the main novelty here is the rapid evolution of sex-biased expression over shorter evolutionary intervals than previously documented, although this is not statistically supported. The other main findings, detailed below, are somewhat overstated.

(1) In the introduction, the authors conflate gametic sex, which is indeed largely binary (with small sperm, large eggs, no intermediate gametic form, and no overlap in size) with somatic sexual dimorphism, which can be bimodal (though sometimes is even more complicated), with a large variance in either sex and generally with a great deal of overlap between males and females. A good appraisal of this distinction is at https://doi.org/10.1093/icb/icad113. This distinction in gene expression has been recognized for at least 20 years, with observations that sex-biased expression in the soma is far less than in the gonad.

For example, the authors frame their work with the following statement:<br /> "The different organs show a large individual variation in sex-biased gene expression, making it impossible to classify individuals in simple binary terms. Hence, the seemingly strong conservation of binary sex-states does not find an equivalent underpinning when one looks at the gene-expression makeup of the sexes"

The authors use this conflation to set up a straw man argument, perhaps in part due to recent political discussions on this topic. They seem to be implying one of two things. a) That previous studies of sex-biased expression of the soma claim a binary classification. I know of no such claim, and many have clearly shown quite the opposite, particularly studies of intra-sexual variation, which are common - see https://doi.org/10.1093/molbev/msx293, https://doi.org/10.1371/journal.pgen.1003697, https://doi.org/10.1111/mec.14408, https://doi.org/10.1111/mec.13919, https://doi.org/10.1111/j.1558-5646.2010.01106.x for just a few examples. Or b) They are the first to observe this non-binary pattern for the soma, but again, many have observed this. For example, many have noted that reproductive or gonad transcriptome data cluster first by sex, but somatic tissue clusters first by species or tissue, then by sex (https://doi.org/10.1073/pnas.1501339112, https://doi.org/10.7554/eLife.67485)<br /> Figure 4 illustrates the conceptual difference between bimodal and binary sexual conceptions. This figure makes it clear that males and females have different means, but in all cases the distributions are bimodal.

I would suggest that the authors heavily revise the paper with this more nuanced understanding of the literature and sex differences in their paper, and place their findings in the context of previous work.

(2) The authors also claim that "sexual conflict is one of the major drivers of evolutionary divergence already at the early species divergence level." However, making the connection between sex-biased genes and sexual conflict remains fraught. Although it is tempting to use sex-biased gene expression (or any form of phenotypic dimorphism) as an indicator of sexual conflict, resolved or not, as many have pointed out, one needs measures of sex-specific selection, ideally fitness, to make this case (https://doi.org/10.1086/595841, 10.1101/cshperspect.a017632). In many cases, sexual dimorphism can arise in one sex only without conflict (e.g. 10.1098/rspb.2010.2220). As such, sex-biased genes alone are not sufficient to discriminate between ongoing and resolved conflict.

(3) To make the case that sex-biased genes are under selection, the authors report alpha values in Figure 3B. Alpha value comparisons like this over large numbers of genes often have high variance. Are any of the values for male- female- and un-biased genes significantly different from one another? This is needed to make the claim of positive selection.

Reviewer #2 (Public Review):

The manuscript by Xie and colleagues presents transcriptomic experiments that measure gene expression in eight different tissues taken from adult female and male mice from four species. These data are used to make inferences regarding the evolution of sex-biased gene expression across these taxa. The experimental methods and data analysis are appropriate; however, most of the conclusions drawn in the manuscript have either been previously reported in the literature or are not fully supported by the data.

There are two ways the manuscript could be modified to better strengthen the conclusions.

First, some of the observed differences in gene expression have very little to no effect on other phenotypes, and are not relevant to medicine or fitness. Selectively neutral gene expression differences have been inferred in previous studies, and consistent with that work, sex-biased and between-species expression differences in this study may also be enriched for selectively neutral expression differences. This idea is supported by the analysis of expression variance, which indicates that genes that show sex-biased expression also tend to show more inter-individual variation. This perspective is also supported by the MK analysis of molecular evolution, which suggests that positive selection is more prevalent among genes that are sex-biased in both mus and dom, and genes that switch sex-biased expression are under less selection at the level of both protein-coding sequence and gene expression.

As an aside, I was confused by (line 176): "implying that the enhanced positive selection pressure is triggered by their status of being sex-biased in either taxon." - don't the MK values suggest an excess of positive selection on genes that are sex-biased in both taxa?

Without an estimate of the proportion of differentially expressed genes that might be relevant for broader physiological or organismal phenotypes, it is difficult to assess the accuracy and relevance of the manuscript's conclusions. One (crude) approach would be to analyze subsets of genes stratified by the magnitude of expression differences; while there is a weak relationship between expression differences and fitness effects, on average large gene expression differences are more likely to affect additional phenotypes than small expression differences. Another perspective would be to compare the within-species variance to the between-species variance to identify genes with an excess of the latter relative to the former (similar logic to an MK test of amino acid substitutions).

Second, the analysis could be more informative if it distinguished between genes that are expressed across multiple tissues in both sexes that may show greater expression in one sex than the other, versus genes with specialized function expressed solely in (usually) reproductive tissues of one sex (e.g. ovary-specific genes). One approach to quantify this distinction would be metrics like those used defined by [Yanai I, et al. 2005. Genome-wide midrange transcription profiles reveal expression-level relationships in human tissue specification. Bioinformatics 21:650-659.] These approaches can be used to separate out groups of genes by the extent to which they are expressed in both sexes versus genes that are primarily expressed in sex-specific tissue such as testes or ovaries. This more fine-grained analysis would also potentially inform the section describing the evolution/conservation of sex-biased expression: I expect there must be genes with conserved expression specifically in ovaries or testes (these are ancient animal structures!) but these may have been excluded by the requirement that genes be sex-biased and expressed in at least two organs.

There are at least three examples of statements in the discussion that at the moment misinterpret the experimental results.

The discussion frames the results in the context of sexual selection and sexually antagonistic selection, but these concepts are not synonymous. Sexual selection can shape phenotypes that are specific to one sex, causing no antagonism; and fitness differences between males and females resulting from sexually antagonistic variation in somatic phenotypes may not be acted on by sexual selection. Furthermore, the conditions promoting and consequence of both kinds of selection can be different, so they should be treated separately for the purposes of this discussion.

The discussion claims that "Our data show that sex-biased gene expression evolves extremely fast" but a comparison or expectation for the rate of evolution is not provided. Many other studies have used comparative transcriptomics to estimate rates of gene expression evolution between species, including mice; are the results here substantially and significantly different from those previous studies? Furthermore, the experimental design does not distinguish between those gene expression phenotypes that are fixed between species as compared to those that are polymorphic within one or more species which prevents straightforward interpretation of differences in gene expression as interspecific differences.

The conclusion that "Our results show that most of the genetic underpinnings of sex differences show no long-term evolutionary stability, which is in strong contrast to the perceived evolutionary stability of two sexes" - seems beyond the scope of this study. This manuscript does not address the genetic underpinnings of sex differences (this would involve eQTL or the like), rather it looks at sex differences in gene expression phenotypes. Simply addressing the question of phenotypic evolutionary stability would be more informative if genes expressed specifically in reproductive tissues were separated from somatic sex-biased genes to determine if they show similar patterns of expression evolution.

Reviewer #3 (Public Review):

This manuscript reports some interesting and important patterns. The results on sex-bias in different tissues and across four taxa would benefit from alternative (or additional) presentation styles. In my view, the most important results are with respect to alpha (fraction of beneficial amino acid changes) in relation to sex-bias (though the authors have made this as a somewhat minor point in this version).

The part that the authors emphasize I don't find very interesting (i.e., the sexes have overlapping expression profiles in many nongonadal tissues), nor do I believe they have the appropriate data necessary to convincingly demonstrate this (which would require multiple measures from the same individual).

This study reports several interesting patterns with respect to sex differences in gene expression across organs of four mice taxa. An alternative presentation of the data would yield a clearer and more convincing case that the patterns the authors claim are legitimate.

I recommend that the authors clarify what qualifies as "sex-bias".

Reviewer #1 (Public Review):

Summary:

The title states "IL-2 enhances effector function but suppresses follicular localization of CD8+ T cells in chronic infection" which data from the paper show but does not seem to be the major goal of the authors. As stated in the short assessment above, the goal of this work seems to connect IL-2 signals, mostly given exogenously, to the differentiation of progenitor T cells (TPEX) that will help sustain effector T cell responses against chronic viral infection (TEX/TEFF). The authors mostly use chronic LCMV infection in mice as their model of choice, Flow cytometry, fluorescent microscopy, and some in vitro assays to explore how IL2 regulates TPEX and TEX/TEFF differentiation. Gain and loss of functions experiments are also conducted to explore the roles of L2 signaling and BLIMP-1 in regulating these processes. Lastly, a loose connection of their mouse findings on TPEX/TEX cells to a clinical study using low-dose IL-2 treatment in SLE patients is attempted.

Strengths:

(1) The impact of IL-2 treatment of TPEX/TEX differentiation is very clear.

(2) The flow cytometry data are convincing and state-of-the-art.

Weaknesses:

(1) The title appears disconnected from the major focus of the work.

(2) The number of TPEX cells is not changed. IL2 treatment increases the number of TEFF and the proportion of TPEX is lower suggesting it does not target TPEX formation. The conclusion about an inhibitory role of IL2 treatment on TPEX formation seems therefore largely overstated.

(3) Are the expanded TEX/TEFF cells really effectors? Only GrB and some cell surface markers are monitored (44, 62L). Other functions should be included, e.g., CD107a, IFNg, TNF, chemokines - Tbet?

(4) The rationale for IL2 treatment timing is unclear. Seems that this is given at the T cell contraction time and this is interesting compared to the early treatment that ablate TPEX generation. Maybe this should really be explored further?

(5) The TGFb/IL6/IL2 in vitro experiment does not bring much to the paper.

(6) The Figure 2 data try to provide an explanation for a prior lack of difference in viral titers after IL2 treatment. It is hard to be convinced by these tissue section data as presented. It also begs the question of how the host would benefit from the low dose IL-2 treatment if IL-2 TEFF are not contributing to viral control as a result of their inappropriate localization to viral reservoirs.

(7) It is unclear what the STA5CA and BLIMP-1 KO experiments in Figure 3 add to the story that is not already expected/known.

(8) The connection to the low-dose IL2 treatment in SLE patients is very loose and weak. This version is likely not the ligand that preferentially signals to CD122 either. SLE is different from a chronic viral infection and the question of timing seems critical from all the data shown in this manuscript. So it is very difficult to make any robust link to the mechanistic data.

(9) It is really unclear what the take-home message is. IL-2 is signaling via STAT5 and BLIMP1 is also a known target as published by many groups including this one, and these results are more than expected. The observation that TEFF may be differentially localized in the WP area is interesting but no mechanisms are really provided (guessing CXCR5 but again expected). Also, all these observations are highly dependent on the timing of IL2 administration which is fascinating but not explored at all. It also limits significance since underlying mechanisms are unknown and we do not know when such treatment would have to be given.

Reviewer #2 (Public Review):

This study utilized the LCMV Docile infection model, which induces chronic and persistent infection in mice, leading to T cell exhaustion and dysfunction. Through exogenous IL-2 fusion protein treatment during the late stage of infection, the researchers found that IL-2 treatment significantly enlarges the antigen-specific effector CD8 T cells, expanding the CXCR5-TCF1- exhausted population (Tex) while maintaining the size of the CXCR5+TCF1+ precursors of exhausted T cell population (Tpex). This preservation of the Tpex population's self-renewing capacity allows for sustained T cell proliferation and antiviral responses.

The authors discovered a dual effect of IL-2 treatment: it decreases CXCR5 expression on Tpex cells, restricting their entry into the B cell follicle. This may explain why IL-2 treatment has little impact on overall viral control. However, this finding also suggests a potential application of IL-2 treatment for autoimmune diseases, as it can suppress specific immune responses within the B cell follicle. Using imaging-based approaches, the team provided direct evidence that IL-2 treatment shifts the viral load to concentrate within the B cell follicle, correlating with the observed decrease in CXCR5 expression.

Further, the researchers showed that ectopic expression of constitutively active STAT5, downstream of IL-2 induced cytokine signaling, in P14 TCR transgenic T cells (specific for an LCMV epitope), drove the T cell population toward the CXCR5- Tex phenotype over the CXCR5+ Tpex cells in vivo. Additionally, abrogating Blimp1, upregulated by active IL-2-phosphorylated STAT5 signaling, restored the CXCR5+ Tpex population.

Building on these results, the researchers used an engineered IL-2 fusion protein, ANV410, targeting the beta-chain of the IL-2 receptor CD122, which successfully replicated their earlier findings. Importantly, the Tpex-sustaining effect of IL-2 was only observed when treatment was administered during the late stage of infection, as early treatment suppressed Tpex cell generation. Immune profiling of SLE patients undergoing low-dose IL-2 treatment showed a similar reduction in the CXCR5+ Tpex cell population.

This study provides compelling data on the physiological consequences of IL-2 treatment during chronic viral infection. By leveraging the chronic and persistent LCMV Docile infection model, the researchers identified the temporal effects of IL-2 fusion protein treatment, offering strategic insights for therapies targeting cancer and autoimmune diseases.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Ghazi et reported that inhibition of KRASG12C signaling increases autophagy in KRASG12C expressing lung cancer cells. Moreover, the combination of DCC 3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C driven lung cancer cell proliferation in vitro and tumor growth in vivo. Additionally, in genetically engineered mouse models of KRASG12C driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Additionally, this study found that LKB1 deficiency diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer to the combination treatment, perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas.

Strengths:

Both human cancer cells and mouse models were employed in this study to illustrate that inhibiting ULK1/2 could enhance the responsiveness of KRASG12C lung cancer to sotorasib. This research holds translational importance.

Weaknesses:

The revised manuscript has addressed most of my previous concerns. However, I still have one issue: the sample size (n) for the GEMM study in Figures 4E and 4F is too small, despite the authors' explanation. The data do not support the conclusion due to the lack of significant difference in tumor burden. Additionally, the significance labels in Figure 4E are not clearly explained.

Reviewer #1 (Public Review):

Summary:

The authors profile gene expression, chromatin accessibility and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over a 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T cell activation.

This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as explore the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.

Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.

I thank the authors for their revisions in response to my initial review. The revised version now includes a more comprehensive comparative analysis of different datasets and V2G approaches and discusses the potential sources of differences in the results. Most significantly, the authors have now included an interesting comparison of their methodology with the popular ABC technique and outlined the key limitations of ABC relative to their method and other (Capture) Hi-C-based V2G approaches.

Reviewer #2 (Public Review):

Summary:

There is significant interest in characterizing the mechanisms by which genetic mutations linked to autoimmunity perturb immune processes. Pahl et al. collect information of dynamic accessible regions, genes, and 3D contacts in primary CD4+ T cell samples that have been stimulated ex vivo. The study includes a variety of analyses characterizing these dynamic changes. With TF footprinting they propose factors linked to active regulatory elements. They compare the performance of their variant mapping pipeline that uses their data versus existing datasets. Most compelling there was a deep dive into additional study of regulatory elements nearby the IL2 gene. Finally, they perform a pharmacological screen targeting several genes they suggest are involved in T cell proliferation.

Strengths:

- The work done characterizing elements at the IL2 locus is impressive.

Weaknesses:

- There are extensive studies performed on resting and activated immune cell states (CD4+ T cells and other cell types) and some at multiple time points or concentrations of stimuli that collect ATAC-seq and/or RNA-seq. Several analyses performed in published studies were similarly performed in this study. I expected the authors to at least briefly mention published studies and whether their conclusions generally agree or disagree. Are the same dynamic regulatory regions or genes identified upon T cell activation? Are the same TF footprints enriched in these dynamic regulatory elements? In the revision, I appreciate that the authors now include additional data from several studies that I had initially suggested for the purposes of nominating disease genes in their precision-recall analysis.

Reviewer #3 (Public Review):

Summary:

This paper used RNAseq, ATACseq, and Hi-C to assess gene expression, chromatin accessibility, and chromatin physical associations for native CD4+ T cells as they respond to stimulation through TCR and CD28. With these data in hand, the author identified 423 GWAS signals to their respective target genes, where most of these were not in the proximal promoter, but rather distal enhancers. The IL-2 gene was used as an example to identify new distal cis regulatory regions required for optimal IL-2 gene transcription. These distal elements interact with the proximal IL2 promoter region. When the distal enhancer contained an autoimmune SNP, it affected IL-2 gene transcription. The authors also identified genetic risk variants that were associated to genes upon activation. Some of these regulate proliferation and cytokine production, but others were novel.

Strengths:

This paper provides a wealth of data related to gene expression after CD4 T cells are activated through the TCR and CD28. An important strength of this paper is that these data were intensively analyzed to uncover autoimmune disease SNPs in cis acting regions. Many of these could be assigned to likely target genes even though they often are in distal enhancers. These findings help to provide a better understanding concerning the mechanism by which GWAS risk elements impact gene expression.

Another strength to this study was the proof-of-principle studies examining the IL-2 gene. Not only were new cis acting enhancers discovered, but they were functionally shown to be important in regulating IL-2 expression, including susceptibility to colitis. Their importance was also established with respect to such distal enhancers harboring disease relevant SNPs, which were shown to affect IL-2 transcription.

The data from this study were also mined against past Crispr screens that identified genes that control aspects of CD4 T cell activation. From these comparisons, novel genes were identified that function during T cell activation.

Weaknesses:

A weakness from this study is that few individuals were analyzed, i.e., RNAseq and ATACseq (n=3) and HiC (n=2). Thus, the authors may have underestimated potentially relevant risk associations by their chromatin capture-based methodology. This might account for low overlap of their data with the eQTL-based approach or the HIEI truth set.

The authors explain that the low overlap is not due to few GWAS associations by HiC. The expanded discussion in the revised manuscript provides a framework to help explain inherent differences between these methods that may contribute to the low overlap.

Impact:

This study indicates that defining distal chromatin interacting regions help to identify distal genetic elements, including relevant variants, that contribute to gene activation.

Reviewer #1 (Public Review):

Summary:

The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated.

Strengths:

(1) They have done a better job explaining the rationale for their experiments thought-out.

(2) The use of molecular scissors in their construct represents a creative approach to examine inter-domain interactions. Appropriate controls were included.

(3) From my assessment, the experiments are well-conceived and executed.

(4) The authors do a better job of highlighting the question being addressed experimentally.

Reviewer #2 (Public Review):

In the revised manuscript, the authors tried to address some of my comments from the previous round of review. Notably, they have performed some additional ITC experiments where protein precipitation is not an issue to probe interactions between PARKIN and different domains. In addition, they have toned down some of the language in the text to better reflect their data and results. However, I still feel that the manuscript lacks some key answers regarding the relative interactions between p-PARKIN and different domains, as discussed in my previous review. A deeper dive into the underlying biophysical and biochemical features that drive these interactions is important to fully understand the importance of their work. However, this manuscript does provide some interesting potential insights into the mechanisms of PARKIN activation that could be useful for the field moving forward.

Reviewer #3 (Public Review):

Summary:

In their manuscript, Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).

Strengths:

The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.

Weaknesses:

As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.

Following peer review and revision, the claims are still not fully supported by direct evidence. While the experimental system may be necessary and/or convenient given the unique challenges in studying Parkin, it does not directly speak toward the conclusions that the authors make, nor does it provide an accurate representation of biology.

Reviewer #2 (Public Review):

Summary:

This is an exciting paper that explores the in vitro assembly of recombinant alpha-synuclein into amyloid filaments. The authors changed the pH and the composition of the assembly buffers, as well as the presence of different types of seeds, and analysed the resulting structures by cryo-EM.

Strengths:

By doing experiments at different pHs, the authors found that so-called type 2 and type-3 polymorphs form in a pH dependent manner. In addition, they find that type-1 filaments form in the presence of phosphate ions. One of their in vitro assembled type-1 polymorphs is similar to the alpha-synuclein filaments that were extracted from the brain of an individual with juvenile-onset synucleinopathy (JOS). They hypothesize that additional densities in a similar place as additional densities in the JOS fold correspond to phosphate ions.

Comments on the revised version:

This is OK now. I thank the authors for their constructive engagement with my comments.

Reviewer #3 (Public Review):

Summary

The high heterogeneity nature of α-synuclein (α-syn) fibrils posed significant challenges in structural reconstruction of the ex vivo conformation. A deeper understanding of the factors influencing the formation of various α-syn polymorphs remains elusive. The manuscript by Frey et al. provides a comprehensive exploration of how pH variations (ranging from 5.8 to 7.4) affect the selection of α-syn polymorphs (specifically, Type1, 2 and 3) in vitro by using cryo-electron microscopy (cryo-EM) and helical reconstruction techniques. Crucially, the authors identify two novel polymorphs at pH 7.0 in PBS. These polymorphs bear resemblance to the structure of patient-derived juvenile-onset synucleinopathy (JOS) polymorph and diseased tissue amplified α-syn fibrils. The revised manuscript more strongly supports the notion that seeding is a non-polymorph-specific in the context of secondary nucleation-dominated aggregation, underscoring the irreplaceable role of pH in polymorph formation.

Strengths

This study systematically investigates the effects of environmental conditions and seeding on the structure of α-syn fibrils. It emphasizes the significant influence of environmental factors, especially pH, in determining the selection of α-syn polymorphs. The high-resolution structures obtained through cryo-EM enable a clear characterization of the composition and proportion of each polymorph in the sample. Collectively, this work provides a strong support for the pronounced sensitivity of α-syn fibril structures to the environmental conditions and systematically categorizes previously reported α-syn fibril structures. Furthermore, the identification of JOS-like polymorph also demonstrates the possibility of in vitro reconstruction of brain-derived α-syn fibril structures.

Weaknesses

All my previous concerns have been resolved to my satisfaction.

Reviewer #1 (Public Review):

In this study, the authors introduced an essential role of AARS2 in maintaining cardiac function. They also investigated the underlying mechanism that through regulating alanine and PKM2 translation are regulated by AARS2. Accordingly, a therapeutic strategy for cardiomyopathy and MI was provided. Several points need to be addressed to make this article more comprehensive:

(1) Include apoptotic caspases in Figure 2B, and Figure 4 B and E as well.

(2) It would be better to show the change of apoptosis-related proteins upon the knocking down of AARS2 by small interfering RNA (siRNA).

(3) In Figure 5, the authors performed Mass Spectrometry to assess metabolites of homogenates. I was wondering if the change of other metabolites could be provided in the form of a heatmap.

(4) The amounts of lactate should be accessed using a lactate assay kit to validate the Mass Spectrometry results.

(5) How about the expression pattern of PKM2 before and after mouse MI. Furtherly, the correlation between AARS2 and PKM2?

(6) In Figure 5, how about the change of apoptosis-related proteins after administration of PKM2 activator TEPP-46?

Reviewer #2 (Public Review):

Summary:

The authors aimed to elucidate the role of AARS2, an alanyl-tRNA synthase, in mouse hearts, specifically its impact on cardiac function, fibrosis, apoptosis, and metabolic pathways under conditions of myocardial infarction (MI). By investigating the effects of both deletion and overexpression of AARS2 in cardiomyocytes, the study aims to determine how AARS2 influences cardiac health and survival during ischemic stress.

The authors successfully achieved their aims by demonstrating the critical role of AARS2 in maintaining cardiomyocyte function under ischemic conditions. The evidence presented, including genetic manipulation results, functional assays, and mechanistic studies, robustly supports the conclusion that AARS2 facilitates cardiomyocyte survival through PKM2-mediated metabolic reprogramming. The study convincingly links AARS2 overexpression to improved cardiac outcomes post-MI, validating the proposed protective AARS2-PKM2 signaling pathway.

This work may have a significant impact on the field of cardiac biology and ischemia research. By identifying AARS2 as a key player in cardiomyocyte survival and metabolic regulation, the study opens new avenues for therapeutic interventions targeting this pathway. The methods used, particularly the cardiomyocyte-specific genetic models and ribosome profiling, are valuable tools that can be employed by other researchers to investigate similar questions in cardiac physiology and pathology.

Understanding the metabolic adaptations in cardiomyocytes during ischemia is crucial for developing effective treatments for MI. This study highlights the importance of metabolic flexibility and the role of specific enzymes like AARS2 in facilitating such adaptations. The identification of the AARS2-PKM2 axis adds a new layer to our understanding of cardiac metabolism, suggesting that enhancing glycolysis can be a viable strategy to protect the heart from ischemic damage.

Strengths:

(1) Comprehensive Genetic Models: The use of cardiomyocyte-specific AARS2 knockout and overexpression mouse models allowed for precise assessment of AARS2's role in cardiac cells.

(2) Functional Assays: Detailed phenotypic analyses, including measurements of cardiac function, fibrosis, and apoptosis, provided evidence for the physiological impact of AARS2 manipulation.

(3) Mechanistic Insights: This study used ribosome profiling (Ribo-Seq) to uncover changes in protein translation, specifically highlighting the role of PKM2 in metabolic reprogramming.

(4) Therapeutic Relevance: The use of the PKM2 activator TEPP-46 to reverse the effects of AARS2 deficiency presents a potential therapeutic avenue, underscoring the practical implications of the findings.

Weaknesses:

(1) Species Limitation: The study is limited to mouse and rat models, and while these are highly informative, further validation in human cells or tissues would strengthen the translational relevance.

(2) Temporal Dynamics: The study does not extensively address the temporal dynamics of AARS2 expression and PKM2 activity during the progression of MI and recovery, which could offer deeper insights into the timing and regulation of these processes.

Reviewer #3 (Public Review):

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

(1) WGA staining showed obvious cardiomyocyte hypertrophy in the AARS2 cKO heart. Whether AARS affects cardiac hypertrophy needs to be further tested.

(2) The authors observed that AARS2 can improve myocardial infarction, and whether AARS2 has an effect on other heart diseases.

(3) Studies have shown that hypoxia conditions can lead to mitochondrial dysfunction, including abnormal division and fusion. AARS2 also affects mitochondrial division and fusion and interacts with mitochondrial proteins, including FIS and DRP1, the authors are suggested to verify.

(4) The authors only examined the role of AARS2 in cardiomyocytes, and fibroblasts are also an important cell type in the heart. Authors should examine the expression and function of AARS2 in fibroblasts.

(5) Overexpression of AARS2 can inhibit the production of mtROS, and has a protective effect on myocardial ischemia and H/ R-induced injury, and the occurrence of iron death is also closely related to ROS, whether AARS protects myocardial by regulating the occurrence of iron death?

(6) Please revise the English grammar and writing style of the manuscript, spelling and grammatical errors should be excluded.

(7) Recent studies have shown that a decrease in oxygen levels leads to an increase in AARS2, and lactic acid rises rapidly without being oxidized. Both of these factors inhibit oxidative phosphorylation and muscle ATP production by increasing mitochondrial lactate acylation, thereby inhibiting exercise capacity and preventing the accumulation of reactive oxygen species ROS. The key role of protein lactate acylation modification in regulating oxidative phosphorylation of mitochondria, and the importance of metabolites such as lactate regulating cell function through feedback mechanisms, i.e. cells adapt to low oxygen through metabolic regulation to reduce ROS production and oxidative damage, and therefore whether AARS2 in the heart also acts in this way.

Reviewer #1 (Public Review):

Tsai and Seymen et al. investigate associations between RTE expression and methylation and age and inflammation, using multiple public datasets. Compared to the previous round of review, the text of the manuscript has been polished and the phrasing of several findings has been made clearer and more precise. The authors also provided ample discussion to the prior reviewer comments in their rebuttal, including new analyses. All these changes are in the correct direction, however, I believe that part of the content of the rebuttal should be incorporated in the main text, for reasons that I will outline below.

Both reviewers found the reliance on microarray expression data to detract from the study. The authors argued that their choices are supported by existing publications which performed a similar quantification of TE expression using microarray data. It could still be argued that (as far as I can tell) Reichmann et al. used a substantially larger number of probes than this study, as a consequence of starting from different arrays, however, this is a minor point which the authors do not need to address. It is still undeniable that including the validation with RNA-seq data performed in the rebuttal would strengthen the manuscript. I especially believe that many readers would want to see this analysis be prominent in the manuscript, considering that both reviewers independently converged on the issue with microarray expression data. Personally, I would have included an RNA-seq dataset next to the microarray data in the main figures, however, I understand that this would require considerable restructuring and that placing RNA-seq data besides array data might be misleading. Instead, I would ask that the authors include their rebuttal figures R1 and R2 as supplementary figures.<br /> I would suggest introducing a new paragraph, between the section dedicated to expression data and the one dedicated to DNA methylation, mentioning the issues with microarray data (Some of which were mentioned by the reviewers and other which were mentioned by the authors in the discussion and introduction) to then introduce the validation with RNA-seq data.

Figure R3 is also a good addition and should be expanded to include the GTP and MESA study and possibly mentioned in the paragraph titled "RTE expression positively correlates with BAR gene signature scores except for SINEs."

"In this study, we did not compare MESA with GTP etc. We have analysed each dataset separately based on the available data for that dataset. Therefore, sacrificing one analysis because of the lack of information from the other does not make sense. We would do that if we were after comparing different datasets. Moreover, the datasets are not comparable because they were collected from different types of blood samples."

Indeed, the datasets are not compared directly, but the associations between age, BER and TE expression for each dataset are plotted and discussed right next to each other. It is therefore natural to wonder if the differences between datasets are due to differences in the type of blood sample or if they are a consequence of the different probe sets. Using a common set of probes would help answer that question.

Reviewer #1 (Public Review):

Summary:

This work focuses on the structure and regulation of the Anaphase-Promoting Complex/Cyclosome (APC/C), a large multi-subunit ubiquitin ligase that controls the onset of chromosome segregation in mitosis. Previous high-resolution structural studies have uncovered numerous structural features and regulatory mechanisms of the human APC/C, but it has remained unclear if these mechanisms are conserved in other model eukaryotes. To address this gap in our understanding, the authors employed cryo-electron microscopy to generate structural models of APC/C from the budding yeast S. cerevisiae, a key model organism in cell cycle analysis. In their comparison of the human and yeast complexes, the authors uncover many conserved structural features that are documented here in detail, revealing widespread similarities in the fundamental structural features of the enzyme. Interestingly, the authors also find evidence that two of the key mechanisms of human APC/C regulation are not conserved in the yeast enzyme. Specifically:

(1) The ubiquitin ligase activity of the APC/C depends on its association with a co-activator subunit such as CDH1 or CDC20, which serves both as a substrate-binding adaptor and as an activator of interactions with the E2 co-enzyme. Previous studies of the human APC/C revealed that co-activator binding induces a conformational change that enables E2 binding. In contrast, the current work shows that this E2-binding conformation already exists in the absence of a co-activator in the yeast enzyme, suggesting that the enhancement of E2 binding in yeast depends on other, as yet undiscovered, mechanisms.

(2) APC/C phosphorylation on multiple subunits is known to enhance APC/C activation by the CDC20 co-activator in mitosis. Previous studies showed that phosphorylation acts by promoting the displacement of an autoinhibitory loop that occupies part of the CDC20-binding site. In the yeast enzyme, however, there is no autoinhibitory loop in the CDC20-binding site, and there is no apparent effect of APC/C phosphorylation on co-activator binding sites. Thus, phosphorylation activates the yeast CDC20-APC/C by unknown mechanisms.

Strengths:

The strength of this paper is that it provides a comprehensive analysis of yeast APC/C structure and how it compares to previously determined human structures. The article systematically unwraps the key features of the structure in a subunit-by-subunit fashion, carefully revealing the key features that are the same or different in the two species. These descriptions are based on a thorough overview of past work in the field; indeed, this article serves as a concise review of the key features, conserved or otherwise, of APC/C structure and regulation.

Weaknesses:

No significant weaknesses were identified.

Reviewer #2 (Public Review):

Summary:

This paper from the Barford lab describes medium/high-resolution cryo-EM structures of three versions of the S. cerevisiae anaphase-promoting complex/cyclosome (APC/C):

(1) the recombinant apo complex purified from insect cells,

(2) the apo complex phosphorylated in vitro by cyclin-dependent kinase, and

(3) an active APC/C-Cdh1-substrate ternary complex.

The focus of the paper is on comparing similarities and differences between S. cerevisiae and human APC/C structures, mechanisms of activation by coactivator, and regulation by phosphorylation. The authors find that the overall structures of S. cerevisiae and human APC/C are remarkably similar, including the binding sites and orientation for the substrate-recruiting coactivator, Cdh1. In addition, the mechanism of Cdh1 inhibition by phosphorylation appears conserved across kingdoms. However, key differences were also observed that reveal divergence in APC/C mechanisms that are important for researchers in this field to know. Specifically, the mechanism of APC/C-Cdc20 activation by mitotic phosphorylation appears to be different, due to the absence of the key Apc1 autoinhibition loop in the S. cerevisiae complex. In addition, the conformational activation of human APC/C by coactivator binding was not observed in the S. cerevisiae complex, implying that stimulation of E2 binding must occur via a different mechanism in this species.

Strengths:

Consistent with the numerous prior cryo-EM structures of human APC/C from the Barford lab, the technical quality of the structure models is a major strength of this work. In addition, the detailed comparison of similarities and differences between the two species will be a very valuable resource for the scientific community. The manuscript is written very well and allows readers lacking expertise in cryo-EM to understand the important aspects of the conservation of APC/C structure and mechanism across kingdoms.

Weaknesses:

The lack of experimentation in this work to test some of the putative differences in APC/C mechanism (e.g. stimulation of E2 binding by coactivator and stimulation of activity by mitotic phosphorylation) could be considered a weakness. Nonetheless, the authors do a nice job explaining how the structure interpretations imply these differences likely exist, and this work sets the stage nicely for future studies to understand these differences at a mechanistic level. There is enough value in having the S. cerevisiae structure models and the comparison to the human structures, without any additional experimentation.

The validation of APC/C phosphorylation in the unphosphorylated and hyperphosphorylated states is not very robust. Given the lack of significant effects of phosphorylation on APC/C structure observed here (compared to the human complex), this becomes important. A list of phosphorylation sites identified by mass spec before and after in vitro phosphorylation is provided but lacks quantitative information. This list indicates that a significant number of phosphorylation sites are detected in the purified APC/C prior to reaction with purified kinases. Many more sites are detected after in vitro kinase reaction, but it isn't clear how extensively any of the sites are modified. There is reason for caution then, in accepting the conclusions that structures of unphosphorylated and hyperphosphorylated APC/C from S. cerevisiae are nearly identical.

Reviewer #3 (Public Review):

Vazquez-Fernandez et al. present a comprehensive and detailed analysis of the S. cerevisiae APC/C complex, providing new insights into its structure and function. The authors determined the medium-resolution structures of three recombinant S. cerevisiae APC/C complexes, including unphosphorylated apo-APC/C (4.9 Å), the ternary APC/CCDH1-substrate complex (APC/CCDH1:Hsl1 , 4.0 Å), and phosphorylated apo-APC/C (4.4 Å). Prior structures of human, E. cuniculi, S. cerevisiae, and S. pombe APC/C subunits, as well as AlphaFold2 predictions were used to guide model building. Although the determined structures are not sufficient to fully explain the molecular mechanism of APC/C activation and regulation in S. cerevisiae, they provide valuable insights into the similarities and differences with the human complex, shedding light on the conserved and divergent features of APC/C function.

The manuscript synthesizes the structural analysis of the APC/C complex in S. cerevisiae, with literature into a cohesive and clear picture of the complex's structure and function. It is well-written and clear, making the complex biology of the APC/C complex accessible to a wide range of readers. The complex forms a triangular shape, with a central cavity surrounded by two modules: the TPR lobe and the platform module. The TPR lobe consists of three TPR proteins (APC3, APC6, and APC8), which stack on top of each other to form a quasi-symmetric structure. The platform module is composed of the large APC1 subunit, together with APC4 and APC5. The authors also analyzed the structure of several smaller subunits that are involved in regulating the activity of the APC/C complex and showed their structural similarities to and discrepancies from their human counterparts. These subunits, including CDC26/APC12, SWM1/APC13, APC9, and MND2/APC15, form extended, irregular structures that simultaneously contact multiple large globular APC/C subunits.

While the authors report the similarity between the overall structure of S. cerevisiae and human APC/C complexes, they also found two unexpected differences. First, in the S. cerevisiae apo-complex, the E2 binding site on APC11RING is accessible, whereas, in humans, it requires CDH1 binding. Second, a structural element similar to the human APC1 auto-inhibitory segment is missing in S. cerevisiae. In humans, the phosphorylation-dependent displacement of this segment allows CDC20 binding to APC/C. In S. cerevisiae, the binding requires phosphorylation however the structures reported here are suggestive that this could involve a different (presently unknown) mechanism. These structural insights highlight the importance of understanding the species-specific features of APC/C function.

Strengths:

The manuscript does a great job of revealing new structures.

Opportunity for increasing impact: It would have been nice if some functional differences were demonstrated, for example regarding the mechanism of CDC20 binding, and the comparison between apo-APC/C and ternary APC/CCDH1:Hsl1 does not explain the molecular activation mechanism of S. cerevisiae APC/C. Nonetheless, the authors nicely integrate their data with well-established literature on the similarities and differences between yeast and human systems.

Weaknesses:

The authors should cite and discuss Cole Ferguson et al., Mol Cell 2022. This study describes the loss of APC7 in human disease and provides a detailed structural and biochemical examination of the effects of APC7 loss on human APC/C. Given that much of our understanding of APC7 comes from this work, it should be highlighted in the introduction and discussed in depth in light of the new work on S. cerevisiae APC/C.

Reviewer #1 (Public Review):

Summary:

This study investigates how the human brain flexibly adjusts its representations of the world as the environment continually changes. The authors identified regions where the representation continuously drifted across multiple months. They also found that the representation in the parahippocampal cortex could be rapidly influenced by recent environmental inputs.

Strengths:

(1) This study touches upon a crucial but less-explored issue: the relationship between semantic knowledge updating and representation drift in the brain.

(2) This study addresses this issue with a unique dataset in which participants viewed objects embedded in thousands of natural scenes across many fMRI sessions over eight months.

(3) The method for investigating whether the recent inputs could change the neural representation is compelling (i.e., subtracting the backward correlation value from the forward correlation value).

Weaknesses:

(1) Statistical Inference.

(a) Statistical inference is across eight subjects. Low statistical power means high false positive rates.

(b) Multiple comparisons across brain regions were not corrected.

(2) Object Encoding

It is unclear whether the identified brain regions represent the objects (as declared in the manuscript) or the visual features shared by pictures of similar items. Such visual features could be those of the background (e.g., spatial layout or the color tone of the scene), not the objects.

(3) Semantic Content in the MTL

Items with higher levels of semantic association tend to cooccur in the same picture. The results could be driven by the number of pictures shared between each pair of items, not semantic similarity (as declared in the manuscript).

(4) Long-term Drift of Item Representations in the MTL

(a) The results show a long-term representational drift in the brain but provide no evidence suggesting that this long-term neural representational drift reflects the drift in semantic representation. Although the authors used the "semantic" mask defined in the previous step, it does not mean the representation drift in the semantic mask is semantic, and there is doubt whether the "semantic" mask defined in the previous step is really semantic (see the third point).

(b) The beta value of the drift can not be directly compared across regions. Different regions have different sizes and signal-to-noise ratios in the BOLD signal. Their within-item similarity can not be compared directly in the first place.

(5) Recent Structure Rapidly Influences Item Representations in PHC

(a) It is unclear why the authors implement additional modularity analysis instead of directly using the pairwise co-occurrence frequencies among the 80 items, which is more straightforward.

(b) It does not make sense to compare the recent structure to the long-term structure across all 30 sessions because the structure of the posterior sessions cannot influence the current structure updating.

(c) It is unclear how the authors calculate the structure-induced change in the PHC in Figure 7.

Reviewer #2 (Public Review):

Summary:

The authors set out to uncover which brain regions might support the continuous updating of semantic associations thereby showing a system of semantic plasticity. Using fMRI data from participants viewing thousands of natural scene images over 30 recording sessions, they hoped to establish how objects co-occuring with each other within images influences the semantic representations in the human brain that relate to those object concepts.

Strengths:

There is a lot to like about the paper. A major strength of the methods and results is the convincing demonstration of many of the results. This includes showing item representations in the ventral visual pathway and medial temporal lobes (MTL), as we would expect. They also show semantic effects - defined using the word co-occurrence vectors from word2vec, along the posterior and anterior ventral visual pathway and MTL - replicating various past studies. The authors use a creative approach to show that the item representations measured within each session are modulated by the co-occurrence structure in previous trials, becoming more closely related. And that item representations seem to subtly change over the course of the 30 sessions, in that they become less related to each other with increasing distance. However, the semantic effects within each session itself are claimed to remain unchanged.

Weaknesses:

This leads to what I see as a weakness in the study. The conclusions relate to semantic plasticity and the changes in semantic (associative) representations. The drift analyses do appear to show representational changes across the sessions, but this is based on the item representations. The inference is that this is due to an updating of knowledge about the associations each item has had with other items. Yet, in the same regions, the authors suggest that semantic associative effects, as tested using word2vec for each session, remain stable. Doesn't this seem to contradict the claims about semantic plasticity?

Some of this is difficult to unpick as the semantic stability analysis using word2vec in each session is only very briefly mentioned, and the data is not shown (I would include it). So, at present, I feel they show evidence of representational changes but do not show evidence of what the nature of the change is. If the neural representations consistently reflect the long-term semantic associations (which is what word2vec captures), then how does this combine with the drift effects of item representations?

Does it mean that the changes in item representations do not reflect semantic associative knowledge? And reflect some other non-specified type of information (perhaps as the participants are doing an image memory test).

Another potential weakness is the robustness of the drift analysis itself. For the drift analysis, item representations in each session are compared to all other sessions and then averaged according to the number of intervening sessions. This means the data for item representation with a session difference of 1 will be based on 29 data points, a session difference of 2 on 28 data points ... and a session difference of 29 based on 1 data point. So there is a huge imbalance in the amount of data that goes into the analysis for the different numbers of intervening sessions. This leads me to wonder if it could impact the validity of the results. An alternative might be to use 1 datapoint for each session (or a suitable value, I imagine 5 would still give enough data to analyse drifts) and calculate drift, and then repeat this with different partitions of the data to see how stable it is, and if drift is reliably occurring. Alternatively, the analyses they use might have been used and validated previously.

To be clear, I do think this is a very nice study and will have a positive impact on researchers interested in object processing, semantic knowledge, statistical learning, and schemas. But think there are some gaps between what the data shows evidence for, and the ultimate inferences made.

Reviewer #3 (Public Review):

Summary:

This study characterizes the relative stability of semantic representations in the human brain using functional magnetic resonance imaging (fMRI) data. The authors suggest that representations in the early stages of processing within the visual system are stable over hours, weeks, and months, while representations in later stages of processing - within the medial temporal lobe - change more rapidly, sometimes within the span of a single fMRI session.

To make this claim, the authors conduct a series of analyses using a well-established fMRI dataset. This begins with a decoding analysis to identify regions that contain reliable object-specific information. This approach identifies early stages within canonical visual cortices (e.g., primary visual cortex, V1), as well as downstream regions within the medial temporal lobe (MTL); this includes perirhinal cortex (PRC), parahippocampal cortex (PHC), and several subfields within the hippocampal cortex (e.g., CA1). Next, they identify regions that are correlated with "semantic features" associated with these objects, determined using word2vec embeddings of each of these object names. Several regions within the MTL (CA1, PRC, PHC) were significantly correlated with these word2vec embeddings. The authors then turn their analyses to representational change across two different timescales. Between scan sessions, regions at early stages of visual processing (e.g., V1) contain relatively stable representations, while regions within the MTL decreased their auto-correlation across sessions, suggesting that there is increased representational change/drift in the MTL. Finally, the authors demonstrate that there is representational change with PHC within a single scan session - changes that reflect the statistics of visual experiences.

Strengths:

The analyses conducted in this study are solid and creative and they yield compelling theoretical results. Beyond the paper's central claims, this study also highlights the utility of publicly available datasets (i.e., NSD) in exploring and evaluating novel theoretical ideas.

Especially compelling is the combined analysis used to estimate reliable item-level representations, first, and then the long-term drift of item representations (i.e., between sessions). The design choices for modeling the fMRI data (e.g., the cross-validated approach to predicting voxel-level responses) reflect state-of-the-art analysis methods, while the control regions used in these analyses (e.g., V1) provide compelling contrasts to the experimental effects. This makes it clear that the observed representational drift/instability is not present throughout the visual system. These results indicate that this effect is worthy of future experiments, while also providing auxiliary information related to effect size, etc.

Weaknesses:

The concerns outlined here do not challenge the central claim within this study, relating to the relative instability of representations within the MTL as compared to V1. Instead, these concerns focus on whether these representations should be described as "semantic," the importance we should give to the distinction between PHC and other MTC structures, and the lack of systematic analysis in relation to the "gradient" from posterior to anterior regions. In each case, I have provided suggestions as to how these concerns might be addressed. Finally, I've made a note about whether these data should be interpreted in terms of neural "plasticity" given the lack of behavioral change in relation to these fMRI data.

(1) No reason to believe that representations within the MTL are necessarily 'semantic.'

The authors suggest "evoked object representations in CA1, PHC, and PRC are semantic in nature." However, the correlation between fMRI responses and word2vec embeddings-the only evidence for "semantic" representations-is ambiguous. These structures might contain high-dimensional features that are associated with these objects for other reasons; concretely, there might be visual information that is not semantic but relates to the reliable visual properties of these objects (e.g., texture, shape, location in the image). Yet there are no analyses to disambiguate between these alternative accounts. As such, labeling these as "semantic" representations is suggestive but premature. Nonetheless, developing such a control analysis should be relatively straightforward. I outline one possible approach below.

While "semantic" information is a relatively nebulous term in the cognitive neurosciences, contemporary deep-learning methods might offer unambiguous ways to characterize such representations. If we assume that "semantics" relate to the meaning of an object/entity and not the "low-level" sensory attributes related to encoding this information, this leads to a straightforward implementation of object semantics: the reliable variance that can be isolated within the residuals of a sensory encoder. For example, do word2vec embeddings explain variance within the medial temporal lobe above and beyond the variance explained by a vision-only image encoder? Of course, care must be taken to use a visual encoder which is not itself a crystallization of object semantics (e.g., encoders optimized using a classification objective), but this is all very feasible given contemporary computer vision methods. Adding such a control analysis would offer a significant improvement over the current approach, clarifying the nature of the stimulus-driven representations within the medial temporal lobe by disentangling "semantic" properties of reliable visual features.

Additionally, it is not clear whether results from the current "object encoding" analysis and "semantic detection" analysis differ because of underlying differences in representational content in these regions or because of design choices in these analyses themselves. That is, while the object encoding analysis learns a linear projection from a one-hot 80-dimensional vector to hemodynamic responses in each brain region, the semantic detection analysis correlates these predicted hemodynamic responses with word2vec embeddings associated with each of these 80 objects. These different analysis methods result in different outcomes: not all regions identified by the object encoding analysis are also identified in the semantic detection analysis (e.g., hippocampal subfields). It is not clear to what degree these different outcomes are a function of "semantic" information, or are simply a consequence of differences in analytic approaches. It would be useful to know the results by repeating the logic from the object encoding analysis, but instead of 1-hot vectors for each object, use the word2vec embeddings.

(2) Unclear if the differences between PHC and other MTL structures are driven by SNR.

Parahippocampal cortex (PHC) is a region reliably identified by the analyses in this study: PHC is identified in the analysis of item encoding, semantic content, and representational drift across long (between-session) and short (within-session) timescales. Control regions here provide a convincing contrast to PHC in each of these analyses, and so the role of PHC appears clear in these analyses. However, it is unclear how to interpret the difference between PHC and other structures within the MTC - namely, the observation that PHC alone is influenced by representational drift across shorter timescales. It's possible that these effects are common throughout the MTL, but are only evident in PHC because of increased SNR. This concern seems plausible when observing PHC's "encoding success" and "semantic content," both visually and statistically, relative to other MTL structures: the magnitude of PHC's effect appears greater, which could simply be an artifact of PHC's relatively high SNR. In fMRI data, for example, PRC typically has relatively low SNR due to field inhomogeneities related to dropout, due to PRC's relative proximity to the ear canal-which is exacerbated in 7T (vs 3T) scanners, which was the case for the data in this study.

Addressing this concern could be relatively straightforward. For example, including information about the SNR in each respective brain region would be very helpful. If the SNR across brain regions within the MTL is relatively uniform, then this already addresses the concern above. regardless, it would be useful to report the experimental effects in relation, for example, the split-half reliability of signal in each brain region. That is, instead of simply reporting that that results are significant across brain regions, the authors might estimate how reliable the variance is across brain regions, and use this reliable variance as a ceiling which can be used to normalize the amount of variance explained in each analysis. By providing an account of the differences in the reliability/SNR of different regions, we would have a much better estimate of the relative importance of differences in the results reported for different regions within the MTL.

(3) Need for more systematic analysis/visualization of "posterior" vs. "anterior" regions.

The authors report that "Whole-brain analyses revealed a gradient of plasticity in the temporal lobe, with drift more evident in anterior than posterior areas." However, the only contrast provided in the main text is between MTL structures and V1-there is no "gradient" in any of these analyses. There are other regions visualized in Supplemental Figure 3, but there is not a systematic evaluation of the gradient along a "posterior/anterior" axis. It would be helpful to see the results in Figures 3A, 4A, 5A, and 6A to include other posterior visual regions (e.g., V4, LOC, PPA, FFA) beyond V1.

(4) Without behavioral data, not a direct relationship with "stability-plasticity tradeoff"

The results from this study are framed in relation to a "stability-plasticity tradeoff." As argued in this manuscript, this tradeoff is central to animal behavior - our ability to rapidly deploy prior knowledge to respond to the world around us. Given that there are no behavioral measures used in the current study, however, no claims can be made about how these fMRI data might relate to learning, or behavior more generally. As such, framing these results in terms of a stability-plasticity tradeoff is tenuous. "Representational drift," on the other hand, is a term that is relatively agnostic in its relationship with behavior, and aptly describes the results presented here. The authors refer to this term as well. Considering the lack of behavioral evidence, alongside the core findings from these neuroimaging data, "representational stability" or "representational "drift" seems to be a more direct description of the available data than "neural plasticity" or a "stability-plasticity tradeoff."

Reviewer #1 (Public Review):

In their paper, Kalidini et al. investigate why the motor system sometimes coarticulates movements within a sequence. They begin by examining this phenomenon in an optimal feedback controller (OFC) that performs reaching movements to two targets (T1 and T2). They show that coarticulation occurs only when the controller is not required to slow down at T1. When the controller must decelerate at T1, coarticulation does not occur. This observation holds true even though the controller has information about both targets in both scenarios. They test the same experiment on human participants and show that humans also coarticulate the reaches only when they are instructed to treat the first target as a via point. Both in human participants and OFC simulations, whenever the coarticulation is present, the long-latency response to perturbations during the first reach is also informed by the second target- suggesting that the information about the second target is already present in the circuitry that control the long-latency reflex.<br /> All experiments and analyses are standard and clearly explained. Their analysis of long-latency as a measure of coarticulation of sequence items is highly interesting and broadly useful for future experiment design. They successfully demonstrate that one reason the motor system sometimes coarticulates movements is due to high-level instructions on how to execute the sequence. These high-level instructions can, in turn, determine how and to what extent information about future sequence items is utilized by the low-level controller that governs muscle activity. However, the precise interaction between high-level task demands and low-level controllers at the neural tissue level remains an open question.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript the authors examine the question of whether discrete action sequences and coarticulated continuous sequential actions can be produced from the same controller, without having to derive separate control policies for each sequential movement. Using modeling and behavioral experiments, the authors demonstrate that this is indeed possible if the constraints of the policy are appropriately specified. These results are of interest to those interested in motor sequences, but it is unclear whether these findings can be interpreted to apply to the control of sequences more broadly (see weaknesses below).

Strengths:<br /> The authors provide an interesting and novel extension of the stochastic optimal control model to demonstrate how different temporal constraints can lead to either individual or coarticulated movements. The authors use this model to make predictions about patterns of behavior (e.g., in response to perturbations), which they then demonstrate in human participants both by measuring movement kinematics as well as EMG. Together this work supports the authors' primary claims regarding how changes in task instructions (i.e., task constraints) can result in coarticulated or separated movement sequences and the extent to which the subsequent movement goal affects the planning and control of the previous movement.

Weaknesses:<br /> Although this work is quite interesting, it remains unknown whether there is a fundamental distinction between a coarticulated sequence and a single movement passing through a via point (or equivalently, avoiding an obstacle). The notion of a coarticulated sequence brings with it the notion of sequential (sub)movements and temporal structure, whereas the latter can really be treated as more of a constraint on the production of a single continuous movement. The authors suggest that these are not truly different kinds of movements at the level of a control policy, but this remains to be tested experimentally.

It also remains unclear for the theory of optimal feedback control as a whole where and how the cost function and constraints are specified to guide the optimization process. That is, presumably there is the ability for higher-level or explicit description of these constraints, but how they then become incorporated into a control policy remains unclear. With regard to the kind of multi-target constraints proposed here, in typical sequence tasks, while some movements become coarticulated, people also tend to form chunks with distinct chunk boundaries. This presumably means that there is at least some specification of the sequential ordering of these chunks that must exist beyond the control policy and that multiple control policies may still be warranted to execute an entire sequence (otherwise the authors' model might suggest that people can coarticulate forever without needing to exhibit any chunk boundaries). Hence, while the authors fairly convincingly show that a single control policy can lead to separated or coarticulated movements given an appropriate set of constraints, their work does not speak to where or how those constraints are specified, nor to how longer sequences are controlled.

Reviewer #1 (Public Review):

Summary:

The authors sought to investigate the associations of age at breast cancer onset with the incidence of myocardial infarction (MI) and heart failure (HF). They employed a secondary data analysis of the UK Biobank. They used descriptive and inferential analysis including Cox proportional hazards models to investigate the associations. Propensity score matching was also used. They found that Among participants with breast cancer, younger onset age was significantly associated with elevated risks of MI (HR=1.36, 95%CI: 1.19 to 1.56, P<0.001) and HF (HR=1.31, 95% CI: 1.18 to 1.46, P<0.001). the reported similar findings after propensity matching.

Strengths:

The use of a large dataset is a strength of the study as the study is well-powered to detect differences. Reporting both the unmatched and the propensity-matched estimates was also important for statistical inference.

Weaknesses:

The authors have addressed all my previous comments. I have no further comments.

Reviewer #3 (Public Review):

Summary:

Bell and colleagues studied how different splice isoforms of voltage-gated CaV2 calcium channels affect channel expression, localization, function, synaptic transmission, and locomotor behavior at the larval Drosophila neuromuscular junction. They reveal that one mutually exclusive exon located in the fourth transmembrane domain encoding the voltage sensor is essential for calcium channel expression, function, active zone localization, and synaptic transmission. Furthermore, a second mutually exclusive exon residing in an intracellular loop containing the binding sites for Caβ and G-protein βγ subunits promotes the expression and synaptic localization of around ~50% of CaV2 channels, thereby contributing to ~50% of synaptic transmission. This isoform enhances release probability, as evident from increased short-term depression, is vital for homeostatic potentiation of neurotransmitter release induced by glutamate receptor impairment, and promotes locomotion. The roles of the two other tested isoforms remain less clear.

Strengths:

The study is based on solid data that was obtained with a diverse set of approaches. Moreover, it generated valuable transgenic flies that will facilitate future research on the role of calcium channel splice isoforms in neural function.

Weaknesses:

(1) Based on the data shown in Figures 2A-C, and 2H, it is difficult to judge the localization of the cac isoforms. Could they analyze cac localization with regard to Brp localization (similar to Figure 3; the term "co-localization" should be avoided for confocal data), as well as cac and Brp fluorescence intensity in the different genotypes for the experiments shown in Figure 2 and 3 (Brp intensity appears lower in the dI-IIA example shown in Figure 3G)? Furthermore, heterozygous dIS4B imaging data (Figure 2C) should be quantified and compared to heterozygous cacsfGFP/+.

(2) They conclude that I-II splicing is not required for cac localization (p. 13). However, cac channel number is reduced in dI-IIB. Could the channels be mis-localized (e.g., in the soma/axon)? What is their definition of localization? Could cac be also mis-localized in dIS4B? Furthermore, the Western Blots indicate a prominent decrease in cac levels in dIS4B/+ and dI-IIB (Figure 1D). How do the decreased protein levels seen in both genotypes fit to a "localization" defect? Could decreased cac expression levels explain the phenotypes alone?

(3) Cac-IS4B is required for Cav2 expression, active zone localization, and synaptic transmission. Similarly, loss of cac-I-IIB reduces calcium channel expression and number. Hence, the major phenotype of the tested splice isoforms is the loss of/a reduction in Cav2 channel number. What is the physiological role of these isoforms? Is the idea that channel numbers can be regulated by splicing? Is there any data from other systems relating channel number regulation to splicing (vs. transcription or post-transcriptional regulation)?

(4) Although not supported by statistics, and as appreciated by the authors (p. 14), there is a slight increase in PSC amplitude in dIS4A mutants (Figure 2). Similarly, PSC amplitudes appear slightly larger (Figure 3J), and cac fluorescence intensity is slightly higher (Figure 3H) in dI-IIA mutants. Furthermore, cac intensity and PSC amplitude distributions appear larger in dI-IIA mutants (Figures 3H, J), suggesting a correlation between cac levels and release. Can they exclude that IS4A and/or I-IIA negatively regulate release? I suggest increasing the sample size for Canton S to assess whether dIS4A mutant PSCs differ from controls (Figure 2E). Experiments at lower extracellular calcium may help reveal potential increases in PSC amplitude in the two genotypes (but are not required). A potential increase in PSC amplitude in either isoform would be very interesting because it would suggest that cac splicing could negatively regulate release.

(5) They provide compelling evidence that IS4A is required for the amplitude of somatic sustained HVA calcium currents. However, the evidence for effects on biophysical properties and activation voltage (p. 13) is less convincing. Is the phenotype confined to the sustained phase, or are other aspects of the current also affected (Figure 2J)? Could they also show the quantification of further parameters, such as CaV2 peak current density, charge density, as well as inactivation kinetics for the two genotypes? I also suggest plotting peak-normalized HVA current density and conductance (G/Gmax) as a function of Vm. Could a decrease in current density due to decreased channel expression be the only phenotype? How would changes in the sustained phase translate into altered synaptic transmission in response to AP stimulation?

(6) Why was the STED data analysis confined to the same optical section, and not to max. intensity z-projections? How many and which optical sections were considered for each active zone? What were the criteria for choosing the optical sections? Was synapse orientation considered for the nearest neighbor Cac - Brp cluster distance analysis? How do the nearest-neighbor distances compare between "planar" and "side-view" Brp puncta?

(7) Cac clusters localize to the Brp center (e.g., Liu et al., 2011). They conclude that Cav2 localization within Brp is not affected in the cac variants (p. 8). However, their analysis is not informative regarding a potential offset between the central cac cluster and the Brp "ring". Did they/could they analyze cac localization with regard to Brp ring center localization of planar synapses, as well as Brp-ring dimensions?

(8) Given the accelerated PSC decay/ decreased half width in dI-IIA (Fig. 5Q), I recommend reporting PSC charge in Figure 3, and PPR charge in Figures 5A-D. The charge-based PPRs of dI-IIA mutants likely resemble WT more closely than the amplitude-based PPR. In addition, miniature PSC decay kinetics should be reported, as they may contribute to altered decay kinetics. How could faster cac inactivation kinetics in response to single AP stimulation result in a decreased PSC half-width? Is there any evidence for an effect of calcium current inactivation on PSC kinetics? On a similar note, is there any evidence that AP waveform changes accelerate PSC kinetics? PSC decay kinetics are mainly determined by GluR decay kinetics/desensitization. The arguments supporting the role of cac splice isoforms in PSC kinetics outlined in the discussion section are not convincing and should be revised.

(9) Paired-pulse ratios (PPRs): On how many sweeps are the PPRs based? In which sequence were the intervals applied? Are PPR values based on the average of the second over the first PSC amplitudes of all sweeps, or on the PPRs of each sweep and then averaged? The latter calculation may result in spurious facilitation, and thus to the large PPRs seen in dI-IIB mutants (Kim & Alger, 2001; doi: 10.1523/JNEUROSCI.21-24-09608.2001).

(10) Could the dI-IIB phenotype be simply explained by a decrease in channel number/ release probability? To test this, I propose investigating PPRs and short-term dynamics during train stimulation at lower extracellular Ca2+ concentration in WT. The Ca2+ concentration could be titrated such that the first PSC amplitude is similar between WT and dI-IIB mutants. This experiment would test if the increased PPR/depression variability is a secondary consequence of a decrease in Ca2+ influx, or specific to the splice isoform.

(11) How were the depression kinetics analyzed? How many trains were used for each cell, and how do the tau values depend on the first PSC amplitude? Time constants in the range of a few (5-10) milliseconds are not informative for train stimulations with a frequency of 1 or 10 Hz (the unit is missing in Figure 5H). Also, the data shown in Figures 5E-K suggest slower time constants than 5-10 ms. Together, are the data indeed consistent with the idea that dI-IIB does not only affect cac channel number, but also PPR/depression variability (p. 9)?

(12) The GFP-tagged I-IIA and mEOS4b-tagged I-IIB cac puncta shown in Figure 6N appear larger than the Brp puncta. Endogenously tagged cac puncta are typically smaller than Brp puncta (Gratz et al., 2019). Also, the I-IIA and I-IIB fluorescence sometimes appear to be partially non-overlapping. First, I suggest adding panels that show all three channels merged. Second, could they analyze the area and area overlap of I-IIA and I-IIB with regard to each other and to Brp, and compare it to cac-GFP? Any speculation as to how the different tags could affect localization? Finally, I recommend moving the dI-IIA and dI-IIB localization data shown in Figure 6N to an earlier figure (Figure 1 or Figure 3).

Reviewer #1 (Public Review):

Summary:

The manuscript by Bell et. al. describes an analysis of the effects of removing one of two mutually exclusive splice exons at two distinct sites in the Drosophila CaV2 calcium channel Cacophony (Cac). The authors perform imaging and electrophysiology, along with some behavioral analysis of larval locomotion, to determine whether these alternatively spliced variants have the potential to diversify Cac function in presynaptic output at larval neuromuscular junctions. The author provided valuable insights into how alternative splicing at two sites in the calcium channel alters its function.

Strengths:

The authors find that both of the second alternatively spliced exons (I-IIA and I-IIB) that are found in the intracellular loop between the 1st and 2nd set of transmembrane domains can support Cac function. However, loss of the I-IIB isoform (predicted to alter potential beta subunit interactions) results in 50% fewer channels at active zones and a decrease in neurotransmitter release and the ability to support presynaptic homeostatic potentiation. Overall, the study provides new insights into Cac diversity at two alternatively spliced sites within the protein, adding to our understanding of how regulation of presynaptic calcium channel function can be regulated by splicing.

Weaknesses:

The authors find that one splice isoform (IS4B) in the first S4 voltage sensor is essential for the protein's function in promoting neurotransmitter release, while the other isoform (IS4A) is dispensable. The authors conclude that IS4B is required to localize Cac channels to active zones. However, I find it more likely that IS4B is required for channel stability and leads to the protein being degraded, rather than any effect on active zone localization. More analysis would be required to establish that as the mechanism for the unique requirement for IS4B.

Reviewer #2 (Public Review):

This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones. Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There are some outstanding questions regarding the mechanisms underlying the changes in Cac localization and function, and some additional suggestions are listed below for the authors to consider in strengthening this study. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

Reviewer #2 (Public Review):

Summary:

This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.

Strengths:

The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent directed cell migration without affecting ligand-binding itself or some other ligand-induced signaling pathways. The authors have used a relatively broad set of experiments to validate and demonstrate the effects of the drug. Importantly, the authors also test AGR1.137 in vivo, using a zebra fish model of tumorigenesis and metastasis. A relatively strong inhibitory effect of the compound is reported.

Weaknesses:

The authors have been able to significantly strengthen their data from the first submission. The content of this manuscript is pretty solid, although studies in mammalian model systems are naturally needed in the future to better assess the promise of the compound.

Reviewer #2 (Public Review):

Summary:

This paper addresses an important computational problem in learning and memory. Why do related memory representations sometimes become more similar to each other (integration) and sometimes more distinct (differentiation)? Classic supervised learning models predict that shared associations should cause memories to integrate, but these models have recently been challenged by empirical data showing that shared associations can sometimes cause differentiation. The authors have previously proposed that unsupervised learning may account for these unintuitive data. Here, they follow up on this idea by actually implementing an unsupervised neural network model that updates the connections between memories based on the amount of coactivity between them. The authors use their modeling framework to simulate three recent empirical studies, showing that their model captures aspects of these findings that are hard to account for with supervised learning.

Overall, this is a strong and clearly described work that is likely to have a positive impact on computational and empirical work in learning and memory. While the authors have written about some of the ideas discussed in this paper previously, a fully implemented and openly available model is a clear advance that will benefit the field. It is not easy to translate a high-level description of a learning rule into a model that actually runs and behaves as expected. The fact that the authors have made all their code available makes it likely that other researchers will extend the model in numerous interesting ways, many of which the authors have discussed and highlighted in their paper.

Strengths:

The authors succeed in demonstrating that unsupervised learning with a simple u-shaped rule can produce results that are qualitatively in line with the empirical reports. In each of the three models, the authors manipulate stimulus similarity (following Chanales et al.), shared vs distinct associations (following Favila et al.), or learning strength (a stand-in for blocked versus interleaved learning schedule; following Schlichting et al.). In all cases, with hand-tuning of additional parameters, the authors are able to produce model representations that fit the empirical results, but that can't easily be accounted for by supervised learning. Demonstrating these effects isn't trivial and a formal modeling framework for doing so is a valuable contribution. Overall, the work is very thorough. The authors investigate many different aspects of the learning dynamics (learning rate, oscillation strength, hidden layer overlap etc) in these models and produce several key insights. Of particular value are their demonstrations that when differentiation occurs, it occurs very quickly and asymmetrically and results in anti-correlated representations, as well as the distinction between symmetric and asymmetric integration in their model. The authors thoroughly acknowledge the relative difficulty of producing differentiation in their models relative to integration, and are now more clear about why they don't necessarily view this as mismatch with the empirical data. The authors are also more clear about the complicated activation dynamics in their model and why critical ranges for some parameters can't be given -- the number of interacting parameters mean that there are many combinations that could produce the critical activation dynamics and thus the same result. Despite this complexity, the paper is very clearly written; the authors do a good job of both formally describing their model as well as giving readers a high level sense of how many of their critical model components work.

Weaknesses:

Though the u-shaped learning rule is essential to this framework, the paper doesn't do any formal investigation of this learning rule or comparison with other learning rules. The authors do have a strong theoretical interest in this rule as well as experimental precedent for testing this rule, which they now thoroughly discuss in the paper. Still, a stronger argument in support of the non monotonic plasticity hypothesis could have been made by comparing this learning rule to alternatives. Additionally, the authors' choice of strongly prewiring associations makes it difficult to think about how their model maps onto experimental contexts where associations are only weakly learned. However, the authors thoroughly acknowledge why this was necessary and discuss this limitation in the paper.

Reviewer #1 (Public Review):

Ritvo and colleagues present an impressive suite of simulations that can account for three findings of differentiation in the literature. This is important because differentiation-in which items that have some features in common, or share a common associate are less similar to one another than are unrelated items-is difficult to explain with classic supervised learning models, as these predict the opposite (i.e., an increase in similarity). A few of their key findings are that differentiation requires a high learning rate and low inhibitory oscillations, and is virtually always asymmetric in nature.

This paper was very clear and thoughtful-an absolute joy to read. The model is simple and elegant, and powerful enough to re-create many aspects of existing differentiation findings. The interrogation of the model and presentation of the findings were both extremely thorough. The potential for this model to be used to drive future work is huge.

The authors have been very responsive to my previous reviews and I have no further concerns and identify no major weaknesses.

Reviewer #3 (Public Review):

This paper proposes a computational account for the phenomenon of pattern differentiation (i.e., items having distinct neural representations when they are similar). The computational model relies on a learning mechanism of the nonmonotonic plasticity hypothesis, fast learning rate and inhibitory oscillations. In the revised paper, the authors justified the initialization of the model, added empirical evidence supporting the use of two turning points in the NMPH function and provided details of the learning mechanisms of the model. The relatively simple architecture of the model makes its dynamics accessible to the human mind. Furthermore, using similar model parameters, this model produces simulated data consistent with empirical data of pattern differentiation. The authors also provide insightful discussion on the factors contributing to differentiation as opposed to integration.

Reviewer #1 (Public Review):

Using a combination of cutting-edge high-resolution technologies (expansion microscopy, SIM, and CLEM) and biochemical approaches (in vitro translocation of actin filaments, cargo uptake assays, and drug treatment), the authors revisit and update previous results about TbMyo1 and TbACT in the bloodstream form (BSF) of Trypanosoma brucei. They show that a great part of the myosin motor is cytoplasmic but the fraction associated with organelles is in proximity to the endosomal system and in glycosomes. In addition, they show that TbMyo1 can move actin filaments in vitro and visualize for the first time this actomyosin system using specific antibodies, a "classical" antibody for TbMyo1, and a chromobody for actin. Finally, using latrunculin A, which sequesters G-actin and prevents F-actin assembly, the authors show the delocalization and eventually the loss of the filamentous actin signal and the concomitant loss of the endosomal system integrity.<br /> Overall this well-conducted and convincing study paves the way toward the elucidation of the role of an actomyosin system in the maintenance of the endosomal network in T. brucei.

Strengths:

The work is of high quality and uses advanced technologies to determine the involvement TbMyo1 and actin in the integrity of the endosomal system. The conclusions are not over-interpreted and are supported by the experimental results and their quantification.

Weaknesses:

Although disruption of the actomyosin system using either the actin-depolymerizing drug latrunculin A or the TbMyo1-RNAi cell line established an effect on the endosomal system integrity, it remains to understand how this occurs mechanistically and what are the intracellular components involved.

Reviewer #2 (Public Review):

The study by Link et al. advances our understanding of the actomyosin system in T. brucei, focusing on the role of TbMyo1, a class I myosin, within the parasite's endosomal system. Using a combination of biochemical fractionation, in vitro motility assays, and advanced imaging techniques such as correlative light and electron microscopy (CLEM), this paper demonstrates that TbMyo1 is dynamically distributed across early and late endosomes, the cytosol, is associated with the cytoskeleton, and a fraction has an unexpected association with glycosomes. Notably, the study shows that TbMyo1 can translocate actin filaments at velocities suggesting an active role in intracellular trafficking, potentially higher than those observed for similar myosins in other cell types. This work not only elucidates the spatial dynamics of TbMyo1 within T. brucei but also suggests its broader involvement in maintaining the complex architecture of the endosomal network, underscoring the critical role of the actomyosin system in a parasite that relies on high rates of endocytosis for immune evasion.

A key strength of the study is its exceptional rigor and successful integration of a wide array of sophisticated techniques, such as in vitro motility assays, and advanced imaging methods, e.g. CLEM. This combination of approaches underscores the study's comprehensive approach to examining the ultrastructural organization of the trypanosome endomembrane system. The application of functional data using inhibitors, such as latrunculin A for actin depolymerization, further strengthens the study by providing insights into the dynamics and regulatory mechanisms of the endomembrane system. This demonstrates how the actomyosin system contributes to cellular morphology and trafficking processes. Furthermore, the discovery of TbMyo1 localization to glycosomes introduces a novel aspect to the potential roles of myosin I proteins within the cell, particularly in the context of organelles analogous to peroxisomes. This observation not only broadens our understanding of myosin I functionality but also opens up new avenues for research into the cell biology of trypanosomatids, marking a significant contribution to the field.

A significant initial weakness was the reliance on spatial association data to infer functional relationships without direct demonstration of biochemical activities in vivo. The authors have since addressed this by including new evidence from TbMyo1 RNAi cell lines and EM data that show the effects of TbMyo1 depletion on cellular ultrastructure. The authors' responses and additional data reinforce their initial conclusions and address previous concerns. Several new, elegant hypotheses are proposed in the discussion that warrant further investigation to fully understand TbMyo1's interactions and regulatory mechanisms in vivo.

Reviewer #3 (Public Review):

Summary:

In this work, Link and colleagues have investigated the localization and function of the actomyosin system in the parasite Trypanosoma brucei, which represents a highly divergent and streamlined version of this important cytoskeletal pathway. Using a variety of cutting-edge methods, the authors have shown that the T. brucei Myo1 homolog is a dynamic motor that can translocate actin, suggesting that it may not function as a more passive crosslinker. Using expansion microscopy, iEM, and CLEM, the authors show that MyoI localizes to the endosomal pathway, specifically the portion tasked with internalizing and targeting cargo for degradation, not the recycling endosomes. The glycosomes also appear to be associated with MyoI, which was previously not known. An actin chromobody was employed to determine the localization of filamentous actin in cells, which was correlated with the localization of Myo1. Interestingly, the pool of actomyosin was not always closely associated with the flagellar pocket region, suggesting that portions of the endolysomal system may remain at a distance from the sole site of parasite endocytosis. Lastly, the authors used actin-perturbing drugs to show that disrupting actin causes a collapse of the endosomal system in T. brucei, which they have shown recently does not comprise distinct compartments but instead a single continuous membrane system with subdomains containing distinct Rab markers.

Strengths:

Overall, the quality of the work is extremely high. It contains a wide variety of methods, including biochemistry, biophysics, and advanced microscopy that are all well deployed to answer the central question. The data is also well quantitated to provide additional rigor to the results. The main premise, that actomyosin is essential for the overall structure of the T. brucei endocytic system, is well supported and is of general interest, considering how uniquely configured this pathway is in this divergent eukaryote and how important it is to the elevated rates of endocytosis that are necessary for this parasite to inhabit its host.

Comments on revised version:

The revised manuscript has addressed the main issue, the lack of TbMyo1 functional data that was brought up during the first round of review. I find it interesting that Myo1 depletion has what appears to be a limited effect on endocytosis while producing a similar fragmentation of the endocytic pathway to what is seen with the LatA treatments. As CCV remains in both LatA treatments and TbMyo1 RNAi, it seems apparent that the organization of the endocytic pathway is not required for at least basal levels of endocytosis.

My other points were well addressed by the rebuttal. I am satisfied with the update.

Reviewer #1 (Public Review):

Summary:

This research used cell-based signaling assay and Gaussian-accelerated molecular dynamics (GaMD) to study peptide-mediated signaling activation of Polycystin-1 (PC1), which is responsible for the majority of autosomal dominant polycystic kidney disease (ADPKD) cases. Synthetic peptides of various lengths derived from the N-terminal portion of the PC1 C-terminal fragment (CTF) were applied to HEK293T cells transfected with stalkless mouse CTF expression construct. It was shown that peptides including the first 7, 9, and 17 residues of the N-terminal portion could activate signaling to the NFAT reporter. To further understand the underlying mechanism, docking and peptide-GaMD simulations of peptides composed of the first 9, 17, and 21 residues from the N-terminal portion of the human PC1 CTF were performed. These simulations revealed the correlation between peptide-CTF binding and PC1 CTF activation characterized by the close contact (salt bridge interaction) between residues R3848 and E4078. Finally, a Potts statistical model was inferred from diverged PC1 homologs to identify strong/conserved interacting pairs within PC1 CTF, some of which are highly relevant to the findings from the peptide GaMD simulations. The peptide binding pockets identified in the GaMD simulations may serve as novel targets for design of therapeutic approaches for treating ADPKD.

Strengths:

(1) The experimental and computational parts of this study complement and mostly support each other, thus increasing the overall confidence in the claims made by the authors.

(2) The use of exogenous peptides and a stalkless CTF in the GaMD is a step forward compared to earlier simulations using the full CTF, CTF mutants, or the stalkless CTF alone. And it led to findings of novel binding pockets.

(3) Since the PC1 shares characteristics with the Adhesion class of GPCRs, the approaches used in this work may be extended to other similar systems.

Weaknesses:

(1) Only results for selective peptides (p9, p17 p21) binding with the protein were shown. It would be interesting to see the interaction between some (if not all) of the other peptides with the protein.

(2) The convergence of the simulations is not very good. The results should be interpreted more qualitatively rather than quantitively because large variations in the free energy profile were seen between different replicates. Although these simulations might have identified representative low-energy binding conformations of the peptides, whether they have explored all possible conformations is still a question.

Reviewer #2 (Public Review):

Summary:

This manuscript, "Activation of Polycystin-1 Signaling by Binding of Stalk-derived Peptide Agonists", by Miao and coworkers. The autosomal dominant polycystic kidney disease (ADPKD) is a major form of polycystic kidney disease (PKD). To provide better treatment and avoid side effects associated with currently available options, the authors investigated an interesting GPCR, polycystin-1 (PC1), as a potential therapeutic target. In vitro and in silico studies were combined to identify peptide agonists for PC1 and to elucidate their roles in PC1 signaling. Overall, regarding the significance of the findings, this work described valuable peptide agonists for PC1 and the combined in vitro and in silico approach can be useful to study a complex system like PC1. However, the strength of the evidence is incomplete, as more experiments are needed as controls to validate the computational observations. The work appears premature.

Strengths:

(1) This work first described the experimental discovery of short peptides designed to mimic the stalk region of PC1, followed by computational investigation using docking and MD simulations. PC1 is a complex membrane protein and an emerging target for ADPKD, but it can be challenging to study. The knowledge and the peptide discovery can be valuable and useful to understand the mechanism and potential modulation of PC1.<br /> (2) The authors published the mechanistic study of PC1 and identified key interacting residues such as N3074-S3585 and R3848-E4078, using very similar techniques (PNAS 2022, 119(19), e2113786119). This work furthers this research by identifying peptides that are stalk mimics for PC1 activation.<br /> (3) Eight peptides were designed and tested experimentally first; three were computationally studied with docking and GaMD simulations to understand their mechanism (s).

Weaknesses:

(1) The selectivity of the peptides between PC1 and PC2 remains unknown in this revision.

Overall, my comments were mostly addressed properly.

Reviewer #3 (Public Review):

Summary:

The authors demonstrate the activation of Polycystin-1 (PC1), a G-protein coupled receptor, using small peptides derived from its original agonist, the stalk TA protein. In the experimental part of the study, the authors performed cellular assays to check the peptide-induced reactivation of a mutant form of PC1 which does not contain the stalk agonist. The experimental data is supported by computational studies using state-of-the-art Gaussian accelerated Molecular Dynamics (GaMD) and bioinformatics analysis based on sequence covariance. The computer simulations revealed the mechanistic details of the binding of the said peptides with the mutant PC1 protein and discovered different bound, unbound, and intermediate conformations depending on the peptide size and sequence. Due to the use of reliable and well-established molecular simulation algorithms and the physiological relevance of this protein autosomal dominant polycystic kidney disease (ADPKD) make this work particularly valuable.

Strengths:

This work is exploratory and its goal is to establish that small peptides can be used to probe the PC1 signaling process. The authors have provided sufficient evidence to justify this claim. Their GaMD simulations have produced free-energy landscapes that differentiate the interaction of PC1 with three different synthetic peptides and demonstrate the associated conformational dynamics of the receptor protein. Their trajectory analysis and sequence covariance analysis could identify residue-specific interactions that facilitate this process. The authors also performed residue-wise and total interaction energy calculations to substantiate their findings.

Weaknesses:

The reported free energy landscapes are not fully converged. But they are still sufficient to gain biological insight.

Reviewer #1 (Public Review):

Summary:

In this study, Djebar et al. perform a comprehensive analysis of mutant phenotypes associated with the onset and progression of scoliosis in zebrafish ciliary transition zone mutants rpgrip1l and cep290. They determine that rpgrip1l is required in foxj1a-expressing cells for normal spine development, and that scoliosis is associated with brain ventricle dilations, loss of Reissner fiber polymerization, and the loss of 'tufts' of multi-cilia surrounding the subcommissural organ (the source of Reissner substance). Informed by transcriptomic and proteomic analyses, they identify a neuroinflammatory response in rpgrip1l and cep290 mutants that is associated with astrogliosis and CNS macrophage/microglia recruitment. Furthermore, anti-inflammatory drug treatment reduced scoliosis penetrance and severity in rpgrip1l mutants. Based on their data, the authors propose a feed-forward loop between astrogliosis, induced by perturbed ventricular homeostasis, and immune cells recruitment as a novel pathogenic mechanism of scoliosis in zebrafish ciliary transition zone mutants.

Strengths:

- Comprehensive characterization of the causes of scoliosis in ciliary transition zone mutants rpgrip1l and cep290<br /> - Comparison of rpgrip1l mutants pre- and post-scoliosis onset allowed authors to identify specific phenotypes as being correlated with spine curvature, including brain ventricle dilations, loss of Reissner fiber, and loss of cilia in proximity to the subcommissural organ<br /> - Elegant genetic demonstration that increased urotensin peptide levels do not account for spinal curvature in rpgrip1l mutants<br /> - The identification of astrogliosis and Annexin over-expression in glial cells surrounding diencephalic and rhombencephalic ventricles as being correlated with scoliosis onset and severe curve progression is a very interesting finding, which may ultimately inform pathogenic mechanisms driving spine curvature

Weaknesses:

- The fact that cilia loss/dysfunction and Reissner fiber defects cause scoliosis in zebrafish is already well established in the literature, as is the requirement for cilia in foxj1a-expressing cells<br /> - Neuroinflammation has already been identified as the underlying pathogenic mechanism in at least 2 previously published scoliosis models (zebrafish ptk7a and sspo mutants)<br /> - Anti-inflammatory drugs like aspirin, NAC and NACET have also previously been demonstrated to suppress scoliosis onset and severe curve progression in these models<br /> Therefore, although similar observations in rpgrip1l and cep290 mutants (as reported here) add to a growing body of literature that supports a common biological mechanism underlying spine curvature in zebrafish, novelty of reported findings is diminished.<br /> - Although authors demonstrate that astrogliosis and/or macrophage or microglia cell recruitment are correlated with scoliosis, they do not formally demonstrate that these events are sufficient to drive spine curvature. Thus, the functional consequences of astrogliosis and microglia infiltration remain uncertain.<br /> - Authors do not investigate the effect of anti-inflammatory treatments on other phenotypes they have correlated with spinal curve onset (like ventricle dilation, Reissner fiber loss, and multi-cilia loss around the subcommissural organ). This would help to identify causal events in scoliosis.

Reviewer #2 (Public Review):

Summary:

The manuscript by Djebar et al investigated the role and the underlying mechanism of the ciliary transition zone protein Rpgrip1l in zebrafish spinal alignment. They showed that rpgrip1l mutant zebrafish develop a nearly full penetrance of body curvature at juvenile stages. The mutant fish have cilia defects associated with ventricular dilations and loss of the Reissner fibers. Scoliosis onset and progression are also strongly associated with astrogliosis and neuroinflammation, and anti-inflammatory drug treatment prevents scoliosis in mutant zebrafish, suggesting a novel pathogenic mechanism for human idiopathic scoliosis. This study is quite comprehensive with high quality data, and the manuscript is well written, providing important information on how the ciliary transition zone protein functions in maintaining the zebrafish body axis straightness.

Strengths:

Very clear and comprehensive analysis of the mutant zebrafish.

Reviewer #1 (Public Review):

This manuscript by Tyler and colleagues describes a thorough analysis of IR-induced changes in nascent RNA transcripts, and a genome-wide screening effort to identify the responsible proteins. The findings extend previous work describing DNA damage-induced transcriptional repression from DNA breaks in cis to bulk genomic DNA damage. A significant discovery is the inability of arrested cells to undergo DNA damage-induced gene silencing, which, at least at the rDNA locus, is attributed to an inability to mediate ATM-induced transcriptional repression. While the findings add to our knowledge of how DNA damage affects gene expression, there are several limitations to the current study that remain inadequately addressed. In addition, some of the proposed conclusions seem speculative and should be marked as such, omitted or experimentally supported.

Two major concerns were as follows and have been addressed as outlined in the authors' response to this review:

(1) The CIRSPR screen designed to detect regulators of damage-induced transcriptional repression is based on EU incorporation following a 7-day selection of stable knockout cells. As the authors point out, cell cycle arrest reduces rDNA transcription on its own. The screen, which assesses changes in sgRNA distribution in EU high cells, is thus likely to be dominated by factors that affect cell cycle progression. This is exemplified in the analyses of top hits related to neddylation. The screen's limitations in terms of identifying DDR effectors of damage-induced silencing needs to be clearly stated.

(2) The authors confirm previous findings of DNA damage-induced repression of rDNA and histone gene transcription. The authors propose that these highly transcribed genes are more susceptible to silencing than the bulk of protein coding genes and propose a global damage-induced signaling event that is independent of DNA breaks in cis. While this is possible, it is not demonstrated in this manuscript, and the authors should acknowledge alternative explanations. For example, the loci found to be repressed by bulk IR are highly repetitive gene arrays that tend to form nuclear sub-compartments (nucleoli, histone bodies). As such, their likelihood of being in the vicinity of DNA damage is high, at least for a fraction of gene copies. The findings, therefore, remain consistent with cis-induced silencing. Moreover, silencing may spread through the relevant nuclear sub-compartments, consistent with the formation of DNA damage compartments described recently (PMID: 37853125).

Other comments - also addressed in the authors' response:

(1) The statement that silencing is due to transcription initiation rather than elongation is not sufficiently supported by the data. Could equivalent nascent transcript reduction not be the result of the suppression of elongating RNA PolII? To draw the proposed conclusion, the authors would need to demonstrate that RNA PolII initiation is altered, using RNA PollII ChIP and/or analysis of relevant RNA PolII phosphorylation patterns.

(2) The lack of rDNA silencing in arrested cells is interesting, though the underlying mechanism remains unclear. To further corroborate the proposed defect in ATM-mediated signaling, the authors should look directly at ATM and Treacle phosphorylation upstream of TOPBP1.

(3) The "change in relative heights of the EU low (G1) and EU high (S/G2) peaks" in Figs 5D, 5E and 6B is central to the proposed model of transcriptional changes being affected by cell cycle arrest. These differences should be visualized more clearly and quantified across independent experiments. Ideally, cell cycle stage should be dissected as in Fig. 2B. How do the authors envision cell cycle arrest triggers the defect in transcriptional silencing?

Reviewer #2 (Public Review):

In this manuscript, the authors attempted to study mechanisms of transcription inhibition in cells treated with IR. They observed that unlike transcription inhibition induced by UV damage that depends on histone chaperone HIRA, IR induced transcription inhibition is independent on HIRA. Through a CRISPR/Cas9 screen, they identified protein neddylation is important for transcription inhibition. By sequencing nascent RNA, they observed that down-regulated transcripts upon IR treatment are largely highly transcribed genes including histone genes and rDNA.

This study utilized comprehensive approaches to fill in knowledge gap of IR-induced transcription inhibition.

Comments on current version:

The revised manuscript largely addressed my concerns.

Reviewer #2 (Public Review):

Summary:

In the manuscript by Oestreicher et al, the authors use patch-clamp electrophysiology, immunofluorescent imaging of the cochlea, auditory function tests, and single-unit recordings of auditory afferent neurons to probe the unique properties of calcium signaling in cochlear hair cells that allow rapid and sustained neurotransmitter release. The calcium binding proteins (CaBPs) are thought to modify inactivation of the Cav1.3 calcium channels in IHCs that initiate vesicle fusion, reducing the calcium-dependent inactivation (CDI) of the channels to allow sustained calcium influx to support neurotransmitter release. The authors use knockout mice of Cabp1 and Cabp2 in a double knockout (Cabp1/2 DKO) to show that these molecules are required for enabling sustained calcium currents by reducing CDI, enabling proper IHC neurotransmitter release. They further support their evidence by re-introducing Cabp2 using injection of AAV containing the Cabp2 sequence into the cochlea, which restores some of the auditory function and reduces CDI in patch-clamp recordings.

Strengths:

Overall the data is convincing that Cabp1/2 is required for reducing CDI in cochlear hair cells, allowing their sustained neurotransmitter release and sound encoding. Figures are well-prepared, recordings are careful and stats are appropriate, and the manuscript is well written. The discussion appropriately considers aspects of the data that are not yet explained and await further experimentation.

Weaknesses:

There are some sections of the manuscript that pool data from different experiments with slightly different conditions (wt data from a previous paper, different calcium concentrations, different holding voltages, tones vs clicks, etc). This makes the work harder to follow and more complicated to explain. However, the major conclusion, that that cabp1 and 2 work together to reduce calcium dependent inactivation of L-type calcium channels in cochlear inner hair cells, still holds and is well supported. Another minor weakness is that the authors used injections of AAV containing sequences for Cabp2, but do not present data from sham surgeries. In most cases, the improvement of hearing function with AAV injection is believable and should be attributed to the cabp2 function. However, in at least one instance (Figure 4B), the results of the AAV injection experiments may be overinterpreted - the authors show that upon AAV injection, the hair cells have a much longer calcium current recovery following a large, long depolarization to inactivate the calcium channels. Without comparison to a sham surgery, it is not known if this result could be a subtle result of the surgery or indeed due to the Cabp2 expression. The authors have added text acknowledging this, as appropriate.

Reviewer #1 (Public Review):

Summary:

This manuscript dissects the contribution of the CaBP 1 and 2 on the calcium current in the cochlear inner hair cells. The authors measured the calcium current inactivation from the double knock-out CaBP1 and 2 and show that both proteins contribute to the voltage-dependent and calcium-dependent inactivation. Synaptic release was reduced in the double KO. As a consequence, the authors observed a depressed activity within the auditory nerve. Taken together, this study identifies a new player that regulates the stimulation-secretion coupling in the auditory sensory cells.

Strengths:

In this study, the authors bring compelling evidence that CaBP 1 and 2 are both involved in the inactivation of the calcium current, from cellular up to system level and by taking care to probe different experimental conditions such as different holding potentials and by rescuing the phenotype with the re-expression of CaBP2. Indeed, while changing the holding potential worsen the secretion, it completely changes the kinetics of the inactivation recovery. It alerts the reader that probing different experimental conditions that may be closer to physiology are better suited to uncover any deleterious phenotype. This gave pretty solid results.

Weaknesses:

Although this study clearly points that CaBP1 is involved in the calcium current inactivation, it is not clear how CaBP1 and CaBP2 act together (but this is probably beyond the scope of the study). Another point is that the authors re-express CaBP2 to largely rescue the phenotype in the double KO but no data are available to know whether the re-expression of both CaBP1 and CaBP2 would achieve a full recovery and what would be the effect of the sole re-expression of CaBP1 in the double KO.

Reviewer #3 (Public Review):

Summary:

The authors attempted to unravel the role of the Ca2+-binding proteins CaBP1 and CaBP2 for the hitherto enigmatic lack of Ca2+-dependent inactivation of Ca2+ currents in sensory inner hair cells (IHCs). As Ca2+ currents through Cav1.3 channels are crucial for exocytosis, the lack of inactivation of those Ca2+ currents is essential for the indefatigable sound encoding by IHCs. Using a deaf mouse model lacking both CaBP1 and CaBP2, the authors convincingly demonstrate that both CaBP1 and CaBP2 together confer a lack of inactivation, with CaBP2 being far more effective. This is surprising given the mild phenotype of the single knockouts, which has been published by the authors before. Re-admission of CaBP2 through viral gene transfer into the inner ear of double-knockout mice largely restored hearing function, normal Ca2+ current properties, and exocytosis.

Comments on the revised version:

The authors improved the quality of the figures as requested.

Reviewer #1 (Public Review):

Summary:

In this work, Wang and colleagues used Drosophila-Serratia as a host-microbe model to investigate the impact of the host on gut bacteria. The authors showed that Drosophila larvae reduce S. marcescens abundance in the food likely due to a combination of mechanical force and secretion of antimicrobial peptides. S. marcescens exposed to Drosophila larvae lost virulence to flies and could promote larval growth similar to typical Drosophila gut commensals. These phenotypic changes were reflected in the transcriptome and metabolome of bacteria, suggesting that the host could drive the switch from pathogenicity to commensalism in bacteria. Further, the authors used single-cell bacterial RNA-seq to demonstrate the heterogeneity in gut bacterial populations.

Strengths:

This is a valuable work that addresses an important question of the impact of the host on its gut microbes. The authors could convincingly demonstrate that gut bacteria are strongly affected by the host with important consequences for both interacting partners. Moreover, the authors used state-of-the-art bacterial single-cell RNA-seq to reveal heterogeneity in host-associated commensal populations.

Overall most parts of the study are solid and clear.

Reviewer #3 (Public Review):

In this study, Wang and coworkers established a model of Drosophila-S. marcescens interactions and thoroughly examined host-microbe bidirectional interactions. They found that:

(1) Drosophila larvae directly impact microbial aggregation and density;<br /> (2) Drosophila larvae affect microbial metabolism and cell wall morphology, as evidenced by reduced prodigiosin production and EPS production, respectively;<br /> (3) Drosophila larvae attenuate microbial virulence;<br /> (4) Drosophila larvae modulate the global transcription of microbes for adaptation to the host;<br /> (5) Microbial single-cell RNA sequencing (scRNA-seq) analysis revealed heterogeneity in microbial pathogenicity and growth;<br /> (6) AMPs are key factors controlling microbial virulence phenotypes.

Taken together, they concluded that host immune factors such as AMPs are directly involved in the pathogen-to-commensal transition by altering microbial transcription.

In general, in this revised version, I feel that the authors addressed all the points raised in the previous review process. Specifically, they demonstrated that sub-lethal doses of antibiotics such as kanamycin or ampicillin is sufficient to induce the virulence switch in S. marcescens. Furthermore, by testing IMD pathway mutant animals, they concluded that AMP plays a major role in the commensal-to-pathogen transition. In summary, I appreciate the authors' efforts, and I am satisfied with the revision.

Reviewer #1 (Public Review):

This study addresses the temporal patterning of a specific Drosophila CNS neuroblast lineage, focusing on its larval development. They find that a temporal cascade, involving the Imp and Syb genes changes the fate of one daughter cell/branch, from glioblast (GB) to programmed cell death (PCD), as well as gates the decommissioning of the NB at the end of neurogenesis.

Reviewer #2 (Public Review):

Guan and colleagues address the question of how a single neuroblast produces a defined number of progeny, and what influences its decommissioning. The focus of the experiments are two well-studied RNA-binding proteins: Imp and Syp. The Authors find that these factors play an important role in determining the number of neurons in their preferred model system of VNC motor neurons coming from a single lineage (LinA/15) by separate functions taking place at specific stages of development of this lineage: influencing the life-span of the LinA neuroblast to control its timely decommissioning and functioning in the Late-born post-mitotic neurons to influence cell death after the appropriate number of progeny is generated. The post-mitotic role of Imp/Syp in regulating programmed-cell death (PCD) is also correlated with a specific code of key transcription factors that are suspected to influence neuronal identity, linking the fate of neuronal survival with its specification. This paper addresses a wide scope of phenotypes related to the same factors, thus providing an intriguing demonstration of how the nervous system is constructed by context-specific changes in key developmental regulators. The bulk of conclusions drawn by the authors are supported by careful experimental evidence, and the findings are a useful addition to an important topic in developmental neuroscience.

Reviewer #3 (Public Review):

This study by Guan and co-workers focuses on a model neuronal lineage in the developing Drosophila nervous system, revealing interesting aspects about: a) the generation of supernumerary cells, later destined for apoptosis; and, b) new insights into the mechanisms that regulate this process. The two RNA-binding proteins, Imp and Syp, are shown to be expressed in temporally largely complementary patterns, their expression defining early vs later born neurons in this lineage, and thus also regulating the apoptotic elimination. Moreover, neuronal 'fate' transcription factors that are downstream of Imp and signatures of early-born neurons, can also be sufficient to convert later born cells to an earlier 'fate', including survival. The authors provide solid evidence for most of their statements, including the temporal windows during which the early and the later-born motoneurons are generated by this model lineage, how this relates to patterns of cell death by apoptosis and that mis-expression of early-born transcription factors in later-born cells can be sufficient to block apoptosis (part of, and perhaps indicative of the late-born identity). Other studies have previously outlined analogous, mutually antagonistic roles for Imp and Syp during nervous system development in Drosophila, in different parts and at different stages, with which the working model of this study aligns. Overall, this study adds to and extends current working models and evidence on the developmental mechanisms that underlie temporal cell fate decisions.

Reviewer #1 (Public Review):

This study introduces an innovative method for assessing the mean kurtosis, utilizing the mathematical foundation of the sub-diffusion framework. In particular, a new fitting technique that incorporates two different diffusion times is proposed to estimate the parameters of the sub-diffusion model. The evaluation of this technique, which generates kurtosis maps based on the sub-diffusion framework, is conducted through simulations and the examination of data obtained from human subjects.

The authors have revised the manuscript to address the initial critiques. However, there appears to be some confusion regarding the following responses.

"The comment "... using the new sub-diffusion model -an approximation of the DKI-based signal expression..." is a bit misleading. In fact we propose that the reverse interpretation is the more suitable way to view the relationship: the DKI model is a degree-2 approximation of the sub-diffusion model, as in eq. (7).<br /> We appreciate the suggestion. However, unfortunately, it is not appropriate to generate data with the DKI model, as the maximum b-value is limited to 2000~3000s/mm^2 and hence the DKI model cannot represent diffusion MRI signals from a full spectrum of b-values. A key strength of our proposed model is that it removes this limitation. "

The main motivation of this study is to investigate the feasibility of the sub-diffusion model, which was proposed in Yang et al., NeuroImage 2022, to provide fast and robust estimation of kurtosis model parameters. I understand that mathematically, the DKI model can be written as a degree two approximation of the sub-diffusion model. However, the hypothesis is that the proposed sub-diffusion model can be used to obtain practically useful mapping of mean kurtosis. Therefore, unless the authors use a different parameter or phenomenon as the "true" or "ground-truth kurtosis," this study examines whether the sub-diffusion model parameters can serve as an approximation to the conventional DKI parameters.

With the current simulation study design, 1) the data is generated by the proposed sub-diffusion model, 2) the "ground-truth" or "true" D* and K* are computed based on the proposed equality (Eq.7); 3) and then the data is fit with the conventional DKI model and also with the proposed sub-diffusion model. Since the data is generated by the proposed model, and the ground truth (or true) values calculated by the proposed equality, as expected, the fitted kurtosis values by the sub-diffusion model match better with the simulated ones compared to the conventional DKI model.

Furthermore, as the authors noted, the sub-diffusion model eliminates the restriction on b-value selection, allowing for DWI data acquisition with higher b-values. However, it is unclear how the new K* and D* values, calculated directly from the sub-diffusion model using a higher b-value DWI protocol, are superior to the K and D values from the conventional DKI model, which uses a DWI protocol limited to b-values of 2000-3000 s/mm². In clinical practice, b-values of 2000-3000 s/mm² are generally considered "high b-value."

Reviewer #2 (Public Review):

Summary:

The authors present an interesting technique for analysis of diffusion magnetic resonance images (dMRI) using a sub-diffusion model of the diffusion process. They show that the results of their technique when fitted to dMRI with two diffusion times provide robust diffusion coefficient and kurtosis measures.

Strengths:

The measures provided by the sub-diffusion technique are robust and can be reliably estimated from short dMRI data acquisitions. This is potentially useful in application to clinical studies.

Weaknesses:

The authors do not fully demonstrate that their D* and K* measures are not affected by diffusion time. Potential limitations of the technique are not considered.

This reviewer suggests that the paper would benefit from considerations of the limitations of the applied techniques. This would include consideration of:<br /> (i) The use of the sub-diffusion model in the simulation studies - there are circular arguments that should be considered.<br /> (ii) The time dependence of D* and K*. This is because the human data provided in Tables 3 and 4 (for Δ=19ms and Δ=49ms) seem to show that<br /> D* and K* are time dependent.

With respect to the second point this reviewer acknowledges the authors' argument that when the fitting is performed over the higher dimensional space that includes multiple diffusion times then this leads to a more robust estimation of sub-diffusion measures. However, the authors only include two diffusion times in their in-vivo human analysis (Δ=19ms and Δ=49ms) so it is not possible for them to show here that different pairs of diffusion times lead to invariant D* and K* values. This is a limitation of the study as the authors show there is time dependence of D* and K* in tables 3 and 4 (when the model is fitted to single diffusion times). Potentially the larger apparent time dependence of K* in white matter compared to grey matter (tables 3 and 4) could lead to the tissue specific differences in root mean squared error shown in Figure 7.

This reviewer requests that the authors discuss their results more clearly with respect to these potential limitations and include some discussion of their single (and multiple) diffusion time results (for D_SUB and K*) in comparison with the time dependent DKI literature.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript entitled A Modified BPaL Regimen for Tuberculosis Treatment<br /> replaces Linezolid with Inhaled Spectinamides by Malik Zohaib Ali et al. is an extension of previous studies by this group looking at the new drug spectinamide 1599. The authors directly compare therapy with BPaL (bedaquiline, pretomanid, linezolid) to a therapy that substitutes spectinamide for linezolid (BPaS). The Spectinamide is given by aerosol exposure and the BPaS therapy is shown to be as effective as BPaL without adverse effects. The work is rigorously performed and analyses of the immune responses are consistent with curative therapy.

Strengths:<br /> 1) This group uses 2 different mouse models to show the effectiveness of the BPaS treatment.<br /> 2)Impressively the group demonstrates immunological correlates associated with Mtb cure with the BPaS therapy.<br /> 3)Linezolid is known to inhibit ribsomes and mitochondria whereas spectinaminde does not. The authors clearly demonstrate the lack of adverse effects of BPaS compared to BPaL.

Weaknesses:<br /> 1) Although this is not a weakness of this paper, a sentence describing how the spectinamide would be administered by aerosolization in humans would be welcomed.

Reviewer #2 (Public Review):

Summary:<br /> Replacing linezolid (L) with the preclinical development candidate spectinamide 1599, administered by inhalation, in the BPaL standard of care regimen achieves similar efficacy, reduces hematological changes and por-inflammatory responses.

Strengths:<br /> The authors not only measure efficacy but also quantify histological changes, hematological responses and immune responses, to provide a comprehensive picture of treatment response and the benefits of the L to S substitution.

The authors generate all data in two mouse models of TB infection, each reproducing different aspects of human histopathology.

Extensive supplementary figures ensure transparency.

Weaknesses:<br /> Articulation of objectives and hypotheses can be improved, as suggested below.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, the authors sought to evaluate whether the novel TB drug candidate, spectinamide 1599 (S), given via inhalation to mouse TB models, and combined with the drugs B (bedaquiline) and Pa (pretomanid), would demonstrate similar efficacy to that of BPaL regimen (where L is linezolid). Because L is associated with adverse events when given to patients longterm, and one of those is associated with myelosuppression (bone marrow toxicity) the authors also sought to assess blood parameters, effects on bone marrow, immune parameters/cell effects following treatment of mice with BPaS and BPaL. They conclude that BPaL and BPaS have equivalent efficacy in both TB models used and that BPaL resulted in weight loss and anemia (whereas BPaS did not) under the conditions tested, as well as effects on bone marrow.

Strengths:<br /> The authors used two mouse models of TB that are representative of different aspects of TB in patients (which they describe well), intending to present a fuller picture of the activity of the tested drug combinations. They conducted a large body of work in these infected mice to evaluate efficacy and also to survey a wide range of parameters that could inform the effect of the treatments on bone marrow and on the immune system. The inclusion of BPa controls (in most studies) and also untreated groups led to a large amount of useful data that has been collected for the mouse models per se (untreated) as well as for BPa - in addition to the BPaS and BPaL combinations which are of particular interest to the authors. Many of these findings related to BPa, BPaL, untreated groups etc corroborate earlier findings and the authors point this out effectively and clearly in their manuscript. To go further, in general, it is a well written and cited article with an informative introduction.

Weaknesses:<br /> The authors performed a large amount of work with the drugs given at the doses and dosing intervals stated, but there is no exposure data available at this time. The authors intend to evaluate exposure-effect relationships in future work. An understanding of the exposures at which the efficacy and adverse effects are seen will assist in the translation of these findings to the clinic.<br /> In addition, it is always challenging to interpret findings for combinations of drugs and for now, the data available cannot attribute confidence to the weight loss seen for only the BPaL group to L specifically, as opposed to a PK interaction leading to an elevated exposure and weight loss due to B or Pa. It is not yet possible, then to state that what is seen are "L-associated AEs" - this is assumed only.<br /> The evaluations of activity in the BALB/c mouse model as well as the spleens of the Kramnik model resulted in CFU below/at the limit of detection so comparisons between BPaL and BPaS cannot be made and so the conclusion of equivalent efficacy in BALB/c is not supported with the data shown. There is no BPa control in the BALB/c study, therefore it is not possible to discern whether L or S contributed to the activity of BPaL or BPaS. The same is true for the assessment of lesions - unfortunately, there was no BPa control meaning that even where equivalency is seen for BPaL and BPaS, the reader is unable to deduce whether L or S made a contribution to this activity.<br /> Although these weaknesses limit what we can learn from the current body of data, the authors note that further studies will be done to increase understanding of the points above.

Reviewer #3 (Public Review):

Summary:

ImmCellTyper is a new toolkit for Cytometry by time-of-flight data analysis. It includes BinaryClust, a semi-supervised clustering tool (which takes into account the prior biological knowledge), designed for automated classification and annotation of specific cell types and subpopulations. ImmCellTyper also integrates a variety of tools to perform data quality analysis, batch effect correction, dimension reduction, unsupervised clustering, and differential analysis.

Strengths:

The proposed algorithm takes into account the prior knowledge.<br /> The results on different benchmark indicates competitive or better performance (in terms of accuracy and speed) depending on the method.

Reviewer #1 (Public Review):

Summary:

Insects inhabit diverse environments and have neuroanatomical structures appropriate to each habitat. Although the molecular mechanism of insect neural development has been mainly studied in Drosophila, the beetle, Tribolium castaneum has been introduced as another model to understand the differences and similarities in the process of insect neural development. In this manuscript, the authors focused on the origin of the central complex. In Drosophila, type II neuroblasts have been known as the origin of the central complex. Then, the authors tried to identify those cells in the beetle brain. They established a Tribolium fez enhancer trap line to visualize putative type II neuroblasts and successfully identified 9 of those cells. In addition, they also examined expression patterns of several genes that are known to be expressed in the type II neuroblasts or their lineage in Drosophila. They concluded that the putative type II neuroblasts they identified were type II neuroblasts because those cells showed characteristics of type II neuroblasts in terms of genetic codes, cell diameter, and cell lineage.

Strengths:

The authors established a useful enhancer trap line to visualize type II neuroblasts in Tribolium embryos. Using this tool, they have identified that there are 9 type II neuroblasts in the brain hemisphere during embryonic development. Since the enhancer trap line also visualized the lineage of those cells, the authors found that the lineage size of the type II neuroblasts in the beetle is larger than that in the fly. They also showed that several genetic markers are also expressed in the type II neuroblasts and their lineages as observed in Drosophila.

Weaknesses:

I recommend the authors reconstruct the manuscript because several parts of the present version are not logical. For example, the author should first examine the expression of dpn, a well-known marker of neuroblast. Without examining the expression of at least one neuroblast marker, no one can say confidently that it is a neuroblast. The purpose of this study is to understand what makes neuroanatomical differences between insects which is appropriate to their habitats. To obtain clues to the question, I think, functional analyses are necessary as well as descriptive analyses.

Reviewer #2 (Public Review):

The authors address the question of differences in the development of the central complex (Cx), a brain structure mainly controlling spatial orientation and locomotion in insects, which can be traced back to the neuroblast lineages that produce the Cx structure. The lineages are called type-II neuroblast (NB) lineages and are assumed to be conserved in insects. While Tribolium castaneum produces a functional larval Cx that only consists of one part of the adult Cx structure, the fan-shaped body, in Drosophila melanogaster a non-functional neuropile primordium is formed by neurons produced by the embryonic type-II NBs which then enter a dormant state and continue development in late larval and pupal stages.

The authors present a meticulous study demonstrating that type-II neuroblast (NB) lineages are indeed present in the developing brain of Tribolium castaneum. In contrast to type-I NB lineages, type-II NBs produce additional intermediate progenitors. The authors generate a fluorescent enhancer trap line called fez/earmuff which prominently labels the mushroom bodies but also the intermediate progenitors (INPs) of the type-II NB lineages. This is convincingly demonstrated by high-resolution images that show cellular staining next to large pointed labelled cells, a marker for type-II NBs in Drosophila melanogaster. Using these and other markers (e.g. deadpan, asense), the authors show that the cell type composition and embryonic development of the type-II NB lineages are similar to their counterparts in Drosophila melanogaster. Furthermore, the expression of the Drosophila type-II NB lineage markers six3 and six4 in subsets of the Tribolium type-II NB lineages (anterior 1-4 and 1-6 type-II NB lineages) and the expression of the Cx marker skh in the distal part of most of the lineages provide further evidence that the identified NB lineages are equivalent to the Drosophila lineages that establish the central complex. However, in contrast to Drosophila, there are 9 instead of 8 embryonic type-II NB lineages per brain hemisphere and the lineages contain more progenitor cells compared to the Drosophila lineages. The authors argue that the higher number of dividing progenitor cells supports the earlier development of a functional Cx in Tribolium.

While the manuscript clearly shows that type-II NB lineages similar to Drosophila exist in Tribolium, it does not considerably advance our understanding of the heterochronic development of the Cx in these insects. First of all, the contribution of these lineages to a functional larval Cx is not clear. For example, how do the described type-II NB lineages relate to the DM1-4 lineages that produce the columnar neurons of the Cx? What is the evidence that the embryonically produced type-II NB lineage neurons contribute to a functional larval Cx? The formation of functional circuits could rely on larval neurons (like in Drosophila) which would make a comparison of embryonic lineages less informative with respect to understanding the underlying variations of the developmental processes. Furthermore, the higher number of progenitors (and consequently neurons) in Tribolium could simply reflect the demand for a higher number of cells required to build the fan-shaped body compared to Drosophila. In addition, the larger lineages in Tribolium, including the higher number of INPs could be due to a greater number of NBs within the individual clusters, rather than a higher rate of proliferation of individual neuroblasts, as suggested. What is the evidence that there is only one NB per cluster? The presented schemes (Fig. 7/12) and description of the marker gene expression and classification of progenitor cells are inconsistent but indicate that NBs and immature INPs cannot be consistently distinguished.

The main difference between Tribolium and Drosophila Cx development with regard to the larval functionality might be that Drosophila type-II NB lineage-derived neurons undergo quiescence at the end of embryogenesis so that the development of the Cx is halted, while a developmental arrest does not occur in Tribolium. However, this needs to be confirmed (as the authors rightly observe).

Reviewer #3 (Public Review):

Summary:

In this paper, Rethemeier et al capitalize on their previous observation that the beetle central complex develops heterochronically compared to the fly and try to identify the developmental origin of this difference. For this reason, they use a fez enhancer trap line that they generated to study the neuronal stem cells (INPs) that give rise to the central complex. Using this line and staining against Drosophila type-II neuroblast markers, they elegantly dissect the number of developmental progression of the beetle type II neuroblasts. They show that the NBs, INPs, and GMCs have a conserved marker progression by comparing to Drosophila marker genes, although the expression of some of the lineage markers (otd, six3, and six4) is slightly different. Finally, they show that the beetle type II neuroblast lineages are likely longer than the equivalent ones in Drosophila and argue that this might be the underlying reason for the observed heterochrony.

Strengths:

- A very interesting study system that compares a conserved structure that, however, develops in a heterochronic manner.

- Identification of a conserved molecular signature of type-II neuroblasts between beetles and flies. At the same time, identification of transcription factors expression differences in the neuroblasts, as well as identification of an extra neuroblast.

- Nice detailed experiments to describe the expression of conserved and divergent marker genes, including some lineaging looking into the co-expression of progenitor (fez) and neuronal (skh) markers.

Weaknesses:

- Comparing between different species is difficult as one doesn't know what the equivalent developmental stages are. How do the authors know when to compare the sizes of the lineages between Drosophila and Tribolium? Moreover, the fact that the authors recover more INPs and GMCs could also mean that the progenitors divide more slowly and, therefore, there is an accumulation of progenitors who have not undergone their programmed number of divisions.

- The main conclusion that the earlier central complex development in beetles is due to the enhanced activity of the neuroblasts is very handwavy and is not the only possible conclusion from their data.

- The argument for conserved patterns of gene expression between Tribolium and Drosophila type-II NBs, INPs, and GMCs is a bit circular, as the authors use Drosophila markers to identify the Tribolium cells.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions: Based on the above, I believe that the authors, despite advancing significantly, fall short of identifying the reasons for the divergent timing of central complex development between beetle and fly.

Reviewer #1 (Public Review):

Summary:

Hahn et al use bystander BRET, NanoBiT assays, and APEX2 proteomics to investigate endosomal signaling of CCR7 by two agonists, CCL19 and CCL21. The authors suggest that CCR7 signals from early endosomes following internalisation. They use spatial proteomics to try to identify novel interacting partners that may facilitate this signaling and use this data to specifically enhance a Rac1 signaling pathway. Many of the results in the first few figures showing simultaneous recruitment of Barr and G proteins by CCR7 have been shown previously (Laufer et al, 2019, Cell Reports), as has signaling from endomembranes, and Rac1 activation at intracellular sites. The new findings are the APEX2 proteomics studies, which could be useful to the scientific community. Unfortunately, the authors only follow up on a single finding, and the expansion of this section would improve the manuscript.

Strengths:

(1) The APEX2 resource will be valuable to the GPCR and immunology community. It offers many opportunities to follow up on findings and discover new biology. The resource could also be used to validate earlier findings in the current manuscript and in previous manuscripts. Was there enrichment of early endosomal markers, Barr and Gi as this would provide further evidence for their earlier claims regarding endosomal signaling? Previous studies have suggested signaling from the TGN, so it is possible that the different ligands also direct to different sites. This could easily be investigated using the APEX2 data.

(2) The results section is well written and can be followed very easily by the reader.

(3) Some findings verify previous studies (e.g. endomembrane signalling). This should be acknowledged as this shows the validity of the findings of both studies.

Weaknesses:

(1) The findings are interesting although the studies are almost all performed in HEK293 cells. I understand that these are commonly used in GPCR biology and are easy to transfect and don't express many GPCRs at high concentrations, but their use is still odd when there are many cell-lines available that express CCR7 and are more reflective of the endogenous state (e.g. they are polarised, they can perform chemotaxis/ migration). Some of the findings within the study should also be verified in more physiologically relevant cells. At the moment only the final figure looks at this, but findings need to be verified elsewhere.

(2) The authors acknowledge that the kinetic patterns of the signals at the early endosome are not consistent with the rates of internalisation. They mention that this could be due to trafficking elsewhere. This could be easily looked at in their APEX2 data. Is there evidence of proximity to markers of other membranes? Perhaps this could be added to the discussion. Similarly, previous studies have shown that CCR7 signaling may involve the TGN. Was there enrichment of these markers? If not, this could also be an interesting finding and should be discussed. It is also possible that the Rab5 reporter is just not as efficient as the trafficking one, especially as in later figures the very convincing differences in the two ligands are not as robust as the differences in trafficking.

(3) In the final sentence of paragraph 2 of the results the authors state that the internalisation is specific to CCR7 as there isn't recruitment to V2R. I'm not sure this is the best control. The authors can only really say it doesn't recruit to unrelated receptors. The authors could have used a different chemokine receptor which does not respond to these ligands to show this.

(4) The miniGi-Barr1 and imaging showing co-localisation could be more convincing if it was also repeated in a more physiological cell line as in the final figure. Imaging of CCR7, miniGi, and Barr1 would also provide further evidence that the receptor is also present within the complex.

(5) The findings regarding Rac1 are interesting, although an earlier paper found similar results (Laufer et al, 2019, Cell Reports), so perhaps following up on another APEX2-identified protein pathway would have been more interesting. The authors' statement that Rac1 is specifically activated, and RhoA and Cdc42 are not, is unconvincing from the current data. Only a single NanoBiT assay was used, and as raw values are not reported it is difficult for the reader to glean some essential information. The authors should show evidence that these reporters work well for other receptors (or cite previous studies) and also need evidence from an independent (i.e. non-NanoBiT or BRET) assay.

(6) At present, the studies in Figure 7 do not go beyond those in the previous Laufer et al study in which they showed blocking endocytosis affected Rac1 signalling. The authors could show that Rac1 signalling is from early endosomes to improve this, otherwise, it could be from the TGN as previously reported.

Reviewer #2 (Public Review):

Summary:

This manuscript describes a comprehensive analysis of signalling downstream of the chemokine receptor CCR7. A comprehensive dataset supports the authors' hypothesis that G protein and beta-arrestin signalling can occur simultaneously at CCR7 with implications for continued signalling following receptor endocytosis.

Strengths:

The experiments are well controlled and executed, employing a wide range of assays using - in the main - CCR7 transfectants. Data are well presented, with the authors' claims supported by the data. The paper also has an excellent narrative which makes it relatively easy to follow. I think this would certainly be of interest to the readership of the journal.

Weaknesses:

Since the authors show a differential enrichment of RhoGTPases by CCR7 stimulation with CCL19 versus CCL21, I think that they also need to show that the Gi/o coupling of HEK-292-CCR7-APEX2 cells to both CCL19 and CCL21 is not perturbed by the modification. Currently, the authors only show data for CCL19 signalling, which leaves the potential for a false negative finding in terms of CCL21 signalling being selectively impaired. This should be relatively easy to do and should strengthen the authors' conclusions.

The authors conclude the discussion by suggesting that their findings highlight endosomal signalling as a general mechanism for chemokine receptors in cell migration. I think this is an overreach. The authors chose several studies of CXC chemokine receptors to support their argument that C-terminal truncation or mutation of the C-terminal phosphorylation sites impairs endocytosis and chemotaxis (refs 40-42). However, in some instances e.g. at the related chemokine receptor CCR4, C-terminal removal of these sites impairs endocytosis but promotes chemotaxis (Nakagawa et al, 2014); Anderson et al, 2020). I therefore think that either the final statement needs to be tempered down or the counterargument discussed a little.

References:

Anderson, C. A. et al. A degradatory fate for CCR4 suggests a primary role in Th2 inflammation. J Leukocyte Biol 107, 455-466 (2020).

Nakagawa, M. et al. Gain-of-function CCR4 mutations in adult T cell leukaemia/lymphoma. Journal of Experimental Medicine 211, 2497-2505 (2014).

Reviewer #1 (Public Review):

Summary:

In this study, Nandy and colleagues examine neural, physiological and behavioral correlates of perceptual variability in monkeys performing a visual change detection task. They used a laminar probe to record from area V4 while two macaque monkeys detected a small change in stimulus orientation that occurred at a random time in one of two locations, focusing their analysis on stimulus conditions where the animal was equally likely to detect (hit) or not-detect (miss) a briefly presented orientation change (target). They discovered two behavioral and physiological measures that are significantly different between hit and miss trials - pupil size tends to be slightly larger on hits vs. misses, and monkeys are more likely to miss the target on trials in which they made a microsaccade shortly before target onset. They also examined multiple measures of neural activity across the cortical layers and found some measures that are significantly different between hits and misses.

Strengths:

Overall the study is well executed and the analyses are appropriate (though several issues still need to be addressed as discussed in Specific Comments).

Weaknesses:

My main concern with this study is that, with the exception of the pre-target microsaccades, the correlates of perceptual variability (differences between hits and misses) appear to be weak, potentially unreliable and disconnected. The GLM analysis of predictive power of trial outcome based on the behavioral and neural measures is only discussed at the end of the paper. This analysis shows that some of the measures have no significant predictive power, while others cannot be examined using the GLM analysis because these measures cannot be estimated in single trials. Given these weak and disconnected effects, my overall sense is that the current results provide limited advance to our understanding of the neural basis of perceptual variability.

Reviewer #2 (Public Review):

Strengths:

The experiments were well-designed and executed with meticulous control. The analyses of both behavioural and electrophysiological data align with the standards in the field.

Weaknesses:

Many of the findings appear to be subtle differences and incremental compared to previous literature, including the authors' own work. While incremental findings are not necessarily a problem, the manuscript lacks clear statements about the extent to which the dataset, analysis, and findings overlap with the authors' prior research. For example, one of the main findings, which suggests that V4 neurons exhibit larger visual responses in hit trials (as shown in Fig. 3), appears to have been previously reported in their 2017 paper.

Furthermore, the manuscript does not explore potentially interesting aspects of the dataset. For instance, the authors could have investigated instances where monkeys made 'false' reports, such as executing saccades towards visual stimuli when no orientation change occurred, which allows for a broader analysis that considers the perceptual component of neural activity over pure sensory responses. Overall, lacking broad interest with the current form.

Reviewer #1 (Public Review):

The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further.

After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phase relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirp that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause a doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.

Finally, the reviewer is concerned about this sentence in the rebuttal - "The methods section has been edited to clarify the approach (not yet)". This section is unfinished, which suggests that it is difficult to explain the modeling results from a logical point of view. Thus the reviewer's major concern from the previous review remains unresolved. To summarize, the model calculates field strengths at an instant in time and integrates over time with a 500 ms window. This window is 10 times longer than the small chirps, while the longer chirps cover a much larger proportion of the window. Yet, the small chirps have a bigger impact on discriminability than the longer chirps. The authors should attempt to explain this seemingly contradictory result. This remains a major issue because this analysis was the most direct evidence that chirping could impact localization accuracy.

Reviewer #2 (Public Review):

Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing wave-like electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that should have a great impact on the field.

The authors provide convincing evidence that chirps may function in homeoactive sensing. In particular, the evidence showing increased chirping in more cluttered environments and a relationship between chirping and movement are especially strong and suggestive. Their evidence arguing against a role for chirps in communication is not as strong. However, based on an extensive review of the literature, the authors conclude, I think fairly, that the evidence arguing in favor of a communication function is limited and inconclusive. Thus, the real strength of this study is not that it conclusively refutes the communication hypothesis, but that it calls this hypothesis into question while also providing compelling evidence in favor of an alternative function.

In summary, although the evidence against a role for chirps in communication is not as strong as the evidence for a role in active sensing, this study presents very interesting data that is sure to stimulate discussion and follow-up studies. The authors acknowledge that chirps could function as both a communication and homeactive sensing signal, and the language arguing against a communication function is appropriately measured. A given electrical behavior could serve both communication and homeoactive sensing. I suspect this is quite common in electric fish (not just in gymnotiforms such as the species studied here, but also in the distantly related mormyrids), and perhaps in other actively sensing species such as echolocating animals.

Reviewer #3 (Public Review):

Summary:<br /> This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish as well as objects.

Strengths:<br /> The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry. The BEM modelling also convincingly predicts how the electric image of a receiver conspecific on a sending fish is enhanced by a chirp.

The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference in frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction. The authors carefully submit that the new putative echolocation function of chirps is not mutually exclusive with a possible communication function.

These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

Reviewer #1 (Public Review):

The present study provides a phylogenetic analysis of the size prefrontal areas in primates, aiming to investigate whether relative size of the rostral prefrontal cortex (frontal pole) and dorsolateral prefrontal cortex volume vary according to known ecological or social variables.

I am very much in favor of the general approach taken in this study. Neuroimaging now allows us to obtain more detailed anatomical data in a much larger range of species than ever before and this study shows the questions that can be asked using these types of data. In general, the study is conducted with care, focusing on anatomical precision in definition of the cortical areas and using appropriate statistical techniques, such as PGLS.

I have read the revised version of the manuscript with interest. I commend the authors for including the requested additional analyses. I believe these highlight some of the major debates in the field, such as the relationship between absolute and relative brain size of areas. Providing a full description of the data will help this field be more open about these issues. All too often, debates between different groups focus on narrow anatomical or statistical arguments, and having all the data here is important.

I do not agree with some of the statements of the other reviewers regarding development. Clearly, evolution works for a large part by tinkering (forgive the sense of agency) with development, but that does not mean that looking at the end result cannot provide insights. Ultimately, we will look at both phylogeny and ontogeny within the same framework, but the field is not quite there yet.

As I said before, I do believe this is a positive study. I am happy that we as a field are using imaging data to answer more wider phylogenetic questions. Combining detailed anatomy, big data, and phylogenetic statistical frameworks is an important approach.

Reviewer #1 (Public Review):

Summary:

This study further develops the potential of in vitro granulomas to study host-pathogen interactions in tuberculosis. It uses a human-based cellular model and a collection of M. tuberculosis isolates representative of the pathogen's diversity. It provides important methodologic information and some findings that help in defining protective responses in TB.

Strengths:

A strength of the study is the multitude of parameters addressed across different M. tuberculosis strains and donors. The inclusion of several strains of the same lineage shows that intra-lineage diversity is also relevant, illustrating how complex it is to model the immune response to M. tuberculosis.

Weaknesses:

A weakness of the study is that although several interesting findings are reported and a hypothesis proposed, the work is mainly descriptive and correlative. Some functional data based on the current observations would strengthen the findings.

Reviewer #2 (Public Review):

Summary:

This manuscript reports a comparison of microbial traits and host response traits in a laboratory model of infected granuloma using Mtb strains from different lineages. The authors report increased bacillary growth and granuloma formation, inversely associated with T cell activation that is characterized by CXCL9, granzyme B, and TNF expression. They therefore infer that these T cell responses are likely to be host-protective and that the greater virulence of modern Mtb lineages may be driven by their ability to avoid triggering these responses.

Strengths:

The comparison of multiple Mtb lineages in a granuloma model that enables evaluation of the potential role of multiple host cells in Mtb control offers a valuable experimental approach to studying the biological mechanisms that underpin differential virulence of Mtb lineages that have been previously reported in clinical and epidemiological studies.

Weaknesses:

The study is rather limited to descriptive observations and lacks experiments to test causal relationships between host and pathogen traits. Some of the presentation of the data is difficult to interpret, and some conclusions are not adequately supported by the data.

Reviewer #3 (Public Review):

Summary:

In "CXCL9, granzyme B, and TNF-α orchestrate protective in vitro granulomatous responses across Mycobacterium tuberculosis complex lineages", Arbués and colleagues describe the impact of mycobacterial genetic diversity on host-infection phenotypes. The authors evaluate Mtb infection and contextualize host responses, bacterial growth, and metabolic transitioning in vitro using their previously established model of blood-derived, primary human cells cultured on a collogen/fibronectin matrix. They seek to demonstrate the effectiveness of the model in determining mycobacterial strain-specific granuloma-dependent host-pathogen interactions.

Strengths and weaknesses:

Understanding the way mycobacterial genetic diversity impacts granuloma biology in tuberculosis is an important goal. One of this work's strengths is the use of primary human cells and two constituents of the pulmonary extracellular matrix to model Mtb infection. The authors and others have previously shown that Mtb-infected PBMC aggregates share important characteristics with early pulmonary TB granulomas (Arbues et al., Bio Protoc, 2020, PMID: 3659472; Guirado et al., mBio, 2015, PMID: 25691598; Kapoor et al., PloS One, 2013, PMID: 23308269). The use of multiple genetically distinct strains of Mtb defines this work and further bolsters its potential impact. However, the study is not comprehensive as lineages 6 and 7 are not tested. Experiments are primarily descriptive, and the methodologies are conventional. Correlative relationships are the manuscript's focus and functional validation is not conducted. Convoluted data presentation hampers the readers' ability to effectively evaluate many of the findings for significance. The effect sizes are generally small and most quantitative data are unitless. A further weakness of the study is a lack of any in vivo modeling.

Achievement of aims, and support for conclusions:

The main aim of this work is to extend an in vitro granuloma model to the study of a large collection of well-characterized, genetically diverse representatives of the mycobacterium tuberculosis complex (MTBC). I believe that they accomplish that aim. The work does investigate MTBC infection of aggregated PBMCs using three strains each of Mtb lineages 1-5 and H37Rv, which is not a trivial undertaking. The experimental aims are to show that MTBC genetic diversity impacts the growth and dormancy of granuloma-bound bacteria and, the host responses of granulomatous aggregation as well as macrophage apoptosis, lymphocyte activation, and soluble mediator release within granulomas. A lack of basic descriptive statistics for raw data makes it difficult to determine if benchmarks for most of the experimental aims have been reached. Although the methodologies employed should have been able to test most of these aims. The title's conclusion that CXCL9, granzyme B, and TNF orchestrate a protective granulomatous response is not tested and is not supported by the findings. Those molecules are not a focus of the work, their effects are not investigated effectively and their relationship to the granulomatous response is not determined. The authors' conclusions regarding their results are a mixed bag. Their conclusion that lineage impacts growth within granulomas is likely true and the data as presented reflect such a relationship. However, even without the basic descriptive statistics needed to evaluate the data supporting that claim, the methods employed for bacterial collection call into question whether all Mtb plated for CFU assay resided within granulomatous aggregates. Their conclusions regarding lineage's impact on dormancy are not supported, as their findings demonstrate that assays for dormancy identify replicating bacteria as being dormant. Their conclusion that strain diversity results in a spectrum of granulomatous responses in their model system is strongly supported by the results. Their conclusion that strain diversity impacts macrophage apoptosis is supported by the data but a relationship of apoptosis to the granulomatous response is not effectively evaluated. Their conclusion that lymphocyte activation is associated with reduced mycobacterial growth as an aspect of granulomas is well supported in the literature and a negative correlation between T cell activation and growth is supported by their results.

Impact on the field:

The authors contribute some valuable insights, particularly in Figure 3 and supplementary Figures 1 and 2, where data is more accessible to critique. Their identification of donor-dependent aggregation phenotypes by mycobacterial strain has the potential to enable future reverse-genetic screens for human and Mtb loci that contribute to granulomatous inflammation. Their model is a higher echelon relative to others in the field, but I don't believe that it possesses all of the necessary tissue and cellular components to effectively replicate the formation of granulomas in nature. The bulk of the data in its current form is not of high value to the community, but I think it has the potential to contribute additional novel insights if panels that display descriptive statistics are added to the figures.

Reviewer #1 (Public Review):

Summary:

The manuscript by Choi and co-authors presents "P3 editing", which leverages dual-component guide RNAs (gRNA) to induce protein-protein proximity. They explore three strategies for leveraging prime-editing gRNA (pegRNA) as a dimerization module to create a molecular proximity sensor that drives genome editing, splitting a pegRNA into two parts (sgRNA and petRNA), inserting self-splicing ribozymes within pegRNA, and dividing pegRNA at the crRNA junction. Among these, splitting at the crRNA junction proved the most promising, achieving significant editing efficiency. They further demonstrated the ability to control genome editing via protein-protein interactions and small molecule inducers by designing RNA-based systems that form active gRNA complexes. This approach was also adaptable to other genome editing methods like base editing and ADAR-based RNA editing.

Strengths:

The study demonstrates significant advancements in leveraging guide RNA (gRNA) as a dimerization module for genome editing, showcasing its high specificity and versatility. By investigating three distinct strategies-splitting pegRNA into sgRNA and petRNA, inserting self-splicing ribozymes within the pegRNA, and dividing the pegRNA at the repeat junction-the researchers present a comprehensive approach to achieving molecular proximity and reconstituting function. Among these methods, splitting the pegRNA at the repeat junction emerged as the most promising, achieving editing efficiencies up to 76% of the control, highlighting its potential for further development in CRISPR-Cas9 systems. Additionally, the study extends genome editing control by linking protein-protein interactions to RNA-mediated editing, using specific protein-RNA interaction pairs to regulate editing through engineered protein proximity. This innovative approach expands the toolkit for precision genome editing, demonstrating the feasibility of controlling genome editing with enhanced specificity and efficiency.

Weaknesses:

The initial experiments with splitting the pegRNA into sgRNA and petRNA showed low editing efficiency, less than 2%. Similarly, inserting self-splicing ribozymes within pegRNA was inefficient, achieving under 2% editing efficiency in all constructs tested, possibly hindered by the prime editing enzyme. The editing efficiency of the crRNA and petracrRNA split at the repeat junction varied, with the most promising configurations only reaching 76% of the control efficiency. The RNA-RNA duplex formation's inefficiency might be due to the lack of additional protein binding, leading to potential degradation outside the Cas9-gRNA complex. Extending the approach to control genome editing via protein-protein interactions introduced complexity, with a significant trade-off between efficiency and specificity, necessitating further optimization. The strategy combining RADARS and P3 editing to control genome editing with specific RNA expression events exhibited high background levels of non-specific editing, indicating the need for improved specificity and reduced leaky expression. Moreover, P3 editing efficiencies are exclusively quantified after transfecting DNA into HEK cells, a strategy that has resulted in past reproducibility concerns for other technologies. Overall, the various methods and combinations require further optimization to enhance efficiency and specificity, especially when integrating multiple synthetic modules.

Reviewer #2 (Public Review):

Choi et al. describe a new approach for enabling input-specific CRISPR-based genome editing in cultured cells. While CRISPR-Cas9 is a broadly applied system across all of biology, one limitation is the difficulty in inducing genome editing based on cellular events. A prior study, from the same group, developed ENGRAM - which relies on activity-dependent transcription of a prime editing guide RNA, which records a specific cellular event as a given edit in a target DNA "tape". However, this approach is limited to the detection of induced transcription and does not enable the detection of broader molecular events including protein-protein interactions or exposure to small molecules. As an alternative, this study envisioned engineering the reconstitution of a split prime editing guide RNA (pegRNA) in a protein-protein interaction (PPI)-dependent manner. This would enable location- and content-specific genome editing in a controlled setting.

The authors explored three different design possibilities for engineering a PPI-dependent split pegRNA. First, they tried splitting pegRNA into a functional sgRNA and corresponding prime editing transRNA, incorporating reverse-complementary dimerization sequences on each guide half. This approach, however, resulted in low editing efficiency across 7 different designs with various complementary annealing template lengths (<2% efficiency). They also tried inserting a self-splicing ribozyme within the pegRNA, which produces a functional pegRNA post-transcriptionally. The incorporation of a split-ribozyme, dependent on a PPI, could have been used to reconstitute the split pegRNA in an event-controlled manner. However again, only modest levels of editing were observed with the self-splicing ribozyme design (<2%). Finally, they tried splitting the pegRNA at the repeat:anti-repeat junction that was used to join the original dual-guide system comprised of a crRNA and tracrRNA, into a single-guide RNA. They incorporated the prime editing features into the tracrRNA half, to create petracrRNA. Dimerization was initially induced by different complementary RNA annealing sequences. Using this design, they were able to induce an editing efficiency of ~28% (compared to 37% efficiency using a positive control epegRNA guide).

Having identified a suitable split pegRNA system, they next sought to induce the reconstitution of the two halves in a PPI-dependent manner. They replaced the complementary RNA annealing sequences with two different RNA aptamers (MS2 and BoxB). MS2 detects the MCP protein, while BoxB detects the LambdaN protein. Close proximity between MCP and LambdaN would thus bring together the two split pegRNA halves, creating a functional pegRNA that would enable prime editing at a specific target site. They demonstrated that they could induce MCP-BoxB proximity by fusing them to different dimerizing protein partners: 1) constitutive epitope-nanobody/antibody pairs such as scFv/GCN4 or NbALFA/ALFA-Tag; 2) split-GFP; or 3) chemically-induced protein pairs such as FKBP/FRB or ABI/PYL. For all of these approaches, they could achieve between ~20-60% normalized editing efficiency (relative to positive control editing levels with epegRNA). Additional mutation of the linkers between the RNA and aptamers could increase editing efficiency but also increase non-specific background editing even in the absence of an induced PPI.

Additional applications of this overall strategy included incorporating the design with different DNA base editors, with the most promising examples shown with the base editors CBE4max and ABE8. It should be noted that these specific examples used a non-physiological LambdaN-MCP direct fusion protein as the "bait" that induced reconstitution of the two halves of the guideRNA, rather than relying on a true induced PPI. They also demonstrated that the recently reported RADARS strategy could be incorporated into their system. In this example, they used an ADAR-guide-RNA to drive the expression of a LambdaN-PCP fusion protein in the presence of a specific target RNA molecule, IL6. This induced LambdaN-PCP protein could then reconstitute the split peg-RNAs to drive prime editing. To enable this last application, they replaced the MS2 aptamer in their pegRNA with the PP7 aptamer that binds the PCP protein (this was to avoid crosstalk with RADARS, which also uses MS2/MCP interaction). Using this strategy, they observed a normalized editing efficiency of around 12% (but observed non-specific editing of around 8% in the absence of the target RNA).

Strengths:

The strengths of this paper include an interesting concept for engineering guide RNAs to enable activity-dependent genome editing in living cells in the future, based on discreet protein-protein interactions (either constitutively, spatially, or chemically induced). Important groundwork is laid down to engineer and improve these guide RNAs in the future (especially the work describing altering the linkers in Supplementary Figure 3 - which provides a path forward).

Weaknesses:

In its current state, the editing efficiency appears too low to be applied in physiological settings. Much of the latter work in the paper relies on a LambdaN-MCP direction fusion protein, rather than two interacting protein pairs. Further characterizations in the future, especially varying the transfection amounts/durations/etc of the various components of the system, would be beneficial to improve the system. It will also be important to demonstrate editing at additional sites; to characterize how long the PPI must be active to enable efficient prime editing; and how reversible the reconstitution of the split pegRNA is.

Reviewer #1 (Public Review):

Summary:

The main conclusion of this manuscript is that the mediator kinases supporting the IFN response in Downs syndrome cell lines represent an important addition to understanding the pathology of this affliction.

Strengths:

Mediator kinase stimulates cytokine production. Both RNAseq and metabolomics clearly demonstrate a stimulatory role for CDK8/CDK19 in the IFN response. The nature of this role, direct vs. indirect, is inferred by previous studies demonstrating that inflammatory transcription factors are Cdk8/19 substrates. The cytokine and metabolic changes are clear-cut and provide a potential avenue to mitigate these associated pathologies.

Weaknesses:

This study revealed a previously undescribed role for the CKM in splicing. The previous identification of splicing factors as substrates of CDK8/CDK19 is also intriguing. However, additional studies seem to be necessary in order to attach this new function to the CKM. As the authors point out, the changes in splicing patterns are relatively modest compared to other regulators. In addition, some indication that the proteins encoded by these genes exhibit reduced levels or activities would support their RNAseq findings.

Seahorse analysis is normally calculated with specific units for oxygen consumption, ATP production, etc. It would be of interest to see the actual values of OCR between the D21 and T21 cell lines rather than standardizing the results. This will address the specific question about relative mitochondrial function between these cells. Reduced mitochondrial function has been associated with DS patients. Therefore, it would be important to know whether mitochondrial function is reduced in the T21 cells vs. the D21 control. Importantly for the authors' goal of investigating the use of CDK8/19 inhibitors in DS patients, does CA treatment reduce mitochondrial function to pathological levels?

Reviewer #2 (Public Review):

Summary:

In this manuscript, Cozzolino et al. demonstrate that inhibition of the Mediator kinase CDK8 and its paralog CDK19 suppresses hyperactive interferon (IFN) signaling in Down syndrome (DS), which results from trisomy of chromosome 21 (T21). Numerous pathologies associated with DS are considered direct consequences of chronic IFN pathway activation, and thus hyperactive IFN signaling lies at the heart of pathophysiology. The collective interrogation of transcriptomics, metabolomics, and cytokine screens in sibling-matched cell lines (T21 vs D21) allows the authors to conclude that Mediator kinase inhibition could mitigate chronic, hyperactive IFN signaling in T21. To probe the functional outcomes of Mediator kinase inhibition, the authors performed cytokine screens, transcriptomic, and untargeted metabolomics. This collective approach revealed that Mediator kinases establish IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Mediator kinase inhibition suppresses cell responses during hyperactive IFN signaling through inhibition of pro-inflammatory transcription factor activity (anti-inflammatory effect) and alteration of core metabolic pathways, including upregulation of anti-inflammatory lipid mediators, which served as ligands for specific nuclear receptors and downstream phenotypic outcomes (e.g., oxygen consumption). These data provided a mechanistic link between Mediator kinase activity and nuclear receptor function. Finally, the authors also disclosed that Mediator kinase inhibition alters splicing outcomes.

Overall, this study reveals a mechanism by which Mediator kinases regulate gene expression and establish that its inhibition antagonizes chronic IFN signaling through collective transcriptional, metabolic, and cytokine responses. The data have implications for DS and other chronic inflammatory conditions, as Mediator kinase inhibition could potentially mitigate pathological immune system hyperactivation.

Strengths:

(1) One major strength of this study is the mechanistic evidence linking Mediator kinases to hyperactive IFN signaling through transcriptional changes impacting cell signaling and metabolism.

(2) Another major strength of this study is the use of sibling-matched cell lines (T21 vs D21) from various donors (not just one sibling pair), and further cross-referencing with data from large cohorts, suggesting that part of the data and conclusions are generalizable.

(3) Another major strength of this study is the combined experimental approach including transcriptomics, untargeted metabolomics, and cytokine screens to define the mechanisms underlying suppression of hyperactive interferon signaling in DS upon Mediator kinase inhibition.

(4) Another major strength of this study is the significance of the work to DS and its potential impact on other chronic inflammatory conditions.

Weakness:

(1) Genetic evidence linking the mentioned nuclear receptors to activation of an anti-inflammatory program upon Mediator kinase inhibition could improve the definition of the mechanism and overall impact of the work.

(2) Page 5 states that "Mediator kinases broadly regulate cholesterol and fatty acid biosynthesis and this was further confirmed by the metabolomics data", but a clear mechanistic explanation was lacking. Likewise, the data suggest but do not prove, that altered lipid metabolites influence the function of nuclear receptors to regulate an anti-inflammatory program in response to Mediator kinase inhibition (p. 6), despite the fact the gene expression changes elicited by Mediator kinase inhibition tracked with downstream metabolic changes.

(3) The figures are outstanding but dense.

(4) Figure 6 (PRO-Seq). The authors refer to pro-inflammatory TFs (e.g. NF-kB/RelA). It is not clear whether the authors have specifically examined TF binding at enhancers or more broadly at every region occupied by the interrogated TFs?

Reviewer #1 (Public Review):

Summary:

I have previously reviewed this manuscript as a submission to another journal in 2022. My recommendations here mirror those of my prior suggestions, now with further added details.

This manuscript describes the identification and isolation of several phage from deep sea isolates of Lentisphaerae strains WC36 and zth2. The authors observe induction of several putative chronic phages with the introduction of additional polysaccharides to the media. The authors suggest that two of the recovered phage genomes encode AMGs associated with polysaccharide use. The authors also suggest that adding the purified phage to cultures of Pseudomonas stutzeri 273 increased the growth of this bacteria due to augmented polysaccharide use genes from the phage.

Strengths:

Interesting isolate of deep sea Lentisphaerae strains which will undoubtedly further our understanding of deep sea microbial life.

The revisions have addressed the weaknesses raised in the previous review.

Reviewer #2 (Public Review):

Summary:

This paper investigates deep-sea bacteriophage systems which appear to employ a chronic replication mechanism that is induced or enhanced by polysaccharide addition. Some preliminary evidence for the potential role of auxiliary metabolic genes in aiding phage and/or host proliferation is also provided. The hypothesis being tested is fully supported with solid and convincing evidence and the findings are potentially generalizable with implications for our understanding of polysaccharide-mediated virus-host interactions and carbon cycling in marine ecosystems more broadly.

Strengths:

This paper synthesizes sequencing and phylogenic analyses of two Lentisphaerae bacteria and three phage genomes; electron microscopy imaging of bacterial/phage particles; differential gene expression analyses; differential growth curve analyses, and differential phage proliferation assays to extract insights into whether laminarin and starch can induce both host growth and phage proliferation. The data presented convincingly demonstrate that both host culture density and phage proliferation increase as a result having host, phage, and polysaccharide carbon source together in culture.

Weaknesses:

The AMG-centered elements of the article would be strengthened by more "mechanistic" experiments focusing on identifying "HOW" the polysaccharide processing, transport, and metabolism genes are being used by the phages to either directly increase viral infection/replication or else to indirectly do so by supporting the growth of the host (via mutualism). The concept of "selfishness" in bacterial systems and its potential role in viral life cycles could be more developed. Selfish bacteria are active throughout the water column of the ocean. ISME COMMUN. 3, 11 (2023) (see for instance https://doi.org/10.1038/s43705-023-00219-7) and such "selfish" bacteria sequester metabolizable polysaccharides in their periplasm to advantage (https://www.nature.com/articles/ismej201726). It is plausible that phages may be either hijacking such polysaccharide sequestration mechanisms to improve infectivity and ENTRY or else helping their hosts to grow and proliferate so they can reap the benefits of simply having more hosts to infect. The current work does not clearly distinguish between these two distinct mechanistic possibilities. The paper would be strengthened by a more detailed/clear discussion of this possibility.

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #2 (Public Review):

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

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

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

Strengths:

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

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

Oustanding questions that could lead to interesting follow-up work:

Like all great pieces of research, this article makes it clear where current limitations lie and therefore opens up opportunities for future work.

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

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

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

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

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

(6) More broadly, with the type of objectives that the authors assume the inputs fulfill, some M1 properties that lead to strong preparation - notably, limited readout controllability - may not be favorable for control in general, so it would be interesting if other objectives and assumptions could robustly lead to strong preparation under more general M1 properties.'

Reviewer #3 (Public Review):

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

As discussed in the prior round of reviews, the central constraint that the authors use is a mathematically tractable stand-in for a range of plausible (but often trickier to define and evaluate) constraints, such as simplicity of inputs (or inputs being things that other areas could provide). The manuscript now embraces this fact more explicitly and also gives some results showing that other constraints (such as on the derivative of activity, which is one component of complexity) can have the same effect. The manuscript also now discusses and addresses a modest weakness of the previous manuscript: the preparatory activity in their simulations is often overly complex temporally, lacking the (rough) plateau typically seen for data. Depending on your point of view, this is simply 'window dressing', but from my perspective it was important to know that their approach could yield more realistic-looking preparatory activity.

The most recent version of the manuscript also has a useful section in the Discussion on the topic of preparation when there is no external delay, which I found helpful given prior behavioral and physiological studies arguing that preparation can 1) be very brief, but 2) is always present. These findings mesh nicely with the authors' central result that preparation is a good network strategy, and that it would thus be normative for there to be at least a brief interval of preparation even when not imposed externally.

Reviewer #2 (Public Review):

Summary:

The authors investigated systemic inflammation induced by LPS in various tissues and also examined immune cells of the mice using tight junction protein-based PDZ peptide. They explored the mechanism of anti-systemic inflammatory action of PDZ peptides, which enhanced M1/M2 polarization and induced the proliferation of M2 macrophages. Additionally, they insisted the physiological mechanism that inhibited the production of ROS in mitochondria, thereby preventing systemic inflammation.

Strengths:

In the absence of specific treatments for septic shock or sepsis, the study demonstrating that tight junction-based PDZ peptides inhibit systemic inflammation caused by LPS is highly commendable. Whereas previous research focused on antibiotics, this study proves that modifying parts of intracellular proteins can significantly suppress symptoms caused by septic shock. The authors expanded the study of localized inflammation caused by LPS or PM2.5 in the respiratory tract to systemic inflammation, presenting promising results. They not only elucidated the physiological mechanism by identifying the transcriptome through RNA sequencing but also demonstrated that PDZ peptides inhibit the production of ROS in mitochondria and prevent mitochondrial fission. This research is highly regarded as an excellent study with potential as a treatment for septic shock or sepsis.

Weaknesses:

(1) They Focused intensively on acute inflammation for a short duration instead of chronic inflammation.

(2) LPS was used to induce septic shock but administrating actual microbes such as E.coli would yield more accurate results.

(3) The authors used pegylated peptides, but future research should utilize the optimized peptides to derive the optimal peptide, and further, PK/PD studies are also necessary.

Reviewer #1 (Public Review):

The manuscript introduces a bioinformatic pipeline designed to enhance the structure prediction of pyoverdines, revealing an extensive and previously overlooked diversity in siderophores and receptors. Utilizing a combination of feature sequence and phylogenetic approaches, the method aims to address the challenging task of predicting structures based on dispersed gene clusters, particularly relevant for pyoverdines.

Predicting structures based on gene clusters is still challenging, especially pyoverdines as the gene clusters are often spread to different locations in the genome. The revised manuscript has much improved in clarity and reproducibility. I believe that the method is not yet applicable to all NRPS in general and that there is a clear scalability issue when talking about Big Data. However, the method is highly useful for specific NRPS families such as the pyoverdines, so the manuscript presents a useful bioinformatic pipeline for pyoverdine structure prediction, showcasing a commendable exploration of siderophore diversity.

Reviewer #2 (Public Review):

Pyoverdines, siderophores produced by many Pseudomonads, are one of the most diverse groups of specialized metabolites and frequently used as model systems. Thousands of Pseudomonas genomes are available, but large scale analyses of pyoverdines are hampered by the biosynthetic gene clusters (BGCs) being spread across multiple genomic loci and existing tools' inability to accurately predict amino acid substrates of the biosynthetic adenylation (A) domains. The authors present a bioinformatics pipeline that identifies pyoverdine BGCs and predicts the A domain substrates with high accuracy. They tackled a second challenging problem by developing an algorithm to differentiate between outer membrane receptor selectivity for pyoverdines versus other siderophores and substrates. The authors applied their dataset to thousands of Pseudomonas strains, producing the first comprehensive overview of pyoverdines and their receptors and predicting many new structural variants.

The A domain substrate prediction is impressive, including the correction of entries in the MIBiG database. Their high accuracy came from a relatively small training dataset of A domains from 13 pyoverdine BGCs. The authors acknowledge that this small dataset does not include all substrates, and correctly point out that new sequence/structure pairs can be added to the training set to refine the prediction algorithm. The workflow unfortunately cannot differentiate between different variants of Asp and OHOrn. To validate their predictions, they elucidated structures of several new pyoverdines, and their predictions performed well. The authors tested their workflow on Burkholderiales A domains and had good results, suggesting it can be used on other taxa. Skimming through the source code and data, the algorithm itself appears to be sound and a clear improvement over existing tools for pyoverdine BGC annotation.

Predicting outer membrane receptor specificity is likewise a challenging problem and the authors have made a promising achievement by finding specific gene regions that differentiate the pyoverdine receptor FpvA from FpvB and other receptor families. Their predictions were not tested experimentally, but the finding that only predicted FpvA receptors were proximate to the biosynthesis genes lends credence to the predictive power of the workflow. The authors find predicted pyoverdine receptors across an impressive 468 genera, an exciting finding for expanding the role of pyoverdines as public goods beyond Pseudomonas. However, whether or not these receptors can actually recognize pyoverdines (and if so, which structures!) remains to be investigated.

In all, the authors have assembled a rich dataset that will enable large scale comparative genomic analyses. This dataset could be used by a variety of researchers, including those studying natural product evolution, public good eco/evo dynamics, and NRPS engineering.

Reviewer #2 (Public Review):

Summary:

One of the greatest challenges for the spliceosome is to be able to repress the many cryptic splice sites that can occur in both the intronic and exotic sequences of genes. Although many studies have focused on cryptic signals in introns (because of their common involvement in disease) the question still remained open as to the factors that repress cryptic exons in exons. Because exons are normally much shorter than introns, in many cases the problem does not exist. However, in human genes a significant proportion of exons can be considerably longer than the average 150 nt length and this raises the question of how cryptic splicing can be prevented in long exons. To address this question, the authors have focused on the possible role played by an ancient mammalian RBD protein called RBMX. Using a combination of high-throughput and classic splicing methodologies, they have shown that there is a class of RBMX-dependent ultra-long exons connected where the RBMX, RBMXL2 and RBMY paralogs have closely related functional activity in repressing cryptic splice site selection.

Strengths:

In general, the present work sheds light on what has been a rather understudied process in splicing research. The use of iCLIP and RNA-seq data has not only allowed to identify the long exons where cryptic splicing is prevented by the RBMX proteins but has also allowed to identify a network of genes mostly involved in genome stability and transcriptional control where these proteins seem to play a prominent role. This can therefore also shed additional information on the way splicing has shaped evolutionary processes in the mammalian lineage and will therefore be of interest to many researchers in this field.

Weaknesses:

There are no major weaknesses, although some specific aspects of the findings could be addressed more in-depth in the recommendations to authors.

Reviewer #1 (Public Review):

Summary:

The article by Siachisumo, Luzzi and Aldalaquan et al. describes studies of RBMX and its role in maintaining proper splicing of ultra-long exons. They combine CLIP, RNA-seq, and individual example validations with manipulation of RBMX and its family members RBMY and RBMXL2 to show that the RBMX family plays a key role in maintaining proper splicing of these exons.

I think one of the main strengths of the manuscript is its ability to explore a unique but interesting question (splicing of ultra-long exons), and derive a relatively simple model from the resulting genomics data. The results shown are quite clean, suggesting that RBMX plays an important role in proper regulation of these exons. The ability of family members to rescue this phenotype (as well as only particular domains) is also quite intriguing and suggests that the mechanisms for keeping these exons properly spliced may be a quite important and highly conserved mechanism.

The revised manuscript addresses many of my earlier critiques and does an effective job of arguing that RBMX plays a large-scale role in regulating splicing of long exons. I think there are obvious open questions for future work (the mechanism of how RBMX/RBMXL2 achieve this splicing control is perhaps hinted at but not fully explored here), but I think the article provides an intriguing analysis of the role of RBMX that will activate interesting future studies.

Reviewer #3 (Public Review):

The manuscript by Siachisumo et al builds upon a previous publication from the same group of collaborators that showed that depletion of mouse RBMXL2 leads to a block in spermatogenesis associated with mis-splicing, particularly of large exons in genes associated with genome stability (Ehrmann et al Elife 2019). RBMXL2 is an RNA-binding protein and an autosomal retrotransposed paralog of the X-chromosomally encoded RBMX. RBMXL2 is expressed during meiosis when RBMX and the more distantly related RBMY (on the Y chromosome) are silenced. It is therefore an appealing hypothesis that RBMXL2 might provide cover for RBMX function during meiosis. To address this hypothesis the authors analysed the transcriptomic consequences of RBMX depletion by RNA-Seq in human cells (MDA-MB-231 and existing RNA-Seq data from HEK293 cells), complemented by iCLIP to analyze the binding targets of FLAG-tagged RBMX in HEK293 cells. The findings convincingly demonstrate that - like RBMXL2 - RBMX mainly acts as a splicing repressor and that it particularly acts to protect the integrity of very long ("ultra-long") exons, defined as those over 1000 nt. Upon RBMX depletion, many of these exons are shortened due to the use of cryptic 5' and/or 3' splice sites. Moreover, affected genes are particularly enriched for functions associated with genome integrity - indeed "comet assays" show that RBMX depletion leads to DNA damage defects. Strikingly, RNA-Seq analysis showed that overexpression of RBMXL2 is able to complement the majority of splicing changes caused by RBMX depletion, particularly those involving ultra-long exons. In a smaller scale experiment RBMY was also able to complement effects of RBMX knockdown upon three target events in the ETAA1, REV3L and ATRX genes.

In addition to these core findings the manuscript also includes some experiments that begin to address more mechanistic questions, such as the potential for RBMX to sterically block access of spliceosome components to splice site elements, and preliminary structure-function analyses of RBMX showing that its RRM domain is not necessary for splicing regulatory activity on the ETAA1, REV3L and ATRX target events.

In summary, this manuscript provides clear and convincing evidence to support the role of RBMX in somatic cells as a repressor of cryptic splice sites in ultra-long exons, mirroring the function of RBMXL2 in meiotic cells. It therefore demonstrates how the RBMX/RBMXL2/RBMY family perform a key role in protecting the transcriptomic integrity of ultra-long exons.

Reviewer #2 (Public Review):

Summary:

Overall this is an interesting innovative study that examines chromatin accessibility in an inhibitory iPSC model of Dravet Syndrome. The authors detect a potential intriguing development defect in the patient-specific neurons, however the correlation with gene expression or protein abundance is not compelling and the variability of the data is still difficult to determine.

Strengths:

(1) This is a novel and interesting study that aims to investigate the epigenetic changes that occur in a sodium channel model of epilepsy, these are oft ignored, but also an interesting area for future therapeutics.

(2) The paper is well written with good graphics and flow.

(3) With caveats noted below, there is an intriguing developmental defect in GABAergic neuron differentiation in this model. It would be interesting to see how this correlated with the expression of SCN1A, and I was surprised this was not addressed in the manuscript via RNA/protein abundance, nor how the absence of a sodium channel can accelerate differentiation when a priori I might expect the opposite (as less 'neuronal' signal)

(4) There is exploratory analysis that VPA alters chromatin accessibility at an individual-specific level. Though it was not noted if any of the DS patients,

Weaknesses addressed:

(1) Representative images for cell-identity markers are now shown for D19 and D65.

(2) The methods now state that three differentiations were performed.

(3) The authors address a possible role for cell death in data obtained from their cultures by assessing viability with trypan blue staining.

(4) Some features of ATAC signal normalization and enrichment analysis have been better documented.

(5) Some of the variability in key results is better documented.

Weaknesses poorly or not addressed:

(1) Although the authors include prior RNAseq data and report on qPCR measurements for SCN1A (Supp Fig 1)these do not on the surface appear to agree, with the RNAseq showing little apparent difference between patients and controls, while the qPCR seems to show a two-fold difference at D65. This is likely a misleading artifact of normalizing PCR expression to that at D0 when the gene is not expressed but has mildly different low levels in patients and controls. No measurement of the protein product or its function is included. This is a major weakness that casts doubt on the core hypothesis that epigenetic changes play a key causal role in Dravet syndrome.

(2) Although some QC on ATAC is described, QC performed on iPSC lines, i.e. karyotype/CNV analysis and confirmation of genotypes is not described in the paper.

(3) The authors describe a method for trying to diminish variability but do not adequately explain this method or how much variability remains in many of their measures.

(4) Given that VPA would be administered in patients with fully mature inhibitory neurons, it is difficult to determine the biological relevance of these findings.