347 Matching Annotations
  1. Last 7 days
    1. Reviewer #3 (Public Review):


      This is a tools paper that describes an open source software package, BonVision, which aims to provide a non-programmer-friendly interface for configuring and presenting 2D as well as 3D visual stimuli to experimental subjects. A major design emphasis of the software is to allow users to define visual stimuli at a high level independent of the actual rendering physical devices, which can range from monitors to curved projection surfaces, binocular displays, and also augmented reality setups where the position of the subject relative to the display surfaces can vary and needs to be adjusted for. The package provides a number of semi-automated software calibration tools to significantly simplify the experimental job of setting up different rigs to faithfully present the intended stimuli, and is capable of running at hardware-limited speeds comparable to and in some conditions better than existing packages such as Psychtoolbox and PsychoPy.

      Major comments:

      While much of the classic literature on visual systems studies have utilized egocentrically defined ("2D") stimuli, it seems logical to project that present and future research will extend to not only 3D objects but also 3D environments where subjects can control their virtual locations and viewing perspectives. A single software package that easily supports both modalities can therefore be of particular interest to neuroscientists who wish to study brain function in 3D viewing conditions while also referencing findings to canonical 2D stimulus responses. Although other software packages exist that are specialized for each of the individual functionalities of BonVision, I think that the unifying nature of the package is appealing for reasons of reducing user training and experimental setup time costs, especially with the semi-automated calibration tools provided as part of the package. The provisions of documentation, demo experiments, and performance benchmarks are all highly welcome and one would hope that with community interest and contributions, this could make BonVision very friendly to entry by new users.

      Given that one function of this manuscript is to describe the software in enough detail for users to judge whether it would be suited to their purposes, I feel that the writing should be fleshed out to be more precise and detailed about what the algorithms and functionalities are. This includes not shying away from stating limitations -- which as I see it, is just the reality of no tool being universal, but because of that is one of the most important information to be transmitted to potential users. My following comments point out various directions in which I think the manuscript can be improved.

      The biggest point of confusion for me was whether the 3D environment functionality of BonVision is the same as that provided by virtual spatial environment packages such as ViRMEn and gaming engines such as Unity. In the latter software, the virtual environment is specified by geometrically laying out the shape of the traversable world and locations of objects in it. The subject then essentially controls an avatar in this virtual world that can move and turn, and the software engine computes the effects of this movement (i.e. without any additional user code) then renders what the avatar should see onto a display device. I cannot figure out if this is how BonVision also works. My confusion can probably be cured by some additional description of what exactly the user has to do to specify the placement of 3D objects. From the text on cube mapping (lines 43 and onwards), I guessed that perhaps objects should be specified by their vectorial displacement from the subject, but I have very little confidence in my guess and also cannot locate this information either in the Methods or the software website. For Figure 5F it is mentioned that BonVision can be used to implement running down a virtual corridor for a mouse, so if some description can be provided of what the user has to do to implement this and what is done by the software package, that may address my confusion. If BonVision is indeed not a full 3D spatial engine, it would be important to mention these design/intent differences in the introduction as well as Supplementary Table 1.

      More generally, it would be useful to provide an overview of what the closed-loop rendering procedure is, perhaps including a Figure (different from Supplementary Figure 2, which seems to be regarding workflow but not the software platform structure). For example, I imagine that after the user-specified texture/object resources have been loaded, then some engine runs a continual loop where it somehow decides the current scene. As a user, I would want to know what this loop is and how I can control it. For example, can I induce changes in the presented stimuli as a function of time, whether this time-dependence has to be prespecified before runtime, or can I add some code that triggers events based on the specific history of what the subject has done in the experiment, and so forth. The ability to log experiment events, including any viewpoint changes in 3D scenes, is also critical, and most experimenters who intend to use it for neurophysiological recordings would want to know how the visual display information can be synchronized with their neurophysiological recording instrumental clocks. In sum, I would like to see a section added to the text to provide a high-level summary of how the package runs an experiment loop, explaining customizable vs. non-customizable (without directly editing the open source code) parts, and guide the user through the available experiment control and data logging options.

      Having some experience myself with the tedium (and human-dependent quality) of having to adjust either the experimental hardware or write custom software to calibrate display devices, I found the semi-automated calibration capabilities of BonVision to be a strong selling point. However I did not manage to really understand what these procedures are from the text and Figure 2C-F. In particular, I'm not sure what I have to do as a user to provide the information required by the calibration software (surely it is not the pieces of paper in Fig. 2C and 2E..?). If for example, the subject is a mouse head-fixed on a ball as in Figure 1E, do I have to somehow take a photo from the vantage of the mouse's head to provide to the system? What about the augmented reality rig where the subject is free to move? How can the calibration tool work with a single 2D snapshot of the rig when e.g. projection surfaces can be arbitrarily curved (e.g. toroidal and not spherical, or conical, or even more distorted for whatever reasons)? Do head-mounted displays require calibration, and if so how is this done? If the authors feel all this to be too technical to include in the main text, then the information can be provided in the Methods. I would however vote for this as being a major and important aspect of the software that should be given air time.

      As the hardware-limited speed of BonVision is also an important feature, I wonder if the same ~2 frame latency holds also for the augmented reality rendering where the software has to run both pose tracking (DeepLabCut) as well as compute whole-scene changes before the next render. It would be beneficial to provide more information about which directions BonVision can be stressed before frame-dropping, which may perhaps be different for the different types of display options (2D vs. 3D, and the various display device types). Does the software maintain as strictly as possible the user-specified timing of events by dropping frames, or can it run into a situation where lags can accumulate? This type of technical information would seem critical to some experiments where timings of stimuli have to be carefully controlled, and regardless one would usually want to have the actual display times logged as previously mentioned. Some discussion of how a user might keep track of actual lags in their own setups would be appreciated.

      On the augmented reality mode, I am a little puzzled by the layout of Figure 3 and the attendant video, and I wonder if this is the best way to showcase this functionality. In particular, I'm not entirely sure what the main scene display is although it looks like some kind of software rendering — perhaps of what things might look like inside an actual rig looking in from the top? One way to make this Figure and Movie easier to grasp is to have the scene display be the different panels that would actually be rendered on each physical panel of the experiment box. The inset image of the rig should then have the projection turned on, so that the reader can judge what an actual experiment looks like. Right now it seems for some reason that the walls of the rig in the inset of the movie remain blank except for some lighting shadows. I don't know if this is intentional.

    2. Reviewer #2 (Public Review):

      BonVision is a package to create virtual visual environments, as well as classic visual stimuli. Running on top of Bonsai-RX it tries and succeeds in removing the complexity of the above mentioned task and creating a framework that allows non-programmers the opportunity to create complex, closed loop experiments. Including enough speed to capture receptive fields while recording different brain areas.

      At the time of the review, the paper benchmarks the system using 60Hz stimuli, which is more than sufficient for the species tested, but leaves an open question on whether it could be used for other animal models that have faster visual systems, such as flies, bees etc.

      The authors do show in a nice way how the system works and give examples for interested readers to start their first workflows with it. Moreover, they compare it to other existing software, making sure that readers know exactly what "they are buying" so they can make an informed decision when starting with the package.

      Being written to run on top of Bonsai-RX, BonVision directly benefits from the great community effort that exists in expanding Bonsai, such as its integration with DeepLabCut and Auto-pi-lot. Showing that developing open source tools and fostering a community is a great way to bring research forward in an additive and less competitive way.

    3. Reviewer #1 (Public Review):

      In this project, the authors set out to create an easy to use piece of software with the following properties: The software should be capable of creating immersive (closed loop) virtual environments across display hardware and display geometries. The software should permit easy distribution of formal experiment descriptions with minimal changes required to adapt a particular experimental workflow to the hardware present in any given lab while maintaining world-coordinates and physical properties (e.g. luminance levels and refresh rates) of visual stimuli. The software should provide equal or superior performance for generating complex visual cues and/or immersive visual environments in comparison with existing options. The software should be automatically integrated with many other potential data streams produced by 2-photon imaging, electrophysiology, behavioral measurements, markerless pose estimation processing, behavioral sensors, etc.

      To accomplish these goals, the authors created two major software libraries. The first is a package for the Bonsai visual programming language called "Bonsai.Shaders" that brings traditionally low-level, imperative OpenGL programming into Bonsai's reactive framework. This library allows shader programs running on the GPU to seamlessly interact, using drag and drop visual programming, with the multitude of other processing and IO elements already present in numerous Bonsai packages. The creation of this library alone is quite a feat given the complexities of mapping the procedural, imperative, and stateful design of OpenGL libraries to Bonsai's event driven, reactive architecture. However, this library is not mentioned in the manuscript despite its power for tasks far beyond the creation of visual stimuli (e.g. GPU-based coprocessing) and, unlike BonVision itself, is largely undocumented. I don't think that this library should take center stage in this manuscript, but I do think its use in the creation of BonVision as well as some documentation on its operators would be very useful for understanding BonVision itself.

      Following the creation of Bonsai.Shaders, the authors used it to create BonVision which is an abstraction on top of the Shaders library that allows plug and play creation of visual stimuli and immersive visual environments that react to input from the outside world. Impressively, this library was implemented almost entirely using the Bonsai visual programming language itself, showcasing its power as a domain-specific language. However, this fact was not mentioned in the manuscript and I feel it is a worthwhile point to make. The design of BonVision, combined with the functional nature of Bonsai, enforces hard boundaries between the experimental design of visual stimuli and (1) the behavioral input hardware used to drive them, (2) the dimensionality of the stimuli (i.e. 2D textures via 3D objects), (3) the specific geometry of 3D displays (e.g. dual monitors, versus spherical projection, versus head mounted stereo vision hardware), and (4) automated hardware calibration routines. Because of these boundaries, experiments designed using BonVision become easy to share across labs even if they have very different experimental setups. Since Bonsai has integrated and standardized mechanisms for sharing entire workflows (via copy paste of XML descriptions or upload of workflows to publicly accessible Nuget package servers), this feature is immediately usable by labs in the real world.

      After creating these pieces of software, the authors benchmarked them against other widely used alternatives. IonVisoin met or exceeded frame rate and rendering latency performance measures when compared to other single purpose libraries. BonVision is able to do this while maintaining its generality by taking advantage of advanced JIT compilation features provided by the .NET runtime and using bindings to low-level graphics libraries that were written with performance in mind. The authors go on to show the real-world utility of BonVision's performance by mapping the visual receptive fields of LFP in mouse superior colliculus and spiking in V1. The fact that they were able to obtain receptive fields indicates that visual stimuli had sufficient temporal precision. However, I do not follow the logic as to why this is because the receptive fields seem to have been created using post-hoc aligned stimulus-ephys data, that was created by measuring the physical onset times of each frame using a photodiode (line 389). Wouldn't this preclude any need for accurate stimulus timing presentation?

      Finally the authors use BonVision to perform one human psychophysical and several animal VR experiments to prove the functionality of the package in real-world scenarios. This includes an object size discrimination task with humans that relies on non-local cues to determine the efficacy of the cube map projection approach to 3D spaces (Fig 5D). Although the results seem reasonable to me (a non-expert in this domain), I feel it would be useful for the authors to compare this psychophysical discrimination curve to other comparable results. The animal experiments prove the utility of BonVision for common rodent VR tasks.

      In summary, the professionalism of the code base, the functional nature of Bonsai workflows, the removal of overhead via advanced JIT compilation techniques, the abstraction of shader programming to high-level drag and drop workflows, integration with a multitude of input and output hardware, integrated and standardized calibration routines, and integrated package management and workflow sharing capabilities make Bonsai/BonVision serious competitors to widely-used, closed-source visual programming tools for experiment control such as LabView and Simulink. BonVision showcases the power of the Bonsai language and package management ecosystem while providing superior design to alternatives in terms of ease of integration with data sources and facilitation of sharing standardized experiments. The authors exceeded the apparent aims of the project and I believe BonVision will become a widely used tool that has major benefits for improving experiment reproducibility across laboratories.

    4. Evaluation Summary:

      Increasingly, neuroscience experiments require immersive virtual environments that approximate natural sensory motor loops while permitting high-bandwidth measurements of brain activity. BonVision is an open-source graphics programming library that allows experimenters to quickly implement immersive 3D visual environments across display hardware and geometry with automated calibration and integration with hundreds of different neural recording technologies, behavioral apparatuses, etc. BonVision standardizes sharing complex, closed-loop visual tasks between labs with vastly different equipment, provides a concrete and easy way to do so, and should be of interest to a wide array of visual systems neuroscientists.

  2. Feb 2021
    1. Author Response:

      Reviewer #1 (Public Review):

      1) Figure 2E: it is surprising that the potentiation shown in WT mice is not longer lasting. Under the experimental conditions used here, plasticity seems to be biased towards depression. In the methods, the authors state that they use 2mM Calcium and 1mM Magnesium in their external saline. A recent study (Titley et al., J. Physiol. 597, 2019) has demonstrated that under realistic conditions (incl. an ion milieu of 1.2 mM Calcium and 1mM Magnesium), LTP results under most conditions - even those involving climbing fiber co-stimulation - while LTD only results from prolonged complex spike firing. Optimally, the authors would establish a real LTP control in their WT mice (using conditions as described in Titley et al or similar) and test for changes in the mutants. As LTP is not the focus of this paper and this might be out of the scope of this work, it should be acceptable to leave it as it is, but this caveat should at least be discussed.

      The experimental conditions and the strain of mice used to test potentiation and LTD at PF/PCs are indeed different from the ones used in Titley et al.(2019), (divalent concentration, internal solution, holding potential, CF stimulation frequency). We acknowledge that more physiological conditions may have slightly shifted plasticity toward more potentiation. However, our main goal was to compare WT vs KO genotype and we think our results, albeit under non strict physiological conditions, demonstrate that depression mechanisms are strongly impaired in SUSD4 KO mice. We have amended the results section page 5.

      2) Figure 3: The climbing fiber physiology is described in detail, but what is missing is a characterization of potential changes in the complex spike waveform, recorded in current-clamp mode. This should certainly be provided. This is important as it has been shown that changes in the complex spike waveform affect the probability for LTD induction (Mathy et al., Neuron 62, 2009). The CF-EPSC is a rather indirect measure.

      As in Mathy et al. 2009 we used repeated CF stimulation to ensure LTD induction. As requested, we have included in our supplementary data the analysis of the complex spike waveform recorded in current-clamp mode during this protocol (Figure S7 and results section page 6). We have measured the same parameter as in Titley et al. (2019) and Mathy et al. (2009), the number of spikelets, spikes induced by repeated CF stimulation, and did not find any difference between Susd4 WT controls and Susd4 KO Purkinje cells.

      3) Is synaptic pruning at parallel fiber synapses impaired in the SUSD4 mutants? The LTD deficit is quite obvious. In the light of the role of autophagy in pruning, and the molecular similarity between LTD and pruning, it would be of interest to see whether activity-dependent pruning at these synapses is altered. This aspect is somewhat addressed by the VGLuT1 measures shown in Figure 2, but should be discussed in more depth.

      As noted by Reviewer #1, our morphological measurements do not reveal any significant difference in the number of PF synapses in Susd4 WT versus Susd4 KO Purkinje cells. This is also supported by the input-output curve in Figure 2C that does not reveal any difference in Purkinje cell responses to PF stimulation in the absence of Susd4. However, we agree that our data do not exclude a transient effect of Susd4 mutations on synaptic pruning during development, in particular at earlier stages when PF pruning is likely more important (third postnatal week, Ichikawa et al. PNAS 2016).

      Reviewer #2 (Public Review):

      Major comments:

      In Figure 1 localization images are shown using exogenous protein. Can the authors visualize endogenous protein?

      Unfortunately, we have tested many antibodies against SUSD4, commercial ones and custom-made ones. None gave satisfactory results for the detection of the endogenous SUSD4 protein, as assessed by immunohistochemistry in sections from control and SUSD4 KO mice. This is a classical problem in our field and is stated in the text (line 167 page 4).

      It appears that SUSD4 is expressed in multiple brain regions, even at higher levels than the cerebellum. The authors should provide a good explanation for why deficits in the KO do not affect other functions, and seem to preferentially affect cerebellar functions.

      We never intended to convey the message that cerebellar functions are preferentially affected in this mouse mutant. We have chosen to analyze in depth the phenotype of Susd4 loss-of-function in the cerebellar system, because of the many advantages of this model (as stated in page 3 introduction) and the high expression of Susd4 in cerebellar Purkinje cells. We have changed the text to make this point clearer: 1) in the results section at the level of the transition between the behavior results and the rest (page 5); 2) in the discussion page 11.

      Figure 4: immunofluorescence data are not very convincing.

      We have changed the panel in figure 4D to better highlight the partial colocalization between GluA2 and SUSD4 in cultured Purkinje cells.

      Figure 5: The use of the word "could" is not supporting a strong conclusion. The authors should demonstrate whether SUSD4 DOES indeed regulate GluA2.

      We have changed the title of Figure 5 and changed the text in the result section accordingly.

      Reviewer #3 (Public Review):

      Specific comments:

      1) Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Figure 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

      We agree with Reviewer 3 that it would be important to localize SUSD4 at this particular synapse. However, the morphology of this synapse is rather peculiar with big CF boutons contacting several thorny spines on proximal dendrites (cf. Sotelo and Dusart 2009). In our experience, it is very difficult to interpret immunofluorescence data for postsynaptic localization at this synapse as the thorny spines are very close to the dendrite, and most often the images are not very conclusive.

      2) Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

      The mean baseline surface GluA2 levels are not significantly different between WT and KO slices (results added in figure S8). We agree with Reviewer 3 that we cannot exclude an effect on endocytosis of GluA2 given that, despite the same magnitude of change on average, the statistical test shows a significant effect for controls, but not for KO. We have changed the text accordingly page 7.

      3) Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

      We have changed the panel in Figure 4D to better highlight the partial colocalization between GluA2 and SUSD4 in cultured Purkinje cells.

      4) Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

      We agree with Reviewer 3 that PVRL3aplha used as a control in the experiment in figure 5D only controls for non-specific interactions with the anti-HA beads and not for non-specific interactions of highly overexpressed membrane proteins. To control for this, we have added an experiment (Figure S9) in which we co-expressed SEP-GluA2 together with HA-SUSD4 or PVRL3alpha but this time performed co-immunoprecipitation using anti-GFP beads to pull down SEP-GluA2. We probed the extracts by immunoblot for either GluA2, HA or PVRL3alpha. In both cases SEP-GluA2 was readily immunoprecipitated. PVRL3alpha was not co-immunoprecipitated at all, while HA-tagged SUSD4 was. This result further supports a specific interaction between SEP-GluA2 and HA-SUSD4 in transfected HEK293 cells.

      5) Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

      We have modified the graph to show GluA2 in the IP normalized to the input and relative to the HA-SUSD4 construct (Figure 5D). We have added the quantifications of the GluA2 input amounts in the different conditions (Figure S11). We have also modified the text to clarify the presentation of these co-immunoprecipitation results (page 9).

      6) Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

      This has been corrected.

    2. Reviewer #3 (Public Review):

      In this study from the Selimi lab, Gónzalez-Calvo and colleagues investigate the role of the uncharacterized complement family protein SUSD4. SUSD4 is expressed at the time of cerebellar synaptogenesis and localizes to dendritic spines of Purkinje cells. Susd4 KO mice show impaired motor learning, a cerebellum-dependent task. Susd4 KO mice display impaired LTD and facilitated LTP at parallel fiber (PF)-Purkinje cell (PC) synapses, indicating altered synaptic plasticity in the absence of Susd4. Climbing fiber (CF)-Purkinje cell synapses show largely normal basal transmission, with the exception of larger quantal EPSCs. Immunohistochemical analysis shows a small decrease in the proportion of CF/PC synapses lacking GluA2. As their data indicates a role for SUSD4 in regulation of postsynaptic GluA2 content at cerebellar synapses, they next explored the molecular mechanism by which SUSD4 might do so. Activity-dependent degradation of GluA2 does not occur in the absence of SUSD4. Affinity purification of proteins associated with recombinant SUSD4 identifies ubiquitin ligases as well as several proteins involved in AMPAR turnover. Finally, the authors show that SUSD4 can bind GluA2 in HEK cells, and that SUSD4 can bind the ubiquitin ligase NEDD4, but that these two interactions are not dependent on each other.

      This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

      Specific comments:

      1) Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Fig. 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

      2) Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

      3) Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

      4) Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

      5) Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

      6) Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

    3. Reviewer #2 (Public Review):

      The authors show that SUSD4 is expressed throughout the brain and is abundant in cerebellar dendrites and spines. Mice with deletion of SUSD4 have motor coordination and learning deficits, along with impaired LTD induction. The also attempt to show that GluA2 AMPA subunits are misregulated, but that is not as convincing. They find Nedd1, along with many other proteins in a proteomic screen for SUSD4 interactors, and try to explain the phenotypes through the regulation of GluA2 degradation by Nedd4 through SUSD4. These are potentially interesting findings, but very preliminary at this point. While the electrophysiology is good, the mechanistic studies are incomplete.

      Major comments:

      In Figure 1 localization images are shown using exogenous protein. Can the authors visualize endogenous protein?

      It appears that SUSD4 is expressed in multiple brain regions, even at higher levels than the cerebellum. The authors should provide a good explanation for why deficits in the KO do not affect other functions, and seem to preferentially affect cerebellar functions.

      Figure 4: immunofluorescence data are not very convincing.

      Figure 5: The use of the word "could" is not supporting a strong conclusion. The authors should demonstrate whether SUSD4 DOES indeed regulate GluA2.

      Overall, while the electrophysiology seems fine, the mechanistic studies are preliminary and speculative at this point.

    4. Reviewer #1 (Public Review):

      This is a highly interesting manuscript by Gonzalez-Calvo et al., describing the involvement of the CCP domain containing protein SUSD4 in the degradation of GluA4 receptors at cerebellar synapses. The novelty of this work lies in the specificity of this degradation pathway. In comparison, synaptic proteins involved in AMPA receptor endocytosis, such as GRIP1 and PICK1, play a role in multiple trafficking processes. In addition, CCP domain proteins play a role in synaptic pruning, which is closely related to LTD. We will return later to this point.

      The paper will certainly enrich the field and further our understanding of cellular plasticity in the cerebellum. These are exciting findings that should be published. I have three relatively minor comments:

      1) Figure 2E: it is surprising that the potentiation shown in WT mice is not longer lasting. Under the experimental conditions used here, plasticity seems to be biased towards depression. In the methods, the authors state that they use 2mM Calcium and 1mM Magnesium in their external saline. A recent study (Titley et al., J. Physiol. 597, 2019) has demonstrated that under realistic conditions (incl. an ion milieu of 1.2 mM Calcium and 1mM Magnesium), LTP results under most conditions - even those involving climbing fiber co-stimulation - while LTD only results from prolonged complex spike firing. Optimally, the authors would establish a real LTP control in their WT mice (using conditions as described in Titley et al or similar) and test for changes in the mutants. As LTP is not the focus of this paper and this might be out of the scope of this work, it should be acceptable to leave it as it is, but this caveat should at least be discussed.

      2) Figure 3: The climbing fiber physiology is described in detail, but what is missing is a characterization of potential changes in the complex spike waveform, recorded in current-clamp mode. This should certainly be provided. This is important as it has been shown that changes in the complex spike waveform affect the probability for LTD induction (Mathy et al., Neuron 62, 2009). The CF-EPSC is a rather indirect measure.

      3) Is synaptic pruning at parallel fiber synapses impaired in the SUSD4 mutants? The LTD deficit is quite obvious. In the light of the role of autophagy in pruning, and the molecular similarity between LTD and pruning, it would be of interest to see whether activity-dependent pruning at these synapses is altered. This aspect is somewhat addressed by the VGLuT1 measures shown in Figure 2, but should be discussed in more depth.

    5. Evaluation Summary:

      The reviewers agreed that this is a very interesting paper that demonstrates the involvement of a specific protein degradation pathway in a form of synaptic plasticity in the cerebellum. The strength of the work results from its innovative character. The authors show that SUSD4 is expressed throughout the brain and is abundant in cerebellar dendrites and spines. Mice with deletion of SUSD4 have motor coordination and learning deficits, along with impaired LTD induction. This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      In this article, Gregory Grecco and colleagues developed a novel translational mouse model of prenatal methadone exposure (PME) that closely resembles the opioid exposure experienced by pregnant women living with opioid use disorder and treated with methadone maintenance pharmacotherapy. The article delineates the impact of prenatal methadone exposure on physical development and motor behavior of the next generation male and female progeny. The authors also relied on a combination of electrophysiological, immunohistochemical and volumetric MRI imaging approaches to investigate the mechanisms underlying PME-derived phenotypes in male and female offspring. Overall, PME produced changes in motor function, motor coordination and growth in progeny. These phenotypes were accompanied by changes in the electrophysiological properties and density of neurons in the primary motor cortex of offspring raised by opioid-exposed dams.

      One of the stated goals by the authors was to develop a mouse model that closely mirrored exposure and dosing regimens in clinical populations living with opioid use disorder in order to increase the translational value of the findings outlined in this report. One of the strengths of the article is the experimental design and the longitudinal nature of the studies. The dams were first treated with oxycodone, a commonly abused pain killer to mimic this condition in patients living with SUD. 5 days prior to mating, the animals were switched to methadone to model maintenance pharmacotherapy that is commonly used in SUD patients. The doses of oxycodone and methadone were carefully selected to mimic as closely as possible the suspected exposure experienced by pregnant women and their unborn offspring. The authors demonstrated that the concentrations of methadone and related metabolites were present in the plasma, brain and placentas of dams and offspring in the opioid-treated group during gestation, parturition and up to one week after birth. Another strength of the study was the fact that the authors convincingly demonstrated a lack of change in maternal behavior in the opioid-treated dams, which could have been a major confounding factor. The dams exposed to oxycodone and methadone did develop dependence to opioids as expected, however the amount and nature of maternal care delivered to their offspring was not affected by oxycodone and methadone exposure. This critical finding enabled the authors to delve further into the biological underpinnings of the observed phenotypes. The offspring produced by opioid-exposed dams showed some phenotypes consistent with neonatal opioid withdrawal syndrome (NOWS) in humans, including hyperthermia and twitches or jerks. Together, these findings demonstrate that the authors were successful in creating a novel model of prenatal opioid use and methadone maintenance in mice.

      Overall, both males and females produced by opioid-treated dams had lower body weight and length during development and through adolescence. Bone volume was also lower in PME offspring compared to controls at 1 week of age, an effect that dissipated by adolescence in PME progeny. Locomotor activity was reduced at P1 and increased at P7 and P21. Interestingly, ultra sonic vocalization emitted by pups when separated from their mothers, was highest for PME females compared to all groups and this increase in calls also coincided with increased activity. PME offspring also had delays in demonstrated coordinated motor behaviors such as acquisition of surface righting, forelimb grasp and cliff aversion during the early stages of development. Prepulse inhibition, a measure of sensorimotor gating was not disrupted by PME.

      At the anatomical level, the largest impact of PME was found in the primary motor region of the cortex, where cell density was reduced particularly in the upper cortical layers. Next, the authors probed the properties of cells and circuits in primary motor cortex and found reduced firing rates in response to injected currents in PME animals compared to controls. The input resistance of these cells was also diminished in the PME group. Together, these findings suggest that the number of cells may be reduced by PME in primary motor cortex and that the remaining neurons are not able to fire as effectively, resulting in blunted transmission within this brain region. Lastly, the authors stimulated local synaptic inputs to M1 using glutamate uncaging and found that the neural circuits connecting the top layers of M1 to layer 5 are enhanced in PME animals.

      Overall, the authors identified some electrophysiological correlates of altered motor function and coordination produced by a novel prenatal opioid exposure model and regimen. This article had several strengths highlighted above but also included some areas of potential improvement. The authors included both sexes in many of their analyses but it is not always clear when the sex of the offspring were combined in the analyses and/or whether sex was always included as a factor in the many endpoints described in the paper. The authors acknowledge some of the limitations of their model in better understanding OUD in pregnant women. Including the caveat that many women do not switch to maintenance therapy prior to conception would be worth mentioning. Moreover the use of buprenorphine has increased in recent years and methadone is not the only maintenance therapy available. Lastly, the electrophysiological recordings do not exactly coincide with some of the overt phenotypes reported: at P21, the PME animals are hyperactive but the time window does not match with the coordination deficits reported. Overall, these minor weaknesses detracted only slightly from the overall impact and value of the reported findings.

    2. Reviewer #2 (Public Review):

      This manuscript establishes a novel rodent model for prenatal methadone exposure and characterizes various aspects of neurodevelopment in the offspring. Given the global opioid crisis and the rampant rise of drug use by pregnant mothers and incidence of neonatal abstinence/opioid withdrawal syndrome, there is a critical need to determine potential outcomes for children born with this condition. In their model, the investigators use mice that are already taking oxycodone and switched to methadone treatment prior to becoming pregnant, which is a major translational advantage compared to other models where opioid dosing does not start until sometime mid-gestation. The experimental design also included a wide variety of measurable endpoints, including physical development, sensorimotor behavioral tasks, vocalizations, brain imaging, circuit electrophysiology, and histology; this comprehensive approach allows for synthesis of the results that has traditionally been difficult to find in this field, given the vast differences in species, dosing paradigms, etc. Sex differences were also considered, which is especially important given what is known about varying rates of NOWS between males and females. The text is very well-written, including detailed descriptions of statistical analysis.

      Despite overall enthusiasm for the study and its findings, there are some concerns regarding the brain volume analyses as well as potential stress confounds with the experimental design. The analysis of structural differences measured by volumetric MRI showed that there were no appreciable differences across grey matter structures with PME (Supp. Fig. 9). This was surprising, given that regional decreases in brain volume are a consistent finding with prenatal drug-exposed offspring (Yuan et al., 2014 [DOI 10.1038/jp.2014.111]; Sirnes et al., 2017 [DOI 10.1016/j.earlhumdev.2017.01.009]; Nygaard et al., 2018 [DOI 10.1016/j.ntt.2018.04.004]). Traditionally, these deficits tend to be more true for white matter than grey, though the authors do not indicate whether this was investigated.

      The opioid dosing protocol required twice-daily subcutaneous injections for at least 3 weeks (possibly longer, but it was difficult to determine from the text when exactly the treatments were halted). The effects of maternal/prenatal stress, even in the vehicles, cannot be discounted. The authors rightly noted this caveat in the Discussion, but it remains a critical concern in this otherwise well-designed study.

    3. Reviewer #1 (Public Review):

      The authors have succeeded in their attempt to develop and characterize a rigorous preclinical model of prenatal methadone exposure secondary to pre-pregnancy prescription opioid use. The model is a technical advance in terms of the opioid exposure being consistent throughout pregnancy and the outcome measures of methadone impact are rigorous. Many aspects neurodevelopment and key physiological processes are assessed and key knowledge is provided about the effects of prenatal methadone exposure on physical development, sensorimotor behavior and neuronal properties.

      Major strengths include the thoroughness and rigor of analyses and the multiple body systems study. In addition, scientific questions are approached using physical, biochemical and behavioral assessments to fully characterize the effects of prenatal methadone exposure.

      The strengths of this paper outweighs the weaknesses. Weakness are minor and include an incomplete assessment or discussion of whether withdrawal in the postnatal period may explain the pathophysiology described and changes in circuitry. Similarly, white matter analyses are not included MRI assessments confining the results to gray matter brain regions.

    4. Evaluation Summary:

      This work studied mice that had already taken oxycodone that then were switched to methadone treatment prior to becoming pregnant, to model prenatal methadone exposure (PME). The experimental design featured a study of a wide array of measures in the next generation progeny: including physical development, sensorimotor behavior, vocalizations, brain imaging, electrophysiology, and histology. All three reviewers agreed this work provides a novel, thorough, and highly clinically-relevant model of PME that has high value to the field of neuroscience of addictions and developmental neuropharmacology.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      By applying modern viral tracing methods, this paper described in detail extensive input-output connections of Gad1Cre+, VgatCre+, or Ntsr1Cre+ IntA projection neurons.

      Because diverse neurons are intermingled in a small region, it is generally challenging to isolate specific excitatory or inhibitory neurons and their circuits in the cerebellar nucleus.

      The authors focused on IntA of CN and demonstrated that 1) both inhibitory (Gad1Cre+ and/or VgatCre+) and excitatory (Ntsr1Cre+) neurons comprise extensive input-output connections with many extracerebellar regions, and 2) inhibitory circuits are functionally distinct from excitatory circuits on the basis of projection targets. This work could provide insights into diversity of inhibitory IntA neurons, and thus could be an interesting addition to the field's expanding efforts to identify cell types of CN, their input-output connections, and their functions.

      However, interpreting the data is difficult because of technical challenges. Critically, the main conclusion could be compromised by experimental artifacts, which need better characterization. In addition, the text could be revised to make it more accessible to a broad audience.

    2. Reviewer #2 (Public Review):

      Judd et al. systematically examine the input/output connectivity of discrete excitatory and inhibitory neuronal subpopulations in the cerebellar interposed anterior nucleus (IntA) using conditional AAV and rabies virus mapping strategies. The authors first define distinctions in the output connectivity of excitatory and inhibitory neurons in the IntA nucleus, and describe a surprisingly much wider projection pattern by inhibitory neurons than previously thought. They also characterize distinctions in projection pattern between identifiable subtypes of IntA inhibitory neurons as well as distinctions in morphology of their terminal fields. The authors next explore the input connectivity of excitatory and inhibitory neurons in the IntA nucleus and found that excitatory output neurons receive fewer, but more organized inputs than inhibitory output neurons, and that many output targets provide reciprocal connections with the CN.

      In general, the output analysis is strong and there are only a few questions about interpretation of the distinctions of projections by different subtypes of IntA inhibitory neurons. For instance, the distribution of the initial targeting within the cerebellar nuclei, cerebellar cortex and outside the cerebellum was not analyzed in Ntsr1-Cre and Gad1-Cre similar to the analysis performed for the intersectional output analysis. Clarification on whether and how the distinctions in projections could be due to variability in the specificity of the initial targeting or recombination ability of the two mouse Cre-lines is needed to strengthen interpretation of the different projections patterns observed. As for the input analysis using rabies, there were two major issues identified.

      First, the use of conditional GFP-labeled G protein and the use of rabies that is also GFP potentially confounds analysis of input cells.

      Next, the low number of starter cells is a concern and the identity of starter cells outside the cerebellar nuclei in Ntsr1-Cre and Gad1-Cre is vague and needs to be clarified. This is important for interpretation of whether input structures observed project specifically into the CN or also into the cerebellar cortex, and whether distinctions observed in number of input structures may reflect amount of starter cells in each Cre line.

    3. Reviewer #1 (Public Review):

      In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make widespread extra-cerebellar projections relies on the assumption that the Cre-lines used in the study are able to specifically/exclusively mark to those inhibitory neurons – these details were not fully worked out in this study.

    4. Evaluation Summary:

      Judd and colleagues use a combination of mouse genetics and viral marking to expand the extra-cerebellar map of projections. These data will impact our understanding of how the cerebellum contributes to behavior and in general how brain function is packaged at the anatomical level. These data will not only impact cerebellar scientists but also those workers interested in how inter-regional brain connectivity is organized and how fine input-output circuit relationships are structured.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2, and #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      The manuscript by Sando et al. describes experiments directed at unraveling how latrophilins (Lphns) orchestrate synapse formation. Lphns are a unique family of adhesion molecules harboring extensive extracellular N-terminal domains with several known interacting motifs coupled to the classical 7 transmembrane architecture of G-protein coupled receptors. In recently published work from the Sudhof group, Lphns were shown to play a surprising postsynaptic role in synapse formation onto CA1 pyramidal neurons with Lphn2 and 3 important for perforant path and Schaffer collateral synapse formation respectively (Sando et al., Anderson et al). However, it remains unclear whether G-protein signaling through Lphns is important for their role as synapse organizers.

      To address this issue, the authors use conditional knockout/rescue approaches to convincingly demonstrate an essential role of the GPCR domain of Lphns 2 and 3 both in vitro and in vivo. Replacing the intracellular 3rd loop of the GPCR domain (which is essential for G-protein activation) of either Lphn2 or 3 fails to rescue reduced synapse number in the knockout background (nor does deleting the entire GPCR domain). Thus it appears that cell adhesion properties alone are not sufficient for Lphn-mediated synapse formation. The experiments appear to be robust and convincing and the conceptual advance of Lphn-mediated GPCR signaling during synapse formation is substantial. I have a few specific points outlined below, but overall the authors use a nice combination of imaging, electrophysiology and rabies virus-based synaptic connectivity measurements to support their conclusions. Naturally, I'd like to know more details about the signaling requirement (e.g. how is Lphn signaling spatially compartmentalized compared to other GPCRs present, which G-protein(s) Lphns couple to, how/when/whether GPCR signaling is regulated by ligand engagement etc.) but these questions seem better suited to a separate study.

    2. Reviewer #2 (Public Review):

      This manuscript by Sando and Sudhof addresses whether GPCR activity of latrophilin2 and 3 is necessary for the role of these proteins in synapse formation. The key findings are:

      — the generation and validation of mutants that lack transmembrane and intracellular domains (but are GPI-anchored instead), the lack only intracellular domains, or that contain all domains but lack GPCR-activity. All mutants work properly in cell aggregation assays and appear to be localized normally when overexpressed in wild type neurons. This also led to the development of an elegant PKA-phosphorylation reporter assay.

      — in cultured latrohphilin 3 knockout neurons, latrophilin3 expression restores a decreased synapse density and mini-frequency, but the GPI-anchored, truncated or inactive versions do not restore these parameters.

      — in vivo/hippocampal brain slices, latrophilin2 knockout impaired perforant path but not Schaffer collateral transmission onto CA1 neurons, and rescue required latrphilin2 GPCR activity. Conversely, Latrophilin3 knockout impaired Schaffer collateral but not perforant path transmission onto CA1 neurons, and rescue required latrophilin3 GPCR activity.

      — finally, monosynaptic tracing confirmed that latrophilin3 knockout reduced inputs onto CA1 starter neurons, and rescue again required GPCR activity.

      Altogether, the data are rigorously acquired, the paper is well written, and the finding that GPCR activity is necessary for latrophilins' role is both surprising and important. It is also elegant, as coupling cell-adhesion directly to signal transduction via a single molecule for synapse formation is a compelling way to drive synaptic assemblies. Naturally, the question arises how compartmentalized GPCR-signaling then instructs synapse formation, a topic that will undoubtedly require and attract more research. This is an exciting manuscript that will inspire new research on compartmentalized GPCR signaling at the synapse. Given the central importance of surface trafficking and localization within spines for the conclusions, better description of experimental procedures and quantification, and possibly additional data would clearly strengthen this point.

    3. Reviewer #1 (Public Review):

      The general thesis of the work, provided by the authors, is the demonstration that latrophilins 2 and 3 function as classical GPCRs at the synapse and that this activity is necessary for synapse formation at a specific synapse within the hippocampus. The topic is interesting and important for several reasons. First, the knowledge of GPCRs at synaptic connections is focused largely on neurotransmitter receptors in the literature – metabotropic GluR and AChR as well as neuromodulatory neurotransmitter receptors (NPY, Seratonin etc). The mechanism demonstrated in this work concerns the function of a GPCR receptor system that could confer specificity to synapse formation.

      The effect sizes that are documented throughout this work are large, giving this reviewer confidence that the effects are robust and will be reproducible and, more importantly, are indeed a biological mechanism related to synapses.

      The other major strength of the work is that the studies in neuronal cell culture are recapitulated in vivo providing additional confidence in the validity and importance of the work. Indeed, the concept of specificity requires this type of in vivo work as the identity of synapses in culture systems can not be readily determined.

      A further strength is the rational and implementation of three mutant receptors that are used to dissect the signaling modalities of these receptors, validated for their effects on the protein and then used as rescue constructs in synaptogenesis assays.

    4. Evaluation Summary:

      The main finding that GPCR activity is necessary for latrophilins' role in synapse formation is both surprising and important. This work will inspire new research on compartmentalized GPCR signaling at the synapse.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

    1. Reviewer #3 (Public Review):

      The authors herein have nicely dissected the role of RNF43 in WNT5A signaling in mammalian cells, with a focus in the context of melanoma. They show that RNF43 inhibits WNT5A activity by ubiquitinating and thereby marking for proteasomal degradation multiple proteins involved in WNT5A signal transduction (i.e., VANGL2). The authors have performed the study in a thorough manner.

    2. Reviewer #2 (Public Review):

      In the present manuscript Radaszkiewicz et al. analyze the role of Ring Finger Protein 43 (RNF43) in inhibiting the noncanonical WNT5A pathway. The authors demonstrate that RNF43 can interact with proteins involved in the WNT5A pathway, including ROR1, ROR2, VANGL1 and VANGL2. Specifically, they propose that RNF43 induces: i) VANGL2 ubiquitination and proteasomal degradation and ii) clathrin-dependent internalization of the ROR1 receptor. Considering the role of the WNT5A pathway in melanoma metastasis and resistance to targeted therapy, the authors further explore the role of RNF43 in melanoma invasion and resistance to vemurafenib. The authors ultimately conclude that RNF43 can prevent invasion and resistance to targeted therapy by inhibiting the WNT5A pathway. The data supporting the interaction between RNF43 and proteins involved in the WNT5A pathway are pretty rigorous. However, the study would benefit from additional experiments in the context of RNF43's role in invasion and resistance to targeted therapy in melanoma. Overall, the techniques utilized in the manuscript are appropriate, however additional cell lines and in vivo studies are strongly recommended to strengthen the manuscript.

    3. Reviewer #1 (Public Review):

      The authors present data suggesting that RNF43 affects WNT5a signaling through turnover of ROR1 and ROR2 receptors on the cell surface. The strengths of this work are the many overexpression, knockdown and mutant cell lines the authors use to delineate specific protein interactions and localizations. The authors have done a good job of analyzing the interaction of multiple proteins within the Wnt signaling pathways to determine how RNF43 affects expression of proteins associated with non-canonical Wnt signaling. The weakness of this study is that most of these protein interactions were performed in 293 cells and not in melanoma cell lines. One melanoma cell line was used to relate the protein interactions studied in 293 cells to signaling in melanoma. The authors present data that suggest RNF43 decreases invasion and proliferation of melanoma cells in vitro. Analyzing the role of RNF43 in invasion, proliferation and signaling in more than one melanoma cell line would strengthen the authors conclusions about the role of RNF43 in Wnt5A signaling in melanoma.

    4. Evaluation Summary:

      Radaszkiewicz and collaborators describe RNF43 as a novel negative regulator of WNT5A-induced signaling in human cells. They demonstrate that RNF43 can interact with proteins in this pathway, namely ROR1, ROR2, VANGL1 and VANGL2. Specifically, they find that, through these interactions, RNF43 can suppress invasive properties of melanoma cells, as well as the development of resistance to BRAF V600E inhibitor. The experiments are well done and well explained; however, they were performed only in an in vitro setting and with a very limited number of cell lines.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      P2X2 receptor channels do not have a canonical voltage-sensor, yet they display profound voltage-dependence especially when activated by physiologically relevant low ATP concentrations. Understanding the mechanisms of this voltage dependence is not an easy undertaking because there are neither similar proteins as precedent nor clear indications from available structures. In this manuscript, Andriani and Kubo incorporated Anap into 96 residues (separately) in P2X2 receptor channels and performed a comprehensive scanning using voltage-clamp fluorometry technique to probe structural changes during ATP- and voltage-dependent gating. Out of the 96 residues, the authors only observed voltage-dependent fluorescence intensity (F) changes at A337 and I341 in the TM2 domain. The changes are fast and linear, consistent with them being electrochromic effect. When an additional mutant K308R is introduced, the authors were able to detect a small slow and voltage-dependent F change at A337, which could potentially result from structural rearrangements at this position. With a P2X2 model built upon the hP2X3 open state structure, they also proposed that A337 interacts with F44 in TM1, and this interaction is important for activation. The amount of work involved in this study is impressive. The data presented are of good quality. Most conclusions drawn from the results are reasonable and backed with good evidence.

      Overall, the identification of a converged electric field around A337 and I341 is new and intriguing. Previously reported functional results and available high resolution P2X receptor structures all suggest that residues A337 and I341 are facing TM1 and they are accessible to Ag+ when mutated to Cys. It is conceivable that the "voltage-sensor" in P2X2 receptor channels involve ion filled crevices between TM1 and TM2 in the membrane. This work is of great value for understanding how membrane proteins sense voltages.

    2. Reviewer #2 (Public Review):

      P2X2 activation depends on both ATP binding and voltage. However, the voltage sensor of P2X2 is not elucidated. This manuscript describes the study of voltage dependent conformational changes of P2X2 using voltage clamp fluorometry of the fluorescent unnatural amino acid Anap that substituted P2X2 amino acid residues. 96 positions in different structural domains were scanned by substituting with Anap, and voltage dependent fluorescence signals were detected only at two positions, A337 and I341 in the TM2 domain. A fast and linear voltage dependence of fluorescence suggested that the membrane voltage converged at and around these two positions. With a mutation K308R that was supposed to enhance voltage dependent conformational changes, Anap at the A337 position showed a time and voltage dependent fluorescence. The authors concluded that this result indicated a voltage dependent conformational change. Structure guided mutations suggested that F44 in TM1 might move to interact with A337 in response to voltage. In this study the fluorescence signals were small, but the authors made a great effort and managed to obtain the data that are convincing. The experiments were well designed and the manuscript is clearly reasoned. Considering that among all the positions that were tested only at the two positions in the TM2 segment Anap showed voltage dependent fluorescence, and that the F44 mutations abolished voltage dependence of the P2X2 currents, the conclusion that voltage converges at the A337/I341/F44 and induces a conformational change seems to be well supported.

    3. Reviewer #1 (Public Review):

      The study aims to determine the mechanism of voltage-sensing in P2X2 receptor. These receptors are primarily activated by ligand, ATP but their activity is also regulated to some extent by voltage even though they lack a canonical voltage-sensing domain. To address this question, the authors introduce unnatural fluorescent amino acid throughout the structure of the P2X2 receptor. The interaction between excited state dipole and electric fields can cause shift in the fluorescence emission and excitation spectra. For a given probe, the extent of these shifts are directly proportional to the strength of the electric field. The authors exploit this phenomenon to determine the strength of the electric field in the various regions of the P2X2 receptor. The underlying premise is that the regions which sense the largest electric field are likely to be the primary sensors of membrane voltage.


      The approach to localize the putative voltage-sensing region is novel and maybe broadly applicable to other voltage-regulated channels which lack canonical voltage-sensors.

      Unnatural amino acid, ANAP was introduced and tested at 96 positions in the structure of P2X2 receptor. This is an insane amount of work and has to be a tour de force.


      The main limitation of this approach is that ANAP is not going to be incorporated with equal efficiency at all sites and therefore, it is likely that some of the potential where the electric field is strong may remain undetected.

      Overall, using ANAP scanning approach, they were able to identify couple of sites in TM2 helix which exhibits large electrochromic signals. Furthermore, they find that the interaction between Ala 337 and Phe44 is critical for voltage-dependent response. These studies lay the groundwork for further investigations of the mechanism of voltage-sensing these physiologically important ion channels.

    4. Evaluation Summary:

      This study will be of broad interest to ion channel researchers interested in understanding the fundamental mechanisms of voltage-sensing. The researchers use a novel approach to determine the mechanism of voltage-sensing in a channel that lacks a canonical voltage-sensing domain.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      The authors describe a method for fitting a simple, separable function of contrast and cone excitation to a set of fMRI data generated from large, unstructured chromatic flicker stimuli that drive the L- and M- cone photoreceptors across a range of amplitudes and ratios. The function is of the form of a scaled ellipse – hereafter referred to as a 'Quadratic Color Model' (QCM). The QCM fits 6 parameters (ellipse orientation, ellipse elongation, and 4 parameters from a non-linear, saturating (Naka-Rushton) contrast response curve. The QCM fits the dataset well and the authors compare it (favorably) to a 40-parameter GLM that fits each separate combination of chromatic direction and contrast separately.

      The authors note three things that 'did not have to be true' (and which are therefore interesting):

      1) The data are well-fit by a separable ellipse+contrast transducer - consistent with the idea that the underlying neuronal computations that process these stimuli combine relatively independent L-M and L+M contrast.

      2) The short axis of the QCM tends to align with the L-M cone contrast directing (indicating that this direction is one of maximum sensitivity and the L+M direction (long axis) is least sensitive. This finding is qualitatively consistent with psychophysical measurements of chromatic sensitivity.

      3) Fit parameters do not change much across the cortical surface – and in particular they are relatively constant with respect to eccentricity.

      This is a technically solid paper – the data processing pipeline is meticulous, stimuli are tightly-calibrated (the ability to apply cone-isolating stimuli to fovea and periphery simultaneously is an impressive application of the 56-primary stimulus generator) and the authors have been careful to measure their stimuli before and after each experimental session. I have a few technical questions but I am completely satisfied that the authors are measuring what they think they are measuring.

      The analysis, similarly, is exemplary in many ways. Robust fitting procedures are used and model performance and generalizablility are evaluated with a leave-run-out and leave-session-out cross validation procedures. Bootstrapped confidence intervals are generated for all fits and analysis code is available online.

      The paper is also useful: it summarises a lot of (similar) previous findings in the fMRI color literature going back to the late 90s and points out that they can, in general, be represented with far fewer parameters than conditions. My main concerns are:

      1) Underlying mechanisms: The QCM is a convenient parameterization of low spatial-frequency, high temporal-frequency L-M responses. It will be a useful tool for future color vision researchers but I do not feel that I am learning very much that is new about human color vision. The choice to fit an ellipse to these data must have been motivated at least in part by inspection. It works in this case (possibly because of the particular combination of spatial and temporal frequencies that are probed) but it is not clear that this is a generic parametric model of human color responses in V1. Even very early fMRI data from stimuli with non-zero spatial frequency (for example, Engel, Zhang and Wandell '97) show response envelopes that are ellipse-like but which might well also have additional 'orthogonal' lobes or other oddities at some temporal frequencies.

      2) Model comparison: The 40-parameter GLM model provides a 'best possible' linear fit and gives a sense of the noisiness of the data but it feels a little like a strawman. It is possible to reduce the dimensionality of the fit significantly with the QCM but was it ever really plausible that the visual system would generate separate, independent responses for each combination of color direction and contrast? I suspect that given the fact that the response data are not saturating, it would be possible to replace the Naka-Rushton part of the model with a simple power function, reducing the parameter space even further. It would be more interesting to use the data to compare actual models of color processing in retina/V1 and, potentially, beyond V1.

      3) Link to perception. As the authors note, there is a rich history of psychophysics in this domain. The stimuli they choose are also, I think, well suited to modelling in the sense that they are likely to drive a very limited class of chromatic cells in V1 (those with almost no spatial frequency tuning). It is a shame therefore that no corresponding psychophysical data are presented to link physiology to perception. The issue is particularly acute because the stimulus differs from those typically used in more recent psychophysical experiments: it flickers relatively quickly and it has no spatial structure. It may, however, be more similar to the types of stimuli used prior to the advent of color CRTs : Maxwellian view systems that presented a single spot of light.

    2. Reviewer #2 (Public Review):

      The goal of this work is to advance knowledge of the neural bases of color perception. Color vision has been a model system for understanding how what we see arises from the coordinated action of neurons; detailed behavioral measurements revealed color vision's dependence upon three types of photoreceptors (trichromacy) and three second stage retinal circuits that compute sums and differences of the cone signals (color opponency). The processing of color at later, cortical stages has remained poorly understood however, and studies of human cortex have been hampered by methodologies that abandoned the detailed approach. Typical past work simply compared neural responses in two conditions, the presentation of colorful (formally, chromatic) vs grayscale (luminance) images. The present work returns to the older tradition that proved so successful.

      The project's specific goals were to measure functional MRI responses in human cortex to a large range of colors, and equally importantly, capture the pattern responses with a quantitative model that can be used to predict response to many additional colors with just a few parameters. The reported work achieved these goals, establishing both a comprehensive data set and a modeling framework that together will provide a strong basis for future investigations. I would not hesitate to query the data further or to use the QCM model the paper provides to characterize other data sets.

      The strengths of the work include its methodological rigor, which gives high confidence that the goals were achieved. Specifically:

      1) The visual presentation equipment was uniquely sophisticated, allowing it to correct for possible confounds due to differences in photoreceptor responses across the retina.

      2) The testing of the model was quite rigorous, aided by distinct replications of the experiment planned prior to data collection.

      3) The fMRI methods were also state of the art.

      The work was well-situated within the literature, comparing its findings to past results. The limitations and assumptions of the present work were also clearly stated, and conclusions were not overstated.

      Weaknesses of the current draft are relatively minor, however, I believe:

      1) The data could be presented in a way to make them more comparable to prior fMRI work, e.g. by using percent change units in more places, comparing the R^2 of model fits reported here to those reported in other papers, and explaining and exploring how the spatially uniform stimuli, used here but not in other fMRI studies, limited responses in visual areas beyond V1.

      2) Comparison between the two models, the GLM and QCM is not quite complete.

      3) The present results are not discussed in context with past results using EEG, and Brouwer and Heeger's model of fMRI responses to color.

      4) Implications of the basic pattern of response for the cortical neurons producing the data are discussed less than they could be.

    3. Reviewer #1 (Public Review):

      This manuscript presents new data and a model that extend our understanding of color vision. The data are measurements of activity in human primary visual cortex in response to modulations of activity in the L- and M-cone photoreceptors. The model describes the data with impressive parsimony. This elegant simplification of a complex data set reveals a useful organizing principle of color processing in the visual cortex, and it is an important step towards construction of a model that predicts activity in the visual cortex to more complex visual patterns.

      Strengths of the study include the innovative stimulus generation technique (which avoided technical artifacts that would have otherwise complicated data interpretation), the rigor of experimental design, the clear and even-handed data presentation, and the success of the QCM.

      The study could be improved by a more thorough vetting of the QCM and additional discussion on the biological substrate of the activation patterns.

    4. Evaluation Summary:

      This paper will be of interest to neuroscientists who study relationships between visual stimuli and their cortical representation, particularly, but not exclusively, those who use functional imaging techniques. The experiments are carefully designed, the dataset is substantial, and a model is presented that describes the data with very few parameters.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      Myotonia congenita is a heritable disorder of muscle fiber excitability in which a severe reduction of the resting chloride conductance (gCl, CLCN1 mutations) produces susceptibility to involuntary after-contractions and transient weakness. Fifty years ago, Bryant, Adrian and colleagues showed that loss of > 50% of gCl is sufficient to cause myotonic bursts of after-discharges. Much less is known about the mechanistic basis for the transient weakness (several seconds, up to 1 minute) that occurs with initial contractions after rest. This study elegantly confirms what has long been suspected; that sustained depolarization of the resting potential is the basis for the transient weakness. The experimental approach employed several new techniques to achieve this demonstration. First, the use of repeated in situ contraction tests every 4 sec (Fig. 1) clearly shows the coincidence of myotonia and transient weakness, both of which exhibit warm-up. This animal model for the transient weakness in a low gCl state was essential for the success of this study. Secondly, the remarkably stable measurements of membrane potential (Vm), without the need to apply a holding current to achieve the normal resting potential (Figure 2) is necessary to convincingly demonstrate the plateau depolarizations are a consequence of the myotonic condition, and not a stimulation artifact. Moreover, a severe reduction of fiber excitability was directly demonstrated by application of brief current pulses during the plateau depolarization (Figure 2E). Third, the authors have used the ncDHPR mouse (non-conducting CaV1.1) to show the Ca current has some role in prolonging the duration of the plateau. This is an important contribution because the sluggish, low-amplitude Ca current in skeletal muscle has not previously been implicated in the pathogenesis of myotonia. Finally, the authors built upon their recent work showing ranolazine suppresses myotonia in low gCl muscle to also show this drug abolishes the plateau potential. Taken together, this excellent study provides the most definitive experimental evidence to date for the mechanistic basis of transient weakness in myotonia congenita and also suggests ranolazine may be beneficial for prophylactic management.

      Major Points:

      1) The major experimental limitation that prevented prior studies from establishing the mechanism for the transiently reduced excitability and weakness in MC was the concern that plateau depolarizations frequently occur as an artifact in studies of skeletal muscle membrane potential (e.g. secondary to leakage current from electrode impalement or failure to completely suppress contraction with motion-induced damage). The authors are to be commended for including many records of Vm (absolutely necessary for this publication) and for explicitly stating that a holding current was not applied to maintain Vrest. The confidence of these observation could be further increased by addressing these questions:

      — Were recordings excluded from the analysis if the plateau potential was not followed by a subsequent return to Vrest? Was a criterion used to define successful return to the resting potential?

      — If fibers that failed to repolarize were excluded, was this a frequent or a rare event, and importantly, was the likelihood of failure different for control versus myotonic fibers?

      2) The data clearly show a large variance for the duration of the plateau potential (e.g. horizontal extent of data in Figure 3B), which is interesting and may provide additional insights on the balance of currents that contribute to this phenomenon. The authors also point out that the distribution was skewed toward briefer plateau periods for the 9-AC model than the adr mouse. It is suggested this difference may be a consequence of life-long reduced gCl in adr mice with some chronic compensation versus the acute block of ClC-1 in the 9-AC model. What about the possibility that the reduction of gCl is more severe in the adr fibers than in 9-AC treated animals? A residual Cl current could foreshorten the duration of the plateau potential. Another question with regard to the variable duration of the plateau potential is a "duration of 0". In other words, as shown in Fig 3C, how frequently was the absence of a PP encountered?

      3) The possibility that activity-dependent accumulation of myoplasmic Ca may contribute to the PP is suggested (page 9 line 175), but this is not further commented upon in the Discussion. Namely, is the reduction of PP duration in ncDHPR fibers proposed to be a consequence of less inward charge movement or of less myoplasmic Ca accumulation (i.e. is it a balance of ionic currents or an intracellular signaling factor)? Moreover, with regard to an activity-dependent process that influences the likelihood and/or duration of the PP, the authors quantify the "mean firing rate" and the "mean membrane potential", both quantified during the preceding myotonic burst. Both of these factors may contribute to an activity-dependent process, but another factor has been omitted; namely the duration of the antecedent myotonic run. It would be interesting to test whether the duration of the myotonic burst had an influence on the PP.

    2. Reviewer #2 (Public Review):

      The manuscript by Myers et al provides new insight into the mechanism of transient muscle in myotonia congenita, a question that has escaped understanding since its first description over >40 years ago. The authors use a complementary set of approaches (including measurements of in situ muscle force production, membrane voltage and ion currents) to determine the membrane conductances that underlie transient weakness in muscle from both genetic (Clc1-/- adr mice) and pharmacologic (9-AC-treated WT mice) models of myotonia congenita. The authors utilize a combination of a non-conducting Cav1.1 mouse and treatment with ranolazine to dissect the relative contribution of Cav1.1 and persistent Nav1.4 conductances, respectively, to sustained plateau membrane depolarizations observed following myotonic runs, which are proposed to underlie the transient weakness observed following myotonic runs.

    3. Reviewer #1 (Public Review):

      Patients with myotonia congenita caused by loss-of-function mutations in ClC-1 experience muscle stiffness (due to hyperexcitability) as well as transient muscle weakness. This study examines the mechanisms underlying the transient muscle weakness seen myotonia congenita. The authors show that a ClC-1 null mouse exhibits the transient weakness after muscle stimulation observed in humans. Current clamp recordings of muscle fibers from ClC-1-null mice showed indicated myotonia after electrical stimulation that often terminated in a plateau potential for varying periods, during which the muscle was unexcitable, before repolarization to the resting membrane potential. The myotonia and plateau potentials could be recapitulated in wild type muscle fibers with acute pharmacological inhibition of ClC-1. Experiments in fibers from a non-conducting Cav1.1 knockin mouse indicated Ca2+ influx is important for sustaining, but not initiating, plateau potentials. Ranolazine blocked both the myotonia and development of a plateau potential in isolated muscle fibers, as well as the in vivo transient muscle weakness observed in ClC-1-null mice, implicating Na+ persistent inward currents through Nav1.4 (NAPIC) as the molecular mechanism.

      Overall, the experiments presented in this work are well-executed and the results convincing. While the role of NAPIC in the development of myotonia in ClC mice has been previously reported this work provides the new insight that it is also responsible for the development of plateau potentials that underlie muscle weakness in myotonia congenita.

    4. Evaluation Summary:

      Patients with myotonia congenita (or Becker disease) experience episodes of transient muscle weakness but the reasons underlying this phenomenon are unknown. This study provides the most definitive experimental evidence to date for the mechanistic basis of transient weakness in myotonia congenita and also suggests ranolazine may be beneficial for prophylactic management.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2, and #3 agreed to share their names with the authors.)

    1. Joint Public Review:

      The presented manuscript takes a very comprehensive look at the molecular underpinnings of the differential outcomes of IL-27 and IL-6 signaling. Both cytokines engage GP130 as a cellular receptor, however while IL-6 uses homodimers of this signal transducing receptor, IL-27 signals through a heterodimer of GP130 and IL-27Ra. Both receptor complexed lead to the phosphorylation and activation of STAT1 and STAT3 and, hence, to a similar transcriptional program. Strikingly, however, IL-27 responses lean more towards an anti-inflammatory nature (suppressing Th17 and supporting Treg responses), and IL-6 stimulates a classical inflammatory response (inhibiting Treg differentiation, supporting Th17 generation). The presented study deals with elucidating this functional pleiotropy of similar or identical signal transducers.

      The authors follow a comprehensive and elaborated approach, combining in vitro experiments in cell lines and human Th1 cells with (phospho-)proteomics, transcriptome sequencing and mathematical modeling, which gives rise to an impressive data set presented in this manuscript. The large body of experimental work is complemented by mathematical modelling of the signaling pathway(s), which is used to discriminate feasibility of distinct hypothesis in terms of mechanisms behind differential STAT activation.

      The major finding of the study is that IL-27, at least in certain cells (Th-1), leads to the stronger and more sustained activation of STAT1 as compared to IL-6, and that this higher activation of STAT1 is the basis of the differential transcriptional result. The subsequent -omics analyses support differences in signaling outcome between IL-6 and IL-27, and provide an interesting data base for the community. Finally, data re-analysis in a cohort of patients suffering from the autoimmune disease Systemic lupus erythematosus (SLE), reproduced the effects expected by the mathematical model, potentially pointing to differences in their response to different cytokines.

      Overall, the extensive and complex study presents a comprehensive analyses of IL-6 and IL-27 signaling, puzzling together pieces that may have been around before but not put into meaningful context. It provides a compelling overall idea and model of how cytokine receptors make differential use of STAT proteins.

    2. Evaluation Summary:

      This study is a great example of an elaborate combination of experimental and mathematical analyses to examine an intriguing, pleiotropic immunological signaling pathway. While a good number of individual aspects of this signaling pathway have been studied and reported before, the present work pieces together many pieces and succeeds to present a conclusive and comprehensive model of this particular cytokine system. The main conclusions are well supported by the presented data and the manuscript will be of interest and relevance for the study of many other cytokine signaling pathways, being of broad relevance for immunologists and cell biologists.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

    1. Reviewer #3 (Public Review):

      Lee et al. report results from an fMRI experiment with repeated viewings of a single movie clip, finding that different brain regions come to anticipate events to different degrees. The findings are brief but a potentially very interesting contribution to the literature on prediction in the brain, as they use rich movie stimuli. This literature has been limited as it has typically focused on fixed short timescales of possible anticipation, with many repetitions of static visual stimuli, leading to only one possible time scale of anticipation. In contrast, the current video design allows the authors to look in theory for multiple timescales of anticipation spanning simple sensory prediction across seconds to complex social dynamics across tens of seconds.

      The authors applied a Hidden Markov Model to multivoxel fMRI data acquired across six viewings of a 90 second movie. They fit a small set of components with the goal of capturing the different sequentially-experienced events that make up the clip. The authors report clusters of regions across the brain that shift in their HMM-identified events from the first viewing of the movie through the (average of the) remaining 5 viewings. In particular, more posterior regions show a shift (or 'anticipation') on the order of a few seconds, while more anterior regions show a shift on the order of ~10 seconds. These identified regions are then investigated in a second way, to see how the HMM-identified events correspond to subjective event segmentation given by a separate set of human participants. These data are a re-analysis of previously published data, presenting a new set of results and highlighting how open sharing of imaging data can have great benefits. There are a few important statistical issues that the authors should address in a revision in order to fully support their arguments.

      1) The authors report different timescales of anticipation across what may be a hierarchy of brain regions. However, do these timescales change significantly across regions? The paper rests in part on these differences, but the analyses do not yet actually test for any change. For this, there are multiple methods the authors could employ, but it would be necessary to do more than fit a linear model to the already-reported list of (non-independently-sorted) regions.

      2) The description of the statistical methods is unclear at critical points, which leads to questions about the strength of the results. The authors applied the HMM to group-averaged fMRI data to find the neural events. Then they run statistical tests on the difference in the area-under-the-curve (AUC) results from first to other viewings. It seems like they employ bootstrap testing using the group data? Perhaps it got lost, but the methods described here about resampling participants do not seem to make sense if all participants contributed to the results. Following this, they note that they used a q < 0.05 threshold after applying FDR for the resulting searchlight clusters, but based on their initial statement about the AUC tests, this is actually one-tailed? Is the actual threshold for all these clusters q < 0.10? That would be quite a lenient threshold and it would be hard to support using it. The authors should clarify how these statistics are computed.

      3) Regarding the relationship to annotated transitions, the reported difference in correlations at zero lag don't tell the story that the authors wish they tell, and as such it does not appear that they support the paper. While it is interesting to see that the correlation at zero lag in the initial viewing is often positive in the independently identified clusters, the fact that there is a drop in correlation on repeated viewings doesn't, in itself, mean that there has been a shift in the temporal relationship between the neural and annotated events. A drop in correlation could also occur if there was just no longer any correlation between the neural and annotated events at any lag due to noisy measurements, or even if, for example, the comparison wasn't to repeated viewings but to a totally different clip. The authors want to say something about the shift in in the waveform/peak, but they need to apply a different method to be able to make this argument.

      4) Imaging methods with faster temporal resolution could reveal even earlier reactivation, or replay, of the movies, that would be relatively invisible with fMRI, and the authors do not discuss relevant recent work. E.g. Michelmann et al. 2019 (Nat Hum Beh) and Wimmer et al. 2020 (Nat Neuro) are quite relevant citations from MEG. Michelmann et al. utilize similar methods and results very similar to the current findings, while Wimmer et al. use a similar 'story' structure with only one viewing (followed by cued retrieval) and find a very high degree of temporal compression. The authors vaguely mention faster timescale methods in the discussion, but it would be important to discuss these existing results, and the relative benefits of these methods versus the benefits and limitations of fMRI. It would be interesting and puzzling if there were multiple neural timescales revealed by different imaging methods.

      5) The original fMRI experiment contained three conditions, while the current results only examine one of these conditions. Why weren't the results from the two scrambled clip conditions in the original experiment reported? Presumably there were no effects observed, but given that the original report focused on a change in response over time in a scrambled video where the scrambled order was preserved across repetitions, and the current report also focuses on changes across viewings, it would be important to describe reasons for not expecting similar results to these new ones in the scrambled condition.

    2. Reviewer #2 (Public Review):

      Aly et al. investigated anticipatory signals in the cortex by analysing data in which participants repeatedly watched the same movie clip. The authors identified events using an HMM-based data-driven event segmentation method and examined how the timing of events shifted between the initial and repeated presentation of the same video clip. A number of brain regions were identified in which event timings were shifter earlier in time due to repeated viewing. The main findings is that more anterior brain regions showed more anticipation than posterior brain regions. The reported findings are very interesting, the approach the authors used is innovative and the main conclusions are supported by the results and analyses. However, many cortical regions did not show any anticipatory effects and it is not clear why that is. In part, this may be due to a number of suboptimal aspects in the analysis approach. In addition, the analyses of behavioural annotations are open to multiple interpretations.

      Methods and Results:

      1) The paper shows that across multiple regions in the cortex, there is significant evidence for anticipation of events with repeated viewing. However, there are also many areas that do not show evidence for anticipation. It is not clear whether this is due to a lack of anticipation in those areas, or due to noise in the data or low power in the analyses. There are two factors that may be causing this issue. First, the data that were used are not optimal, given the short movie clip and relatively low number of participants. Second, there are a number of important issues with the analyses that may have introduced noise in the observed neural event boundaries (see points 2-4 below).

      2) Across all searchlights, the number of estimated events was fixed to be the same as the number of annotated events. However, in previous work, Baldassano and colleagues (2017) showed that there are marked differences between regions in the timescales of event segmentation across the cortex. Therefore, it may be that in regions such as visual cortex, that tends to have very short events, the current approach identifies a mixture of neural activity patterns as one 'event'. This will add a lot of noise to the analysis and decrease the ability of the method to identify anticipatory event timings, particularly for regions lower in the cortical hierarchy that show many more events than tend to be observed in behavioural annotations.

      3) If I understand correctly, the HMM event segmentation model was applied to data from voxels within a searchlight that were averaged across participants. Regular normalization methods typically do not lead to good alignment at the level of single-voxels (Feilong et al., 2018, Neuroimage). Therefore, averaging the data without first hyperaligning them may lead to noise due to functional alignment issues within searchlights.

      4) In the analyses the five repeated viewings of the clips were averaged into a single dataset. However, it is likely that participants' ability to predict the upcoming information still increased after the first viewing. That is especially true for perceptual details that may not have been memorised after watching the clip once, but will be memorised after watching it five times. It is not clear why the authors choose to average viewings 2-6 rather than analyse only viewing 6, or perhaps even more interesting, look at how predictive signals varied with the number of viewings. I would expect that especially for early sensory regions, predictive signals increase with repeated viewing.

      5) In the analyses of the alignment between the behavioural and neural event boundaries, the authors show the difference in correlation between the initial and repeated viewing without taking the estimated amount of anticipation into account. I wonder why the authors decided on this approach, rather than estimating the delay between the neural and behavioural event boundaries. The finding that is currently reported, i.e. a lower correlation between neural and annotated events in the repeated viewing condition, does not necessarily indicate anticipation. It could also suggest that with repeated viewing, participants' neural events are less reflective of the annotated events. Indeed the results in figure 5 suggest that the correlations are earlier but also lower for the repeated viewing condition.

      6) To do the comparison between neural and annotated event boundaries, the authors refit the HMM model to clusters of significant voxels in the main analysis. I wonder why this was done rather than using the original searchlights. By grouping larger clusters of voxels, which cover many searchlights with potentially distinct boundary locations, the authors may be introducing noise into the analyses.


      7) To motivate their use of the HMM model, the authors state that: "This model assumes that the neural response to a structured narrative stimulus consists of a sequence of distinct, stable activity patterns that correspond to event structure in the narrative." If neural events are indeed reflective of the narrative event structure, what does it mean if these neural events shift in time? How does this affect the interpretation the association between neural events and narrative events?

    3. Reviewer #1 (Public Review):

      In this study, Lee et al. reanalyzed a previous fMRI dataset (Aly et al., 2018) in which participants watched the same 90s movie segment six times. Using event-segmentation methods similar to Baldassano et al. (2017), they show that event boundaries shifted for the average of the last 5 viewings as compared to the first viewing, in some regions by as much as 12 seconds. Results provide evidence for anticipatory neural activity, with apparent differences across brain regions in the timescale of this anticipation, in line with previous reports of a hierarchy of temporal integration windows.

      – One of the key findings of the paper – long-timescale anticipatory event reinstatement – overlaps with the findings of Baldassano et al., 2017. However, the previous study could not address the multiple time scales/hierarchy of predictions. Considering that this is the novel contribution of the current study, more statistical evidence for this hierarchy should be provided.

      – The current hierarchy of anticipation is closely linked to (and motivated by) previous studies showing evidence of a hierarchy of temporal integration windows. Indeed, the question of the study was "whether this hierarchy also exists in a prospective direction". This question is currently addressed somewhat indirectly, by displaying above-threshold brain regions, but without directly relating this hierarchy to previous findings of temporal integration windows, and without directly testing the claimed "posterior (less anticipation) to anterior (more anticipation) fashion" (from abstract).

      – The analysis is based on averaging the data of the 5 repeated viewings and comparing this average with the data of the first viewing. This means that the repeated viewing condition had much more reliable data than the initial viewing condition. This could potentially affect the results (e.g. better fit to HMM). To avoid this bias, the 5 repeated viewings could be entered separately into the analysis (e.g., each separately compared to the first viewing) and results averaged at the end. Alternatively, only the 6th viewing could be compared to the first viewing (as in Aly et al., 2018).

      – Correlation analysis (Fig 6). "we tested whether these correlations were significantly positive for initial viewing and/or repeated viewing, and whether there was a significant shift in correlation between these conditions". It was not clear to me how we should interpret the correlation results in Figure 6. Might a lower correlation for repeated viewing not also reflect general suppression (e.g. participants no longer paying attention to the movie)? Perhaps comparing the correlations at the optimal lag (for each cluster) might help to reduce this concern; that is, the correlation difference would only exist at lag-0.

      – Correlation analysis (Figure 6). "For both of these regions the initial viewing data exhibits transitions near the annotated boundaries, while transitions in repeated viewing data occur earlier than the annotated transitions" How was this temporal shift statistically assessed?

      – Not all clusters in Figure 2/6 look like contiguous and meaningful clusters. For example, cluster 9 appears to include insula as well as (primary?) sensorimotor cortex, and cluster 4 includes both ventral temporal cortex and inferior parietal cortex/TPJ. It is thus not clear what we can conclude from this analysis about specific brain regions. For example, the strongest r-diff is in cluster 4, but this cluster includes a very diverse set of regions.

      – In previous related work, the authors correlated time courses within and across participants, providing evidence for temporal integration windows. For example, in Aly et al., 2018 (same dataset), the authors correlated time courses across repeated viewings of the movie. Here, one could similarly correlate time courses across repeated viewings, shifting this time course in multiple steps and testing for the optimal lag. This would seem a more direct (and possibly more powerful) test of anticipation and would link the results more closely to the results of the previous study. If this analysis is not possible to reveal the anticipation revealed here, please motivate why the event segmentation is crucial for revealing the current findings.

    4. Evaluation Summary:

      The study addresses a topic that is timely and of general interest. The findings represent a potentially very interesting contribution to the important question of how the brain comes to predict the future, in particular lifelike sequences of events. However, some of the main conclusions would require further statistical support.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

    1. Reviewer #3 (Public Review):

      In a previous study, the authors had shown that germline tumors that accumulate in the C. elegans gonad because of the lack the RNA binding translational repressor GLD-1, have an increased propensity to differentiate and express somatic proteins in response to ER stress induced by tunicamycin or the absence of the TRK kinase protein tfg-1 (a process the authors call GED). Using this as a model, here, the authors investigate the mechanisms by which the abnormal nuclei accumulate in the tumorous gonad of glp-1 animals by manipulating genes in the soma and germline.

      The key message of this paper is, then, the identification of neurons and neuromodulators that suppress or enhance this accumulation of abnormal germline cells in the glp-1 germline. While the results of this analysis could potentially provide an interesting advance, the validity of the many of the conclusions are difficult to evaluate because of limitations posed by the experimental methods and ambiguity in defining the GED.


      A key issue is the identity of the abnormal germline cells that accumulate in glp-1 gonads. Modulation of the neuronal circuits examined (FLP-6, serotonin, cholinergic) change the germline, alter ovulation rates, modulate somatic gonad contraction rates etc. in wild-type animals. The effects of these circuits on a glp-1 germline are not known, but some of the same effects are likely to continue even if germ cells turned tumorous. Therefore, how neurons and neuromodulators alter the accumulation of abnormal cells in the gonad may or may not be surprising or novel, based on what is actually happening to these cells (the phenotype scored as GED). However, this is unclear as all the abnormal effects on the germline are assessed using DAPI at some steady state. Therefore, GED (ectopic differentiation) needs to be better demonstrated separate from the simple accumulation of abnormal nuclei, which could happen for a number of different reasons.


      One strength of this paper is the identification of the neuropeptide FLP-6 as a suppressor of GED and a possible RIDD target. However, there is insufficient analysis conducted to fully support this claim.

    2. Reviewer #2 (Public Review):

      Levi-Ferber and colleagues showed in their previous paper that ER stress regulates germline transdifferentiation in a way that is IRE-1 dependent, but XBP-1 independent. An open question at that time was how IRE-1 activation could mediate this signaling. The authors present several experiments in this manuscript that support the idea that neuronal Ire-1 can cell non-autonomously control germline differentiation through regulation of the neuropeptide FLP-6. Mechanistically, the authors characterize that FLP-6 is a target of IRE-1 RIDD activity. This is the first demonstration of RIDD in C. elegans, an important finding given that no RIDD targets have yet been identified in this organism. Using a wide range of mutants, the authors were also able to identify a neuronal circuit that can control the germline ectopic differentiation (GED) phenotype, involving the sensory neuron ASE, the interneuron AIY, and the motor neuron HSN. The data presented in the manuscript are sound, the mapping of a pivotal three-neuron circuit is impressive, and the findings are likely to be of high interest to a broad readership. However, some more evidence is required to support some of the conclusions made, in particular the characterization of flp-6 as a substrate for RIDD.

    3. Reviewer #1 (Public Review):

      In this manuscript, Levi-Ferber et al use C elegans to study how germline cells maintain pluripotency and avoid GED (germline ectopic differentiation) before fertilization. The authors previously showed that activation of the ER stress sensor Ire1 (but not its major downstream target Xbp1) enhances GED, and here they explore the mechanism of this effect.

      The authors convincingly – and surprisingly – show that the Ire1-mediated GED increase results not from Ire1 activity in the germline but in the nervous system, specifically in certain sensory neurons. Worms lacking a specific neuropeptide (FLP-6) or a particular neuron that produces this peptide (ASE) also displayed increased GED. Although FLP-6 deficiency did not induce ER stress, ER stress did lead to a reduction of FLP-6 transcript (and protein) levels in an Ire1-dependent manner, suggesting this RNA is a target of Regulated Ire1-dependent decay (RIDD). The authors then go on to map out the signaling cascade that begins with FLP6 reduction in ASE by Ire1 and is transmitted to the gonad via an ASE-AIY-HSN circuit, including serotonin produced by HYE.

      This paper is quite interesting and for the most part the data are very convincing and support the model. The demonstration that Ire1 and the ER stress response have non-cell autonomous effects is of particular interest, and is very well supported here. The description of this circuit linking particular neurons and signaling molecules to gonad pluripotency is also very strong.

      A weakness of the paper is the link between RIDD of FLP6 and the disruption of this circuit. The data presented do clearly support the model. However, additional information would strengthen this considerably. The authors show that FLP6 mRNA levels are reduced in Ire1+ but not Ire-/- animals subjected to ER stress. They also show that GED results from the nuclease activity of Ire1 in the ASE; and that loss of FLP6 can also induce a similar effect. However, they do not show as clearly that Ire1's effects on GED are mediated primarily through FLP6.

    4. Evaluation Summary:

      The authors of this manuscript previously showed that ER stress, and in particular the ER stress sensor Ire1, regulates transdifferentiation in C. elegans, leading to the ectopic differentiation of germline cells. In this follow-up manuscript, the authors present several lines of evidence supporting the idea that Ire1 modules these effects through degrading a novel mRNA substrate flp6. The authors identify the neurons and neuromodulators that affect accumulation of abnormal germline cells. The reviewers agreed that the discovery that flp6 is a regulated Ire-1-dependent decay target in C. elegans, and the demonstration of a non-cell-autonomous effect of Ire1 activity, are novel and likely to be of interest to a broad readership. However, more evidence is required to support some of the main conclusions.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      This is a very interesting and well conducted study that addresses a question of crucial importance and will make a very valuable contribution to the literature. The question of the vulnerability of newly generated oligodendrocytes in an inflamed environment has not previously been examined with anything like the sophistication of the current series of experiments. The paper is excellent and the data convincing. I only have a few relatively minor issues that the authors might want to consider.

      The first results section on sephin1 in EAE is a little confusing. If I have understood the rationale correctly, it is to activate the ISR to protect oligodendrocytes, newly generated from OPCs, in the face of a hostile inflammatory environment. If that is correct, then perhaps this could be explained more explicitly, and the concluding sentence re-worded so as not to give the impression that sephin-1 is able to enhance remyelination (which I realise is not what is stated but is the conclusion that might be drawn).

      The effect of the BZA-sephin combination of g ratio of remyelinated axons is very interesting. This could, of course, be because the process is accelerated with this combination rather than enhanced given that g ratios in the CC will eventually return to normal after cuprizone induced demyelination (eg Stidworthy et al., Brain Pathology 2003). This could perhaps be addressed in the discussion.

      The authors could make the point in the discussion that regenerative medicines are very unlikely to be given in the absence of effective drug-mediated suppression of aggrieved inflammation.

    2. Reviewer #2 (Public Review):

      This is an interesting paper showing that prolonging the integrated stress response provides protection to oligodendrocytes in the presence of an inflammatory cytokine. For their experiments, the authors use the cuprizone model in transgenic mice overexpressing IFNg in an inducible manner in combination with a genetic and pharmacological approach to enhance the integrated stress response. The experiments are well conducted and the results clearly presented in the text. The Popko lab has previously demonstrated in a series of papers the importance of the integrated stress response for oligodendrocyte function. The novel aspect of this work is that targeting the integrated stress response requires a neuroinflammtory environment for the protective effects to occur.

      It is important to improve the introduction. As written it is not clear what was known before and how this paper goes beyond the existing literature.

      The rational for combining for combining BZA and Seph needs to be explained.

      The figures and legends could be improved according to the following suggestions:

      The evidence that Sephin1 promotes remyelination in the EAE model shown in Figure 1 is only based on differences in g-ratio with the overall number of myelinated axons being unchanged. It is difficult to make conclusion based on these results. It is difficult to obtain accurate g-ratios in lesions. Maybe the authors could extend the analysis by performing histology and counting the number of oligodendrocytes.

      Figure 2 contains only a scheme. Figure 2 should be combined with Figure 3. In addition, a scheme showing the time line of the cuprizone treatment and recovery from the treatment would be helpful. I assume W0 is at the time of treatment, W5 after 5 weeks of cuprizone and W8 represents 5 weeks of cuprizone and 3 weeks of recovery. If yes, it is not clear why the ASPA cell count shown it not reduced between W0 and W5. The numbers seem to be similar for W0, W5 and W8 in the absence of IFNg. In addition, the comparison shown in Figure 3 are incomplete. W0 is only shown without IFNg but not with. Does IFNg affect ASPA number in the absence of cuprizone?

      Panel B and C in Figure 5 could be combined to be able to compare the analyses and to evaluate the recovery of cell number by Seph at W8. The number of mice per group is borderline (only 3 mice).

      Same issue as above: Panel B and C in Figure 6 should be combined and a multiple comparison should be performed between W0, W5 and W8.

      The rational for combining BZA and Seph as shown in Figure 8 should be explained in the text. The figure and legends should be improved to clarify at which time point the analyses were performed. The panel number stated in the legends do not match with what is shown in the figure. I assume the analyses were done at W8. Only g-ratios change, whereas the number of ASPA cells and amount of myelinated axons are not affected by the combined treatment. The interpretation of this result is not easy, and the emphasis of this result should be removed from the abstract.

    3. Reviewer #1 (Public Review):

      Drs. Chen and colleagues report that augmentation of the integrated stress response (ISR) increases the oligodendrocytes and myelination during recovery after experimental demyelination in the presence of inflammation. Homozygous GADD43 KO mice or Sephin1 are used, respectively, to genetically and pharmacologically augment the ISR. Sephin1 treatment in mice with experimental autoimmune encephalomyelitis (EAE) shows increased remyelination in the spinal cord after inflammatory demyelination. Cuprizone administration to GFAP/tre;TRE/IFN-gamma double transgenic mice produced corpus callosum demyelination and CNS inflammation, with release of interferon-gamma initiated by removal of doxycycline from the drinking water. GADD43 KO did not change overall severity of cuprizone demyelination based on loss of oligodendrocytes and demyelination in corpus callosum after 5 weeks of cuprizone with ectopic interferon-gamma. The authors state that GADD43 KO enhanced the recovery of oligodendrocytes and remyelination during the 3 weeks after removal of cuprizone from the diet, but an incorrect figure prevents evaluation of this result. In double transgenic mice, with initiation of CNS inflammation, but without the GADD43 null mutation, pharmacologically enhancing the ISR with Sephrin1, increased recovery of oligodendrocytes and remyelination at 3 weeks after removal of cuprizone from the diet. These effects of genetically or pharmacologically enhancing ISR were not observed in the absence of ectopic interferon-gamma. Genetic and pharmacologic enhancement of the ISR did not appear to significantly alter the progenitor or microglial response to cuprizone demyelination. The combination of Sephin1 with bazedoxifene (BZA) enhanced the oligodendrocyte density and remyelination during the recovery period to a similar extent as either treatment alone. The authors provide several results supporting their interpretation that augmenting the ISR can overcome inhibitory effects of inflammation to enhance oligodendrocyte density and remyelination. Clarifications of the methods, correction of missing data, and additional experiments are needed to support the authors' conclusions that the potentially significant findings that combination of Sephin1 and BZA protects remyelinating oligodendrocytes and promotes remyelination even in the presence of inflammation.

      Major concerns:

      1) The experimental design and interpretation of the results would be strengthened by examining an indicator of the ISR to allow the reader to interpret the extent of ISR activation and the effect of the genetic and pharmacologic modulators of the ISR. This analysis would be particularly helpful in the corpus callosum in conditions with and without cuprizone.

      2) Cuprizone is started at 6 weeks of age which is designated as week 0 (W0). The studies use W0 for comparison to the treatment groups that are analyzed at W5 or W8. The authors refer to W0 as pre-lesion or baseline levels, which is appropriate. The authors' statements related to the vehicle condition are appropriate as is. However, it is not clear why the W8 age-match (non-cuprizone and non-IFN-gamma) was not used to more directly interpret the extent of recovery. Using W0, the comparison is 6 versus 14 weeks of age. Myelinated axons continue to significantly increase during this age interval in mice.

      3) The data graphed in panel 3C for the KO genetic prolongation of the ISR is exactly the same and the data graphed in panel 5C for the Seph pharmacologic enhancement of the ISR. The graph in 3C is actually labeled for Seph and so must have been inadvertently inserted when the graph of the KO data was intended.

      4) The combined Sephin1/BZA treatment does not appear to work through remyelination, based on the definition of thinly myelinated axons (g-ratio >0.8) as used by the authors. The authors state that the data shows the after cuprizone demyelination, mice treated with Sephin1/BZA "reached myelin thickness levels comparable to pre-lesion levels" and "restored myelin thickness to baseline levels". To support this interpretation, the authors would need to include analysis of the Sephin1/BZA mice at 5 weeks of cuprizone to show that the combined treatment, which is initiated at 3 weeks of cuprizone, did not protect oligodendrocytes or reduce demyelination during weeks 3-5 of cuprizone and Sephin1/BZA treatment.

      5) Conditions during which augmenting ISR is protective of mature oligodendrocytes or protecting remyelinating oligodendrocytes should be more clearly presented in the Discussion. The prior EAE results are reported as protecting mature oligodendrocytes. The results (Figures 3B and 5B) show that genetically or pharmacologically augmenting the ISR did NOT protect from mature oligodendrocyte loss at 5W cuprizone. The results (Figure 5B) show increased oligodendrocytes at 8W cuprizone. The current results are interpreted as protecting remyelinating oligodendrocytes, which are presumably mature as well.

    4. Evaluation Summary:

      This is an elegantly conducted study showing integrated stress response (ISR) contributes to protection of oligodendrocytes in the remyelination process in the setting of an inflammatory environment. The authors use both genetic (GADD43KO) and pharmacological approaches (Sephin1) to study ISR in demyelination animal models. The data are convincing and have important clinical implications for multiple sclerosis and other diseases. The reviewers agree that revisions are needed for the sake of presentation, clarity, rationale, and interpretation of datasets.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      In the article "Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA", the authors describe the function of FPA as a mediator of premature cleavage and polyadenylation of transcripts. They also focused their study on NLR-encoding transcripts, as that was their most novel observation, describing an additional layer of control.

      In general, the article is well written and clear. The experimental design is good, they didn't seem to over-interpret the results, the controls were solid, and the nanopore data were quite informative for their work. It is rather descriptive, but the results will be helpful for those working on NLRs, and demonstrate the utility of bulk long-read transcript data. The authors were able to string together a number of descriptive observations or vignettes into an informative paper. Overall, it is solid science.

      One minor complaint is that the authors don't focus on NLRs starting on line 436, and then they have extensive results on NLRs; by the time I got to the discussion, I'd forgotten about the early focus on the M6A. While the first part of the article is necessary, I would suggest a more concise results section to give the paper more focus on the NLR control (since that is emphasized in the abstract and the title of the manuscript).

    2. Reviewer #2 (Public Review):

      Parker et al attempted to show that the FPA protein functions to regulate the widespread premature transcription termination of the Arabidopsis NLR genes. Using in vivo interaction proteomic-mass spectrometry, FPA was shown to co-purified with the mRNA 3' end processing machinery. Metagene analysis was used to show that FPA co-localized with Pol II phosphorylated at Ser2 of the CTD heptad repeat at the 3' end of Arabidopsis genes. Using a combination of Illumina RNA-Seq, Helicos, and nanopore DRS technologies, FPA was found to affect RNA processing by promoting poly(A) site choice, and hence controls the processing of NLR transcripts whereas such process is independent of IBM1.

      Overall, it is a potentially important research. The data is rich and could be useful. However, the biological stories described are not thoroughly supported by the data presented, especially when the authors tried to touch on several aspects without some important validations and strong connections among different parts. Some special comments are provided below:

      1) The title of this manuscript is "The expression of Arabidopsis NLR immune response genes is modulated by premature transcription termination and this has implications for understanding NLR evolutionary dynamics". Therefore, the readers will expect some functional connections between the FPA and the novel NLR isoforms due to premature transcription termination. However, the transcript levels of plant NLR genes are under strict regulation (e.g. Mol. Plant Pathol. 19:1267). Since the functions of NLR genes are related to effector-triggered immunity, it is more important to study the function of FPA on premature transcription termination when the plants are challenged with pathogens. In this manuscript, most transcript analyses are based on samples under normal growth conditions. It is therefore a weak link between the genomic studies and the functional aspects. For instance, it is more important to identify unique NLR isoforms produced upon pathogen challenges that are regulated by FPA. The authors will need to provide some of these data to fill this gap.

      2) Since the function of FPA is to regulate NLR immune response genes, we should expect a change in plant defense phenotype in FPA loss-of-function mutants. Could the authors provide more information on this? On the contrary, in line 728 of this manuscript, the authors found that at least for some pathogens, "loss of FPA function does not reduce plant resistance". It is not consistent with the hypothesis that FPA is important to regulate NLR immune response genes.

      3) Furthermore, the authors mentioned in lines 729-731 "Greater variability in pathogen susceptibility was observed in the fpa-8 mutant and was not restored by complementation with pFPA::FPA, possibly indicating background EMS mutations affecting susceptibility." Does it mean that fpa-8 contains other mutations? Will these additional mutations complicate the results of the RNA processing? Could the authors outcross the fpa-8 mutation to a clean background?

      4) In line 318, the authors found 285 and 293 APA events in the fpa-8 mutant and the 35S::FPA:YFP construct respectively, but only 59 loci (line 347) exhibited opposite APA events (about one fifth). The low overlapping frequency suggests that some results could be false positive.

      5) In line 732-736: "In contrast, 35S::FPA:YFP plants exhibited a similar level of sporulation to the pathogen-sensitive Ksk-1 accession (median 3 sporangiophores per plant). This suggests that the premature exonic termination of RPP7 caused by FPA has a functional consequence for Arabidopsis immunity against Hpa-Hiks1." It is contradictory to the statement in line 728 that "loss of FPA function does not reduce plant resistance". Is it possible that overexpression of FPA:YFP had generated an artificial condition that is not related to the natural function of FPA?

      6) The fpa-8 mutant has a delayed flower phenotype (Plant Cell 13:1427). Could the 35S::FPA:YFP fusion protein construct reverse this phenotype and the plant defense response phenotype? It is important to interpret the data when the 35S::FPA:YFP construct was used to represent the overexpression of FPA.

      7) Under the subheading "FPA co-purifies with the mRNA 3' end processing machinery". The results were based on in vivo interaction proteomics-mass spectrometry. MS prompts to false positives and will need proper controls and validations. Have the authors added the control of 35S:YFP instead of just the untransformed Col-0? At least for the putative interacting partners in Figure 1A, could the authors perform validations of some important targets, using techniques such as reverse co-IP, or to show direct protein-protein interaction between FPA to a few of the important targets by in vitro pull-down, BiFC, or FRET, etc.

      8) In Fig. 3, the data show that the last exon of the FPA gene is missing in the FPA transcripts generated from the 35S::FPA:YFP construct. Will the missing of this exon affect the function of the transcript and the encoded protein?

      9) The function of FPA is still ambiguous. There was a quantitative shift toward the selection of distal poly(A) sites in the loss-of-function fpa-8 mutant and a strong shift to proximal poly(A) site selection when FPA is overexpressed (35S::FPA:YFP) in some cases (Fig. 3, Fig. 5, Fig. 8). But the situation could be kind of reversed in other cases (Fig. 6). What is the mechanism behind it?

      10) Under the subheading: "The impact of FPA on NLR gene regulation is independent of its role in controlling IBM1 expression". IBM1 is a common target of FPA and IBM2. Indeed, FPA and IBM2 share several common targets (Plant Physiol. 180:392). It may be more meaningful to compare the impact of FPA and IBM2 on NLR gene instead.

      11) In lines 423-425, the authors described "Consistent with previous reports, the level of mRNA m6A in the hypomorphic vir-1 allele was reduced to approximately 10% of wild-type levels (Parker et al., 2020b; Ruzicka et al., 2017) (Figure 4 - supplement 3)." This data could not be found.

      12) In line 426: "However, we did not detect any differences in the m6A level between genotypes with altered FPA activity." Which data is this statement referring to?

    3. Reviewer #1 (Public Review):

      The manuscript by Parker and colleagues presents an extensive body of work on characterizing the role of FPA in the choice of polyadenylation sites in transcripts of A. thaliana. Investigation on the mechanistic details that FPA engages on the mRNA processing was first initiated with the in vivo pull-down followed by LC-MS/MS, which revealed the its protein interactome relevant for 3'-end processing. The main dataset pertaining to the manuscript title comes from the comparative transcriptome analysis of Col-0, fpa-8 mutant and the overexpressor of FPA, 35S:FPA:YFP. The strength of this work lies in the use of nanopore DRS by demonstrating the layers of FPA-dependent transcripts, including its own, and its comparison to datasets by Illumina RNA-Seq and Helicos DRS. The systematic analysis uncovered unexpected complexity in the A. thaliana NLR transcriptome under the control of FPA and thus delivers a new insight on NLR biology. Several studies anecdotally have reported the importance of using genomic DNA, but not a single cDNA species, for addressing full functionality of NLR genes. Recent advances in NLRome sequencing from multiple genomes of a species and NLR structure/function studies also highlight the importance of understanding modular nature of NLR. As alluded with the modular diversity of NLRs kept in the genomes of a species in recent studies, NLR genes are prone to reshuffle in the genome to generate different variants, including partial entities with the loss of some parts of the proteins or even chimeras, supposedly maximizing the repertoire for defense. This work adds the level of transcript diversity on that of genomic diversity; FPA, an essential factor for transcription termination determinant, targets numerous NLRs to control the layers of NLR transcriptome of an individual plant. Although it is yet to be clarified for the regulatory significance of FPA-mediated NLR transcript changes under biotic or abiotic conditions, the authors succeeded in employing fine genetic schemes utilizing FPA-defective vs. -overexpressing lines along with long-read nanopore DRS technology for the first time to uncover the breadth of differential transcript generation focused on 3'-end choices. This work is timely and impactful for NLR research owing to the above-mentioned recent advances in NLR field.

      As this work is the first of its kind in utilizing nanopore DRS to address NLR transcriptome, several technical concerns can be addressed to corroborate the claims made in the manuscript, which authors can find in the following section (1-8). Regarding the organization of the manuscript, the authors may consider to rebalance the two parts: FPA interactome vs. FPA targets and NLRs. Overall, the manuscript can be seen as combining two stories; first to characterize FPA function in 3'-end processing of transcripts inferred by interacting proteomes and meta-analysis of ChIP-seq data; second part includes detailed analysis of NLR transcripts and others. Although the first half of the analysis is a necessary prelude to the following NLR analysis, the current title and academic novelty mainly lies, or were intended by the authors, on the NLR analysis. However, current manuscript has relatively enlarged section of the first with NLR analysis packed into a series of supplementary dataset. If authors wishes to opt for highlighting NLR analysis, the following suggestions would help (9-14).

      1) Earth mover distance (EMD) has been applied to identify a locus with alternative polyadenylation. What is the basis of using EMD value of 25 as a cutoff? According to Figure 4 B,D, EMD can range from 0-4000. One would also wonder if the distance unit equals bp. In addition, EMD values of some genes (e.g. FPA and representative NLRs) can be specified in the main dataset so that significance of the cut-off values shall be appreciated.

      2) Regarding the manual annotation of alternatively polyadenylated NLR genes (L1160-): Genes with alternative polyadenylation were identified and the ending location was supported when there were minimum four DRS reads. It would be relevant to provide the significance of "the four" based on read coverage statistics, for example, with average read number covering an annotated NLR transcript with the specification of an average size.

      3) Figure 4E shows that Ilumina-RNAseq dataset detects the number of loci with a different order of magnitude compared with the other two methods. Reference-agonistic pipeline shall be appreciated, however, the method engaged might have elevated the counting of paralogous reads mapped to different locations than they should be. Along with paralogous read collapsing, this is always a problem with tandemly repeated genes, such as NLRs by and large. For example, NLR paralogs in a complex cluster with conserved TIR/NBS but diversified LRRs would have higher coverage in the first two domains but drop in the diversified parts. The authors need to specify their bioinformatic consideration to avoid such problems.

      Although the tone of the Illumina read section was careful and the main 3'-end processing conclusion was made by nanopore DRS, the authors are also advised to clearly state the limitation of using Illumina-RNAseq to address alternative polyadenylating sites at the beginning of the section, for example what to be maximally taken out from Figure 4 E and 4F. This will give relative weights to each dataset generated by different methods. One advantage of using Illumina data would be that the expression level changes can be associated with changes in processing, it seems.

      4) At the RPP7 locus, At1g58848 is identical in sequences with At1g59218 as is At1g58807 with At1g59214 (two twins in the RPP7 cluster by tandem duplication). It would be good to check whether the TE At1g58889 readthrough indeed occurs in the sister duplicate with a potential TE in the downstream of At1g59218. If not, it can be used as an example of duplication and neofunctionalization through an alternative polyadenylation site choices.

      5) HMM search shall be revisited to confirm if they are to detect the TIR domain. Given that a large proportion of NLRs in A. thaliana carry TIR at their N-terminal ends and the specified examples included TIR-NLR, it is surprising to see no TIR domain in Figure 5.

      6) L659-668: how does the new data relate to the previously TAIR annotated At1g58602.1 vs At1g58602.2 (Figure 6, Inset 1)? It would be good to see these clearly stated in the main text as compared to newly identified ones. From the nanopore profiling, At1g58602.2 appears to be the dominant form.

      7) One thing to note is that in the overexpressor of which Hiks1 R is suppressed, there was hardly any At1g58602.1 produced in addition to the large reduction of At1g58602.2. Thus, relative functional importance of the two transcripts shall be discussed in line with the Hpa resistance data. Accordingly, L740-741 phrasing shall be revised to include the possibility of absolute or relative "depletion" of functional transcript(s) contributing to the compromise in Hpa resistance.

      8) It would be necessary to state in the main text the implication of phosphorylation on the two Ser residues on Pol II at L245. A clear description distinguishing the effect of the two phosphorylation and the specificity of the antibodies is desirable, as the data was interpreted as if the two sites made differences, such that Ser2 was heavily emphasized (e.g. subtitle). Albeit low level, Ser5 data also shows an overlap with FPA ChIP-seq coverage at the 3' end. If there is a statistical significance to be taken account to interpret the coverage, please state it. Given that elongation occurs progressively, I wonder how much should be taken out from the distinction.

      9) Figures presentation for RPP4 and RPP7 are great in detailing the FPA-dependent NLR transcript complexity. To make the functional link more evident, the authors may consider bringing up parts of the Figure 5-supplement to a main Figure to detail the revised annotation of NLRs. Given recent advances in NLR structure and function studies, extra domain fusion, fission and truncated versions of NLRs require a great deal of attention. For example, potential functional link to the NMD-mediated autoimmunity and revised annotation of At5g46470 (RPS6) needs a clear visual guidance preferably with a main figure (Figure 5-Supplement 3).

      10) The section "FPA controls the processing of NLR transcripts" includes dense information and can be broken down to several categories. To this end, Supplement File 3 (NLR list) shall be revised to deliver the categorical classes and further details and converted to a main table.

      For NLR audience, for example, it would be important to associate the information to raw reads to assess where the premature termination would occur. At least, the ways to retrieve dataset or to curate the termination sites shall be guided.

      On the contrary, there is no need to include other genes in Figure 4 Sup4-8 under this section. They are not NLRs.

      11) Figure 7 and IBM1 section can be spared to the supplement.

      12) The list of "truncated NLR transcripts" in particular, either by premature termination within protein-coding or with intronic polyadenylation, should be made as a main table. The table can be preferably carrying details in which degree the truncation is predicted to be made. With current sup excel files, it is difficult to assess the breadth of the FPA effect on the repertoire of NLRs and their function. This way, functional implication of differential NLRs transcriptome can be better emphasized.

      13) FPA-mediated NLR transcript controls, as to promote transcript diversity, is expected to exert its maximum effect if FPA level or activity is subject to the environmental stresses, such as biotic or abiotic stresses. The discussion on effectors targeting RNA-binding proteins (L909-918) is a great attempt in broadening the impact of this research. In addition, if anything is known to modulate FPA activity, such as biotic or abiotic stresses or environmental conditions, please include in the discussion.

      14) NLR transcript diversity as source of cryptic variation contributing to NLR "evolution" is an interesting concept, however, evolutionary changes require processes of genic changes affecting transcript layers or stabilizing transcriptome diversity. In the authors' proposition in looking into accessions, potential evolutionary processes can be further clarified.

    4. Evaluation Summary:

      In this study, the authors examined the function of the RNA-binding protein FPA through analyzing its protein interactome and its global impact on gene expression using a combined approach of Nanopore DRS, Helicos DRS, and short-read Illumina RNA-Seq. The combined datasets and new computational approaches developed by the authors permitted them to identify the predominant role of FPA in promoting poly(A) site choice. The authors further revealed that FPA mediates widespread premature cleavage and polyadenylation of transcripts of NLR genes, important plant immune regulators. Overall, this study suggests that control of transcription termination processes mediated by FPA provides an additional layer of the regulatory dynamics of NLRs in plant immune responses.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      The manuscript of Anchimiuk and colleagues investigates the mechanism of translocation of Bacillus subtilis SMC-ScpAB, a well characterized bacterial condensin. First, the authors use several SMC constructs where the coil-coiled region has been extended and /or the hinge exchanged and test what are the effects on growth and on the organization of the chromosome. They find highly altered conformations for most of the mutants. Particularly, these altered SMCs are unable to bridge two arms in the presence of the naturally-occurring parS sequences. Interestingly, they are partially able to restore arm pairing if a single parS sequence is provided.

      Next, the authors used Chipseq to compare the binding pattern of wildtype SMC and SMC-CC425 (a mutant with an extended coil coiled region and a different hinge). They observe that the binding of wt-SMC is only midly affected by removal of most parS sequences, whilst that of the mutant is highly affected. In time-lapse experiments where ParB is depleted and then re-expressed, the authors show that in a strain with a single parS wt-SMC loads in the origin region and then redistributes over the chromosome while the mutant can only partially achieve redistribution and to a large extent remains concentrated on the origin region.

      The authors then use wt-SMC and investigate how the conformation of the chromosome changes with two different parS sites located in different positions. They observe that each parS site is able to produce arm-pairing. They observe a decrease in the strength of arm pairing when both parS sites are present.

      Finally, the authors increase the expression level of wt-SMC, and observe decreased levels of arm-pairing in the presence of all the naturally-occurring parS sites. More normal levels of arm-pairing are observed when only one parS is present, despite the higher wt-SMC levels. When two parS sites are introduced, more complex structures appear in the contact map.

      These observations are new, interesting and intriguing. However, there are multiple possible interpretations, models and mechanism that are not discerned by the data presently presented in the manuscript.

      At times, there seem to be inconsistencies in their interpretation of results, and at times the models proposed do not seem well supported by data.

      Finally, the presentation of previous models and results from the literature could be improved.

      Major issues:

      In Fig. 1 the authors make several mutant SMC constructs with larger or shorter arms and different hinges and use Hi-C to explore the changes in 3D chromosome organization. Is it not clear to me why the arc is still visible in the mutants, nor what happens to the overall organization of the chromosome in the mutants? Is chromosome choreography normal?

      In Fig. 1C the authors show that strains with parS-359 only display a secondary diagonal and conclude "chromosome arm alignment was comparable to wild-type". A quantification of the degree of pairing for each mutant normalized by the wild-type is necessary to evaluate the degree of pairing and its dependence on genomic distance to the origin.

      In Fig. 2, the authors use HiC and chip-seq to quantify the effects of changes in SMC arm length on chromosome organization and SMC genomic distributions. It would be important to verify that the expression levels of these SMC mutants are the same as wt, as as they show in Fig. 4 changes in protein levels can change also 3D chromosome organization.

      In Fig. 2C, what is the distribution of SMC at t0? Showing this result would support their claim that SMC can load in absence of ParB.

      In Fig. 2C it is claimed that SMC-CC425 moves at a slower rate than WT. Can the authors provide a quantification?

      In Fig. 2, the authors focused on one of the mutants with longer SMC arms (CC425) and performed HiC and Chip-seq in time-lapse after induction of ParB in a ParB-depleted culture. These experiments clearly establish that SMC-CC425 can redistribute from the origin and can achieve arm pairing but to a lesser extent than the WT. The authors speculate that a slower translocation rate and/or a faster dissociation rate explain the experiments. However, other possibilities exist: for instance that the mutant SMC is defective at passing through road-blocks (highly expressed genomic regions, e.g rRNA sites) or at managing collisions with RNAP/ DNAP/ other SMCs, it makes different higher-order complexes than wt-SMC, etc. This could could be due to the change in the length of the SMC, or to the use of a hinge/coiled-coil region different from that of the wt-SMC. Thus, I am not convinced that the text explores all the possible models or that the data shown discerns between any of them.

      In Fig. 3B, the authors show that use of two parS-opt sites at -304kb and -9kb lead to the formation of two secondary diagonals. They argue that these can be rationalized in terms of the diagonals formed by the strains harboring single parS-opt (either -9kb or -304kb). However, I cannot see how these can happen at the same time! If a cells makes arm pairing from -9kb then it cannot make it from -304kb right? I do not understand either how the authors can conclude from these experiments that ParS may act as unloading sites for SMC. Again, the authors are speculating over mechanisms that are not really tested.

      If parS sites triggered the unloading of SMCs, then one would assume that ~5-6 natural parS sites in the origin region are unloading the SMC complexes loaded at other parS sites? This makes little sense to me, or there is something I clearly do not understand in their explanations.

      In their text, the authors explain that "A small but noticeable fraction of SMC complexes however managed to translocate towards and beyond other parS sites apparently mostly unhindered". I am confused as to where is the evidence supporting this statement. I do not think the ensemble Hi-C experiments provided in Fig. 3 can provide conclusive evidence for this.

      The authors often hypothesize on a mechanism, but then assume this mechanism is correct. For instance, the disruption in the secondary diagonals in Fig. 3B when experiments are performed with two parS sites are initially hypothesized to be due to roadblocks (e.g with highly transcribed regions) or to collisions between SMCs loaded at different parS sites. These possibilities cannot be discerned from their data. However, the authors then assume that collisions is what is going on (e.g. paragraph in lines 274-284). I think they should provide evidence on what is producing the changes in the secondary diagonals in mutants with two ParS sites.

      Why is the ChIP-seq profile for a strain with all the natural parS sites and for a strain with only parS-9kb the same? even with the same peaks at the same locations? Does this mean that SMC peaks do not require the presence of parS? But, then SMCs do not load equally well in all naturally occurring parS sites? This is then in contradiction to their assumption that parS cannot be selectively loaded?

      Do we really know that it is a single SMC ring that is responsible for translocation? The authors assume so in their models and interpretations, but if it were not the case it could drastically modify the mechanisms proposed. For instance, SMC may be able to load on a ParS site without pairing arms (i.e. only one dsDNA strand going through the SMC ring).

      In Fig. 2C-D it is shown that a large fraction of wildtype SMC and SMC-CC425 accumulate at the origin region at early time points (Fig. 2C) however this does not seem to lead to an increased Hi-C signal in the origin region (compare early time points to the final t60). Also, despite small amounts of wt-SMC in the chromosome at the latter time points, the intensity of the secondary diagonal is very strong. Why is this? These results would be consistent with many SMCs loading at the origin region but only a fraction of them being responsible for arm-pairing. Is this not in contradiction to their assumption that SMCs pair two dsDNA arms when they load?

      The authors state that: "If SMC-CC425 indeed fails to juxtapose chromosome arms due to over-enrichment in the replication origin region, collisions may be rare in wild-type cells because of a high chromosome residence time and a limited pool of soluble SMC complexes, resulting in a small flux of SMC onto the chromosome. If so, artificially increasing the flux of SMC should lead to defects in chromosome organization with multiple parS sites but not with a single parS site (assuming that most SMC is loaded at parS sites)". However, this assumption seems inconsistent with their results in Fig. 2 that show that the peaks of SMC do not change upon removal of most parS sites.

      I am a bit confused about the interpretation of the results in Fig. 4D. The authors talk about 'loop contacts' and point to the secondary diagonal (yellow ellipses). But these are not loop contacts, but rather contacts between arms that have surpassed the two parS sequences, right? Also, it is not clear what they mean by paired-loop contacts (red ellipse). Do they mean contacts between the two loops originating at parS-359 and parS-334? If this where the case, then it means SMCs are bridging more than two dsDNA segments? Or that there are multimers of SMC linking together? Or that and SMC can circle one arm from one loop and another from the other...? But in this case, how can it load? For me it is very unclear what these experiments really mean. The explanations provided by the authors seem again highly hypothetical.

    2. Reviewer #2 (Public Review):

      In this manuscript, Anchimiuk et al reported that B. subtillis SMC can collide with each other, and that the collision is modulated by several factors including the number, strength, distribution of parS sites, the residence time of SMC on DNA, the translocation rate, and the cellular abundance of SMC. The authors suggested that these parameters are fine-tuned in the wild-type B. subtillis to minimize SMC collision. In my opinion, the finding is interesting, the experimental setup is creative, and the experiments were beautifully executed. Arguably, these experiments can only be performed in B. subtilis since parAB- and the insertion of another parS site at the mid-arm are not detrimental to cell viability (in Caulobacter crescentus, insertion of another parS mid-arm affects chromosome segregation, hence cell viability severely). Furthermore, the rare set of arm-modified SMCs from the Gruber lab also gives this manuscript a unique mechanistic angle. Given the available data, the conclusion of the manuscript is safe. I especially appreciate that the authors did not bias towards the model of SMC traversing each other by Z-loop formation.

    3. Reviewer #1 (Public Review):

      The authors investigate the role of Condensin and its loading in ensuring appropriate chromosome dynamics in the model organism Bacillus subtilis. The data are of high quality and generally support the ultimate conclusions.

      The demonstration of collisions between ectopically-loaded Condensin and their negative impact on cellular viability are important insights, particularly in light of the recent single-molecular in vitro experiments demonstrating the ability of 2 Condensins to pass one another and thereby form Z-structures on DNA.

      The main caveat is that the work lacks direct quantization of the levels of chromosome-associated Condensin—inclusion of experiments to evaluate this parameter would go a long way to validating (or refuting) the authors' conclusions.

    4. Evaluation Summary:

      This manuscript presents some intriguing data to support the notion that B. subtilis cells have tuned a variety of parameters related to SMC loading and translocation to ensure that individual complexes do not collide. This is likely an important but poorly understood aspect of condensins/SMCs, and as such represents a valuable contribution to the field and should be of interest to a broad set of readers.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      The authors of this manuscript combine electrophysiological recordings, anatomical reconstructions and simulations to characterize synapses between neurogliaform interneurons (NGFCs) and pyramidal cells in somatosensory cortex. The main novel finding is a difference in summation of GABAA versus GABAB receptor-mediated IPSPs, with a linear summation of metabotropic IPSPs in contrast to the expected sublinear summation of ionotropic GABAA IPSPs. The authors also provide a number of structural and functional details about the parameters of GABAergic transmission from NGFCs to support a simulation suggesting that sublinear summation of GABAB IPSPs results from recruitment of dendritic shaft GABAB receptors that are efficiently coupled to GIRK channels.

      I appreciate the topic and the quality of the approach, but there are underlying assumptions that leave room to question some conclusions. I also have a general concern that the authors have not experimentally addressed mechanisms underlying the linear summation of GABAB IPSPs, reducing the significance of this most interesting finding.

      1) The main novel result of broad interest is supported by nice triple recording data showing linear summation of GABAB IPSPs (Figure 4), but I was surprised this result was not explored in more depth.

      2) To assess the effective radius of NGFC volume transmission, the authors apply quantal analysis to determine the number of functional release sites to compare with structural analysis of presynaptic boutons at various distances from PC dendrites. This is a powerful approach for analyzing the structure-function relationship of conventional synapses but I am concerned about the robustness of the results (used in subsequent simulations) when applied here because it is unclear whether volume transmission satisfies the assumptions required for quantal analysis. For example, if volume transmission is similar to spillover transmission in that it involves pooling of neurotransmitter between release sites, then the quantal amplitude may not be independent of release probability. Many relevant issues are mentioned in the discussion but some relevant assumptions about QA are not justified.

      3) The authors might re-think the lack of GABA transporters in the model since the presence and characteristics of GATs will have a large effect on the spread of GABA in the extracellular space.

      4) I'm not convinced that the repetitive stimulation protocol of a single presynaptic cell shown (Figure 5) is relevant for understanding summation of converging inputs (Figure 4), particularly in light of the strong use-dependent depression of GABA release from NGFCs. It is also likely that shunting inhibition contributes to sublinear summation to a greater extent during repetitive stimulation than summation from presynaptic cells that may target different dendritic domains. The authors claim that HCN channels do not affect integration of GABAB IPSPs but one would not expect HCN channel activation from the small hyperpolarization from a relatively depolarized holding potential.

    2. Reviewer #2 (Public Review):

      The authors present a compelling study that aims to resolve the extent to which synaptic responses mediated by metabotropic GABA receptors (i.e. GABA-B receptors) summate. The authors address this question by evaluating the synaptic responses evoked by GABA released from cortical (L1) neurogliaform cells (NGFCs), an inhibitory neuron subtype associated with volume neurotransmission, onto Layer 2/3 pyramidal neurons. While response summation mediated by ionotropic receptors is well-described, metabotropic receptor response summation is not, thereby making the authors' exploration of the phenomenon novel and impactful. By carrying out a series of elegant and challenging experiments that are coupled with computational analyses, the authors conclude that summation of synaptic GABA-B responses is linear, unlike the sublinear summation observed with ionotropic, GABA-A receptor-mediated responses.

      The study is generally straightforward, even if the presentation is often dense. Three primary issues worth considering include:

      1) The rather strong conclusion that GABA-B responses linearly summate, despite evidence to the contrary presented in Figure 5C.

      2) Additional analyses of data presented in Figure 3 to support the contention that NGFCs co-activate.

      3) How the MCell model informs the mechanisms contributing to linear response summation.

      These and other issues are described further below. Despite these comments, this reviewer is generally enthusiastic about the study. Through a set of very challenging experiments and sophisticated modeling approaches, the authors provide important observations on both (1) NGFC-PC interactions, and (2) GABA-B receptor mediated synaptic response dynamics.

      The differences between the sublinear, ionotropic responses and the linear, metabotropic responses are small. Understandably, these experiments are difficult – indeed, a real tour de force – from which the authors are attempting to derive meaningful observations. Therefore, asking for more triple recordings seems unreasonable. That said, the authors may want to consider showing all control and gabazine recordings corresponding to these experiments in a supplemental figure. Also, why are sublinear GABA-B responses observed when driven by three or more action potentials (Figure 5C)? It is not clear why the authors do not address this observation considering that it seems inconsistent with the study's overall message. Finally, the final readout – GIRK channel activation – in the MCell model appears to summate (mostly) linearly across the first four action potentials. Is this true and, if so, is the result inconsistent with Figure 5C?

      Presumably, the motivation for Figure 3 is that it provides physiological context for when NGFCs might be coactive, thereby providing the context for when downstream, PC responses might summate. This is a nice, technically impressive addition to the study. However, it seems that a relevant quantification/evaluation is missing from the figure. That is, the authors nicely show that hind limb stimulation evokes responses in the majority of NGFCs. But how many of these neurons are co-active, and what are their spatial relationships? Figure 3D appears to begin to address this point, but it is not clear if this plot comes from a single animal, or multiple? Also, it seems that such a plot would be most relevant for the study if it only showed alpha-actin 2-positive cells. In short, can one conclude that nearby, presumptive NGFCs co-activate, and is this conclusion derived from multiple animals?

      The inclusion of the diffusion-based model (MCell) is commendable and enhances the study. Also, the description of GABA-B receptor/GIRK channel activation is highly quantitative, a strength of the study. However, a general summary/synthesis of the observations would be helpful. Moreover, relating the simulation results back to the original motivation for generating the MCell model would be very helpful (i.e. the authors asked whether "linear summation was potentially a result of the locally constrained GABAB receptor - GIRK channel interaction when several presynaptic inputs converge"). Do the model results answer this question? It seems as if performing "experiments" on the model wherein local constraints are manipulated would begin to address this question. Why not use the model to provide some data – albeit theoretical – that begins to address their question?

      In sum, the authors present an important study that synthesizes many experimental (in vitro and in vivo) and computational approaches. Moreover, the authors address the important question of how synaptic responses mediated by metabotropic receptors summate. Additional insights are gleaned from the function of neurogliaform cells. Altogether, the authors should be congratulated for a sophisticated and important study.

    3. Reviewer #1 (Public Review):

      This manuscript by Gabor Tamas' group defines features of ionotropic and metabotropic output from a specific cortical GABAergic cell cortical type, so-called neurogliaform cells (NGFCs), by using electrophysiology, anatomy, calcium imaging and modelling. Experimental data suggest that NGFCs converge onto postsynaptic neurons with sublinear summation of ionotropic GABAA potentials and linear summation of metabotropic GABAB potentials. The modelling results suggest a preferential spatial distribution of GABA-B receptor-GIRK clusters on the dendritic spines of postsynaptic neurons. The data provide the first experimental quantitative analysis of the distinct integration mechanisms of GABA-A and GABA-B receptor activation by the presynaptic NGFCs, and especially gain insights into the logic of the volume transmission and the subcellular distribution of postsynaptic GABA-B receptors. Therefore, the manuscript provides novel and important information on the role of the GABAergic system within cortical microcircuits.

    4. Evaluation Summary:

      This manuscript provides quantitative information of the integration of GABA-A and GABA-B receptor inhibitory responses in cortical pyramidal neurons induced by a presynaptic GABAergic neuron type called neurogliaform cell (NGFC). Experimental and modeling data suggest that NGFCs converge onto postsynaptic neurons with sublinear summation of ionotropic GABA-A potentials and linear summation of metabotropic GABA-B potentials probably due to a preferential spatial distribution of GABA-B receptor-GIRK clusters on the dendritic spines of postsynaptic neurons. The data represent an attempt to gain insights into the logic of GABA volume transmission within cortical microcircuits.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #2 (Public Review):

      In "Evolution of cytokine production capacity in ancient and modern European populations", Dominguez-Andrés et al. collect a large amount of trait association data from various studies on immune-mediated disorders and cytokine production, and use this data to create polygenic scores in ancient genomes. They then use the scores to attempt to test whether the Neolithic transition was characterized by strong changes in the adaptive response to pathogens. The impact of pathogens in human prehistory and the evolutionary response to them is an intriguing line of inquiry that is now beginning to be approachable with the rapidly increasing availability of ancient genomes.

      While the study shows a commendable collection of association data, great expertise in immune biology and an interesting study question, the manuscript suffers from severe statistical issues, which makes me doubt the validity and robustness of their conclusions. I list my concerns below, in rough order of how important I believe they are to the claims of the paper:

      — In addition to the magnitude of an effect away from the null, P-values are a function of the amount of data one has to fit a model or test a hypothesis. In this case, the authors have vastly more data after the Neolithic Revolution than before, and so have much higher power to reject the null hypothesis of "no relationship to time" after the revolution than before. One can see this in the plots the authors provided, which show vastly more data after the Neolithic, and consequently a greater ability to fit a significant linear model (in any direction) afterwards as well.

      — The authors argue that Figure S2 makes their results robust to sample size differences, but showing a consistency in direction before and after downsampling in the post-neolithic samples is not enough, because:

      1) you still lack power to detect changes in direction before the Neolithic.

      2) even for the post-Neolithic, the relationship may be in the same direction but no longer significant after downsampling. How much the significance of the linear model fit is affected by the downsampling is not shown.

      — The authors chose to test "relationship between PRS with time" before and after the Neolithic as a way to demonstrate that "the advent of the Neolithic was a turning point for immune-mediated traits in Europeans". A more appropriate way to test this would be creating a model that incorporates both sets of scores together, accounts for both sample size and genetic drift in the change of polygenic scores, and shows a significant shift occurs particularly in the Neolithic, rather in any other time period, instead of choosing the Neolithic as an "a priori" partition of the data. My guess is that one could have partitioned the data into pre- and post-Mesolithic and gotten similar results, largely due to imbalances in data availability.

      — The authors only talk about partitions before and after the Neolithic, but plots are colored by multiple other periods. Why is the pre- and post-Neolithic the only transition that is mentioned?

      — Extrapolating polygenic scores to the distant past is especially problematic given recent findings about the poor portability of scores across populations (Martin et al. 2017, 2019) and the sensitivity of tests of polygenic adaptation to the choice of GWAS reference used to derive effect size estimates (Berg et al. 2019, Sohail et al. 2019). In addition to being more heavily under-represented, paleolithic hunter-gatherers are the most differentiated populations in the time series relative to the GWAS reference data, and so presumably they are also the genomes for which PGS estimates built using such a reference would have higher error (see, e.g. Rosenberg et al. 2019). Some analyses showing how believable these scores are is warranted (perhaps by comparing to phenotypes in distant present-day populations with equivalent amounts of differentiation to the GWAS panel).

      — In multiple parts of the paper, the authors mention "adaptation" as equivalent to the patterns they claim to have found, but alternative hypotheses like genetic drift are not tested (see e.g. Guo et al. 2018 for a review of methods that could be used for this).

      — 250 kb window is too short a physical distance for ensuring associated loci that are included in the score are not in LD, and much shorter than standard approaches for building polygenic scores in a population genomic context (e.g. see Berg et al. 2019, Berisa et al. 2016). Is this a robust correction for LD?

      — If one substitutes dosage with the average genotyped dosage for a variant from the entire dataset, then one is biasing towards the partitions of the dataset that are over-represented, in this case, post-Neolithic samples.

      — It seems from Figure 2, that some scores are indeed very sensitive to the choice of P-value cutoff (e.g., Malaria, Tuberculosis) and to the amount of missing data (e.g. HIV). This should be highlighted in the main text.

      — Some of the score distributions look a bit strange, like the Tuberculosis ones in Figure 2, which appear concentrated into particular values. Could this be because some of the scores are made with very few component SNPs?

    2. Reviewer #1 (Public Review):

      This paper focuses on the role of historical evolutionary patterns that lead to genetic adaptation in cytokine production and immune mediated diseases including infectious, inflammatory, and autoimmune diseases. The overall goal of this research was to track the evolutionary trajectories of cytokine production capacity over time in a number of patients with different exposure to infectious organisms, infectious disease, autoimmune and inflammatory diseases using the 500 Functional Genomics cohort of the Human Functional Genomics Project. The identified cohort is made up of 534 individuals of Western European ancestry. Much of this focus is on the impact and limitations of certain datasets that they have chosen to use such as the "average genotyped dosage" to be substituted for missing variants and data interpretation. Moreover, some data pairings in the data set are not complete or had varying time points . Similarly, a split was done to look at before and after the Neolithic era and the linear regression correspond to those two eras. However, the authors do not comment or show the data to demonstrate why they choose that specific breakpoint as opposed to looking at every historical era transition, i.e., from early upper paleolithic to late upper paleolithic to Mesolithic to Neolithic to post-Neolithic to modern. Lastly, the authors should highlight additional limitations of this current study in terms of the generalizability to other populations or to clearly state that this is limited to the European population at the specified latitude and longitudes used.

    3. Evaluation Summary:

      Dominguez-Andrés et al. collect a large amount of immune-related trait association data from a cohort made up of 534 individuals of Western European ancestry. The goal was to track the evolutionary trajectories of cytokine production capacity over time in a number of patients with different exposure to infectious organisms, infectious disease, autoimmune and inflammatory diseases, using the 500 Functional Genomics cohort of the Human Functional Genomics Project. From this analysis it was hypothesized that the Neolithic transition was characterized by strong changes in the adaptive response to pathogens in human biology. Overall, the manuscript is interesting but could be improved by significant enhancements to statistical methodology.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

    1. Reviewer #3 (Public Review):

      In zebrafish embryo development the surface epithelium, the enveloping layer (EVL), proliferates and migrates along with the yolk sac during epiboly. This process requires the simultaneous proliferation and migration of cells, which must undergo cell shape changes. Co-ordination of these processes is regulated by proliferation, whereby cell number and shape perturb tissue-scale forces necessary for epiboly. This paper investigates explicitly the importance of successful cytokinesis, through abscission of cytokinetic bridges, on regulating these forces and epiboly progression. They show that Rab25, a GTPase belonging to the Rab11 subfamily, regulates abscission through endomembrane trafficking in the EVL. Through their detailed analysis of cellular-level phenotypes, including qualitative and quantitative approaches, this paper presents convincing evidence for this novel role of Rab25. The authors should be congratulated on excellent time-lapse movies of cytokinesis in early zebrafish development.

    2. Reviewer #2 (Public Review):

      The authors examined the role of Rab25 during cell division within a developing epithelia. Strikingly, they found that the RabGTPase, Rab25, localized to mitotic structures such as centrosomes and cytokinetic midbodies in dividing cells of the developing zebrafish embryo. They went on to create maternal-zygotic Rab25a and Rab25b mutant embryos where they clearly demonstrate that apical cytokinetic bridges fail to undergo abscission leading to anisotropic cell morphologies that likely contribute to a delayed epiboly.

      The major strengths of this study is the clear cell biology defects found in a developing embryo that lead to downstream developmental defects (delayed epiboly). The rab25 localization is beautiful. The examination of the viscoelastic properties is also compelling. The main improvements would be to expand upon the spatio-temporal localization of Rab25a and Rab25b during cell division at different stages of epiboly, present Rab11 localization patterns in the Rab25 mutant embryos, and clearly demonstrate that changes in viscoelasticity are also in their multinucleated cells that occur in Rab25 mutant conditions. These additions will help the authors support their conclusions that Rab25 localization/regulation of endomembranes (potentially recycling endosomes) regulates abscission and subsequently the viscoelastic properties of the developing tissue.

      This study has identified novel roles for Rab25 in cytokinesis/abscission and opens the doors for examining it in regulating mitotic centrosome function. It is paradigm shifting in that it creates a new way to think about Rab25 and potentially its relationship with Rab11 and recycling endosomes during division in the early embryo.

    3. Reviewer #1 (Public Review):

      In the manuscript by Willoughby et al. the authors examine the role of Rab25 in early embryogenesis in zebrafish. They implicate Rab25 activity in abscission and show various defects including delayed epiboly and altered cell behaviors associated with defective acting dynamics. This is an interesting and well-written paper that uses reverse genetics and microscopy to analyze the function of Rab25, a GTPase previously implicated in membrane recycling, in vivo. Their work illustrates how defects in cytokinesis affect epiboly and establish an interesting link to acto-myosin regulation of the mechanical properties of the EVL. While these pehnotypes are described and demonstrated clearly, the implication of membrane recycling is not fully supported in the present work. It is also unclear whether Rab25 plays a role in oogenesis that may account for some of the observed phenotypes.

    4. Evaluation Summary:

      Willoughby et al. examine the role of Rab25 in early embryogenesis in zebrafish. They implicate Rab25 activity in abscission and show various defects including delayed epiboly and altered cell behaviors associated with defective acting dynamics. Overall, this is an interesting and well-written paper. However, there are a number of important controls that are missing and some connections such as the implication of membrane recycling that require stronger experimental validation.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

    1. Reviewer #2 (Public Review):

      This manuscript set out to address several outstanding questions concerning the impact of 'eusocial' behaviour in mammals, here represented by the experimental model of the Damaraland mole-rat, on skeletal remodelling. Specifically, the transition to breeding status (queen) for some individuals in the colony is accompanied by changes that support high fecundity. The authors investigate the extent to which changes are localised in the skeleton and the underlying regulatory changes that are associated with these morphological features. The paper is well-written, the experiments have been planned thoughtfully and described carefully, and the panel figures convey information without over-crowding. Overall, I thoroughly enjoyed reading this manuscript, which represents as a multi-pronged approach to advancing understanding of the unusual biology and phenotype of queen mole rats.

    2. Reviewer #1 (Public Review):

      The authors provide a novel case-study of the skeletal consequences of queen-only breeding in Damaraland mole-rats, one of the few eusocial mammals. Out of a population of adults, a queen will be selected as the sole female to breed with a male, and the non-breeders will provide support in the highly cooperative society. Once selected, a new queen will undergo a rapid skeletal transformation in which lumbar vertebrae expand. Supporting closely-timed pregnancies and lactation, mineral reserves will be excavated by bone-specific macrophages along the inner, or endosteal, lining of some limb bones. Unlike most other mammals, the skeletons of queens do not typically recover to their pre-pregnancy phenotype as rapid sequential pregnancies continually erode the limbs, leaving them vulnerable to fracture.

      To understand the molecular mechanisms driving these phenotypic changes associated with breeding in queens, the authors artificially selected queens in captivity, recreated a eusocial society, and then tracked gene expression along with skeletal phenotypes throughout breeding cycles. After lumbar expansion in queens had completed only long bones showed gene expression consistent with breeding status. Specifically, results showed upregulation of differentiation and activity of bone-specific macrophages, call osteoclasts. These cells liberate minerals from bone and make components of the extracellular matrix available metabolism and development of embryos.

      To understand if these changes were driven by the presence of sex-steroids, multiple cell types were harvested from the marrow of lumbar vertebrae and limb bones and treated with estradiol. No significant effect was found. Data, therefore, suggest that mechanisms shaping the postcranial skeleton were not consequences of sex-steroid mediated signaling pathways.

      Non-recoverable bone loss in queens is unusual among mammals and is a vulnerability that potentially limits the number of pups a queen can produce. Vulnerable queens may therefore be protected in cooperative societies in which non-breeders can work more and offer queens more rest.

      This study furthers the field of skeletal biology by exploring how enduring bone resorption contributes to the greater fecundity of one of the world's few eusocial mammals but has a potentially life-long consequence on limb performance and fracture resistance. The authors weave together multiple lines of evidence to better illustrate the enormous and rapid changes that occur as a female ascends to queen status, and what she sacrifices to build her colony. Results offer compelling and transdisciplinary insights into an extreme skeletal strategy and the impact of this work can be bolstered by only minor changes.

    3. Evaluation Summary:

      This manuscript takes a deep dive into the skeletal effects of burrowing and eusocial Damaraland mole rats. By exploring the genetic and skeletal consequences of breeding restricted to a single queen with multiple and closely-timed pregnancies and lactation, this study offers a compelling story that will bolster textbooks on skeletal biology, mammalian evolution, and ethology. The results show the molecular mechanisms driving adaptive plasticity within the unusually expanded lumbar spine and thin limb bones of queens are an adaptive consequence of breeding status.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #3 (Public Review):

      The manuscript by Turner et al. employs a transcriptome-wide approach to study the effects of mutants of the 3'-end processing machinery and the anti-cancer drug cordycepin (3' deoxyadenosine) on alternative poly(A) site selection in budding yeast to better understand alternative polyadenylation (APA) mechanism(s). In particular, poly(A) test sequencing (PAT-seq), a 3'-end focused deep sequencing technique, is employed to determine cleavage/poly(A) site choice in seven mutants of the core 3'-end processing machinery – three cleavage factor IA (CFIA) mutants (rna14-1, pcf11-2, clp1-pm), one cleavage factor IB (CFIB) mutant (nab4-1), and three cleavage and polyadenylation factor (CPF) mutants (ysh1-13, fip1-1, pap1-1). Six of the 3'-end processing factor mutants exhibit increased distal poly(A) site usage and lengthening of 3'-UTRs, with rna14-1 and pcf11-2 showing the greatest effect, but clp1-pm exhibiting little effect. Notably, 3511/7091 genomic annotations (49.5%) have two or more poly(A) sites and 422 genes have significantly changed poly(A) sites in all the 3'-end processing factors mutants except clp1-pm. APA is also examined in 41 genes in a full spectrum of 3'-end processing mutants (22) using a multiplexed poly(A) test (mPAT) method and most of the mutants alter poly(A) site choice, with a predominant shift to distal site usage. In addition, APA analysis of cells treated with cordycepin using PAT-seq indicates that cordycepin alters poly(A) site choice in 1959 genes, with predominant distal cleavage site usage and lengthening of 3'-UTRs. Cordycepin is also shown to increase nucleotide abundance. Interestingly, impairment of transcription elongation, using mycophenolic acid (MPA), which reduces GTP levels, or an RNA polymerase II mutant, rpb1-H1085Y, in cells treated with cordycepin promotes proximal poly(A) site usage and shorter 3'-UTRs, reversing the effects of cordycepin. Finally, comparison of genes altered in APA by cordycepin to a dataset of yeast nucleosome occupancy suggests that 3'-end nucleosome positioning and length of intergenic regions in convergent genes correlates with cordycepin responsiveness. The data presented in the paper suggest a kinetic model for cleavage/poly(A) site selection in yeast that involves a balance between the concentration/availability of the cleavage and polyadenylation machinery and transcription elongation rate.

      The strengths of the study include the generation of transcriptome-wide datasets for poly(A) site usage in numerous mutants of evolutionarily conserved, essential cleavage and polyadenylation factors using the PAT-seq method. In addition, the study indicates that almost 50% of the annotated genes in budding yeast exhibit alternative polyadenylation. The study also indicates that impairment of numerous 3'-end processing factors, irrespective of subcomplex, predominantly causes an increase in distal poly(A) site usage and lengthening of 3'-UTRs. Interestingly, the study also suggests that the choice of poly(A) site is regulated by the availability of cleavage and polyadenylation factors and transcription elongation. Finally, the study shows that anticancer drug cordycepin causes transcriptome-wide changes in alternative polyadenylation, predominantly elevating distal poly(A) site usage.

      The weaknesses of the study revolve around basing some conclusions solely on the transcriptome-wide data without additional small-scale experiments. In addition, the effects of 3'-end processing mutants and cordycepin on alternative polyadenylation have been examined in two different strain backgrounds, which could impact direct comparisons of the data. The proposed kinetic model for cleavage site choice in yeast seems only to be tested in cells treated with cordycepin.

      Overall, the authors achieved their aims of providing greater insight into the mechanism of alternative polyadenylation and its links to transcription and more understanding of the biological effects of cordycepin in cells. At present, most of the conclusions are supported by the results, but some conclusions require additional experiments.

      This study will be of enormous interest to the RNA processing field and to the wider community, especially given that alternative polyadenylation regulates so many aspects of mRNA function, the 3'-end processing factors studied are evolutionary conserved, and cordycepin is an anti-cancer agent.

    2. Reviewer #2 (Public Review):

      The authors investigated how alternative polyadenylation (APA) is modulated in yeast using appropriate transcriptomic methodologies.

      The authors found that mutants for mRNA 3' end formation factors and cordycepin treatment alter alternative polyadenylation in the same manner, generating transcripts with longer 3'UTRs, due to a switch to distal polyadenylation sites (PAS). Most mutants analyzed cause a PAS switch, in particular mutants for RNA14, PCF11, YSH1, FIP1, NAB4 and PAP1. They also found that MPA and a rpb1 mutant, with a slower transcription elongation rate, reverts the cordycepin effect of distal PAS selection. This implies that in yeast, as in higher organisms, APA is modulated by RNAPII elongation. There is nucleosome depletion in the 3' end of convergent genes that undergo cordycepin-driven APA alterations, which is a new finding.

      On the basis of their data, the authors propose a kinetic model for APA in yeast that is regulated by the concentration of core mRNA 3' end factors and nucleotide levels, which in turn modulates RNAPII elongation. This integrative model has been already described in higher organisms, but not in yeast, and overall this study covers an impressive body of work that makes an important contribution to the field.

      1) The authors show that cordycepin have the same effect in APA as most of the 3' end factors mutants used, but there is a lack of integration between the two sets of PAS-seq data. The cordycepin APA effect may be due to decreased expression of mRNA 3' end factors but this hypothesis was not fully explored. Treating those mRNA 3' end mutants with cordycepin could shed some light on this.

      2) A new role for SEN1 in APA for a subset of protein coding was observed. The SEN1 mechanism could be clarified if the authors show that SEN1 is within the subset of convergent genes analyzed, and also if SEN1 expression changes upon cordycepin treatment.

    3. Reviewer #1 (Public Review):

      The authors set out to test a variety of factors that could impact poladenylation site (PAS) selection in yeast. To that end, they rigorously tested a collection of temperature-sensitive mutations in polyadenylation machinery components and utilized a custom 3'-end sequencing method to assess PAS selection genome-wide. The most common result associated with polyadenylation machinery dysfunction was global switching to a more distal PAS. Further, the authors test an interesting phenomenon of cordecypin-induced switching to the distal PAS and reveal through metabolomics that enhanced nucleotide biosynthesis may be the root cause. The enhanced nucleotide pools was found to alter elongation rate leading to alterations in PAS choice. Finally, the authors find that convergent genes are influenced by the nucleosome landscape to impact APA events.

      Overall, this is a rigorous and thorough study that brings together multiple regulatory components that impact PAS selection. The model presented by the authors is supported by their work and provides the field with a clear picture of the complex nature of cleavage and polyadenylation in yeast.

    4. Evaluation Summary:

      This study aims to provide a comprehensive analysis of factors governing polyadenylation site selection in yeast. Overall, the authors reveal that multiple but distinct inputs including polyadenylation machinery integrity, transcription elongation rate, nucleotide availability and chromatin landscape all contribute to controlling cleavage and polyadenylation.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Reviewer #4 (Public Review):

      The authors have studied the effects of microstimulation in a single subject with 2 microelectrode arrays in the somatosensory cortex. They aimed to investigate the how altering frequency, current amplitude and train duration affected the elicited percepts. They report three new findings:

      1) Increasing stimulus frequency did not increase the intensity of the percept, in fact there was frequency selectivity of cortical regions and these were somewhat topographically organized on the cortical surface.

      2) The intensity of the subject's responses were similar using suprathreshold (higher) currents but using lowest electrical currents (perithreshold) required higher frequencies for detection similar to other somatosensory brain regions.

      3) Frequency-intensity variation could evoke different types of sensations, with higher frequencies more likely to evoke tingle or buzz (less natural), and lower frequencies eliciting more pressure, tap, or touch (more natural type sensations).

      The major strength of this work is the detailed testing performed over multiple sessions through the same microelectrodes, demonstrating consistent effects. It provides new methods to alter sensations by changing the parameters of stimulation to optimize the type of percept that they are trying to produce.

    2. Reviewer #3 (Public Review):

      Microstimulation of the somatosensory cortex is a very promising approach to restore sensory feedback in disabled people. Hughes and colleagues performed cortical microstimulation experiments in a spinal cord injured subject to characterize the relationship between the stimulation parameters (frequency and amplitude) and the perceived sensation (type and intensity). This type of experiment is very important to better understand the potentials and limits of this approach. The results achieved by the authors are very interesting and can represent a first step towards the development of more effective and personalized approaches to restore sensory feedback. These results need to be confirmed with additional subjects and during closed-loop experiments.

    3. Reviewer #2 (Public Review):

      This study induced tactile percepts through microstimulation via two multi-electrode arrays implanted over a quadriplegic's primary somatosensory hand region. The report focuses on manipulation of the stimulation frequency of microstimulation, though further manipulations were tested and are briefly reported.

      For different stimulation sites, the perceived intensity was highest at different stimulation frequencies. This result contradicted the expectation that higher stimulation frequency would be related to higher perceived intensity. This expectation derived from previous work in non-human primates that showed lower detection thresholds for higher-frequency stimulation. The authors show that the same result is obtained in their human patient, suggesting that differences exist between near- and supra-threshold perceived stimuli and that, accordingly, generalizing from non-human primate work has its traps.

      The authors grouped stimulation sites according to optimal stimulation frequency into low, intermediate, and high frequency preferring sites. These three classes were spatially clustered, and related to different patterns of reported perceptual qualities (such as vibration, pressure etc).

      The paper's results are important for practical developments of sensory feedback in brain-machine interfaces. Understanding the perceptual result of brain stimulation requires reports by human participants, as underlined by the differences uncovered here between near- and supra-threshold stimulation. They furthermore reveal new aspects of the cortical organisation of primary somatosensory cortex.

      The conclusion of clustered patches sensitive to specific frequencies is tentative. As an inherent limitation of intracranial recordings, the total number of stimulation sites is small, and some electrodes did not produce significant results, further reducing the number of analysable sites. Therefore, it is possible that stimulation doesn't truly fall into three distinct clusters (even if such clustering is statistically supported with the current data set), but are actually continuous or divide into a larger number of classes. Notably, this critique does not invalidate the main finding that different patches of cortex show specific frequency preferences.

    4. Reviewer #1 (Public Review):

      This manuscript reports data from unique experiments in which a paralysed person reported sensations evoked by microstimulation of the somatosensory cortex. The main emphasis of this paper is on the effects of increase in stimulation frequency. It was discovered that depending on the electrode used, the peak intensity was felt at different frequencies. Accordingly, the electrodes and stimulation sites were divided into three groups-Low, Intermediate and High frequency preferring. Overall, it was noticed that in most electrodes increasing stimulation frequency beyond about 100 Hz led to less intense sensation. Without knowing the exact somatosensory circuits involved in processing, the connection with recently discovered human vibrotactile psychophysics phenomena and cortical recordings in mice are speculative, but are in close agreement with the current observation and thus the manuscript would benefit from expanding discussion on this. I personally don't think there is any contradiction with non-human primate studies, as the authors state, rather it should be viewed as a significant extension to those studies and warrants viewing them in a new light.

      A very interesting observation is that three types of frequency-intensity effects are associated with different perceptual qualities. However, types of seemingly distinct sensations might be attributed to semantics describing sensation of periodic stimulation at different intensities. Subjective reports of one subject are very valuable to set future directions for this kind of investigation, but may not be enough to generalise those findings just yet.

      The location of electrodes belonging to three different frequency-intensity effect groups appeared to be not at random, but whether it reflects cortical organisation or some other factors like systematic variation in electrode depth might have influenced the result, needs to be confirmed. Only a small number of electrodes was tested - 8 in the Medial Array and 11 in the Lateral Array.

      Three frequency-intensity effect group electrodes also differed in median intensity reported across all frequencies, which cautions that the reported perceptual quality differences at least partly might be attributed to the overall level of intensity sensation. It has to be noted that the overall frequency-intensity response profile did not change by changing the stimulation current, however some shifts seems to be present. Alternatively, such frequency-intensity effect profiles represent circuits tuned to detection of specific features of stimuli. This possibility is indeed very intriguing.

      As those experiments performed on a human subject with implanted electrodes are absolutely unique, the data are exceptionally interesting regardless of limitations generalising those findings. Unlike animal experiments humans can describe sensations evoked by cortical microstimulation so there is no substitution for these experiments and every piece of evidence is highly valuable. These results give ground for new hypotheses to better understand how the somatosensory system works and generate ideas for designing future human psychophysics and animal model experiments. From a practical point of view, it is exceptionally valuable for informing the design of stimulation protocols for bidirectional brain-computer interfaces (BCIs).

    5. Evaluation Summary:

      This paper characterizes percepts evoked by micro-stimulating the somatosensory cortex of a human participant. The study provides some new insight into the organization of the human somatosensory cortex and represents an important step in providing more effective somatosensory feedback for brain-machine interface users.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      Hallast and coworkers identify a potentially novel complex Y chromosome structural rearrangement that is associated with male infertility in a carefully phenotyped European cohort. The authors interrogate the Y chromosome AFZc region in 1190 Estonian idiopathic male infertility cases of varying severity and 1134 controls (healthy young men or proven fathers). They replicate partial AFZc deletions and replicate a known gr/gr deletion association with comparable effect sizes. After conditioning on gr/gr deletion status, they identify an association with secondary b2/b4 duplications on case status, but with no accompanying observed effect on andrological sub-phenotypes.

      The authors identify multiple non-syntenic DAZ/CDY1 deletion patterns that are consistent with a large inversion followed by deletion. The authors further infer that this putative inversion is fixed in a Y chromosome sub-lineage. Based on population haplotype frequency estimates they infer that a surprisingly large number of individuals harbouring the r2/r3 inversion have a subsequent deletion. They show through detailed phenotyping shows that r2/r3 inversion+deletion cases in their cohort have more severe disease.


      1) Despite being a very common disease, idiopathic infertility is severely understudied, due in large part to difficulties in sample acquisition. More generally, sex chromosome genetic associations for common disease as a whole are understudied owing to their structural complexity and other technical issues. The authors should be applauded for attempting to overcome these challenges.

      2) The putative finding of a large-effect common variant conferring risk to a common genetic disease is of great interest. The authors leverage the advantages of a logistically coherent health care system. The level of phenotypic detail of andrological parameters in both cases and controls is impressive and aid in biological interpretation of the genetic findings. For example, the distinction between azoo- versus oligozoo-spermia shed light on a potential meiotic disease aetiology. The endocrine values add important context.

      3) The authors imply that the combination of the inversion+deletion risk allele favours a meiotic failure disease aetiology as opposed to a gene dosage aetiology. This is a potentially disruptive finding.


      1) The authors do not replicate their association, raising the possibility of a false positive finding.

      2) The study is underpowered to reliably detect variants of small effect, and underpowered in general. This is a common challenge in reproductive genetics.

      3) The logical inferences (as opposed to direct measurement) made by the authors are elegant but add substantial uncertainty to the findings. Most notably, cytogenetic or long-read sequencing based validation of the inversion genotype would strengthen confidence in the study considerably.

      If the genetic association is robust and the allele frequency estimates are well calibrated, the implications of this work are considerable. The locus could become a genetic biomarker for infertility. The locus could potentially account for a huge amount of variance in polygenic risk associated with infertility. The findings also raise a fascinating evolutionary conundrum as to how an allele associated with such an evolutionarily destructive phenotype could occur at such high frequencies. The authors briefly raise the possibility of age-dependent effects, but with extremely sparse data.

    2. Reviewer #2 (Public Review):

      Hallast et al. have performed an extensive genetic analysis of a cohort of men with idiopathic infertility, for whom many have accompanying phenotypic data in the form of andrological parameters. The complex genetic architecture of repeating sequences on the Y chromosome gives rise to recurrent AZFc deletions that affect male infertility. However, while partial deletions of AZFc are reasonably frequent, they have less clear phenotypic effects. gr/gr and b2/b3 deletions seem to be a risk factor for spermatogenic impairment in some populations but are fixed in others. Hallast et al. focus on these partial AZFc deletions in a reference cohort and a cohort with idiopathic infertility from the same geographic population, characterising further structural and sequence variation, Y-chromosomal haplogroups, and gene dosage.

      While the gr/gr deletion is present in the reference group of individuals with normal andrological parameters, Hallast et al. show that this deletion is enriched among patients, with 2.2-fold increased susceptibility to infertility. As observed in other European populations, the prevalence of b2/b3 deletion was similar in the reference group and the patient group, suggesting that it is not a spermatogenic impairment risk factor for this Estonian population either.

      A quarter of Estonian gr/gr deletion carriers belonged to the Y chromosomal haplogroup R1a1-M458. Within this Y haplogroup, an inversion has occurred that promotes subsequent deletion, likely causing severe spermatogenic failure in the majority of carriers as this complex rearrangement is enriched 8.6-fold in individuals with severe spermatogenic impairment.

      Some major strengths of this paper are the size of the groups recruited (1,190 patients and 1,134 reference individuals) from a single national population, the extensive accompanying andrological data, and the genomic characterisation of many individuals to elucidate the relationship between specific structural variants and effects on fertility.

      The discovery of the fixed inversion infertility risk factor on a specific Y haplogroup is a useful contribution that could aid genetic counselling efforts through carrier identification and risk mitigation.

      However, I am seeking clarity on multiple testing correction for microdeletion association with specific andrological parameters. Besides this, the main conclusions of this paper are supported by other data presented.

    3. Reviewer #1 (Public Review):

      In this study, Hallast and colleagues performed a detailed genetic analysis of the AZFc region of the Y-chromosome in a large cohort of 1190 Estonian men with idiopathic infertility and >1100 controls from the same population. They focused on partial deletions of the AZFc regions, because their clinical significance remains controversial and published reports are often contradictory. The authors performed a comprehensive genetic analysis, which in addition to a standard AZFc deletion protocol with gene dosage of the key AZFc genes, included also Y-haplogroup determination and re-sequencing of the retained DAZ, BPY2 and CDY genes. The authors showed that gr/gr deletions were enriched in infertile men, thus confirming that this deletion is a risk factor for impaired spermatogenesis. An important novel finding is identification of a previously unknown structural variant: a long r2/r3 inversion, which likely destabilizes two palindromes and leads to deletions. This variant is fixed in the Y lineage R1a1-M458, which is common in some Central European populations. In the Estonian study group, nearly all patients with this variant and a gr/gr deletion, had a severe impairment of spermatogenesis. The authors mentioned that the variant largely 'destroys' two palindromes, P1 and P2. One would like to see more discussion what are the structural and functional consequences - e.g. are any loci for e.g. non-coding RNA affected by a deletion in men with this inversion in comparison to those without?

      The authors also speculated in the discussion that deletion on this background might lead to progressive worsening of the reproductive phenotype. This is based on just one control individual, a young man with borderline reproductive parameters, and corroborating this hypothesis would require further studies, including repeated evaluation of the same individuals over a long period of time.

      This is a high quality study, performed by collaborators from the UK and Estonia, with an excellent track record in the analysis of the Y-chromosome structure and evolution, and in reproductive genetics and clinical andrology, respectively. The data presentation and figures are very informative and convincing. Among the strengths of the study, I have to emphasise a detailed phenotypic evaluation of the study subjects, including several parameters of testis function, semen analysis, and reproductive hormone profiles. Hence, the results and conclusions are valuable and add to the understanding of the consequences of the partial AZFc deletions. The authors also provided useful guidelines how to identify men with this variant in labs performing genetic analysis of infertile couples.

    4. Evaluation Summary:

      This study presents extensive genetic analysis of a relatively large cohort of men with idiopathic infertility, with considerable accompanying andrological phenotypic data. Through careful step-by-step investigations, an inversion variant is identified as a risk factor for subsequent deletion variants that can lead to substantially increased risk of impaired spermatogenesis, on an age-structured basis, relative to non-carriers. This work will be of particular interest to the reproductive genetics field, but also has wide ranging implications for colleagues interested in common disease genetics, meiosis, structural variation, dosage sensitivity, and sex chromosome evolution. As part of the most comprehensive investigation of AZFc micro-deletions and structural variation to date, the authors have identified a novel structural variant of the Y-chromosome that predisposes to spermatogenic failure and provided clear guidelines for genetic counseling.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    1. Joint Public Review:

      Hsiang-Chun Chang et al. investigated the role of ALR, component of the mitochondrial MIA40/ALR protein import apparatus, in cytosolic Fe/S cluster biogenesis performing loss-of-function (silencing) and gain-of-function (over-expression) experiments with MEFs (mouse embryonic fibroblast) and HEK293 (human embryonic kidney) cells. They find that downregulation of ALR impairs maturation of cytosolic Fe/S cluster proteins, while activities of mitochondrial Fe/S cluster proteins such as complex I and II are unaffected. Furthermore by reducing ALR expression cells up-regulate cellular iron transporter transferrin receptor 1 (Tfrc) and consequently cellular iron levels increase. The authors reveal that ALR down-regulation post-transcriptionally regulates Trfc through stabilization of Trfc mRNA mediated by IRP1, which is activated by absence of its mature Fe/S cluster. Additionally they demonstrate that only over- expression of full-length ALR, mainly located in the mitochondria and not the cytosolic short from ALR can reverse cytosolic Fe/S cluster maturation and therefore IRP1 activity and cellular iron levels. In the last part of their manuscript the authors present evidence about the mechanism by which ALR carries out this function. They find that ALR enables mitochondrial import of ABCB8 but not ABCB7, two mitochondrial proteins involved in the maturation of cytoplasmic Fe/S clusters. This transport into mitochondria requires functional MIA40/ALR in the IMS and further the TIM23 complex to the inner mitochondrial membrane. ABCB8 interacts directly with MIA40 by 5 cysteines (difulfide bond formation) and therefore these conserved cysteins are necessary for recognition and binding, which is not the case for ABCB7. These data add an interesting view on how ALR expression is linked to Fe/S cluster protein maturation, cellular iron homeostasis and their potential impact on related dieases.

      The strength of the manuscript are the well designed and performed experiments presenting evidence of how mitochondrial function of ALR is linked to the sulfur redox homeostasis and cellular iron regulation. Interestingly, reduction in cytsolic Fe/S cluster maturation and therefore increased cellular iron levels is also associated with increased sensitivity of cells to oxidative stress and this might be a plausible explanation for the previously described impact of full length ALR expression on oxidative stress in various disease models (PMID: 30579845).

      The drawn conclusions that the mechanistic studies about the role of ALR for Fe/S cluster maturation and cellular iron uptake may parallel the disease phenotype of patients with mutations in ALR gene GFER may be in parts speculative. The reported ALR mutations are varying and result either in partial functional or truncated protein expression (PMID: 20593814, PMID: 25269795). ALR is expressed in several isoforms (varying between two or three depending on the organ) of different size (15kDa, 21kDa, 23kDa). Most of the data showing the short form ALR (15kDa) solely in the cytosol and the full length ALR (23 kDa) as wells a second immuno-reactive band of 21 kDa ALR, both in cytosol and mitochondria (PMID: 30579845). While over-expressing full length ALR the authors show in the manuscript higher expression level in the cytosol than in the mitochondria fraction (w-blot, which is not reflected in the graph of Fig. S3 B). It was reported earlier that continuous over-expression of full length ALR in mammalian cells leads to the accumulation of full length ALR not only in the mitochondria but also in the cytosol (PMID: 23676665), which is also in agreement to observations of cytosolic occurrence of full length ALR (see above). This raises the question whether the conclusions made in the manuscript may be due to its cytosolic accumulation rather than or in addition to its mitochondrial localization. The presented study refers at several points to a study by Lange et al 2001 demonstrating that ALR rescues cytoplasmic Fe/S cluster maturation defects in Erv1- null yeast. There has been contradictory evidence published about the role of ALR in the maturation and export of cytosolic Fe-S cluster proteins. Lange et al. claimed that ALR interacts with Atm1 (an ABC transporter in the inner membrane of the mitochondria) and facilitates the export of Fe-S proteins to the cytosol. However, later it was suggested that, in yeast cells, ALR plays neither a direct nor an indirect role in cytosolic Fe-S cluster assembly and iron homeostasis. It is claimed that Iron homeostasis is independent of Erv1/Mia40 function in various yeast strains (Erv1 mutant) and that the finding by Lange et al. is based on only one Erv1 mutant strain, mainly due to strongly decreased glutathione (GSH) levels (PMID: 26396185).

      Additionally, this statement is reinforced by a study in human cells, demonstrating that depletion of ALR does not impact the maturation of cytosolic Fe-S proteins assembled via the CIA pathway (PMID: 25012650). Furthermore, this study in mammalian cells has pointed out the role of ALR in exporting MitoNEEt to the outer mitochondrial membrane (OMM). MitoNEEt is a Fe-S protein that is synthesized in the mitochondrial matrix. Upon synthesis, MitoNEEt translocates through the inner membrane of the mitochondria by ABCB7 and then through the IMS by ALR to the OMM where it contributes to cell proliferation (PMID: 25012650).

    2. Evaluation Summary:

      This is an interesting manuscript and experiments generally make their point on Alr effects. However, additional data would strengthen the paper with respect to the relative roles of cytoplasmic vs mitochondrial isoforms as would mitochondrial function studies.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      These authors report the identification of the function of a genetic determinant (dev1, formerly ydcO ) carried by the ICEBs1 element that increases fitness of the host strain by delaying the entry into the normal developmental pathway leading to biofilm formation and ultimately sporulation, such that the subpopulation expressing the product of dev1 increases in a mixed pool. An interesting novel aspect of the dev1 system is that it is co-regulated with ICEBs1 conjugation, and thus is only activated when the host strain is a minority of a mixed population; in this scenario the Dev1+ subpopulation is essentially cheating on the Dev-. Since expression of the Dev1 phenotype in an entire population would likely cause a crash, the ICE- population density-dependent regulation ensures that the fitness advantage disappears before the crash can occur. I think that the gene is interesting and this report adds a significant aspect to our understanding of the biology and evolution of ICE elements. Overall I am positive about this paper.

    2. Reviewer #2 (Public Review):

      This manuscript provides convincing evidence that the ICEBs1 conjugative element confers a fitness advantage on the model bacterium B. subtilis during biofilm formation and sporulation. This effect is frequency dependent and is effected in large measure via an element gene, named devI, by an unknown mechanism that probably decreases the concentration of Spo0A-P. The data are well presented and successfully make the case for a fitness advantage conferred by the mobile element during biofilm formation and sporulation. It is likely that a mechanistic exploration of DevI will follow and will provide another facet to the regulation of Spo0A, a gift that keeps on giving.

      Delaying sporulation in a mixed culture confers an advantage for the delayers. This has been convincingly shown. But I wonder about the effects in a clonal population of cells carrying ICEBs1 in competition with a null population. I appreciate that the delay in sporulation is transient, as pointed out in lines 404-407. But a delay of a few hours may be critical in this type of competition between populations as resources become limiting. This is presumably why sporulation is so exquisitely regulated on so many levels and in response to many external an internal signals. If so, ICEBs1 would have a deleterious effect and the element might be in danger of extinction. I suppose that an analogous discussion could be considered for biofilm formation.

    3. Reviewer #1 (Public Review):

      Mobile genetic elements like phages, transposons, plasmids, and conjugative elements are widespread in prokaryotes and confer important traits to their hosts, including antibiotic resistance and virulence. In this study, the authors convincingly demonstrate that the mobile element ICEBs1 of Bacillus subtilis confers a fitness advantage to its host by delaying entry into metabolically costly developmental processes (biofilm formation and sporulation). The gene devI is identified as being responsible for delaying initiation of development, but the mechanistic basis for this could be further explored. Their results show that, in addition to conferring novel phenotypes, mobile elements exert influence by tuning existing host pathways, a paradigm that could be extended to many other prokaryotes.


      The paper is written very clearly, the experimental data is convincing, the interpretations and conclusions are justified by the data.

      The authors implemented clever genetic approaches to quantitatively compare the fitness of strains harboring or lacking ICEBs1 in co-culture. I appreciated the use of the conjugation mutant (comEK476E) to prevent ICE transfer that would confound the analysis. Similarly, the authors genetically separate the developmental pathways under which ICEBs1 confers an advantage (biofilm formation and sporulation), by deleting the spo0A promoter under sigH control to prevent sporulation but retain biofilm formation. Finally, to assess the contribution of ICE-encoded genes to fitness, the authors take advantage of a "locked-in" ICE variant (∆attR, oriT*) that cannot excise and replicate - thereby eliminating the confounding variable of gene dosage from ICE replication.

      As mentioned above, the effects of ICEBs1 on development set an important precedent for how mobile genetic elements interact with their hosts. They are often regarded as autonomous elements, but the authors provide an example of how these elements can influence host pathways.

      Suggestions for improvement:

      The authors show that the gene devI is necessary and sufficient for ICE-mediated delay of development initiation. Gene expression analyses suggest this delay affects the earliest stages of development (genes under control of spo0A, the master regulator of sporulation, are affected). I think the authors could investigate the mechanism of spo0A inhibition in more detail. Which aspect of spo0A function is affected by DevI? Starvation sensing, spo0A expression, activation of upstream kinases (KinA?), phosphorelay, or binding of Spo0A~P to promoters?

      Ectopically expressed DevI (Fig 5) seemed to have a stronger inhibition of sporulation than ICEBs1 alone (Figure 2) - does the constitutively expressed protein block rather than delay sporulation? I wonder if the authors would like to comment on how, in the wild-type ICEBs1 context, DevI activity is eventually overcome by cells that eventually do sporulate after a delay. Furthermore, will cells that successfully transfer ICEBs1 be relieved of DevI-mediated sporulation inhibition?

      The data in Fig 4 suggest that devI is not the only ICEBs1-encoded factor providing a fitness advantage. Do the unknown factor(s) also delay development, or do they work via other mechanisms: i.e. does the ∆devI mutant have a sporulation delay? Any idea what the other factors might be (from bioinformatics for example)?

    4. Evaluation Summary:

      All the reviewers were in agreement that this is an exceptionally rigorous paper that sets an important precedent for how mobile genetic elements can influence host biology.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      In "KLF10 integrates circadian timing and sugar signaling to coordinate hepatic metabolism", Anthony Ruberto and colleagues characterize the role of the transcription factor KLF10 in circadian transcription and the transcriptional and physiological responses to hexose sugars in mouse hepatocytes. They confirm earlier reports that Klf10 is expressed rhythmically in mouse liver, with peak expression at ZT9. They show that Klf10 expression is induced by glucose and fructose and that hepatocyte-specific deletion of Klf10 exacerbates hyperglycemic and hepatosteatotic responses to 8 weeks of elevated sugar consumption. They use RNA sequencing and ChIP sequencing to define the complement of Klf10 target genes in hepatocytes and how they are regulated by glucose and fructose. Together their data support a model in which KLF10 limits the transcriptional induction of rate-limiting enzymes involved in gluconeogenesis and lipogenesis in response to elevated sugar consumption, thus mitigating the pathophysiological impact of high sugar diets. The experiments are mostly well designed, presented, and interpreted but several points require additional investigation and/or clarification. While the current manuscript suggests an integration of circadian timing and sugar signaling by KLF10, additional experiments to establish how some of the molecular and physiological effects are modulated by time of day are needed to better support that claim.


      This study uses a combination of genetic, biochemical, and physiological approaches to investigate the hepatocyte-specific function of the transcription factor KLF10. Deletion of KLF10 specifically in hepatocytes distinguishes this study from other related work. Further, the characterization of global daily gene expression patterns in mouse liver is well designed and analyzed and establishes that hepatocyte-specific deletion of Klf10 remodels daily rhythms of gene expression in the liver. The combination of that analysis with ChIP sequencing provides powerful evidence to establish the hepatocyte-specific KLF10-dependent transcriptome and highlights its targeting of rate-limiting enzymes in lipogenic pathways. Together, the molecular and physiological analyses in this study provide compelling evidence that KLF10 plays a protective role in the context of excessive sugar consumption by limiting lipogenic gene expression pathways and thereby suppressing hepatic steatosis.


      In its present form, this study does not thoroughly connect the in vitro and in vivo findings and misses the opportunity to fully characterize the role of KLF10 in circadian regulation of lipogenesis in response to excessive sugar consumption in vivo. It is unclear whether the concentrations of glucose and fructose used to stimulate primary hepatocytes are similar to those experienced in response to the dietary stimulus in vivo and there is no examination of the impact of sucrose on Klf10 expression or downstream gene expression. This omission complicates the interpretation of the response to the combined sugar stimulus in vivo, especially in light of a recent report that KLF10 deletion protects against hepatosteatosis caused by consumption of a high sucrose diet. It also does not examine how time of day influences KLF10-dependent gene regulation in response to sugar consumption. Without these analyses, it falls short of connecting the circadian and sugar-response pathways through KLF10.

    2. Reviewer #2 (Public Review):

      This study builds on a previously published paper from this group showing that KLF10 is under circadian control, and it in turn affects the oscillation of a set of metabolic genes in the liver. While the previous study utilized a systemic Klf10 KO mouse model, here, Ruberto et al. generated a conditional hepatocyte-specific Klf10 KO mouse model (Klf10Δhep).

      The authors find that the absence of hepatocyte KLF10 alters the circadian oscillation of a number of metabolic genes. In response to sugar consumption, Klf10Dhep mice demonstrate exacerbated adverse effects as well as significantly increased hepatic expression of many glycolysis, gluconeogenesis, and lipogenesis related genes. They conclude that Klf10 normally acts as a "transcriptional brake" to protect animals against the effects of high sugar consumption and show via ChIP-seq that KLF10 is present at a wide range of metabolic genes, particularly at those involved in acetyl-CoA metabolism. The findings are interesting, particularly in the context of the burgeoning burden of metabolic disease and its relation to high sugar consumption, and are supported by the experimental findings.

    3. Reviewer #1 (Public Review):

      Ruberto et al. utilize hepatocyte-specific Klf10 knock-out mice to demonstrate expression changes of rhythmic transcripts, highlighting dysregulated glucose and lipid metabolism as an enriched gene set. They demonstrate that KLF10 is necessary for proper glycemic control in mice and that KLF10 coordinates suppression of metabolic gene expression in the liver in response to high sugar diet. The authors corroborate their findings by analyzing gene expression changes of primary hepatocytes stimulated with fructose and high glucose. Finally, the authors identify KLF10 target genes using ChIP-seq and validate Acss2 and Acacb as target genes suppressed in mice following a high sugar diet. Novel aspects of this work include the metabolic characterization of a hepatocyte-specific Klf10 knock-out mouse, identification of KLF10 target genes in hepatocytes using ChIP-seq, and description of circadian transcript expression with Klf10 loss.

    4. Evaluation Summary:

      This paper will be of interest in the fields of circadian biology and metabolic physiology. It provides a molecular mechanism for protection against development of fatty liver in response to high sugar consumption. Quality data support the key claims of the paper in each of the main research areas (circadian biology and metabolism) but additional efforts are needed to integrate the two parts. The current study does not thoroughly connect the in vitro and in vivo findings and misses the opportunity to fully characterize the role of KLF10 in circadian regulation in response to excessive sugar consumption.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

    1. Reviewer #3 (Public Review):

      The authors sought to directly compare manipulations of different signaling pathways for their ability to induce cell cycle activation and proliferation in cardiomyocytes from various species and maturation levels. The manipulation consisted of peristent lentiviral expression of beta catenin, cyclin D2, rat Erbb2, human Erbb2, and Yap8SA.

      A major strength of this study is that it shows that most of above expressions appeared to induce negative feed-back responses at the post-transcriptional level to limit protein overexpression, illustrating how difficult it is to manipulate cardiomyocyte proliferation. By contrast, human Erbb2 did induce prominent proliferative effects in both rat and human cardiomyocytes. However, this finding has been shown before. The novelty here is limited to interspecies differences of the effects of Erbb2 overexpression. Multiple studies in oncology have shown that Erbb2 overexpression increases cell proliferation and is sufficient to induce cancer growth. It has also been shown that transient overexpression of Erbb2 in vivo in the heart results in dedifferentiation and proliferation of cardiomyocytes. The observation that Erbb2 overexpression induces cardiomyocyte dedifferentiation alongside mitosis is not unexpected; in general, stimuli that induce cardiomyocyte proliferation also induce cardiomyocyte dedifferentiation and sarcomere disassembly.

      In this study, in a 3D model of rat neonatal cardiobundles Erbb2 overexpression also led to formation of a necrotic core. It also led to loss of sarcomeres and contractile force and tissue stiffening. These effects appeared to be mediated by mTOR-independent, Erk-dependent mechanisms. Although experiments in this study are of a high technical level, and results interesting, the likely impact of this work is minor. Indeed, the overall picture of Erbb2-induced pathologic hypertrophy is likely related to the applied methodologies, i.e., a persistent as opposed to temporally controlled Erbb2 overexpression and the use of an avascular 3D model lacking the cellular complexity of the intact heart.

    2. Reviewer #2 (Public Review):

      This manuscript by Nicholas Strash et al. compares the effects of several potential mitogens on cell cycle of the two most used in vitro models of cardiomyocytes (CMs): neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell (hiPSC)-derived CMs. In addition, they use a 3D model of NRVMs as a model that represents more mature, non-proliferating CMs. The work is interesting for researchers working in the field of cardiac regeneration and provides the first direct comparison of several potential mitogens. The inclusion of several in vitro models to account for potential species differences strengthens the data. The results support previously published findings and the main conclusions are supported by the data presented.

      The authors used a 3D model, cardiobundles made from NRVMs, as a more mature CM model. However, these cardiobundles still had a considerable number of CMs in active cell cycle in basal conditions. Whether this reflects true proliferation or the postnatal multinucleation process of rat cardiomyocytes, is unclear. Furthermore, post-mitotic human CMs were not studied. These can be obtained from hiPSC-CMs by prolonged culture or using metabolic stimuli as shown by Mills et al. 2017 (PNAS).

      The authors demonstrate that the known mitogenic pathway for CMs, Erbb2-mediated signalling, promotes cell cycle activation in 2D cultures or NRVMS and hiPSC-CMs as well as in 3D cardiobundles. Although cell cycle activity was clearly induced, no actual proof of cytokinesis has been presented. For the cardiobundle work, it remains unclear if the increase in cross-sectional size of cardiobundles induced by Erbb2 signalling is due to increased number of CMs or increased size of CMs. Both the physiological ligand of Erbb3, Neuregulin-1, and the downstream ERK pathway are known to induce CM hypertrophy (see for example Zurek et al. 2020 Circulation; Bueno and Molkentin 2002 Circ Res).

      The data analysis and statistics raise some concerns, which require clarification. First, the N numbers are really big and according to the Table 1 it is unclear if they all indeed represent independent samples. For example, one field in a monolayer (Table 1, definition of n in Figures 1J, 1P, 4C, 4E, 4G) should not be considered to represent n=1, if several images were analysed from the same sample and/or if several technical replicates (samples prepared from the same cell isolation or differentiation and treated similarly) were analysed. Only samples from separate differentiations or cell isolations should be considered as representatives of n and the results from technical replicates should be averaged to form the n=1 data. Second, the selection of statistical tests is a concern. It is unclear if the data were analysed for equal variances before selecting the test (parametric vs. non-parametric). It is also unclear why the authors carried out multiple t tests instead of using ANOVA or its variations, which are generally considered more suitable for multiple comparisons.

    3. Reviewer #1 (Public Review):

      This manuscript titled "Human Erbb2-induced Erk Activity Robustly Stimulates Cycling and Functional Remodeling of Rat and Human Cardiomyocytes" directly compared a number of previously identified candidate mitogenic genes in different cardiomyocytes and different maturity status and investigated the pathway involved. The authors found that the human Erbb2 triggers the strongest proliferative effect in both human-induced Pluripotent Stem Cells and Neonatal Rat Ventricular Myocyte, and was associated with the Erk pathway. The authors then proved this association by demonstrating that inhibition with Mek inhibitor and Erk inhibitor attenuates the human Erbb2-induced response. In addition, the authors found that Yap8SA failed to trigger proliferation in the cardiomyocyte tested due to negative feedback loop. Thus, this study provides helpful information regarding the relative effectiveness of a number of candidate genes.


      — This study investigates five candidate genes in different species and different maturation status of cardiomyocyte. In each setting, all genes are studied. Therefore, direct comparison regarding their effectiveness can be made.

      — Furthermore, this study demonstrated the mechanism on how the differing responses arose, providing in-depth information.


      — Although this study showed induced proliferation of cardiomyocyte following candidate genes expression, the authors did not present sufficient proof that the function would improve. Cardiomyocyte harbor differing functions and parameters that represents it should ideally be investigated.

    4. Evaluation Summary:

      This paper will be of interest to scientists in the field of regenerative medicine. The authors compare effects of persistent lentiviral expression of various mitogens in cardiomyocytes in vitro. Technically experiments are of a very high standard, but the data are somewhat difficult to translate to the in vivo situation. The statistical analyses would have to be robust and sufficient for the conclusions to be supported by the data.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

    1. Joint Public Review:

      The manuscript by Liu and colleagues is a very elegant study demonstrating the emergence of ectopic beta cells after beta cell specific ablation in zebrafish pancreas in a context in which vascularization of the larvae was altered in either npas4l mutants or etv2 morphants. Provocatively, the authors demonstrate the mesodermal origin of ectopic and functional beta cells using 2 mesodermal mapping strategies. This study is very well conducted with appropriate controls and rigorous statistical analyses. This study will likely impact the field of pancreas regeneration providing a novel source for beta cells within the adjacent mesodermal tissue.

    2. Evaluation Summary:

      This is an elegant study demonstrating the emergence of mesoderm-derived beta-like cells following beta-cell ablation in an endothelial cell deficient context. These findings will be of interest to scientists in the areas of regeneration and reprogramming, as they reveal a previously unknown degree of germ layer plasticity in the embryo. In the long term the study has potential impact in the diabetes field, as it reveals a novel path for redirecting somatic cells into insulin-producing cells in an in vivo context.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

    1. Joint Public Review:

      Although sensory neurons are thought to be the primary detectors of environmental stimuli in skin, it is more and more appreciated that non-neuronal cell types also play important roles. Previous work from the Stucky group (and others) has shown stimulation of optical excitation of keratinocytes can evoke action potentials in sensory neurons and behavioral responses suggesting functional connectivity. Earlier work from the Stucky group provided evidence that keratinocytes are thermosenstive and required for normal temperature sensation.

      Here, they look into whether these cells are also important for mechanosensation. Using K14-Cre-dependent conditional KO mice, functional assays and behavioral analysis, Moehring and collaborators report that the mechanosensitive channel Piezo1 is expressed in keratinocytes in mice and humans and claim that it contributes to normal touch sensation. The in vitro data convincingly show that keratinocytes have mechanically evoked currents mediated by Piezo1. Interestingly, this work shows that recruitment of epidermal, non-neuronal Piezo1 by mechanical stimulation of keratinocytes could contribute significantly to touch through activation of cutaneous sensory fibers (mechanoreceptors). Specifically, they provide evidence that removing Piezo1 from keratinocytes reduces the frequency of spiking in select types of sensory neurons to punctate and dynamic touch stimuli. Finally, they supply quite surprising data documenting significant behavioral deficits in Krt-conditional knockout mice.

      Overall, this work provides an intriguing series of observation and potentially fundamental discovery. However, concerns remain as to how the relatively subtle differences in the skin-nerve recordings result in such profound behavioral effects? Similarly, it is hard to understand how loss of the related channel Piezo2 in sensory neurons completely abolishes many touch responses if mechanosensitivity of keratinocytes is sufficient to evoke touch behaviors (as their experiments applying Yoda-1 to the hindpaw of mice would suggest). Altogether, this work suggests a novel role for epidermal Piezo1 in normal touch but the key neuro-epithelial signaling remains to be identified.

    2. Evaluation Summary:

      This manuscript is of broad interest to readers in the field of somatosensation. The identification that a common type of skin cell responds to mechanical force using a specific molecular receptor called Piezo1 is an important contribution to our understanding of mechanotransduction. A combination of conditional gene knockout with physiological and behavioral assays provides intriguing evidence that communication between skin and nerves is important for normal touch sensation, a conclusion that if further supported by additional data could become a fundamental discovery.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      The authors clearly demonstrate the effectiveness of optimized tools to generate precise C to T point mutations in zebrafish F0 embryos. The demonstrate germline transmission and an associated mutation for one mutation. There is sufficient data for members of the community to consider adopting these tools to generate mutation in their own laboratories

    2. Reviewer #2 (Public Review):

      Rosello et al. present very compelling evidence that Cytosine base editors can be used to introduce G:C to A:T base conversions with high efficiency in zebrafish. Furthermore, they describe engineering and validation of a base editor targeting the NAA PAM sequence. Finally, they have developed a potential novel model of the Noonan syndrome. The manuscript represents an important and much needed advance in precision genome editing in the zebrafish model system.

    3. Reviewer #1 (Public Review):

      The manuscript by Rosello et al., describes the application of cytosine base editing to efficiently introduce known and predictable mutations into disease genes in vivo in zebrafish, and examine signaling pathways and model disease. The majority of the data presented is analysis of editing precision and efficiency in somatically targeted embryos, with one example of a precise edited germline allele recovered. A direct comparison of the cytosine base editor BE4 and an improved version ancBE4max indicates both are highly efficient at somatic base editing. ancBE4max reduces alteration of bases outside the base editing window, and the data suggests loci for which BE4 base editing has failed can be targeted with ancBe4max. The authors demonstrate efficient base editing in embryos at multiple cancer genes (up to 91%), introducing activating mutations into oncogenes and nonsense mutations in a number of tumor suppressors. A S33L allele was introduced into the b-catenin gene ctnnb1 to activate the wnt signaling pathway as evidenced by expression of the wnt reporter Tg(tcf:GFP). Another novel aspect of this study is that the authors have expanded base editing target site selection by switching out the ancBe4max SpCas9 PAM-interacting motif domain with the domain from Spymac, which recognizes an NAA PAM. ancBe4maxSpymac editing efficiency was modest (16-19%). The method reported here has strong potential for effective combinatorial mutagenesis to map complex genetic interactions that underly disease pathogenesis. Overall, this study demonstrates cytosine base editing is an efficient and powerful method for introducing precise in vivo edits into the zebrafish genome.

    4. Evaluation Summary:

      The manuscript by Rosello et al. represents a major advance in implementation of cutting-edge genome editing methodologies in the zebrafish. The study seeks to describe optimized tools for precise base editing in zebrafish and to demonstrate their effective application. Overall, this study demonstrates that cytosine base editing is an efficient and powerful method for introducing precise in vivo edits into the zebrafish genome, and will be of interest to scientists who use zebrafish and other genetic systems to model human development and disease.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #2 (Public Review):

      The manuscript "Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways" by Bowerman and colleagues describes a study of archaeasome dynamics combining molecular simulations, cryo-EM, and sedimentation velocity analytical ultracentrifugation. How chromatin evolved is a fundamental question in biology, marking a striking departure from the bacterial nucleoid. Indeed, ever since the first description of archaeal nucleosomes and histones HmfA/B (Sandman and Reeves mid-80s) from thermophilic archaea, this question has fascinated and puzzled the field.

      Recent work from the Luger lab figured out the organization of these archaeal chromatin fibers as a continuous loop structure. Here, the authors extend this question further. MD analyses show that Arc90 has two preferred states (closed and flexible ends), but the same 5T5K structure on 120 or 180 bp of DNA prefer a single state (closed). Sedimentation velocity analytical ultracentrifugation showed that Arc207 sediments slower than the H3 mononucleosome, implying that that Arc207 has a shape with higher anisotropy, resulting in excessive drag compared to a mononucleosome. Subsequently, high-resolution cryoEM showed that at least two distinct classes for Arc207 exist, where one class represents a 5-mer and another class represent a 7-mer. The latter has a unique shape in that the 7-mer forms an L-shape (or open clam) with a 3-mer hinging on a 4-mer.

      Overall, these data provide exciting structural insights into how archaeal chromatin is folded up at its basic unit level, which the authors describe as most fittingly as a "slinkie". Because so little is known about how nucleosomes evolved during the transition from archaea to eukaryotes, we found this interdisciplinary report well written and with compelling data, that will be of interest to the chromosome biology field at large. We suggest a minor revision in which a few technical points are addressed.


      1) The cryoEM data showed two main groups of particles: 5-mer protecting 150 bp and a 7-mer protecting either 90bp or 120bp. A few times in the manuscript (both in the results and discussion section) the authors mention a 30-bp MNase digestion ladder is observed. The Mnase data should be included, as this provides evidence that the structures observed by cryoEM indeed represent physiological structures, especially if strong discrete bands are observed at 90, 120, and 150 bp.

      2) The two main classes found by cryoEM give the impression that adding dimers results in altered structures. The 7-mer shows an angled structure, which is interpreted as an open structure. The 5-mer shows a more uniform structure, which is interpreted as a closed structure. The former structure protects the full length of DNA on which HTkA histones were reconstituted, whereas the latter might be an incomplete reconstitution or a partially disassembled structure. It also raises the question if the length of the DNA is a limiting factor. What if HTkA was reconstituted on 170 bp or 307 bp instead? Would this in turn only permit the formation of the 5-mer on the 170 bp construct and two 5-mers on the 307 bp construct? The authors should consider addressing this point because the reconstitution might be constrained by the length of the DNA construct used. Indeed, a related topic might be AT content- what does archaeal DNA look like from the perspective of DNA sequence for chromatin (Jon Widom's group had a ChIPSeq paper on this a few years ago, just after his untimely passing).

      3) In the discussion the authors cite that in one archaeal species the Mg2+ concentration is ~120 mM, more than a magnitude greater than that tested in Figure 5. What happens to reconstituted archaeasomes at higher Mg+? This is relevant because in vivo, archaea are thought to have 10x the concentration of Mg+ (amongst other ions) relative to us humble eukaryotes who would probably die of kidney failure at those ionic concentrations. Indeed at high ionic conditions, eukaryotic chromatin can be made to precipitate out of solution (for e.g. 10mM Mg+, 3M NaCl). An AUC assay with higher Mg2+ concentrations seems a doable and physiologically relevant addition to the ms that would strengthen it. It is relevant to consider that in vivo structure in these halophilic and thermophilic organisms might be dependent on the concentration of various salts and temperature, it would be nice to read the authors' thoughts on this issue.

    2. Reviewer #1 (Public Review):

      While I am not sufficiently qualified to comprehensively assess the molecular dynamics simulations, all interpretations seem careful and remain within the described limitations of the various metrics that the authors report.

      The experiments are well executed; the results are presented clearly and interpreted carefully. This is a rigorous and important biophysical study that provides a solid foundation for the investigation of archaeal genome biology. The authors' new findings raise interesting questions, and although addressing them is outside the scope of this study, the article would perhaps benefit from a more detailed discussion of the biological implications of the results. The manuscript does not indicate whether the cryo-EM maps and atomic models were deposited in the EMDB and PDB. I strongly encourage the authors to do that: it would add a lot of value not only for the readers of this study, but also for the wider structural biology community.

    3. Evaluation Summary:

      In their manuscript titled "Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways", Bowerman et al. use an elegant combination of cryo-EM, analytical ultracentrifugation and molecular dynamics simulations to investigate the structure and dynamics of archaeal histone — DNA complexes, termed archaeasomes to distinguish them from eukaryotic nucleosomes. This study builds upon the crystal structure of an archaeasome and the functional analysis of its disruption recently published by the same group (Mattiroli et al, 2017) by analyzing the dynamics of this complex and discussing how these dynamics could relate to archaeal genome biology. How chromatin evolved is a fundamental question in biology, marking a striking departure from the bacterial nucleoid. This current manuscript describes a rigorous biophysical study that not only provides substantial new insights into archaeal genome biology but also raises intriguing questions for future study. This manuscript will therefore no doubt be of interest not only to the archaeal research community but also to the field of chromatin biology.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      This is a well written and elegant study from a collaboration of groups carrying out models based on high resolution imaging. I think the study also serves as a prime example for where modeling and simulation bring added value in the sense that the insights revealed in the study would not likely be gained through other methods.

      1) As the authors point out, clinical studies have revealed that the fibrotic burden in ESUS patients is similar to those with aFib. The question is why then, do so few ESUS patients exhibit clinically detectable arrhythmias with long-term monitoring. The authors hypothesize and their data support the notion that while the substrate is prime for pro-arrhythmia in ESUS patients, a lack of triggering events may explain the differences between the two groups.

      2) I think the authors could go further in describing why this is surprising. Generally, severe fibrosis is thought to potentially serve as a means or mechanism for pro-arrhythmic triggers. This is because damage to cardiac tissue typically results in calcium dysregulation. When calcium overload occurs in isolated fibrotic tissue areas, or depolarization of the resting membrane potential due to localized ischemia allows for ectopic peacemaking, we might expect that the diseased/fibrotic tissue is itself the source of arrhythmia generation. I think the novel finding here is that this notion may be a simplification, and the sources of arrhythmia generation may be more complex and may need to come from outside the areas of fibrosis. I think this is a big deal.

    2. Reviewer #2 (Public Review):

      Bifulco et al. performed a large-scale in silico study to test whether the spatial fibrosis distribution measured via LGE-MRI in 45 patient with embolic stroke of undetermined source (ESUS) as compared to the distribution in 45 atrial fibrillation (AFib) patients without stroke leads to differences in reentrant arrhythmia inducibility of dynamics.

      1) This study comprises a high number of simulations and is one of the computational electrophysiology studies that covers the most anatomical and structural variability on the atrial level. In their comprehensive analysis, Bifulco et al. answered their question and found no pronounced differences in arrhythmia inducibility and dynamics between ESUS and AFib models. It would be interesting to learn how the spatial fibrosis distributions compare in terms of the previously suggested features density and entropy (Zahid et al.). This might also influence the statements in L170/L207.

      2) The authors chose to exclude patients with stroke from the AFib group, the reasons for this choice are not entirely clear. The same holds for the fact that the ESUS models included AFib-induced electrophysiological remodeling even though these patients have not been diagnosed with AFib (by definition).

      3) An acknowledged limitation of the study is the assumption of fixed conduction velocity and action potential duration/effective refractory period. Bifulco et al. base this assumption on previous studies by the group (e.g. L312), which, however, concluded that reentrant driver locations and inducibility are sensitive to changes of action potential and conduction velocity (Deng et al.). For conduction velocity, wider ranges have been reported since the publication of the supporting reference (35) in 1994, e.g. Verma et al.; Roney et al.

      4) The number of pacing sites is rather low for a comprehensive in silico arrhythmia inducibility test but likely a good balance of coverage and computational feasibility considering that the primary goal of this research was to check whether the two groups of models show differences when undergoing the same (but not necessarily exhaustive) protocol.

      5) The discussion does a good job in putting the results into context. Two interesting observations that deserve more attention are that i) the Inducibility Score was always higher for AFib vs. ESUS (Figure 6A, no statistical test performed). However, this did not translate to a difference in silico arrhythmia burden (inducibility). ii) Reentrant drivers were about twice as likely to localize to the left pulmonary veins than the right pulmonary veins in the AFib models (Figure 6D).

      6) The study succeeded in answering the question it posed in the sense that no marked difference was found between the ESUS and AFib models. This leads to the question what the stroke-inducing mechanism is in the ESUS patients. A hypothesis for future work could be that the fibrotic infiltrations in the ESUS patients reduce the hemodynamic efficacy of the left atrium and render clot formation (e.g. in the atrial appendage) more likely in this way.

      7) The negative finding in this study (no difference between groups) does not naturally allow us to draw clinical implications for diagnosis or stratification. Additional ways to put the hypothesis proposed by the authors (fewer arrhythmogenic triggers in the ESUS patients) to test could be to consider readouts/surrogate measures of the autonomic nervous system.

    3. Reviewer #1 (Public Review):

      Previous research showed a close link between sub-clinical AFib (Atrial Fibrillation) and ESUS (Embolic Stroke of Undetermined Source). As such, current established clinical care for ESUS patients is long-term monitoring for evidence of AFib and anticoagulant treatment for an individual with high risk for AFib. Nevertheless, questions are still unanswered about who the individuals with high-risk for ESUS are and how to properly identify this population.

      This research tries to identify the fibrotic properties of ESUS patients and its pro-arrhythmic potential using computational modeling of patient's left atria reconstructed from cardiac LGE-MRI (Late-Gadolinium Enhanced Magnetic Resonance Imaging). Ultimately, their results of the comparison between left atria of ESUS and AFib patients revealed that the fibrotic substrate that could induce arrhythmia in ESUS and AFib patients are indistinguishable, raising more questions that would need to be addressed in further studies.

      This study uses a sophisticated personalized computational modeling approach that has been validated in previously published papers. This study is also well designed, clearly written, with robust data and proper statistical analysis.

      What is left unclear is what is unique about the fibrotic substrate in ESUS patients in comparison to AFib patients in the future.

    4. Evaluation Summary:

      In this manuscript, the authors try to answer an important clinical question about the previously observed connection between embolic stroke of undetermined source (ESUS) and atrial fibrillation (AF). Using cutting-edge personalized computational modeling of left atrium from both ESUS and AFib patients, the researchers try to understand why the fibrotic substrate found in both ESUS and AFib patients causes arrhythmia in the latter group but not the former. Their study concludes that the intrinsic capacity to sustain arrhythmias of fibrosis found in ESUS and AFib atrium are identical. The key claims of the manuscript are well supported by the data, and the modeling methodology is largely appropriate.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

    1. Reviewer #3 (Public Review):

      In this manuscript, Sheng et al. have demonstrated that Langerhans cells (LCs) do not exit the skin both under steady-state conditions and after skin sensitization, using newly generated DC-SIGN DT mice and others. In addition, through a combined use of genetic fate mapping and novel inducible LC ablating mouse models, they show that the originally described lymph node LC fraction is actually an independent LClike cell population that originates from the dermis, not from the epidermis. Moreover, these LClike cells, which are replaced over time by bone marrow-derived counterparts, are characterized by their slow turnover rate and trafficking to the LN. This study contains novel and important findings. This reviewer has several comments on the manuscript.

      Major comments:

      1) The functional roles of LClike cells remains unclear, especially in the relationship with conventional LCs. Thus far it has been considered that LCs play essential roles in OVA-induced atopic dermatitis like models. But it remain unclear whether conventional LC s or LC like cells play important roles.

      2) The authors stated that a fraction of CD11bhiF4/80hi cells co-expressed CD326 and CD207 are detected in the dermis (Fig. 1b, upper panel), which is likely to be derived from the epidermis. But it remains unclear whether this subset is just a contamination through the separation process or a truly migratory one from the epidermis. The authors can demonstrate clear localization of LCs in the epidermis, LClike cells, and LCs in the dermis.

      3) Related to the above question, the authors claim that LCs can emigrate from the epidermis to the dermis. Given that LCs can emigrate from the epidermis to the dermis by transmigrating through the basement membrane, why LCs cannot migrate into the lymphatic vessels. LCs are known to express CCR7 highly that is important for migrating into the lymph nodes from the skin. What is the functional (APC, migration, etc) difference between LCs and LClike cells?

      4) I agree that the novel subset exists as CD207+CD326+ LClike cells in the dermis, which is different from conventional LC. But the term, "LC-independent" CD207+CD326+ LClike cells, which the authors used often through the manuscript, is a bit confusing, because it is not totally clear whether LClike cells are completely independent of LCs or not. It would be informative if the authors can demonstrate whether LClike cells contain bierbeck granules (this is also a hallmark of LCs) or not, since bierbeck granule-positive cells were detected in the LN (https://pubmed.ncbi.nlm.nih.gov/4758275).

      5) The authors can discuss the relationship between LClike cells and short lived LCs that were previously described (https://pubmed.ncbi.nlm.nih.gov/23159228).

      6) Where do LClike cells locate by FACS plots analysis using CD11b and CD103?

    2. Reviewer #2 (Public Review):

      The authors used several approaches to define a discrete population of Langerhans cell-like (LC-like) dendritic cells (DC) in the dermis of mice. By flow cytometry, these cells expressed langerin/CD207 and EpCAM/CD236 and were found in the CD103-CD11b- fraction of dermal cells. It was also shown that LC-like cells, rather than LC, are the main contributors to CD103- langerin+ cells in the lymph node. By single cell RNAseq of dermal cells they clustered with Langerhans cells but lacked expression of DC-SIGN/CD209. Fate-mapping with Kitmercremer/Rosa26loxPSTOPloxPeYFP showed a similar origin to other dermal DC, with no yolk sac signal as observed in LC. Bone marrow chimeras, however, indicated a much slower turnover compared with other dermal DC. Independence from LC and other DCSIGN+ DC was also demonstrated by unchanged kinetics during continuous ablation of DCSIGN+ fractions in a DTR model.

      The results explain the expression of langerin on two fractions of dermal DC observed several years ago (CD103+ and CD103-). The demonstration that epidermal LC do not contribute to LN populations in the steady state is completely unexpected and raises an important question of the in vivo function of the LC-like subset. LC-like cells appear to be related to LC but a more in-depth analysis of their gene expression differences compared with LC would be interesting. Also, their potential relationships with other DC (cDC1 or cDC2), was not defined. Langerin expression by human cDC2 is well described and possibly correlated with these observations in mice. Finally, the turnover of LC-like cells was slow, yet they contributed as many langerin+ cells to the resting lymph node as cDC1, which turnover quickly. Proliferation in situ might explain this observation but there were no data on this.

    3. Reviewer #1 (Public Review):

      In the days of the COVID-19 pandemic vaccines, mechanisms of vaccine administration are important and of broad interest. Vaccines are most often given into the skin. Antigen-presenting cells of the skin are responsible for eliciting the immune response in draining lymph nodes. Langerhans cells, the dendritic cell variant of the epidermis, are one of these cutaneous antigen presenting cells that are believed to do this job. They migrate from the skin, the site of antigen/vaccine uptake to the draining lymph nodes, where lymphocytes are located and where the immune reaction will be initiated. With their sophisticated experiments, the authors challenge this view. They use leading edge methodology (mouse models) that strongly suggest that there may be yet another subset of skin antigen presenting cells, that is responsible for carrying antigen from skin to lymph — at least in the steady-state skin. This population resides in the dermis (the connective tissue part of skin), as opposed to the classical Langerhans cells, which sit in the epidermis. This may be relevant to the maintenance of immunologic tolerance to innocuous substances in the absence of an overt inflammation. The data suggest that Langerhans cells may not play the crucial role they were thought to play. This is certainly a conceptual advance that — like always in science, especially when experimental systems are complex, as they are here — needs to be underpinned by future studies. In the long run, it will be very interesting (but much more difficult to study) to see whether this also holds true for human skin.

    4. Evaluation Summary:

      The present study uses innovative approaches to further our knowledge of skin immunobiology. The corresponding results explain the expression of langerin on two fractions of dermal DC (CD103+ and CD103-) observed several years ago. The demonstration that epidermal LC do not contribute to LN populations in the steady state is completely unexpected and raises important questions about the in vivo function of the LC-like subset unveiled in the present study.

      (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

    1. This manuscript is in revision at eLife

      The decision letter after re-review, sent to the authors on February 2 2021, follows.


      The reviewers concur that this article offers an interesting conclusion regarding optimal foraging and chemosensory valence. However, they also agree that it would benefit from a second round of revision, aiming at an improved precision of language and a better discussion of the assumptions of the model and experimental conclusions.

      Public Review 1:

      The authors present experiments that demonstrate how C. elegans worms bias their foraging decisions depending on feeding history and sensory cues (here, called pheromones) that reflect the density of worms. Navigational preference for these sensory cues is found to change from attractive to repulsive depending on the time at which worms leave a food patch, and additional experiments that condition worms under different combinations of conditions (with/without the sensory cues, with/without food, with/without repellent) indicate that associative learning is involved in this inversion of preference. A mathematical model is provided to argue that this inversion represents an optimal foraging strategy that is also evolutionarily stable.

      Public Review 2:

      The authors use the nematode C. elegans to reveal how animals associate social signals with specific contexts and modify their behaviors. Specifically, they show that C. elegans leaving a food patch are attracted to pheromonal cues, while those leaving later are repelled from pheromones. The authors using a behavioral model to suggest that the switch from attraction to repulsion is likely due to a change in learning. This study links learning with social signals providing a framework for further analysis into the underlying neuronal pathways.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 29 2021, follows.


      This paper builds on recent studies that have made the connection between chronic endothelial damage and cellular senescence among endothelial cells in PH. Here, using a transgenic mouse that expresses in endothelial cells a dominant negative form of the TRF2 protein needed for telomere maintenance, the authors induce cellular senescence in the endothelium and show that these mice demonstrate worse PH characteristics following exposure to chronic hypoxia. They go on to test the effect of this dominant negative protein on human pulmonary artery endothelial cells in vitro and show that transfected ECs increase expression of secreted and surface-bound signaling molecules, and that when co-cultured in direct contact with pulmonary artery smooth muscle cells the SMCs increase their proliferation, an effect blocked by pharmacologic inhibition of Notch signaling. Notch blockade in vivo attenuates pulmonary hypertension in both transgenics and wild type controls. These data provide an intriguing framework for understanding how endothelial damage alters signaling to neighboring cells in the vascular wall and provides further evidence that Notch signaling plays a key role in the development of PH vasculopathies.

      Essential Revisions

      1) It's unclear where within the pulmonary vasculature the TRF2DN transgene is expressed, and therefore which vessels are effected by senescence. That the transgene is driven by a well-established endothelial promoter (VEcad) is not sufficient to demonstrate universal expression. Especially in the case of a transgene whose expression is intended to result in chromosomal abnormalities, DNA damage and a halt to cell proliferation, significant mosaicism is to be expected. In situ hybridization with probes specific to TRF2DN or an antibody stain that specifically recognizes the transgenic protein on lung tissue sections would address this problem. Both representative images and a careful characterization of the classes of arteries (subdivided by diameter, for example), capillaries, veins, and lymphatics that express the transgene and with which levels of mosaicism would be ideal.

      2) Validation of the EC expression changes, specifically of the Notch ligands identified as upregulated in vitro, need to be validated in situ to ensure they are upregulated in the endothelium of arteries where the PH phenotype (increased muscularization, increased SMC proliferation) is observed in this model. Whole lung dissociation followed by enrichment for CD146+ ECs will result in an overwhelming number of capillary ECs and a tiny number of artery ECs (Figure 3E). Similarly for the in vivo validation of Notch reception in SMCs through qPCR for indicators of Notch reception (Figure 3F, 4I) - this experiment was done on whole lung lysate and does not demonstrate increased expression of these genes in the artery wall. In situ hybridization with probes specific to TRF2DN or an antibody stain that specifically recognizes the transgenic protein on lung tissue sections would address this problem. Both representative images and a careful characterization of the classes of arteries (subdivided by diameter, for example), capillaries, veins, and lymphatics that express the transgene and with which levels of mosaicism would be ideal.

      3) The method by which PAs are identified (Figure 1D, 4F) and the metrics by which artery muscularization from images of tissue sections is quantitated (Figure 1F, 1H, 4H) are somewhat unclear and appear to be made from very few fields from an unspecified number of animals. There appears to be significant variance in artery response to hypoxia (comparing Figure 1E with vehicle in 4G), which is not a problem and very much to be expected, but means there must be absolute clarity in how the data for graphs summarizing imaging data were obtained. A supplementary figure with representative images demonstrating how arteries were scored would be very helpful. The number of independent mice for each analysis must appear either in the figure legends or in the relevant sections of the methods. A reader's understanding of how the PAs were identified in Figure 1D and 4F would be helped by using a vascular specific antibody stain. And a supplementary figure with a large panel of artery images from Tg and Wt animals before and after hypoxia exposure, with and without DAPT, so the reader can grasp the range of effects on vessels in each case would be immensely helpful.

      4) Please describe the in vivo relevance of endothelial progeria induced by decreased TERF2 function in patients with PAH.

      5) While endothelial senescence leads to decreased proliferation and apoptosis of EC, which have been shown to occur in PAH, clonal proliferation of EC is also a hallmark of advanced disease in PAH. The study does not comment on this varied phenotype of EC in the pulmonary circulation in PAH patients and the relationship of senescence of EC to SMC migration.

      6) Increased levels of Jag1 have been linked to excess proliferation in several cancer cell lines. In the context of senescence with decreased EC proliferation, increase in Jag1/Jag2 levels is surprising and the paper does not comment on this phenotype as being distinct from cancer cells.

      7) The mechanism for increased notch ligand expression in response to progeria was indirectly addressed with 5-Aza studies which presumably leads to inhibition of DNA methylation. However, it is unclear how this inhibits increase in notch ligand expression. In the discussion, the authors mention (Line 17, page 10) that DNA hypomethylation promotes specific transcriptional programs as a result of senescence. However, 5-Aza prevented the induction of Notch ligand expression in senescent EC (Suppl Fig 2). The discussion of these results needs further clarification. It is unclear what specific epigenetic modifications occur to increase the expression of Jag1/2 and Dll4 in senescence associated changes.

      8) The study did not report whether aorta and other systemic vessels demonstrate senescence changes in endothelial cells-endothelial progeria in the TG mice would involve all vasculature. Presumably, vascular remodeling was limited to the lung, given the unique response of the lung to hypoxia. However, examination of a systemic vascular bed would have strengthened the conclusions of the study. Do the EC and SMC derived from aorta or coronary vessels show similar responses in vitro compared to human PAEC with DN-TERF2 transfection?

  3. Jan 2021
    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on December 14 2020, follows.


      This study examined osmolarity-dependent dendritic signaling in oxytocin magnocellular neurosecretory cells (OT-MNCs). The authors show that repetitive depolarizations evoke larger calcium responses in proximal dendrites relative to distal dendrites. When these neurons were exposed to hyperosmotic stimuli, the distal calcium responses were found to be inhibited to a greater extent compared to proximal dendritic calcium responses. Propagation of glutamate evoked depolarizations from the dendrite towards the soma were also found to be reduced following increases in osmolarity. These effects of hyperosmotic stimuli are likely mediated by changes in membrane resistance of dendrites. A non-selective blocker of the channels, ruthenium red, blocked these effects of hyperosmolarity, indicating the non-selective cation channels (e.g. TRPV types) may be responsible.

      All three reviewers agreed that the finding is potentially important and could address fundamental questions about MNC dendritic physiology. However, the reviewers identified a number of technical concerns, as summarized below. These concerns need to be addressed for further consideration.

      Essential Revisions

      1) The title and abstract are not exactly reflecting what this study is about. The title of the paper is "Dendritic membrane resistance modulates activity-induced Ca2+ influx in oxytocinergic magnocellular neurons of mouse PVN". However, dendritic membrane resistance is never actually measured. As such, a title that does not mention membrane resistance may be more appropriate. Also, the purpose and rationale of this study are not clearly communicated in the abstract and introduction. The implication to the regulation of soma-dendritic release of oxytocin, but not hyperosmotic responses, was mentioned in Introduction, while the entire Results and Discussion sections are about hyperosmotic stress.

      2) Figure 3: The reviewers believe that stimulation paradigm is not physiological (neurons voltage-clamped at -70 mV with repetitive voltage steps to +50 mV for 5 ms). It is important to show that action potentials in the current clamp, instead of the +50mV voltage step in the voltage-clamp, can produce similar signals.

      3) A major focus of the manuscript is on Ca2+ elevations in MNC dendrites. However, the authors have not performed the essential experiments to identify what the Ca2+ entry/release pathways are. It is important to show that Ca2+ is through voltage-gated Ca2+ channels for their main conclusions. In addition, it should also be established whether dendritic propagation is active or passive.

      4) It is essential to report the effect of the osmotic stimulus alone on dendritic resting Ca2+, as this would affect the interpretation of the Ca2+ data.

      5) Figure 8: What is the effect of RR on proximal EPSCs? This information is needed to interpret the effect of RR on distal EPSCs. It would be required to also test the effect of RR on the modulation of Ca2+ responses in distal dendrites to see their effects on the dendritic conductance.

      Statistical handling:

      Please provide the statistical methods (t-test, 2-way ANOVA with Hom-Sidak corrections, 2-way repeated-measures ANOVA, etc.) used for each measurement in the text or figure legend (not just in the method section). For repeated measures ANOVA, please indicate how measurements were repeated.

      For the statistics of sex differences (Fig. 2-1, 4-1 etc), it is required to use 3-way ANOVA to assess variability by cells, animals, and sex. The number of males and females used is not clear in some cases, but it appears that only 2 females and 2 males are used (Line 203-204). If this is the case, the statistical comparisons between males and females are not meaningful and should be removed.

    1. Reviewer #3:

      The glypicans Dally and Dlp have important roles in morphogen signaling, and this work is of particular interest for me because it significantly advances our understanding of the multiple roles they appear to have in signal processing, signal presentation and signal reception. It is unfortunate that most of the literature has presented results and phenotypes in simplistic or simple-minded ways that do not recognize the different roles or the glypicans, or do not take experimental approaches that might distinguish them. This work of the Guerrero lab is an exception, as it is an important contribution to understanding these different roles, especially given the additional complexity introduced by the role of cytonemes. If its thoroughness and in-depth analysis are typical of work from this lab, so is the challenging presentation that makes understanding it so difficult. My recommendation to the authors is to clearly describe the different roles that have been attributed to the glypicans and for every experiment they present, clearly articulate how the results might implicate or distinguish any or several of them.

      Although the figures are excellent, the manuscript is not well-written and would benefit from a rewrite.

    2. Reviewer #2:

      This manuscript interrogates function of Ihog and Boi adhesion molecules in cytoneme-based transport of the Hedgehog morphogen in Drosophila. The cell biology of how cytonemes are regulated to deliver morphogen signals is not yet well understood, so the work addresses an important topic that will be of interest to a broad audience. However, much of the study refines previous work from the same group to provide only a modest advance in understanding of how Ihog impacts cytoneme behavior.

      The authors use genetic strategies in Drosophila to investigate how Ihog and Boi influence cytoneme dynamics. They find that the two proteins act differently with regard to cytoneme function. Boi effects are not exhaustively analyzed, but a number of genetic experiments are performed to interrogate Ihog. The authors reveal that the extracellular domains of Ihog interact with the glypicans Dally and Dlp to stabilize cytonemes that originate from Ihog over-expressing cells. Knockdown of Ihog does not alter cytoneme dynamics.

      The most novel aspect of the study - that Boi functions differently than Ihog in cytonemes - is, unfortunately, not expanded upon. Some experiments lack controls or are presented in a manner that prevents clear interpretation of results.

      Key points to be addressed:

      Figure 1: Null alleles and RNAi silencing are used interchangeably to reduce Ihog, Boi, Dally and Dlp function in vivo. Results between methods are directly compared. Oftentimes, controls are not included to confirm the level of knockdown following RNAi. If possible use null alleles due to consistency. However, if this is not possible due to experimental reasons, give an explanation and state impact in the discussion.

      Ihog levels decrease following loss of Dally or Dlp and Boi levels appear to increase following knockdown of Ihog, Dally, or Dlp. These stability changes have previously been reported. The mechanism is not clear, so should have been investigated here - especially the increased Boi protein level. How does this occur? Is stabilization occurring at the protein level or is gene expression changing? Is this a compensatory upregulation?

      Based upon the supplement for Figure 2, it looks like the Ihog truncation mutants show variable stability. Might this be affecting the extent to which they alter Dally or Dlp stability? The western blot data are presented as crops of single bands adjacent to crops of a molecular weight ladder. Blots should be shown as intact images, preferable with all variants compared across a single gel with a loading control. As presented, relative stability/expression levels are impossible to assess.

      Figures 3-4: Ihog mutant transgenes are tagged with either HA or RFP. Best to be consistent with tags when mutant function is being directly compared. Given that the HA tag is a small epitope and the RFP is a protein tag, they may differentially alter protein functionality. To be consistent it would be preferable to use the same tags. However, if this is not possible due to experimental reasons, the technical implication can also be mentioned in the discussion.

      Figure 5: Investigation of histoblast cytonemes reaching into ttv, botv mutant clones: The ability of cytonemes to invade double mutant clones is altered only under the engineered situation of glypican dysfunction combined with Ihog over-expression. From this, it is concluded that Ihog is acting with glypicans to stabilize cytonemes. This may be the case, but they ability to see it only under an engineered situation of compound mutation plus Ihog over-expression leads this review to question the physiological relevance of the observation. Of similar concern is that the authors state the ability of Ihog over-expressing cell cytonemes to cross small vs. large ttv, botv clones differs. The difference is very difficult to appreciate from the results presented.

      Figure 6: The apparent functional difference between Ihog and Boi in the ability to stabilize cytonemes is potentially very interesting, but is not investigated, which limits the advance of the current study.

    3. Reviewer #1:

      In the article "Glypicans specifically regulate Hedgehog signalling through their interaction with Ihog in cytonemes" Simon et al. elucidate the function of Glypicans in Hh transport via cytonemes. The manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog. Ihog - in turns - stabilises Hh cytonemes through its interaction with Glypicans to establish the Hh gradient in the wing imaginal disc. The authors further carried out an extensive molecular analysis of Ihog and identified the relevant domains within the protein required for interactions with Glypicans, Patched, and Hh. In general, this is a very thorough, detailed analysis of Ihog function. The images and videos are excellent. However, prior publication, there are two major criticisms, which needs to be addressed, in my opinion.

      Firstly, the first part of the manuscript, the molecular analysis of Ihog (Fig.1-4) seems to be detached from the second cytoneme-focussed part (Fig. 5, 6). Independent evidence is needed to show support for the idea that the Ihog-Gly mediated stabilisation of cytonemes is responsible for the expansion of the signalling gradient. Are the static cytonemes involved in a flattened gradient or are the receiving cells just sensitised for Hh? Can cytonemes be (de-) stabilised w/o interfering with Hh components to untangle these observations? The authors write "Intriguingly, the same Ihog domains that regulate cytoneme dynamics are those also involved in the recruitment of Hh ligand, glypicans and the reception complex."

      My concern is that cytoneme dynamics and Hh gradient formation could be two parallel, independent events -> one needs to show this interdependency in a clear way. I could imagine an analysis of the consequences when Ihog is overexpressed, and cytoneme formation is inhibited (by other means). Consistently, could one stabilise cytonemes in an Ihog-reduced background and analyse gradient formation?

      Secondly, the authors demonstrate an effect of Ihog alterations on the formation of the gradient. However, what is the physiological relevance? What are the consequences of Ihog/Gly-mediated cytoneme stabilisation and gradient formation on tissue patterning and wing formation? If this is not possible to show experimentally, this needs to be discussed.

    4. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 12 2021, follows.


      In summary, this manuscript elucidates the function of Glypicans in Hh transport via cytonemes. The reviewers felt that that the manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog, which stabilises cytonemes to establish the Hh gradient in the wing imaginal disc. A molecular analysis of Ihog domains was well executed.

      Although the manuscript provides an in-depth analysis, the reviewers believe that the presentation of the data is rather challenging for the readers. The authors need to clearly describe the different roles that have been attributed to the glypicans and for every experiment presented, a clear explanation of the impact of the results is needed e.g. Figure 5. In addition, the stability of Ihog and Boi by altered Glypican levels and their ability to stabilize cytonemes needs to be investigated. Finally, linking the Ihog analysis to cytoneme stability analysis needs improvement.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on December 17 2020, follows.


      We feel that the major conclusions are right but the manuscript and story is not quite clear enough at present and there is a lack of deeper cellular and molecular mechanistic understanding of these phenomena to distinguish this work from the previous published studies. That ASC senescence impairs adipogenic differentiation capacity, has been previously reported in eLife in 2015 (doi: 10.7554/eLife.12997). For example, you concluded that adipose derived progenitor cells from older adults have higher potential to become senescence, which impaired adipogenesis. The percentage of senescent cells in adipose tissues is low, but the mechanism of how they could significantly affect adipose tissue functions is unknown. Is this through paracrine effects? or cross talk with other immune cells? etc.

      Essential Revisions

      1) Although the authors found that ASC senescence is associated with mitochondrial dysfunction and oxidative stress, it the nature of the links between these cellular events is unclear. It is well-known that mitochondrial dysfunction can directly lead to senescence. If the authors meant to prove that ASC senescence causes early adipocyte mitochondrial dysfunction, more evidence is required.

      2) It has already been reported that ASC senescence impairs adipogenic differentiation capacity, in Elife in 2015 (doi: 10.7554/eLife.12997). Furthermore, although the authors found that metformin prevents the onset of senescence and associated dysfunctions in ASCs, it has been shown in many publications that metformin is a senomorphic drug that can reduce the senescence-associated secretory phenotype. So it is not surprising that metformin can block the effects of senescent ASCs. Also regarding the increased adipogenesis by metformin, it has been reported that metformin can directly regulate adipogenic transcription factors, such as peroxisome proliferator-activated receptor (PPARγ), CCAAT/enhancer binding protein α (C/EBPα). As such, sufficient novelty is lacking at this point, and would require demonstration of causal links among these cellular events.

      3) Several conclusions need to be smoothed out and discussed in more detail. Methods must be described with more details, especially with regard to fat depot digestion (type of collagenase, concentration of collagenase, amount of tissue used for the digestion, are cell yields similar between young and old adipose tissue? Number of plated ASC?). The authors must consider that the term ASC is nowadays related to Adipose stromal cells and not Stem cells. As described in the introduction and method sections, ASC are stromal cells that adhere to plastic including fibroblast, smooth muscle cells, pericytes, endothelial cells, resident macrophages, preadipocytes and progenitors. This must be discussed since distribution and repartition of stromal cells are modulated with aging. The term "adipocyte" must be changed to "differentiated ASC" because adipocytes are characterized by unique lipid droplet (not the case here). The title must be modified. Senescence is related to ASC and not to adipocytes.

      4) Figure 1: It is unclear why the authors conclude about they are recapitulating in vivo aging. If so, one might expect that senescent "young ASC" phenotype may recapitulate the one of "old ASC" with a time lag, what is not the case for all the studied parameters. For example, the % of bgal cells is equivalent between P7 old cells and P11 young cells what is also true for P16, P21 and prelamin A but not for reactive oxygen species or mitochondrial potential. The authors must discuss this point.

      5) Figure 2: Was Cell number at confluency controlled and similar between "young" and "old" ASC? Since post-confluent mitosis are necessary for adipogenesis, one might speculate that the decreased adipogenesis might be related to less cell number and proliferation.

      6) Figure 3 and 4: Cells were treated from P3 with metformin. Do the authors consider potential "resistance" effect? When taking into account the large number of individuals treated with metformin, is there any evidence of an impact of metformin treatment on age-related loss in subcutaneous adipose tissue? Finally, inhibition of senescence may lead to cancer development. The authors must discuss this point.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 18 2021, follows.


      This is an exhaustive study of different phenotypes associated with Histone H3-G34 mutations in a fission yeast model. Because mutations at this site occur in certain human cancers, teasing apart their different phenotypes in a model system helps to understand their potential effects in pathology. The phenotypes vary widely, suggesting a key role for this residue in a variety of genome maintenance functions.

      The authors systematically examine histone modifications, transcription, and use genetic and cytological assays to measure genome stability. The phenotypes vary widely, suggesting a key role for this residue in a variety of genome maintenance functions. Direct extrapolation to human cells is limited due to the absence of multiple H3 variants in fission yeast, and the absence of the PRC1/2 pathway. However, this is balanced by the rapid and thorough analysis of numerous variants that is enabled in this model system.

      It is not possible to draw a simple model as there is little consistency in the phenotypes. This suggests that the G34 residue independently affects multiple activities. These will require laborious efforts to tease out.

      Essential Revisions

      This is overall a technically very well done paper with a variety of methods to examine different mutations in H3-G34. The strength is the consistent approach applied to numerous mutations. However, as there is no single response, it's rather descriptive overall. We have no major concerns about the data, but feel that the conclusions need to be tempered in two areas where the assays were not direct.

      1) In the absence of NHEJ repair assays it needs to be noted that conclusions about NHEJ proficiency based on drug sensiutivty are indirect.2)

      2) The authors imply that the H3G34 mutants affect the activity of the Set2 H3K36 methyltransferase. In the absence of an in vitro H3K36 methylation assay on the mutant histones with recombinant or affinity purified Set2 the authors need to note that this conclusion is speculative as they have not measured it directly.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on December 29 2020, follows.


      This paper describes very clearly a set of experiments to assess collateral sensitivity to certain antibiotics that is created by carriage of beta-lactam (incl carbapenenem) resistance plasmids in E. coli. This addresses one of the limitations of existing literature on CS, which typically focuses on the effects of resistance point mutations, which are clinically less significant. By documenting multiple ways that this CS is real and selectable and to a degree generalizable across genetic backgrounds, this is an important contribution in showing that CS is a real phenomoenon for clinically important resistance mechanisms.

      Essential Revisions

      1) The primary screen of 'antibiotics x plasmids' to identify collateral sensitivity, presented in Figure 1B, lacks an analysis of the statistical significance of results. Supplementary data shows that measurements of MIC are a little too noisy to robustly identify 2-fold changes with only 4 or 5 replicates. Defining "significant" as "mean more than 2x" is not adequate. Using a power calculation derived from the data in the manuscript, a sample size should be determined to have a 90 (or other high) % chance of detecting a 2x difference given the variability observed between assays, and then they should be done. Ideally this would be for all organism-plasmid pairs, but at least for the ones that the preliminary screen found a mean of 2x for.

      2) Recommendation (not required for acceptance, but please temper claims of clinical relevance if not done): The comparative killing data should be repeated in competition. This is technically more challenging but I believe not more so than the comparative growth curves. This would establish as proof of principle that a mixed population could be purified of plasmid-bearers by CS. Without this, the clinical relevance will still remain speculative. Also, two reviewers initially misread these as competition assays. The text and legend should emphasize that these are separate populations

      3) (Not required for acceptance but suggestion for future work:) The presented work is solid but, as pointed out by the authors, there is no mechanistic explanations for the observations. It would be highly interesting to know if the collateral effects are due to specific genes (OXA-48 would be a good place to start) and/or if the observed effects are due to the plasmid backbone.

      4) The experiment in Figure 3 demonstrates the exploitation of collateral sensitivity to preferentially inhibit plasmid-bearing bacteria. The terminology in this section refers to 'eradicate', 'mortality' etc, but in practice, the experiment defines survival as OD>0.2 after ~24 hours. It seems likely that in the 'non-surviving' conditions, waiting another day or two would show regrowth of some bacteria in these conditions.We don't think this requires any change to the experiment, only how the results are described: they show preferential inhibition of growth, not eradication. A more patient approach to identifying regrowth would be necessary to definitively state that these bacterial populations have been eradicated. Suggest tempering claims.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 11 2021, follows.


      Adiponectin is a key adipokine, and much of our knowledge about this molecule has come from the Scherer lab. It is well known that adiponectin promotes improved insulin sensitivity and glucose tolerance, along with anti-inflammatory effects, which can be followed by decreased fibrosis. In this paper the Authors use loss and gain of function mouse models to explore whether the beneficial effects of adiponectin on metabolism can be translated into greater healthspan or lifespan. They show that lifespan decreases in adiponectin KOs and increases in the transgenic (ΔGly) mice. The expected effects on glucose metabolism, insulin sensitivity, inflammation, and fibrosis are also demonstrated.

      Essential Revisions

      1) Given the known significant effects of adiponectin on metabolic fitness, the effects on healthspan which the Authors observed in their 2 models, was expected. However, while median survival time is definitely less in the APN-KOs and greater in the ΔGly mice, the effects are relatively modest compared to other longevity studies. Any increase in lifespan is a good thing, particularly when accompanied by a corresponding increase in healthspan. We would've hoped for greater effects on lifespan than those observed but even modest effects are worthwhile. The Authors should comment in their discussion on this point. In other words, it would be good to know the Authors' thinking as to why these impressive effects on glucose, insulin, inflammation, fibrosis, etc. do not lead to even greater effects on lifespan. Also, is there any information on the causes of mortality in the WT vs. KOs that might point to why lifespan is decreased?

      2) APN-KO clearly leads to impaired glucose tolerance, but it is a bit surprising why insulin levels aren't increased, which is the typical metabolic response to insulin resistance.

      3) Can the Authors please comment on adipose tissue mass in the KOs, particularly if they have any information on subq fat?

      4) In Figure 3, they show increased staining for ATMs with Mac2 in the KOs. What about the expression of other inflammatory gene markers, such as those shown in Figure 3G for the liver?

      5) With respect to hepatic effects, this paper shows increased inflammation in the liver in APN-KOs. However these gene expression patterns are in total liver tissue, and it would be helpful to understand the origin of these inflammatory markers. Are they from Kupffer cells, monocyte-derived macrophages, etc. In a similar vein, various fibrosis marker genes are increased in total liver from the APN-KOs. Most likely these expression differences reflect stellate cell effects. Do the Authors have any information on the effect of adiponectin on stellate cell function. Although fibrosis-related genes are elevated in the APN-KO, is there histologic evidence of increased fibrosis in the liver sections?

      6) The Authors suggest that the increased inflammation in the liver is the cause of the increased fibrosis. Presumably they think that the immune cells in the liver are signaling to stellate cells to produce this effect. Is this the scenario the Authors propose. If so, it should be made more explicit and corroborated by histologic staining of hepatic fibrosis.

      7) It would be of interest to know the extent of inflammation in the kidneys with APN-KO, beyond Mac2 staining (Figure 3D).

      8) In the results in the ΔGly mice, is the enhanced lifespan statistically significant. Unless we are misreading it, the p value suggests it is not. Also, why have only study chow fed mice and not HFD mice in the transgenics, as they did in KOs?

      9) ITTs are shown in Figure 4G, but the basal glucose values are different between the 2 groups. Can the Authors also present the data normalized to the basal value to determine whether the kinetics of the curve are different?

      10) The resulting changes in tissue fibrosis are clearly important when thinking about healthy tissue function. It would help if the authors could show histologic staining for collagen deposition in the various tissues, particularly liver and kidney. Although it might be asking for too much if the they don't already have this information, it would also be useful to know which cell types within the various tissues are responsible for the changes in inflammatory markers and collagen related genes. This could also be discussed.

      11) From an aesthetic point of view there is a certain lack symmetry in this paper, since some of the measurements made in the KOs are not performed in the transgenics and HFD was not utilized in the transgenics either.

      12) Much of the data could be predicted from studies by them or the other investigators in the field (Nature Med. 8, 731 (2002), J. Biol. Chem. 277, 25863 (2002), J. Biol. Chem. 277, 34658 (2002), J. Biol. Chem. 278, 2461 (2003), Endocrinology 145, 367 (2004), J. Biol. Chem. 281, 2654 (2006), Am. J. Physiol. Endocrinol. Metab. 293, 210 (2007), J. Clin. Invest. 118, 1645 (2008) . IT would be helpful if authors could provide insights into the life-promoting mechanism by adiponectin that has not been clarified so far.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 5 2021, follows.


      In this manuscript, Olive and colleagues used a genetic screen to identify Complex I (CI) of the electron transport chain (ETC) as a regulator of IFNg-mediated gene expression in macrophages. They attribute this role of CI to effects on the activity of the JAK-STAT pathway downstream of the IFNg receptor.

      While a potential link between CI activity and the activity of the JAK-STAT pathway would be interesting, the reviewers think that additional analyses are needed to substantiate this claim and rule out alternative interpretations.

      Essential Revisions

      1) Lines 204-205: The authors find that sgRNAs targeting other complexes of the ETC, including CIII and CIV, had no effect on the ability of IFNg to stimulate expression of cell surface markers. How do the authors interpret these findings, since CI does not work in isolation in the ETC and is rather dependent on CIII and CIV activity?

      2) How does IFNg stimulation affect oxidative metabolism as assessed by Seahorse? In order to corroborate the authors' conclusions regarding activity of individual ETC complexes (point 1 above), Seahorse analysis of individual complexes is also advised.

      3) The authors do some limited analyses in human MDMs to suggest that their findings in the mouse macrophage cell line can be generalized to other macrophage populations. It would be great if the analyses in the human MDMs could be extended to further strengthen the generality of their central findings.

      4) Fig 6D: Not clear whether similar exposures were used in different panels. Would be better to load samples in the same gel so that the same exposure can be used and a direct comparison between conditions can be made.

      5) Fig 6D: Does acute treatment with rotenone (but not inhibitors of other ETC complexes) have similar effects in reducing JAK-STAT signaling as knockdown of CI subunits? If not, then stable, long-term knockdown of CI subunits may have some effect independent of respiration in influencing JAK-STAT signaling (for example, on expression of some component of the JAK-STAT pathway). This interpretation could also explain why knockdown of other components of the ETC do not have similar effects to CI. Rotenone treatment could be tried (and compared with inhibitors of other ETC complexes), and if the data are different from knockdown of CI subunits, then related data in the study could be re-interpreted and conclusions modified.

      6) In Fig. 3H a key control is missing. What about survival of the cells when the import of the only energy substrate is blocked?

      7) The authors could consider placing their findings in the context of the broader literature. (As just one example, Ivashkiv Nat Imm 2015 described a role for mTORC1 and metabolism in IFNg-mediated transcriptional and translational regulation in macrophages.) This would increase the impact of their findings.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 7 2021, follows.


      In this study, Olive and colleagues used a genetic screen to identify new regulators underpinning the ability of the cytokine IFNg to upregulate MHC class II molecules, of relevance to our understanding of how macrophages are activated by IFNg to confer host defense during microbial infection. They identified the signaling protein GSK3b, and MED16, a subunit of the Mediator complex previously implicated in gene induction.

      Essential Revisions

      1) Experimental treatment with IFNg may not be physiological. In key experiments, authors should try co-culture with activated NK cells +/- IFNg neutralization. A dose and time response curve of IFNg treatment may be valuable in key experiments.

      2) Comparison to cells not stimulated with IFNg is needed in key experiments. Comparison to WT cells is needed in Fig 5A,B.

      3) Stimulation with Type I IFN and other PAMPs in key experiments, as comparison to the effects of IFNg and to broaden the relevance of their findings.

      4) More insight into how IFNg signaling interfaces with GSK3 and MED16 is needed (e.g. role of mTORC1 pathway in regulating GSK3).

      5) Can the authors extend their data to an in vivo setting?

      6) Can the authors clarify the relative roles of GSK3a and GSK3b? For example, how do the authors explain the lack of a robust phenotype in Fig 3B-F?

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on December 17 2020, follows.


      In the paper, the authors used metabolomics to identify Valine and TDCA as metabolites depleted in diet-induced obesity (DIO) and replenished after sleeve gastrectomies (SGx) in mice. Intraperioneal injection of these two metabolites mimics many of the benefits of SGx, including weight loss, reduced adipose stores and insulin sensitivity. These benefits are related to Val/TDCA's ability to reduce food intake without altering locomotor activity, leading to a negative energy balance. Val/TDCA injection eliminated the fasting-associated rise in hypothalamic MCH expression in obese mice, and central injections of recombinant MCH blunted weight loss induced by Val/TDCA. Overall, this paper reports interesting and surprising observations related to the impact of metabolomic disturbances in obesity, and suggests a role for Val and/or TDCA in regulating food intake through MCH.

      Essential Revisions

      1) It is unclear from the data whether the effects are derived from valine, TDCA, or both. Both reviewers felt that any reader would want to see experiments where either of these metabolites is injected alone.

      2) No quantitative metabolite concentration values are provided anywhere, making it difficult to evaluate the robustness of the data. How much do the levels of TDCA and valine change with SGx in mice and humans, and what levels are achieved with the injections of these metabolites in the mice?

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 10 2021, follows.


      The reviewers agree that this is an interesting and useful contribution for understanding LQ extinctions, and that it is generally well-presented. It shows that the factors that increase extinction risk are de-coupled from the factors that eventually lead to extinction and thus in its timing. However, the reviewers also note that although the modelling approach is novel, it is reliant on datasets that are biased and at times these biases are not well-accounted for. Because much of the conclusions drawn from the modelling could already be drawn from existing records and using literature that is glossed over here, attention to that literature should be improved and the contributions beyond the megafauna debate should be emphasized. Furthermore, the authors should take care to improve clarity in the framing of the models, the presentation and interpretation of results, figures, and discussion.

      Essential Revisions

      1) ADDITIONAL ANALYSES (no additional data collection). The reviewers had specific concerns about the effects of sampling on the extinction chronology and the influence of body mass on a number of things (recovery potential, life history/demographic correlates, etc). Specifically, the analytical issues that present the biggest problems revolve around sampling uncertainty and body mass correlation. The former could be addressed by introducing some sensitivity tests. These could be directed towards chronological biases (how does removing one date affect the confidence intervals?), as well as geographical sampling biases (how does removing a region affect the trends?). The latter in particular would be important in the claims of a continental trend. It is also possible that biases are a function of taxon sampling. There are an increasing number of small mammal Pleistocene extinctions being recognized in Australia, and it is unclear if these follow the same trends as the megafauna. If so, that would indeed remove the body size issues.


      (i) appears to assume that only human hunting will differentially affect demographically sensitive species. However, novel or extreme climate change can also affect such species (e.g. Selwood, K.E., McGeoch, M.A. and Mac Nally, R., 2015. The effects of climate change and land‐use change on demographic rates and population viability. Biological Reviews, 90(3), pp.837-853.)

      (ii) this mechanism is predicated on using a modelling result [ref. 25] as data. It also makes the bold claim that species inhabiting certain habitats are less accessible to human hunters without any consideration of the archaeological or modern record on this point (e.g. Roberts, P., Hunt, C., Arroyo-Kalin, M., Evans, D. and Boivin, N., 2017. The deep human prehistory of global tropical forests and its relevance for modern conservation. Nature Plants, 3(8), pp.1-9; Fa, J.E. and Brown, D., 2009. Impacts of hunting on mammals in African tropical moist forests: a review and synthesis. Mammal Review, 39(4), pp.231-264).

      (iv) many of the supporting references here do not seem like logical choices for this argument. E.g. [28] refers to coral-reef fishes. Moreover, this hypothesis conflicts with much modern data showing that extinction risk and body size are correlated under climate and environmental change (e.g. Cardillo, M., Mace, G.M., Jones, K.E., Bielby, J., Bininda-Emonds, O.R., Sechrest, W., Orme, C.D.L. and Purvis, A., 2005. Multiple causes of high extinction risk in large mammal species. Science, 309(5738), pp.1239-1241. Liow, L.H., Fortelius, M., Bingham, E., Lintulaakso, K., Mannila, H., Flynn, L. and Stenseth, N.C., 2008. Higher origination and extinction rates in larger mammals. Proceedings of the National Academy of Sciences, 105(16), pp.6097-6102. Tomiya, S., 2013. Body size and extinction risk in terrestrial mammals above the species level. The American Naturalist, 182(6), pp.E196-E214.)


      The major weakness in this manuscript is in the discussion. The authors should be very clear in their discussion that their model does not indicate that demographic factors had no part in extinct events per se, but rather that they don't explain extinction chronology. Extinction chronologies reflect a number of different factors and processes, but they don't take away from the fact that certain life history traits can make a species more likely to go extinct from those factors.

      The authors seem to argue that demographics don't explain the megafaunal extinction in the Sahul, but in fact, their results suggest that they do; the only thing demographics by themselves don't explain is the chronology. Extinction risk as determined by demographic susceptibility is highly related to body mass and generation time (which in turn is also affected by body mass) but differential survival (timing of extinction) is determined by factors such as geographic range size, dispersal ability, access to refugia, and behavioral and morphological adaptations against hunting, and the ability to survive catastrophic events. A reiteration of this point would be beneficial to the clarity of this otherwise well written manuscript.

      The authors clearly (and elegantly) show that extinct species, which were all large, and had long generation times, had demographic traits that made them more susceptible to extinction. This is evident in figures 3 and 4. However, in the discussion, in lines 301-303, they state that no demographic trends explain the extinction. This is not supported by the results. While the timing of when species go extinct doesn't correlate with demographic susceptibility, the peculiar nature of the extinction-a large size biased extinction-is explained by demographic factors, and is a phenomenon that has been explored in a global analysis by Lyons et al. 2016 Biol. Lett. Therefore, demographic trends DO explain why certain species go extinct, while others survive. The authors should be careful when they say that "that no obvious demographic trends can explain the great Sahul mass extinction event"; instead, they should re-iterate that no obvious demographic trend explains the extinction chronology.

      4) MORE CAREFUL DISCUSSION OF RESULTS RELATIVE TO LITERATURE. The authors further go on to suggest that their results suggest that the extinctions were random, but the size-selectivity clearly shows that the extinctions were in fact not random with respect to body size.Their analyses do show that the rate of extinction doesn't exceed background to the same degree that it's been suggested in prior studies, and this is something that researchers need to explore further. Also, the authors raise an important point in lines 309-311 that human hunting could have interacted with demographic susceptibility, something that Lyons et al. 2016 Biol. Lett. show, and the results of the present study should be discussed in light of the 2016 paper.

      They also raise an important point in lines 312-320 that behavioral or morphological adaptations may have allowed some seemingly "high risk" species to persist despite anthropogenic pressure. These model "mis-matches" have been reported by Alroy 2001 Science as well in a multispecies overkill simulation. It would be beneficial to discuss the present results within the context of other examples of model mismatches, such as those from Alroy 2001.

      In lines 353-358, the authors once again state that their results show no clear relationship between body-mass and demographic disadvantage, despite clearly showing these relationships in Figures 3 and 4, and even stating as much in the beginning of the discussion. The plots clearly show that large bodied taxa were at a demographic disadvantage. There is a difference between explaining why certain taxa go extinct vs. why they go extinct at a certain point in time, and this should be made clear. The authors are correct in stating that demographic factors don't explain the relative extinction chronology, i.e. when species go extinction relative to each other, but they do explain why large species go extinct, and why these extinctions take place after human arrival. Moreover, generation length, which is also correlated with demographic susceptibility, is highly correlated with body mass (Brook and Bowman 2005 Pop. Ecol), once again showing that body mass-related effects do help explain the extinctions.

      The authors rightfully point out earlier in the discussion that spatial variation, local climates, ecological interactions, etc. all influence how and why a particular population disappears. Extinction chronologies reflect a number of different factors and processes, but they don't take away from the fact that certain life history traits can make a species more likely to go extinct from those factors. Large proboscideans like mammoths had a high risk of extinction based on life history traits, but managed to survive on island refugia into the mid-Holocene. Similar other examples exist, and show that extinction chronologies can vary vastly.

      Therefore, the lack of correlation can be explained by these factors, and the authors need to expand on these in their discussion, perhaps if possible, by giving specific examples. They should be more careful in their discussion by clearly distinguishing drivers of extinction risk, and how these drivers can be de-coupled from timing, but at the same time providing a good explanation for the biological factors leading to the extinction. Here again the authors should consider the work of Brook and Bowman and Lyons et al.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 7 2021, follows.


      This manuscript describes a detailed investigation of the sigma-1 receptor, with an emphasis on the effects of membrane cholesterol content. The authors report that sigma-1 receptor clusters in cholesterol-rich microdomains in the endoplasmic reticulum (ER), contributing to its previously-described localization at mitochondria-associated ER membranes. A series of reconstitution experiments show cholesterol-dependent clustering of the sigma-1 receptor, an effect which is modulated by membrane thickness and drug-like ligands of the receptor. These findings are supplemented by an investigation of the effects of sigma-1 receptor on IRE1a signaling, leading to the finding that sigma-1 knockout attenuates IRE1a function.

      Essential Revisions

      The reviewers agreed that the manuscript was likely to be of broad interest and addresses important biological questions surrounding the poorly understood sigma-1 receptor. However, concerns were raised regarding a number of points that need to be addressed in order for the manuscript to be suitable for publication. Specifically:

      Most of the imaging experiments throughout the manuscript are interpreted only qualitatively, and many of these show relatively minor differences. See "MINOR POINTS" below for a list of specific examples. Objective quantitative analysis should be provided wherever possible. Any subjective assessments should be conducted using blinding to avoid introduction of bias.

      The connection between the biological effects on IRE1a activation and cholesterol-dependent clustering is relatively indirect. The reviewers agree that additional experimental data should be provided to further assess the validity of the authors' proposed model. For example, inclusion of rescue experiments in sigma-1 knockout cells using the cholesterol-binding mutants would help to strengthen the connection between IRE1a function and membrane cholesterol content. Similarly, disruption of cholesterol-rich domains by addition of beta-cyclodextrin could provide additional evidence to support the model. In addition, testing the effects of ligands in the cellular imaging experiments would strengthen the link between in vitro biophysical experiments and cellular physiology.

      A related issue is that cholesterol binding is not tested explicitly for certain sigma-1 receptor mutants, potentially confounding interpretation of experimental data. These include experiments where alterations were made to the S1R sequence, with results interpreted in light of S1R no longer being able to bind cholesterol. Two specific places where this issue arises are:

      1) Studies described on pages 6-7 and shown in Figure 3B where wild-type sigma-1 receptor is compared to S1R-Y201S/Y206S, S1R-Y173S, S1R-4G, and S1R-W9L/W11L. These mutations had differential effects on receptor distribution that were attributed to alterations in cholesterol binding without confirming the changes in cholesterol binding. This is particularly relevant for the explanation given for why S1R-W9L/W11L fails to cluster in both cells and the cholesterol supplemented GUV system, while the S1R-4G mutant exhibited cholesterol-induced clustering in the GUV system but not in cells (page 7, lines 27-31).

      2) Another example is the membrane thickness experiment described at the top of page 8 and shown in Figure 4A. Shortening the S1R by deletion of 4 aa in the TM region produced a sigma-1 receptor that exhibited a more diffuse distribution when expressed in HEK293 cells. The authors appear to be attributing this only to the decreased length of the sigma-1 receptor transmembrane domain. However, it seems feasible (based on their other data) that if this construct fails to bind cholesterol, the same result would be observed. Confirming that the truncated sigma-1 receptor does in fact bind cholesterol would strengthen the argument being made here.

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 5 2021, follows.


      Your work analyzes the impact of the INPP5E inositol lipid-5 phosphatase on immune synapse formation and function. INPP5E is a cilium enriched protein. Although T cells do not display primary cilia, previous work by several laboratories showed that several ciliary proteins are involved in immunological synapse formation and in T cell activation and your work intends to further this view. Although the work has potential for publication in eLife, it requires essential additional data to support the central claims of the paper. Each reviewer raised substantive concerns (see below) that need to be resolved experimentally. For instance, experiments involving knockout in primary T cells will need to be performed. A better time series will also help deciding in what process the INPP5E protein is involved in. Moreover, imaging data should be quantified more precisely to assess spatial and dynamic differences.

      Reviewer #1:

      An important aspect of mature synapse formation is signal termination and ... effector responses, such as secretion of cytokines, exosomes and CD40L on synaptic ectosomes (Huse et al, 2006; Mittelbrunn et al, 2011). The demonstration of ESRCT function in both TCR signal termination and CD40L release to B cells on synaptic ectosomes likely involves inositol lipids that lack phosphorylation on the 5' position.

      It might make sense for the author to investigate a synapse effector function like degranulation of CD8 or CD40L transfer of synaptic ectosome in CD4 T cells as these effector functions actually link into synapse formation more directly than bulk IL-2 secretion.

      The ESCRT machinery is also highly entwined with ciliary biology and several ESCRT components important for signal termination and effector function will also require PIP metabolism.

      Reviewer #2:

      Interesting similarities between the primary cilium and the immunological synapse have been noted and investigated extensively over the last few years. In this context and beyond, the role of phosphatidylinositol lipids in the organisation of the immunological synapse and T cell function has been extensively investigated. Here Chiu et al. add to these topics by investigating INPP5E, a primary cilium-associated 5' phosphatidylinositol lipid phosphatase that can use PIP3, PI(4,5P)P2 and PI(3,5)P2 as substrates, in T cell activation. The authors show that INPP5E is recruited to the interface of a T cell with an activating antigen presenting cell. INPP5E binds to TCRzeta, ZAP-70 and Lck. INPP5E knockdown reduces TCR recruitment to the T cell/APC interface, clearance of PI(4,5)P2 from the centre of the interface, and TCR and ZAP-70 phosphorylation. These findings are consistent with the large body of existing work on the role of phosphatidylinositol lipids in the organisation of the immunological synapse and T cell function and, therefore, don't constitute a conceptual advance. Nor do they provide new mechanistic insight into phosphatidylinositol lipids in T cell activation. The data add another molecule to the existing body of work.

      In the first two figures Chiu et al. show that a number of cilium-associated proteins, including INPP5E are recruited to the interface of a Jurkat cells with a Raji B cell presenting superantigen. Such recruitment is not surprising. On the contrary, because of the reorientation of the MTOC to the centre of the cellular interface and the accompanying shift of the nucleus to the back of the T cell to create more cytoplasmic space at the interface, most proteins associated with vesicular trafficking shift their subcellular distribution towards the interface. Only data showing spatial or temporal distinctions in such recruitment within the small cytoplasmic space underlying the T cell/APC interface could provide interesting new insight. Reduced detection of INPP5E interface recruitment after INPP5E knockdown could be trivially caused by the worse signal to staining background noise ratio (Fig. 2A-E). The STORM data showing that INPP5E interface recruitment occurs in the T cell not the APC are welcome. However, spatial and temporal features provided by the higher resolution of these experiments are not explored.

      In the investigation of the contribution of different INPP5E domains to its interface recruitment the representative imaging data in Fig. 3A suggest that substantial quantitative differences exist. The '% conjugate with recruitment' metric doesn't capture such differences. Some form of a recruitment index as used in other parts of the manuscript would be more powerful. A more complex picture of INPP5E domain contributions to INPP5E interface recruitment is likely to emerge.

      The immunological synapse is a highly dynamic structure. TCR interface recruitment and PI(4,5)P2 clearance in response to various manipulations of PI turnover are only analysed at a single time point. A dynamic picture should provide more insight. For example, interface recruitment of the TCR may be consistently impaired, delayed or shifted in time. Reduced interface recruitment of the TCR upon overexpression of PIP5Kgamma (Fig. 5D, E) has already been described in the cited Sun et al. reference. This should be acknowledged.

      In Fig. 6E, the authors show a small reduction in IL-2 secretion in Jurkat cells stimulated with anti-CD3/CD28 upon knockdown of INPP5E. As INPP5E is expected to exert its functional effects through the control of the spatiotemporal organisation of the immunological synapse, activation of Jurkat cells with APCs would be more appropriate.

      The knockdown efficiency of INPP5E should be quantified.

      Reviewer #3:

      The work is fully performed in Jurkat cells, which a very good and widely used model to investigate T cell activation, yet, not perfect. Actually, in the case of events related with phosphoinositide function, Jurkat cells present a strong caveat. These cells lack the Phosphoinositide phosphatase PTEN, therefore having altered phosphoinositide turnover.

      Therefore, as a first critical point, the authors should confirm most of the central data of this work in primary T cells. They should also discuss this point, since it might bias some of their data.

      Additional points needing attention are detailed below.

      1) Regarding data in Fig 1D, the authors say the they find INPP5E localized with the centriole in the absence of SEB stimulation. The pattern shown is in the picture is very diffuse and blurry, not showing at all a centriole pattern.

      It seems to be more visible in Fig S1. The authors should replace Fig1D panel by a better "quality" picture if they wish to convey that message.

      2) What do the authors mean with "number of events" in the figures ? Please explain or replace by another term or means of quantification. If it means counting conjugates with INPP5E recruited "by visual observation", it would be much more appropriated to quantify fluorescence enrichment at the synapse making a ratio.

      It is also bizarre to plot "pairs" which are all at 100%. What does that mean?

      3) In Fig 2 D, E the authors observe by TIRF the presence of INPP5E at the planar pseudosynapse. They do in parallel TCRz. It would be interesting to better take advantage of that type of microscopy images to also quantify the impact of INPP5E on TCRz recruitment and to assess co-localization between INPP5E and TCRz using Pearse corelation on images with a very good resolution. From that image they look like they do not co-localize at all.

      4) The reasoning of the authors in Fig 2 H is somehow strange: "Since the distribution of INPP5E signals mostly appear at the T cell-APC contact site, it was necessary to examine whether INPP5E belonged to T or B cells" Although they use dSTORM the resolution of the image is not single molecule as they claim, but relatively large clusters. Moreover, they say that INPP5E is inside the T cell while TCRz is at the plasma membrane. In that image there are spots labelled far on the B cell. Moreover, it has been shown by several authors that TCRz largely occupies intracellular vesicular compartments. So the conclusion is not accurate. Finally, they claim that the overlap in some regions is suggestive possible interactions. The overlap is really minimal and in zones of clustering. So the comment is far from accurate. A proper colocalization analysis in TIRF_dSTORM images of INPP5E and TCRz quantified by Pearson correlation would be much more appropriate and accurate.

      By the way, the authors could use panel F of T cells transfected with Flag-INPP5E that relocalizes to the synapse to say that INPP5E in T cells relocalizes to the synapse.

      5) Fig 4A: The strongest interactor with INPP5E seems to be Lck, rather than TCRz. It would be interesting to also assess the effect of INPP5E silencing on Lck recruitment at the synapse.

      Is there a mistake in labeling IP in horizontal and IP in vertical. I guess one of them should be IB (immunoblotted / Western blot). Please clarify and correct if necessary. Same in B, there is labelled IP-Flag everywhere, is one of them input? Please clarify/correct if mistaken.

      The term INPP5E "interacted" with TCRz, ZAP and Lck in the text (line 168-169) is not fully correct here, since these molecules make complexes during TCR activation. The term "co-immunoprecipitated" would be more accurate here.

      Fig 4D Not clear here why the authors use cells transfected with TCRz-GFP while to conclude that INPP5E is required for exogenous CD3z clustering, they could just stain for endogenous TCR.

      Fig 6B: If the authors normalized the pProtein band density with respect to the total same protein, the Y axis should be expressed as band density ratio rather than "optical intensity (a.u.)"

    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on January 4 2021, follows.


      In this manuscript, the authors have generated a new mouse model for the severe disease, Ataxia Telangiectasia (A-T). They introduce null mutations in Atm onto the background of mice that are somewhat sensitized since they also harbor mutations in the Aptx gene. The outcome is the mice show a set of phenotypes that are strikingly similar to symptoms seen in human patients. These include cerebellar degeneration, cancer, and immune system abnormalities. The also deliver small molecule readthrough (SMRT) compounds into tissue explants and show that such a manipulation can restore the production of ATM protein. The success in producing an Atm model with cerebellar degeneration is a compelling advance as this particular phenotype has been incredibly difficult to reproduce in animal models. The authors perform an interesting set of analyses to confirm that the other important features of the disease are also present in their mice. This paper has broad interest to multiple fields including neuroscience, cancer, and immunology.

      Essential Revisions

      1) It is not clear how progressive the cerebellar degeneration is. What is the spatiotemporal pattern of degeneration? Please consider the lobule by lobule effects over time.

      2) For the electrophysiology, what stage cells have you recorded from? That is, what was the structure of the Purkinje cells that you recorded? If the cells look really "normal" but fire abnormally, then please comment on how they are being affected. If the morphology is abnormal, then please explain what defects you see and how they might impact function. Essentially, the authors need to disentangle cell autonomous effects and non-cell autonomous effects with more clarity. That is, are you studying the "dying" cells or the cells that that escaped the genetic defect?

      3) Are both the Atm and the Aptx genes expressed in all (or the same) Purkinje cells? What is the experimental evidence?

      4) Please provide more context and rationale for Aptx in the abstract. As it stands, its mention comes out of nowhere.

      5) In the Introduction, please provide more information as to why previous studies/models might have failed to produce severe Atm-related cerebellar phenotypes.

      6) In the Introduction, the rationale for the choice of paring the Atm mutations with defects in the Aptx gene is unclear. Are they in the same pathway? Are the genes located in close proximity to one another? There are many issues that need to be discussed.

      Related to above, ATM and APTX, while involved in DDR, are involved in parallel pathways-ATM in DNA double stranded break repair, and APTX in single stranded break repair. Homozygous mutations in APTX causes human ataxia (AOA1), but there is nothing to indicate an intersection mechanistically between AT and AOA1. One could just as well call the AT-APTX double mutation a model of AOA1. As indicated above, please expand on the rationale of the experimental design.

      Also, are there more single stranded DNA breaks? Double stranded DNA breaks? Is there a sequestration of SS DNA break repair components including PARP1? How are the changes in PC firing related to DDR (it would be worthwhile for the authors to examine the following papers Hoch et al. Nature. 2017 Jan 5;541(7635):87-91, Stoyas et al. Neuron 2020 Feb 19;105(4):630-644) to give insight into studies that can explore mechanism for DDR and changes in cerebellar morphology/function.

      Therefore, the authors need to address whether single vs double stranded break repair is present and the authors could do a better job of linking the change in PC firing to DNA damage.

      7) Figure 2B: Apologies if I am missing something, but I do not understand the reason or explanation for what determines the probability of survival for the green, gold, and orange traces (the three severe cases in the graph). That is, why is the gold so strong?

      8) How come rotarod was not used as a test? This is a standard motor behavior test that is useful for comparing across animal models and studies.

      9) Related to above, why not use in vivo recordings? I can understand using slice recordings to tackle the biophysical and intrinsic mechanisms, although the authors did not do that. It seems to me that extracellular recordings would have been more informative in the in vivo, awake context.

      10) The authors picked specific regions of the cerebellum to target their slice recordings, which is perfectly reasonable. But why did you pick these regions? Please provide a full justification and discussion for the importance of these particular lobules in relation to what you are trying to solve.

      11) Given the use of slice recordings and that Purkinje cell degeneration is a key aspect of the phenotype, it would be very compelling if the authors showed some filled cells. As it stands, it is very hard to appreciate what the severity of neuropathology actually looks like, especially in relation to what the functional defects are teaching us.

      12) The authors state that "The largest differences were detected in the anterior [38.6{plus minus}3.4 Hz (n=187) vs. 88.1{plus minus}1.8 Hz (n=222)] and posterior [46.9{plus minus}1.9 Hz (n=175) vs. 84.1{plus minus}2.4 Hz (n=219)] medial cerebellum [1-way ANOVA, p<0.0001; Fig. 4B]." Okay, but what does this mean? What is your interpretation for why these regions were more heavily impacted (cell sensitivity based on circuit architecture, gene expression and protein make-up, neuronal lineage?) and how might it impact the phenotype?

      13) The authors state and reference "Previous studies in mouse models of heritable ataxia indicate that physiological disruption in PN firing not only includes changes in frequency but also affects its regularity (Cook, Fields, and Watt 2020)." I agree with having this reference, but what about other models of ataxia? There are a number of other excellent models that should be discussed.

      14) Purkinje cell firing data (figure 4B) should not be averaged across all of the ages, as this is not standard practice, and would be akin to averaging all behavior across ages. I think the data in fig. 4C suffices. If you want to compare across lobules on one graph, simply choose a particular age (perhaps when behavioral changes are first observed?) or at the oldest age.

      15) Why examine Purkinje cell firing deficits in different lobules but not make that distinction for Purkinje cell loss? The Purkinje cell loss analysis focussed on the areas with most pronounced firing deficits but this means that we don't know whether the cells that fire abnormally are the only ones that die. Also see point #2 above.

      16) Figure 4E and related text: Please provide a much more extensive set of images to show the cerebellar pathology. 1) Please show views of the different lobules to demonstrate the pattern of degeneration. 2) Please show different ages to show the progression of degeneration. 3) Please show higher power images of the Purkinje cells to clearly demonstrate their morphology.

      17) The authors need to need provide more data for what is actually happening in relation to cell death. Why not perform Tunel or caspase staining etc.? The authors must show that there are actually acellular gaps where cells have died, or some other indication that cell death has occurred or is occurring.

      18) Also in relation to the Purkinje cell degeneration, what do the dendrites look like? What about the axons? Do you see any torpedoes or axonal regression?

      19) In regards to the cerebellar degeneration, what happens to the other cell types in the cerebellar cortex? Are they intact? What about the cerebellar nuclei?

      20) The authors state "Of interest, APTX deficiency by itself had the greatest effect on the loss of DN4 cells...". Okay, but it is hard to see what this means for A-T as a disease. Interesting as it is, what is the relevance of this gene and these findings to the actual disease?

      21) Please provide a more extensive description and rationale for why this explant system was chosen.

  4. Dec 2020
    1. This manuscript is in revision at eLife

      The decision letter after peer review, sent to the authors on December 14 2020, follows.


      This study addresses an important topic of broad ecological interest and provides important insights into the role of local-scale processes in shaping patterns of species diversity, aiming to (i) assess if there is a global latitudinal diversity gradient (using alpha diversity) of rocky shore organisms and its functional groups and, (ii) whether there are any large scale or local environmental predictors of richness patterns. The strength of this paper is the global coverage of studies analyzed, showing for the first time that rocky shore richness does not appear to peak in the tropics - in contrast to many other studies of marine and terrestrial systems. These outcomes are not specific for rocky intertidal systems, with an increasing number of studies showing that the search for global ecological patterns may be elusive. While sampling in the tropics and the polar regions is poor (acknowledged by the authors), this should be viewed as a call for further research in these regions - not as a weakness of the paper per se. There are also some reservations on how the analysis has been conducted, including the lack of standardization of sampling effort and other details (e.g., size of sampling units) to derive a comparable measure of diversity across sites.

      Public Review

      The latitudinal gradient of diversity has been studied and confirmed in many aquatic and terrestrial habitats and species across the globe. In the vast majority of cases, richness increases towards the tropics. Using an impressive global dataset of latitudinal diversity gradients in 433 rocky intertidal assemblages of algae and invertebrates from the Arctic to the Antarctic, Thyrring and Peck show that rocky shore ecosystems may not follow this general pattern. The authors show that there is no clear latitudinal gradient for rocky shore organisms using alpha diversity - as posited by prevailing theories - although some functional groups exhibit contrasting patterns. Diversity within functional groups of predators, grazers and filter-feeders decreased towards the poles, whereas the opposite was observed for macroalgae. Correlation with environmental drivers highlighted the importance of local-scale processes in driving spatial patterns of diversity in rocky intertidal assemblages. The paper is well written and the many of the analyses are well done, but there is the concern, which the authors acknowledge, that sampling within tropical latitudes is sparse and needs to be carefully considered when interpreting the results of this paper.

      The work can be improved in the following manner:

      1) The relevant data to standardize species richness may not be available from the primary literature. However, it should be possible to employ relevant standardization methods within the 5{degree sign} latitudinal bands in which the data have been aggregated. An analysis based on standardized data, at least for the more data-rich latitudinal bands, must be added.

      2) Employ models that allow assessing unimodality, which is stated but untested. At the bare minimum, a quadratic relationship with latitude should be included in the GLMM. As implemented here, the GLMM employed to relate diversity to latitude can only detect linear trends, but not unimodal patterns and the mid-latitude peak suggested by LOESS for the northern hemisphere. To provide a formal test for unimodality, models with or without a quadratic term could be contrasted using standard model comparison procedures. Alternatively, GAM could be used to evaluate nonlinear effects.

      3) Clarify whether p-values are relevant or not. As is, it is confusing. For example, the legend of Table 1 mentions p-values, but these are not reported. Materials and Methods indicate that 95% confidence intervals are used to take decisions on null hypotheses, suggesting that p-values are not used in the analysis (lines 436-439). Nevertheless, p-values are reported in Table 2.

      4) Provide a rationale for distinguishing between canopy and other algal forms (the distinction is compelling, but it is not explained).

      5) We like the conclusion on the importance of local-scale processes. This should be placed in the context of previous studies that have quantified patterns and processes at multiple scales reaching the same conclusion.