Banning these books undermines the efforts towards equity for black authors, as It limits their market reach.

Educators and parents who value diversity should stand up to these bans. Publicly supporting these books promotes the importance of diverse literature.

Challenges to these books can negatively impact sales mostly in regions where these books are being complained about by parents. Its important to keep an eye on these trends and make a plan to keep these titles profitable.

people's experiences, thoughts, and emotions are always sought after by other people who experience the same thing as them.

famous people who did a lot for their communities. Their stories are important

I think teaching these hard topics is important to the development of children and how the view themselves and their peers. They should be taught this stuff to see the signs of injustice

important

https://web.archive.org/web/20240611050132/https://jentery.github.io/ts200v2/notes.html

An interesting read turning a range of #psts issues into specific reflection questions to ask at the start of a tech project. With references to the background concepts. From 2018. #toread

Vgl [[Techpledge 20190917062614]] as the 'political' phrasing of same.

Delete this. this is not needed and can just make the user of the file confuse.

Can you add a short paragraph outlining why this is the oh-so-important first step?

Can you come up with an impressive numbers section as a quick reference point for the achievements you've helped your clients secure? How many clients you've supported to their dream role? How many deals were above the original offer? What is the average percentage of increase? How long on avarage?

Have you been featured in the media or on recognisable podcasts? If so, I'd include a log bar here similar to the one you have on your home page.

Can you add an intro section above this section (which again I think is FAB!)?

Something that hooks the reader and makes them feel seen. It would be talking about some of the situations they might have been facing to get them here, some of the common challenges that lead into your insider perspective as an agency recruiter and in-house recruiter.

I'd speak to the frustration they're feeling, and then lead into the issues causing that frustration (which you cover in this section)

Including the values is a smart move, I would just move them to the bottom of your about page just above the services section.

Love the tagline!

What I would include is a H1 reference to what you do/how people would Google you so you give yourself another chance to rank and get in front of people looking for your services

Something like 'The Job Search Strategist to help you find your inevitable edge'

From the perspective of organizing collaborative activities, I believe "coopetition" could be a solution, avoiding the limitations of exclusively using collaborative methods. Coopetition, a blend of competition and cooperation, describes the simultaneous competitive and collaborative relationships between individuals who work together to optimize their gains.

A balanced environment of coopetition, such as intragroup collaboration with intergroup competition, can encourage students to refine and expand upon their ideas collaboratively in successive rounds of annotations while fostering a healthy competitive atmosphere where they strive to make the most insightful contributions.

Our team conducted a critical review of studies on coopetition in educational settings (Chen et al., 2023), which may provide valuable insights for designing effective social annotation activities.

Chen, M., Lv, C., Wang, X., Li, L., & Yang, P. (2023). A Critical Review of Studies on Coopetition in Educational Settings. Sustainability, 15(10), 8370.

According to the Community of Inquiry (CoI) theory, these suggestions could help learners perceive a strong teaching presence. This refers to the perceived design embedded in the tool that facilitates students' social annotation and enhances knowledge construction.

This is atest from Sheeraz

Need to tie this into the cleaning robot example above

test

fix

How does this tie to previous or material in quick guide

potential spot for screencast

Maybe a "Need help? Watch this" sort of thing.

This is the main takeaway from this page, right?

I think reframing this as... Let GLOW do the work for you! or something like that might work.

Would you like a 10-second screencast to illustrate this?

people centered problem

people centered problem

This page isn't loading for me. 6/10/24 at 5pm.

If others have this problem, I suggest making this a link to an external resource. The link works for me.

I'd be willing to make an optional deep-dive video into how a structured home page aligns with UDL principles!

While this might be an understandable question for a piece of data journalism or data art, this does not feel like a safe assumption to make of readers of a scholarly book. For example, when I started reading this chapter, the main question I had was: "what is the main argument of this chapter, and how does it relate to the overarching story that the book is trying to tell?"

Because of this disconnect, I found the opening two paragraphs largely confusing because I wasn't sure how to interpret the anecdotes or contextualize them as part of a broader argument. As others may have written in their feedback, I think this is a symptom of a difference between the expository, observation-first writing style of humanities disciplines vs the style more common in VIS of thesis statement first, then supporting the thesis with argumentation/observational evidence.

This is a general feeling I have about the chapter titles, but I would love for them to be a bit more descriptive of the main idea of the chapter.

For example, I know the team refers to this chapter colloquially as "the labor chapter". I don't think the fact that this chapter is about labor is apparent from the main chapter title, nor does it feel easy to remember and map the chapter titles back onto their contents.

Just to make a case for why I think this is important: as a reader, I will probably read this book and later on want to cite it. I will probably think "oh there was that interesting paragraph about XYZ" and then go to the table of contents to try to remember which chapter it was in. I feel like it might be easier to go through that search process of the titles are more descriptive of each chapter's argument.

Interesting, I'm still reading but are there other places in this book where we discuss measurement and not just representation?

This was one of the core tensions that folks brought up when we were discussing the first two chapters in group meeting --- to what extent are critiques in this book directed at graphic representations and their use, vs are they directed at the idea of data and quantification more broadly?

This is super interesting. I'd love for this idea to not be buried in the middle of a paragraph (and also slowly unpacked further) because I also see the potential here to connect some of the threads of this chapter into an overarching story.

To me, the language of "alternative future" suggests a design process that explores alternate options in parallel. I wonder if to make this more explicit it might be interesting to see the early design work (mentioned earlier and attributed to Tanvi and Shiyao) and show some of your discussions of pros and cons and unpack those tensions explicitly for readers. I feel like in contrast to a visualization of commits, it might be helpful to bring home one of the points of the book which is that design decisions matter, and that different choices of representation relate to their social context in different ways. It could be a way to teach readers by example how to do this kind of design critique and reflection.

this is going to confuse visualization practitioners, who will think you mean something like the Grammar of Graphics or Vega-Lite

How does Listening to Images specifically contribute this understanding? In other words, why do we need Listening to Images to understand this rather than some other theoretical construct being enough, like Haraway?

This feels like a really interesting statement that goes by very quickly. I think it is worth unpacking and dwelling on for much longer, because to me it feels core to the argument that is underneath the surface of this chapter.

Though you say you're taking a page from Du Bois, my impression from reading the previous chapter is that part of the problem with Du Bois' work is that we know his students contributed a lot but the nature of those contributions is totally lost to time (indeed, it took you some work to even recover their names from records).

So it feels to me like this chapter is trying to do better than Du Bois by specifically recording the names and contributions of the people who worked on this book. I think there could be a lot more to say about why you think that's important and what the implications of that choice are --- what is made different by this inclusion.

I'm not sure what I'm taking away from this paragraph, which reads as a list of pretty standard tools. What is the goal of foregrounding the specific tools?

One question I had: If the outcome is determined by manner of use rather than the specifics of the tools, then is it relevant what the tools are? Could different tools have been used to the same ends?

There's an article by Lisa Nakamura that defines "digital work" as "the work of the hand and its digits"

May be good to edit captions so that the email reads correctly.

This can be one of the most laborious parts of facilitating or being in a position of leadership, yet is it a critical step. Ensuring all stakeholders feel they are a part of the solution makes the work in carrying it out more efficient in the long run.

Even though it is summer, I was reading about simple machines in physics before school ended. Many of my questions are similar, and the author was able to answer most of my questions. My textbook is better written, as it is intended to go very in depth.

The crux or summary of this page is that instead of using the elements of a list to use a for-loop for iteration, you can use the 'range' function.

My living room, the foyer at my school, and my kitchen table are all great. The foyer at school is the best because it has the least distractions.

I can use the Cornel method.

Preparing, reading, capturing key ideas, and reviewing. Reading will take the most time because it is the most tedious.

references penalties in the 2001 Act - so this is just a command/authority decree type.

goal

evidence?

Three ways to make up for notes is to ask a friend who takes notes, research the topic online using reliable sources, or ask the teacher if the topic is going to be on the test because if not, you may not even need notes.

The benefit of a laptop is that it has many tools and features to make note taking easier. The problems are that you have the potential to accidentally lose the notes and it is much easier to become distracted on a computer.

The Cornell method lets you write more information and it is a better way to memorize the important things.

Using the Cornell method, I can retain information more efficiently and focus much better.

They emphasize the word or definition, repeat the material, or just tell you straight forward that the information is important.

You should sit in or near the front of the classroom so that the teacher can see that you are paying good attention to the lesson.

To prepare yourself for active listening, you should get your mind in the right place and get yourself in the right space.

I would consider my friend to be an excellent listener. If something is going on in my life, he cares and shows it in his body language by doing things like making eye contact. A poor listener would be my grandmother, as when I start talking, she won't pay attention and makes it clear that she'd rather be the one talking.

I have good eye contact, but sometimes I zone out a little bit.

So is the hero's journey something that is more common in western stories, and Michael Wesch was confused by the stories in Papa New Guinea because they didn't follow this typical pattern? Or is the hero's journey universal?

interesting

Important

@Dave, this is great!

• Metaphor of Knowledge and Innocence in "The Time Machine"
• "HG Wells' 'The Time Machine' describes a world populated by two races, the Eloi and the Morlocks."

• "The Eloi live in a paradise they don't fully understand, and sometimes what they don't know comes up and bites them."

• Foundations of Software Engineering

• "As software engineers, we build on foundations that we don't understand...we sit atop 60 years of accreted abstractions."

• "At some point, we have to be content with what we know and build upwards."

• Hierarchical Structures in Problem Solving

• "We're taught ways to cope with the fact that we don't understand the systems that we build in their entirety, and chief among them is the hierarchy or the tree."

• "For some problems, this is a completely accurate representation... but the systems we build look more like this (diagram of connected components)."

• Polite Fiction and Model Limitations

• "We spend a lot of our time pretending that these two things (simple hierarchies and complex systems) are the same... it's a necessary story because otherwise, we can't make progress."

• "It's easy to forget that the model we have is not the world as it actually is."

• Historical Example: Scientific Forestry

• "Scientific forestry is roughly the process of going from a natural forest to a monoculture for efficient lumber production."

• "The bureaucrat understood that his model was not the forest as it actually was... he made the world conform to his understanding of it."

• Systems Thinking and Complexity

• "Gerald Weinberg defines a space with two axes: complexity and randomness."

• "Heuristic devices are lossy ways of looking at the world... effective models but not true models."

• Mid-Century Critical Theory and Heuristic Devices

• "Deleuze and Guattari talk about the interplay between the state (hierarchical structures) and the Nomad (local understanding)."

• "They describe 'rhizomatic' structures versus 'arborescent' or tree-like structures."

• Borges' Parable: The Map and the Territory

• "In Borges' story 'On Exactitude in Science,' a map the size of the empire becomes useless."

• "We use maps because they are reductive... as we become more reductive, the utility of the map falls off."

• Modern Urban Design and Control

• "Baron Haussmann's redesign of Paris included wide roads to prevent slums from seceding."
• "Le Corbusier's sketches for the new center of Moscow had no concern for the context."

• "Brasilia's construction showed the failure of a completely planned city, leading to disconnected spaces and 'Brasília' syndrome."

• Pattern Language and Quality without a Name

• "Christopher Alexander's 'A Pattern Language' and 'A Timeless Way of Building' describe a holistic quality in buildings."

• "Alexander emphasizes that this quality is contextual and cannot be reduced."

• Software Design Patterns and Habitability

• "The Design Patterns book tries to give universal constructs, missing the contextual nature of Alexander's ideas."

• "Richard Gabriel's 'Patterns of Software' focuses on habitability... the degree to which you can make a codebase your own and adapt to changes."

• Timeless Knowledge versus Cunning

• "The Greeks had two words for knowledge: 'techne' (timeless knowledge) and 'metis' (cunning)."

• "In practice, everything depends on the domain and our experience, not on universal truths."

• Conclusion and Broader Lessons

• "We should plan for the eventuality that our models and systems will be wrong."
• "The goal is to inform intuition and provide a vocabulary for discussing complex systems and their trade-offs."

I like the section headers here at the top!

Meditation, daydreaming, internal visualization, as well as communication with the self are all intrapersonal communication styles.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

This manuscript uses C. elegans as a model to interrogate the effects of autism-associated variants of previously unknown function in the RNA-binding protein RBM-26/RBM27.

Despite its potential impact, there are several concerns related to the technical rigor and specificity of the observed effects.

Major concerns: 1. The effects on PLM are interesting, but why was this neuron selected for study? Was this a lucky guess or are other axons also affected? It is important to clarify whether the effects of RBM-26 are specific to this neuron or act pleiotropically across many or all neurons. According to CeNGEN, rbm-26 is strongly expressed in the well-characterized neurons ASE, PVD, and HSN. Are there morphological defects in these neurons, or others? As a note, there are also functional assays for these neurons (salt sensing, touch response, and egg laying, respectively).

We have added new data to the supplemental materials showing that loss of rbm-26 function also causes the beading phenotype in the axons and dendrites of the PVD neuron (Figure S4 and lines 196-199). We have focused on the PLM neuron because our preliminary studies indicated that it had a higher penetrance of axon defects relative to the PVD neuron. Moreover, we observed expression of endogenously tagged RBM-26 in the PLM neuron (Figure 3A-C and lines 210-215).

Similarly, the choice of the MALSU homolog seemed like a shot in the dark. It is ranked 46th (out of 63 genes) for fold-enrichment following RBM-26 pull-down, and 9th for p-value. Were any of the mRNAs with greater fold-enrichment or smaller p-values examined further? It is important to determine whether many or all of these interacting genes are overexpressed in the absence of RBM-26 and whether they are also required for the phenotypic effects of RBM-26 mutants, or if the MALSU homolog is special.

We have clarified our reasoning for selecting the MALS-1 ortholog of MALSU1 for further study (see lines 283-284 and Table S2). Amongst binding partners with human orthologs, MALS-1 was by far the top ranked candidate. The adjusted p-value for MALS-1 was 0.0008. The next smallest adjusted p-value was two orders of magnitude larger (0.028 for dpy-4). Moreover, the log2fold fold enrichment for MALS-1 was 1.98, about the same as the largest (ACADS with 2.13). Nonetheless, we agree that some of the other interactors may also be of interest and have thus included them in the supplemental table S2. Although these other potential binding partners are outside the scope of this study, we expect that future studies by ourselves or others may focus on the roles of these other binding partners.

In addition to the specificity controls mentioned above, positive and negative controls are needed throughout the results. While each of these may be relatively minor by itself, as a group they raise questions about the technical rigor of the study. Briefly these include: Fig 1C. Missing loading controls and negative control (rbm-26 null allele). Additional exposures should be included to show whether RBM-26(P80L) protein or the lower band for RBM-26(L13V) are present at all, relative to the null allele.

We have added no-stain loading controls to figure 1C. We have also switched to using ECL detection, which is much more sensitive and reveals faint bands for RBM-26(P80L) and additional faint bands for RBM-26(L13V). In addition, we have included a longer exposure for the blot (Figure S1). We are unable to test the null, as we can only produce a limited number of small maternally rescued progeny, thereby precluding western blot analysis.

Fig 2. Controls to distinguish overextension of PLM axon from posterior mispositioning of ALM cell body are needed. Quantification of PLM axon lengths in microns (or normalized to body size) with standard deviation, not error of proportion, should be shown. Measurement of "beading phenotype" should be more rigorous, see for example the approach in Rawson et al. Curr. Biol. 2017 https://doi.org/10.1016/j.cub.2014.02.025 . The developmental stage examined, and the reason for choosing that stage, should be described for this and all figures.

We have added new data that shows PLM axon length relative to body length for each of the RBM-26 mutants (Figure S2 and lines 183-185). These results indicate that the PLM axon has a larger axon length to body length ration, suggesting that the PLM/ALM overlap phenotype is a result of PLM axon overextension. For most experiments, we retain penetrance, as this has been standard practice in the field and allows for a much larger sample size (see examples listed below). We have also added examples of how the beading phenotype was measured (Figure S3). Moreover, we have now analyzed this phenotype and others at multiple developmental stages (Figures 2D-H and Table S1). In general, we have conducted experiments at the L3 stage because the rbm-26(null) mutants don't survive past this stage. However, for many of our experiments we have also included additional stages as well. We have added this explanation to the methods section of phenotype analysis and also at various locations throughout the text. We have also labeled all graphs to clearly indicate the developmental stages and included.

10.1038/s41467-019-12804-3 Article by laboratory of Brock Grill

10.1371/journal.pgen.1002513 Article by laboratory of Ian Chin-Sang

doi.org/10.1073/pnas.1410263111 Article by laboratory of Chun-Liang Pan

10.1016/j.neuron.2007.07.009 Article by laboratory of Yishi Jin

doi.org/10.1523/JNEUROSCI.5536-07.2008 Article by laboratory of William Wadsworth

Fig 3. Controls without auxin and with neuronal TIR1 expression alone should be included. Controls demonstrating successful RBM-26 depletion, in larvae as well as in embryos at the time of PLM extension, should be included (weak embryonic depletion might explain why the overextension phenotype is only 14% instead of 40% as in the null). According to CeNGEN, rbm-26 expression in PLM is barely detected, thus depletion with a PLM-specific TIR1 should also be tested. To confirm the authors' identification of the cell marked "N" as the PLM cell body, co-expression of rbm-26 and a PLM-specific marker should be added. Rescue of the rbm-26 mutants with neuronal (and PLM-only) expression should be included to test sufficiency in PLM, and as a further control for potential artifacts of the AID system.

We have added new data showing that an endogenously tagged RBM-26::Scarlet protein is expressed in the PLM neuron (Figure 3A-C). Moreover, we have added rescue experiments, showing that a Pmec-7::rbm-26::scarlet transgene can rescue the beading phenotype and the PLM/ALM overlap phenotype (Figure 3 F-G). We have also added controls without auxin (Figure S7) __and without the rbm-26::scarlet::aid gene (Figure S8). We have added a new figure showing auxin-mediated depletion of RBM-26::Scarlet::AID in the PLM neuron (Figure S10)__. We examined auxin-mediated depletion at the L3 stage for consistency with our auxin-mediated phenotypic experiments. Moreover, these were done at the L3 stage for consistency with other experiments that included the rbm-26(null) mutants, which don't survive past this stage.

In general, auxin-mediated knockdown tends to be hypomorphic in neurons. This is likely due to the fact that the neuronal TIR1 driver is expressed at much lower levels relative to the other drivers. In addition, the lower penetrance observed in auxin-mediated PLM/ALM overlap phenotype could reflect the fact that this phenotype resolves by the L4 stage in the hypomorphic mutants. For example, in P80L mutants at the L3 stage we see only about a 20% penetrance of the PLM/ALM overlap phenotype (relative to about 15% in auxin-mediated knockdown).

Fig 4. More rigorous quantification of the distribution of mitochondria along the axon should be included, not only total number, and it should be clarified what region of the axon the images are taken from. Including the AID-depletion strain with and without auxin would further add to the sense of rigor. For the mitoTimer experiments, why is RBM-26(L13V) not included and why do wild-type values differ ~5-fold between experiments (despite error bars being almost non-existent)? A more rigorous approach to standardizing imaging conditions may be needed. Positive controls using compounds that affect oxidation should be included. Measurements of individual mitochondria with standard deviations should be shown, rather than aggregate averages with error of proportion.

We have changed our methodology for measuring mitochondria, so that we now report the density of mitochondria in the axon (number per 100µm), (Figure 4E-F). We agree that this method is much better than counting the total number of mitochondria per axon, as it corrects for differences in body length and axon length). We also now include data for the whole axon (Figure 4E), proximal axon (Figure 4G), and distal axon (Figure 4H). These data suggest that the mitochondrial density defects occur in the proximal axon but not in the distal axon. Using the null allele, we have also examined the timing of mitochondria defects in the axon and report that the defects begin in the L1 stage and continue throughout larval development (Figure 4F). Individual datapoints have been added for all graphs in Figure 4.

For the mitoTimer experiments (Figure 5), we have added data for L13V and have added the individual datapoints to the graph. In the prior version, the values did not differ 5-fold between experiments with the same stage, rather the different graphs were from different stages (as noted in the figure legends/main text) and the L4 stage has much more oxidation than the L2 stage. To clear this up, we have added labels to the graphs to indicate the stages for each experiment. We have also added new data, so that we now show results for the L2, L3, and L4 stages for all three rbm-26 mutants (see Figure 5C-E). We didn't test the L1 stage because the signal was not sufficient for accurate quantitation.

Fig 5. Additional positive and negative controls should be added, including additional rbm-26 alleles, the AID-tagged strain with and without auxin, and a rescued mutant.

The old Figure 5 has become Figure 6 in the new version. We have added the rbm-26(L13V) allele to each experiment, (Figure 6B-D). We have also added the loading controls for the western blot along with quantification for 3 biological replicates of the western blot analysis (Figure 6D). We agree that these additions significantly strengthen the data because they show that two independent alleles of rbm-26 cause very substantial increase in the expression of mals-1 at both the mRNA and protein levels. We did not do these experiments with the rescuing transgene or with the AID-tagged strain because these experiments are done on whole worm lysates, whereas the AID-tagged and rescuing transgene are neuron-specific.

Fig 6. Controls showing whether the Scarlet-tagged protein is functional are needed, to rule out dominant negative or toxicity-related effects.

This is Figure 7 in the new version. For this experiment, we are showing that overexpression of MALS-1 does cause defects. The idea is that excessive amounts of MALS-1 causes deleterious effects to the mitochondria. In fact, these defects could be considered as dominant negative or toxic. We considered the possibility of crossing the Pmec-7::mals-1::scarlet transgene with rbm-26; mals-1 double mutants. However, this does not seem workable, because the single copy Pmec-7::mals-1::scarlet transgene produces the phenotypes at penetrances that are similar to what we observe in rbm-26; mals-1 double mutants. We concede that the results of the overexpression experiments in Figure 7 are limited when considered in isolation. However, we think that they are meaningful when considered in combination with the results on the mals-1;rbm-26 double mutants in Figure 8.

Fig 8. Controls for other mitochondrial components need to be included. It is important to determine if the decrease in ribosomes is specific or reflects a general decrease in mitochondria. If there are fewer mitochondria as suggested in Fig. 4, then of course mitochondrial ribosomal protein levels are also reduced. Additional rbm-26 alleles should be included here as well. Is this effect dependent on the MALSU homolog?

This is Figure 8D-E in the new version. We have added new data showing that the decrease in MRPL-58 expression that is caused by the rbm-26(P80L) mutation is dependent on MALS-1. We concede that these experiments cannot be used to determine anything about the mitoribosomes per se, but rather serve as an alternative way of testing the effect of rbm-26 on mitochondria. We have revised the text accordingly (lines 355-357). Given these limitations we have elected not to try additional mitochondrial markers and have also not included additional rbm-26 alleles for this experiment.

Finally the authors should address concerns about image manipulation, which amplify the concerns about technical rigor outlined above. The image in Fig. 2A appears to have a black box placed over the lower-right portion of the field to hide some features. Black boxes also appear to have been placed over the tops of images in Fig. 4B and 4D and at the left of Fig. 6A, 6B, and 6C. While these manipulations probably do not affect the conclusions, they further undermine confidence in data integrity and experimental rigor.

We have corrected all of these image processing errors. The box in 2A was for the purpose of squaring off a corner that was clipped during image rotation. The boxes in Figures 4 and 6 (of the prior version) were added to give space for labels (without obscuring image features). We have now used alternative methods to accomplish the same goals. For example, in Figures 4-D we have placed the labels outside of the images.

Minor points. 1. C. elegans nomenclature conventions should be followed: - C. elegans gene names have three or four letters, thus the MALSU homolog cannot be named "malsu-1". Please have new gene names approved by WormBase BEFORE submitting for publication http://tazendra.caltech.edu/~azurebrd/cgi-bin/forms/gene_name.cgi

We have changed malsu-1 to mals-1. In addition, both mals-1 and mrpl-58 have now been approved by wormbase and will be listed on the website upon its next update.

• If two sequential CRISPR edits are made on the same gene then they should be listed as a compound allele, such as rbm-26(cue22cue25)

We have updated our gene names to reflect this convention.

• Genes on the same chromosome should not be separated with a semicolon, for example rbm-26(cue40) K12H4.2(syb6330)

We have updated our gene names to reflect this convention.

Describing the defects as "neurodevelopmental" is misleading in the case of axon beading or degeneration. Similarly, there is no evidence for an "axon targeting" defect as stated in the abstract.

We have revised such that instead of referring to degeneration phenotypes as neurodevelopmental, we now refer to axon degeneration phenotypes that occur during development. For example, in the abstract we now say, "These observations reveal a mechanism that regulates expression of a mitoribosomal assembly factor to protect against axon degeneration during neurodevelopment.

Regarding targeting defects, this was meant to refer to the misplacement of the PLM axon tip (which contains electrical synapses). However, our subsequent analysis has revealed that these defects are transient in P80L and L13V mutants, as they resolve by the L4 stage. The rbm-26 null axon development defects do not resolve, though these mutant die prior to the L4 stage. Given these findings, we have decided not to use the term of targeting defects. Instead, we now refer to this as an axon tiling defect or PLM/ALM overlap phenotype.

In Fig. 5A, the symbol that appears to correspond to F59C6.15 (lowest p-value) is a different size than the others and is colored as ncRNA, whereas WormBase annotates this gene as snoRNA.

This error has been corrected.

In the Introduction, the last sentences of the first two paragraphs should be varied ("However, little is known about the [...] mechanisms that protect [...] during neurodevelopment.")

This has been done.

Why is RBM-26 protein running as a doublet at both sizes?

We have improved our western blotting methodology by using 12% gel, allowing for better resolution. We have also switched from colorimetric detection to ECL detection, allowing for greater sensitivity. In our new blots, we identify 6 different RBM-26 protein bands. We don't know the reason for these bands, but speculate that they are the result of post-translational processing (148-150).

When showing the RBM-26 expression pattern (Fig. 3) please include a lower-magnification image of the entire animal.

This has been done (Figure S6)

It is confusing to refer to the RNA IP experiments as an "unbiased screen", which in C. elegans typically refers to a genetic screen.

We now refer to this as a "biochemical screen".

The relationship between axon overextension, beading, and mitochondrial localization is not clear. What causal connection between these is being proposed? The causal connections between these phenotypes, if any, should be clarified experimentally. For example, if the axon extension defects develop before mitochondrial localization defects, then it is unlikely that mitochondrial defects cause axon overextension.

We have added new data showing that the reduction in mitochondrial density within the axon begins during the L1 stage and increases throughout larval development (Figure 4F). We have also added additional data showing that the increase in mitochondrial oxidation is weak in the L2 stage and surges in the L3 stage (Figure 5C-E), coincident with the beginning of the axon degeneration phenotypes. We propose (lines 383-391) that a low level of mitochondrial defects is present in L1 larvae, giving rise to the axon tiling defects. In the L3 stage there is a surge in excessive mitochondrial oxidation, giving rise to the axon degeneration phenotypes. We have added a new section to the discussion that addresses the relationship between defects in axon development and axon degeneration (lines 375-405).

Please explain how to interpret the difference in axon beading in the two deletion alleles of the MALSU homolog (axon beading defects in tm12122 but not in syb6330). Is syb6330 not a null allele? Or are the defects in tm12122 due to other mutations in this strain background?

One likely reason for this difference is that tm12122 is predicted to cause a partial deletion of the mals-1 coding sequence, whereas the syb6330 is a full deletion. Thus, the tm12122 could be acting as a dominant negative. In fact, prior work on the MALSU1 ortholog has indicated that this protein is subject to interference by a dominant negative construct (see Rorbach et al, Nucleic Acids Res 2012). Nonetheless, we cannot rule out the possibility of a linked second mutation in tm12122. However, since we have found similar phenotypes and genetic interactions with both alleles, we can conclude that these phenotypes and interactions are due to loss of MALS-1, rather than a second mutation.

Are mitochondria reduced in number or mislocalized? If they are reduced in number, is this due to altered balance of fission/fusion?

We have adjusted our methods for quantifying mitochondria and have also analyzed the proximal vs distal axon (Figure 4). We find that the density of mitochondria is decreased in the proximal axon, but not in the distal axon. We speculate that this might reflect a higher demand on mitochondria in the proximal axon, due to a higher amount of trafficking activity in the proximal axon (lines 255-257). We propose that the loss of RBM-26 causes dysfunction in mitochondria. Since fission and fusion are mechanisms that can help to repair damaged mitochondria, it is likely that they would be involved in the phenotypes that we observe.

In Fig. 3A-D, please keep the labels in the same position in all panels and do not alter brightness settings between single-color and merged panels.

These images have been moved to the supplemental data section (Figure S5). We have adjusted the labels as suggested. We have not changed the brightness settings, as they were already the same in all panels. However, the blue signal in the merged panel does obscure some of the red signal, giving an appearance of an alteration in color balance.

The claim that rbm-26 acts cell-autonomously requires PLM-specific depletion and rescue experiments.

We have added new data indicating that a Pmec-7::rbm-26::scarlet transgene can rescue the beading phenotype (Figure 3F-G).

**Referees cross-commenting** I appreciate the use of the consultation session to resolve differences between reviewers, but in this case I fully agree with the content and tone of all the comments from the other reviewer -- I think our remarks are very well aligned!

Reviewer #1 (Significance (Required)):

The study engineers autism-associated variants in conserved residues of RBM27 into the C. elegans homolog RBM-26 and identifies neuronal phenotypes potentially relevant to autism and a potential molecular mechanism involving regulation of mitochondrial ribosome assembly.

The key claims of the study are 1} that autism-associated variants in RBM-26 decrease its protein expression; 2} that impaired RBM-26 function leads to a variety of defects in development and maintenance of a single neuron called PLM, including altered axonal localization of mitochondria; 3} that RBM-26 normally binds the mRNA for the C. elegans homolog of MALSU, a mitochondrial ribosomal assembly factor; 4} that loss of RBM-26 leads to overexpression of the MALSU homolog; and 5} that MALSU is required for some of the deleterious effects on the PLM neuron seen in RBM-26 mutants.

This study will be of interest to the autism research community because it bolsters the idea that variants in RBM27 are likely to disrupt gene function and to affect neuronal health. It will also be of interest to the broader cell biology community because it suggests an interesting potential nucleus-to-mitochondria signaling mechanism, in which a nuclear RNA-binding protein might regulate assembly of mitochondrial ribosomes.

My field of expertise is developmental biology in C. elegans.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary In this manuscript, the authors studied an ASD-associated gene, rbm-26 in neuronal morphology using the touch receptor neuron PLM in C. elegans, and found that loss-of-function rbp-27 causes overextension and the formation of bulb-like structures in the axon. Using UV-crosslinking RNA immunoprecipitation and RNA-Seq, they identify malsu-1 as a target of rbm-26. Genetic analyses suggest malsu-1 likely functions downstream of rbm-26 in controlling the PLM morphology. Major comments:

• The authors describe RBM27 is associated with ASD and ID while they only cite SFARI paper that describes a weak association of RBM27 to ASD. The appropriate referenced that show link between RBM27 and ID should be provided. The link with ID was an error. We had meant to say "ASD or other neurodevelopmental disorders." This has been corrected.

• SFARI database only has three (P79L, R190Q, G348D) mutations listed as ASD-associated. Where are other mutations L13V and R455H, particularly L13V that the authors used to generate the C. elegans mutant come from? Are they associated with intellectual disabilities? The others came from the devovo-DB. We have added a reference for this database and have also added the primary source references for each of the five de novo variants (see line 121).

• The authors should be very careful when describing 'gene X causes Y diseases'. Many (if not all) of the examples described in this manuscript are disease-associated genes without validation to be causal genes. We have revised accordingly. For example on lines 433-435, we now say," For example, mutations in the EXOSC3, EXOSC8 and EXOSC9 are thought to cause syndromes that include defects in brain development such as hypoplasia of the cerebellum and the corpus callosum". We have decided to use the phrase "thought to cause" because three of the five referenced articles on these genes use titles that indicate causation.

• The authors refer PLM axon beading and overextension phenotypes to 'axon degeneration and targeting defects'. The authors must provide additional evidence of axon degeneration (see below). Also the term 'targeting defects' is misleading as the authors did not examine if overextension of the PLM axon causes targeting defects. At least they should examine some synaptic markers. To provide more evidence of degeneration we have analyzed several additional phenotypes at multiple developmental stages (Figure 2 and Table S1). Regarding targeting defects, this was meant to refer to the misplacement of the PLM axon tip (which contains electrical synapses). However, our subsequent analysis has revealed that these defects are transient in P80L and L13V mutants, as they resolve by the L4 stage. The rbm-26 null axon development defects do not resolve, though these mutant die prior to the L4 stage. Given these findings, we have decided not to use the term of targeting defects. Instead, we now refer to this as an axon tiling defect or PLM/ALM overlap phenotype.

• Neuronal phenotypes (axon overextension and beading) should be examined at different developmental timepoints (larval, young adult, and aged animals) to test if these phenotypes are indeed degenerative instead of developmental defects. We have included new data to observe all of these phenotypes at multiple developmental time points (Figure 2 and Table S1).

• The authors use the blebbing (beading) phenotype in the axon as the sole evidence of neurodegenerative properties of the PLM neuron. A more thorough analysis of this phenotype as done by others (Pan PNAS 2006) must be provided to support the authors' claim that this phenotype represents neurodegeneration. We have included new data on multiple degenerative phenotypes in axons including: blebbing, beading, waviness and breaks (Table S1).

• The number of beads per axon should be quantified to better represent the severity of rbm-26 mutant. Individual samples should be plotted in the quantification instead of showing the percentage of animals. We have added data on the density of beads in rbm-26(null), rbm-26(P80L), and rbm-26(L13V) mutants (Figure S3). For most experiments we have decided to use penetrance to measure axon degeneration because this is a standard in the field and allows for a larger sample size. For examples please see:

10.1523/JNEUROSCI.1494-11.2012 (Toth et al, 2012)

https://doi.org/10.1016/j.cub.2014.02.025 (Rawson et al, 2014)

10.1073/pnas.1011711108 (Pan et al, 2012)

https://doi.org/10.7554/eLife.80856 (Czech et al, 2023)

https://doi.org/10.1016/j.celrep.2016.01.050 (Nichols et al, 2016)

• Based on the single gel image in Fig. 1C with no loading control, the P80L mutant appears to have no protein expression. How is the P80L viable while the null mutant is lethal? The authors should quantify the protein expression levels from multiple blots with proper loading controls. If P80L mutation is introduced into RBM-26::mScarlet strain can it cause depletion of the signal in vivo? We have added new data showing that the RBM-26::Scarlet signal is diminished by the P80L mutation in vivo (Figure 1E-F). We have also added quantification from 3 biological replicate blots (Figure 1D). Finally, we have improved the sensitivity of our blots by using ECL detection and also show various exposures to highlight the fainter bands (Figures 1C and S1). Therefore, we are now able to detect low level expression of RBM-26(P80L) mutant protein. It is likely that the low level of RBM-26(P80L) and RBM-26(L13V) seen on western blots is sufficient to prevent the lethal phenotype.

• 'Moreover, loss of either the SPTBN1 or ADD1 genes causes a neurodevelopmental syndrome that includes autism and ADHD' References are missing, and as described above, be extra careful when indicating causality. Very few genes are known to cause ASD and ADHD. We have added the citations for this work (line 81). We also note that the titles for both of the cited articles indicate causation. To be on the safe side we have revised this line to say, "Moreover, loss of either the SPTBN1 or ADD1 genes are thought to cause a neurodevelopmental syndrome that includes autism and ADHD"

• Fig. 3E F, the authors should use the strains that express TIR1 specifically in the touch receptor neurons to argue cell autonomous function of RBM-26. Alternatively, the authors may conduct PLM neuron-specific rescue experiments to test the sufficiency. We have added new data indicating that a Pmec-7::rbm-26::scarlet transgene can rescue the beading phenotype and the PLM/ALM overlap phenotype (see Figure 3F-G).

• 'Loss of RBM-26 causes mitochondria dysfunction in axons.' The authors did not examine mitochondria function in axons. They only examined the number of mitochondria, and ROS production in the soma. The authors should provide additional evidence to support the idea that elevated ROS production in the soma is due to mitochondrial dysfunction in axons. Also, the authors should use both P80L and L13V for this experiment, and indicate individual datapoint as dots. Here, they quantified at the L4 stage, which the authors should justify. We have added the L13V data to this experiment and now show the individual data points. In addition, we have now conducted this analysis at the L2, L3 and L4 stages (Figure 5C-E). We have also revised the text to indicate that loss of rbm-26 function causes mitochondrial dysfunction in the cell body which could potentially cause a reduction of mitochondria in the axon (see lines 100-101 and 268-270). We speculate that mitochondria in the axon are also dysfunctional. However, the mitoTimer signal is not bright enough in axons to allow for quantification.

• Figure 5B and C: the authors should also use L13V to quantify malsu-1 mRNA and protein level, and include quantifications in panel C (from multiple blots). This is Figure 6 in the new version. We have added new data for expression of mals-1 mRNA and protein in rbm-26(L13V) mutants (Figure 6B-D). We have also included quantifications from 3 biological replicates (Figure 6D).

• In the rbm-26 mutant, the number of mitochondria is reduced, while the amount of MALSU-1 protein is increased. If MALSU-1 is specifically localized at mitochondria in wild type, where does the excessive MALSU-1 go in the rbm-26 mutants? Quantification of MALSU-1 signal intensity should be provided. Our Pmec-7::mals-1::scarlet transgene uses the tbb-2 3'UTR and causes an overexpression phenotype. To address the question posed by the reviewer, we would need to express MALS-1 at endogenous levels. Given that endogenous levels of MALS-1 are very low, it is unlikely that we would be able to visualize its expression. Nonetheless, as a way to address this question we have attempted to create a single copy Pmec-7::mals-1::scarlet transgene that utilizes the mals-1 endogenous 3'UTR. We have tried multiple approaches for generating this construct, but all have failed, likely due to sequence complexities within the mals-1 3'UTR. While we cannot say where the extra MALS-1 protein goes, we think that it is likely overloaded into the remaining mitochondria and could also be in the cytosol as well.

• Figure 7C: malsu-1 knockout mutants exhibit PLM overextension phenotype, which is not consistent with their model. The authors should discuss this in detail. We have added a paragraph to the discussion explaining that mitochondria function could be disrupted by either MALS-1 overexpression or by MALS-1 loss of function (lines 471-480).

• 'To validate these findings, we also repeated these experiments with an independent allele of malsu-1, malsu-1(tm12122) and found similar results (Fig. 7A-C).' The malsu-1(tm12122) exhibits beading phenotype and more severe overextension phenotype which the authors must describe and discuss more carefully. One likely reason for this difference is that tm12122 is predicted to cause a partial deletion of the mals-1 coding sequence, whereas the syb6330 is a full deletion. Thus, the tm12122 could be acting as a dominant negative. In fact, prior work on the MALSU1 ortholog has indicated that this protein is subject to interference by a dominant negative construct (see Rorbach et al, Nucleic Acids Res 2012). Nonetheless, we cannot rule out the possibility of a linked second mutation in tm12122. However, since we have found similar phenotypes and genetic interactions with both alleles, we can conclude that these phenotypes and interactions are due to loss of MALS-1, rather than a second mutation (albeit at a slightly different penetrance). We have added these considerations to the results section (lines 342-244).

• Figure 8: The authors should include data from L13V, malsu-1 and rbm-26; malsu-1 mutants. Quantification from multiple blots should be provided. This is Figure 8D in the new version. We have added the malsu-1 and rbm-26;malsu-1 double mutants to this experiment. We have also added quantification from multiple biological replicate blots. As pointed out by the other reviewer, we think that this experiment does not give specific information about mitoribosomes, but is an alternative approach to looking at the reduction in mitochondria. Given this limitation and considering that we have added L13V data to the mitochondria experiment in Figure 8B, we have elected not to add additional data on L13V to the western blot experiment in Figure 8D

Minor comments: • 'Consistent with a role for mitochondria in neurodevelopmental disorders, some of these disorders include a neurodegenerative phenotype.' Why is it consistent to have neurodegenerative phenotypes if mitochondria is associated with neurodevelopmental disorders? A better explanation would help.

We have changed this sentence to, "Some neurodevelopmental syndromes feature neurodegenerative phenotypes that occur during neuronal development."

• L13V is generally more severe in axon overextension phenotype than P80L while protein level is more abundant. The authors should discuss about this. We have also added a time course for the PLM/ALM overlap phenotype mutants (Figure 2D). This new data shows that the PLM/ALM overlap is quite similar overall between the P80L and L13V mutants. Both of these mutations cause an increase in PLM/ALM overlap in early larval development that is resolved by the L4 stage. The P80L phenotype resolves slightly sooner for reasons that are unknown. This could reflect differences in expression within the PLM that are not reflected in the whole worm lysate. This could also be due to a slight difference in the genetic background or other stochastic factors. The key point is that these two independent alleles cause similar phenotype overall, indicating that this phenotype is the result of loss in RBM-26 function.

• Fig. 2E, F: 'Beading refers to focal enlargement or bubble-like lesions which were at least twice the diameter of the axon in size.' How are the diameters of axons measured? A more detailed quantification method, and examples of measurement should be provided. We have added example measurements to the supplemental section (Figure S3). Additional detail on the measurements are in the Methods section (lines 517-518).

• Figure 3: The authors should also include low-magnification images to show where RBM-26 is expressed. The current image does now allow identifying cells. The transgene that labels the nuclei of hypodermis should be indicated in the manuscript. Specifically, the expression of the RBM-26 in the PLM should be shown. We have added a low magnification image (Figure S6) and have also added images of endogenously tagged RBM-26:Scarlet in the PLM (Figure 3A-C). The transgenic label for the hypodermis has been added to the legend of Figure S5.

• Figure 3: 'Tissue specific degradation of RBM-26::SCARLET::AID was achieved due to cell-type specific TIR-1 driver lines (see methods for details).' This information is not provided in the method section. This information has been added to methods section, "Auxin proteindegredation"

• Fig. 4 E. Values from individual samples should be indicated as dots. Representative images of P80L and L13V should be included. Conduct quantifications at adult stage as the authors use in other quantifications, or justify use of specific developmental stage (L3) they used. Figure 4 has become Figures 4 and 5 in the revised version. We have updated the graphs to include dots for individual data points. We have added quantifications of the mitoTImer experiments for the L2, L3 and L4 stages (Figure 5C-E). We note that our other experiments were done at the L1, L2, L3 and L4 and adult stages. The mitoTimer signal is not sufficient at the L1 stage for quantification. At the adult stage, the red signal becomes saturated. We have added representative images for mitoTimer in P80L and L13V mutants (Figure S9).

• The genes malsu-1 and mrpl-58 are not listed on wormbase. If the authors would like to designate names to these gene, they should clearly indicate that along with the sequence name. We have changed malsu-1 to mals-1. In addition, both mals-1 and mrpl-58 have now been approved by wormbase and will be listed on the website upon its next update.

• The authors found that MRPL-58 amount is reduced in rbm-26 mutants (which require additional verifications). This can be explained by the fact that axonal mitochondria number is reduced in the rbm-26 mutants. How did the authors confirm that the reduction in MRPL-58 level is due to the disruption of mitoribosome assembly? This is Figure 8D-E in the new version. We have added new data showing that the decrease in MRPL-58 expression that is caused by the rbm-26(P80L) mutation is dependent on MALS-1. We concede that these experiments cannot be used to determine anything about the mitoribosomes per se, but rather serve as an alternative way of testing the effect of rbm-26 on mitochondria. We have revised the text accordingly (lines 355-357).

• 'MALSU-1 is a mitoribosomal assembly factor that functions as part of the MALSU1:LOR8F8:mtACP anti-association module [37-39].' I don't think these are known for C. elegans MALSU-1. We have revised to, "MALS-1 is an ortholog of the MALSU1 mitoribosomal assembly factor that functions as part of the MALSU1:LOR8F8:mtACP anti-association module"

• 'Moreover, our results also suggest that disruption of this process can give rise to neurodevelopmental disorders.' I feel this is a quite a bit of stretch.

This has been replaced with, "Therefore, we speculate that human RBM26/27 could function with the RNA exosome complex to protect against neurodevelopmental defects and axon degeneration in infants." (lines 371-373)

**Referees cross-commenting** Yes, many of our comments overlap, and I fully agree with all comments from the other reviewer too. Reviewer #2 (Significance (Required)):

I found the manuscript interesting particularly the use of innovative techniques in identifying the target of RBM-26, The genetic analyses of rbm-26 and malsu-1 generally support the authors main conclusions that rbm-26 inhibits malsu-1 and be of potential interest to basic neuroscientists and cell biologists. However, the current manuscript looked premature which made my reading experience less pleasant. The phenotypic analyses is superficial compared to works similar to this work, which are insufficient to support the authors' claim of 'axon degeneration and targeting defects'. A number of issues listed above should be addressed before this manuscript is published. The reviewer's expertise: neurodevelopment in model organisms.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors studied an ASD-associated gene, rbm-26 in neuronal morphology using the touch receptor neuron PLM in C. elegans, and found that loss-of-function rbp-27 causes overextension and the formation of bulb-like structures in the axon. Using UV-crosslinking RNA immunoprecipitation and RNA-Seq, they identify malsu-1 as a target of rbm-26. Genetic analyses suggest malsu-1 likely functions downstream of rbm-26 in controlling the PLM morphology.

Major comments:

• The authors describe RBM27 is associated with ASD and ID while they only cite SFARI paper that describes a weak association of RBM27 to ASD. The appropriate referenced that show link between RBM27 and ID should be provided.
• SFARI database only has three (P79L, R190Q, G348D) mutations listed as ASD-associated. Where are other mutations L13V and R455H, particularly L13V that the authors used to generate the C. elegans mutant come from? Are they associated with intellectual disabilities?
• The authors should be very careful when describing 'gene X causes Y diseases'. Many (if not all) of the examples described in this manuscript are disease-associated genes without validation to be causal genes.
• The authors refer PLM axon beading and overextension phenotypes to 'axon degeneration and targeting defects'. The authors must provide additional evidence of axon degeneration (see below). Also the term 'targeting defects' is misleading as the authors did not examine if overextension of the PLM axon causes targeting defects. At least they should examine some synaptic markers.
• Neuronal phenotypes (axon overextension and beading) should be examined at different developmental timepoints (larval, young adult, and aged animals) to test if these phenotypes are indeed degenerative instead of developmental defects.
• The authors use the blebbing (beading) phenotype in the axon as the sole evidence of neurodegenerative properties of the PLM neuron. A more thorough analysis of this phenotype as done by others (Pan PNAS 2006) must be provided to support the authors' claim that this phenotype represents neurodegeneration.
• The number of beads per axon should be quantified to better represent the severity of rbm-26 mutant. Individual samples should be plotted in the quantification instead of showing the percentage of animals.
• Based on the single gel image in Fig. 1C with no loading control, the P80L mutant appears to have no protein expression. How is the P80L viable while the null mutant is lethal? The authors should quantify the protein expression levels from multiple blots with proper loading controls. If P80L mutation is introduced into RBM-26::mScarlet strain can it cause depletion of the signal in vivo?
• 'Moreover, loss of either the SPTBN1 or ADD1 genes causes a neurodevelopmental syndrome that includes autism and ADHD' References are missing, and as described above, be extra careful when indicating causality. Very few genes are known to cause ASD and ADHD.
• Fig. 3E F, the authors should use the strains that express TIR1 specifically in the touch receptor neurons to argue cell autonomous function of RBM-26. Alternatively, the authors may conduct PLM neuron-specific rescue experiments to test the sufficiency.
• 'Loss of RBM-26 causes mitochondria dysfunction in axons.' The authors did not examine mitochondria function in axons. They only examined the number of mitochondria, and ROS production in the soma. The authors should provide additional evidence to support the idea that elevated ROS production in the soma is due to mitochondrial dysfunction in axons. Also, the authors should use both P80L and L13V for this experiment, and indicate individual datapoint as dots. Here, they quantified at the L4 stage, which the authors should justify.
• Figure 5B and C: the authors should also use L13V to quantify malsu-1 mRNA and protein level, and include quantifications in panel C (from multiple blots).
• In the rbm-26 mutant, the number of mitochondria is reduced, while the amount of MALSU-1 protein is increased. If MALSU-1 is specifically localized at mitochondria in wild type, where does the excessive MALSU-1 go in the rbm-26 mutants? Quantification of MALSU-1 signal intensity should be provided.
• Figure 7C: malsu-1 knockout mutants exhibit PLM overextension phenotype, which is not consistent with their model. The authors should discuss this in detail.
• 'To validate these findings, we also repeated these experiments with an independent allele of malsu-1, malsu-1(tm12122) and found similar results (Fig. 7A-C).' The malsu-1(tm12122) exhibits beading phenotype and more severe overextension phenotype which the authors must describe and discuss more carefully.
• Figure 8: The authors should include data from L13V, malsu-1 and rbm-26; malsu-1 mutants. Quantification from multiple blots should be provided.

Minor comments:

• 'Consistent with a role for mitochondria in neurodevelopmental disorders, some of these disorders include a neurodegenerative phenotype.' Why is it consistent to have neurodegenerative phenotypes if mitochondria is associated with neurodevelopmental disorders? A better explanation would help.
• L13V is generally more severe in axon overextension phenotype than P80L while protein level is more abundant. The authors should discuss about this.
• Fig. 2E, F: 'Beading refers to focal enlargement or bubble-like lesions which were at least twice the diameter of the axon in size.' How are the diameters of axons measured? A more detailed quantification method, and examples of measurement should be provided.
• Figure 3: The authors should also include low-magnification images to show where RBM-26 is expressed. The current image does now allow identifying cells. The transgene that labels the nuclei of hypodermis should be indicated in the manuscript. Specifically, the expression of the RBM-26 in the PLM should be shown.
• Figure 3: 'Tissue specific degradation of RBM-26::SCARLET::AID was achieved due to cell-type specific TIR-1 driver lines (see methods for details).' This information is not provided in the method section.
• Fig. 4 E. Values from individual samples should be indicated as dots. Representative images of P80L and L13V should be included. Conduct quantifications at adult stage as the authors use in other quantifications, or justify use of specific developmental stage (L3) they used.
• The genes malsu-1 and mrpl-58 are not listed on wormbase. If the authors would like to designate names to these gene, they should clearly indicate that along with the sequence name.
• The authors found that MRPL-58 amount is reduced in rbm-26 mutants (which require additional verifications). This can be explained by the fact that axonal mitochondria number is reduced in the rbm-26 mutants. How did the authors confirm that the reduction in MRPL-58 level is due to the disruption of mitoribosome assembly?
• 'MALSU-1 is a mitoribosomal assembly factor that functions as part of the MALSU1:LOR8F8:mtACP anti-association module [37-39].' I don't think these are known for C. elegans MALSU-1.
• 'Moreover, our results also suggest that disruption of this process can give rise to neurodevelopmental disorders.' I feel this is a quite a bit of stretch.

Referees cross-commenting Yes, many of our comments overlap, and I fully agree with all comments from the other reviewer too.

Significance

I found the manuscript interesting particularly the use of innovative techniques in identifying the target of RBM-26, The genetic analyses of rbm-26 and malsu-1 generally support the authors main conclusions that rbm-26 inhibits malsu-1 and be of potential interest to basic neuroscientists and cell biologists. However, the current manuscript looked premature which made my reading experience less pleasant. The phenotypic analyses is superficial compared to works similar to this work, which are insufficient to support the authors' claim of 'axon degeneration and targeting defects'. A number of issues listed above should be addressed before this manuscript is published.

The reviewer's expertise: neurodevelopment in model organisms.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

This manuscript uses C. elegans as a model to interrogate the effects of autism-associated variants of previously unknown function in the RNA-binding protein RBM-26/RBM27.

Despite its potential impact, there are several concerns related to the technical rigor and specificity of the observed effects.

Major concerns:

1. The effects on PLM are interesting, but why was this neuron selected for study? Was this a lucky guess or are other axons also affected? It is important to clarify whether the effects of RBM-26 are specific to this neuron or act pleiotropically across many or all neurons. According to CeNGEN, rbm-26 is strongly expressed in the well-characterized neurons ASE, PVD, and HSN. Are there morphological defects in these neurons, or others? As a note, there are also functional assays for these neurons (salt sensing, touch response, and egg laying, respectively).
2. Similarly, the choice of the MALSU homolog seemed like a shot in the dark. It is ranked 46th (out of 63 genes) for fold-enrichment following RBM-26 pull-down, and 9th for p-value. Were any of the mRNAs with greater fold-enrichment or smaller p-values examined further? It is important to determine whether many or all of these interacting genes are overexpressed in the absence of RBM-26 and whether they are also required for the phenotypic effects of RBM-26 mutants, or if the MALSU homolog is special.
3. In addition to the specificity controls mentioned above, positive and negative controls are needed throughout the results. While each of these may be relatively minor by itself, as a group they raise questions about the technical rigor of the study. Briefly these include:

Fig 1C. Missing loading controls and negative control (rbm-26 null allele). Additional exposures should be included to show whether RBM-26(P80L) protein or the lower band for RBM-26(L13V) are present at all, relative to the null allele.

Fig 2. Controls to distinguish overextension of PLM axon from posterior mispositioning of ALM cell body are needed. Quantification of PLM axon lengths in microns (or normalized to body size) with standard deviation, not error of proportion, should be shown. Measurement of "beading phenotype" should be more rigorous, see for example the approach in Rawson et al. Curr. Biol. 2017 https://doi.org/10.1016/j.cub.2014.02.025 . The developmental stage examined, and the reason for choosing that stage, should be described for this and all figures.

Fig 3. Controls without auxin and with neuronal TIR1 expression alone should be included. Controls demonstrating successful RBM-26 depletion, in larvae as well as in embryos at the time of PLM extension, should be included (weak embryonic depletion might explain why the overextension phenotype is only 14% instead of 40% as in the null). According to CeNGEN, rbm-26 expression in PLM is barely detected, thus depletion with a PLM-specific TIR1 should also be tested. To confirm the authors' identification of the cell marked "N" as the PLM cell body, co-expression of rbm-26 and a PLM-specific marker should be added. Rescue of the rbm-26 mutants with neuronal (and PLM-only) expression should be included to test sufficiency in PLM, and as a further control for potential artifacts of the AID system.

Fig 4. More rigorous quantification of the distribution of mitochondria along the axon should be included, not only total number, and it should be clarified what region of the axon the images are taken from. Including the AID-depletion strain with and without auxin would further add to the sense of rigor. For the mitoTimer experiments, why is RBM-26(L13V) not included and why do wild-type values differ ~5-fold between experiments (despite error bars being almost non-existent)? A more rigorous approach to standardizing imaging conditions may be needed. Positive controls using compounds that affect oxidation should be included. Measurements of individual mitochondria with standard deviations should be shown, rather than aggregate averages with error of proportion.

Fig 5. Additional positive and negative controls should be added, including additional rbm-26 alleles, the AID-tagged strain with and without auxin, and a rescued mutant.

Fig 6. Controls showing whether the Scarlet-tagged protein is functional are needed, to rule out dominant negative or toxicity-related effects.

Fig 8. Controls for other mitochondrial components need to be included. It is important to determine if the decrease in ribosomes is specific or reflects a general decrease in mitochondria. If there are fewer mitochondria as suggested in Fig. 4, then of course mitochondrial ribosomal protein levels are also reduced. Additional rbm-26 alleles should be included here as well. Is this effect dependent on the MALSU homolog? 4. Finally the authors should address concerns about image manipulation, which amplify the concerns about technical rigor outlined above. The image in Fig. 2A appears to have a black box placed over the lower-right portion of the field to hide some features. Black boxes also appear to have been placed over the tops of images in Fig. 4B and 4D and at the left of Fig. 6A, 6B, and 6C. While these manipulations probably do not affect the conclusions, they further undermine confidence in data integrity and experimental rigor.

Minor points.

1. C. elegans nomenclature conventions should be followed:
   • C. elegans gene names have three or four letters, thus the MALSU homolog cannot be named "malsu-1". Please have new gene names approved by WormBase BEFORE submitting for publication http://tazendra.caltech.edu/~azurebrd/cgi-bin/forms/gene_name.cgi
   • If two sequential CRISPR edits are made on the same gene then they should be listed as a compound allele, such as rbm-26(cue22cue25)
   • Genes on the same chromosome should not be separated with a semicolon, for example rbm-26(cue40) K12H4.2(syb6330)
2. Describing the defects as "neurodevelopmental" is misleading in the case of axon beading or degeneration. Similarly, there is no evidence for an "axon targeting" defect as stated in the abstract.
3. In Fig. 5A, the symbol that appears to correspond to F59C6.15 (lowest p-value) is a different size than the others and is colored as ncRNA, whereas WormBase annotates this gene as snoRNA.
4. In the Introduction, the last sentences of the first two paragraphs should be varied ("However, little is known about the [...] mechanisms that protect [...] during neurodevelopment.")
5. Why is RBM-26 protein running as a doublet at both sizes?
6. When showing the RBM-26 expression pattern (Fig. 3) please include a lower-magnification image of the entire animal.
7. It is confusing to refer to the RNA IP experiments as an "unbiased screen", which in C. elegans typically refers to a genetic screen.
8. The relationship between axon overextension, beading, and mitochondrial localization is not clear. What causal connection between these is being proposed? The causal connections between these phenotypes, if any, should be clarified experimentally. For example, if the axon extension defects develop before mitochondrial localization defects, then it is unlikely that mitochondrial defects cause axon overextension.
9. Please explain how to interpret the difference in axon beading in the two deletion alleles of the MALSU homolog (axon beading defects in tm12122 but not in syb6330). Is syb6330 not a null allele? Or are the defects in tm12122 due to other mutations in this strain background?
10. Are mitochondria reduced in number or mislocalized? If they are reduced in number, is this due to altered balance of fission/fusion?
11. In Fig. 3A-D, please keep the labels in the same position in all panels and do not alter brightness settings between single-color and merged panels.
12. The claim that rbm-26 acts cell-autonomously requires PLM-specific depletion and rescue experiments.

Referees cross-commenting I appreciate the use of the consultation session to resolve differences between reviewers, but in this case I fully agree with the content and tone of all the comments from the other reviewer -- I think our remarks are very well aligned!

Significance

The study engineers autism-associated variants in conserved residues of RBM27 into the C. elegans homolog RBM-26 and identifies neuronal phenotypes potentially relevant to autism and a potential molecular mechanism involving regulation of mitochondrial ribosome assembly.

The key claims of the study are 1} that autism-associated variants in RBM-26 decrease its protein expression; 2} that impaired RBM-26 function leads to a variety of defects in development and maintenance of a single neuron called PLM, including altered axonal localization of mitochondria; 3} that RBM-26 normally binds the mRNA for the C. elegans homolog of MALSU, a mitochondrial ribosomal assembly factor; 4} that loss of RBM-26 leads to overexpression of the MALSU homolog; and 5} that MALSU is required for some of the deleterious effects on the PLM neuron seen in RBM-26 mutants.

This study will be of interest to the autism research community because it bolsters the idea that variants in RBM27 are likely to disrupt gene function and to affect neuronal health. It will also be of interest to the broader cell biology community because it suggests an interesting potential nucleus-to-mitochondria signaling mechanism, in which a nuclear RNA-binding protein might regulate assembly of mitochondrial ribosomes.

My field of expertise is developmental biology in C. elegans.

I'm having a little bit of trouble with this argument because—although the subsequent text tries to explain it—I'm not exactly sure how (or persuaded that) a shuffle addresses the issues that this section opened with (empty space in timelines, teleology, etc.).

Although shuffled, the images are still arrayed out horizontally. And, given that the text is written in English, I read the images left-to-right in the manner of a linear timeline rather than the web of timelines in step 2 of this scrolly. (And I didn't realize until reading further, that I could actually interact with the images; and even then, it took me a very long time to realize I could drag the images around; perhaps switching the mouse cursor to be the "grabby hand" might make that clearer?)

I think I'd also be more persuadable if the writing was a bit more critical in its discussion of its design choices—eg, besides reading too self-assuredly like shuffle was the right call, I am very curious about what tradeoffs the shuffle is forced to make. For instance, doesn't the shuffled display make it hard to trace particular (alternate) paths through history? It seems to (perhaps necessarily, because it must display things on a 2D computer display) flatten things out.

make the prompt titles bolded or in larger print to separate them from the responses. Also please add some pictures, copies of certificates you completed, projects or inservices, etc. throughout the document.

Pique my interest

remind you of something you’ve learned in another course

Raise questions

eLife assessment

This valuable work explores the utility of using analyte ratios for improved biological interpretation in a MALDI MSI workflow. The evidence supporting the conclusions is however incomplete, as relevant controls are missing and the novelty of the study has been exaggerated, omitting discussion of key relevant background. The work would be of interest to the mass spectrometry community.

Reviewer #1 (Public Review):

Cheng et al explore the utility of analyte ratios instead of relative abundance alone for biological interpretation of tissue in a MALDI MSI workflow. Utilizing the ratio of metabolites and lipids that have complimentary value in metabolic pathways, they show the ratio as a heat map which enhances the understanding of how multiple analytes relate to each other spatially. Normally, this is done by projecting each analyte as a unique color but using a ratio can help clarify visualization and add to biological interpretability. However, existing tools to perform this task are available in open-source repositories, and fundamental limitations inherent to MALDI MSI need to be made clear to the reader. The study lacks rigor and controls, i.e. without quantitative data from a variety of standards (internal isotopic or tissue mimetic models for example), the potential delta in ionization efficiencies of different species subtracts from the utility of pathway analysis using metabolite ratios.

Reviewer #2 (Public Review):

Summary:

In the article, "Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging" the authors describe their software package in R for visualizing metabolite ratio pairs. I think the novelty of this manuscript is overstated and there are several notable issues with the figures that prevent detailed assessment but the work would be of interest to the mass spectrometry community.

Strengths:

The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding of metabolomics data and enhance the identification of critical metabolites.

Weaknesses:

The authors are missing several references and discussion points, particularly about SIMS MSI, where ratio imaging has been previously performed.

There are several misleading sentences about the novelty of the approach and the limitations of metabolite imaging.

Several sentences lack rigor and are not quantitative enough.

The figures are difficult to interpret/ analyze in their current state and lack some critical components, including labels and scale bars.

因果が逆のような気がします

原文 Many packages, however, are kept out of the standard library so they can be developed on a timeline independent of the language itself.

代案

しかし、多くのパッケージは標準ライブラリに含まれておらず、言語自体とは独立したスケジュールで開発できます。

ここからの4行の訳の代案を考えてみました。

原文 Python doesn’t include pytest by default, so you’ll learn to install external libraries. Knowing how to install external libraries will make a wide variety of well-designed code available to you. These libraries will expand the kinds of projects you can work on immensely.

代案

pytestはPythonにはデフォルトでは含まれていないので、外部ライブラリのインストール方法についても学びます。外部ライブラリをインストールするということは、さまざまな洗練されたコードが手に入るということです。これらのライブラリによって取り組めるプロジェクトの種類が格段に増えます。

代案の補足

Knowing how to～を「～方法を知ることで」と書くと翻訳調になるので、方法を知る＝できると意訳しました。 また、取り組めるthe kinds of projectがすごく拡張する、ということで、「できることを大きく拡張」のところを「種類が格段に増える」に変えました。

eLife assessment

This valuable study uses convincing state-of-the-art neuroimaging analyses to characterise whole-brain networks during reward-based motor learning. This work motivates future research to dissociate why the observed changes in neural connectivity occur and how they support reward-based motor learning. The study is highly relevant for researchers at the intersection of decision-making and sensorimotor learning.

Reviewer #1 (Public Review):

This important study uses a wide variety of convincing, state-of-the-art neuroimaging analyses to characterize whole-brain networks and relate them to reward-based motor learning. During early learning, the authors found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks. During late learning, they observed the opposite pattern. It remains to be seen whether these changes reflect generic changes in task engagement during learning or are specific to reward-based motor learning. This study is highly relevant for researchers interested in reward-based motor learning and decision-making.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is compelling and goes beyond the current state of the art. The results are of interest to neuroscientists working on motor control and reward-based learning.

Comments on revised version:

The revision has produced a stronger manuscript. Thank you for your thorough responses to the comments and concerns.

Reviewer #3 (Public Review):

Summary:

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field.

Strengths:

This manuscript is written in a clear and comprehensible way. It introduces a rather novel technique of assessing connectivity across lower-dimensional manifold which has hitherto not been applied in this way to the question of reward-based motor learning. Thus, this presents a unique viewpoint on understanding how the brain changes with motor learning. I particularly enjoyed the combination of connectivity-based, followed by further scrutiny of seed-based connectivity analyses, thus providing a more comprehensive viewpoint. Now it also has added a more comprehensive report on the behavioural changes of learning, and the added statistical quantification, which is useful.

Weaknesses:

The main weakness of the manuscript is the lack of direct connection between the reported neural changes and behavioural learning. Namely, most of the results could also be explained by changes in attention allocation during the session, or changes in movement speed (independent of learning). The authors acknowledge some of these potential confounds and argue that factors like attention are important for learning. While this is true, it is nonetheless very limiting if one cannot ascertain whether the observed effects are due to attention (independent of learning) or attention allocated in the pursuit of learning. The only direct analysis linking behavioural changes to neural changes is based on individual differences in learning performance, where the DAN-A shows the opposite trend than group level effects, which they interpret as differences given the used higher-resolution parcellation. However, it could be that these learning effects are indeed much smaller and subtler compared to more dominant group-level attention effects during the task. The lack of a control condition in the task limits the interpretability of results as learning-related.

Author Response

We appreciate the thoughtful comments provided by the editor and reviewers. We were pleased to hear that they appreciated our work's contribution to the field of motor learning as well as our use of state-of-the-art analysis techniques.

We are currently preparing a comprehensive revision of our manuscript to address several of the recommendations of the reviewers. It is our belief that this revision will not only strengthen our paper but also help clarify several areas that were highlighted by the reviewers.

To address the concerns regarding potential confounds in our experimental design, we will be providing a more detailed justification and rationale for the experimental design and analysis choices made during our study. It appears that some reviewers’ comments may stem from misunderstandings concerning certain details of our task and we will carefully revise these sections to ensure that the design and purpose of the study are unambiguous. We will also be improving our characterizations of subjects’ learning behavior, which we believe will clarify some of the reviewers comments and enhance the overall rigor of our analyses. Lastly, we will be dealing with all concerns related to the statistical quantification of our results.

We appreciate the opportunity to improve our manuscript for eLife and are eager to provide a revision that satisfies the majority of the reviewers’ recommendations

eLife assessment

This study represents a valuable step toward understanding how brain connectivity changes during reward-based motor learning. However, the evidence presented is incomplete. On one hand, the study leverages state-of-the-art techniques to examine brain connectivity; on the other hand, there are potential confounds in the experimental design, some omissions in statistical quantification, and at times, a lack of clarity about the methods used and the motor learning mechanisms being isolated.

Reviewer #1 (Public Review):

The authors investigated how global brain activity varied during reward-based motor learning. During early learning, they found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks; during late learning, they found the opposite pattern. Individual learning performance varied only with changes in the dorsal attention network. The authors certainly used a wide variety of valuable, state-of-the-art techniques to interrogate whole-brain networks and extract the key components of learning behavior. However, the findings are incomplete, tempered by potential confounds in the experimental design. As such, the underlying claim regarding how these networks jointly support reward-based motor learning is unclear.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is highly compelling and goes beyond the current state-of-the-art, but it is incomplete with respect to examining different signatures of learning, clarifying probed learning processes, and investigating changes in all relevant subcortical structures is incomplete and would benefit from more rigorous approaches. With the rationale and data presentation strengthened this paper would be of interest to neuroscientists working on motor control and reward-based learning.

Reviewer #3 (Public Review):

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field. The evidence supporting the presented findings is strong, though at times lacking rigorous statistical quantification. Nevertheless, there are several issues that require attention and clarification.

eLife assessment

This study presents an important finding on the splicing regulatory function of RBM7 and its functional impact in breast cancer metastasis. The evidence supporting the claims of the authors is solid, although the inclusion of more delineation of how RBM7 regulates NF-kB and coordinates splicing would have strengthened the study. The work will be of interest to scientists working on breast cancer.

Reviewers’ Comments:

Reviewer #1 (Remarks to the Author):

Summary:

Fang Huang et al found that RBM7 deficiency promotes metastasis by coordinating MFGE8 splicing switch and NF-kB pathway in breast cancer by utilizing clinical samples as well as cell and tail vein injection models.

Strengths:

This study uncovers a previously uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis, and provides evidence supporting RBM7 function in splicing regulation. These findings facilitate the mechanistic understanding of how splicing dysregulation contributes to metastasis in cancer, a direction that has increasingly drawn attention recently, and provides a potentially new prognostic and therapeutic target for breast cancer.

We thank the reviewer for appreciating the novelty and importance of this study, and have provided new data to address the following concerns raised by the reviewer.

Weaknesses:

This study can be strengthened in several aspects by additional experiments or at least by further discussions. First, how RBM7 regulates NF-kB, and how it coordinates splicing and canonical function as a component of NEXT complex should be clarified. Second, although the roles of MFGE8 splicing isoforms in cell migration and invasion have been demonstrated in transwell and wound healing assays, it would be more convincing to explore their roles in vivo such as the tail vein injection model. Third, the clinical significance would be considerably improved, if the therapeutic value of targeting MFGE8 splicing could be demonstrated.

We’re thankful for the constructive suggestions. A preliminary study on the mechanism by which RBM7 regulates NF-kB pathway is already underway. We found RBM7 depletion remarkably promoted the expression of IL-1β as judged by qPCR and ELISA assays (new Figure S5G- S5I, also see below). IL-1β, commonly known as a pro-inflammatory cytokine, could bind to IL-1R and initiate a multistage enzymatic reaction that triggers the activation of NF-κB pathway (Axel Weber, 2010) (Qing Guo, 2024). Thus we speculated that the upregulation of IL-1β might be a causal factor in RBM7-depletion-induced activation of NF-kB signaling. It will be interesting to determine the complete molecular mechanism in our future study. In addition, we performed a co-IP experiment and found that RBM7 could interact with RNA splicing factor SF3B2, a component of spliceosomal U2 snRNP complex (new Figure S6B, also see below). Consistent with the AS regulation of MFGE8 by RBM7, the depletion of SF3B2 also promoted exon7 skipping, implying a cooperative effect of the two proteins in regulating MFGE8 splicing (new Figure S6C-6D, also see below). This is in concert with a previous study that RRM domain of RBM7 could bind a proline-rich segment within SF3B2 (Falk, Finogenova et al., 2016). The interaction mode with strong similarity to RBM7RRM–ZCCHC8Proline interaction in the NEXT complex indicated mutually exclusive binding of SF3B2 and ZCCHC8 to RBM7. Thus, RBM7 appears to play dual, but not conflicting, roles during RNA processes depending on its interaction with the spliceosome or exosome (see line 427-437 in the new manuscript).

Author response image 1.

The mRNA levels of IL-1β in MDA-MB-231 or BT549 cells with stable RBM7 knockdown or control vector were examined by qRT-PCR approach.

Author response image 2.

Supernatants from RBM7-knockdown MDA-MB-231 or BT549 cells were collected and protein expression of IL-1β was measured by ELISA kit.

Author response image 3.

The knockdown efficiency of RBM7 in two breast cancer cell lines were determined by qRT-PCR approach.

Author response image 4.

Immunoprecipitation assay was performed in breast cancer cells expressing HA-RBM7 and Flag-SF3B2 or empty vector. The Flag-tagged precipitated complexes and lysates were analyzed through western blotting.

Author response image 5.

The splicing shift of MFGE8 upon SF3B2 knockdown in breast cancer cells was examined by RT-PCR approach. The mean ± SD of PSI values derived from three independent replicates is shown.

Author response image 6.

The SF3B2 knockdown efficiency was examined by qRT-PCR.

To further corroborate the roles of two MFGE8 isoforms in cell invasion, we have performed Fluorescent Gelatin Degradation Assays for investigating invadopodia formation. Consistent with the transwell assay results, MFGE8-L up-regulation suppressed breast cancer cells invasion through a layer of extracellular matrix, whereas breast cancer cells with ectopic expression of MFGE8-S acquired enhanced ability to degrade matrix and invasion (new Figure 5B, also see below). In addition, to determine the therapeutic value of targeting MFGE8 splicing, we transfected triple-negative breast cancer cells with ASOs targeting RBM7-binding motif and examined the potential impact on cell aggressiveness. The results showed an obvious increase in exon7-skipped variant of MFGE8 as compared to the scramble negative control ASOs, meanwhile, the migrative and invasive ability of breast cancer cells treated with splice-targeting ASOs was significantly boosted (new Figure 6B and S5B, also see below), further suggesting that RBM7-knockdown stimulated aggressiveness of breast cancer at least partially relies on splicing switch of MFGE8.

Author response image 7.

Gelatin degradation assay was performed to test the effect of RBM7 knockdown on invadopodia function. 10000 cells were plated onto FITC-gelatin substrates (Green) and cultured for 48 h. Representative images are shown (red, Cy3-phalloidin; blue, DAPI) and the degraded areas were quantified by Image J software. Scar bars= 50 μm. P values were determined by one-way ANOVA with Tukey's multiple comparison test (n = 3).

Author response image 8.

Representative transwell analysis of migrative/invasive capability of breast cancer cells transfected with 500 nM ASO directed against RBM7-binding region in MFGE8 pre-mRNA. P values were determined by one-way ANOVA with Tukey's multiple comparison test.

Author response image 9.

RT-PCR quantification of two MFGE8 isoforms after transfecting breast cancer cells with 500 nM ASO directed against RBM7-binding region in MFGE8 pre-mRNA. P values were calculated by one-way ANOVA with Tukey's multiple comparison test.

The minor concerns

(1) Several figure legends do not match with the images, for example, Figure 2K, Figure 4, Figure 7D, and 7E, and the description of Fiure 7F is missing in the text.

As suggested by the reviewer, we have checked all of the figure legends carefully and corrected all of the misinterpretation.

(2) The statistical methods for Figure1A and Figure1B should be indicated.

As suggested by the reviewer, we have included the statistical methods for Figure1A and 1B in Figure1 legend. Data in Figure 1A and 1B are presented as means ± SD and P values were obtained by Mantel-Cox log-rank test.

(3) The molecular weight of the proteins in the Western Blot images should be marked.

As suggested by the reviewer, we have added the molecular weight of proteins in all of the western blot images.

(4) The sequences where RBM7 binds on MFGE8 RNA should be clearly indicated.

We thank the reviewer for this question. We analyzed the sequence of alternative exon 7 and the motifs nearby its 5’ or 3’ splice sites, and found two RBM7 potentially binding motifs are positioned in proximal to the pseudo 3’ splice site. Subsequent RT-PCR for the precipitation in RIP assays confirmed RBM7 could bind to the upstream sequence containing 5’-UUUCUU-3’ motifs adjacent to intron6/exon7 junction of MFGE8 cassette exon, but not another region nearby it. To pinpoint the location for the potential cis-element for AS regulation by RBM7, we designed antisense oligonucleotides (ASOs) to block RBM7 potentially binding sites (UUUCUU). As shown in revised Figure 4F, when compared to scramble ASO, targeting ASOs contributed to the exclusion of exon7. Additionally, we constructed an exogenous MFGE8 splicing reporter containing exon 6-8 and partial intron sequences to determine the binding site for AS regulation by RBM7. The depletion of RBM7 still induced the splicing shift of the minigene reporter by elevating MFGE8-S variant. While the binding motif UUUCUU was removed or mutated, RBM7 failed to affect the splicing outcomes of MFGE8 (new Figure S3C, also see below). Due to its close proximity to 3’ splice site, UUUCUU residues bound by RBM7 is very likely to participate in spliceosome assembly at the upstream 3’ splice site of exon7, which may explain why disruption of the motif led to almost complete exon7 skipping. The above data suggested that RBM7 regulated the exon skipping of MFGE8 by binding to UUUCUU located six nucleotides upstream of the 3’ splice-site of exon7.

Author response image 10.

Upper: the red line in diagram indicates ASOs targeting region which contains UUUCUU; down: MCF7 and MDA-MB-231 cells were transfected with ASOs targeting MFGE8 pre-mRNA for 48h and then applied for RT-PCR identification. P values were determined by one-way ANOVA with Tukey's multiple comparison test.

Author response image 11.

Upper: MFGE8 min-splicing reporters with mutation in the RBM7 binding site or a non-specific binding were generated and shown in cartoon; down: RT-PCR assays were performed to identify the splicing outcomes of MFGE8 reporter while RBM7 was depleted in breast cancer cells.

(5) Some typos, graphic errors, and sentences are hard to understand and need to be corrected, such as lines 80-81, 249-250, line 221 "motfs", line 319 "RBM4". Please carefully proofread and revise the entire manuscript.

As suggested by the reviewer, we have corrected typos and graphic errors mentioned above. In addition, this manuscript was also extensively edited to improve grammar and sentence structure.

(6) Define the abbreviations when they first appear, such as MFGE8-L, RBM, etc.

We thank the reviewer for raising this point. We have defined the abbreviations when firstly presented in the manuscript.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

Strengths:

Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

Weaknesses:

However, there are some controls missing, and the data presentation needs to be improved. The writing of the manuscript needs some grammatical improvements because some of the wording might be confusing.

We thank the reviewer for the positive comments on this work, and have addressed all the concerns raised by the reviewer.

Specific comments:

(1) Figure 2. The figure legend is missing for Figure 2C, which caused many mislabels in the rest of the panels. The labels in the main text are correct, but the authors should check the figure legend more carefully. Also in Figure 2C, it is not clear why the authors choose to examine the expression of this subset of genes. The authors only refer to them as "a series of metastasis-related genes", but it is not clear what criteria they used to select these genes for expression analysis.

We thank the reviewer for raising this question. We have included the figure legend for Figure 2C and improved other figure legends throughout the article. For the second question, since gene ontology analysis of RNA-seq data in RBM7-depleted breast cancer cells showed that a series of differentially expressed genes were enriched in metastasis-associated processe, we identified the expression of this subset of genes in breast cancer cells in the presence or absence of RBM7 by heatmap differential analysis based on qRT-PCR results. To clarify this point and address the reviewer’s concern, we have improved the relevant description of this part (see line 174-180 in the new manuscript).

(2) Line 218-220. The comparison of PSI changes in different types of AS events is misleading. Because these AS events are regulated in different mechanisms, they cannot draw the conclusion that "the presence of RBM7 may promote the usage of alternative splice sites". For example, the regulators of SE and IR may even be opposite, and thus they should discuss this in different contexts. If they want to conclude this point, they should specifically discuss the SE and A5SS rather than draw an overall conclusion.

We are thankful for the reviewer’s valuable comment. According to the suggestion, we have removed the overall conclusion and corrected to discuss in SE and A5SS.

(3) In the section starting at line 243, they first referred to the gene and isoforms as "EFG-E8" or "EFG-E8-L", but later used "EFGE8" and "EFGE8-L". Please be consistent here. In addition, it will be more informative if the authors add a diagram of the difference between two EFGE8 isoforms in terms of protein structure or domain configuration.

As suggested by the reviewer, we keep using the name “MFGE8-L” for the canonical MFGE8 isoform and “MFGE8-S” for the truncated isoform in this manuscript. In addition, to clarify the structural basis for the different tumor invasion-related functions of two MFGE8 isoforms, we have included a diagram of their domain configuration in new Figure S4F and predicted protein structure in new Figure S4G. The details in the revised manuscript are given below:

Author response image 12.

Schematic diagram of the domain composition of two MFGE8 isoforms. Upper: the full-length variant with exon7 indicated by yellow square; down: the truncated variant with exon7 skipping.

Author response image 13.

The model structure of two MFGE8 isoforms was implemented using SwissModel software. The F5/8 type C2 protein domain excluded from MFGE8-S variant was marked in red.

(4) Figure 7B and 7C. The figures need quantification of the inclusion of MFGE exon7 (PSI value) in addition to the RT-PCR gel. The difference seems to be small for some patients.

As suggested by the reviewer, we have included the relative quantification of PSI for endogenous MFGE8 in breast cancer patients and found increased proportion of exon7 exclusion in most tumor samples when compared to normal tissues (case#1: 86:94; case#2: 84:86; case#3: 79:85; case#4: 63:75; case#5: 69:93; case#6: 71:80) (new Figure 7B, also see below). On the other hand, we have expanded the number of metastatic breast cancer cases and quantified the the AS events within MFGE8 by analyzing the PSI values. The lymph node metastases contain a higher proportion of MFGE8 variant with skipped exon7 in comparison with paired primary tumor tissues (case#1: 80:95; case#2: 86:97; case#3: 84:90; case#4: 70:78; case#5: 83:89) (Figure 7C). This is coherent with decreased RBM7 expression levels found in breast cancer with lymph node metastasis.

Author response image 14.

The splicing alteration of MFGE8 in 6 pairs of primary breast cancer tissues and adjacent normal tissues was examined using RT-PCR. The quantification of PSI vales was based on relative band intensities using Image J software.

Author response image 15.

The splicing alteration of MFGE8 in primary breast cancer tissues and corresponding lymph node metastases was identified by RT-PCR assays. The quantification of PSI vales wa determined by Image J software.

Minor comments:

The writing in many places is a little odd or somewhat confusing, I am listing some examples, but the authors need to polish the whole manuscript more to improve the writing. 1. Line 169-170, "...followed by profiling high-throughput transcriptome by RNA sequencing", should be "followed by high-throughput transcriptome profiling with RNA sequencing". 2. Line 170, "displayed a wide of RBM7-regulated genes were enriched...", they should add a "that" after the "displayed" as the sentence is very long. 3. Line 213, "PSI (percent splicing inclusion)" is not correct, PSI stands for "percent spliced in". 4. Line 216-217, the sentence is long and fragmented, they should break it into two sentences. 5. Line 224, the "tethering" should be changed to "recognizing". There is a subtle difference in the mechanistic implication between these two words. 6. Line 250, should be changed to "...in the ratio of two MFGE8 isoforms".

We thank the detailed comments from the reviewer. The points mentioned above has been addressed one by one and this manuscript was also extensively edited to improve grammar and sentence structure for better understanding.

Reference

Axel Weber PW, Michael Kracht* (2010) Interleukin-1 (IL-1) Pathway. SCIENCESIGNALING.

Qing Guo1, Yizi Jin1,2, Xinyu Chen3, Xiaomin Ye4, Xin Shen5, Mingxi Lin1,2, Cheng Zeng1,2, Teng Zhou1,2 and Jian Zhang1,2 (2024) NF-κB in biology and targeted therapy: new insights and translational implications. Signal Transduction and Targeted Therapy.

Falk S, Finogenova K, Melko M, Benda C, Lykke-Andersen S, Jensen TH, Conti E (2016) Structure of the RBM7–ZCCHC8 core of the NEXT complex reveals connections to splicing factors. Nature Communications.

eLife assessment

This study presents a rather valuable finding on the RBM7 function in spicing regulation and uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis. The evidence supporting the claims of the authors is solid. The work will be of broad interest to clinicians, medical researchers and scientists working on breast cancer.

Reviewer #1 (Public Review):

Fang Huang et al found that RBM7 deficiency promotes metastasis by coordinating MFGE8 splicing switch and NF-kB pathway in breast cancer by utilizing clinical samples as well as cell and tail vein injection models.

This study uncovers a previously uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis, and provides evidence supporting RBM7 function in splicing regulation. These findings facilitate the mechanistic understanding of how splicing dysregulation contributes to metastasis in cancer, a direction that has increasingly drawn attention recently, and provides a potentially new prognostic and therapeutic target for breast cancer.

Reviewer #2 (Public Review):

In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

eLife assessment

This study provides important findings based on compelling evidence demonstrating that females and males have different strategies to regulate energy consumption in the brain in the context of low energy intake. While food deprivation reduces energy consumption and visual processing performance in the visual cortex of males, the female cortex is unaffected, likely at the expense of other functions. This study is relevant for scientists interested in body metabolism and neuroscience.

assuring that the treatment in a research study is conducted consistently and reliably.

elf-efficacy is an individual's belief in their capacity to act in the ways necessary to reach specific goals. T

eLife assessment

This manuscript provides important information about the influence of TOR signaling pathway on development and aflatoxin production in the plant and human fungal pathogen Aspergillus flavus. Compared to an earlier version, the authors have addressed most of the concerns of the reviewers, including the convincing demonstration of the essential TOR pathway in this fungus by constructing a xylose promoter mutant strain.

Reviewer #1 (Public Review):

This paper reports the useful discovery of the roles and signaling components of the TOR pathway in vegetative growth, sexual development, stress response, and aflatoxin production in Aspergillus flavus.

While I acknowledge the authors' effort in conducting Southern blot analysis to address my prior concern regarding the presence of dual copies of torA and tapA, I find their current resolution inadequate. Specifically, the simple deletion of the respective result sections for torA and tapA significantly impacts the overall significance of this study. The repeated unsuccessful attempts to generate correct mutants only offer circumstantial evidence, as technical issues may have been a contributing factor. Therefore, instead of merely removing these sections, it is essential for the authors to present more compelling experimental data demonstrating that torA and tapA are indeed vital for the viability of A. flavus. Such data would enhance the overall significance of this study.

Reviewer #2 (Public Review):

In this study, authors identified TOR, HOG and CWI signaling network genes as modulators of the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation. They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially that the conserved site K19 of FKBP3 plays a key role in regulating aflatoxin biosynthesis. In general, the study involved a heavy workload and the findings are potentially interesting and important for understanding or controlling the aflatoxin biosynthesis. However, the findings have not been deeply explored and the conclusions mostly are based on parallel phenotypic observations.

Author response:

The following is the authors’ response to the previous reviews.

Reviewer #1 (Public Review):

While I acknowledge the authors' effort in conducting Southern blot analysis to address my prior concern regarding the presence of dual copies of torA and tapA, I find their current resolution inadequate. Specifically, the simple deletion of the respective result sections for torA and tapA significantly impacts the overall significance of this study. The repeated unsuccessful attempts to generate correct mutants only offer circumstantial evidence, as technical issues may have been a contributing factor. Therefore, instead of merely removing these sections, it is essential for the authors to present more compelling experimental data demonstrating that torA and tapA are indeed vital for the viability of A. flavus. Such data would enhance the overall significance of this study.

We agree and appreciate reviewer's important comments on our manuscript. In this version, we address this issue by providing additional experimental data to further support the importance of torA and tapA in the viability of A. flavus. We conducted additional experiments to generate more compelling evidence regarding the essential role of torA and tapA in the growth and development of A. flavus. We constructed a mutant strain (xylPtorA) using an xylose-inducible promoter, which allows for conditional induction with the addition of xylose (Lines 204-238, page 10).

Due to the unsuccessful construction of TapA knockout strains and xylose promoter replacement strains, we used homologous recombination to replace the original promoter with the gpdA strong promoter for overexpression of tapA (OE::tapA). We thank reviewer for highlighting this important aspect, and we revise our manuscript accordingly to enhance its overall significance (Lines 277-297, page 13). We are grateful for the opportunity to enhance our manuscript and believe these revisions provide a more comprehensive understanding of the roles of torA and tapA in A. flavus.

Reviewer #1 (Recommendations For The Authors):

Minor comments

Lines 421-423 and 465-466: these sentences are grammatically awkward. Please rephrase them.

Thank you for your feedback on our manuscript. We conducted additional experiments, so we have removed the sentence from the manuscript to maintain coherence and avoid redundancy.

Reviewer #2 (Public Review):

In this study, authors identified TOR, HOG and CWI signaling network genes as modulators of the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation. They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially that the conserved site K19 of FKBP3 plays a key role in regulating aflatoxin biosynthesis. In general, the study involved a heavy workload and the findings are potentially interesting and important for understanding or controlling the aflatoxin biosynthesis. However, the findings have not been deeply explored and the conclusions mostly are based on parallel phenotypic observations.

Thank you for your constructive comments on our manuscript. In response to your comments, we have conducted additional experiments, including the construction of a xylose promoter mutant strain and an overexpression strain. We have also expanded the discussion section to provide a more comprehensive analysis of our findings in the context of existing literature. Thank you again for your insightful feedback, which has been instrumental in improving the quality of our work. (Lines 464-469, page 22).

Reviewer #2 (Recommendations For The Authors):

Point 1: Our findings revealed that both the tor and tapA genes are present in double copies in our strains, which guided our decision to construct single-copy deletion strains using homologous recombination However, the tor gene in A. flavus exhibited varying copy numbers, as was confirmed by absolute quantification PCR at the genome level (Table S1). However, it is hard to understand for Table S1: Estimation of copy number of tor gene in A. flavus toro and sumoo stand for the initial copy number, and the data are graphed as the mean {plus minus} 95%confidence limit. CN is copy number. As indicated in the Methods, Using sumo gene as reference, the tor and tapA gene copy number was calculated by standard curve. In Table S1 of WT, for tor gene, CN value is1412537 compared to 1698243 in tor+/-, for the reference gene sumo,794328 compared to1584893, how these data could support copy gene numbers of tor?

Thank you for your insightful comments. We understand the confusion with the data presented in Table S1 regarding the copy number estimation of the torA gene in A. flavus. We apologize for not providing a clear explanation for the data in the table. Quantitative real-time PCR (qPCR) is widely used to determine the copy number of a specific gene. It involves amplifying the gene of interest and a reference gene simultaneously using specific primers and probes. By comparing the amplification curves of the gene of interest and the reference gene, we can estimate the relative copy number of the gene.

To address your concern and provide more accurate information, we have re-performed the copy number analysis using southern blot. Southern blot analysis allows for the direct estimation of gene copy number by hybridizing genomic DNA with a specific probe for the gene. This method provides more reliable and accurate results in determining gene copy numbers. We discovered that the A. flavus genome contains a single copy of the torA gene. Consequently, we conducted additional experiments to elucidate its function. Specifically, we generated strains with a xylose-inducible promoter system to modulate the expression of torA (Lines 204-238, page 10).

Point 2: In response: For the knockout of the FRB domain, we used the homologous recombination method, but because tor genes are double-copy genes, there are also double copies in the FRB domain. Despite our efforts, we encountered challenges in precisely determining the location of the other copy of the tor gene. I could not understand these consistent data, why not for using sequencing?

Thank you for your valuable feedback. We determined again and confirmed that the torA gene is a single copy. So we removed this part of the results to avoid any ambiguity or potential misinterpretation.

Point 3: Response in Due to the large number of genes involved, we did not perform a complementation experiment. If there were no complementation data, how to demonstrate data are solid?

Thank you for your important suggestion. We understand that complementation experiments are commonly used to validate gene deletions. Therefore, to ensure the reliability of our data, we have conducted supplementary experiments on specific gene deletions, such as Δ_sitA_-C and Δ_ppg1_-C. Thank you again for your positive comments and valuable suggestions, which have significantly contributed to enhancing the quality of our manuscript (Lines 320-322, page 15).

Point 4: Acknowledge the confusion? We acknowledge the confusion in our presentation and will ensure that accurate genetic nomenclature is used consistently

Thank you for your comments on our manuscript. We recognize the importance of precise and consistent use of genetic nomenclature, as it is critical for the clarity and integrity of our research findings. We have carefully reviewed the sections of our manuscript where genetic terms were used and have made the necessary corrections to ensure that all nomenclature is accurate and used consistently throughout the text.

Point 5: In the revised version of new manuscript, southern blotting was carried out and found only one copy was existed for tested genes at last. Thus, whole manuscript conclusions should be changed. In addition, Reviewer 1 suggestion for using Illumina-sequence strategy, their tor and tapA mutants could be verified whether they are aneuploid?

We would like to express our gratitude for your insightful comments and suggestions. Following the new experimental data obtained from Southern blotting, we have identified that only one copy of the tested genes exists, and we have revised our conclusions throughout the manuscript. This has led to a significant reinterpretation of our results and a reassessment of the implications for our study. Based on this result, we designed and constructed strains with the tor gene under the control of a xylose-inducible promoter. This approach allows for the conditional expression of the tor gene. Thank you once again for your meticulous review (Lines 204-238, page 10).

eLife assessment

This important study contributes to the understanding of how parafoveal words are neurally processed during naturalistic sentence reading. Convincing evidence is provided that the MEG response to a word can be modulated by the semantic congruency of a parafoveal target word. The study addresses a classic question in reading using a new Rapid Invisible Frequency Tagging (RIFT) technique, which can separately monitor the neural processing of multiple words during sentence reading.

Reviewer #1 (Public Review):

Summary:

The study investigates parafoveal processing during natural reading, combining eye-tracking and MEG techniques, building upon the RIFT paradigm previously introduced by Pan et al. (2021).

The manuscript is well-written with a clear structure, and the data analysis and experimental results are presented in a lucid manner.

Comments on revised version:

I am satisfied with the revisions made by the authors. I believe the study introduces a new research paradigm to the field.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review):

The study investigates parafoveal processing during natural reading, combining eye-tracking and MEG techniques, building upon the RIFT paradigm previously introduced by Pan et al. (2021). Overall, the manuscript is well-written with a clear structure, and the data analysis and experimental results are presented in a lucid manner.

The authors have addressed the issues I raised in the previous round of review to my satisfaction. However, I still have two concerns that require the authors' consideration.

Firstly, the similarity between the RIFT analysis process in this study and traditional ERP analysis could lead readers to equate RIFT with components like N400, potentially influencing their interpretation of the results. Although the author's response has somewhat clarified my queries, I seek confirmation: does RIFT itself signify "visual attention" or the "allocation of attentional resources to the flickering target words" (line 208) in this study? While this may not be pivotal, as it primarily serves as an indicator to evaluate whether contextual congruity can indeed modulate the RIFT response rather than indicating early parafoveal semantic integration, I recommend that the authors explicitly address this point in the manuscript, maybe in the discussion section, to enhance reader comprehension of the article's rationale.

Secondly, regarding the study's conclusions, there appears to be an overemphasis in stating that "semantic information ... can also be integrated with the sentence context ..." (line 21-22). As raised by Reviewer 2 (Major Point 1) and acknowledged by the authors in the limitations of the revised manuscript (lines 403-412), the RIFT effect observed likely stems from local congruency. Therefore, adjusting the conclusion to "integrated with previous context" may offer a more precise reflection of the findings.

We appreciate the positive comments from the Reviewer.

In response to the first concern, we have rephrased the sentence (Line 207-209 in the revised manuscript) to clarify that RIFT measure visual attention : “Moreover, as RIFT directly measures visual attention, the left-skewed RIFT response curve suggests that more visual attention is allocated towards the flickering target words before fixating on them, aligning with the left-to-right order of reading English.”

Regarding the second concern, we have addressed the issue by modifying “sentence context” to “previous context” in both the Abstract (Line 18 and Line 22) and the Discussion section (Line 314 and Line 361) of the revised manuscript.

eLife assessment

The authors utilized scRNAseq profiling of NSCLC patient tumor samples to generate useful insights into the determinants of ICI responsiveness in NSCLC patients. While some of the findings add weight to the current literature, the analysis is incomplete due to the small cohort size and occasional departures from recognized subtype markers. This study would benefit from external cohorts to both validate the findings and to justify the statistical analysis undertaken.

Reviewer #1 (Public Review):

Summary:

The authors study the variability of patient response of NSCLC patients on immune checkpoint inhibitors using single-cell RNA sequencing in a cohort of 26 patients and 33 samples (primary and metastatic sites), mainly focusing on 11 patients and 14 samples for association analyses, to understand the variability of patient response based on immune cell fractions and tumor cell expression patterns. The authors find immune cell fraction, clonal expansion differences, and tumor expression differences between responders and non-responders. Integrating immune and tumor sources of signal the authors claim to improve prediction of response markedly, albeit in a small cohort.

Strengths:

- The problem of studying the tumor microenvironment, as well as the interplay between tumor and immune features is important and interesting and needed to explain the heterogeneity of patient response and be able to predict it.

- Extensive analysis of the scRNAseq data with respect to immune and tumor features on different axes of hypothesis relating to immune response and tumor immune evasion using state-of-the-art methods.

- The authors provide an interesting scRNAseq data set linked to outcomes data.

- Integration of TCRseq to confirm subtype of T-cell annotation and clonality analysis.

- Interesting analysis of cell programs/states of the (predicted) tumor cells and characterization thereof.

Weaknesses:

- Generally, a very heterogeneous and small cohort where adjustments for confounding are hard. Additionally, there are many tests for association with outcome, where necessary multiple testing adjustments would negate signal and confirmation bias likely, so biological takeaways have to be questioned.

- RNAseq is heavily influenced by the tissue of origin (both cell type and expression), so the association with the outcome can be confounded. The authors try to argue that lymph node T-cell and NK content are similar, but a quantitative test on that would be helpful.

- The authors claim a very high "accuracy" performance, however, given the small cohort and lack of information on the exact evaluation it is not clear if this just amounts to overfitting the data.

- Especially for tumor cell program/state analysis the specificity to the setting of ICIs is not clear and could be prognostic.

- Due to the small cohort with a lot of variability, more external validation is needed to be convincingly reproducible, especially when talking about AUC/accuracy of a predictor.

Reviewer #2 (Public Review):

Summary:

The authors have utilised deep profiling methods to generate deeper insights into the features of the TME that drive responsiveness to PD-1 therapy in NSCLC.

Strengths:

The main strengths of this work lie in the methodology of integrating single-cell sequencing, genetic data, and TCRseq data to generate hypotheses regarding determinants of IO responsiveness.

Some of the findings in this study are not surprising and well precedented eg. association of Treg, STAT3, and NFkB with ICI resistance and CD8+ activation in ICI responders and thus act as an additional dataset to add weight to this prior body of evidence. Whilst the role of Th17 in PD-1 resistance has been previously reported (eg. Cancer Immunol Immunother 2023 Apr;72(4):1047-1058, Cancer Immunol Immunother 2024 Feb 13;73(3):47, Nat Commun. 2021; 12: 2606 ) these studies have used non-clinical models or peripheral blood readouts. Here the authors have supplemented current knowledge by characterization of the TME of the tumor itself.

Weaknesses:

Unfortunately, the study is hampered by the small sample size and heterogeneous population and whilst the authors have attempted to bring in an additional dataset to demonstrate the robustness of their approach, the small sample size has limited their ability to draw statistically supported conclusions. There is also limited validation of signatures/methods in independent cohorts, no functional characterisation of the findings, and the discussion section does not include discussion around the relevance/interpretation of key findings that were highlighted in the abstract (eg. role of Th17, TRM, STAT3, and NFKb). Because of these factors, this work (as it stands) does have value to the field but will likely have a relatively low overall impact.

Related to the absence of discussion around prior TRM findings, the association between TRM involvement in response to IO therapy in this manuscript is counter to what has been previously demonstrated (Cell Rep Med. 2020;1(7):100127, Nat Immunol. 2017;18(8):940-950., J Immunol. 2015;194(7):3475-3486.). However, it should be noted that the authors in this manuscript chose to employ alternative markers of TRM characterisation when defining their clusters and this could indicate a potential rationale for differences in these findings. TRM population is generally characterised through the inclusion of the classical TRM markers CD69 (tissue retention marker) and CD103 (TCR experienced integrin that supports epithelial adhesion), which are both absent from the TRM definition in this study. Additional markers often used are CD44, CXCR6, and CD49a, of which only CXCR6 has been included by the authors. Conversely, the majority of markers used by the authors in the cell type clustering are not specific to TRM (eg. CD6, which is included in the TRM cluster but is expressed at its lowest in cluster 3 which the authors have highlighted as the CD8+ TRM population). Therefore, whilst there is an interesting finding of this particular cell cluster being associated with resistance to ICI, its annotation as a TRM cluster should be interpreted with caution.

Author response:

We appreciate the comprehensive reviews and would like to address the critiques and suggestions provided by both reviewers. We will make significant revisions to the manuscript to address these concerns. These include a more cautious interpretation of our results, an expanded discussion on key findings, additional analyses for TRM characterization, and a clearer outline of future validation efforts. We believe these changes will enhance the clarity and robustness of our study, and we hope they meet the reviewer’s expectations.

Reviewer 1:

Weaknesses:

(1) Heterogeneous and small cohort:

Increasing the cohort size is not feasible due to resource constraints. We acknowledge the challenges posed by the heterogeneous and small cohort, which complicate adjustments for confounding. We will apply multiple testing corrections to transparently assess and accurately report the robustness of our findings in the revision.

(2) Influence of tissue of origin on RNAseq:

We agree that RNAseq results can be heavily influenced by the tissue of origin. While immune cell composition in the normal lung tissues and lymph nodes is quite different, we found that in tumor tissues and metastatic lymph nodes, these differences diminish and common features dominate. Although we depicted this data in the supplementary figure 1, we did not provide a quantitative test in the original submission. In the revision, we will perform additional quantitative tests to compare immune cell composition across different tissue origins. These tests will provide a more precise understanding of the cellular composition and support our argument regarding the similarity of tumor-sculpted microenvironment. We will include these results and detailed methodologies in the revision.

(3) Accuracy performance and overfitting:

We acknowledge the concern regarding the high “accuracy” performance potentially indicating overfitting. We will clarify the evaluation methods used and moderate our claims regarding accuracy in the revision.

(4) Specificity of the tumor cell program/state analysis to the setting of ICIs:

The comment suggests that the tumor programs in our study may not be specific to the ICI group but rather prognostic in lung cancer. We acknowledge this possibility as we performed comparisons between responders and non-responders (with different cut-offs) to find common trends and interpreted them in terms of their association with ICI. In the revision, we will test the prognostic association of the tumor programs using public lung cancer data.

(5) More external validation needed:

We recognize the importance of external validation for reproducibility. While increasing the cohort size is not feasible, we will propose future directions for validation using larger, independent cohorts and potential experimental validations.

Reviewer 2:

Weaknesses:

(1) Small sample size and heterogeneous populations:

Increasing the cohort size is not feasible due to resource constraints. We acknowledge the challenges posed by the heterogeneous and small cohort, which complicate adjustments for confounding. We will apply multiple testing corrections to transparently assess and accurately report the robustness of our findings in the revision.

(2) Limited validation of signatures/ methods in independent cohorts:

We recognize the importance of external validation for reproducibility. While increasing the cohort size is not feasible, we will propose future directions for validation using larger, independent cohorts and potential experimental validations.

(3) Lack of functional characterization and discussion on key findings:

We appreciate the feedback regarding the need for functional characterization and a more thorough discussion of key findings on the roles of specific cell populations and genes. In the revised manuscript, we will expand the discussion section to include in-depth analysis of these findings and their relevance to the study. This includes a detailed interpretation of how these factors contribute to the immune response and potential implications for therapy.

(4) TRM findings and marker selection:

We understand the concern regarding the association between TRM involvement in response to IO therapy, which appears counter to previous demonstrations. It is indeed important to note that we employed alternative markers for TRM characterization. Our choice of markers was based on transcriptional references relevant to our study. However, we agree that classical TRM markers such as CD69 and CD103, which were absent in our definition, are critical for accurate TRM identification. To address this, we will include a detailed rationale for our marker selection and acknowledge the limitations of our TRM characterization. We will include additional analyses using classical TRM markers where possible and incorporate these findings into the revision. This will provide a clearer understanding of our TRM population and its role in the immune response to IO therapy.

minor comment: would be nice to see a chart title

there is a well-quoted line from Shneiderman that continues this logic:

There are many visual design guidelines but the basic principle might be summarized as the Visual Information Seeking Mantra: Overview first, zoom and filter, then details-on-demand Overview first, zoom and filter, then details-on-demand

Shneiderman, Ben. "The eyes have it: A task by data type taxonomy for information visualizations." The craft of information visualization. Morgan Kaufmann, 2003. 364-371.

a skeptic might say here: "but how much of this epistemological claim is also driven by a material shift?"

D3 did not have to be made by using data to manipulate the DOM -- it would be helpful to see this point made more explicit in your argument!

links?

this paragraph also gave me pause (I haven't read pass this yet, so you might address my concerns in the next section) but there is a lot of vis research about decision making with visualizations that offer both a similar and different perspective on the intersection of visualization and decision making. Dimara & Stasko 2021 offers a good place to start on that discussion.

Dimara, Evanthia, and John Stasko. "A critical reflection on visualization research: Where do decision making tasks hide?." IEEE Transactions on Visualization and Computer Graphics 28.1 (2021): 1128-1138.

when it comes to the audience of the book, this is one of the statements that might strike a vis researcher as different. I don't mean to imply that it is unfounded, but rather, vis doesn't have the same history of unpacking context in this way to justify design decisions. i find this deeply interesting.

a follow-up comment, I wish the topic did not change so quickly from uncertainty to "for whom this knowledge is useful". I find myself wanting more to understand your argument.

it would be nice to see the old title and notes about scale through the scrollytelling story

minor comment: could the text for the toggle change? for example, show footnotes -> hide footnotes.

at the moment I have trouble knowing what state the toggle is in

it would be nice to have this highlighted as quote text or bold or alt titles for the chapters

flagging in context to the previous comments that call for a deemphasis of Tufte in the historical narrative of visulization

eLife assessment

The current version of the study presents important findings on how the RelA/Stat3-dependent gene program in the liver influences intestinal homeostasis. The evidence supporting the conclusions is solid, with new data added compared to an earlier version of the study. The work will be of interest to scientists in gastrointestinal research fields.

Reviewer #2 (Public Review):

Summary:

Singh and colleagues employ a methodic approach to reveal the function of the transcription factors Rela and Stat3 in the regulation of the inflammatory response in the intestine.

Strengths of the manuscript include the focus on the function of these transcription factors in hepatocytes and the discovery of their role in the systemic response to experimental colitis. While the systemic response to induce colitis is appreciated, the cellular and molecular mechanisms that drive such systemic response, especially those involving other organs beyond the intestine are an active area of research. As such, this study contributes to this conceptual advance. Additional strengths are the complementary biochemical and metabolomics approaches to describe the activation of these transcription factors in the liver and their requirement - specifically in hepatocytes - for the production of bile acids in response to colitis.

In this revised version, the authors have addressed previously raised questions.

Reviewer #3 (Public Review):

Summary:

The authors try to elucidate the molecular mechanisms underlying the intra-organ crosstalks that perpetuate intestinal permeability and inflammation.

Strengths:

This study identifies a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases via the gut liver axis using both murine models and human samples.

Weaknesses:

(1) The mechanism by which DSS administration induces the activation of the Rela and Stat3 pathways and subsequent modification of the bile acid pathway remains clear. As the authors state, intestinal bacteria are one candidate, and this needs to be clarified. I recommend the authors investigate whether gut sterilization by administration of antibiotics or germ free condition affects 1. the activation of the Rela and Stat3 pathway in the liver by DSS-treated WT mice and 2. the reduction of colitis in DSS-treated relaΔhepstat3Δhep mice.

(2) It has not been shown whether DSS administration causes an increase in primary bile acids, represented by CDCA, in the colon of WT mice following activation of the Rela and Stat3 pathways, as demonstrated in Figure 6.

(3) The implications of these results for IBD treatment, especially in what ways they may lead to therapeutic intervention, need to be discussed.

The above weakness points have been resolved by the revision and additional experiments.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This important study reveals the RelA/Stat3-dependent gene program in the liver influences intestinal homeostasis. The evidence supporting the conclusions is compelling, although some additional experiments will strengthen the study. The work will be of interest to scientists in gastrointestinal research fields.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, the authors showed that activation of RelA and Stat3 in hepatocytes of DSS-treated mice induced CYPs and thereby produced primary bile acids, particularly CDCA, which exacerbated intestinal inflammation.

Strengths:

This study reveals the RelA/Stat3-dependent gene program in the liver influences intestinal homeostasis.

Our reply: We thank the reviewer for the positive feedback and for appreciating the strength of our study.

Weaknesses:

Additional evidence will strengthen the conclusion.

(1) In Fig. 1C, photos show that phosphorylation of RelA and Stat3 was induced in only a few hepatocytes. The authors conclude that activation of both RelA and Stat3 induces inflammatory pathways. Therefore, the authors should show that phosphorylation of RelA and Stat3 is induced in the same hepatocytes during DSS treatment.

Our reply: The reviewers have raised a pertinent issue in Figure 1, as later on in our study we suggest that the combined activation of Rela and Stat3 is critical for aggravating the colitogenic phenotype in the murine model.

To address this issue, we have co-stained the fixed liver tissue of untreated and DSS-treated wild type mice with p-RelA (Ser536) and p-Stat3(Ser727) antibodies. Author response image 1 below shows the single staining for p-Rela (Ser536), pStat3 (Ser727), DAPI (to demarcate the nuclei) and merged image (p-Rela + pStat3).

Author response image 1.

Further, the signal intensity of p-RelA (Ser536) and p-Stat3(Ser727) per nuclei was calculated and plotted as a box plot. It is evident that the median of p-Rela and p-Stat3 signal intensity in DSS-treated samples is more than that of the control samples, suggesting that the majority of the treated hepatocytes have the presence of both p-Rela and p-Stat3 in the nuclei.

Author response image 2.

Further, we calculate the number of nuclei in the DSS-treated samples which are above the 90th percentile of the control samples (data has been provided in Author response table 1 below). We also calculate the percentage overlap of p-Rela to p-Stat3 and vice versa in Author response table 1 below.

Author response table 1.

Together our analysis concludes that indeed there is an activation of Rela and Stat3 in the same hepatocytes to generate the downstream effect that we observe in our study post-DSS treatment.

(2) In Fig. 5, the authors treated mice with CDCA intraperitoneally. In this experiment, the concentration of CDCA in the colon of CDCA-treated mice should be shown.

Our reply: We have experimentally examined if the CDCA supplemented intraperitoneally at the experimental dose used in our study, is reaching the colon or not. To quantify colonic CDCA we have performed targeted mass spectrometric studies and the data has been provided as a bar plot below.

Author response image 3.

It is evident from the plot that the CDCA levels are significantly higher in mice supplemented with CDCA as compared to their corresponding control (where only the vehicle was supplemented). The data has been added to the supplementary section S5b and the main text has been modified accordingly.

Reviewer #2 (Public Review):

Singh and colleagues employ a methodical approach to reveal the function of the transcription factors Rela and Stat3 in the regulation of the inflammatory response in the intestine.

Strengths of the manuscript include the focus on the function of these transcription factors in hepatocytes and the discovery of their role in the systemic response to experimental colitis. While the systemic response to induce colitis is appreciated, the cellular and molecular mechanisms that drive such systemic response, especially those involving other organs beyond the intestine are an active area of research. As such, this study contributes to this conceptual advance. Additional strengths are the complementary biochemical and metabolomics approaches to describe the activation of these transcription factors in the liver and their requirement - specifically in hepatocytes - for the production of bile acids in response to colitis.

Our reply: We express our gratitude to the reviewer for recognizing and appreciating the mechanistic insight provided by our work, and for considering it valuable in advancing conceptual understanding in the relevant field.

Some weaknesses are noted in the presentation of the data, including a comprehensive representation of findings in all conditions and genotypes tested.

Our reply: We thank the reviewer for the query and we have suitably modified the figures for a comprehensive representation of the findings, as described below:

● In Figure 2C, we have added the control alcian blue stained samples to clarify that there were no qualitative differences in the mucin levels observed in the relaΔhepstat3Δhep as compared to the wild type mice.

● We have also modified the figure 2D for a better presentation of the data.

● We have included histopathological analysis for the relaΔhepstat3Δhep mice in Figures S3a and S3b, following a format similar to the wild-type data previously provided as Figure S1a and S1b.

● For Figure 5C, the corresponding untreated samples with and without CDCA supplementation have been provided in the supplementary section Figure S5e.

● For Figure 2E, 3E, and 4C - the RT-qPCR data of the DSS-treated samples is plotted relative to their corresponding control samples, hence we only display two conditions in the bar plot. We have accordingly modified the figure legend for better clarity.

Reviewer #3 (Public Review):

Summary:

The authors try to elucidate the molecular mechanisms underlying the intra-organ crosstalks that perpetuate intestinal permeability and inflammation.

Strengths:

This study identifies a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases via the gut-liver axis using both murine models and human samples.

Our reply: We thank the reviewer for appreciating the therapeutic potential of our work.

Weaknesses:

(1) The mechanism by which DSS administration induces the activation of the Rela and Stat3 pathways and subsequent modification of the bile acid pathway remains clear. As the authors state, intestinal bacteria are one candidate, and this needs to be clarified. I recommend the authors investigate whether gut sterilization by administration of antibiotics or germ-free condition affects 1. the activation of the Rela and Stat3 pathway in the liver by DSS-treated WT mice and 2. the reduction of colitis in DSS-treated relaΔhepstat3Δhep mice.

Our reply: We thank the reviewer for bringing up the aspect of gut microbiota in imparting colitis in our mice model. In accordance with reviewer's recommendation, we have sterilized the gut by administration of antibiotics, to evaluate if the intestinal bacteria are an important component leading to the activation of Rela and Stat3 pathway in the liver of DSS-treated WT mice or not.

(a) A brief schematic representation of the experimental design has been provided below and the detailed description of the methods has been described in supplementary methods.

Author response image 4.

Extract of liver tissues from mice treated with DSS for 6 days with/without prior antibiotic treatment were probed with p-Stat3 (Ser727) to examine the activation status of the hepatic Stat3 pathway. We observe that the signals for p-Stat3 (Ser727) are comparatively reduced post antibiotic treatment as evident from the blot below. p-Stat3 (Ser727) was a prominent activation signal at Day 6 DSS treatment that we have observed in Figure 1D,E.

Author response image 5.

These studies suggest that the activation status of Stat3 activation is hampered by antibiotic treatment and considering that Rela and Stat3 have to coordinate activity, presumably the downstream activation will be modulated upon gut sterilization. However, it should be appreciated that a sterilized gut is not likely to be physiologically relevant and intestinal bacteria along with bile acid levels would modulate Rela/Stat3 pathways.

b) It is likely that the hepatic deficiency of Rela and Stat3 may have modified the gut microbiome in relaΔhepstat3Δhep mice because of the altered bile composition. Moreover, the gut microbiota is a key component that guides the outcome of colitis. Hence, future studies are important to examine the role of the gut microbiome in imparting resistance in relaΔhepstat3Δhep mice, to colitogenic insults.

(2) It has not been shown whether DSS administration causes an increase in primary bile acids, represented by CDCA, in the colon of WT mice following activation of the Rela and Stat3 pathways, as demonstrated in Figure 6.

Our reply: In order to address the query, we would kindly like to request the reviewers to look at figure 4B where we show an increase in the CDCA levels of the colonic tissue, which is corresponding to our CDCA levels in the liver tissue (figure 4A) thus indicating that it may be driven by the hepatic Rela and Stat3 pathways.

(3) The implications of these results for IBD treatment, especially in what ways they may lead to therapeutic intervention, need to be discussed.

Our reply: We are grateful to the reviewer for bringing this topic for discussion.

Until now, only immunosuppressive agents and immunomodulators have been conventionally considered as therapeutic measures to manage IBD. However, with increasing research on the role of hepatic bile acid metabolism during experimental colitis, its potential cannot be undermined in the clinical setting. The potential of bile acids as a therapeutic target has been harnessed in the past; bile acid sequestrants have been utilized as a treatment for hyperlipidemia 46. Remedies like fecal microbial transplantation, which serve to normalize the bile acid ratios in the gut, are emerging as potential therapeutics in the last decade for IBD 47, 40. However, the potential of altering hepatic bile metabolism has remained unexplored for IBD, possibly due to a lack of mechanistic insight. Towards this, our work demonstrates the pro-inflammatory potential of CDCA during colitis following the activation of the Rela/Stat3 pathway. The suppression of Rela/Stat3-induced CDCA could provide beneficial effects in IBD patients while protecting the basal bile acid levels (through FXR signaling). Thus our studies identify a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases. Another approach could be the use of bile acid sequestrants, which will temporarily decrease the levels of primary bile acids in the colon until the proinflammatory pathways are dampened as a combinatorial therapy alongside existing treatments.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Minor:

Fig. 4C should be Fig. 4D and vice versa.

Our reply: We have swapped Fig. 4C and Fig. 4D and corresponding changes have been incorporated in the main text.

Reviewer #2 (Recommendations For The Authors):

Please make note of the following specific comments

The immunostainings for phosphorylated p-Rela and STAT3 are unclear. Is there nuclear translocation of these phosphorylated transcription factors? Can the authors enumerate the percentage of cells in which nuclear translocation (presumably in hepatocytes) is detected?

Our reply: We apologize that immunostainings for phosphorylated p-Rela and STAT3 are unclear to the reviewers. Here we have tried our best to make the data clear by analyzing the stained section and plotting them.

To start with, we have co-stained the fixed liver tissue of untreated and DSS-treated wild type mice with p-RelA (Ser536) and p-Stat3(Ser727) antibodies, below we have provided a representative image used for analysis. To demarcate the nuclear boundary of the hepatocytes DAPI was used and the signal intensity for p-RelA (Ser536) and p-Stat3(Ser727) was quantified using ZenBlue software.

Author response image 6.

Below we have provided the box plot for the calculated nuclear intensities in the control (untreated) and DSS-treated samples for p-Rela and p-Stat3. We can clearly see that the median of p-Rela and p-Stat3 signal intensity in DSS-treated samples is more than that of the control samples, suggesting that the majority of the treated hepatocytes have the translocation of p-Rela and p-Stat3 in their nuclei.

Author response image 7.

The figure legends for Figures 2C and D are flipped. Please correct.

Our reply: Thank you for pointing it out, our apologies for the error and we have corrected the figure 2 accordingly.

For all H&E stainings, the authors should include histological scoring disease severity.

Our reply: Thank you for the query put forward, histological scoring to quantify the qualitative data obtained through microscopy is given below. Dot plot for the histological scoring of the H&E data for untreated and DSS-treated colon samples, we have referred to the scale described by Ren Y et al. 2019 (doi: 10.1038/s41598-019-53305-z) to score the sections.

Author response image 8.

We have added the dot plot to supplementary figure 2d, also the method applied for the above analysis has been described in the supplementary method section.

Please include Alcian Blue Staining in non-DSS treated WT and rel/stat3 double cKO mice.

Our reply: Thank you for pointing this out, we have added the Alcian Blue Staining of non-DSS treated WT and rel/stat3 double KO mice to figure 2C

For Figure 3C, can the authors indicate in the figure itself which bile acid is being represented (not only in the Figure legend)?

Our reply: Thank you for the suggestion we have indicated the respective bile acid in Figure 3C for better understanding.

As these data are from untargeted metabolomics, were other bile acids detected?

Our reply: This is a part of a separate study conducted by our collaborator, and will form a part of a new manuscript which will be focussed on human studies.

Can the authors validate the downregulation of key enzymes shown in Figure 3D, E at the protein level?

Our reply: We agree with the reviewer’s comment, that mRNA levels are not critical determinants of activation of any pathway, rather an indicator of probable activation. In that scenario, the estimation of protein levels is more determinative. But taking into consideration that we have the metabolomic data in subsequent figures (as in Figure 4 A, B) supporting our findings in Figure 3D, E, this makes RT-qPCR data a more robust indicator of an activated hepatic bile acid biosynthesis machinery.

The figure legends for Figures 4C and D are flipped. Please correct.

Our reply: Taking into consideration the suggestions by reviewer 1 we have swapped Fig. 4C and Fig. 4D and corrected the legend placement accordingly, thank you for pointing this out.

Also, please include representative images for the data represented in 4C.

Our reply: Thank you for the query, we have already added the representative images of confocal microscopy as figure S4.

Figure 5B should indicate that the data presented is from double cKO mice.

Our reply: We have indicated that the colon length data is from double KO animals in figure to make the visual representation clear for the readers, thank you for the concern.

Please correct typos: "entrocytic" and "Untread" in Figure Legend 5.

Our reply: Thank you for pointing out the error in the Legend, we apologize for the error in these errors we have corrected Figure 5.

Figure S4 includes a dataset (qPCR for Mmp3) that is not described. Neither Figure S4 nor S5 are described in the text.

Our reply: Thank you for the query, firstly we have already added Figure S4 and S5 to the text, our apologies that it has not been properly highlighted.

Secondly, the data for RT-qPCR for Mmp3 has been removed from supplementary figures as it may not be very relevant to the study.

Overall, the manuscript should be edited to ensure the correct use of English. Please also note that the last name of the first author seems to be missing in the main text.

Our reply: Thank you for the suggestion we have re-checked the manuscript for the probable errors and rectified them. The first author has a single name (with no surname) and we would like to correct that during the final print of the manuscript.

Reviewer #3 (Recommendations For The Authors):

(1) The authors need to show if DSS treatment affects the serological or histological changes in the liver of relaΔhepstat3Δhep mice.

Our reply: To address that, we have analyzed key serological markers of liver damage as well as looked into tissue histology.

The pathophysiological parameters of the liver of DSS treated relaΔhepstat3Δhep mice has been added to the revised manuscript as figure S3a and S3b. Here we show that the serological parameters are within the physiological range upon DSS treatment (Author response image 9a). Besides, the histological parameters remain unaltered as compared to the control tissue (Author response image 9b).

Cumulatively, both at the tissue level and functional level, there is not much effect of DSS

treatment on liver of relaΔhepstat3Δhep mice.

Author response image 9.

(2) It is recommended to use a second model to verify if this phenomenon is applicable to colitic status in general.

Our reply: We appreciate the query put forward, this is an ongoing study and we hope to examine further the role of hepatic RelA and Stat3 in TNBS-induced colitis model and in T cell transfer model of colitis.

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eLife assessment

This work critically evaluates several widely-used assays of transcriptional responses to water limitation in Arabidopsis grown on defined agar-solidified media and, finding inconsistent responses in root transcriptome responses, introduces a new 'hard agar' assay with more consistent responses. The work is valuable as a simple and alternative experimental system that would enable high-throughput genetic screening (and GWAS) to assess the impacts of environmental perturbations on transcriptional responses in various genetic backgrounds. Within this scope, the work is solid, though the debate about whether field-level physiological inferences can be made from such assays remains.

Reviewer #2 (Public Review):

This manuscript describes new methodology to study low water potential (drought) stress responses in agar plates. They devote considerable effort in comparing transcriptome data among various previously published experimental systems, examining how different approaches of reducing water potential impact the Arabidopsis root and shoot transcriptome. Each method purported to reduce water potential in plate-grown seedlings has a different effect on Arabidopsis root transcriptome responses, which is problematic for the field. In this reviewer's view, differences in transcriptome are not as important, and often not as informative as measurement of physiological parameters, which they do very little of in their study.

The focus on transcriptome data to the almost complete exclusion of other types of data is a symptom of a broader over-emphasis on the transcriptome that is quite prevalent in plant science now. We measure transcriptomes because we can, not because it is inherently the most informative thing to do. The important thing is protein amount, and even more so protein activity/function, which we know has an imperfect, at best, correlation with transcript level. This reviewer acknowledges that using Arabidopsis transcriptomics is a commonly employed method, and as such, the outcomes of this study will hold value for a broad audience, even if largely as a cautionary tale. If transcriptomics is used to identify candidate genes for future investigations, an approach that has had some success, then appropriate cautions should be taken in translating expectations about gene, protein, and phenotypic responses in field conditions.

Reviewer #3 (Public Review):

This work compares transcriptional responses of shoots and roots harvested from four plate-based assays that aim to simulate drought and from plants subjected to water deficit in pots using the model plant Arabidopsis thaliana with the goal to select a plate-based assay that best recapitulates transcriptional changes that are observed during water-deficit in pots. For the plate-based assays polyethylene glycol (PEG), mannitol, and sodium chloride (salt) treatments were used as well as a 'hard agar' assay which was newly developed by the authors. In the 'hard agar' assay, less water was added to the solid components of the media leading to an increase in agar strength and nutrient concentration. Plants in pots were grown on vermiculite with the same nutrient mix as used in the plates and drought was induced by withholding watering for five days.

The authors observed a good directional agreement of differential expressed genes for shoots between the plate assays on the vermiculite drying experiment. However, less directional agreement was observed for differential expressed genes of roots, except for their newly developed 'hard agar' assay which had good directional agreement. Testing whether the increase in agar strength or more concentrated nutrients are attributed to this, they found that both factors contributed to the effect of the 'hard agar'. Arabidopsis ecotypes that showed a stronger reduction in shoot size when grown on the 'hard agar' tended to have a lower fitness according to an external study which may indicate that the 'hard agar' assay simulates physiological relevant conditions.

The work highlights that transcriptional responses for simulated drought on plates and drought caused by water deficit are highly variable and dependent on the tissues that are observed. The authors demonstrate that transcriptomics can be used to select a suitable plate assay that most closely recapitulates drought through water deficit for plants grown in pots. Interestingly their newly developed 'hard agar' assay provides an alternative to traditional plate-based assays with improved directional agreement of differential expressed genes in roots in comparison to plants experiencing water deficit in vermiculite. It is promising that the impact of 'hard agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

Author response

The following is the authors’ response to the previous reviews

eLife assessment 

This work is an attempt to establish conditions that accurately and efficiently mimic a drought response in Arabidopsis grown on defined agar-solidified media - an admirable goal as a reliable experimental system is key to conducting successful low water potential experiments and would enable high-throughput genetic screening (and GWAS) to assess the impacts of environmental perturbations on various genetic backgrounds. The authors compare transcriptome patterns of plant subjected to water limitation imposed with different experimental systems. The work is valuable in that it lays out the challenges of such an endeavor and points out shortcomings of previous attempts. There was concern, however, that a purely gene expression-based approach may not provide sufficient physiologically relevant information about plant responses to drought, and therefore, despite improvements from a previous version, the new methodology championed by this work remains inadequate.   

Molecular biologists who study drought stress must make choices about which assays to use in their investigation. Serious resources and effort are put into their endeavor, and choice of assay matters. Our manuscript’s goal was largely practical: to guide molecular biologists employing transcriptomics in their choice of drought stress assay, and thus help ensure their work will discover transcriptional signatures of importance, and not those that may be an artifact from lowering water potential using chemical agents on agar plates.  

We examine how different approaches of reducing water potential impact the Arabidopsis root and shoot transcriptome. Our manuscript shows that each method of reducing water potential has a different effect on Arabidopsis root transcriptome responses. We acknowledge that drought stress induces a complex physiological response, and can vary depending on the method used. However, by comparing across assays, we find instances where a gene is downregulated by low water potential in one assay, and upregulated by low water potential in another assay. We feel it is only natural to question why this could be, and to hypothesize that it may be caused by secondary effects caused by the way low water potential is imposed.  We note that comparative transcriptomics has been a standard approach for decades. We take it as the reviewer’s opinion that it may not be insightful, but it does not factually impact our findings. 

Reviewer #2 (Public Review): 

This manuscript purports to develop a new system to study low water potential (drought) stress responses in agar plates. They make numerous problematic comparisons among transcriptome datasets, particularly to transcriptome data from a vermiculite drying experiment which they inappropriately present as representing an authentic "drought response" to the exclusion of all other data. For some reason, which the reviewer cannot fully understand, the authors seem intent on asserting the superiority of their experimental system to all others. They do not succeed in this and such an effort is ultimately a disservice to the field of drought research as a whole. 

While they devote considerable effort in comparing transcriptome data among various experimental systems, the potentially more informative experiment at the end of the manuscript of testing growth responses of a number of Arabidopsis accessions is only done for their "LW" system. The focus of this manuscript on transcriptome data to the almost complete exclusion of other types of data which is a symptom of a broader over-emphasis on transcriptome that unfortunately is quite prevalent in plant science now. It is worth reminding that for protein coding genes, which constitute the vast majority of genes, transcriptome data is a proxy measurement. The really important thing is protein amount, and even more so protein activity/function, which we know has an imperfect, at best, correlation with transcript level. We measure transcriptomes because we can, not because it is inherently the most informative thing to do. The author's quixotic quest to see if the transcriptomes of different stress treatments match is of limited value and further diminished by their misleading presentation of one particular transcriptome data set (from their vermiculite drying experiments) as somehow a special data set that everything else must be evaluated against. This study sheds no new light on how to do relevant drought (low water potential) experiments in the lab. 

Although the reviewer acknowledges that the authors have made some effort to respond to previous comments, the fundamental flaws remain and the present version of this study is little improved from the first submission. 

One challenge faced by the drought community is establishing consensus regarding the definition of drought itself. According to the criteria followed by the reviewer, any method leading to a reduction in water potential qualifies as drought stress. However, the findings presented in this manuscript demonstrate that transcriptional responses in roots vary considerably across five different methods of reducing water potential. This indicates that beyond responding to a change in water potential itself, root transcriptomes will also respond to the specific way low water potential is introduced. We believe this variability is of interest to the drought research community. 

Of the five methods we explore, we hold the view that the gene expression changes induced by vermiculite drying as the most analogous to the expression signatures Arabidopsis would exhibit in response to low water potential in the natural environment. In contrast, we posit that Arabidopsis grown on agar plates - where the root system is exposed to air and light, and where water potential is lowered using chemical agents - may contain gene expression signatures plant molecular biologists may not find particularly relevant. However, we acknowledge that this is our opinion, and will make this more explicit on our revised text. 

More broadly, we believe that the reviewer’s observation regarding the ‘over-emphasis’ on transcriptomics that is prevalent within the plant science community justifies, rather than diminishes, the work presented here. If transcriptomics is a commonly employed method, then we anticipate that the outcomes of this study will hold value for a broad audience. Such researchers are likely not only using transcriptomics as a proxy measure for protein abundance, as the reviewer suggests, but also because it is one of the more straightforward genomic techniques biologists can use to identify candidate genes that may be chosen for further scrutiny. 

Reviewer #3 (Public Review): 

Comments on revised version: 

Specific previous criticisms that were addressed are: 

(1) that gene expression changes were only compared between the highest dose of each stress assay. In the revised version, the authors changed their framework and are now using linear modelling to detect genes that display a dose response to each specific treatment. I agree that this might be a more robust approach to selecting genes that are specific to a certain treatment. 

(2) that concentrations of PEG, mannitol, NaCl, and the "low water" agar which were chosen are not comparable in regards to their specific osmotic component. I appreciate that the authors measured the osmotic potential of each treatment. It revealed that both PEG and NaCl at their highest concentration had a much more negative osmotic potential compared to the other treatment. The authors claim that using ANCOVA they did not detect any significant differences between the treatments (lines 113, 114). I do believe that ANCOVA is not the appropriate test in this case. ANCOVA has an assumption of linearity, while the dose response between concentration and osmotic potential is non-linear. This is particularly evident for PEG (Steuter AA. Water potential of aqueous polyethylene glycol. Plant Physiol. 1981 Jan;67(1):64-7. doi: 10.1104/pp.67.1.64.). Since the treatments are not the same at the highest level, I think this could have effects on the validity of comparisons by linear model. One approach could be to remove the treatment level with the highest concentration and compare the results or adjust the treatments to the same osmolarity. 

(3) that only two biological replicates were collected for RNA sequencing which makes it impossible to know how much variance exists between samples. The authors added a third replicate in the revised version for most treatments. However, some treatments still have only two replicates, which cannot be easily seen from the text or the figure. I would prefer that those differences are pointed out. 

(4) that the original manuscript did not explore what effect the increase of agar and nutrient concentration in the "low water" agar had on water potentials. The authors conducted additional experiments showing that changes in water potential were exclusively caused by changes in the nutrient concentration (Figure 2-figure supplement 5; lines 222-224). However, the increase in agar strength had also some effect on gene expression. While this is not further discussed in the text, I believe this effect of agar on gene expression could be similar to root responses to soil compaction. 

(5) That the lower volume of media in the "low water" agar could have an effect on plants. The authors compared these effects in Figure 2-figure supplement 7. They claim that "different volumes of LW agar media do not play a significant part in modulating gene expression". While I can see that they detected 313 overlapping DEGs, there were still 146 and 412 non-overlapping DEGs. The heatmap in subpanel E also shows that there were differences in particular in the up-regulated genes. My conclusion would be that the change in volume does play a role and this should be a consideration in the manuscript. 

We thank the reviewer for their suggestions. We plan to resubmit the manuscript reflecting the requested changes. Specifically, we will: 

-       We will detail more thoroughly the effects of agar volume on gene expression changes elicited by LW agar treatment. 

-       We will investigate whether the tensile stress introduced by hard agar is similar to soil compaction by an analysis with existing literature. 

-       Assess more rigorously the suitability of the ANCOVA model for assessing water potential changes of different media types.

Author response:

eLife assessment

This useful study shows how genetic variation is associated with fecundity following a period of reproductive diapause in female Drosophila. The work identifies the olfactory system as central to successful diapause with associated changes in longevity and fecundity. While the genetic screening and methods used are solid, the approach to assessing diapause is incomplete and could benefit from additional orthogonal experiments.

Response: We agree that, as with most studies, additional follow-up work will be informative.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

Response: Thank you for the suggestions. We screened 193 lines and we will add that information to the methods.

Additionally, we will add the heritability estimate of the post-diapause fecundity trait.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

Response: We agree that it is possible that the ability to endure cool temperatures per se may influence post-diapause fecundity. However, cool temperature is the essential diapause-inducing condition in Drosophila, so it is not obvious how to separate those effects experimentally, and we agree that phenotypic variation in the cool-sensitivity trait itself could be significant in regulating diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K).

Response: Our definition of successful diapause is the ability to produce viable adult progeny post-diapause, which requires that the flies enter, maintain, and exit diapause, alive and fertile. We will restate our conclusion to say that Dip-γ and sbb are required for post-diapause fecundity.

In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity.

Response: We interpret those results to mean that those RNAi lines were not effective since Dip-γ and sbb are known to be essential.

Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

Response: Ubiquitous expression of RNAi lines #27049 or #KK104056 was lethal, so we included the tubGAL80ts repressor to prevent RNAi from taking effect during development. Flies had to be shifted to 30 °C to inactivate the repressor and thereby activate the RNAi. At 30 °C, fecundity of the controls (GFP RNAi lines #9331, KK60102) were also lower (average non-diapause fecundity = 12 and 19 respectively) and similar to #27049 or #KK104056. We also assessed the knockdown using Repo GAL4 and nSyb GAL4 and did not find a significant difference/decline in the non diapause fecundity for #27049 and #KK104056 as compared to a nonspecific RNAi control (#54037).

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

We agree with the reviewer, and in Supplemental Table S3 we list top-associated SNPs in order from the lowest (most significant) p-value. Most of the top-associated genes from this analysis were uncharacterized CG numbers for which there were insufficient tools available for validation purposes. Nevertheless, there is overlap amongst the highly significant genes by p-value and those with multiple SNPs. Amongst the top 15 genes with multiple associated SNPsCG18636 & CR15280 ranked 3rd by p-value, CG7759 ranked 4th, CG42732 ranked 10th, and Drip ranked 30th (all above the conservative Bonferroni threshold of 4.8e-8) while three Sbb-associated SNPs also appear in Table 3 above the standard e-5 threshold.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

Response: The 2001 paper by Tatar et al. reports the number of eggs laid after 3, 6, or 9 weeks in diapause conditions. Thus the diapause conditions used in this study (35 days or 5 weeks) are neither short nor long, rather intermediate. Does the reviewer have a specific concern?

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

Response: We have unambiguously defined successful diapause as the ability to produce viable adult progeny post-diapause. Other groups have measured % of flies that arrest ovarian development or % of post-diapause flies with mature eggs in the ovary, or # eggs laid post-diapause; however we suggest that # of viable adult progeny produced post-diapause is more meaningful than the other measurements from the point of view of perpetuating the species.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

Response: The reviewer has a point. We carried out the lifespan study under two different conditions: either by removing the antenna and moving the flies directly to 10 °C or by removing the antenna and allowing a “wound healing” period prior to moving the flies to 10 °C (out of concern that the flies might have died quickly because wound healing may be impaired at 10 °C). In both cases, lifespan was shortened. We will add a discussion of the technical limitations of this experiment.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

Response: It is unclear to us why the reviewer has such a negative opinion of measuring post-diapause fecundity, specifically the ability to produce viable progeny post-diapause. The value of this measurement seems obvious from the point of view of perpetuating the species.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

Response: The reviewer has not explained why his/her opinion is so negative.

branch number BR015634 at the address Westbrook Centre, Milton Road, Cambridge, CB4 1YG, UK.

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

eLife assessment

This useful study shows how genetic variation is associated with fecundity following a period of reproductive diapause in female Drosophila. The work identifies the olfactory system as central to successful diapause with associated changes in longevity and fecundity. While the genetic screening and methods used are solid, the approach to assessing diapause is incomplete and could benefit from additional orthogonal experiments.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K). In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity. Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

The first highlight

I am a bit divided about whether this last screen is needed based on the chapter's narrative. On the one hand, based on my own visualisation training, I appreciate annotation on the insights.

But at the same time, it comes across as simplifying Peabody's charts to make them more intuitive to read and find insights/patterns out of. From this chapter's argument, I understand that it is the experiential/hard won process of doing that should give that insight instead.

The references from here on have some issues (seem wrong or don't appear)

Some sense of scale would be useful here. Especially as the text describes murals and textbooks (and later on workbooks).

Can this contrast be seen on the image shown on the right? Otherwise it's a bit confusing (since the visualisation starts from 1601).

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

(1) The modeling process is outlined, but an explanation of why Maxent (Phillips & Dudík, 2008) was chosen for SDMs and why the specified predictor variables were used could provide additional context. This clarity would help readers understand the rationale behind the methodology.

In L.558-571 (Predictor variables subsection), we added the explanation about predictor variables as follows:

“Predictors encompass a range of environmental variables recognized to impact species distribution (Table 3): land use (Newbold et al., 2015), climate (bioclim variables (Booth et al., 2014)), vegetation (Abe, 2018), lithology (Ott, 2020) and elevational range (Udy et al., 2021). Additionally, categorical variables representing known biogeographic regions, reflecting geological history, were included. We applied  Blakiston's Line —Tsugaru straits dividing the northern and main islands of Japan (i.e., Hokkaido and Honshu islands)— reflecting a significant historical migration barrier for mammals and birds (Dobson, 1994; Saitoh et al., 2015). Due to the distinct fauna (Wepfer et al., 2016; Yamasaki, 2017), we also specified oceanic islands (i.e. Ogasawara and Daito isles) which have never been connected with the Asiatic continents. Continuous environmental variables were transformed into linear, quadratic and hinge feature classes to illustrate nonlinear associations between environments and species occurrence (Phillips et al., 2017). The regularisation multiplier was set at 2.5, falling within the established optimal range of 1.5 to 4 (Elith et al., 2010; MorenoAmat et al., 2015).”

In L.614-618 (Modelling subsection), we explain why we chose MaxEnt:

“To model species distributions from presence-only data, several algorithms have been utilised, including generalised additive models, random forest, and neural networks (Norberg et al., 2019; Valavi et al., 2022). In our study, we opted for MaxEnt (Phillips and Dudík, 2008) due to its high estimation accuracy and relatively low computational burden (Valavi et al., 2022).”

(2) While the study outlines a manual reidentification process by experts for wild individuals, it might be beneficial to elaborate on the criteria or expertise level of these experts. This transparency ensures the reliability of the reidentification process. Reply

In L.519-523, we added description about experts as follows:

“These experts have professional backgrounds, serving as a technician at a prefectural research institute (fish), highly-experienced field survey conductors (plants and insects, respectively), a post-doctoral researchers (amphibians and reptiles, and mammals, respectively), and a museum curator (mollusks) specialising in the focal taxa.”

(3) The analysis of the effects of data type (Biome+Traditional data or Traditional survey data) on BI is comprehensive. However, a brief discussion on the potential implications of these effects on the study's overall conclusions could add depth to the interpretation.

We enforced our discussion about the causes and consequences of improved modelling accuracy. 

In L.276-282, we argued about the causes: 

“Therefore, incorporating Biome data could significantly enhance modelling accuracy in urban and suburban landscapes, which are typically underrepresented in traditional survey data. As pseudo-absences are selected based on search effort, our models utilise numerous pseudoabsences from these areas. Consequently, this might lead to better estimation of species absence in such areas, not just presence, resulting in an overall increase in model accuracy across a wider range of species.”  

In L.370-387, we argued how improved modelling accuracy may help build naturepositive society as follows:

“By blending data from traditional surveys and communities, we improved the accuracy of species distribution estimates. This enhanced estimation lays the groundwork for more precise subsequent analyses. For instance, estimated distributions will be useful in selecting new protected areas or areas with OECMs (Other Effective area-based Conservation Measures: allowing a wider range of land use as long as biodiversity and ecosystem services are sustained/improved). Using estimated distributions of each species, hotspots of species or evolutionary diverse taxa can be inferred. Such sites will be good candidates for protected areas (Jones et al., 2016) or OECMs (Shiono et al., 2021). Further, estimated distributions can be used as input for spatial conservation prioritisation tools (e.g. Marxan (Ball et al., 2009)). 

In our experience, stakeholders—including corporate social responsibility managers and conservation practitioners—often seek the list of species potentially inhabiting their locations. Due to the uncertainty of SDMs and their thresholding into presence/absence, on-site surveys remain essential for assessing biodiversity status. SDMs can make such surveys costeffective by screening important locations for on-site assessment (e.g., Locate phase in TNFD framework) and narrowing down the target species for surveying. Improved estimation through SDMs can mitigate risks associated with their use in society and enable more informed decisionmaking for conservation efforts.”

Following the editorial policy, we have reorganised our supplementary materials as follows:

-        Formerly Supplementary File 1 - Remains unchanged.

-        Formerly Supplementary File 2 - Transferred into the main text, in the subsection "Filtering suspicious occurrence record in Biome data" in the Methods section, and Table 2. Citations remain as Supplementary File 2.

-        Formerly Supplementary File 3 - Remains unchanged.

-        Formerly Supplementary File 4 - Transferred into "Figure 3—figure supplement 1".

-        Formerly Supplementary File 5 - Transferred into Figure 4.

-        Formerly Supplementary File 6 - Transferred into the main text, in the subsection "Predictor variables" in the Methods section and Table 3.

-        Formerly Supplementary File 7 - Transferred into the main text, in the subsection "Pseudo-absence reflecting search effort" in the Methods section and Figure 5.

-        Formerly Supplementary File 8 - Transferred into the main text, in the subsection "Model evaluation" in the Methods section and Figure 6.

-        Formerly Supplementary File 9 - Renamed as Supplementary File 4.

eLife assessment

This important study presents findings of great practical value, offering fresh insights into natural species distributions across Japan. By combining multiple data sources (including those from non-academic sectors, aka citizen scientists), the manuscript also presents a compelling new tool that can be used to aid conservation agendas, detect species distribution changes, and testing of ecological theories.

Reviewer #1 (Public Review):

Summary:

The study presented by Atsumi et al. is about using smartphone-driven, community-sourced data to enhance biodiversity monitoring. The idea is to leverage the widespread use of smartphones to gather data from the community quickly, contributing to a more comprehensive understanding of biodiversity. The authors discuss the importance of ecosystem services linked to biodiversity and the threats posed by human activities. It emphasizes the need for comprehensive biodiversity data to implement the Kunming-Montreal Global Biodiversity Framework. The 'Biome' mobile app, launched in Japan, uses species identification algorithms and gamification to gather over 6 million observations since 2019. While community-sourced data may have biases, incorporating it into Species Distribution Models (SDMs) improves accuracy, especially for endangered species. The app covers urban-natural gradients uniformly, enhancing traditional survey data biased towards natural areas. Combining these sources provides valuable insights into species distributions for conservation, protected area designation, and ecosystem service assessment.

Strengths:

The use of a smartphone app ('Biome') for community-driven species occurrence data collection represents an innovative and inclusive approach to biodiversity monitoring, leveraging the widespread use of smartphones. The app has successfully accumulated a large volume of species occurrence data since its launch in 2019, showcasing its effectiveness in rapidly gathering information from diverse locations. Despite challenges with certain taxa, the study highlights high species identification accuracy, especially for birds, reptiles, mammals, and amphibians, making the 'Biome' app a reliable tool for species observation. The integration of community-sourced data into Species Distribution Models (SDMs) improves the accuracy of predicting species distributions. This has implications for conservation planning, including the designation of protected areas and assessment of ecosystem services. The rapid accumulation of data and advancements in machine learning methods open up opportunities for conducting time-series analyses, contributing to the understanding of ecosystem stability and interaction strength over time. The study emphasizes the collaborative nature of the platform, fostering collaboration among diverse stakeholders, including local communities, private companies, and government agencies. This inclusive approach is essential for effective biodiversity assessment and decision-making. The platform's engagement with various stakeholders, including local communities, supports biodiversity assessment, management planning, and informed decision-making. Additionally, the app's role in fostering nature-positive awareness in society is highlighted as a significant contribution to creating a sustainable society.

Weaknesses:

While the studies make significant contributions to biodiversity monitoring, they also have some weaknesses. Firstly, relying on smartphone-driven, community-sourced data may introduce spatial and taxonomic biases. The 'Biome' app, for example, showed lower accuracy for certain taxa like seed plants, molluscs, and fishes, potentially impacting the reliability of the gathered data. Furthermore, the effectiveness of Species Distribution Models (SDMs) relies on the assumption that biases in community-sourced data can be adequately accounted for. The unique distribution patterns of the 'Biome' data, covering urban-natural gradients uniformly, might not fully represent the diversity of certain ecosystems, potentially leading to inaccuracies in the models. Moreover, the divergence in data distribution patterns along environmental gradients between 'Biome' data and traditional survey data raises concerns. The app data shows a more uniform distribution across natural-urban gradients, while traditional data is biased towards natural areas. This discrepancy may impact the representation of certain ecosystems and influence the accuracy of Species Distribution Models (SDMs). While the integration of 'Biome' data into SDMs improves accuracy, the study notes that controlling the sampling efforts is crucial. Spatially-biased sampling efforts in community-sourced data need careful consideration, and efforts to control biases are essential for reliable predictions.

eLife assessment

The paper illustrates a valuable approach to generating TCR transgenic mice specific for known epitopes. There is some solid evidence for the efficacy of this approach, although only limited evidence is provided that the TCR clone in question successfully recapitulates the functional features of the endogenous response to the same antigen, and the claim that this method is superior to more traditional clone selection methods is incompletely substantiated by the data presented.

Reviewer #1 (Public Review):

Summary:

Debeuf et al. introduce a new, fast method for the selection of suitable T cell clones to generate TCR transgenic mice, a method claimed to outperform traditional hybridoma-based approaches. Clone selection is based on the assessment of the expansion and phenotype of cells specific for a known epitope following immune stimulation. The analysis is facilitated by a new software tool for TCR repertoire and function analysis termed DALI. This work also introduces a potentially invaluable TCR transgenic mouse line specific for SARS-CoV-2.

Strengths:

The newly introduced method proved successful in the quick generation of a TCR transgenic mouse line. Clone selection is based on more comprehensive phenotypical information than traditional methods, providing the opportunity for a more rational T cell clone selection.

The study provides a software tool for TCR repertoire analysis and its linkage with function.

The findings entail general practical implications in the preclinical study of a potentially very broad range of infectious diseases or vaccination.

A novel SARS-CoV-2 spike-specific TCR transgenic mouse line was generated.

Weaknesses:

The authors attempt to compare their novel method with a more conventional approach to developing TCR transgenic mice. In this reviewer's opinion, this comparison appears imperfect in several ways:

• Work presenting the "traditional" method was inadequate to justify the selection of a suitable clone. It is therefore not surprising that it yielded negative results. More evidence would have been necessary to select clone 47 for further development of the TCR transgenic line, especially considering the significant time and investment required to create such a line.

• The comparison is somewhat unfair, because the methods start at different points: while the traditional method was attempted using a pool of peptides whose immunogenicity does not appear to have been established, the new method starts by utilising tetramers to select T cells specific for a well-established epitope.

• Given the costs and time involved, only a single clone could be tested for either method, intrinsically making a proper comparison unfeasible. Even for their new method, the authors' ability to demonstrate that the selected clone is ideal is limited unless they made different clones with varying profiles to show that a particular profile was superior to others.

In my view, there was no absolute need to compare this method with existing ones, as the proposed method holds intrinsic value.

While having more data to decide on clone selection is certainly beneficial, given the additional cost, it remains unclear whether knowing the expression profiles of different proteins in Figure 2 aids in selecting a candidate. Is a cell expressing more CD69 preferable to a cell expressing less of this marker? Would either have been effective? Are there any transcriptional differences between clonotype 1 and 2 (red colour in Figure 2G) that justify selecting clone 1, or was the decision to select the latter merely based on their different frequency? If all major clones (i.e. by clonotype count) present similar expression profiles, would it have been necessary to know much more about their expression profiles? Would TCR sequencing and an enumeration of clones have sufficed, and been a more cost-effective approach?

Lastly, it appears that several of the experiments presented were conducted only once. This information should have been explicitly stated in the figure legends.

Reviewer #2 (Public Review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning, and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that are already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers, and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity, and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in a variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.

eLife assessment

This manuscript uses public datasets of myelodysplastic syndrome (MDS) patients to undertake a multi-omics analysis of clinical, genomic, and transcriptomic datasets. Useful findings are provided by way of interesting correlations of specific mutations with inflammation and differing clinical outcomes. While the evidence is extensive and interesting, it remains incomplete in the absence of pipeline validation and addressing the potential confounding factors present in the datasets used. When these issues are addressed, this will be of substantive value to hematologists and clinical immunologists.

Reviewer #1 (Public Review):

In their manuscript, Gerlevik et al. performed an integrative analysis of clinical, genetic and transcriptomic data to identify MDS subgroups with distinct outcomes. The study was based on the building of an "immunoscore" and then combined with genotype and clinical data to analyze patient outcomes using multi-omics factor analysis.

Strengths: Integrative analysis of RNA-seq, genotyping and clinical data

Weaknesses: Validation of the bioinformatic pipeline is incomplete

Major comments:

(1) This study considered two RNA-seq data sets publicly available and generated in two distinct laboratories. Are they comparable in terms of RNA-seq technique: polyA versus rRNA depletion, paired-end sequencing, fragment length?

(2) Data quality control (figure 1): the authors must show in a graph whether the features (dimensions) of factor 1 were available for each BMMNC and CD34+ samples.

(3) How to validate the importance of "immunoscore"? If GSEA of RNA-seq data was performed in the entire cohort, in the SF3B1-mutated samples or SRSF2-mutated samples (instead of patients having a high versus low level of factor 1 shown in Sup Fig. 4), what would be the ranking of Hallmarks or Reactome inflammatory terms among the others?

(4) To decipher cell-type composition of BMMNC and CD34+ samples, the authors used van Galen's data (2019; supplementary table 3). Cell composition is expressed as the proportion of each cell population among the others. Surprisingly, the authors found that the promonocyte-like score was increased in SF3B1-mutated samples and not in SRSF2-mutated samples, which are frequently co-mutated with TET2 and associated with a CMML-like phenotype. Is there a risk of bias if bone marrow subpopulations such as megakaryocytic-erythroid progenitors or early erythroid precursors are not considered?

(5) Figures 2a and 2b indicated that the nature of retrotransposons identified in BMMNC and CD34+ was different. ERVs were not detected in CD34+ cells. Are ERVs not reactivated in CD34+ cells? Is there a bias in the sequencing or bioinformatic method?

(6) What is the impact of factor 1 on survival? Is it different between BMMNC and CD34+ cells considering the distinct composition of factor 1 in CD34+ and BMMNC?

(7) In Figure 1e, genotype contributed to the variance of in the CD34+ cell analyses more importantly than in the BMMNC. Because the patients are different in the two cohorts, differences in the variance could be explained either by a greater variability of the type of mutations in CD34 or an increased frequency of poor prognosis mutations in CD34+ compared to BMMNC. The genotyping data must be shown.

(8) Fig. 2a-b: Features with high weight are shown for each factor. For factor 9, features seemed to have a low weight (Fig. 1b and 1c). However, factor 9 was predictive of EFS and OS in the BMMNC cohort. What are the features driving the prognostic value of factor 9?

(9) The authors also provided microarray analyses of CD34+ cell. It could be interesting to test more broadly the correlation between features identified by RNA-seq or microarrays.

(10) The authors should discuss the relevance of immunosenescence features in the context of SRSF2 mutation and extend the discussion to the interest of their pipeline for patient diagnosis and follow up under treatments.

Reviewer #2 (Public Review):

The authors performed a Multi-Omics Factor Analysis (MOFA) on analysis of two published MDS patient cohorts-1 from bone marrow mononuclear cells (BMMNCs) and CD34 cells (ref 17) and another from CD34+ cells (ref 15) --with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, Retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to attempt to identify the latent factors with significant impact on MDS prognosis.

SF3B1 was found to be the only mutation among 13 mutations in the BMMNC cohort that indicated a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34+ cohort. The MOFA factor representing inflammation showed a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases showed a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Also, MOFA identified RTE expression as a risk factor for MDS. They proposed that this work showed the efficacy of their integrative approach to assess MDS prognostic risk that 'goes beyond all the scoring systems described thus far for MDS'.

Several issues need clarification and response:

(1) The authors do not provide adequate known clinical and molecular information which demonstrates prognostic risk of their sample cohorts in order to determine whether their data and approach 'goes 'beyond all the scoring systems described thus far for MDS'. For example, what data have the authors that their features provide prognostic data independent of the prior known factors related to prognosis (eg, marrow blasts, mutational, cytogenetic features, ring sideroblasts, IPSS-R, IPSS-M, MDA-SS)?

(2) A major issue in analyzing this paper relates to the specific patient composition from whom the samples and data were obtained. The cells from the Shiozawa paper (ref 17) is comprised of a substantial number of CMML patients. Thus, what evidence have the authors that much of the data from the BMMNCs from these patients and mutant SRSF2 related predominantly to their monocytic differentiation state?

(3) In addition, as the majority of patients in the Shiozawa paper have ring sideroblasts (n=59), thus potentially skewing the data toward consideration mainly of these patients, for whom better outcomes are well known.

(4) Further, regarding this patient subset, what evidence have the authors that the importance of the SF3B1 mutation was merely related to the preponderance of sideroblastic patients from whom the samples were analyzed?

(5) An Erratum was reported for the Shiozawa paper (Shiozawa Y, Malcovati L, Gallì A, et al. Gene expression and risk of leukemic transformation in myelodysplasia. Blood. 2018 Aug 23;132(8):869-875. doi: 10.1182/blood-2018-07-863134) that resulted from a coding error in the construction of the logistic regression model for subgroup prediction based on the gene expression profiles of BMMNCs. This coding error was identified after the publication of the article. The authors should indicate the effect this error may have had on the data they now report.

(6) What information have the authors as to whether the differing RTE findings were not predominantly related to the differentiation state of the cell population analyzed (ie higher in BM MNCs vs CD34, Fig 1)? What control data have the authors regarding these values from normal (non-malignant) cell populations?

(7) The statement in the Discussion regarding the effects of SRSF2 mutation is speculative and should be avoided. Many other somatic gene mutations have known stronger effects on prognosis for MDS.

eLife assessment

This useful study draws on published single-cell and spatial transcriptomic data of colon cancer liver metastasis to clarify the pro- and anti-tumorigenic properties of NK cells. The authors discover increased GZMK+ resting NK cells in the tumor tissue and reduced abundance of KIR2DL4+ activated NK cells. However, the evidence is currently incomplete, as the models used to validate the hypothesis and claims are inadequate and lack important controls.

Reviewer #1 (Public Review):

Summary:

Mao and colleagues re-analysed published spatial, bulk and single-cell transcriptomic datasets from primary colorectal cancers and colorectal-cancer-derived liver metastases. The analyses of paired cancer and non-cancer tissue samples showed that T cells are enriched in tumour tissue, accompanied by a reduction in the fraction of NK cells in the cancer tissue transcriptional datasets. Furthermore, authors claim that tumour tissue has a higher fraction of GZMK+ (resting) NK cells and suggest a correlation between the presence of these cells and poorer prognosis for cancer patients. In contrast, the increased frequency of KIR2DL4+ (activated) NK cells correlates with improved survival of cancer patients.

Strengths:

The authors performed a comprehensive analysis of published datasets, integrating spatial and single-cell transcriptomic data, which allowed them to discover the enrichment of GZMK+ NK cells in cancer tissues.

Weaknesses:

Despite their thorough analysis, the authors did not provide sufficient experimental evidence to support their claim that GZMK+ NK cells contribute to a worse prognosis for cancer patients or promote cancer progression. The terms resting and activated NK cells are used without properly defining the characteristics of these populations other than the gene expression of a handful of genes. Furthermore, the criteria used to quantify the NK cell population in spatial data is not entirely clear. While one can visually observe an increased fraction of GZMK+ NK cells compared to KIR2DL4+ NK cells in cancer tissues, no quantification is shown. They did not present any preclinical (animal model) or clinical data suggesting a causal relationship between NK cells and tumour growth. Thus, while a correlation may exist between the presence of GZMK+ NK cells and poorer tumour prognosis, causation cannot be claimed based on the available evidence. Furthermore, the in vitro data provided is limited to a single NK cell line derived from a lymphoma patient, which does not fully represent the diversity and functionality of human NK cells. Moreover, the in vitro experiments suffer from a lack of required controls and inadequate methodology.

Reviewer #2 (Public Review):

Summary:

This manuscript investigates the role of the abundant NK cells that are observed in colon cancer liver metastasis using sequencing and spatial approaches in an effort to clarify the pro and anti-tumourigenic properties of NK cells. This descriptive study characterises different categories of NK cells in tumour and tumour-adjacent tissues and some correlations. An attempt has been made using pseudotime trajectory analysis but no models around how these NK cells might be regulated are provided.

Strengths:

This study integrates multiomics data to attempt to resolve correlates of protection that might be useful in understanding NK cell diversity and activation.

Weaknesses:

While this work is interesting, the power of such studies is in taking the discovered information and applying this to other cohorts to determine the strength and predictive power of the genes identified. It is also clear that these 'snapshots' analysed poorly take account of the dynamic temporal changes that occur within a tumour. It would have been good to see a proposed model of NK cell regulation as it might occur in the tumour (accounting for turnover and recruitment) beyond the static data.

for - progress - progress trap

Could you please precise the total cost definition, especially the CAPEX annualization factor

eLife assessment

The authors develop a self-returning self-avoiding polymer model of chromosome organization and show that their framework can recapitulate at the same time local density and large-scale contact structural properties observed experimentally by various technologies. The presented theoretical framework and the results are valuable for the community of modelers working on 3D genomics. The work provides solid evidence that such a framework can be used, is reliable in describing chromatin organization at multiple scales, and could represent an interesting alternative to standard molecular dynamics simulations of chromatin polymer models.

Reviewer #1 (Public Review):

Carignano et al propose an extension of the self-returning random walk (SRRW) model for chromatin to include excluded volume aspects and use it to investigate generic local and global properties of the chromosome 3D organization inside eukaryotic nuclei. In particular, they focus on chromatin volumic density, contact probability, and domain size and suggest that their framework can recapitulate several experimental observations and predict the effect of some perturbations.

Strengths:

• The developed methodology is convincing and may offer an alternative - less computationally demanding - framework to investigate the single-cell and population structural properties of 3D genome organization at multiple scales.

• Compared to the previous SRRW model, it allows for investigation of the role of excluded volume locally.

• They perform some experiments to compare with model predictions and show consistency between the two.

Weaknesses:

• The model is a homopolymer model and currently cannot fully account for specific mechanisms that may shape the heterogeneous, complex organization of chromosomes (TAD at specific positions, A/B compartmentalization, promoter-enhancer loops, etc.).

• By construction of their framework, the effect of excluded volume is only local and larger-scale properties for which excluded volume could be a main actor (formation of chromosome territories [Rosa & Everaers, PLoS CB 2009], bottle-brush effects due to loop extrusion [Polovnikov et al, PRX 2023], etc.) cannot be captured.

• Apart from being a computationally interesting approach to generating realistic 3D chromosome organization, the method offers fewer possibilities than standard polymer models (eg, MD simulations) of chromatin (no dynamics, no specific mechanisms, etc.) with likely the same predictive power under the same hypotheses. In particular, authors often claim the superiority of their approach to describing the local chromatin compaction compared to previous polymer models without showing it or citing any relevant references that would show it.

• Comparisons with experiments are solid but are not quantified.

Impact:

Building on the presented framework in the future to incorporate TAD and compartments may offer an interesting model to study the single-cell heterogeneity of chromatin organization. But currently, in this reviewer's opinion, standard polymer modeling frameworks may offer more possibilities.

Reviewer #2 (Public Review):

Summary:

The authors introduce a simple Self Returning Excluded Volume (SR-EV) model to investigate the 3D organization of chromatin. This is a random walk with a probability to self-return accounting for the excluded volume effects. The authors use this method to study the statistical properties of chromatin organization in 3D. They compute contact probabilities, 3D distances, and packing properties of chromatin and compare them with a set of experimental data.

Strengths:

(1) Typically, to generate a polymer with excluded volume interactions, one needs to run long simulations with computationally expensive repulsive potentials like the Weeks-Chanlder-Anderson potential. However, here, instead of performing long simulations, the authors have devised a method where they can grow polymer, enabling quick generation of configurations.

(2) Authors show that the chromatin configurations generated from their models do satisfy many of the experimentally known statistical properties of chromatin. Contact probability scalings and packing properties are comparable with Chromatin Scanning Transmission Electron Microscopy (ChromSTEM)  experimental data from some of the cell types.

Weaknesses:

This can only generate broad statistical distributions. This method cannot generate sequence-dependent effects, specific TAD structures, or compartments without a prior model for the folding parameter alpha. It cannot generate a 3D distance between specific sets of genes. This is an interesting soft-matter physics study. However, the output is only as good as the alpha value one provides as input.

eLife assessment

This potentially useful study introduces an orthogonal approach for detecting RNA modification, without chemical modification of RNA, which often results in RNA degradation and therefore loss of RNA molecules. The approach might be of particular interest for sites where modifications are rare. However, the false positive and false negative rates are currently unclear, leaving the evidence for broad applicability of the method incomplete.

Reviewer #1 (Public Review):

The detection sensitivity and accuracy are unclear.

In this manuscript, Zhou et al describe a deaminase and reader protein-assisted RNA m5C sequencing method. The general strategy is similar to DART-seq for m6A sequencing, but the difference is that in DART-seq, m6A sites are always followed by C which can be deaminated by fused APOBEC1 to provide a high resolution of m6A sites, while in the case of m5C, no such obvious conserved motifs for m5C sites exist, therefore, the detection resolution is much lower. In addition, the authors used two known m5C binding proteins ALYREF and YBX1 to guide the fused deaminases, but it is not clear whether these two binding proteins can bind most m5C sites and compete with other m5C binding proteins.

It is well known that two highly modified m5C sites exist in 28S RNA and many m5C sites exist in tRNA, the authors should validate their methods first by detecting these known m5C sites and evaluate the possible false positives in rRNA and tRNA. In mRNA, it is not clear what is the overlap between the technical replicates. In Figures 4A and 4C, they detected more than 10K m5C sites, and most of them did not overlap with sites uncovered by other methods. These numbers are much larger than expected and possibly most of them are false positives. Besides, it is not clear what is the detection sensitivity and accuracy since the method is neither single base resolution nor quantitative. There are no experiments to show that the detected m5C sites are responsive to the writer proteins such as NSUN2 and NSUN6, and the determination of the motifs of these writer proteins.

Reviewer #2 (Public Review):

The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications for the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods - like DRAM-seq, that allow the mapping of m5C independently of ex-situ RNA treatment with chemicals - are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question of whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

Comments:

(1) The use of two m5C reader proteins is likely a reason for the high number of edits introduced by the DRAM-Seq method. Both ALYREF and YBX1 are ubiquitous proteins with multiple roles in RNA metabolism including splicing and mRNA export. It is reasonable to assume that both ALYREF and YBX1 bind to many mRNAs that do not contain m5C.

To substantiate the author's claim that ALYREF or YBX1 binds m5C-modified RNAs to an extent that would allow distinguishing its binding to non-modified RNAs from binding to m5C-modified RNAs, it would be recommended to provide data on the affinity of these, supposedly proven, m5C readers to non-modified versus m5C-modified RNAs. To do so, this reviewer suggests performing experiments as described in Slama et al., 2020 (doi: 10.1016/j.ymeth.2018.10.020). However, using dot blots like in so many published studies to show modification of a specific antibody or protein binding, is insufficient as an argument because no antibody, nor protein, encounters nanograms to micrograms of a specific RNA identity in a cell. This issue remains a major caveat in all studies using so-called RNA modification reader proteins as bait for detecting RNA modifications in epitranscriptomics research. It becomes a pertinent problem if used as a platform for base editing similar to the work presented in this manuscript.

(2) Since the authors use a system that results in transient overexpression of base editor fusion proteins, they might introduce advantageous binding of these proteins to RNAs. It is unclear, which promotor is driving construct expression but it stands to reason that part of the data is based on artifacts caused by overexpression. Could the authors attempt testing whether manipulating expression levels of these fusion proteins results in different editing levels at the same RNA substrate?

(3) Using sodium arsenite treatment of cells as a means to change the m5C status of transcripts through the downregulation of the two major m5C writer proteins NSUN2 and NSUN6 is problematic and the conclusions from these experiments are not warranted. Sodium arsenite is a chemical that poisons every protein containing thiol groups. Not only do NSUN proteins contain cysteines but also the base editor fusion proteins. Arsenite will inactivate these proteins, hence the editing frequency will drop, as observed in the experiments shown in Figure 5, which the authors explain with fewer m5C sites to be detected by the fusion proteins.

(4) The authors should move high-confidence editing site data contained in Supplementary Tables 2 and 3 into one of the main Figures to substantiate what is discussed in Figure 4A. However, the data needs to be visualized in another way than an Excel format. Furthermore, Supplementary Table 2 does not contain a description of the columns, while Supplementary Table 3 contains a single row with letters and numbers.

(5) The authors state that "plotting the distribution of DRAM-seq editing sites in mRNA segments (5'UTR, CDS, and 3'UTR) highlighted a significant enrichment near the initiation codon (Figure 3F).", which is not true when this reviewer looks at Figure 3F.

(6) The authors state that "In contrast, cells expressing the deaminase exhibited a distinct distribution pattern of editing sites, characterized by a prevalence throughout the 5'UTR.", which is not true when this reviewer looks at Figure 3F.

(7) The authors claim in the final conclusion: "In summary, we developed a novel deaminase and reader protein assisted RNA m5C methylation approach...", which is not what the method entails. The authors deaminate As or Us close to 5mC sites based on the binding of a deaminase-containing protein.

(8) The authors claim that "The data supporting the findings of this study are available within the article and its Supplementary Information." However, no single accession number for the deposited sequencing data can be found in the text or the supplementary data. Without the primary data, none of the claims can be verified.

eLife assessment

In this manuscript, the authors describe a new AlphaFold2 pipeline called PabFold that can represent a useful tool for identifying linear antibody epitopes (B-cell epitopes) for different antigens. This information can be used in the selection of different reagents in competitive ELISA assays which can save time and reduce costs. Several questions, however, remain and the study is currently incomplete.

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Reviewer #2 (Public Review):

Summary:

The authors showed the applicability and usefulness of a new AlphaFold2 pipeline called PabFold, which can predict linear antibody epitopes (B-cell epitopes) that can be helpful for the selection of reagents to be applied in competitive ELISA assay.

Strengths:

The authors showed the accuracy of the pipeline to identify correctly the binding epitope for three different antibody-antigen systems (Myc, HA, and Sars-Cov2 nucleocapsid protein). The design of scFvs from Fab of the three antibodies to speed up the analysis time is extremely interesting.

Weaknesses:

The article justifies correctly the findings and no great weaknesses are present. However, it could be useful for a broader audience to show in detail how pLDDT was calculated for both Simple-Max approach (per residue-pLDDT) and Consensus analysis ( average pLDDT for each peptide), with associated equations.

远离Bug,可读性高，易扩展，易维护。

JLKHG