Dear Dr Demontis,

Thank you very much for submitting your Research Article entitled 'A global transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila.' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Hongyan Wang, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript by Graca et al., titled A global transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila does most of what is suggested by the title. The authors do a fairly large and systematic RNAi screen with the goal of identifying novel transcription factors that regulate muscle growth to better understand the mechanisms that underly muscle atrophy and hypertrophy. In the opinion of this reviewer, the manuscript is exceptionally well written, provides clear and easy to interpret figures, and puts forth a mechanisms by which Glycolysis is regulated for the purpose of muscle growth. Although there are some revisions that are necessary to make this manuscript suitable for publication they are relatively minor in scope.

Questions and Comments

1. I personally would not use the word “global” in the title. As stated, 447 out of 708 transcription factors were investigated. Global to me suggests that all transcription factors were examined. I’d recommend “large-scale” or “systematic”

2. Figure 1 and the associated methods suggest that larvae were just categorized as large, small, mishapen, or normal. Additional details indicating the threshold for variation from control necessary to categorize a larva would be helpful, particularly for readers who may try to follow up on this work.

3. The methods associated with Figure 1 list the control as “such as white RNAi”. If multiple different controls were used they should all be listed along with the rationale for using each.

4. Figure 2 represents my biggest and most important questions regarding this manuscript.

4A. It seems that only a subset of the hits from the screen were examined in figure 2. I certainly recognize that completing this analysis of a large number of different conditions might be prohibitive. However, I think it is essential that the authors provide a rationale for choosing the hits they follow up on and ignoring the hits that they did not. If in fact all of the hits were examined, it should be stated explicitly and data for all should be provided in the supplement at the very least.

4B. The hemisegment chosen for muscle size analysis will impact the data. Therefore it is critical to know which hemisegments were used. Additionally, and perhaps more importantly, the hemisegments near the anterior and posterior end of the animal are significantly smaller and more variable than the central hemisegments. Based on figure 2A it seems that a subset of muscles from these hemisegments may have been used. The authors should remove these data points and rely on the data from central hemisegments.

5. I hesitate to suggest this because it has the feel of making the paper what I would like it to be rather than what it is. Nevertheless, figure 5 just seems an anticlimactic way to end a very nice manuscript. It is suggestive, along with the transcriptomic data in figure 4, that Deaf1 works through regulation of glycolysis to regulate muscle growth. But, we already knew that disruption of glycolysis would impact muscle size. If the glycolysis disruption experiments could be done in the background of altered Deaf1 and/or GSK3 levels, it would be a more fitting conclusion to this manuscript. Even selecting just two glycolytic enzymes to do in this background would be a much more convincing way to show that glycolysis works downstream of Deaf1 and GSK3 to regulate muscle growth.

Minor Comments

1. Citations vary between author, year and numbers. I presume this will be fixed and is not a concern, rather something I noticed.

2. At the bottom of page 6, the authors cite two papers as evidence that fusion is not responsible for the variations in muscle size. I do believe that this is not a fusion effect, but I do not believe that these specific disruptions were looked at in those papers. If I am correct, the authors should be clear and precise in their language.

3. Regarding the statistical analysis, the authors state that all data points indicate a biological replicate. I think that this is vague and means different things to different researchers. For example, in figure 2 one might assume that each point refers to a different mating of males and virgins whereas another might think it means a different animal from a single mating. A more precise description will be helpful in understanding the variability of the data.

4. The last sentence of the first paragraph on page 4 suggests that the sickle shape is indicative of defective contractile properties. Without something to reference, or data to support this conclusion, the authors should just report their finding.

Reviewer #2: The manuscript by Graca et al. reports the results of a straightforward RNAi screen in Drosophila designed to uncover roles for conserved transcription factors in muscle size control. The authors employed 1114 RNAi lines that targeted 447 transcription factors that showed some similarity to human transcription factors, for knockdown in developing muscles and then noted size defects at larval and adult stages as well as some wing positioning defects that have been noted to result from muscle abnormalities. In all about 12% of the targeted genes generated a phenotype consistent with and effect on muscle growth. The authors then performed followed up proof of principle studies on DEAf1 a conserved factor that was not known to affect muscle growth. Loss of Deaf1 was found to increase muscle size while overexpression led to smaller muscles. These phenotypes where opposite to those exhibited by manipulation of GSK3, a kinase that has been shown in mammal system to associate with and phosphorylate DEAf1. Interestingly transcriptome analysis of Deaf1 verses GSK3 knockdown and overexpression n lead to a strikingly similar regulation profile for the core genes involved in glycolysis consistent with pervious observations that inhibition of normal glycolytic flux leads to smaller muscle size.

Overall, this is a short but informative communication that will provide a useful resource to those that study muscle size control in both Drosophila and mammals. In general, the experimental design and outcomes are well described and the results easy to follow. That said I think there are a few points that the authors should address before publication.

1) There should be some discussion of the limitations of the screen. Obviously, there will be some false positives as well as false negatives. It is a bit hard to know what these limits are but one criterion for a true hit might be that more than one RNAi targeting a given gene gives a similar phenotype. By my count there are ~34 genes in supplement table 1 that satisfy this criterion. I think it would be useful to list these genes in a separate table within the main results and provide some limited discussion about these hits such has home many of these have been previously linked to muscle development. Likewise, it is worth pointing out that those genes with only one identified RNAI hit might simply be off target effects. Using this same data set the authors should also take the opportunity to provide an example of another type of possible false positive. I note that trachealess might be one such gene. It satisfies the two-hit criterion, but it is a big surprise, since as far as I am aware, there is no known role for trachealess in muscle development. Rather it seems to be specific for regulating genes involved in the formation of the insect oxygen delivery system. Obviously, if the mef-2 Gal4 driver has some off target expression at some stage in trachea, then it could explain the small larval size phenotype. This brings up my main concern with this study which is that only one “muscle-specific” driver has been used (understandable to keep the work effort down). However, one imposed limitation of this methodology is that the results are only interpretable if the driver expression is tight with respect to the targeted tissue. Mef-2 is pretty good, but there is a big problem with some versions of this driver which is that it is also strongly expressed in the ipc neurons (at least in late third instar larvae) that produce and release several key insulin-like peptides. It is incumbent on the authors to explicitly state which mef2 driver line they are using and confirm by crossing to a UAS reporter that it is indeed muscle specific and does not show expression in other tissues especially the IPCs. Without such confirmation, using mef-2 as the sole driver in the screen could produce false positives by alterations in insulin signaling which is a central regulator of body size through several mechanisms. Obviously, rescreening all hits with an ipc “specific driver” to rule out possible effects on insulin signaling is a large amount of work. However, if the authors Mef2 Gal4 line is expressed in IPCs, then perhaps they could just rescreen the 34 genes that satisfy the more than one RNAi hit criteria described above to determine if any of the phenotypes in this class might be caused by the off target Mef-2 expression.

2) A second issue is that the Deaf and GSK3 work leads to suggesting that they work in the same pathway but in opposite ways to modulate glycolysis. One simple test to determine their order of action is genetic epitasis. If GSK3’s effect on muscle size and glycolytic enzyme expression is primarily through altering Deaf 1 activity, then muscle directed overexpression of GSK3CA together with Deaf1 RNAi should produce a Deaf1 phenotype (higher levels of glycolytic enzyme expression and larger muscle size. Have the authors tried such an experiment?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Dear Dr Demontis,

Thank you very much for submitting your Research Article entitled 'A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila.' to PLOS Genetics.

The revised manuscript was fully evaluated at the editorial level and by independent peer reviewers. Both reviewers are generally satisfied with the revision, but reviewer 1 has raised additional minor comments on the manuscript.

We therefore ask you to modify the manuscript according to  review 1's recommendations. 

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Hongyan Wang, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised manuscript by Graca et al. addressed my questions and is suitable for publication with the potential of minor modifications to address one question I have.

In the last full paragraph of page 4, the authors compare the data from the Mef2 based RNAi screen to the MhcK based RNAi screen. Specifically, the similarities and the differences are highlighted. However, I question whether strict reliance on the statistical measures is appropriate here. For example, CG6724 is said to reduce larval size in Mef2 but not MhcK. But, the MhcK looks very similar to the Mef2. I do understand that the MhcK data can't be compared to the Mef2 control, but I think that this could be added to the new discussion section on the limitations of the screen. Similarly, Pc and trachealess are listed as "marginally impacted". If the authors are going to stick strictly to statistical measures, they should do so in this case also and list those RNAi lines as having no effect. Finally, the variation in the pdm3 phenotype could also be a point of discussion.

Reviewer #2: The authors have done a very through job of addressing my concerns. I think it is a nice study that will be of interest to many in the muscle biology field.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Dear Dr Demontis,

We are pleased to inform you that your manuscript entitled "A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila." has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Hongyan Wang, Ph.D.

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-00604R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.