Dear Dr Galli,

Thank you for submitting your manuscript entitled "Endomitosis controls tissue-specific gene expression during development" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your revised manuscript back to the previous Review Commons reviewers.

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Kind regards,

Richard

Richard Hodge, PhD

Associate Editor, PLOS Biology

rhodge@plos.org

PLOS

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Dear Dr Galli,

Thank you for submitting a a revised version of your manuscript "Endomitosis controls tissue-specific gene expression during development" for consideration as a Research Article at PLOS Biology. Please accept my sincere apologies for the delays that you have experienced during the peer review process. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by the three original reviewers from Review Commons.

The reviews are attached below. As you can see, the reviewers are generally positive about the revised manuscript. However, Reviewer #2 raises concerns with the auxin-inducible KNL-1 depletion line used to block binucleation in the GFP reporter assays. The reviewer suggests that binucleation is more directly blocked using a CDK-1 depletion line instead. In addition, Reviewer #3 asks for some additional discussions and contextualization of the model presented in the manuscript text.

After discussions with the academic editor, we strongly encourage you to include the auxin-inducible CDK-1 depletion experiment in the revised manuscript, as this would strengthen the accuracy of the conclusions presented. However, we could consider a discussion of the limitations of using the KNL-1 depletion line in the manuscript text if the experiment will take a long time to set up and complete.

In light of the reviews, we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

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https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Richard

Richard Hodge, PhD

Associate Editor, PLOS Biology

rhodge@plos.org

*****************************************************

REVIEWS:

Reviewer #1: Authors have addressed all my original concerns and should be commended on their rigorous study to elucidate the physiology of binucleation in the C.elegans intestinal cells. The manuscript reads well and clearly demonstrates the transcriptional adaption of binucleation and its impact of worm fecundity. This is the first study to date to clearly show in vivo that a cell with multiple nuclei enhances its gene expression compared to a mononucleated cell with equal ploidy.

Reviewer #2: In our original review for van Rijnberk et al, we expressed enthusiasm for the development of methods to perturb endomitosis in C. elegans and the potential importance of binucleation in driving the rapid expression of vitellogenins in intestine cells. In this new version, the authors have addressed the major concerns regarding the link between vitellogenin expression and the different phenotypes observed. While the observed defects in brood size and gene expression are relatively mild, they nonetheless appear to be reproducible.

Interestingly, the authors' data suggest that binucleation is critical for driving rapid gene expression at the larva to adult transition (Fig. 6). To test this idea, the authors developed an assay consisting of a heat-shock promoter driving the expression of GFP. While in controls, GFP is rapidly expressed upon heat-shock, in cells with no endomitosis GFP expression is delayed. These experiments were performed using a line that expressed auxin-inducible KNL-1 degradation to prevent binucleation, but whether the effects observed are a consequence of loss of binucleation or an indirect effect of loss of KNL-1 in chromosome segregation is currently unclear. Considering the importance of this experiment to the overall conclusions of this study, it would be useful to test this idea using a different perturbation that blocks binucleation, such as auxin-inducible CDK-1 depletion. Alternatively, the authors should explain the reason why they chose to use the AID::KNL-1 line for these experiments and not the AID::CDK-1 one.

Minor comment: There is an error in the labelling of Fig. 7a.

Reviewer #3: The authors of the manuscript number PBIOLOGY-D-21-03267R1 entitled "Endomitosis controls tissue-specific gene expression during development" have addressed adequately all my previous comments from their first submission. I have a few of comments/questions.

1. The authors propose that CEH-60 and UNC-62 are limiting VIT-2 in mononucleated intestinal cells. Are they expressed differentially in mononucleated cells or are they suggesting they are translated at lower level in these cells?

2. Other genes downregulated in the intestine include a ribosomal protein (rpl-11.2), and a transcription factor (ets-4) predicted to enable DNA-binding transcription factor activity, RNA polymerase-specific (from WormBase). Lower levels of these two transcripts would be expected to downregulate translation globally and/or RNA polymerase II-specific transcription. How does this possibility reconcile with your findings? Do the authors think it should be addressed in the discussion?

Importantly, the authors do not mention in the discussion or elsewhere that ets-4 transcription factor positively regulates vit-2, vit-3, vit-4 and vit-5, as well as ceh-60. Nor that mutations in ets-4 result in reduced gene expression levels.

3. This is just a comment. It is a bit surprising that overexpression of CEH-60 or UNC-62 completely rescue the phenotypes of animals with mononucleated intestines, given other downregulated genes (spp-5, cpl-1) also affect brood size and/or affect fat content/processing. Should this be a concern or "explained" why might be so?

Dear Dr Galli,

Thank you for submitting your revised Research Article entitled "Endomitosis controls tissue-specific gene expression during development" for publication in PLOS Biology. Please accept my apologies for the delay in getting back to you. I have now obtained advice from the Academic Editor handling your submission, who is satisfied that the reviewer concerns have been fully addressed.

Based on this, we will probably accept this manuscript for publication, provided you address the following data and other policy-related requests that I have provided below (A-D):

(A) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figures, as they are essential for readers to assess your analysis and to reproduce it.

Figure 1E, 1G, 1H, 2A-B, 2D-H, 2K-M, 3A, 4A, 4C-G, 5B-G, 6A, 6D-E, 7A-D, S1B, S1D-E, S2B-D, S3A-F, S4A-G, S5B-E, S6A-B, S7, S8

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

B) Please also ensure that each of the relevant figure legends in your manuscript include information on *WHERE THE UNDERLYING DATA CAN BE FOUND*, and ensure your supplemental data file/s has a legend.

(C) Thank you for depositing the RNA-Sequencing data in the GEO database and for providing the accession number. However, we note that GSE169330 is currently private and is scheduled to be released in September 2022. You should make this data publicly available now for us to be able to process your manuscript for Production.

(D) Please ensure that your Data Statement in the submission system accurately describes where your data can be found and is in final format, as it will be published as written there.

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

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable 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 for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Richard

Richard Hodge, PhD

Associate Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

ORCiD I plosbio.org I @PLOSBiology I Blog

California (U.S.) corporation #C2354500, based in San Francisco

Dear Dr Galli,

On behalf of my colleagues and the Academic Editor, Tom Misteli, I am pleased to say that we can in principle accept your Research Article "Endomitosis controls tissue-specific gene expression during development" for publication in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Richard

Richard Hodge, PhD

Associate Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

ORCiD I plosbio.org I @PLOSBiology I Blog

California (U.S.) corporation #C2354500, based in San Francisco