Dear Dr Fernandes,

Thank you for submitting your manuscript entitled "A Drosophila glial cell atlas reveals a mismatch between transcriptional diversity and morphological diversity", with portable peer reviews from eLife, for consideration as a Methods and Resource article by PLOS Biology. Your revised manuscript, the reviews from eLife, and your response to reviewers have now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise. I am pleased to say that our Academic Editor is largely satisfied by the changes made, and thinks the manuscript would be suitable for publication at PLOS Biology. However, the Academic Editor has suggested that some very minor textual changes should be considered before publication, and we will likely have some data and policy related editorial requests that will need to be address before formal acceptance. We would therefore like to invite a short revision for your paper.

**However, before we can send you our 'minor revision' decision, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks we will send you another email detailing any editorial requests (I anticipate these will be addressable in a very short revision).

To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within three working days, i.e. by Aug 23 2023 11:59PM.

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Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

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Kind regards,

Luke

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

Dear Dr Fernandes,

Thank you for providing the metadata related to your manuscript "A Drosophila glial cell atlas reveals a mismatch between transcriptional diversity and morphological diversity", which is currently under consideration as a Methods and Resources article at PLOS Biology, submitted with portable peer reviews from multiple rounds of review at eLife. As mentioned in my last email, your revised manuscript, and your response to reviewers from eLife, was previously assessed by an Academic Editor who felt that the study was suitable for publication at PLOS Biology with only minor revisions. Below I detail some editorial requests that need to be addressed before we can accept your study.

From the review history, we see that reviewers 2 and 3 from eLife were satisfied by the revision but reviewer 1 had some lingering concerns about the specificity of CG5080-Gal4 as a marker of perineurial glia. We see that that reviewer 1 argued that findings from an earlier report (Beckervordersandforth et al., 2008) suggests that CG5080 is coexpressed with the SPG marker moody - and reviewer 1 felt that more work was needed to demonstrate CG5080 as a marker of PGs.

We understand that you disagree with this interpretation, noting that CG5080 and moody co-expression was actually not directly shown in the previous work. Our Academic Editor largely agrees with your interpretation of the previous study, and s/he thinks the the current data provided is adequate to support your conclusions about perineurial glial cell types. Therefore, based on this assessment, we would not require that this conclusion be removed or experimentally strengthened before publication.

However, as the disagreement comes down to interpretation, and as other readers may have similar questions as reviewer 1, we suggest that you consider adding further discussion regarding reviewer 1's comments to the manuscript. We think it would be helpful to outline in a few sentences detailing why there is a perceived discrepancy. In particular, the the Academic Editor suggests you could expand on this comment from the results "Thus, based on morphology, tiling properties, size and position, these data argue that moody-Gal4 labels the subperineurial glial class, which can be further subdivided into surface-only, surface- and channel-associated, and channel-only morphological categories. By contrast, CG5080- Gal4 labels the perineurial glial class, which can be further subdivided into surface-only and channel associated morphological categories."

**IMPORTANT: As you address this last comment from the Academic Editor, we also ask that you attend to the following editorial requests:

1) TITLE: We would like to suggest a minor tweak to streamline the title. If you agree, we suggest it be changed to: "A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity"

2) DATA AND CODE: Thank you for providing the underlying data and code related to your study as depositions and a supplemental file. I have a couple minor requests related to these datasets:

a. Please update the github link https://github.com/AustinSeroka/2022_stage17_glia, to include a readme file, detailing what the various codes are and where they were applied in the manuscript.

b. Please add a sentence to every figure legend (including supplemental) indicating where the underling data can be found. For example, you can add the sentence "the data underlying this figure can be found at ___" (and then reference the relevant repository/supplemental file)

3) DATA NOT SHOWN: Please note that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript. I detected two instances of this on line 262 and 273. Please either remove mention of these data or provide figures presenting the results and the data underlying the figure(s).

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 are pleased to invite you to address these editorial requests in a revision that we anticipate will not take very long. 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/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. 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,

Luke

Lucas Smith, Ph.D.

Senior Editor,

lsmith@plos.org,

PLOS Biology

Dear Dr Fernandes,

Thank you for your patience while we considered your revised manuscript "A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity" for publication as a Methods and Resources at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors and the Academic Editor.

Based on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, but we would like you to address one remaining issue about the R56F03-Gal4 data. We had mentioned in the previous email that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript.

We note that you have deleted the "data not shown" statement in line 277 but still mention the data. Please either include the data in the manuscript or remove the corresponding reference to this dataset. I didn't see any other references to this dataset apart from the methods section, so you could simply delete the sentence if you do not discuss this dataset anywhere else.

We expect to receive your revised manuscript within one week.

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/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. 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,

Christian Schnell (on behalf of Lucas who will be continue handling your manuscript when he is back in the office next week)

Christian Schnell, Ph.D.

Senior Editor

cschnell@plos.org

PLOS Biology

Lucas Smith, Ph.D.

Senior Editor,

lsmith@plos.org,

PLOS Biology

Dear Dr Fernandes,

Thank you for the submission of your revised Methods and Resources "A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Cody Smith, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed 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, 

Christian Schnell (on behalf of Lucas)

Senior Editor

PLOS Biology

cschnell@plos.org

Lucas Smith, Ph.D., Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org