An unbiased template of the Drosophila brain and ventral nerve cord

John A. Bogovic; Hideo Otsuna; Larissa Heinrich; Masayoshi Ito; Jennifer Jeter; Geoffrey Meissner; Aljoscha Nern; Jennifer Colonell; Oz Malkesman; Kei Ito; Stephan Saalfeld

doi:10.1371/journal.pone.0236495

Peer Review History

Original SubmissionDecember 31, 2019
8 May 2020 Decision Letter - Matthieu Louis, Editor PONE-D-19-36054 An unbiased template of the Drosophila brain and ventral nerve cord PLOS ONE Dear Dr. Bogovic, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by reviewer #1. We would appreciate receiving your revised manuscript by Jun 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, 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. We look forward to receiving your revised manuscript. Kind regards, Matthieu Louis Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including your funding statement; "This work was funded by HHMI." Please expand the acronym “HHMI” (as indicated in your financial disclosure) so that it states the name of your funders in full. This information should be included in your cover letter; we will change the online submission form on your behalf. 3. Please ensure that you refer to Figure 13 in your text as, if accepted, production will need this reference to link the reader to the figure. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript provides a state of the art standardized atlas of the Drosophila central nervous system (including the brain and the VNC) together with a detailed analysis of its quality and public open access availability in comparison to existing standardized brains. The analysis is of outstanding detail and quality, so that the standardized CNS will likely be a highly useful tool for the scientific community. The data are freely available at https://www.janelia.org/open-science/jrc-2018-brain-templates, and the format seems to be open source. The manuscript is well written and I have only minor suggestions for improvement. 1. It would be great to have a brief and simple description of how to use the atlas right up front in the manuscript. It is fantastic that the templates, tranformations, code, and descriptions are all publically available, but a brief paragraph within the manuscript that describes the use of these resources would likely attract many researcher to actually try it out. 2. It is understood that registration of genetically labeled neurons requires to have a standard of non-wildtype Drosophila. However, it might be useful to include a short paragraph of the origin of the respective genetic backgrounds. This would greatly help researchers, that are I not as experienced in Drosophila genetics as the authors are, to judge on potential differences /caveats when they will try to move forward and register data from their own work with different fly strains. 3. It seems a little harsh to state that the existing VNC atlas of Börner is not available at all. Although I have not made the effort to try to find it myself, I know people who have accessed it before. However, it is agreed that it is not as advanced as the one of this study, has not been analyzed nearly as deeply as the work presented, and most importantly, it is not useable with open access software which indeed significantly limits availability to the field. In addition to the high quality, being able to use open source code and software is a compelling advantage of the work presented. 4. A statement about the obvious tissues distortions, especially non-isometric shrinkage, as expected from the fixation, dehydration, and clearing protocols used for histology would be useful. Especially in the light of the expected increasing availability of live imaging data (without fixation etc.). What will users have to be aware of when trying to register functional data? (which would be great in the future) I am not asking for a rigorous analysis here, but a short paragraph provided by these true expert authors would be highly useful. 5. The term “irrelevant sources of variability” needs some additional explanation /justification, especially in the light of the recent study by Linneweber et al. (2020, Science. 367(6482):1112-1119.) which relates morphological variability to behavioral individuality. 6. I agree that the SD of the Jacobian determinant is a highly useful measure for non-isometric shrinkage, as explained nicely in section 4.2.2.. It seems that Hessian matrix norm also measures precisely this, and the reported values for JSD and HMN correlate nicely (table 8). Does this mean that both provide a similarly good means for judging on non-isometric tissue shrinkage? I understood the text like this, but admittedly had some difficulties understanding these sections. It would be nice to explain this somewhat clearer to the non-expert, and maybe also include some judgement on what values become nearly unacceptable for registration of future samples. ****** 6. 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 [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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 note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0236495.r001
Revision 1
22 Jun 2020 Author Response We thank the anonymous reviewer for their comments. We have addressed these in our revision and feel that the clarity and accessibility of the manuscript are improved as a result. Since our initial submission, we have made public a new transformation from our template to a newly released Drosophila electron microscopy dataset, called the "hemibrain". Our changes are highlighed in blue in the ``Revised Manuscript with Track Changes,'' with footnotes indicating the comment each change addresses. A summary of the changes are given below. "It would be great to have a brief and simple description of how to use the atlas right up front in the manuscript. It is fantastic that the templates, transformations, code, and descriptions are all publicly available, but a brief paragraph within the manuscript that describes the use of these resources would likely attract many researcher to actually try it out." We thank the reviewer for the suggestion and considering the accessibility of this work. We have added a brief new section at the end of the introduction (``Usage'') in which we describe the ways in which the resources we have created will be useful to various researchers in their own work. "It is understood that registration of genetically labeled neurons requires to have a standard of non-wildtype Drosophila. However, it might be useful to include a short paragraph of the origin of the respective genetic backgrounds. This would greatly help researchers, that are not as experienced in Drosophila genetics as the authors are, to judge on potential differences /caveats when they will try to move forward and register data from their own work with different fly strains." We have included a short discussion regarding the potential caveats when using this work with Drosophila of different genetic backgrounds, of which be believe there to be very few. As evidence for this, we point out our own internal success in registering many thousands of brains and VNCs from different genetic lines. "It seems a little harsh to state that the existing VNC atlas of Borner is not available at all. Although I have not made the effort to try to find it myself, I know people who have accessed it before. However, it is agreed that it is not as advanced as the one of this study, has not been analyzed nearly as deeply as the work presented, and most importantly, it is not useable with open access software which indeed significantly limits availability to the field. In addition to the high quality, being able to use open source code and software is a compelling advantage of the work presented." We echo the reviewer's appreciation of open data and software. We have softened our language slightly, but feel it is important to recognize this hurdle. A statement about the obvious tissues distortions, especially non-isometric shrinkage, as expected from the fixation, dehydration, and clearing protocols used for histology would be useful. Especially in the light of the expected increasing availability of live imaging data (without fixation etc.). What will users have to be aware of when trying to register functional data? (which would be great in the future) I am not asking for a rigorous analysis here, but a short paragraph provided by these true expert authors would be highly useful. We agree. Our revision includes a short discussion on this point, describing the expected kinds of tissue distortions due to fixation, dehydration, and clearing. We specifically point out the lack of ``ground-truth'' in this regard, and how novel technologies could help, as the reviewer points out. "The term “irrelevant sources of variability” needs some additional explanation/justification, especially in the light of the recent study by Linneweber et al. (2020, Science. 367(6482):1112-1119.) which relates morphological variability to behavioral individuality." The revised manuscript provides more details in the section 5.5 ("Symmetry") clarifying this point, including a reference to the recent related work pointed out by the reviewer. To summarize the additions, we more directly state how analysis can be done to recovery asymmetries after registration to our (symmetric) template, and what analysis to avoid when analyzing asymmetries. "I agree that the SD of the Jacobian determinant is a highly useful measure for non-isometric shrinkage, as explained nicely in section 4.2.2.. It seems that Hessian matrix norm also measures precisely this, and the reported values for JSD and HMN correlate nicely (table 8). Does this mean that both provide a similarly good means for judging on non-isometric tissue shrinkage? I understood the text like this, but admittedly had some difficulties understanding these sections. It would be nice to explain this somewhat clearer to the non-expert, and maybe also include some judgement on what values become nearly unacceptable for registration of future samples." We now more explicitly state our conclusions and recommendation in the main text in a single sentence. The supplement now includes some additional discussion and rationale regarding this topic. Attachments Attachment Submitted filename: Response_To_Reviewers.pdf https://doi.org/10.1371/journal.pone.0236495.r002
9 Jul 2020 Decision Letter - Matthieu Louis, Editor An unbiased template of the Drosophila brain and ventral nerve cord PONE-D-19-36054R1 Dear Dr. Saalfeld, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Matthieu Louis Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: All of my comments have been fully addressed by the revisions made. The work will provide the scientific community with powerful and freely available tools for Drosophila brain atlas rmorphometry ****** 7. 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 https://doi.org/10.1371/journal.pone.0236495.r003
Formally Accepted
20 Jul 2020 Acceptance Letter - Matthieu Louis, Editor PONE-D-19-36054R1 An unbiased template of the Drosophila brain and ventral nerve cord Dear Dr. Saalfeld: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Matthieu Louis Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236495.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .