Dear Dr Liu,

Thank you very much for submitting your Research Article entitled 'Rod-genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor compositions and progressive retinal degeneration' 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 In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts.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,

Anand Swaroop

Guest Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

This manuscript presents interesting findings that complement and expand on another recently published manuscript by Oel et al. The two reviewers have raised important issues. In general, the manuscript has numerous grammatical and spelling mistakes, and many sentences require revision. A better reorganization of the manuscript may allow the authors to focus on key points. In addition, the authors should discuss their findings by incorporating the work from Oel et al and describing distinctions from mammals.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Review uploaded

Reviewer #2: PGENETICS-D-21-01260 Liu et. al “Rod-genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor compositions and progressive retinal degeneration”

This manuscript describes the identification of a gene, mafba, as a novel regulator for nrl-independent rod formation in zebrafish. The authors generated an nrl knockout zebrafish using CRISPR-Cas9, and saw a reduction but not total elimination of rods along with an overgrowth of green cones. There are major and minor comments to be addressed prior to publication. It is strongly recommended that the manuscript be restructured such the main thrust of the data are in the main body of the text and the supplemental data for incremental data for ease of reading.

Major comments:

Abstract/Author Summary

The authors state that “abnormal gene expression caused progressive retinal degeneration and subsequent regeneration in nrl knockout zebrafish.” Since zebrafish are able to regenerate damaged retinas through the Müller glia-derived progenitors (PMCID: PMC2948409), could these be the source of the second wave of rod genesis they mention earlier in the abstract? This is not addressed in the text. Since this is a viable option for data seen, this needs to be addressed and considered.

The authors state that the importance of these findings in zebrafish “highlight the conserved and species-specific regulatory mechanisms of photoreceptor development and maintenance” however the difference between zebrafish photoreceptor development and mammalian photoreceptor development implies they are not a great model for human disease mechanism studies. This is especially true since these results are in contrast with the Nrl knockout mice. From this point, it would be reasonable if the authors restructure their paper such that they are making the point that zebrafish are not a good model for human disease, and they have discovered data underpinning zebrafish development which may not be applicable to human retinal development. This reviewer realizes this is not an exciting or is perhaps controversial, but the data presented here do not correlate with mouse development, and therefore may be different in humans as well. Either way, they do not support the statement above.

“NRL is the most important gene for the occurrence and function of rod cells in mice and humans” – This sort of declarative statement can be refuted and as such care should be taken in overstatements like this throughout the text.

Line 36: “over-grown of” should be replaced with “over-growth of” or “compensation by”

Line 60-61: “breaking the function”? this phrase is confusing

Results

Figure 1

Zebrafish Nrl antibodies couldn’t be used for western or IF. This in concert with an increase in mRNA does not result in confidence that they are getting a true knockout of nrl.

Figure S1

Why are they comparing different ages 1 mpf and 2 mpf WT and KO, respectively?

They have rod nuclei (presumably) in H&E stained sections at 2 mpf in KO but decrease in thickness of PR+RPE layer – is this due to loss of outer segments?

Figure 2/S2

The authors state that thickness of rod OS and number of rod nuclei is significantly reduced – but this is not quantified anywhere in figure2 or S2. Likewise, it is stated on line 163, “we didn’t observe the fluorescence of rods until 7 dpf in KP zebrafish”. Please see figure and legend S4A/B. There are some rods; little, but some.

What is the difference in Rho/anti-Rho labeling? Why do they go back and forth between red and green? This is confusing.

Figures 3 & S3

They state that density of rod OS significantly reduce in nrl-KO zebrafish and show staining in S3 but it is not quantified anywhere in S3.

Same with the number of rod numbers “obviously reduce” on page 10. They never quantify.

In the text, they talk about mRNA levels of cone specific genes being significantly changed and reference 3C, but this is not what 3C shows. I assume they mean 3B.

Figures 4 & S4

A – it is unclear if these are separate animals/in triplicate or duplicate/ or if they’re different time points quantified in B

B – It is difficult to tell which bars go with what WT in the legend. Consider separating by age and comparing WT with KO. S4B should be in the manuscript, not supplemental.

Additionally, the authors state "the fluorescence of every single rod was also significantly lower than WT zebrafish." Is this quantified? It needs to be in order to state this. WT is oversaturated in the image; what do the data look like in sub-saturating conditions?

Line 174/5 states that rods were absent in marginal zones, yet this reviewer sees rods. Indeed, the authors say in the figure legend for S4 that "arrows indicate retinal regions without rods." I don't see that; when I increase the size of the figure in C, I do see what appear to be rods.

The authors state that there is limited distribution and decreased expression of rod opsin on retinal sections of KO at 1 mpg. Is this due to transgene? Transmission electron microscopy should be performed to determine the ultrastructure of the OS in the KO animals. In this case, the OS should be shorter and thinner with a decrease in rhodopsin expression. Additionally, dot blots are warranted to quantitatively show a decrease in rhodopsin expression.

Figure 5

The authors show that mafba mRNA levels are decreased at 2, and 5 months in the nrl KO. If it is compensatory or involved in the “nrl-independent” pathway, why would the mRNA levels be lower?

Figure 6

The authors make the comment that mafba + nrl double knockout eliminates rods (nrl KO alone does not and mafba KO alone dies at 9 dpf). Since it is a global KO of mafba and nrl, could it be that they are affecting the ability of new progenitors being formed from Müller glia etc. Double knockout with a gene that is lethal at 9 dpf could have many off target issues.

**********

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

Attachments

Attachment

Submitted filename: PLosGenetics-Review.docx

Dear Dr Liu,

Thank you very much for submitting your Research Article entitled 'Rod-genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor compositions and progressive retinal degeneration' 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 topic but identified minor issues that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

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,

Anand Swaroop

Guest Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

As indicated by both reviewers, this is a much improved manuscript. Overall, this is an excellent manuscript and the results go farther than what has been shown by Oel et al. In fact, the data are consistent with Kim et al. Dev Cell 2016 suggesting at least two different types of rods. It appears that mafba and nrl have complementary and probably synergistic functions in zebrafish and that NRL became the primary rod determinant in mammals. Changes in regulation of gene expression of these two transcription factors can account for evolutionary differences (evo-devo). Perhaps the authors can discuss some of these aspects in Discussion

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The authors have addressed my major concerns in this revised manuscript. There are two points that I believe should be addressed. First, the authors have expanded their comparison of the previous (overlapping work) of Oel et al. on p. 21-22. However, I feel the text included in the response to the reviewers is more direct and compelling than that in the paper itself. In fact, I suggest the authors incorporate the response directly into the paper. I also think a few sentences in the introduction to signal that there has been similar work published would help the reader and put both studies in perspective. But this may be a matter of taste. Second, the writing has improved, but it still suffers from significant language problems. I recommend that the paper be edited once more, I believe it will dramatically improve the impact.

Reviewer #2: PGENETICS-D-21-01260

This is a much-improved revision of a manuscript describing the identification of mafba as a novel regulator for nrl-independent rod formation in zebrafish. The authors addressed the major and minor concerns posed by the two reviewers. With minor edits, this reviewer agrees this paper should be published in PLOS Genetics.

Minor edits include:

• Lines 181-182: Most likely, the separation between the disks is due to artifacts of transmission electron microscopy. The last sentence implies nrl is directly involved in disk integrity during outer segment turnover. To state this, even with the qualifier “seemingly”, one would need to do cryoelectron tomography to unveil the disk ultrastructure with that level of precision. To avoid having to do this, I would consider deleting this sentence.

• Line 396: change “What’ more important” with “Importantly”

• Line 399: change “tissular” with “tissue”

• Line 432: this sentence should end with “… and retinal degeneration and regeneration in zebrafish.”

**********

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 Liu,

We are pleased to inform you that your manuscript entitled "Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration" 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,

Anand Swaroop

Guest Editor

PLOS Genetics

Gregory Barsh

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-21-01260R2

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.