Dear Dr Svoboda,

Thank you very much for submitting your Research Article entitled 'Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes' to PLOS Genetics. Your 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 welcome a much-revised version of your paper if these issues can be addressed satisfactorily. 

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. For instructions see our guidelines.

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 In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts.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,

Lin He

Associate Editor

PLOS Genetics

Gregory Barsh

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: Taborska et al. examined redundant roles of two small RNA pathways, RNAi and piRNAs, in the silencing of retrotransposons in mouse oocytes. Specifically, this works asked if RNAi activity can explain the lack of apparent germ cell phenotypes in piRNA-deficient mouse oocytes. To address this question, the authors:

1. generated an RNAi pathway mutant mice lacking Dicer protein (Dicer SOM). This is a tighter Dicer mutant allele compared to the one created in their previous study on the oocyte-specific Dicer isoform;

2. characterized phenotypes of the Dicer SOM mice;

3. generated double-mutant Dicer SOM; Mili and control animals;

4. evaluated retrotransposon expression in single and double mutant combinations.

The main finding of this work is that lack of prominent female germ cell phenotypes in the absence of piRNAs cannot be explained by the redundancy with the RNAi pathway. A minor observation is that retrotransposon silencing by the individual small RNA pathways do not overlap completely. As such, this work clarifies the individual and combined contributions of the two small RNA pathways to retrotransposon silencing in the female germline. However, the study represents an incremental advancement in our understanding of retrotransposon regulation in the female germline. Crucially, the primary mechanism(s) that ensures retrotransposon silencing in mouse oocytes remains unknown.

Major comments:

1. Small RNAseq data in figure one is in from Yang et al., 2016. Small RNAseq data should be acquired for single and double mutants to validate putative RNAi targets from mRNA seq, as well as show changes in global production of siRNA and piRNA with mutations.

2. Aside from eliminating truncated Dicer variant, the Dicer SOM mouse doesn't seem to significantly change the overall phenotype compared to Dicer deltaMT, except from few putative RNAi targets and upregulated genes in RNA-seq. Not really significant or novel that this mouse was generated.

3. What is a "genomic checkpoint" that the piRNA pathway relies on? Describe better as this is mentioned twice.

4. Explain better the significance of including comparisons to Ago2, and differences in Dicer vs Ago2 oocyte phenotypes?

Minor comments:

Figure 2B. Missing a loading control.

Figure 2C. Needs labels, what is green and blue? What is the genotype?

Figure 2E. Endogenous control gene for qPCR?

Figure 3B. n oocytes are from how many females?

Figure 3C. Reference for initial set of genes that are sensitive to RNAi pathway?

Figure 3D. Need to validate putative RNAi targets using small RNAseq. Otherwise gene expression differences could be indirect and due to oocyte phenotypes.

Figure 3E. What is the difference between Ago2 mutants from Stein et al., 2015 in orange vs. pink? Triangle vs. square symbols?

Figure 4A. More detailed quantification of primordial follicles vs advanced follicles, maybe there is a difference at that resolution between WT and mutants.

Figure 4D. Better to use RNAseq to analyze abundance of TEs in mutants to eliminate background elements within genes that are changing as a consequence of mutation. Also, how many biological replicates per bar in qRT-PCR?

Reviewer #2: Mammalian PIWI-piRNA pathways have been studied mostly in mouse. Mice express three PIWIs (MIWI/PIWIL1, MIWI2/PIWIL4, and MILI/PIWIL2). All three PIWI proteins are expressed at different stages during spermatogenesis, but only MILI is expressed, albeit weakly, in female germ cells. Deficiency in MIWI, MILI or MIWI2 leads to the activation of transposable elements (TEs) including L1 and IAP elements, and spermatogenesis is arrested at specific stages of spermatogenesis. Mutations in mouse PIWI genes affect the male germline but not the female germline. The authors previously identified an oocyte-specific truncated Dicer isoform, termed Dicero. This Dicer isoform produces endogenous siRNAs and together with AGO2 targets TEs and regulates their activity. These findings have led to assumptions that PIWI-piRNA pathways do not play a role during mouse oogenesis. To address the significance of redundancy of endogenous siRNA and piRNA pathways in oocytes, the authors have generated a mouse model lacking Dicero and then crossed it with MiliDAH mice expressing catalytically inactive MILI. In this study, the authors find that the simultaneous loss of endogenous siRNA and piRNA pathways does not affect oocyte development. The authors also find that endo-siRNA and piRNA pathways suppress some but not all TEs in a redundant manner. This raises the possibility that yet another TE silencing mechanism must operate in female germ cells to maintain genome integrity. Although their findings are themselves not surprising and completely expected, studies of this sort are good references and resources for further comparisons.

Minor comments:

1. The quality of images used in Figure 2C is poor. Its legend lacks important information such as which panel shows what with which antibody.

2. The quality of images used in Figure 3A is also poor.

3. Figure 3C: The authors should add non-targeted genes in this experiment.

4. Figure 4A – C: here the authors analyze ovarian weight, histology and number of fully-grown GV oocytes using mice with age between 8 and 18 weeks. The authors should describe in legends the age in weeks of mice they use for each experiment. In my opinion, the authors should use mice with same age in weeks in these experiments. In addition, the authors’ better present data with male mice in Figure 4D for the reader to understand the degree of contribution of RNAi and piRNA pathways to suppress TEs in female gonads.

Reviewer #3: The manuscript by Taborska et al genetically explores the combined roles of the RNAi and piRNA pathways in the female mouse germ cell development through the use of advanced mouse genetics. The study is designed to address the question of whether the RNAi pathway masks a function for the piRNA pathway in the female mouse germline. An elegant new allele of Dicer is generated that forces the expression of somatic Dicer isoform throughout the entire animal inclusive of the germline. These Dicer-som are viable but present female-specific infertility. The combination of Dicer-som with the catalytically inactive MILI results in the same developmental phenotype but has a distinct molecular phenotype comparted to Dicer-som mice. This stringent genetic experiment and the molecular analysis undertaken substantiate the principal claims of the manuscript. I am intrigued by the data of Figure 5. Overall, I am supportive of publication but have concerns to be addressed:

The upregulation of RNAi targets in figure 4D & F could also be possibly presented in cumulative frequency plots (S-curves). These have traditionally been used for miRNA effects and are quantitative and very easy to understand.

The data presented in Figure 5 is not presented in the results section. This needs to be rectified.

The data presented in Figure 5 is an extremely important observation. I think it should be given some mention in the abstract. I also think the key message from the low levels of L1 transcription in the female germline and minor upregulation of L1 transcripts (compared to the male germline) in the absence of the RNAi and/or piRNA pathways would be substantiated by staining for L1 ORF1 protein. The expectation would be that it should not be detected in the respective mutant genotypes.

I think the discussion should give a sentence or two on why the piRNA-pathway expression in should be retained in the female germline of species that does abundantly express active TEs therein. The piRNA pathway is more than just the PIWI protein but requires the expression of many biogenesis factors (at least >10). Could the expression of the pathway be an insurance policy against a future invasion?

**********

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

Reviewer #3: 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

Reviewer #3: No

* 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. *

Dear Dr Svoboda,

Thank you very much for submitting your Research Article entitled 'Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

The reviewers comments are largely positive at this stage, and there are only a few comments that need to be addressed. We hope to receive a revised manuscript to incorporate these suggestions, before making an editorial decision on the paper. Your revisions should address the specific points made by reviewer 1 and 3.

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 7 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.

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,

Lin He

Associate Editor

PLOS Genetics

Gregory Barsh

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: Overall, I am satisfied with authors' responses.

I still have an issue with the genomic "checkpoint". The current draft describes piRNA clusters as "... specific genomic regions serving as "checkpoints" sensing invading mobile elements". This is not entirely correct since a genomic region senses nothing by itself. If not expressed, its resident sequences are invisible to the piRNA system and the cell. It is an unnecessary invention that will only confuse readers, and I ask authors to refrain from using this term. For example, the same idea can be expressed as follows:

"The piRNA pathway relies on specific genomic regions (piRNA clusters) whose expression primes piRNA production".

Reviewer #2: On the whole it has been improved and the authors have addressed some of the reviewers’ concerns. However, it is already known that the deficiency of PIWI genes results in no discernible phenotype in female mice. In contrast, the authors previously found that the deficiency of the oocyte-specific Dicer variant causes frequent meiotic spindle defects upon resumption of meiosis. Thus, it is still not clear the rationale for why the authors try to examine possible redundancy between RNAi and piRNA pathways in mouse oocytes. The authors had better focus on characterization of Dicersom/som mutant oocytes in my opinion.

Other points:

1. Figure 2 E & F show that expression levels of the full-length Dicer are markedly affected in Dicersom/som mutant oocytes. Why?

2. Figure 3A: the authors should show the figures provided to response to Reviewer #2 comments as supplementary figures.

3. The figure showing L1 ORF1 staining in the authors response to reviewer #3 appears to show that part of chromosomes (DAPI staining) is already excluded from the nucleus in Dicer mutants. This may imply that the deficiency of the Dicer activity already causes chromosome abnormality in early oocyte development and thus, the spindle defects observed are secondary.

4. The figure showing expression levels of piRNA pathway genes in oocytes contains very important information in the field. Thus, the authors should show the figure in the current manuscript. Studies of this sort are good references and resources for further comparisons.

Reviewer #3: All my concerns have been addressed.

**********

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

Reviewer #3: 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

Reviewer #3: No

Dear Dr Svoboda,

We are pleased to inform you that your manuscript entitled "Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes" 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 accept, 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 one way to make 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,

Lin He

Associate 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-19-00984R2

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.