* 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 Hill,

Thank you very much for submitting your Research Article entitled 'Ubiquitin-protein ligase Ubr5 cooperates with Hedgehog signalling to promote skeletal tissue homeostasis' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by three peer reviewers. As you will see, all reviewers are enthusiastic, as are the editors. Based on the review comments and our editorial evaluation, we ask that you prepare a revised version with changes to the presentation to address the reviewers' concerns. We do not think major changes in organization are necessary. 

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.

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,

Gregory S. Barsh

Editor-in-Chief

PLOS Genetics

Gregory Copenhaver

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: attached

Reviewer #2: This is a large amount of work, much of which has been conducted in vivo, in very technically challenging tissues.

The primary aim of the manuscript is to explore the skeletal influence of UBR5, a regulator of the hedgehog pathway. The manuscript describes this and proposes possibilities concerning molecular mechanisms. The authors have previously linked, in a series of papers, this ligase to hedgehog signalling including the regulation of hedgehog ligand expression in the limb where effects on tissue development were minimal.

There is no question that increasing our understand of the regulation of Hh signalling in the mammalian, particularly mature, skeleton is a worthy question as there is increasing confusion particularly regarding the balance of canonical (which has so far received more attention) and non-canonical Hh roles. The authors frame this nicely in the introduction.

General comments

In summary I would say this manuscript adds knowledge to this area, its, perhaps apparently paradoxical and at times slightly superficial and descriptive findings, are worthy of publication. It is extremely important that all information, including that which is mechanistically unresolved or at apparent odds with dogmas, is in the research domain.

Fundamentally there is multi-faceted analysis, sometimes unnecessarily quantitated, to support the major descriptive findings in the mouse backed up by follow-up in vitro studies. Importantly, I am in agreement with the central tenets of the manuscript as summarised, they are supported by the evidence, but I feel that for maximal impact the manuscripts readability could be markedly improved.

This is often points of clarity, improvements to figures and legends etc, but also the authors may wish to consider the salient findings and decluttering the main figures- as in current form the paper is very dense and not easy to follow. The authors are to be commended for a valiant attempt to mechanistically explain the mechanistic links to Hh and PKA signalling in vitro which add support to interpretations of the findings.

As the authors note it is of particular interest that the phenotypes are only apparent in maturing or mature context despite the developmental nature of the pertubation. What do the authors propose might be the reason for this? Does an evolving landscape of Hh signalling account for this and/or are their mechanical inputs into this regulatory axis? Perhaps linkable to locations of ectopic ossification/ failure of cartilage at interface with bone (tears).

I wasn’t clear throughout – are the effects limited to hind limbs? Do you have images of forelimbs?

Was cartilage thickness measured- were effects bias to compartments ? Is cartilage loss a consequence of changes to other soft tissues in limb? Changes to gait – do mice move apparently normally (beyond squat resting position).

Were mice weights normal ? Perhaps previously described.

What were genders of small groups (n=3)

Minors:

I found Figure 1 early panels extremely hard to navigate. Line 108 are ankles shown?

Line 120 authors conveying this shows us where UBR5 is ? - consider re-phrase plain english

Lines 123 beta-gal activity, thus they are derived from embryonic mesenchyme ?

Line 134 different to what?

Lines 135-137 - no comparative controls to aid reader to see 'ectopic' - danger the non-specialist reader is completely lost

Line 145 “joints to reveal”

Concern over n throughout in vivo studies (Limitations should be noted in discussion). No control for cage to cage variability possible with such low n. N could be more easily found for some studies too (key result descriptor- see ARRIVE guidelines). N is often 3 animals ?

Legends are, however, detailed throughout suggesting good accordance with ARRIVE.

At times completely unclear as to n (e.g supplementary Figure 1 legend concerning gender breakdown for C).

Figures – often at times magnifications are not matched (Fig 3H/I) , controls are missing, more labelling would help reader. Quantitation does not immediately tally with images (e.g high density regions in Fig 3B-F, and quantitation in G – 10-15% red?)

K is an example of unnecessary quantitation as also seen in Fig 5G ?

Fig 4 N and O – please label up – what are we looking for? How was the data for 4Q generated?

Commend authors on correct use of Fisher's tests for contingency data.

Reviewer #3: In this manuscript by Mellis and colleagues, they do a very robust characterisation of the role of UBR5 in skeletal homeostasis. Through genetic deletion of UBR5 they show that mice develop profound and progressive cartillage degradation and then metaplasia. Mechanistically, given the activation of GLI following UBR5, suprisingly suppressing smoothened exacerbated the phenotype providing potentially interesting novel mechanism. I think better integration of in vitro mechanism and in vivo phenotypes would improve the paper. However, i think this is an interesting phenotype that is worthy of publication.

Specific comments:

1) I think the phenotyping done in here is excellent and Im happy with this analysis of the paper

2) As the mechanistic analysis is surprising eg the fact that smoothened loss exarcerbated but did not suppress the phenotype, the key i think is how the loss of function of smoothened affected the gli induction.. It is clear that GLI is up in the UBR5 deleting animals so does Smoothened loss inhibit this. This need to be quantified (eg similar to work in figure 7)

3) in terms of the mechanism, have the authors looked at forskolin in vivo?

4) Likewise are the changes in any of the proposed pathways in figure 8E altered in the tissue e.g BRD4 etc...? It would help if the author could integrate their findings 3T3 cells with their in vivo tissue analysis.

**********

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: None

**********

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

Attachments

Attachment

Submitted filename: The paper entitled.docx

Dear Dr Hill,

We are pleased to inform you that your manuscript entitled "Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis" has been editorially accepted for publication in PLOS Genetics. Congratulations!

The revised manuscript was evaluated by members of the editorial board; there is a consensus that the changes to organization and presentation have addressed the concerns raised at the initial round of review.

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,

Gregory S. Barsh

Editor-in-Chief

PLOS Genetics

Gregory Copenhaver

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-20-01820R1

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.