Manuscript ID: 2011-11-15133A

Title: Skin shedding and tissue regeneration in African spiny mice (Acomys)

Response to Reviewers Second Round Comments

We would like to thank the reviewers for their extremely positive comments and for helping to shape our final manuscript into an overall better piece of scientific work. We have addressed the few additional comments made by reviewers 1 and 3 below. We have also done our best to address the editorial suggestions made by the editor.

Specific Response to Referees' comments:

Referee #1 (Remarks to the Author):

1. With the hair follicle work, it would be helpful if the authors could provide a stitched image of a section that encompasses both mature edge of the wound that has pre-existing follicles AND the regenerating part. Then it would be easier to compare and confirm that the structures in figure 3 are regenerating follicles.

We agree with the reviewer that a histological section would strengthen this piece of data. We have included the requested image of the entire wound bed encompassing both mature wound edges and including both pre-existing hair follicles and regenerating ones as Fig. S3e.

2. In the hair follicle work, I don't find the B-catenin staining is very informative in fig3 i,j.S6d and therefore unnecessary.

At the suggestion of the reviewer we have removed the ß-catenin immunohistochemistry from both of these figures and discussion of this material from the text.

3. The histology of the Acomys ear punch showing similar characteristics to blastemas and being distinct from Mus wounds looks fairly convincing. I agree that there is no agreed-upon universal blastema marker. Since it is hard to definitively define a blastema I would suggest to change "demonstrate" in the following sentence to "indicate". Furthermore, in the next sentence, "Coordinated epidermial-mesenchymal communication between the wound epidermis and underlying blastema maintains cell proliferation" is an inference based on the structure of the tissue. This should be indicated.

"Our findings in Acomys demonstrate that these animals form a blastema in response to injury, which in turn directs regeneration of ear tissue. Coordinated epidermal-mesenchymal communication between the wound epidermis and underlying blastema maintains cell proliferation within a pro-regenerative ECM, while a proximal to distal wave of differentiation replaces the missing tissue."

In response to Reviewer 3, who asked for us to tone down our language referencing blastema formation, we removed the sentence in question from the text entirely.

Minor comments:

4. fig 4 n needs to be named as Acomys-would be better to switch m and n considering the labels above f and g.

We thank the reviewer for bringing this our attention. We have made the suggested change in the figure.

5. fig S4 regenerating hair follicles are similar to what?

We agree that the phrasing in both the text and figure legend for Fig. S4 was confusing. We have re-written the sentences in the main text and altered the figure legend to more clearly indicate that regenerating hair follicles go through “similar stages” of hair follicle development and proliferate similarly to developing follicles.

Referee #3 (Remarks to the Author):

In this revised manuscript, the authors only partially addressed my initial comments and my main initial concern regarding the concept of blastema remains.

The authors have now better substantiated their claims concerning de novo hair follicle formation, and these data are much more convincing now.

We would like to thank the reviewer for pointing out the strengths of our additional data.

1. I still find that the concept of blastema that the authors want to push to explain this remarkable regenerative capacity of acronys, is still not substantiated experimentally. The authors used literature from 1950-70 to substantiate their claims and used markers such as ECM proteins expression (eg: TncC, Colagens), proliferation markers, and pSMAD to make their case for blastema formation. However, none of these markers are specific for blastema formation and all these markers are expressed naturally during hair follicle development and normal hair cycle and chondrocyte development. There is no data presented here that demonstrate that acronys regenerate their skin by a cellular process that would resemble to what is happening during salamender regeneration as claims in the abstract. Each of the cellular lineages (epidermis, fibroblast, adipocyte, cartilage and muscle) can be maintained by their own resident progenitors rather than being mediated by a dedifferentiation of differentiated cells that adopt a multipotent progenitor like fate.

The authors should tone down all the claims with the formation of blastema like structure, as it may introduce an important and unnecessary confusion in the filed of regenerative medicine.

In response to the comments raised by the reviewer we have toned down the language used throughout our paper so as to suggest rather than demonstrate that a blastema mayform during Acomys ear hole regeneration. Additionally, we refer now to the structure that forms in Acomys as “similar” to a salamander blastema rather than the “same as”, although we have removed most references to “a blastema forming in Acomys”. In addition, in our abstract we have altered the sentence to which the reviewer refers to now read, “Salamanders capable of limb regeneration form a blastema (a mass of lineage-restricted progenitor cells) following limb loss, and our findings suggest that ear tissue regeneration in Acomysmay proceed through assembly of a similar structure. This sentence cites Kragl and colleagues (see references) who showed that during salamander limb regeneration each cell type is derived from lineage restricted progenitor cells that are not multipotent. Thus the current concept of a blastema is that lineage-restricted progenitor cells are activated (i.e. de-differentiate) and through proliferation replace the missing tissues.

We agree with the reviewer that we do not present data indicating that Acomys regenerate their back skin by formation of a blastema. Our claim is instead specific to the ear. We do not suggest anywhere in our paper that the skin regenerates through blastema formation as skin regeneration in salamanders does not utilize a blastema either.

Editorial Changes

1. We estimate that your manuscript is currently twice as long as our limit for Letters (4 pages) so you will need to shorten it.

We have done our best to reduce the number of pages our MS will take up. We have reduced the size of Figures 1 and 3 to single column figures and both are shorter than a full column. We have cut ~400 words from the original methods and created a methods summary section totaling 265 words. We created a methods section at the end of the manuscript containing the remaining methodological detail. We were able to substantially reduce the text, cutting it by 1600 words. These cuts were possible through a restructuring of the summary paragraph and removal of introductory material in the text. Furthermore, we moved most, if not all, of the technical details to methods, figure legends and supplementary information.

2. Further introductory material in the main text of the paper should not be necessary.

We have removed the majority of this material from the text.

3. Any discussion at the end of the paper should be brief, and not repeat what is written in the initial summary paragraph.

We have some discussion within the paper but have shortened the discussion at the end of the manuscript to 1 paragraph.

4. Authors should ensure that any statistical analysis used is sound and that it conforms to the journal's guidelines.

All statistics conform to journal guidelines and we have included a descriptive statistics section in the Methods section.

5. The text should contain a brief (<300 word) "Methods summary", describing generally the methods used.

We have included a Methods Summary which is now 265 words.

6. Then, at the end of the same text document (after the figure legends), please include a section entitled "Methods", which provides the full, step-by-step instructions that would allow other researchers to replicate the results.

We have now included this section following the figure legends. It is 952 words in length with 3 references following.

7. Our limit for references is 30; we cannot make exceptions.

We have cut our reference list to 28.