Lef1 expression in fibroblasts maintains developmental potential in adult skin to regenerate wounds
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Abstract
Scars are a serious health concern that impacts the clinical outcome and long-term well-being of burn victims and individuals with genetic skin conditions associated with wound healing. In this study using mouse as the model, we identify regenerative factors in neonatal skin that will transform adult skin to regenerate instead of repairing wounds with a scar, without perturbing normal development and homeostasis. We utilized single-cell RNA-sequencing (scRNA-seq) to probe unsorted cells from Regenerating, Scarring, Homeostatic, and Developing skin. Our results revealed a transient regenerative cell type in Developing skin, called papillary fibroblasts, which are defined by the expression of a canonical Wnt transcription factor Lef1. Tissue specific ablation of Lef1 inhibited skin regeneration. Importantly, ectopic expression of Lef1 in dermal fibroblasts did not disrupt development and aging, but primed adult skin to undergo enhanced regeneration. Here, we reveal the possibility of transferring the regenerative abilities of neonatal skin to adult tissue by expressing Lef1 in dermal fibroblasts. Finally, we have generated an expandable web resource with a search function to display gene expression in the context of our scRNA-seq data ( https://skinregeneration.org/ ).
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on August 9 2020, follows.
Summary
Overall this is an interesting paper whose ssRNA seq dataset and experimental analysis of phenotypes provides a valuable resource for investigating gene expression differences associated with key phases of skin development and repair. The enhancement of HF regeneration upon Lef1 overexpression is a striking result and will be of general interest to many fields including developmental, stem cell, and epithelial biologists. The work is well conducted, the results are new, and significant for skin wound healing and HF regeneration, and in sum a good fit for eLife.
Essential Revisions
The overall tone of all reviewers is enthusiastic and favorable, however with very important points raised:
Dermo1-Cre …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on August 9 2020, follows.
Summary
Overall this is an interesting paper whose ssRNA seq dataset and experimental analysis of phenotypes provides a valuable resource for investigating gene expression differences associated with key phases of skin development and repair. The enhancement of HF regeneration upon Lef1 overexpression is a striking result and will be of general interest to many fields including developmental, stem cell, and epithelial biologists. The work is well conducted, the results are new, and significant for skin wound healing and HF regeneration, and in sum a good fit for eLife.
Essential Revisions
The overall tone of all reviewers is enthusiastic and favorable, however with very important points raised:
Dermo1-Cre seems not specific to fibroblasts (and it is non-inducible). Ideally this should be addressed by using an inducible/more specific Cre mouse line. However, as the enhancement of HF regeneration is an exciting finding by itself and a new mouse model is likely out of scope of a revision, this point could be addressed textually by changing the conclusions to reference stromal cells instead of fibroblasts specifically.
The interpretation of the scRNA data should be bolstered with additional analyses. It is important for the authors to revisit the data and figures (including making some improved analysis), and carefully state the actual results and conclusions supporting their claims and following next steps in the manuscript.
a) ScRNA-seq analysis was superficial in relation to regeneration versus repair, especially comparison of the time points that model regeneration and scarring. Does velocity analysis predicting Lef1, or other genes, driving differentiation of one population of fibroblasts into a papillary fibroblast or DP-like state? Do multiple fibroblast subsets follow this trajectory? How do these finding compare between the two wounding time points? Does gene ontology suggest differences within one subcluster of fibroblasts between two conditions or are the major differences in the gene expression profile/function associated with each subcluster? A more complete analysis of this could shed more light on the involvement of fibroblast lineages in regenerative versus reparative healing.
b) From the ssRNA seq analysis the authors state "we identify Developing papillary fibroblasts as a transient cell population that is defined by Lef1 expression.", but this is not clear from the ssRNA seq analysis. In Figure S2, Lef1 expression seems to be largely excluded from cells within the Dpp4 expression cluster (cluster 2), and Dkk1 (Cluster 0), which define the major papillary FB clusters. Can the authors expand upon how the Velocity Analysis identifies different genes than overlaying relative expression levels on the UMAPS?
- Surrounding the claim of a transient papillary fibroblast population (which is an important part in their paper), several parts are unclear;
I.e. they could/should explore the fibroblast populations of all conditions to compare regeneration vs scaring and regeneration vs development (e.g. R2Q2, R3Q5).
Which of the two papillary fibroblast population(s) is/are transient? How to explain the rather minor overlap of Lef1 expression with these two papillary fib populations? Where are the two populations in situ in developing and in regenerating skin?
- Given that the WIHN generates a significant amount of cysts, the authors have to down-tone their statement of "without adverse phenotype". As the authors also refer to Hedgehog-pathway induced de novo HF formation (a model giving rise to tumors and new HFs), they likely meant that their model does not induce apparent tumors (the cysts look different compared to the obvious BCC-like lesions trough Hh-pathway activation) - however the authors totally neglect the fact (don't mention) that the mice apparently develop cysts in addition to HFs in wounds.
Figure 5e,g,j. The regenerated HFs appear very abnormal and cyst-like. The authors state several times in the paper that Lef1 overexpression enhances regeneration without other adverse phenotypes, but these regenerated structure are very abnormal. Are they cancerous? P90 wounds appear to generate a significant amount of cysts; is this representative for all conditions or something more specific for the P90 timepoint?
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