SOX11 and SOX4 drive the reactivation of an embryonic gene program during murine wound repair

Tissue injury induces changes in cellular identity, but the underlying molecular mechanisms remain obscure. Here, we show that upon damage in a mouse model, epidermal cells at the wound edge convert to an embryonic-like state, altering particularly the cytoskeletal/extracellular matrix (ECM) components and differentiation program. We show that SOX11 and its closest relative SOX4 dictate embryonic epidermal state, regulating genes involved in epidermal development as well as cytoskeletal/ECM organization. Correspondingly, postnatal induction of SOX11 represses epidermal terminal differentiation while deficiency of Sox11 and Sox4 accelerates differentiation and dramatically impairs cell motility and re-epithelialization. Amongst the embryonic genes reactivated at the wound edge, we identify fascin actin-bundling protein 1 (FSCN1) as a critical direct target of SOX11 and SOX4 regulating cell migration. Our study identifies the reactivated embryonic gene program during wound repair and demonstrates that SOX11 and SOX4 play a central role in this process.

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Data collection
The microarray slides were scanned with the Agilent Scanner (G2565BA) using Scanner Version C and Scan Control software version A.8.5.1. Data extraction and quality assessment of the microarray data was completed using Agilent Feature Extraction Software Version 11.0.1.1. Data analysis GSEA v3.0 [build: 0160] was used in the gene set enrichment analysis. ChIP-seq reads were mapped to the mouse genome (mm9) using Bowtie2 v2.3. Peak calling and Motif enrichment analysis were carried out with Homer v4.9 (findPeaks -style factor) and (findMotifsGenome.pl), respectively. All gene ontology (GO) enrichment analysis was performed using Metascape v3.0. The Bioconductor package limma v3.5 was used to analyze the microarray data. ImageJ v1.51 was applied to determie the migrated areas in the in vitro wound healing assays. R v3.4.3 and the gmp package were used to calculate the Venn diagram hypergeometric p values.
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April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Microarray and ChIP-seq data from this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE120827 (transcriptional profiles of Sox4cKO, Sox11cKO, and Sox4/11 dcKO mouse keratinocytes and their wild-type control), GSE120826 (transcriptional profiles of Sox4 cKO, Sox11 cKO, and Sox4/11 dcKO mouse epidermis at E16), GSE120824 (transcriptional profiles of murine E13 epidermal cells and P4 epidermal basal cells), GSE120825 (transcriptional profile of SOX11-induced mouse epidermis), and GSE120773 (ChIP-seq) respectively. The source data underlying Figs 1a-c, 3a,c,d, 4a, d-f, 5a-f, 6a,b,h,j, 7a, 8b,c,f,g, 9d,h-j,l and Supplementary Figs 1c, 3, 4a-c and 5b-d are provided as a Source Data file. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Sample size
No sample-size calculation was performed. Sample sizes were chosen based on experiments from published literature.

Replication
Because of variability in the fertilization, implantation and development, it is difficult to predict developmental stage, even using a 6 h mating window. To achieve precise developmental stage, multiple mice were mated for each gestational age. Several litters for each embryonic age were obtained which developmental stage was verified by morphological and histological analysis. In the X-gal exclusion assay, because the skin barrier develops very quickly at E16-17, it might be too early or too late to observe the difference in the epidermal differentiation. To ensure the replicability, we analyzed multiple litters from each mating pair, genotyped all the embryos from each litter, and used the litters in which the wild-type embryos started to acquire the barrier at the dorsal initiation sites (negative by permeability assay). After many trials, we had maximized the replicability in the reepithelization assays in our settings. We used the mold to outline the pattern for the wounds to be made. Particular attention was paid to secure the intactness of the splints till the wounds were harvested. We discarded any samples with the wound beds destroyed during the whole procedures. Biological replicates were used in all experiments and the findings were reproducible.
Randomization Age-matched mice in experimental groups were selected based on their genotypes.
Other samples were not grouped and hence no randomization was performed.

Blinding
Re-epithelialization measurement was done by a staff member who was blinded to group allocation.
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