Differential Expression Of ?6 And ?1 Integrins Reveals Skin Epidermal Cell Behavior And Heterogeneity At Single-Cell Resolution

Skin epidermis is a stratified epithelium that composed of interfollicular epidermis(IFE)and hair follicles(HFs).Integrins are transmembrane receptors that mediate cell-matrix adhesion in skin epidermis,and integrins expression spatially reflect epidermal heterogeneity.It is remained insufficiently illustrated whether differential expression of integrins can indicate epidermal cell behaviors and heterogeneity.Firstly,this study verified the correlation between the differential expression of ?6 and ?1 integrins and the tendency of epidermal cell behaviors,including differentiation,migration and stem cell activation.When epidermal cells were cultured in high concentration of calcium to induce differentiation,both ?6 and ?1 integrins showed downregulation as differentiation proceeded,indicating their downregulation are related to epidermal cell differentiation.Compared to normal epidermis,expression of ?6 integrin significantly increased during wound healing,specifically in the migrating cells in wound area,indicating its upregulation is related to epidermal cell migration.Moreover,RNA sequencing showed that ?6 integrin was upregulated in committed progenitors and early differentiated IFE,while ?1 integrin was downregulated in early differentiated IFE,implicating that differential expression of ?6 and ?1 integrins can reflect the activation and differentiation of epidermal stem cells.Then,in the study of epidermal heterogeneity,nine populations of epidermal cells were sorted based on differential expression of ?6 and ?1 integrins,which were successfully identified as nine main epidermal cell types through RNA sequencing,and the lineage relationship of the IFE populations was confirmed by gene set enrichment analysis.To further investigate epidermal heterogeneity at single-cell resolution,a method that combined differential expression of ?6 and ?1 integrins with GFP tracing was applied,576 single cells were sorted from re-epithelialized wounds of tetOKrt14-H2 BGFP mice.Through RNA sequencing,thirteen epidermal cell types were identified,and the quiescent and intermediate cell states of the stem cell populations in epidermal basal layer and infundibulum were distinguished.These results proved that differential expression of ?6 and ?1 integrins reflects epidermal heterogeneity.This study reveals the relationship between integrins expression and epidermal cell behaviors and heterogeneity,multiple epidermal cell types were identified with their molecular signatures.A sorting strategy based on differential integrins expression and a method of single-cell transcriptomic libraries construction method complied with pre-sorting sequencing were developed,which enabled high throughput and high resolution of cell type characterization in epidermis.This sorting strategy can also be applied to enrich for a certain cell population from skin epidermis.