Molecular and Mechanical Signatures Contributing to Epidermal Differentiation and Barrier Formation

Formation of the skin barrier requires rapid proliferation coupled with differentiation and stratification of the embryonic epidermis. Basal progenitors give rise to progeny throughout development – first to intermediate cells, a transient proliferative suprabasal cell population, and later to spinous cells. Neither the function nor the differentiation trajectory of intermediate cells has been documented. We generated transcriptomes of intermediate and spinous cells and identified specific markers that distinguish these two populations. Further, we found that intermediate cells express a subset of genes in common with granular cells of the epidermis – the terminal living cell type that helps establish the barrier. Lineage tracing revealed that most intermediate cells directly transition to granular cells without expressing markers specific to spinous cells, thus revealing a distinct lineage pathway leading to granular fate. In addition to their transcriptional similarities, intermediate and granular cells both had hallmarks of increased actomyosin contractility. We found that rather than simply lying downstream of cell fate pathways, contractility was sufficient to suppress spinous fate and promote granular gene expression. Together, these data establish the molecular and mechanical characteristics of the developing epidermis that allow this tissue to rapidly develop barrier activity. RNA-seq profiling was performed on epidermal basal cells and their corresponding first layers of suprabasal cells, intermediate cells at E14.5 and spinous cells at E16.5, from mouse embryos. K10-rtTA;TRE-H2B-GFP;K14-RFP pregnant dams were fed doxycycline chow since E12.5. Back skin samples were collected at E14.5 and E16.5, epidermis was dissociated by dispase treatment, and single-cell suspensions were prepared with trypsin+versene. Granular cells were exclude from E16.5 samples using UEA1 lectin labeling. Intermediate cells (K10-GFP⁺;K14-RFP⁻;APC-UEA1⁻), spinous cells (K10-GFP⁺;K14-RFP⁻;APC-UEA1⁻), and basal cells (K10-GFP⁻;K14-RFP⁺) were FACS sorted. RNA was extracted from sorted populations, and three biological replicates per group were collected for sequencing.