Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer

Maintenance of adult tissues requires constant replacement of functional cells that are lost. How highly proliferative stem cell populations undergo differentiation to generate post-mitotic, terminal cell types is poorly understood. Here we combine intravital imaging with molecular insights to investigate how stem cell differentiation is orchestrated in the skin of living mice. We demonstrate that this journey, from initial gene expression changes to movement out of the stem cell compartment, occurs gradually and takes several days to complete. Differentiation-committed cells are capable of proliferating and generating progeny that complete the differentiation trajectory. These divisions are not required to amplify the pool of differentiating cells, but instead occur to protect density in the stem cell layer. Our results thus define epidermal differentiation as a multi-step process, with cell fate commitment temporally preceding the loss of proliferative capacity to satisfy distinct needs within the tissue. Epidermal cell suspensions were isolated from the dorsal skin of two 57-day-old Ivl-CreERT2/R26-tdTomato female mice. Two days prior to cell isolation, these mice were treated with Tamoxifen (10μg/g i.p.) to trace Ivl-expressing cells. Cells were FACS-isolated to traced and non-traced populations of live basal interfollicular epidermis cells (Sytox blue-neg/CD49f+/LY6A+/CD34-neg), which were both captured on a microfluidic Fluidigm C1 system.