SUMMARY During development and regeneration, matrix progenitors undergo terminal differentiation to form the concentric layers of the hair follicle. These differentiation events are thought to require signals from the mesenchymal dermal papilla (DP); however, it remains unclear how DP-progenitor cell interactions govern specific cell fate decisions. Here, we show that the hair follicle differentiated layers are specified asynchronously, with early matrix progenitors initiating differentiation prior to surrounding the DP. Furthermore, these early matrix cells can undergo terminal differentiation in the absence of Shh, BMP signaling, and DP maturation. Whereas early matrix progenitors form the hair follicle companion layer, later matrix populations progressively form the inner root sheath and hair shaft. Together, our findings characterize some of the earliest terminal differentiation events in the hair follicle, and reveal that the matrix progenitor pool can be divided into early and late phases based on distinct temporal, molecular and functional characteristics.

The emergence of hair is a defining event during mammalian skin development, but the cellular mechanisms leading to the opening of the hair follicle canal remain poorly characterized. Our previous studies have shown that early hair buds possess a central column of differentiated keratinocytes expressing Keratin 79 (K79), which marks the future hair follicle opening. Here, we report that during late embryogenesis and early postnatal development, K79+ cells at the distal tips of these columns downregulate E-cadherin, change shape, recede and undergo cell death. These changes likely occur independently of sebaceous glands and the growing hair shaft, and serve to create an orifice for hair to subsequently emerge. Defects in this process may underlie phenomena such as ingrown hair or may potentially contribute to upper hair follicle pathologies including acne, hidradenitis suppurativa and infundibular cysts.

Single cell RNA sequencing (scRNA-seq) has been recently developed to study the gene expression profile of individual cells. This high-resolution transcriptomic analysis is enabling deep insights into biological systems and disease mechanisms. Multiple techniques have been used to capture single cells, including microfluidics platforms, microtiter plates, and droplet-based technologies. These are followed by cell lysis and reverse-transcription (RT) of RNA from the individual cells. Barcoded and multiplexed cDNA are then subjected to high throughput sequencing. To apply this powerful approach for the study of skin biology, we have developed a protocol for the isolation of labeled single cells from neonatal mouse skin. Dispase is used to separate the epidermis from the dermis, with hair follicles remaining in the dermis. Enzymatic digestion then generates single cell suspension, and fluorescently-labeled single cells are sorted into microtiter plates using fluorescence-activated cell sorting (FACS). We have optimized this approach to maximize post-sort cell viability and cDNA purification for scRNA-seq library preparation. Taken together, this approach allows for the collection of high quality scRNA-seq data to analyze the biology of lineage specific cells from neonatal mouse skin.