Molecular Regulation and Cellular Heterogeneity in Skin Repair and Hair Follicle Regeneration
Epithelial tissues are groups of cells, typically tightly adherent to one another, organized as sheets, and lining various tissues. Maintenance of epithelial tissues such as the adult mouse epidermis and hair follicle is largely driven by epithelial stem cells, which have the ability to give rise to the various epithelial cell types that make up these tissues. At a broad level, the focus of this dissertation is multifaceted but aims to add to a better understanding of epithelial stem cell function, regulation, and how these stem cells alter their cellular dynamics (proliferation, differentiation, heterogeneity, and migration) in the context of wound healing.
Chapter 3 focuses on a continuation of previous work from the lab that aims to elucidate the function of Ovol2 in the context of hair follicle (HF) regeneration and epidermal repair. Previous work had explored the function of Ovol2, a known transcriptional repressor of the epithelial to mesenchymal transition (EMT) in the context of mouse epidermal development, but whether Ovol2 has critical functions during the dramatically distinct processes in the adult mouse isn’t clear. As Ovol2 regulates EMT, this work addresses larger questions associated with whether the EMT process itself plays distinct roles in previously underexplored biological processes such as during HF regeneration, a process associated with dynamic epithelial stem cell cellular movements. Cellular movements during epidermal repair have been shown to be largely driven by transcriptional regulators that promote partial-EMT events. Understanding how these regulators both promote the necessary migratory abilities of epidermal cells to facilitate wound closure but maintain cellular adhesions throughout the repair process is not well understood. Beyond the migratory properties associated with epidermal cells during wound healing is their ability to exhibit directional migration. In this work, I show that loss of Ovol2 leads to both defective HF regeneration and wound repair. These defects are associated with enhanced migratory behaviors and loss of directional type movements. I show that the Ovol2-Zeb1 molecular circuit (through Ovol2’s transcriptional repression of Zeb1) is critical for regulating the directional migration of epithelial cells. Although this work provides detailed insight into a critical aspect of repair, wound healing involves many other cell types as well as many other cellular dynamics.
Chapter 4 takes a dramatically different approach, utilizing single cell RNA sequencing (scRNA-Seq) to ask questions at a more global level, directly comparing wounded and un-wounded skin. Isolating total skin during both normal homeostasis and during re-epithelialization, a stage of active wound repair where epidermal cells utilize partial-EMT events to facilitate wound closure, allowed for a whole skin wide characterization and comparison of the key differences between these two states. Here I addressed global changes in cellular heterogeneity, characterizing the major cell types that are present in normal skin and during active wound healing. I then computationally subset epidermal stem cells (or basal cells) for extensive analysis probing for presence of basal cell heterogeneity represented by various cellular states during normal homeostasis. Lineage tracing studies have suggested that there exists some sort of hierarchy within the basal layer of the epidermis but there lacks a global perspective of basal cell heterogeneity. I identify four distinct basal cell states, which are subsequently confirmed in situ. Beyond identification of these different states, we arrange these different states in a lineage hierarchy, identifying a population enriched for Col17a1 and Trp63 as the most stem-like. I then ask a fundamental question of whether this heterogeneity I observed in normal skin during homeostasis is also present during active wound healing. I show that the same cellular states exist but in different proportions, with the expansion of a growth arrested (GA) population, which has distinct migratory, immune, and metabolic characteristics distinct from the other basal cell states.
Overall, this thesis work takes a multi-scale approach, understanding global chances in cellular processes such as epithelial homeostasis and repair as well as taking a more focused approach, elucidating transcriptional regulators that govern cellular dynamics in epithelial stem cells.