Project Summary/Abstract The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the characterization of the different stem cells (SCs) within skin, determining their relative contributions to tissue homeostasis and wound-repair, and elucidating how changes in their niche microenvironments impact these events. Past AR050452 research led to purification of hair follicle (HF) bulge and basal inter-follicular epidermal (Epd) cells and established them as long-lived, self-renewing SCs that function in tissue regeneration and wound-repair. However, both in their biology and their tissue regenerative tasks, these SCs display distinct behaviors predicated by their unique microenvironments (niches). The field still lacks a comprehensive knowledge of the constituents of these niches, the nature of SC:niche interactions, and how they help SCs cope with stressful situations. Past AR050452 research set the foundations to tackle the next key questions: (1). What are dynamics in bulge niche components that drive HFSC behavior during the hair cycle? (2) What are the key niche:HFSC interactions that maintain quiescence and drive tissue (hair) regeneration and how do they differ from the niche: short-lived progeny interactions that drive hair differentiation? (3) How are HFSCs spared during the destructive phase of the cycle when hair growth ceases and most follicle cells below the bulge apoptose? Does eating confer increased SC fitness? (4) How do HFSC:niche interactions change when skin is injured and the SCs become repurposed to repair the wound? How do SCs protect themselves against immune/pathogen attack so that they can orchestrate the re-epithelialization process? (5) How does the natural process of wound-repair differ from the behavior of a SC when it acquires an oncogenic mutation that will ultimately lead it down a path to cancer? To answer these questions, we'll use FACS, single cell spatial transcriptomics and chromatin landscaping, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, wound-induced and tumorigenic environments.