Specification, Molecular Control and Niche Functions of the Hair Follicle Mesenchyme

Project Summary During the hair cycle, growing hair follicles (HF) regress in a phase marked by progenitor death and follicle remodeling (catagen), before a rest period (telogen) and new hair growth (anagen). Signals from the dermal papilla (DP) – a key signaling center – regulate progenitor proliferation and differentiation in the bulb of growing HFs, and induce bulge/germ stem cells (SC) to regenerate fully growing HFs. During catagen regression, the DP needs to relocate from the base of growing HFs to the SC reservoir in the upper HF, traversing the skin several hundred micrometers. How this is accomplished has been unknown for decades. In the first funding period, we have uncovered that the follicle-lining dermal sheath (DS) is a smooth muscle that contracts to physically relocate the DP to reach its essential SC-adjacent position. The dynamic molecular changes that occur in adjacent progenitors and the DS and whether epithelial-mesenchymal crosstalk between them regulates contraction is unknown. Exploring our DS and several other gene signatures suggested an intriguing role of endothelin signaling, a well-known vasoconstriction regulatory pathway, in controlling DS contraction. With several established and newly developed functional assays and novel genetic DS targeting, we have now established the conditions for exploring the dynamic expression of endothelin ligand, receptor and other pathway members in progenitors, DS and DP during both growth and regression; for investigating the functional role(s) of progenitor-derived endothelin in signaling and activating contraction; and for dissecting the downstream pathway mechanism(s). Overall, we will rigorously test the hypothesis that follicle progenitors and the DS engage in epithelial-mesenchymal crosstalk crucial for regulating DS contraction and hair cycle regression. We will precisely map expression localization and timing of key endothelin signaling pathway members through detailed in situ hybridization and immunofluorescence analyses. We will further purify DS and progenitors with novel multicolor isolation methods from growing and regressing follicles and analyze their transcriptomes to define potential signaling crosstalk in general and all components of the endothelin pathway system in particular. We will pharmacologically activate and block endothelin signaling activation in isolated DS cells and microdissected follicles, and determine functional DS contraction in our recently established in vitro and ex vivo live imaging assays. We will then explore the consequences of contraction inhibition in vivo and directly visualize contraction inhibition with intravital imaging. We will perform timed genetic receptor and ligand ablations and assess the impact on follicle regression. Finally, we will define the spatial and temporal calcium signaling dynamics downstream of endothelin pathway activation using calcium reporter mice and activators and inhibitors of Ca2+ channels as well as of the PLC/PKC activation to dissect the major endothelin signaling pathway branches that operate to activate DS contraction. With this work will define a key physiological function of progenitor-DS interaction, which may be useful for hair regeneration approaches, including manipulating follicle regression.