Date of Award
Doctor of Philosophy (PhD)
Adult stem cell (SC) behavior is tightly coordinated by the signals received from the “niche” - the microenvironment that the SCs reside in. Little is known about the role of the niche in SC specification during organ morphogenesis. In particular, the question of whether the niche exists prior to SC specification or whether it is recruited after SC establishment in a developing tissue remains largely unanswered. In addition, the signals responsible for the specification and regulation of SCs during morphogenesis remain unexplored. To answer these questions, I focused my analysis on the earliest stages of hair follicle (HF) morphogenesis. Using immunofluorescence and live imaging, I found that in developing HFs, basal cell divisions are asymmetric and perpendicular to the basement membrane. These divisions result in differential levels of WNT signaling in the daughter cells, with basal cells remaining WNThigh, and suprabasal cells becoming WNTlow. Using in utero lentiviral transduction and genetic mouse models, I created mosaic epidermis with gain- or loss-of-function for WNT signaling to demonstrate that juxtaposition of WNTlow and WNThigh cells is sufficient to confer SOX9+ cell fate to the WNTlow cells. This suggested that the perpendicular asymmetric divisions that I observed in the developing HFs produce the WNT gradient, necessary for the establishment of SOX9+ cells. To further investigate the mechanism behind SOX9+ cell specification, I investigated the signaling patterns of SHH, previously suggested to regulate Sox9 expression. Interestingly, while Shh was expressed exclusively by the WNThigh basal cells, SHH signaling was primarily detected in the suprabasal SOX9+ cells. By inducing the expression of lentivirus-delivered Shh at different stages in morphogenesis, I found that the levels of WNT signaling dictate the responsiveness to SHH. When WNT signaling is low or moderate, cells respond to SHH, resulting in the inhibition of WNT signaling. However, WNThigh cells are resistant to SHH signaling. Thus, in the suprabasal SOX9+ daughters, SHH acts to repress WNT signaling, further boosting the levels of SOX9, while due to high levels of WNT signaling in the basal daughters, they are unable to respond to SHH and remain SOX9-negative. Finally, using Shh-CreER lineage-tracing, I demonstrated that the earliest asymmetric cell divisions of the WNThigh, Shh+ cells produce SOX9+ cells that eventually contribute to the adult stem cell pool. Interestingly, SOX9+ cells are produced only during the early asymmetric cell divisions, while the same Shh+ cells later give rise to various differentiated lineages of the developing HF. Thus, in developing HFs, asymmetric cell divisions produce a WNTlow SOX9+ SC daughter and a WNThigh “niche” cell daughter that produces SHH, necessary to suppress WNT signaling and expand the SOX9+ SCs.
Ouspenskaia, Tamara, "Specification of Stem Cells and Niche During Hair Follicle Morphogenesis" (2017). Student Theses and Dissertations. 335.