Mechanism Analyses Of Wnt/?-catenin Signaling-Mediated Long-Term Stem Cell Fate And Niche Location In Hair Follicle

Adult stem cells sustain tissue and organ regeneration. These cells usually stay at dormant within these tissues and maintain self-renew and multipotency throughout the lifetime of an organism. Adult stem cell originates from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The molecular mechanism of this process remains unclear.Hair follicles (HFs) undergo cycles of regeneration and hair follicle stem cells (HFSCs) locating at the bulge region orchestrate this process. Previous research has suggested that certain embryonic cells were set to become HFSCs before the HFSCs emerge in the adult tissue. However, it remained unclear how this decision was made, and which genes and molecules were involved in this process. By lineage tracing experiment, we discover that long-term HFSCs arise from embryonic progenitor cells occupying a niche location at the upper hair peg. These cells contribute to more than 70% of HFSCs. To investigate how upper hair peg cells acquire the HFSCs fate, we use two-photon laser to precisely ablate SC precursors in hair peg. Following complete ablation, we observe de novo bulge formation at the end of organogenesis and the formed HFSCs are functional as indicated by their ability to support HF down growth. These results indicate niche location in hair peg determines HFSCs fate.To understand the underlying mechanism leading to niche-induced HFSC specification, we profile the expression of mRNA isolated from upper and lower hair peg cells. Based on RNA-seq and bioinformatics analysis, we find genes of the Wnt signaling pathway were enriched prominently in the lower hair peg cells and were uniquely absent from the upper hair peg cells. We active Wnt/p-catenin signaling in cells occupying hair peg niche position and find the HFSC specification is blocked. In the Wnt/?-catenin loss of function mutant, HF development is defective and HFSCs number is expanded to the extent of all remaining cells in HFs. Interestingly, hair peg cells responding to low Wnt signaling could form HFSCs, but these cells were finally exhausted, losing the long-term self-renew potential.Further study indicates attenuation of Wnt/?-catenin signaling is a prerequisite for HFSC specification because it suppresses Sox9, which is required for stem cell formation and quiescence maintaining. After activation of Wnt/?-catenin signaling in upper hair peg cells, the Sox9 expression in these small protrusions is completely absent. In the Wnt/?-catenin loss of function mutant, HFs development is defective but Sox9 expression is expanded to the extent that all remaining cells in HFs.This study elucidates the cascade of events that lead to long-term SC emergence in HFs during organogenesis. HFSCs origin from progenitor cells in upper hair peg, whose niche location determines HFSCs fate. Attenuated Wnt/?-catenin signaling defines HFSCs specification through releasing the suppression of Sox9. This may help future efforts to establish and expand functional adult stem cells for research and clinical organ regeneration in vitro.