Not Black and White: BMP Signaling Drives Melanocyte Differentiation Down Stream of Stem Cell Activation
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy
Tissue stem cells (SCs) maintain, regenerate, and repair the body over the course of an organism’s lifetime. To preserve their long-term function, SCs must exert precise control over their cell state dynamics as they move from quiescence to activation and commit to full differentiation. My graduate research has been centered on investigating the molecular mechanisms that fuel these transitions in melanocyte stem cells (McSCs), a unique neural crestderived SC population located in the hair follicle (HF). Through periodic bouts synchronous with HF cycling, quiescent McSCs become activated to proliferate, giving rise to committed proliferative progeny (McCP) that differentiate into mature pigment-producing melanocytes. The signaling factors and gene expression programs that orchestrate these cellular changes are still incompletely understood. To elucidate new insights into this process, I developed fluorescenceactivated cell sorting strategies to isolate quiescent, activated, and differentiating McSC lineage cells from the mouse skin at discrete stages of the hair cycle. I then performed single cell RNAsequencing (scRNA-seq) to reveal the evolving transcriptional signatures of the lineage with high resolution. Comparative bioinformatic analyses suggested that BMP and WNT signaling increase concomitantly throughout differentiation. I then sought to understand the role of BMP signaling in McSC lineage progression and whether this pathway engages in crosstalk with WNT signaling. To do so, I performed conditional lineage-specific genetic ablation of Bmpr1a to extinguish BMP signaling, which resulted in gray hair. However, McSCs remained intact and functional in these mutant animals, indicating dysfunction in their differentiating progeny. ScRNA-seq and pseudotime analysis of Bmpr1a null cells indicated a block in the differentiation program just downstream of the early McCP stage, and I detected further signs of melanocyte immaturity using immunofluorescence and electron microscopy analyses. Seeking mechanistic understanding, I interrogated changes in transcription factor expression at this blockage point. I found reduced nuclear levels of the master regulator MITF and WNT mediator LEF1. Using WNT mutant mouse models, cell culture systems, and chromatin profiling, I provide evidence demonstrating cooperation of BMP and WNT signaling to trigger complete differentiation of McCP into melanocytes through MITF and LEF1 activity. Altogether, I have generated a thorough characterization of the transcriptional and chromatin landscape changes that temporally define McSC lineage commitment in vivo. My findings underscore a critical role for signaling through BMPR1a to achieve full melanocyte differentiation in the HF. These findings raise intriguing questions about the role of BMP in hair and coat color variation, age-related hair graying, and melanoma initiation and progression.