Investigating the plasticity of the follicular melanocyte stem cell system

Summary: This grant aims to understand the plasticity of melanocyte stem cells (McSCs) and mature melanocytes (Mcs) in adult hair follicles (HF). Our recent study revealed that all McSCs in early anagen undergo partial differentiation and then make the decision to revert to a stem cell (SC) phenotype or go on to full differentiation. This newly described mechanism challenges the previous belief that once the differentiation program is initiated in SCs, it is not reversed in normal homeostasis. Many questions still remain unexplored in this new model, including: What are the steps driving McSC partial differentiation and then either reversion to a SC state or full melanocyte maturation? When does McSC plasticity end? i.e. can fully mature Mcs also undergo dedifferentiation? How does this newly observed plasticity translate to human melanocytes? Addressing these questions will provide an essential platform to advance this new SC paradigm. Further, understanding McSCs in particular is relevant because melanocytes (Mcs) are critical for hair and skin pigmentation, and when dysregulated, they can result in gray hair, skin depigmentation and melanoma. In Aim 1, we will use innovative techniques and tools for live imaging to fully delineate the fate decision process of McSCs. We will track the movement of individual McSCs from the onset of hair growth until their fate is determined using a live imaging system. The regulatory mechanisms governing the ultimate fate of McSCs remain largely unexplored. This is crucial, particularly in light of the understanding that the ultimate fate of SCs involves dedifferentiation from a partially differentiated state within the McSC system. Our live imaging will help us understand how McSCs interact with their environment during early hair growth and when they undergo final fate decisions. In Aim 2, we have found that all McSCs undergo partial differentiation in the onset of the hair follicle growth phase, but they do not go on to full differentiation despite the fact that essential differentiation pathways have been initiated. In examining McSC scRNAseq datasets for regulons, Foxd3, a TF known to repress melanocyte embryonic development, came up in the 1st position. We will use Foxd3 KO and over-expression models to see how its perturbation affects McSC differentiation. We predict that loss-of-function will induce full differentiation in early anagen McSCs, and gain-of-function will promote a more undifferentiated state of McSCs. In Aim 3, we will ask at what stage McSCs are no longer capable of complete dedifferentiation to a SC state. Our preliminary results suggest that mature Mcs retain the potential to undergo full reversion to a SC state in ex vivo assays. We will examine this potential in great detail, and both mouse and human Mcs will be investigated for this potential.