The goals and objectives of this project are to identify and characterize the quiescent melanocyte cell population in the murine hair follicle that previous studies have suggested is the melanocyte stem cell population. This type of adult stem cell is thought to represent the self-renewing cell that generates the melanogenic pigmentary unit during each successive hair cycle. Understanding this cell population is important for a variety of different purposes. Insight into the determinants of quiescent and cellular self-renewal of these cells may provide information useful for elucidating important principles of stem cell behavior. Since the bulge area of murine hair follicle harbors not only melanocyte stem cells but also keratinocyte and neural crest stem cells, knowledge of the signals utilizing by melanocyte stem cells to generate the differentiated cells required for pigmentary unit formation may facilitate the understanding of how different adult stem cell types interact and communicate within a defined stem cell niche. Finally, because of the conservation of cell surface protein expression that has been noted between adult, tissue-specific stem cells and the corresponding cancer stem cell type, it is likely that characterizing the molecular phenotype of melanocyte stem cells, especially cell surface marker expression, will provide information useful for confirming or refuting the existence of melanoma stem cells and dissecting this cellular subset from tumors for experimental analysis. To identify melanocyte stem cells, we are making the assumption that quiescent melanocytes in the lower permanent portion of the hair follicle, the bulge region, possess stem cell properties. This assumption is based upon prior reports that cells in this region expressing certain melanocyte markers exhibit quiescent, or label-retaining, properties, and can renew the follicular pigmentary unit following antibody-mediated ablation of the differentiated melanocyte population. To identify quiescent cells in these prior reports, mice were injected with the nucleoside derivative 5-bromo-2'-deoxyuridine to mark newly synthesized DNA, whose persistence over time indicated the slow-cycling nature of these cells. To identify the quiescent cells in these previous studies, fixation of skin specimens was required, thus rendering the cells non-viable and unsuitable for obtaining RNA in the living state to facilitate characterization of cellular gene expression profiles. One of our goals is to characterize on a large scale the gene expression profile of melanocyte stem cells in order to define a gene signature that describes these cells and differentiates them from other melanocytic cells. We also hope to discover specific markers of this cell type. Hence, for our studies, we are developing a system that utilizes doxycycline-inducible transgenic mice in conjunction with a stable green fluorescent protein (GFP) reporter to label quiescent cells to be isolated in a viable state. This approach is similar to a previously reported study in which a keratinocyte-specific promoter was utilized to isolate keratinocyte stem cells from the murine follicular bulge using fluorescence-activated cell sorting (FACS) for gene expression profiling. In our case, we have generated transgenic mice expressing the tetracycline-regulated transactivator tTA ('Tet-Off') from the dopachrome tautomerase (Dct) promoter, a melanocyte-specific promoter expressed in the previously described melanocyte stem cells. We have identified one founder line, out of a total of 17 screened, which drives successfully expression of a histone 2B (H2B)-GFP fusion protein reporter protein in a doxycycline-regulated manner. When mice are not administered doxycycline, expression of GFP is detected in melanocytic cells in the infundibular, bulge, and lower outer root sheath segment of the adult murine hair follicle. However, following doxycycline administration, the number of GFP-expressing cells is greatly reduced. Follicular GFP-expressing cells under these conditions are generally restricted to the lower permanent portion of the hair follicle, and are located in the CD34-expressing region of the follicle that marks the bulge. We have used FACS to isolate GFP-expressing cells from adult dermal cellular suspensions. RNA obtained from multiple biological replicates of constitutively-expressing GFP cells and label-retaining GFP cells has been amplified and used for microarray analysis. Our collaboration with Aleksandra Michalowska in Dr. Glenn Merlino's group at NCI has led to the identification of over 200 genes that may differ significantly between melanocyte label-retaining cells and their more differentiated counterparts. With this as a foundation, we plan to validate genes, appropriately prioritized, to define a set of genes and their protein products that can be used reliably to identify and isolate melanocyte stem cells. We plan to investigate the function of these cells as melanocyte stem cells using in vivo assays
