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 shown contains the melanocyte stem cell population. The adult melanocyte stem cell is 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 reasons. 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, defining the signals required by melanocyte stem cells will allow us to understand 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 and therapeutic targeting. To identify melanocyte stem cells, we are using 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 (BrdU) 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 to obtain RNA for gene expression profiling. We are characterizing 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 have developed a system that utilizes doxycycline-inducible transgenic mice in conjunction with a stable green fluorescent protein (GFP) reporter to label quiescent cells. 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 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 whose expression differs significantly between quiescent melanocyte label-retaining cells and their non-quiescent counterparts. We chose approximately 35 of the most significantly different and biologically interesting genes for validation by quantitative real-time PCR analysis, using RNA samples derived from different pools of cells than those used for the microarray studies. Biologically independent samples were generated for this validation analysis. Approximately 15 of these genes were found to have greater than 2-fold expression in the quiescent cell subset. Currently, we are focusing on the validation of these genes on the protein level as specific markers of quiescent melanocytes. We are using flow cytometry, fluorescence-activated cell sorting, and in situ immunofluorescence studies to validate these findings. Although these studies are still ongoing, we nonetheless have some intriguing preliminary findings. One of the validated genes encodes a receptor that is also a marker of hematopoietic stem cells and human breast cancer stem cells. Approximately 5 % of quiescent melanocyte label-retaining cells are positive for this marker when analyzed using flow cytometry. This marker also localizes to the bulge area of murine hair follicles in vivo. Further work should establish whether this marker denotes a melanocyte subset with distinctive functional properties. Another gene whose expression is enriched in quiescent melanocytes is a gene expressed in neuronal progenitor cells. Interestingly, this gene is a target of Notch signaling. Since Notch signaling is important in melanocyte stem cell maintenance, identification and validation of this gene may lead to studies assessing mechanistic aspects of its functional significance for melanocyte stem cells. In conjunction with experiments validating markers of cellular quiescence that may represent melanocyte stem cell markers, we are also developing assays permitting us to evaluate melanocyte stem cell behavior. These assays include in vitro assays to evaluate the potential of individual cells to form colonies as well as in vivo assays, using the skin of melanocyte-deficient mice that lack pigment, to evaluate the potential of subsets of quiescent melanocytes to confer pigmentation to hair follicles in a sustained manner.
