Poster presentation, P14

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Notch signaling is dispensable for mature melanocytes but essential for melanocyte stem cells

BACKGROUND: Notch signaling is essential for cell growth, survival and differentiation. We previously showed that signaling through Notch1 (N1) and Notch2 (N2) receptors contributes to the maintenance of melanoblasts and melanocyte stem cells, and that is essential for proper hair pigmentation. The deletion of both receptors results in dose dependent hair graying, due to an elimination of melanocytes and melanocyte stem cells. OBJECTIVE: Investigate the molecular mechanisms involved in N1 and N2 signaling which contribute to melanoblast maintenance and are involved in mature melanocytes. METHODS AND RESULTS: Using a genetic Cre-lox approach we deleted N1 and N2 in melanocytes and melanoblasts (TyrCre::N1lox/lox,N2lox/lox). Subsequently, melanoblasts deficient for both N1 and N2 were isolated by FACS sorting from E16.5 embryos. Melanoblasts, identified as CD117+CD45- cell population, showed a significant reduction in the absolute numbers of cells isolated from N1N2 deficient embryos as compared to littermate control embryos. So far, the downstream target genes of Notch signaling involved in melanoblasts maintenance have not been identified. Therefore, we performed microarray analysis on N1N2 deficient melanoblasts and wild type (WT) controls. The array results are currently validated using qRT-PCR. Since Notch deficient immature melanocytes (P1 to P3) are not viable we utilized an established in vitro assay to generate mature melanocytes carrying N1lox/loxN2lox/lox alleles. Using a lentiviral approach to transduce the mature N1lox/loxN2lox/lox melanocytes with CreERT2 construct, we will be able to identify and evaluate the molecular mechanism involved in Notch signaling in these cells. The deletion of both N1 and N2 was confirmed by Southern blot analysis and deletion PCR. In addition, qRT-PCR results showed a significant downregulation of Hes1. Our results revealed that there is no change in the proliferation rate in both WT and Notch deficient mature melanocytes. We also analyzed WT and Notch deficient melanocytes for changes in cell cycle progression and apoptosis and could not detect a significant difference between the two cell populations assessed. In addition, the cell adhesion properties between WT and N1N2 deficient mature melanocytes were not affected. CONCLUSION: Overall our results show that although Notch signaling is dispensable for the survival of mature melanocytes but it is required for melanocytes during embryonic stages.

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