During the past year, we have focused on three topics in the regulation of melanocyte development and differentiation: (1) understanding the role of neurofibromin in melanocyte development and differentiation, (2) examining survival of otic melanocytes in the Microphthalmia-white heterozygous mouse, a model for human Waardenburg and Tietz syndromes, and (3) developing an inducible system for gene expression in vivo in murine melanocytes. Our progess is as follows:(1) We have worked this year to define better differences between the activity of neurofibromin on the Kit signalling pathway during melanocyte development and during melanocyte differentiation. To confirm our notion that neurofibromin regulates melanogenic gene expression via mechanisms distinct from its effects as a negative regulator of Ras signalling, we have devoted effort to optimizing mouse primary melanocyte culture systems that will permit us to examine this question most fully. To this end, we have utilized cell surface markers that permit us to analyze primary cultures for the presence of melanocytes and to begin sorting primary melanocytes to obtain pure melanocyte cultures. These techniques will be helpful for examining the effects of Ras signalling pathway perturbations on melanogenic gene expression without ambiguities introduced by the presence of contaminating cell populations.(2) Our observation that the survival of otic melanocytes, as opposed to cutaneous melanocytes, is selectively compromised in Microphthalmia-white heterozygous mice prompted us to investigate the effect of skin-derived factors, such as Kit ligand, endothelin-1, and basic FGF that might promote otic melanocyte survival in this genetic background. A set of in vitro experiments using cochlear organ culture suggest that a combination of factors can promote survival of these otic melanocytes, providing additional insight into the determinants of melanocyte survival in cells partially deficient in the melanocyte transcription factor Mitf.(3) We have generated sets of transgenic founder mice expressing the tetracycline transactivators tTA and rtTA from the dopachrome tautomerase (Dct) promoter. We plan to characterize these transgenic lines for efficient and inducible expression and compare them to existing tyrosinase-rtTA mice for the timing of the onset of expression during embryogenesis and for persistent expression in melanocyte stem cells. Following characterization, we plan to use the mice to express activated Ras and activated Ras-signalling pathway components inducibly in mice to understand how Ras modulates melanocyte development and differentiation and to evaluate the use of these mice in murine melanoma models.
