During the past year, we have worked to complete research on two topics in the regulation of melanocyte development and differentiation as the lab undergoes a transition to focus upon melanoma research: (1) understanding the role of neurofibromin in melanocyte development and differentiation, and (2) examining survival of otic melanocytes in the Microphthalmia-white heterozygous mouse, a model for human Waardenburg and Tietz syndromes. Our work on developing an inducible system for gene expression in vivo in murine melanocytes, described in this section last year, is described under Project 2 this year because it is more relevant to our work on malignant melanoma than to work we are completing on melanocyte differentiation.Our progess is as follows:(1) We have worked further this year to define differences between the activity of neurofibromin on the Kit signalling pathway during melanocyte development and during melanocyte differentiation. To test in a more direct way the hypothesis that neurofibromin regulates melanogenic gene expression via its effects upon Ras signalling, we have successfully optimized a system to purify primary mouse melanocytes by flow cytometry. This approach has permitted us to compare directly the effect of neurofibromin haploinsufficiency upon melanogenic gene expression without accounting for variables introduced by the presence of other types of cells in culture. Results from these experiments show that neurofibromin haploinsufficiency, which is associated with Erk activation in cultured melanocytes, increases melanogenic gene expression in primary murine melanocytes. Consistent with this finding, pharmacologic inactivation of Erk activation by inhibition of the upstream kinase Mek decreases melanogenic gene expression in both wild-type and neurofibromin-haploinsufficient melanocytes. These results, in addition to those obtained from primary melanocytes in mixed primary cultures, support a role for Ras-dependent signaling in melanogenic gene expression. Additional results from the genetic crossing of Nf1-deficient mice with murine coat color mutations in the Kit and Mitf genes support a role for neurofibromin in the regulation of melanocyte development in vivo. (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 stem cell factor (SCF)/Kit ligand, endothelin-1 (ET-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. Going further, organ culture experiments with isolated stria vascularis that has been isolated from neonatal mice show that the combination of ET-1 and SCF can promote survival of these Mitf-deficient melanocytes in the strial environment. These results show that environmental factors in or near the murine follicle are likely to be important for promoting the survival of Mitf-deficient melanocytes in this location. This finding may be relevant to the survival of melanocytes in patients with the congenital disorders of pigmentation Waardenburg syndrome and Tietz syndrome.
