The initial experiments will involve primary cultures of neural crest cells isolated from E9.5 mice: one of the investigators (K.S.V.) has extensive experience with establishing neural crest cell cultures from avian and mammalian embryos. Our multiple primary embryonic cell cultures from genotyped individuals for exposure to several different growth factors (17). These studies also demonstrate a role for neurofibromin in the signalling pathway for the receptor tyrosine kinases encoded by the Trk gene family. Since the receptor for Steel factor, which is required for the survival and proliferation of melanocyte precursors (8), is also a receptor tyrosine kinase, our initial experiments with NF1 mutant neural crest cells will focus on the SLF/c-kit signalling pathway. To investigate the migratory and differentiative properties of NF1 mutant melanocyte precursors in vivo, we will transplant mouse NCC onto the dorsolateral NC migration pathway of chick embryos (K.S.V. and J.A. Weston, pers. comm.). Host embryos will continue to develop, and transplanted cells can be identified with mouse-specific probes (18) and visually, as they will presumably pigment in a White Leghorn host chick embryo. Transplantation to the NC migration pathways of chick embryos is an established and informative method to test the developmental potential an dispersive abilities of embryonic cell populations(19).
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