Mutations at the dominant white spotting (W) locus of the mouse have profound effects upon the development of pigment cells, germ cells and hematopoietic cells. The hematopoietic deficiency can be expressed in the affected homozygous W mutant mice (W*/W* mice) as an anemia and a marked deficiency of tissue mast cells. However, immature mast cells can be generated in vitro from hematopoietic cells of either W*/W* mice or the congenic normal (+/+) animals. Mast cells therefore represent the only lineage affected by W mutations which can be expanded in vitro to generate the large numbers of phenotypically homogeneous normal or mutant cells required for detailed functional or biochemical analyses. We and others have shown that W mutant mast cells generated in vitro exhibit predictable abnormalities in proliferation, maturation or survival in vitro or in vivo. For example, W/Wo mast cells derived in vitro, unlike cultured mast cells derived from the congenic normal (+/+) mice, exhibit very limited longevity in suspension culture with IL-3, fail to enter S- phase when placed on 3T3 fibroblasts in vitro in the absence of IL-3 and fail to survive when injected into the peritoneal cavity of W/Wo mice. This evidence is consistent with the possibility that one important function of the W products is to influence mast cell development by regulating interactions between the mast cell lineage and certain stromal cells (such as fibroblasts) in the tissues where mast cell maturation occurs. Recently, we demonstrated that two spontaneous W mutations, W44 and Wx, resulted in disruption of the gene for the c-kit tyrosine kinase receptor. In addition to representing the first example of a germ line mutation in a mammalian proto-oncogene, this finding suggested a molecular basis for the effects of W mutations on mast cell development. However, the mechanisms by which different W mutations produce different effects on mast cell development have not yet been elucidated. For example, while most mice homozygous for mutations at W exhibit variable degrees of mast cell deficiency, W44/W44 mice have normal levels of tissue mast cells. We therefore propose to test the hypothesis that the distinct phenotypic effects of specific W c-kit mutations on mast cell development reflect different consequences of these mutations for c-kit production, structure or function. Specifically, we will characterize the effects of five different W mutations on the structure and expression of the c-kit gene; clone the structural and regulatory elements of the wild-type c-kit gene and use them to produce c-kit expression vectors; examine the effects of specific W mutations on mast cell development; and identify the role of c- kit in this process by manipulating c-kit expression in cultured mast cells from normal and W mutant mice.
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