Nf1 Modulates C-kit Signaling in Mast Cells and Neural Crest Derived Melanocytes in vivo in a Dose Dependent Fashion

Abstract 303 Development Biology Platform, Sunday, 5/2

Neurofibromin, the protein encoded by NF1, functions as a tumor suppressor protein by negatively regulating Ras activity in mammalian cells. Mutations of the NF1 gene cause neurofibromatosis, a disorder characterized by the development of cutaneous melanocyte hyperplasia (cafe au lait spots), neurofibromas containing mast cells, and malignant neoplasms arising from embryonic neural crest cells. Genetic and biochemical data from children with NF1 have demonstrated that loss of heterozygosity (LOH) of the NF1 allele is linked to the development of juvenile myelomonocytic leukemia. Interestingly, no consistent demonstration of LOH has been shown in other tissues affected in NF1, and the function of NF1 at the cellular level in a heterozygous state is unclear. Recently, we have shown that neurofibromin negatively modulates signaling through the c-kit tyrosine kinase receptor (W locus) in murine hematopoietic cells in vitro. To further investigate this in vitro observation, we generated mice with mutations at both the W and Nf1 loci to examine potential interactions in vivo. Mice with germline mutations of the W allele demonstrate mast cell deficiency, anemia and abnormal melanocyte development (white coat color). Mice homozygous for mutations of the murine homologue of NF1 (Nf1) die in utero, and mice heterozygous for the NF1 allele are viable but lack a distinct phenotype. Since the W locus and NF1 locus appear to function along a common developmental pathway, we predicted a partial correction of the W phenotype in mice carrying mutations at both alleles secondary to increased Ras activity with reduced levels of neurofibromin. In comparison to W-/- ; Nf1 +/+ mice, W -/- ; Nfl +/- mice demonstrate a 60-70% restoration of coat color (n = 158), a significant increase in mast cell numbers harvested from peritoneal lavages (574 ± 154 cells/ml vs. 37 ± 24 cells/ml, n = 6), and a six-fold increase in dermal mast cell numbers (n = 6). Cultures for growth of mast cell progenitors from lavages of W mice fail to form colonies in vitro. In contrast, mast cell progenitors from peritoneal lavages of W -/- ; Nf1 +/- mice produce significant numbers of colonies (72 ± 11 colonies/ml, n = 3). Taken together, these results demonstrate that neurofibromin regulates signaling through the c-kit receptor in murine melanocytes and mast cells in vivo and in vitro in a dose dependent fashion. This observation suggests that haploinsufficiency of NF1 and increased output of Ras proteins through the c-kit receptor may significantly contribute to the dominant clinical features associated with neurofibromatosis.

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