Platelet derived growth factors (PDGFs) are required for vascular development, cranial and cardiac neural crest cells, somitic mesoderm, extraembryonic lineages and in the kidney. The mechanisms by which they control cell proliferation, survival and migration have been deciphered in cultured cells, but the identity of the target genes that mediate such pleiotropic functions in development and the adult remain unknown. This proposal focuses on identifying PDGF targets using a novel platform, the gene trap array, in which thousands of cDNAs derived from gene trap disrupted loci in ES cells are spotted on DNA arrays. This approach combines all of the power of DNA array technologies with the possibility of readily generating mutant mice from the frozen ES cell stocks. The physiological roles of a subset of regulated genes will be assessed by deriving mutant mice from the trapped ES cell clones. Target genes will be further characterized by establishing if they are specific for one or the other PDGFR and if they are subject to regulation by other signaling pathways. Conditional gene trap vectors will be generated that allow spatio-temporal elimination of gene activity and the generation of allelic series at the trapped loci. The Gene Trap Array will be expanded to 10,000 mutant ES cell clones and genes mutated by these vectors will be identified by sequencing and their identity will be listed on a web-based platform. Critical components of PDGF signaling will be identified in neural crest cells, using two complementary approaches, the gene trap array or gain-of-function gene trap mutagenesis in a PDGFalphaR sensitized background. Gain-of-function alleles that synergize with a PDGFalphaR mutation will be converted to loss of function alleles to study the normal function of the gene. These studies may shed insight on birth defects associated with abnormal neural crest development, such as cleft palate or DiGeorge Syndrome.
Showing the most recent 10 out of 51 publications
