Study of mouse mutants provides a rational approach to defining complex genetic and environmental factors leading to neural tube defects (NTDs). This program will investigate the cellular and molecular pathogenesis of the crooked tail (Cd) semidominant mutation. Preliminary data indicate that Cd is ameliorated by dietary folate, making it an important model for human NTDs. Linkage analysis has mapped the Cd locus to a 1 cM region of chr. 6, linked to the cell cycle regulatory gene D2 cyclin. The project will examine cellular events leading to brain malformation in Cd, identify the Cd gene defect, and elucidate its molecular consequences. In particular, it will test the hypothesis that Cd arises from defects in neural cell proliferation and neuronal migration. As part of positional cloning efforts, the project will also test the hypothesis that the D2 cyclin gene is mutated in Cd. First, continued investigation of dietary folate will verify and determine the extent of its effect on Cd NTDs and brain development. Second, morphogenesis of individual Cd embryos in utero will be examined in real time by high resolution MRI and brain histogenesis will be defined using markers of neuronal structure and CNS pattern formation. Third, the mechanism of the Cd phenotype will be determined, whether due to altered cell proliferation, neuronal migration, or programmed cell death; the ability of folate to reverse such abnormalities will be investigated.
Aims 1 -3 will provide the foundation of functional analyses of the Cd gene. Fourth, the Cd gene will be sought through positional cloning and testing identified candidates by: (a) linkage analysis to refine the critical region to 0.5 cM, (b) isolation of large insert genomic clones spanning the critical region, (c) cloning cDNAs corresponding to the genomic contigs, (d) analysis of candidate genes and Ests form the region for large and small mutations in Cd mice. To support a mutation as causative, BACs encompassing an entire candidate gene will be introduced into Cd mice to determine whether it will rescue the phenotype. Fifth, the function of Cd will be examined by structural analysis and then by in vitro studies of its molecular interactions with other NTD genes. Study of the genetic molecular and cellular events leading to abnormalities in Cd will expand understanding of NTDs and may provide strategies for prenatal assessment of risk and prevention of human brain maldevelopment.
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