Seven human disorders involving enzyme defects in post-squalene cholesterol biosynthesis have been identified. All are associated with major developmental malformations, demonstrating the importance of the cholesterol metabolic pathway for normal embryonic development. The mechanisms of disease pathogenesis for any of these disorders remain unknown. Nsdhl is a 3beta-hydroxysteroid dehydrogenase involved in the removal of C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in this X-linked gene are associated with the male lethal mouse mutation bare patches (Bpa), as well as most cases of human CHILD syndrome. The long term goal of this research is to understand the role of cholesterol and its biosynthetic intermediates in mammalian developmental processes. During the current funding period, we determined that the male lethality for several murine Nsdhl mutant alleles results from early placental insufficiency, with a lack of invasion and proliferation of fetal vessels derived from allantoic mesoderm. Further, transgenic mice that express an Nsdhl cDNA construct (ploxNsdhl) die perinatally with hydrops, shortened limbs, and placental abnormalities. We hypothesize that signaling by hedgehog or wnt proteins, that undergo covalent cholesterol and/or lipid modification as part of their intracellular processing, is disrupted in Nsdhl deficient cells. To test this hypothesis, the current proposal has 3 specific aims: 1. We will determine the molecular mechanisms associated with the male lethality by characterizing defects for the most severe Bpalli Nsdhl allele and those in the yolk sac vessels and in cultured allantois explants from affected Nsdhf male embryos. 2. We will examine hedgehog and wnt signaling in cultured embryonic fibroblasts from affected embryos, as well as examine lipid rafts and trafficking in the mutant cells. 3. We will examine the higher expression of Nsdhl in the developing axial skeleton and limb bud in normal embryos, in transgenic embryos that express the ploxNsdhl construct, and in embryos containing a chondrocyte-specific targeted Nsdhl allele. Using transgenic mice containing a lacZ reporter under the control of the Nsdhl promoter, we will identify cisacting regulatory elements responsible for the enhanced gene expression in developing chondrocytes.
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