Alcohol abuse during pregnancy can harm fetal development, but genotype-dependent differences in susceptibility have been documented in animal models. The mechanisms of alcohol teratogenesis and the molecular basis of genetically mediated differences in susceptibility are not known. Studies of the genomic response to alcohol can shed light on the molecular mechanisms of pathogenesis of this disorder, and may also provide insight into genetic influences on susceptibility. In this component, the C57BL/6 [B6] inbred mouse strain, known to be susceptible to alcohol-induced teratogenic effects, will be used to identify and confirm sets of genes associated with alcohol-induced teratogenesis during the critical developmental period of early neurulation. The proposed studies will use the embryo explant culture model, which affords precision and control of the timing and dose of the alcohol exposure during specific embryonic stages. Morphological, genomic, proteomic, and confirmatory studies will be used to identify candidate genes regulating pathogenesis in B6 embryos. The embryonic genes contributing to susceptibility to alcohol induced teratogenesis will be identified at two time points (E8+lday; E8+2days) using DNA microarrays and expression proteomics. Differential gene expression will be confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Confirmed genes, in turn, will be localized to tissue sites using in situ hybridization (ISH) to correlate the localization with specific sites of teratogenic defects. Molecular screens will also be performed in DBA/2 (D2) embryos (or in C3H or CBA embryos), known to be resistant to ethanol teratogenesis in utero. Parallel in utero alcohol exposure will be evaluated in each strain to compare to effects in embryo cultures. These will determine whether strains that differ in susceptibility to alcohol teratogenesis in utero (potentially involving both maternal and embryonic factors), also differ in susceptibility to direct effects of ethanol on the embryo (excluding maternal factors). The long-term goal is to identify the pharmacogenomic basis for pathogenesis mechanisms that contribute to genetic differences in embryonic susceptibility to teratogenic effects of ethanol.
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