This research project is designed to increase our understanding of the mechanisms and molecular pathology underlying alcohol-induced dysmorphogenesis. It follows up on our previous identification of selected regions of the brain, otic, and optic primordial as targets of ethanol-induced apoptosis and subsequent birth defects, results of our ongoing microarray analyses, and significant contributions to this field by others. Focusing on Developmental stages in mice that correspond to weeks 3-6 of human embryogenesis and employing in situ hybridization, we will test the hypothesis that within hours following exposure to ethanol, abnormal expression of patterning genes occurs in the embryonic brain, eye and inner ear; changes that presage subsequent dysmorphogenesis. To elucidate pathogenic sequences, the time by which altered patterning is detectable will be compared to that for which apoptosis can initially be identified. Analyses of temporally and regionally-specific alterations in mouse embryogenesis, will be facilitated by utilization of an acute ethanol exposure paradigm, whole embryo culture, and laser confocal imaging. Recognizing that ethanol exposure can interfere with retinoid metabolism and that retinoic acid (RA) regulates gene expression; we will also examine the hypothesis that diminishing RA-dependent gene signaling underlies ethanol's teratogenicity. For this work, we will compare gene expression patterns and patterns of apoptosis in the developing brain, eye and innerear of retinoid-deficient (BMS493-treated)and ethanol-exposed mouse embryos and test RA's ameliorative potential. Additionally, we propose to conduct in vitro and in vivo investigations to test the hypothesis that altered genetic (esp. sonic hedgehog) signaling stemming from ethanol-induced RA deficiency will exacerbate diminished signaling subsequent to cholesterol reduction. These experiments will utilize both pharmacologically (AY9944) and genetically (7 dehydrocholestrol reductase gene modification) - induced cholesterol deficiency, both of which yield malformations consistent with those caused by ethanol. This work is expected to provide important new data relative to factors that may influence sensitivity to ethanol-induced teratogenesis in human populations. It will also establish a foundation for similar analyses to be utilized in following up on other developmental stages and vulnerable tissues.
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