Craniofacial Morphogenesis in Prenatal Alcohol Exposure

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Prenatal alcohol exposure (PAE) is the leading known cause of mental retardation and birth defects. Its teratogenicity originates, in part, through its initiation of apoptosis in critical cell populations. Under previous NIAAA support, we developed a chick embryo model of PAE and showed that ethanol causes the selective apoptosis of the neural crest, an embryonic cell population containing craniofacial and neuronal precursors. Here, we hypothesize that ethanol induces neural crest apoptosis by activating the phospholipase C-dependent release of intracellular Ca2+. This hypothesis is tested using pharmacologic approaches, taking advantage that we can locally target agonists and antagonists of Ca2+ signaling pathways to the premigratory neural crest in ovo, and thus test their ability to attenuate ethanol's effects upon the embryo. Experiments in this proposal test three sub-hypotheses: 1) Acute ethanol induces neural crest apoptosis by stimulating the release of intracellular Ca2+. The requirement for Ca2+ release is tested through the use of fluorescent Ca2+ indicators, Ca2+ ionophores and chelators, and the direct assay of downstream, Ca2+-dependent signaling proteins (CaM kinase, calcineurin). 2) Ethanol mobilizes intracellular Ca2+ and induces apoptosis by activating inositol triphosphate (IP3) receptors. Pharmacologic agonists and antagonists test that the ethanol-released Ca2+ originates from IP3-mediated stores; this IP3 release will be quantified directly. Potential contributions of ryanodine receptors and extracellular Ca2+ also are tested. 3) Ethanol's activation of Phospholipase C (PLC) is responsible for the Ca2+ release and neural crest apoptosis. PLC activation is measured by direct assay and by targeted inhibitors. Contributions of diacylglycerol, receptor-mediated tyrosine kinases, and receptor-mediated G proteins are investigated. these results address the molecular mechanism underlying the neural crest's sensitivity to ethanol, and, thus, contribute to understanding the basis for ethanol's teratogenicity. Results also may provide possible explanations for genetic-based variations in fetal responses to prenatal ethanol exposure.