Craniofacial Morphogenesis in Prenatal Alcohol Exposure

产前酒精暴露（PAE）是已知的导致智力迟钝和出生缺陷的主要原因。其致畸性部分源于其在关键细胞群中引发细胞凋亡。在之前NIAAA的支持下，我们建立了鸡PAE胚胎模型，并表明乙醇导致神经嵴（一种含有颅面和神经元前体的胚胎细胞群）的选择性凋亡。在这里，我们假设乙醇通过激活磷脂酶c依赖的细胞内Ca2+释放来诱导神经嵴凋亡。这一假设是用药理学方法来验证的，利用我们可以局部靶向Ca2+信号通路的激动剂和拮抗剂到卵内的预迁移神经嵴，从而测试它们减弱乙醇对胚胎影响的能力。本实验验证了三个子假设：1)急性乙醇通过刺激细胞内Ca2+的释放诱导神经嵴凋亡。对Ca2+释放的需求是通过使用荧光Ca2+指示剂、Ca2+离子载体和螯合剂，以及下游Ca2+依赖信号蛋白（CaM激酶、钙调磷酸酶）的直接测定来测试的。2)乙醇通过激活三磷酸肌醇（IP3）受体，激活细胞内Ca2+，诱导细胞凋亡。药理激动剂和拮抗剂测试乙醇释放的Ca2+来源于ip3介导的储存；本次IP3发布将直接量化。ryanodine受体和细胞外Ca2+的潜在贡献也进行了测试。3)乙醇激活磷脂酶C （PLC）参与Ca2+释放和神经嵴凋亡。PLC的激活是通过直接测定和靶向抑制剂来测量的。研究了二酰基甘油、受体介导的酪氨酸激酶和受体介导的G蛋白的作用。这些结果阐明了神经嵴对乙醇敏感的分子机制，从而有助于理解乙醇致畸性的基础。结果也可能为胎儿对产前乙醇暴露反应的遗传变异提供可能的解释。

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.