Craniofacial Morphogenesis in Prenatal Alcohol Exposure

产前酒精暴露(PAE)是已知的导致智力低下和出生缺陷的主要原因。它的致畸作用部分源于它在关键细胞群体中启动了细胞凋亡。在NIAAA的支持下，我们建立了PAE的鸡胚模型，并表明乙醇导致神经脊的选择性凋亡，神经脊是一个包含颅面和神经元前体的胚胎细胞群。在这里，我们假设乙醇通过激活依赖于磷脂酶C的细胞内钙释放来诱导神经脊细胞凋亡。这一假说是用药理学方法验证的，利用我们可以将钙信号通路的激动剂和拮抗剂局部靶向卵子中的迁移前神经峰，从而测试它们减弱乙醇对胚胎的影响的能力。这一方案中的实验验证了三个亚假设：1)急性乙醇通过刺激细胞内钙离子的释放而诱导神经脊细胞凋亡。通过使用荧光钙指示剂、钙离子载体和螯合剂，以及直接测定下游依赖钙的信号蛋白(钙调素激酶、钙调神经磷酸酶)来测试对钙释放的需求。2)乙醇通过激活三磷酸肌醇(IP3)受体，激活细胞内钙离子，诱导细胞凋亡。药理激动剂和拮抗剂测试乙醇释放的钙离子来自IP3介导的存储；这种IP3的释放将直接定量。兰尼定受体和细胞外钙离子的潜在贡献也被测试。3)乙醇对磷脂酶C(PLC)的激活参与了细胞内钙离子的释放和神经脊细胞的凋亡。PLC的激活通过直接测定法和靶向抑制物来测量。研究了甘油二酯、受体介导的酪氨酸激酶和受体介导的G蛋白的作用。这些结果解决了神经脊对乙醇敏感的分子机制，从而有助于理解乙醇致畸的基础。这一结果也可能为胎儿对产前酒精暴露的反应的遗传差异提供可能的解释。

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.