Craniofacial Morphogenesis in Prenatal Alcohol Exposure

产前酒精暴露中的颅面形态发生

• 批准号: 6629611
• 负责人: SUSAN M. SMITH
• 金额: $ 31.15万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629611
• 关键词: G protein; apoptosis; biological signal transduction; bone disorder; calcineurin; calcium channel; calcium flux; calmodulin dependent protein kinase; confocal scanning microscopy; craniofacial; diacylglycerols; embryo /fetus toxicology; embryogenesis; enzyme activity; ethanol; fetal alcohol syndrome; high performance liquid chromatography; histogenesis; inositol phosphates; neural crest; pathologic process; phospholipase C; protein tyrosine kinase; thin layer chromatography

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.

产前酒精暴露（PAE）是智力低下和先天缺陷的主要原因。 它的致膜性是由于关键细胞种群中凋亡的一部分而起源。 在先前的NIAAA支持下，我们开发了一种PAE的雏鸡胚胎模型，并表明乙醇会导致神经crest的选择性凋亡，神经crest是一种含有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细胞内Ca2+的磷脂酶C依赖性释放来诱导神经rest凋亡。 该假设是使用药理学方法检验的，利用我们可以将Ca2+信号通路的激动剂和拮抗剂靶向到OVO中的前移民神经波峰，从而测试其衰减乙醇对胚胎的影响的能力。 该提案中的实验测试了三个亚问题：1）急性乙醇通过刺激细胞内Ca2+的释放来诱导神经rest凋亡。 通过使用荧光Ca2+指示剂，Ca2+离子载体和螯合剂以及下游，Ca2+依赖性信号蛋白（CAM激酶，钙调神经酶）的直接测定，对Ca2+释放的需求进行了测试。 2）乙醇通过激活三磷酸肌醇（IP3）受体动员细胞内Ca2+并诱导凋亡。 药理学激动剂和拮抗剂测试乙醇释放的Ca2+源自IP3介导的商店。此IP3版本将直接量化。 还测试了Ryanodine受体和细胞外Ca2+的潜在贡献。 3）乙醇激活磷脂酶C（PLC）是CA2+释放和神经rest凋亡的原因。 PLC激活通过直接测定和靶向抑制剂来测量。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蛋白的贡献。这些结果涉及神经rest对乙醇敏感性的分子机制，从而有助于理解乙醇的致膜性基础。 结果还可能为基于遗传的胎儿反应对产前乙醇暴露的反应提供可能的解释。