Craniofacial Morphogenesis in Prenatal Alcohol Exposure

酒精是一种强有力的神经毒物，产前酒精暴露是导致精神残疾的主要原因。一 受影响的人群是神经嵴。临床相关乙醇水平（20-80 mM）导致神经嵴（NC） 凋亡我们过去在该奖项下的工作表明，细胞凋亡是由乙醇刺激的 细胞内钙（Ca 2+）瞬变源自CPy介导的PLC-IP 3活化。当前 该奖项表明，Ca 2+瞬时激活CaMKII，这反过来又使转录效应子不稳定， β-连环蛋白，其介导经典Wnt信号传导和NC存活。建议的研究直接扩展了这一点 工作，以问“如何β-连环蛋白损失导致神经嵴凋亡？“我们将测试beta- 连环蛋白维持NC细胞粘附和生存因子snail 2（以前称为slug）的表达。我们进一步 证实乙醇介导的β-连环蛋白的缺失引发了NC细胞过早分层，并抑制了NC细胞的凋亡。 snail 2，从而激活NC凋亡。目的1测试snail 2丢失是否直接启动NC凋亡， 它控制凋亡调节因子bcl 2和bax的表达。目的2测试β-连环蛋白在NC中的作用 细胞粘附以及β-连环蛋白缺失是否刺激早熟NC分层和凋亡。目标3 扩展了我们的工作，以测试急性乙醇暴露是否同样使β-连环蛋白及其转录不稳定。 β-连环蛋白控制细胞命运的其他神经元群体，特别是胎儿脑和成人脑中的活性 海马体。最后，我们的ARRA补充执行高通量RNA-Seq的乙醇敏感性和- 耐药NC以鉴定控制NC存活的β-连环蛋白上游的钙依赖性因子。目标4 测试了这些候选基因在控制NC对乙醇的反应中的功效。这项工作是一个逻辑 延长目前的裁决。我们继续使用我们建立的鸡胚模型，该模型复制了 包括人类在内的哺乳动物的酒精反应，已经充分描述了NC的发展，并且适合于 基因操纵。β-连环蛋白失调导致了几种神经病理学，这表明它可能 还调节胎儿和成人的酒精作用。

Alcohol is a potent neurotoxicant and prenatal alcohol exposure is a leading cause of mental disability. One affected population is the neural crest. Clinically relevant ethanoi levels (20-80 mM) cause neural crest (NC) apoptosis. Our past work under this award showed that the apoptosis results from an ethanol-stimulated intracellular calcium (Ca2+) transient originating from the CPy-mediated activation of PLC-IP3. The current award shows that the Ca2+ transient activates CaMKII, which in turn destabilizes the transcriptional effector beta-catenin, which mediates canonical Wnt signaling and NC survival. Proposed studies directly extend this work to ask "How does beta-catenin loss cause neural crest apoptosis?" We will test the hypothesis that beta- catenin maintains NC cell adhesion and expression ofthe survival factor snail2 (formerly slug). We further posit that the ethanol-mediated loss of beta-catenin initiates premature NC cell delamination and suppresses snail2, thus activating NC apoptosis. Aim 1 tests whether snail2 loss directly initiates NC apoptosis because it controls the expression ofthe apoptosis regulators bcl2 and bax. Aim 2 tests the role of beta-catenin in NC cell adhesion and whether beta-catenin loss stimulates precocious NC delamination and apoptosis. Aim 3 extends our work to test if acute ethanoi exposure similarly destabilizes beta-catenin and its transcriptional activity in other neuronal populations in which p-catenin controls cell fate, specifically the fetal brain and adult hippocampus. Finally, our ARRA supplement performs high-throughput RNA-Seq of ethanol-sensitive and - resistant NC to identify calcium-dependent factors upstream of beta-catenin that govern NC survival. Aim 4 tests the efficacy of these candidate genes in controlling NC responses to ethanoi. This work is a logical extension ofthe current award. We continue using our established chick embryo model, which replicates the alcohol responses of mammals including humans, has well-described NC development, and is amenable to genetic manipulation. Beta-Catenin dysregulation contributes to several neuropathologies, suggesting it may also modulate alcohol action in the fetus and the adult.