Craniofacial Morphogenesis in Prenatal Alcohol Exposure

描述（由申请人提供）：酒精是一种强效神经毒物，产前酒精暴露是导致智力迟钝的主要已知原因。一个受影响的群体是神经嵴。我们过去的工作发现，临床相关的乙醇暴露（20-80 mM）引起神经嵴细胞凋亡;一个迄今无法解释的观察是，这种死亡的时间与这些细胞的内源性凋亡相一致。我们目前的裁决确定，乙醇导致细胞内Ca 2+瞬变，这是必要的，足以引起神经嵴凋亡。这种Ca 2+瞬变源于乙醇对Gai 2/3的刺激和GPy介导的PI-PLC的激活。我们最近发现，乙醇及其Ca 2+瞬变快速抑制神经嵴内的经典Wnt/β-连环蛋白信号;这些为神经嵴提供必要的营养支持并支配其形态发生。相反，在神经嵴中由乙醇激活的G蛋白/PI-PLC/Ca通路会聚在一个独特的Wnt通路上，称为非经典Wnt/Ca通路，其使用G蛋白和PI-PLC介导的Ca 2+释放来抑制经典Wnt信号并引起神经嵴的内源性细胞死亡。在这种竞争性的更新测试的假设，乙醇刺激的Ca 2+瞬变是凋亡，因为它抑制了Wnt/S-连环蛋白信号，提供营养支持的神经嵴的研究。我们进一步提出，乙醇诱导的Ca 2+瞬变收敛并激活非经典Wnt/Ca信号，同样抑制3-连环蛋白，导致内源性神经嵴死亡。目的1测试乙醇诱导的Ca 2+瞬变是否抑制了控制神经嵴发育和存活的Wnt/β-连环蛋白信号。我们将研究Wnt/β-连环蛋白提供的营养支持的丧失是否有助于它们的凋亡。目的2测试乙醇对Wnt/β-连环蛋白的抑制是否由钙调蛋白依赖性激酶II（CaMKII）介导，所述钙调蛋白依赖性激酶II将Ca 2+瞬变转化为促凋亡细胞信号。目的3检测乙醇诱导的神经嵴中的Ca 2+瞬变是否破坏了Wnt/β-连环蛋白并导致细胞凋亡，因为它聚集并激活了控制神经嵴同时发生的内源性细胞死亡的Wnt/Ca信号。这些研究是当前奖项的合理延伸。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，具有良好描述的神经嵴发育，并且现在可以使用功能获得或siRNA表达载体的电泳转染进行遗传操作。Wnt活性在脊椎动物中高度保守。它们是不同组织中细胞生长和分化的重要调节因子，这提高了Wnt代表胎儿和成人中酒精作用的新靶点的可能性。

DESCRIPTION (provided by applicant): Alcohol is a potent neurotoxicant, and prenatal alcohol exposure is the leading known cause of mental retardation. One affected population is the neural crest. Our past work found that clinically relevant ethanol exposures (20-80 mM) cause neural crest apoptosis; a hitherto unexplained observation is that the timing of this death coincides with the endogenous apoptosis of these cells. Our current award established that ethanol causes an intracellular Ca2+ transient that is necessary and sufficient to cause neural crest apoptosis. This Ca2+ transient originates from ethanol's stimulation of Gai2/3, and Gpy-mediated activation of PI-PLC. We recently discovered that ethanol and its Ca2+ transient rapidly suppress canonical Wnt/p- catenin signals within neural crest; these provide essential trophic support to neural crest and govern their morphogenesis. Conversely, the G protein/PI-PLC/Ca pathway that is activated in neural crest by ethanol converges on a distinct Wnt pathway, known as the non-canonical Wnt/Ca pathway, that uses G protein and PI-PLC-mediated Ca2+ release to repress canonical Wnt signals and cause the endogenous cell death of neural crest. Studies in this competing renewal test the hypothesis that the ethanol-stimulated Ca2+ transient is apoptotic because it suppresses the Wnt/S-catenin signals that provide trophic support to the neural crest. We further propose that the ethanol-induced Ca2+ transient converges on and activates the noncanonical Wnt/Ca signals that similarly repress 3-catenin to cause endogenous neural crest death. Aim 1 tests whether the ethanol-induced Ca2+ transient suppresses the Wnt/(3-catenin signals that govern neural crest development and survival. We will investigate whether the loss of trophic support provided by Wnt/p-catenin contributes to their apoptosis. Aim 2 tests whether ethanol's suppression of Wnt/p-catenin is mediated by calmodulin-dependent kinase II (CaMKII), which converts the Ca2+ transient into a pro-apoptosis cellular signal. Aim 3 tests whether the ethanol-induced Ca2+ transient in neural crest disrupts Wnt/p-catenin and causes apoptosis because it converges on and activates the Wnt/Ca signals that govern the coincident endogenous cell death of neural crest. These studies are a logical extension of the current award. We continue to use our established chick embryo model, which replicates the alcohol responses of mammals including humans, has well-described neural crest development, and is now accessible for genetic manipulation using electroporetic transfection of gain- of-function or siRNA expression vectors. Wnt activities are highly conserved among vertebrates. They are important regulators of cell growth and differentiation in diverse tissues, raising the possibility that Wnts represent a novel target for alcohol action in the fetus and the adult.