Craniofacial Morphogenesis in Prenatal Alcohol Exposure

描述（由申请人提供）：酒精是一种强效的神经毒物，产前酒精暴露是已知的导致智力迟钝的主要原因。受影响的人群之一是神经嵴。我们过去的研究发现，临床相关的乙醇暴露（20-80 mM）会导致神经嵴凋亡；一个迄今尚未解释的观察结果是，这种死亡的时间与这些细胞的内源性凋亡一致。我们目前的研究发现，乙醇引起细胞内Ca2+瞬变，这是引起神经嵴凋亡的必要和充分条件。这种Ca2+瞬态源于乙醇对Gai2/3的刺激，以及gpy介导的PI-PLC的激活。我们最近发现乙醇及其Ca2+瞬态能快速抑制神经嵴内典型的Wnt/p- catenin信号；这些细胞为神经嵴提供必需的营养支持并控制其形态发生。相反，在神经嵴中被乙醇激活的G蛋白/PI-PLC/Ca通路会聚在一个不同的Wnt通路上，称为非规范Wnt/Ca通路，该通路利用G蛋白和PI-PLC介导的Ca2+释放来抑制规范Wnt信号，导致神经嵴内源性细胞死亡。这一竞争性更新的研究验证了乙醇刺激的Ca2+瞬态是凋亡的假设，因为它抑制了为神经嵴提供营养支持的Wnt/S-catenin信号。我们进一步提出，乙醇诱导的Ca2+瞬间聚集并激活非规范的Wnt/Ca信号，类似地抑制3-catenin导致内源性神经嵴死亡。目的1测试乙醇诱导的Ca2+瞬态是否抑制控制神经嵴发育和存活的Wnt/(3-catenin)信号。我们将研究Wnt/p-catenin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/p-catenin的抑制是否由钙调素依赖性激酶II （CaMKII）介导，CaMKII将Ca2+瞬态转化为促凋亡细胞信号。目的3检测乙醇诱导的神经嵴内Ca2+暂态是否会干扰Wnt/p-catenin并导致细胞凋亡，因为它会聚集并激活控制神经嵴内源性细胞同时死亡的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胎模型，该模型复制了包括人类在内的哺乳动物的酒精反应，已经很好地描述了神经嵴的发育，现在可以使用功能增益或siRNA表达载体的电泳转染进行遗传操作。Wnt的活性在脊椎动物中高度保守。它们是多种组织中细胞生长和分化的重要调节因子，这提高了wnt在胎儿和成人中代表酒精作用新靶点的可能性。

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.