Mechanisms and Pathogenesis of Ethanol-induced CNS Abnormalities in Zebrafish

酒精(乙醇)是一种致畸物质，已知对大脑和颅面发育有多种影响。最近的研究为干扰Sonic hedgehog(Shh)信号作为胎儿酒精谱障碍(FASD)的关键分子事件提供了强有力的支持。然而，Shh干扰的机制尚不清楚，由此导致的异常表型仍未完全确定。在目前的研究中，我们的总体目标是确定乙醇对细胞外基质(ECM)中关键形态发生蛋白(尤其是Shh)功能的干扰是否是乙醇致畸的基础，以及 记录了这种干扰造成的畸形。这项创新和新颖的工作将集中在集聚蛋白，一种主要的基底膜和跨膜热休克蛋白，我们的实验室有丰富的研究经验。它将使用一个模型系统，该系统对识别乙醇的致畸机制--发育中的斑马鱼--具有特殊的前景。此前的研究表明，将斑马鱼胚胎暴露在乙醇中会导致大脑和眼睛畸形，以及行为缺陷。在以前的研究中，我们的实验室已经证明斑马鱼集聚蛋白功能的丧失会导致小眼球。 这似乎是由中断的成纤维细胞生长因子和Shh信号调节的。我们的初步研究还表明，酒精暴露导致斑马鱼眼睛中agrin基因的表达减少，并提示agrin基因敲除和乙醇暴露可能共享一条共同的途径，从而产生乙醇介导的眼部缺陷。因此，这一建议的基本假设是，集聚蛋白是胚胎酒精暴露的中枢神经系统靶标，集聚蛋白功能受到干扰，导致乙醇暴露后正常的Shh和/或FGF信号受到干扰。这一假说将在以下特定目的进行检验：1)检验胚胎酒精暴露和集聚蛋白功能丧失诱导斑马鱼中枢神经系统表型具有可比性的假说；2)检验乙醇诱导集聚蛋白基因表达减少扰乱Shh和/或FGF信号，最终导致与FASD相关的发育异常的假说；以及3)检验由于Shh、FGF和/或集聚蛋白功能受扰而破坏斑马鱼胚胎乙醇暴露扰乱GABA能和多巴胺能神经元分化的假说。最终，这些研究将开始深入了解FASD的分子基础，将斑马鱼作为一种 FASD研究的新模型。

Alcohol (ethanol) is a teratogen known to have diverse effects on brain and craniofacial development. Recent studies provide strong support for interference with Sonic hedgehog (Shh) signaling as a critical molecular event in Fetal Alcohol Spectrum Disorder (FASD). However, the mechanism for Shh interference is unknown and resulting abnormal phenotypes remain incompletely defined. For the current investigation, our overall goal is to determine whether ethanol-mediated interference with the function of key morphogenic proteins (especially Shh) in the extracellular matrix (ECM) underlies ethanol's teratogenicity, as well as documenting the dysmorphology that results from this interference. This innovative and novel work will focus on agrin, a major basement membrane and transmembrane HSPG for which our laboratory has extensive research experience. It will employ a model system that holds particular promise for identification of ethanol's teratogenic mechanisms, the developing zebrafish. Previous studies have shown that exposure of zebrafish embryos to ethanol results in brain and ocular dysmorphology, as well as behavioral defects. In previous studies, our laboratory has shown that loss of agrin function in zebrafish leads to microphthalmia. This appears to be mediated by disrupted Fgf and Shh signaling. Our preliminary studies also demonstrate that agrin gene expression in zebrafish eyes is diminished in response to ethanol exposure, and suggest that agrin knockdown and ethanol exposure may share a common pathway to produce ethanol-mediated ocular defects. Thus, the underlying hypothesis of this proposal is that agrin is a CNS target of embryonic ethanol exposure, with perturbed agrin function resulting in interference with normal Shh and/or Fgf signaling following ethanol exposure. This hypothesis will be tested in the following specific aims: 1) To test the hypothesis that CNS phenotypes induced in the zebrafish by embryonic ethanol exposure and agrin loss-of-function are comparable; 2) To test the hypothesis that ethanol-induced diminution in agrin gene expression disrupts Shh and/or Fgf signaling, ultimately contributing to developmental abnormalities associated with FASD; and 3) To test the hypothesis that embryonic ethanol exposure in zebrafish disrupts GABAergic and dopaminergic neuron differentiation as a consequence of perturbed Shh, Fgf, and/or agrin function. Ultimately, these studies will begin to provide insight into the molecular basis of FASD, using zebrafish as a new model for the study of FASD.