Mechanisms and Pathogenesis of Ethanol-induced CNS Abnormalities in Zebrafish

酒精（乙醇）是一种致畸剂，已知对大脑和颅面发育有不同的影响。 最近的研究提供了强有力的支持干扰声刺猬（Shh）信号作为胎儿酒精谱系障碍（FASD）的关键分子事件。然而，Shh干扰的机制是未知的，所产生的异常表型仍然不完全确定。对于目前的研究，我们的总体目标是确定乙醇介导的对细胞外基质（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性的基础，以及 记录这种干扰导致的畸形。这项创新和新颖的工作将集中在聚集蛋白，一个主要的基底膜和跨膜HSPG，我们的实验室有丰富的研究经验。它将采用一个模型系统，特别是对识别乙醇的致畸机制，发展中的斑马鱼。先前的研究表明，斑马鱼胚胎暴露于乙醇会导致大脑和眼睛的畸形，以及行为缺陷。在以前的研究中，我们的实验室已经表明，斑马鱼聚集蛋白功能的丧失会导致小眼症。 这似乎是由破坏的FGF和Shh信号转导介导的。我们的初步研究还表明，聚集蛋白基因表达在斑马鱼的眼睛减少响应乙醇暴露，并建议，聚集蛋白敲低和乙醇暴露可能有一个共同的途径，以产生乙醇介导的眼部缺陷。因此，该提议的基本假设是聚集蛋白是胚胎乙醇暴露的CNS靶标，其中聚集蛋白功能被扰乱导致乙醇暴露后干扰正常Shh和/或Fgf信号传导。该假设将在以下具体目的中进行检验：1）检验胚胎乙醇暴露和聚集蛋白功能丧失在斑马鱼中诱导的CNS表型是可比的假设; 2）检验乙醇诱导的聚集蛋白基因表达减少破坏Shh和/或Fgf信号传导，最终导致与FASD相关的发育异常的假设;和3）检验斑马鱼胚胎乙醇暴露破坏GABA能和多巴胺能神经元分化作为Shh、Fgf和/或聚集蛋白功能紊乱的结果的假设。 最终，这些研究将开始提供深入了解FASD的分子基础，使用斑马鱼作为一种 为FASD的研究提供了新的模型。

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.