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.
This project is relevant to public health as maternal alcohol use is a leading cause of mental retardation in the western world. This proposal will seek to understand the underlying mechanisms of the pathological basis of maternal alcohol abuse.
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