Agrin Function Associated with Ocular Development is a Target of Ethanol Exposure in Embryonic Zebrafish

BACKGROUND: Alcohol (ethanol) is a teratogen known to affect the developing eyes, face, and brain. Among the ocular defects in fetal alcohol spectrum disorder (FASD) are microphthalmia and optic nerve hypoplasia. Employing zebrafish as an FASD model provides an excellent system to analyze the molecular basis of prenatal ethanol exposure-induced defects because embryos can be exposed to ethanol at defined developmental stages and affected genetic pathways can be examined. We have previously shown that disruption of agrin function in zebrafish embryos produces microphthalmia and optic nerve hypoplasia. METHODS: Zebrafish embryos were exposed to varying concentrations of ethanol in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin function. In situ hybridization was used to analyze ocular gene expression as a consequence of ethanol exposure and agrin knockdown. Morphologic analysis of zebrafish embryos was also conducted. RESULTS: Acute ethanol exposure induces diminished agrin gene expression in zebrafish eyes and, importantly, combined treatment with subthreshold levels of agrin MO and ethanol produces pronounced microphthalmia, markedly reduces agrin gene expression, and perturbs Pax6a and Mbx gene expression. Microphthalmia produced by combined agrin MO and ethanol treatment was rescued by sonic hedgehog (Shh) mRNA overexpression, suggesting that ethanol-mediated disruption of agrin expression results in disrupted Shh function. CONCLUSIONS: These studies illustrate the strong potential for using zebrafish as a model to aid in defining the molecular basis for ethanol's teratogenic effects. The results of this work suggest that agrin expression and function may be a target of ethanol exposure during embryogenesis. Birth Defects Research (Part A) 91:129-141, 2011. (c) 2011 Wiley-Liss, Inc.