Embryonic Ethanol but Not Cannabinoid Exposure Affects Zebrafish Cardiac Development via Agrin and Sonic Hedgehog Interaction

Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both the nervous system and cardiac development are dependent on agrin modulation of sonic hedgehog (shh) and fibroblast growth factor (Fgf) signaling pathways. As both ethanol and cannabinoids impact these signaling molecules, we examined their role on zebrafish heart development. Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 and 2 agonist concentrations in the absence or presence of morpholino oligonucleotides that disrupt agrin or shh expression. In situ hybridization was employed to analyze cardiac marker gene expression. Exposure to cannabinoid receptor agonists disrupted midbrain–hindbrain boundary development, but had no effect on heart development, as assessed by the presence of cardiac edema or the altered expression of cardiac marker genes. In contrast, exposure to 1.5% ethanol induced cardiac edema and the altered expression of cardiac marker genes. Combined exposure to agrin or shh morpholino and 0.5% ethanol disrupted the cmlc2 gene expression pattern, with the restoration of the normal expression following shh mRNA overexpression. These studies provide evidence that signaling pathways critical to heart development are sensitive to ethanol exposure, but not cannabinoids, during early zebrafish embryogenesis.