Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function

Abstract

BACKGROUND Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol’s effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was employed to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8 or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and atonal1a gene expression. CONCLUSIONS These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis.

Developmental age strengthens barriers to ethanol accumulation in zebrafish

“…However, there are significant differences between embryo and embryo + extraembryonic fluid as well as between the two time points of embryos in individual genetic backgrounds. While these results are consistent with the ethanol concentration determined by other groups (Flentke, Klinger, Tanguay, Carvan, & Smith, 2014; Reimers, Flockton, et al, 2004; Zhang et al, 2013), these data suggest that there are two levels of barriers to full ethanol equilibrium with the media. The first is at the level of the chorion, while the second is set up between the embryo and the extraembryonic fluid.…”

“…Zebrafish have been used to examine the teratogenic effects of ethanol (Ali, Champagne, Alia, & Richardson, 2011; Bilotta, Barnett, Hancock, & Saszik, 2004; Blader & Strähle, 1998; Dlugos & Rabin, 2003; Gerlai, Lahav, Gou, & Rosenthal, 2000; Gerlai, Lee, & Blaser, 2006; Li et al, 2007; Lockwood, Bjerke, Kobayashi, & Guo, 2004; McCarthy et al, 2013; Pan, Chatterjee, & Gerlai, 2012; Reimers, Flockton, & Tanguay, 2004; Stockard, 1910; Zhang, Ojiaku, & Cole, 2013; Zhang, Turton, Mackinnon, Sulik, & Cole, 2011). However, there is a lack of consensus regarding ethanol treatment regimens and how these regimens would relate to humans.…”

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis

“…28 Our group used Headspace Gas Chromatography and found tissue levels to be *24%-37%, depending upon the age of the embryo, with older embryos having lower tissue levels. 29 One possible reason for the early disagreement in tissue levels of ethanol may be the very rapid rate at which ethanol levels equilibrate, [28][29][30][31] and therefore, extended or multiple washes would result in an underestimation of the ethanol concentration. Collectively, these studies indicate that the use of ethanol concentrations at or above 2% in the media is likely to result in suprapharmacological levels of ethanol.…”

“…78 Work in chicken, mouse, and zebrafish has shown that ethanol exposure reduces Shh signaling leading to increased CNCC death, as well as disrupted midline and eye development. 30,[79][80][81][82] These ethanol-induced phenotypes can be rescued by injection of shh mRNA. 50 In addition to altering retinoic acid signaling, one alternative (or additional) mechanism by which ethanol attenuates Shh is the disruption of cholesterol modification of Shh needed for proper signaling, as has been found in zebrafish.…”

“…26 Expanding on these studies, the same group has shown that shha interacts with ethanol disrupting GABAergic and glutamatergic neural development. 30 An interaction between ethanol and Shh pathway members has been independently identified in mouse. [92][93][94] Collectively, these studies provide strong evidence for an evolutionarily conserved interaction between ethanol and the Shh pathway.…”