Genetic and bioinformatic approaches to characterize ethanol teratogenesis

Alcohol consumption during pregnancy is the most preventable cause of birth defects, yet approximately 2-5% of children are afflicted with Fetal Alcohol Spectrum Disorders (FASD). FASD describes the complex and highly variable deleterious phenotypes caused by prenatal alcohol exposure. Twin studies suggest a genetic predisposition, contributing to the variation in risk for FASD. Despite this, we lack a basic understanding of 1) the factors that protect or predispose an individual to FASD and 2) how these genetic factors interact in ethanol teratogenesis. Results from a genetic “shelf” screen revealed vangl2, a member of the Wnt/planar cell polarity (PCP) pathway that mediates convergent extension movements that narrow and elongate the body axis, as an ethanol-sensitive genetic locus. Untreated vangl2 mutants displayed a relatively intact craniofacial skeleton. Ethanol-exposed vangl2 heterozygotes and mutants, displayed cyclopean and midfacial defects. To assess the relative level of variation of the transcriptional response to ethanol, I performed single embryo RNA-seq during early embryonic stages. Individual zebrafish embryos were exposed to a subteratogenic dose of 1% ethanol in embryo media. My data suggests that the effect of ethanol is subtle; time is the most important variable driving variation in fold coverage across all samples. Despite this, I find a number of differentially expressed genes in response to ethanol. Transcriptional changes due to ethanol are indicative of increased oxidative stress and ion transport and reduced DNA replication and cell division. Using a bioinformatic approach, I find cyclopamine, a Hedgehog pathway inhibitor, interacts with ethanol. Further genetic analyses shows that ethanol disrupts convergent extension of the mesoderm, which in turn disrupts localization of shh in the axial mesoderm, a signal necessary to separate the eye field. I find this effect to be further exacerbated in the vangl2 mutant background. Together these data yield important insight necessary to advance understanding and treatment for FASD