Gene-environment interactions characterized by single embryo transcriptomics

Gene-environment interactions are likely to underlie most human birth defects. The most common environmental contributor to birth defects is likely prenatal alcohol exposure. Fetal Alcohol Spectrum Disorders (FASD) describes the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem. We first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol- induced midfacial hypoplasia. We performed single-embryo RNA-Seq during early embryonic stages, to assess individual variation to the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network- based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite the fact that ethanol did not alter the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly interact to disrupt midfacial development. Collectively, these results suggest that the midfacial defects in ethanol-exposed vangl2 mutants are due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Consistent with an indirect model, a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface, becomes mispositioned in ethanol-exposed vangl2 mutants. We demonstrate that ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor, blebbistatin. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.