Gene-environment interactions characterized by single embryo transcriptomics

Abstract

Fetal Alcohol Spectrum Disorders (FASD) describes the full range of defects that result from prenatal alcohol exposure. The clinical diagnosis of Fetal Alcohol Syndrome (FAS) is the most severe outcome, characterized by facial dysmorphism and neurological deficits. Gene-ethanol interactions underlie susceptibility to FASD but we lack a clear understanding of the genetic risk factors. 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. However, we have no understanding of the potential mechanism of this sensitization. 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. Transcriptional changes due to ethanol are indicative of increased oxidative stress and ion transport as well as reduced DNA replication and cell division. To identify the pathway(s) that are disrupted by ethanol we used these global changes in gene expression to identify small molecules, often pathway inhibitors, that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. This dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering 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. 3Consistent 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.

Ethanol Effects on Early Developmental Stages Studied Using the Zebrafish

“…Ethanol exposure during development can induce holoprosencephaly (HPE) [ 42 , 43 ]. HPE is characterized by defective rostroventral midline patterning of the forebrain with an array of other abnormalities, including failure of the forebrain to form hemispheres and cyclopia [ 44 , 45 ].…”

“…Studies have also shown that phenotypes produced by embryonic ethanol exposure such as cyclopia and other midfacial defects can arise from cell death of neural crest cells. Furthermore, shh developmental signaling was indirectly displaced by a synergistic interaction between ethanol and cyclopamine, a shh pathway inhibitor [ 43 ]. A rescue experiment using shh mRNA injection into ethanol treated zebrafish embryos reduced ethanol induced phenotypes, indicating that shh signaling is disrupted in FAS [ 47 ].…”