Gene Expression Changes in C57BL/6J and DBA/2J Mice Following Prenatal Alcohol Exposure

Abstract

Background Prenatal alcohol exposure can result in Fetal Alcohol Spectrum Disorder (FASD). Not all women who consume alcohol during pregnancy have children with FASD and studies have shown that genetic factors can play a role in ethanol teratogenesis. We examined gene expression in embryos and placentae from C57BL/6J (B6) and DBA/2J (D2) mice following prenatal alcohol exposure. B6 fetuses are susceptible to morphological malformations following prenatal alcohol exposure while D2 are relatively resistant. Methods Male and female B6 and D2 mice were mated for two hours in the morning, producing four embryonic genotypes: true-bred B6B6 and D2D2, and reciprocal B6D2 and D2B6. On gestational day 9dams were intubated with either 5.8 g/kg ethanol, an is caloric amount of maltose-dextrin, or nothing Four hours later dams were sacrificed and embryos and placentae were harvested. RNA was extracted, labeled and hybridized to Affymetrix Mouse Genome 430 v2 microarray chips. Data were normalized, subjected to analysis of variance and tested for enrichment of gene ontology (GO) molecular function and biological process using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results Several gene classes were differentially expressed in B6 and D2 regardless of treatment, including genes involved in polysaccharide binding and mitosis. Prenatal alcohol exposure altered expression of a subset of genes, including genes involved in methylation, chromatin remodeling, protein synthesis and mRNA splicing. Very few genes were differentially expressed between maltose-exposed tissues and tissues that received nothing, so we combined these groups for comparisons with ethanol. While we observed many expression changes specific to B6 following prenatal alcohol exposure, none were specific for D2. Gene classes up-or down regulated in B6 following prenatal alcohol exposure included genes involved in mRNA splicing, transcription and translation. Conclusions Our study identified several classes of genes with altered expression following prenatal alcohol exposure, including many specific for B6, a strain susceptible to ethanol teratogenesis. Lack of strain specific effects in D2 suggests there are few gene expression changes that confer resistance. Future studies will begin to analyze functional significance of the expression changes.

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

“…Alcohol broadly impacted this pathway, with significant reductions in both nuclear and mitochondrial ribosomal proteins, aminoacyl-tRNA synthases and hydrolases, and components of RNA polymerase I and III. Microarray approaches in mouse embryos of comparable stage similarly flagged decreased ribosome transcripts as differentiating the alcohol response, although they were suppressed in the alcohol-vulnerable strain in one study (B6J vs. DBA/J [18]) and in the alcohol-resistant strain in another (B6N vs. B6J [19]); the discrepancy may reflect differences in dose, exposure route, or analytical platforms. The consistent repression of this gene cluster across models implicates ribosomes in modifying alcohol vulnerability.…”

“…Additional candidates at the pathway level have been revealed though systems-level comparisons of genetically-related strains having differential vulnerability to alcohol-induced teratogenicity. For the headfold-stage mouse embryo, a developmental stage similar to that studied herein, comparisons of strains having differential alcohol sensitivity identified multiple KEGG clusters having altered representation in response to alcohol including methylation, chromatin organization, pentose phosphate pathway, glycolysis / gluconeogenesis, ribosome, mRNA splicing, and proteasome [18,19]. …”

The Genetics of Fetal Alcohol Spectrum Disorders

“…The C57BL/6J mice, one of the original C57 strains, were the first to be shown capable of recapitulating the craniofacial aspects of FAS and are more susceptible to ethanol teratogenesis during neurulation than many other strains, including, DBA/2, short-sleep, long-sleep, A/J, A/lbg, and 129S6 mice (Boehm et al, 1997; Downing et al, 2009; Downing et al, 2012; Sulik et al, 1981). In fact, the C57BL/6J mice are probably the most susceptible of the C57 sub-strains as demonstrated in a recent study comparing the C57BL/6J and C57BL/6N sub-strains during gastrulation-stage exposure, where the 6J mice exhibited ocular defects (rostrally-derived structures) at nearly twice the rate of their 6N counterparts (Dou et al, 2013).…”

Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model

“…However, it should be noted that studies using an in vitro application of ethanol have laid the foundation for this area of research, demonstrating that alcohol alters epigenetic programs (Veazey, Carnahan, Muller, Miranda, & Golding, 2013; Zhou et al, 2011), cell cycle dynamics (Hicks, Middleton, & Miller), cell fate (Kim et al, 2010; Miranda, Santillano, Camarillo, & Dohrman, 2008), Wnt signaling and differentiation (Vangipuram & Lyman, 2012), and transcription factors (Ogony, Malahias, Vadigepalli, & Anni, 2013) during development. Currently, in vivo exposures with subsequent cell culture characterization to derive transcriptome analysis typically deliver high doses of alcohol via intubation or intra-peritoneal injection for an acute period (Downing et al, 2012; Hashimoto-Torii, Kawasawa, Kuhn, & Rakic, 2011). These conditions may result in false positives due either to indirect effects of the high dose of alcohol used or stress of the alcohol administration.…”