Transcriptomic analyses of gastrulation-stage mouse embryos with differential susceptibility to alcohol

Abstract

Genetics are a known contributor to differences in alcohol sensitivity in humans with fetal alcohol spectrum disorders (FASDs) and in animal models. Our study profiled gene expression in gastrulation-stage embryos from two commonly used, genetically similar mouse substrains, C57BL/6J (6J) and C57BL/6NHsd (6N), that differ in alcohol sensitivity. First, we established normal gene expression patterns at three finely resolved time points during gastrulation and developed a web-based interactive tool. Baseline transcriptional differences across strains were associated with immune signaling. Second, we examined the gene networks impacted by alcohol in each strain. Alcohol caused a more pronounced transcriptional effect in the 6J versus 6N mice, matching the increased susceptibility of the 6J mice. The 6J strain exhibited dysregulation of pathways related to cell death, proliferation, morphogenic signaling and craniofacial defects, while the 6N strain showed enrichment of hypoxia and cellular metabolism pathways. These datasets provide insight into the changing transcriptional landscape across mouse gastrulation, establish a valuable resource that enables the discovery of candidate genes that may modify alcohol susceptibility that can be validated in humans, and identify novel pathogenic mechanisms of alcohol. This article has an associated First Person interview with the first author of the paper.

Keywords: Apoptosis; Brain development; Embryo; Fetal alcohol spectrum disorders; Inflammation.

Fig. 1.

Experimental timeline and example of web-based visualization tool. A web tool was created as a resource to allow gene-by-gene exploration of expression patterns across the first 12 h of normal mouse gastrulation in the 6J and 6N strains. (A) Experimental timeline. (B) Comparison of expression of Wdfy1, a gene that significantly differed between the 6J and 6N strains, across time. Single strains can be selected for viewing using the toggles on the left. (C) PAE data can be toggled on and off using the options. Shh expression was impacted by prenatal alcohol exposure (PAE) in the 6J, but not 6N, mice. Figure created with Biorender.com.

Fig. 2.

Immune signaling gene pathways are upregulated in the 6J compared to the 6N strain. (A) Representative image of E7.0 mouse embryo. Embryo highlighted in yellow was dissected from the extraembryonic tissue (EE) for sequencing. (B) Heat map of genes altered in the 6J versus 6N strain at baseline (prior to alcohol administration) on E7.0. Data are expressed as log₂ fold change (Log₂FC). Blue, downregulated genes; red, upregulated genes. n=6/group. (C) Functional profiling of genes differentially expressed in the 6J versus 6N strain at E7.0. n=6/group.

Fig. 3.

Gastrulation-stage alcohol dysregulated more genes in the 6J strain than in the 6N strain 6 h after exposure. (A) Volcano plot of genes significantly dysregulated by alcohol in the 6J mice 6 h after the first dose of alcohol (E7.25). (B) Genes significantly dysregulated by alcohol in the 6N mice 6 h after the first dose of alcohol (E7.25). (C) Heat map comparing 228 genes altered by alcohol in both the 6J and 6N mice at the E7.25 time point. Data are expressed as Log₂FC. Blue, downregulated genes; red, upregulated genes. n=6/group.

Fig. 4.

Functional profiling of biological pathways enriched in the 6J and 6N strains 6 h after alcohol exposure (E7.25). (A) 6J strain. (B) 6N strain. n=6/group.

Fig. 5.

Gastrulation-stage alcohol dysregulated more genes in the 6J strain than in the 6N strain 12 h after exposure. (A) Volcano plot of genes significantly dysregulated by alcohol in the 6J mice 12 h after the first dose of alcohol (E7.5). n=5 vehicle-treated, n=6 PAE. (B) Genes significantly dysregulated by alcohol in the 6N mice 12 h after the first dose of alcohol (E7.5). n=6 vehicle-treated, n=4 PAE. (C) Heat map comparing 228 genes altered by alcohol in both the 6J and 6N mice at the E7.5 time point. Data are expressed as Log₂FC. Blue, downregulated genes; red, upregulated genes. n=6/group.

Fig. 6.

Functional profiling of biological pathways enriched in the 6J and 6N strains 12 h after alcohol exposure (E7.5). (A) 6J strain. (B) 6N strain. n=6/group.

Fig. 7.

Schematic representing a hypothetical mechanism contributing to differences in alcohol sensitivity between the 6J and 6N strains. The Nnt mutation in the 6J strain could affect reactive oxygen species (ROS) breakdown in the mitochondria, leading to higher baseline oxidative stress and inflammation. In the presence of alcohol, 6J mice would undergo increased apoptosis and DNA damage, ultimately resulting in more severe craniofacial and CNS anomalies. GR, glutathione reductase; GSSG, glutathione disulfide; GSH, glutathione; NAD+/NADH, nicotinamide adenine dinucleotide (+ hydrogen); NADP+/NADPH, nicotinamide adenine dinucleotide phosphate; Nnt, nicotinamide nucleotide transhydrogenase.