Scott E. Parnell

Background: Prenatal alcohol exposure can cause fetal alcohol syndrome, but genetic variation can account for some of the observed differential susceptibility to alcohol‐induced birth defects. Here we investigate an inbred mouse strain, 129S1/SvImJ (129S1), for its potential susceptibility to alcohol‐ and Sonic hedgehog pathway inhibitor‐induced craniofacial birth defects and the ways complex genetic factors work to alter susceptibility compared to the highly alcohol‐susceptible strain, the C57BL/6J (B6J). Methods: 129S1 pregnant females were treated with alcohol (two doses of 2.9 g/kg), CP‐55,940 (2.0 mg/kg), vismodegib (40 or 80 mg/kg), or smoothened agonist (12 mg/kg) during gastrulation or neurulation, then fetuses were assessed for defects. Gastrulation‐ and neurulation‐stage embryo gene expression and apoptosis were also analyzed. Results: 129S1 mice are completely resistant to alcohol‐induced craniofacial birth defects despite high blood alcohol concentrations compared to other alcohol‐susceptible mouse strains. This strain is also resistant to craniofacial birth defects after exposure to a synthetic cannabinoid, CP‐55,940, and vismodegib. All three drugs inhibit the Sonic hedgehog (Shh) pathway. 129S1 embryos have higher basal expression of two key Shh‐related mRNAs, Smo and Efcab7 , and lower apoptosis after alcohol compared to B6J embryos. 129S1 embryos are more susceptible to birth defects after treatment with a Shh pathway agonist, smoothened agonist (SAG), than B6J embryos. Conclusions: 129S1 embryos have higher expression of key Shh pathway genes and low apoptosis after alcohol, which may contribute to the alcohol‐resistant phenotype seen in the 129S1/SvImJ strain, adding to the understanding of protective genetic factors in alcohol teratogenesis.

Background: During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later‐life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol‐induced cell death is the pro‐apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation‐stage alcohol exposure.Methods: Male and female Bax^+/− mice mated to produce embryos with full (^−/−) or partial (^+/−) Bax deletions, or Bax^+/+ wild‐type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS‐like facial features.Results: Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol‐induced defects. An RNA‐seq analysis of GD 7 Bax^+/+ and Bax^−/− embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis.Conclusions: Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation‐stage alcohol exposure.

Background: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure.Methods: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53^zdf1 M214K mutation and their wild‐type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation.Results: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol‐exposed fetuses, occurring in nearly 75% of the alcohol‐exposed wild‐type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol‐induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol‐induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion.Conclusions: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol‐induced teratogenesis.

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.

Background: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation.Methods: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA‐seq.Results: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration.Conclusions: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation‐stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol’s actions.