Alcohol exposure during the gastrulation stage of development can disrupt Sonic hedgehog (Shh) signaling and cause eye, craniofacial, and brain defects. One of the genes that regulates Shh signaling is Efcab7, which encodes a protein that facilitates the actions of Smoothened (Smo), a critical component of the Shh pathway. Previous work from our lab has demonstrated that Efcab7 is differentially expressed between two sub-strains of C57BL/6 mice that differ in their sensitivity to gastrulation-stage alcohol exposure. The more alcohol-sensitive C57BL/6 J mice express lower levels of Efcab7 during gastrulation than do the less alcohol-sensitive C57BL/6NHsd mice. The current study examined whether partial or full Efcab7 deletions render mice more sensitive to gastrulation-stage alcohol exposure and affect the sensitivity to other modulators of Shh signaling that cause craniofacial malformations. Efcab7+/- dams were mated with Efcab7+/- sires to produce Efcab7+/+, Efcab7+/-, and Efcab7-/- fetuses. On gestational day 7 (GD 7), they received either alcohol (two doses of 2.9 g/kg, i.p., given 4 hours apart), the Smo antagonist vismodegib (40 mg/kg, or vehicle, p.o.), the Smo agonist SAG (20 mg/kg) or the appropriate vehicles. GD 17 fetuses were collected and examined for ocular and craniofacial dysmorphology. As compared to Efcab7+/+ fetuses, Efcab7-/- fetuses exposed to alcohol or vismodegib treatment had more severe ocular and craniofacial malformations. In contrast, Efcab7-/- fetuses had less severe malformations induced by SAG. These results confirm that Efcab7 can modify responses to Shh agonists and antagonists and further identify Efcab7 as a gene important for the sensitivity to gastrulation-stage alcohol exposure.

Prenatal exposure to alcohol or cannabinoids can produce enduring neurobiological, cognitive, and behavioral changes in the offspring. Furthermore, prenatal co-exposure to alcohol and cannabinoids induces malformations in brain regions associated with reward and stress-related circuitry. This study examined the effects of co-exposure to alcohol and the synthetic cannabinoid (SCB) CP55,940 throughout gastrulation and neurulation in rats on basal corticosterone levels and a battery of behavioral tests during adolescence and alcohol self-administration in adulthood. Importantly, we find that prenatal alcohol exposure (PAE) caused lower baseline corticosterone levels in adolescent males and females. Co-exposure to alcohol + CP produced hyperactivity during open field test in males, but not females. During the two-bottle choice alcohol-drinking procedure, prenatal cannabinoid exposed male and female adolescent rats drank more alcohol than their vehicle-exposed controls. In adulthood, female rats treated with prenatal cannabinoid exposure (PCE), showed an overall total increase in alcohol intake during alcohol self-administration; but this was not found in males. When the reinforcer was changed to a 1% sucrose solution, male rats exposed to PCE, showed a reduced self-administration compared to vehicle-exposed males, potentially indicative of an anhedonic response. This lower self-administration persisted when 20% alcohol was reintroduced to the sucrose solution. Lastly, following an abstinence period, there were no changes due to prenatal drug exposure in either males or females. Overall, these data suggest lasting consequences of prenatal alcohol and cannabinoid exposure during adolescence and adulthood in male and female rats.

Alcohol exposure during the formation and closure of the neural tube, or neurulation (embryonic day [E] 8-10 in mice;-4th week of human pregnancy), perturbs development of midline brain structures and significantly disrupts gene expression in the rostroventral neural tube (RVNT). Previously, alcohol exposure during neurulation was found to alter gene pathways related to cell proliferation, p53 signaling, ribosome biogenesis, immune signaling, organogenesis, and cell migration 6 or 24 h after administration. Our current study expands upon this work by investigating short-term gene expression changes in the RVNT following a single binge-like alcohol exposure during neurulation. Female C57BL/6J mice were administered a single dose of 2.9 g/kg alcohol or vehicle on E9.0 to target mid-neurulation. The RVNTs of stage-matched embryos were collected 2 or 4 h after exposure and processed for RNA-seq. Functional profiling was performed with g:Profiler, as well as with the CiliaCarta and DisGeNet databases. Two hours following E9.0 alcohol exposure, 650 genes in the RVNT were differentially expressed. Functional enrichment analysis revealed that pathways related to cellular metabolism, gene expression, cell cycle, organogenesis, and Hedgehog signaling were down-regulated, and pathways related to cellular stress response, p53 signaling, and hypoxia were up-regulated by alcohol. Four hours after alcohol exposure, 225 genes were differentially expressed. Biological processes related to metabolism, RNA binding, ribosome biogenesis, and methylation were down-regulated, while protein localization and binding, autophagy, and intracellular signaling pathways were up-regulated. Two hours after alcohol exposure, the differentially expressed genes were associated with disease terms related to eye and craniofacial development and anoxia. These data provide further information regarding the biological functions targeted by alcohol exposure during neurulation in regions of the neural tube that give rise to alcohol -sensitive midline brain structures. Disruption of these gene pathways contributes to the craniofacial and brain malformations associated with prenatal alcohol exposure. (c) 2022 Elsevier Inc. All rights reserved.

BACKGROUND:Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex. METHODS:The current study administered either alcohol (2.9 g/kg, two i.p. doses, 4 hr apart) or vehicle to pregnant C57BL/6J females on E7, prior to gonadal sex differentiation, and assessed fetal morphology at E17. RESULTS:Whereas sex did not affect fetal size in controls, alcohol-exposed females were smaller than both control females and alcohol-treated males. Alcohol exposure increased the incidence of eye defects to a similar degree in males and females. Together, these data suggest that females might be more sensitive to the general developmental effects of alcohol, but not effects specific to the craniofacies. Whole transcriptomic analysis of untreated E7 embryos found 214 differentially expressed genes in females vs. males, including those in pathways related to cilia and mitochondria, histone demethylase activity, and pluripotency. CONCLUSION:Gastrulation-stage alcohol induces craniofacial malformations in male and female mouse fetuses at similar rates and severity, though growth deficits are more prevalent females. These findings support the investigation of biological sex as a contributing factor in prenatal alcohol studies.

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.