The teratogenic effects of prenatal ethanol exposure are exacerbated by Sonic Hedgehog or GLI2 haploinsufficiency in the mouse

Abstract

Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh(+/-) or Gli2(+/-) male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two i.p. doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh(+/-) and Gli2(+/-) groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh(+/-) and Gli2(+/-) mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.

Figure 1. Facial dysmorphology rating scale.

Illustrated are a GD 17 fetus having normal facial morphology and 4 fetuses with varying degrees of medial facial deficiency. Numbers assigned to each image (0–4) are scores representing differing degrees of severity of facial dysmorphology. As compared to normal fetuses, those receiving a score of 1 had a notably diminished area of pigmentation between the nostrils (solid arrow) accompanied by reduction in the depth of the normally present median central notch of the upper lip (dashed arrow). A score of 2 was assigned to fetuses that had lost the median lip notch, but still had some remaining pigment at the tip of the nose. Individuals presenting with a single central nostril were assigned a score of 3 and those given a score of 4 had no nostrils.

Figure 2. Effect of treatment and genotype on facial dysmorphology.

To avoid litter bias, the average dysmorphology score from each genotypic group was determined for each litter in the study population. Values represent the mean plus the standard error of litter averages for each genotype and treatment. Brackets indicate p values of ≤ 0.05 as determined by a one-tailed student’s t-test.

Figure 3. Subpopulation of GD17 fetuses exhibiting severe craniofacial phenotypes.

Included in the study population were 9 fetuses with phenotypes not typically observed in wildtype C57BL/6J mice exposed to the employed ethanol exposure paradigm (A-I). Single allele mutations in Shh or Gli2 were detected in 8 of 9 fetuses in this severely affected subpopulation. In addition to varying degrees of upper midfacial deficiency, other notable defects included exencephaly (A), iridial coloboma and microphthalmia (A-D), apparent anophthalmia (E, G, I), agnathia (E), micrognathia (A-D, F-I), and proboscis (I). Median cleft lip was also observed (B, C). Within this subpopulation, fetuses were assigned dysmorphology scores as follows: 2 (A), 3 (B-F), 4 (G-I).

Figure 4. Facial dysmorphology predicts medial forebrain deficiency.

Superior views of dissected brains are shown for a normal fetus (A) and for representative examples of each category of facial dysmorphology (B-E). Medial facial deficiency was associated with increasing hypoplasia of the cerebral cortices (B-E), increasing hypoplasia (B, C) or absence of the olfactory bulbs (D, E), and incomplete division of the forebrain (D, E).