Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model

Abstract

Holoprosencephaly (HPE) is a common and severe human developmental abnormality marked by malformations of the forebrain and face. Although several genetic mutations have been linked to HPE, phenotypic outcomes range dramatically, and most cases cannot be attributed to a specific cause. Gene-environment interaction has been invoked as a premise to explain the etiological complexity of HPE, but identification of interacting factors has been extremely limited. Here, we demonstrate that mutations in Gli2, which encodes a Hedgehog pathway transcription factor, can cause or predispose to HPE depending upon gene dosage. On the C57BL/6J background, homozygous GLI2 loss of function results in the characteristic brain and facial features seen in severe human HPE, including midfacial hypoplasia, hypotelorism and medial forebrain deficiency with loss of ventral neurospecification. Although normally indistinguishable from wild-type littermates, we demonstrate that mice with single-allele Gli2 mutations exhibit increased penetrance and severity of HPE in response to low-dose teratogen exposure. This genetic predisposition is associated with a Gli2 dosage-dependent attenuation of Hedgehog ligand responsiveness at the cellular level. In addition to revealing a causative role for GLI2 in HPE genesis, these studies demonstrate a mechanism by which normally silent genetic and environmental factors can interact to produce severe outcomes. Taken together, these findings provide a framework for the understanding of the extreme phenotypic variability observed in humans carrying GLI2 mutations and a paradigm for reducing the incidence of this morbid birth defect.

Keywords: Birth defects; Gene-environment; Hedgehog signaling; Holoprosencephaly.

Fig. 1.

GLI2 loss of function causes HPE-associated facial dysmorphology. (A-D) B6 Gli2^+/+, Gli2^+/− and Gli2^−/− fetuses at GD15 are shown along with a Gli2^+/+ fetus that had been exposed to 40 mg kg⁻¹ vismodegib (Vis) at GD7.75. Snout width (SW), interocular distance (IOD) and head width (HW) were measured, as illustrated by the dashed lines shown in E. (F) Linear measurements were normalized to Gli2^+/+ control group values and are shown on a semi-log plot. Values represent the mean±s.e.m. (Gli2^+/+ n=6; Gli2^+/− n=9; Gli2^−/− n=6; Gli2^+/++ Vis n=12). *P≤0.05 by one-way ANOVA followed by Tukey's HSD. (G) A neonatal child exhibits the face and brain phenotypes of alobar HPE. A single central nostril, hypotelorism and midfacial hypoplasia co-occur with an undivided forebrain and single ventricle shown by prenatal imaging. Scale bar: 1 mm.

Fig. 2.

Facial dysmorphology in Gli2^−/− fetuses co-occurs with brain abnormalities. (A-D) Animals from the same experimental groups that are described in Fig. 1 are shown. To visualize correlative phenotypes, images of the dorsal surface of the brain along with facial images (inset) of the same animal are shown. Images were captured following Bouin's fixation and have been converted to grayscale. In the Gli2^−/− brain, hypoplasia of the cerebral cortices (cc) and midbrain (mb) was apparent. The olfactory bulbs (ofb) were hypoplastic and abnormally approximated. In the Gli2^+/+ fetus that had been exposed to vismodegib, more severe deficiency of the cerebral cortices and absence of the olfactory bulbs was observed. Scale bar: 1 mm.

Fig. 3.

GLI2 loss of function results in deficiency of medial forebrain and facial tissue. (A-P) Serial histological (hematoxylin and eosin; H&E) images are shown from the same experimental groups described in Figs 1 and 2. Dashed boxes in the low-magnification images on the left provide context for the coronal sections shown in each row. Gli2^−/− and Gli2^+/+ vismodegib-exposed fetuses exhibited absence of nasal septal (ns) cartilage and diminished vomeronasal organs (vo). The midline connective tissue separating the olfactory bulbs (ofb) marked by * was reduced in area in the Gi2^−/− fetus, whereas the olfactory bulbs were absent in the Gli2^+/+ vismodegib-exposed fetus. These fetuses also had severe medial forebrain deficiencies, including absence of the septal region (s) and communicating lateral ventricles (lv). In the Gli2^−/− fetus, attenuation of the third ventricle (tv) and absence of the anterior pituitary (ap) lobe were also apparent. The anterior pituitary was present but hypoplastic in the Gli2^+/+ vismodegib-exposed fetus, which also exhibited subtle attenuation of the third ventricle. (Q-T) A genetic-fate mapping system (Ahn and Joyner, 2005) was used to identify Hh-responsive cell lineages. Embryos that had been exposed to tamoxifen at GD7.75 were sectioned and stained with X-Gal to visualize Hh-responsive cells and their progeny. 100-µM sections were produced at planes comparable to those that were stained with H&E. t, tongue. Scale bars: 0.25 mm.

Fig. 4.

GLI2 loss of function results in altered dorsal-ventral forebrain patterning. Shown are embryos at GD11 of the same experimental groups described in Figs 1-3. (A-D) Frontal images show forebrain and facial morphology. Gli2^−/− and Gli2^+/+ fetuses that had been exposed to vismodegib exhibited hypoplasia and abnormal approximation of the telencephalic vesicles (t), absence of the medial nasal processes (mnp) and a single central nostril (n). (E-T) Embryos were hemisected near the midline and subjected to in situ hybridization for the indicated genes. In the Gli2^+/+ control, Nkx2.1 was expressed in the medial ganglionic eminences (mge) and the ventral aspect of the diencephalon (d), whereas Pax6 was present in the dorsal aspect of the telencephalon and diencephalon. Shh was expressed in the mantle region of the medial ganglionic eminence, along the ventral aspect of the diencephalon, and in the zona limitans intrathalamica (zli). Gli1 expression reflects its paracrine responsiveness to stimulation with secreted SHH. e, eye. Scale bar: 1 mm.

Fig. 5.

Gli2 heterozygosity increases teratogenic sensitivity. Wild-type dams carrying Gli2^+/+ and Gli2^+/− embryos were exposed to 2.5 or 10 mg kg⁻¹ vismodegib at GD7.75. (A-D) Categories of facial morphology are illustrated by GD17 fetuses of the indicated genotype and dose group. Animals that were indistinguishable from controls were assigned ‘normal’ (A). Those with a diminished area of pigmentation between the nostrils were assigned as ‘mild’ (B). Animals with a gross deficiency of the median lip notch but some remaining nasal pigment (np) at the tip of the nose were assigned as ‘moderate’ (C). Those with an absent lip notch (uln) and single central nostril were assigned as ‘severe’ (D). The graph on the left shows the percentage of GD17 fetuses that were classified as having mild, moderate or severe HPE-associated facial dysmorphology. *P<0.05 by Fisher's exact test. n=5 litters per treatment group. Data are mean±s.e.m. (A′-D′) Histologic sections through the cerebral cortices illustrate that the degree of facial dysmorphology corresponded to forebrain abnormalities. Note deficiency of the septal region (s) in B′ and the absent septal region and single communicating lateral ventricle (lv) in C′ and D′. Scale bars: 1 mm.

Fig. 6.

Gli2 heterozygosity diminishes responsiveness to SHH ligand. (A-C) Gli2^+/+, Gli2^+/− and Gli2^−/− mouse embryonic fibroblasts (MEFs) were treated with or without SHH ligand for 48 h. Expression of Gli2 and the conserved Hh target genes Gli1 and Ptc1 were measured by performing real-time reverse transcriptase PCR, and expression was normalized to that of Gapdh. *P<0.05 as determined by Student's t-test for genotype-specific gene expression. Arrows indicate a significant gene-dosage effect, as determined by using a Jonckheere–Terpstra test. Veh, vehicle, DMSO. (D) Gli2^+/+ and Gli2^+/− fibroblasts were treated with or without SHH ligand and with graded concentrations of vismodegib for 48 h. Induction of Ptc1 (SHH relative to vehicle) relative to Gli2^+/+ cells is shown in the semi-log plot. The dashed line shows Ptc1 induction (SHH relative to vehicle) in Gli2^−/− fibroblasts to illustrate a theoretical threshold of teratogenicity. For each graph, values represent the mean±s.e.m. of six replicate experiments.