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

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

Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

<div><p>The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15^th to 24^th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.</p></div

CL/P and CPO associated secondary palate morphology.

<p>In vehicle-exposed embryos at GD14.5 (A) the secondary palatal shelves have approximated and made contact at the midline. In affected cyclopamine-exposed embryos with cleft lip (B), palatal shelves are widely spaced and deficient in width. In vismodegib-exposed embryos (C), secondary palatal shelves have also elevated but are deficient in both length and width. Length (D) and width (E) measurements (arbitrary units), as depicted by the dashed calipers, were made on light microscopy images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120517#pone.0120517.s004" target="_blank">S4 Fig.</a>). Shelf width was determined at 1/3 shelf length from the most rostral aspect. *** p<0.001, **** p<0.0001</p

HPE, CL/P, and CPO associated craniofacial abnormalities.

<p>Bone and cartilage are stained red and blue, respectively. Top row shows coronal view, middle row shows lateral view, and bottom row shows inferior view with mandibles removed and shown to the right. Animals with HPE exhibit a single, small nasal bone (black arrowhead) and a fused premaxilla (black arrow). Animals with CL/P and CPO have increased width between pterygoid and palatine bones compared to vehicle control (white double arrow), and an absent basisphenoid bone, while in those with HPE only the anterior half is ossified (white arrow). Compared to vehicle control, animals with CL/P have shorter mandibles (white arrowhead) and absent vomer and palatal premaxilla processes (white caret). In animals with CPO the vomer is displaced posteriorly (black caret).</p

HPE, CL/P, and CPO associated brain morphology.

<p>Superior views of dissected brains are shown for a vehicle-exposed normal animal (A) and for representative examples of animals with vismodegib-induced HPE (B), cyclopamine-induced CL/P (C), and vismodegib-induced CPO (D). Severe hypoplasia of the cerebral cortices (cc) and olfactory bulb (ofb) absence is apparent in the animal with HPE. In animals with CL/P and CPO, the cerebral cortices appear to be of approximately normal size but the olfactory bulbs are hypoplastic. Serial coronal sections of comparably classified animals are shown in E-P. Notable HPE-associated features include a single central nasal passage with nasal septum (ns) cartilage absence (black arrow), olfactory bulb agenesis (J), and a single telencephalic vesicle (N). Grossly normal division of the olfactory bulbs (K, L) and cerebral cortices (O, P), and apparent forebrain septal (s) region hyperplasia (white arrows) is observed in animals with CL/P and CPO. (t) Tongue, (e) eye, (sp) secondary palate.</p

Additional stage of exposure-dependent phenotypes.

<p>Along with a vehicle-exposed control (A), representative examples of phenotypic outcomes are shown with numbers indicating the gestational stage of acute vismodegib administration. Later exposure was associated with forelimb ectrodactyly, as exhibited bilaterally in fetuses exposed from 9.25 to 9.75 (arrows point to absent fifth digits on the right limb). Kinked tail phenotypes were caused by exposure between GD9.5 and 10.0 (arrowheads). Edema is also apparent in fetuses exposed at GD9.75 and 10.0. For each treatment group the number of litters and fetuses examined, mean litter size and crown-rump length, and the incidence of edema, forelimb ectrodactyly, and kinked tail defects are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120517#pone.0120517.s008" target="_blank">S1 Table</a>.</p

Stage of exposure-dependent facial dysmorphology.

<p>Single doses of vismodegib were administered at discrete time points indicated by tick marks on the x-axis, including: GD7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.625, 8.75, 8.875, 9.0, 9.25, 9.5, 9.75, and 10.0. Cyclopamine was administered by subcutaneous infusion from GD8.25 to ~9.375. Representative examples of distinct face and palate phenotypes are shown, including apparently normal (Normal), HPE, CL/P, and CPO. Note that lateral lip clefts resulting from acute vismodegib exposure typically extended into the primary palate (D’), while those resulting from cyclopamine exposure extended into both the primary and secondary palate (F’). The penetrance of HPE, CL/P, and CPO phenotypes resulting from stage-specific vismodegib exposure is shown in the graph. 5–7 litters were examined for each exposure permutation.</p