Subpopulation of GD17 fetuses exhibiting severe craniofacial phenotypes.

<p>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 <i>Shh</i> or <i>Gli2</i> 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).</p

Facial dysmorphology rating scale.

<p>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.</p

Effect of treatment and genotype on facial dysmorphology.

<p>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.</p

Facial dysmorphology predicts medial forebrain deficiency.

<p>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).</p

Facial dysmorphology scores by treatment and genotype.

<p>Facial dysmorphology scores by treatment and genotype.</p

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

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

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