Volumetric brain abnormalities in cyclopamine-exposed fetuses.

<p>Total brain volumes (inset) were derived following automated skull stripping. Values represent the mean + S.E.M. * p<0.05 compared to the vehicle-exposed control group. For determination of disproportionate differences, the volume of each manually segmented brain region was calculated as a percentage of total brain volume for each animal. Remaining volume includes mid- and hindbrain regions. To illustrate relative changes on the same scale, percent volumes are normalized to mean control values. Values represent the mean ± the S.E.M. ^*p<0.05 compared to the control group.</p

Magnetic resonance microscopy (MRM) enables concurrent visualization of the brain and face of GD17 mouse fetuses.

<p>Forebrain regions, pituitary, and cerebellum were manually segmented from transverse 39 µm MRM sections (A). 3D brain reconstructions were generated by overlaying manually segmented regions with whole-brain masks (B). Reduced opacity of the left cortex and diencephalon allows visualization of the left ventricle, hippocampus, third ventricle, and pituitary. From the same MRM scans, 3D head reconstructions were created, allowing concurrent visualization of the face and brain <i>in situ</i> (C–D). The size of a GD17 mouse fetus can be appreciated when shown in scale with a U.S. penny (E).</p

Cyclopamine-exposed fetuses are not holoprosencephalic.

<p>Along with a vehicle exposed control (A, F, K), representative examples of the cyclopamine-exposed NC (B, G, L), CPO (C, H, M) UL-CLP (D, I, N), BL-CLP (E, J, O), groups are shown. For each example, a coronal MRM section (A–E) showing normal separation between the cerebral hemispheres (arrow) and the secondary palate (arrow head) is shown above a reconstruction of the face and brain (F–J) and a transverse section through the forebrain (K–O). Complete separation of the cerebral hemispheres is evident in each of the reconstructed brains. Transverse sections show normal division of the cerebral cortices with an intact septal region. These images also illustrate deficiency of the pituitary (arrow in G) and olfactory bulbs, and enlargement of the third ventricle (arrow head in M) and septal region in cyclopamine-exposed fetuses. Color-coding in F-O is shown in Fig. 1, where dark red  =  cerebral cortices, light green  =  diencephalon, dark blue  =  septal region, yellow  =  lateral ventricles, orange  =  third ventricle, pink  =  olfactory bulbs, light purple  =  pituitary.</p

Characterization of Subtle Brain Abnormalities in a Mouse Model of Hedgehog Pathway Antagonist-Induced Cleft Lip and Palate

<div><p>Subtle behavioral and cognitive deficits have been documented in patient cohorts with orofacial clefts (OFCs). Recent neuroimaging studies argue that these traits are associated with structural brain abnormalities but have been limited to adolescent and adult populations where brain plasticity during infancy and childhood may be a confounding factor. Here, we employed high resolution magnetic resonance microscopy to examine primary brain morphology in a mouse model of OFCs. Transient <i>in utero</i> exposure to the Hedgehog (Hh) signaling pathway antagonist cyclopamine resulted in a spectrum of facial dysmorphology, including unilateral and bilateral cleft lip and palate, cleft of the secondary palate only, and a non-cleft phenotype marked by midfacial hypoplasia. Relative to controls, cyclopamine-exposed fetuses exhibited volumetric differences in several brain regions, including hypoplasia of the pituitary gland and olfactory bulbs, hyperplasia of the forebrain septal region, and expansion of the third ventricle. However, in affected fetuses the corpus callosum was intact and normal division of the forebrain was observed. This argues that temporally-specific Hh signaling perturbation can result in typical appearing OFCs in the absence of holoprosencephaly—a condition classically associated with Hh pathway inhibition and frequently co-occurring with OFCs. Supporting the premise that some forms of OFCs co-occur with subtle brain malformations, these results provide a possible ontological basis for traits identified in clinical populations. They also argue in favor of future investigations into genetic and/or environmental modulation of the Hh pathway in the etiopathogenesis of orofacial clefting.</p></div

Cyclopamine-induced facial dysmorphology.

<p>From MRM images, extracted facial surfaces are shown for a vehicle-exposed control fetus (A,B), along with three fetuses representative of the phenotypic spectrum observed in the cyclopamine-exposed non-cleft (NC) group (C–H). Snout width (SW), snout length (SL), and mandible length (ML) were measured from facial reconstructions, while interocular distance was measured from coronal MRM sections (inset). For each cyclopamine-exposed group, linear measurements are reported as percent difference relative to the vehicle exposure group. Values represent the mean + S.E.M. * p<0.05 compared to vehicle-exposed control group.</p

Sample size by treatment group.

<p>*18 fetuses were chosen at random for imaging analysis.</p><p>**n = 3 right side UL-CLP, n = 7 left side UL-CLP.</p

Corpus callosum integrity in fetuses with UL-CLP.

<p>Diffusion tensor imaging was used to visualize white matter fiber tracts in GD19 vehicle-exposed (top row) and cyclopamine-exposed fetuses with UL-CLP (bottom row). For each group, one fetus was selected at random from each of three independent litters. The directionality of the brain fiber tracts in the color-coded maps are indicated as follows: red, left/right; blue, inferior/superior; and green, anterior/posterior. In both controls and cyclopamine-exposed fetuses, the corpus callosum (dashed outline), hippocampal commissure (arrow) and anterior commissure (arrowhead) are evident.</p

DSM illustrates unique facial phenotypes resulting from stage-specific ethanol exposure.

<p>(Left) Mean surface shape of the GD7 and GD8.5 exposure groups relative to the control group is shown in the first and second columns respectively, while the GD7 group is directly compared to the GD8.5 ethanol-exposed group in the third column. (A–C) Color-map comparisons reflecting the displacement of mean surface shape for the indicated groups, where red indicates regions most distant and internal, while blue indicates regions most distant and external. Other colors shown in the scales identify intermediate positions. (D–L) Color-map comparisons reflecting the displacement of the indicated mean surface shapes parallel to the three orthogonal axes. Red and blue color intensities reflect displacement in the direction indicated by the corresponding color-coded arrow. Changes are shown at a scale of 1.2 standard deviations. (Right) Snout width (SW) was measured between the most lateral 3^rd row of vibrissae; Median upper lip length (ULL) was measured from the lower edge of the nostrils to the bottom of the upper lip; Facial depth (FD) was measured from the middle of the ear to the top of the philtrum. Values represent the means + the S.E.M. ^*p<0.05 compared to control group.^∧p<0.05 compared to counterpart ethanol exposure group.</p

Scanning electron microscopy illustrates unique brain malformations in GD12 embryos exposed to ethanol at GD7 or GD8.5.

<p>(A–F) Images of specimens hemisected in the coronal (frontal) plane illustrate posterior and anterior aspects of the embryonic brain in control and ethanol-affected groups. (G–I) Additional GD12 embryos were cut to provide a sagittal view of the brain. Notable abnormalities include differences in width of the third ventricle (dashed calipers), and the area from which the septal region will develop (dashed outline in the anterior view and solid calipers in sagittal view). Ganglionic eminences (*).</p

Study population.

<p>Study population.</p