Figure 1.
Magnetic resonance microscopy (MRM) enables concurrent visualization of the brain and face of GD17 mouse fetuses.
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 in situ (C–D). The size of a GD17 mouse fetus can be appreciated when shown in scale with a U.S. penny (E).
Figure 2.
Cyclopamine-induced facial dysmorphology.
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.
Table 1.
Sample size by treatment group.
Figure 3.
Cyclopamine-exposed fetuses are not holoprosencephalic.
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.
Figure 4.
Volumetric brain abnormalities in cyclopamine-exposed fetuses.
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.
Figure 5.
Corpus callosum integrity in fetuses with UL-CLP.
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.