Cyclopamine was administered by subcutaneous infusion that extended from days 8.25 to 9.5 of pregnancy, representing a maternal dosage of 160 mg/kg/d. Cyclopamine exposure induced gross facial malformations in 5 of 14 litters with an approximate intra-litter penetrance of 50%. One affected fetus exhibited unilateral CL while the majority presented with unilateral (n=6) or bilateral CLP (n=12) as previously reported (Lipinski et al., 2008a
; ). Of the 18 fetuses featuring CLP, the medial nasal prominence-derived tissue mass was markedly deficient in two (), and nearly absent in one (). The varying facial morphologies present in these cyclopamine-exposed fetuses are comparable to those occurring in human populations.
Embryonic morphogenesis of cyclopamine-induced CLP
AZ75, a semi-synthetic cyclopamine-analog, demonstrated a four-fold greater potency for Hh signaling inhibition than its parent compound cyclopamine in a ligand-responsive cell line (supplemental Figure 1
), with differences also notable in vivo
. Whereas bolus administration of cyclopamine has been associated with maternal toxicity (Lipinski et al., 2008a
), administration of AZ75 by oral gavage at doses of 40 mg/kg (n=7) and 80 mg/kg (n=4) on day 8.5 of pregnancy produced no apparent toxicity in the dams. In exposed litters, the 80 mg/kg dose caused all of the embryos to resorb. In the 40 mg/kg exposure group, multiple surviving fetuses were found, with gross craniofacial malformations occurring in 6 of 7 litters.
Regarding craniofacial defects, AZ75 administered acutely by oral gavage on day 8.5 of pregnancy resulted in abnormalities that are consistent with overt (alobar and semilobar) HPE. Among six affected litters, approximately 40% of fetuses exhibited hypotelorism (reduced interocular distance) and cebocephaly (small nose with a single nostril) (). In one litter, along with two fetuses exhibiting this phenotype, two littermates presented with median CLP with an almost complete absence of medial nasal prominence-derived tissue ().
To investigate the morphogenesis of cyclopamine-induced CLP, embryos were analyzed at GD11.25, the gestational period closely following that in which the medial nasal, lateral nasal, and maxillary prominences unite to form the upper lip (Reviewed by Jiang et al., 2006
; ). Cyclopamine-affected embryos presented with medial nasal prominences that were too closely situated and for which the lower component – that from which the philtrum and primary palate are derived – was reduced in size and failed to contact both the maxillary and the lateral nasal prominences. Embryos were also analyzed at GD14.0, a time when the maxillary prominence-derived secondary palatal shelves normally begin to elevate to allow midline approximation and fusion. In embryos with cyclopamine-induced CL, the secondary palatal shelves appeared to be of normal size and conformation. However, the midface of affected embryos was slightly wider than normal (compare ), with the anterior aspect of the palatal shelves being too widely spaced (compare ). In severely holoprosencephalic embryos, where the midface is extremely narrow and the primary palate is absent, the secondary palatal shelves were of relatively normal proportion and were prematurely fused anteriorly.
For further detailed analysis, histological sections were produced from GD16.5 fetuses selected by external appearance to represent the predominant phenotype associated with exposure to vehicle (normal), cyclopamine (unilateral and bilateral CLP as depicted in ), and AZ75 (HPE of severity within the range seen in ). In the CLP group, both palatal shelves appeared properly elevated in one fetus, whereas in the other three examined, one shelf was elevated while the contralateral was not (). Additionally, three of the four CLP specimens exhibited agenesis of the Rathke’s pouch-derived anterior lobe of the pituitary gland. For the HPE group, histological sections illustrated that the median facial deficiency reflected that in the forebrain. Olfactory bulb agenesis, rostrally unified cerebral hemispheres (with striatal union), and complete agenesis of the pituitary gland were observed. Additionally, palatal shelves were found to be fused but abnormally highly arched. No apparent changes in histological features of the midbrain and hindbrain were observed in either group.
Histological analysis of CLP- and HPE-associated craniofacial and CNS phenotype
From selected fetuses, 48 μm isotropic MRI sections were reconstructed to provide 3-D visualization of skeletal structure and brain surface morphology. Images representing Normal, CLP, and HPE phenotypes are shown in . While the CLP-associated craniofacial skeletal structure was grossly normal, abnormal fusion of the frontal bones (metopic craniosynostosis) was associated with the HPE phenotype, reflecting clinical observations (Faro et al., 2005).
To quantitatively assess the CLP- and HPE-associated CNS phenotype, a series of linear measurements was employed (adapted from Parnell et al. 2009
). Mid-sagittal brain length, biparietal diameter, transverse cerebellar distance, olfactory bulb length and width, and interocular distance measurements were produced. illustrates linear measurement parameters with corresponding quantitative graphical analysis. This analysis revealed a significant reduction in olfactory bulb length (F(2, 13) = 592.1, p<0.001) and width (F(2, 13) = 473.4, p<0.001) associated with CLP. Holoprosencephalic fetuses exhibited reduced biparietal diameter (F(2, 13) = 13.5, p<0.02) and brain length (F(2, 13) = 10.9, p<0.03), olfactory bulb absence, and severely reduced inter-ocular distance (F(2, 13) = 17.1, p<0.001). Cerebellum width in HPE fetuses was not significantly reduced (F(2, 13) = 3.7, p<0.08).
Segmentation and 3-D reconstruction was used to examine brain morphology associated with the described CLP and HPE phenotypes (). As predicted from linear measurements, a subtle but consistent hypoplasia of the olfactory bulbs was observed in brains of mice with cyclopamine-induced CLP. No other gross morphological differences in these brains were observed. As expected, striking forebrain abnormalities were observed in the holoprosencephalic fetuses; most notably, median union of the cerebral hemispheres accompanied by rostro-caudal foreshortening. While overall brain volume was unchanged in the CLP group, it was significantly reduced in the HPE group (F(2, 13) = 13.6, p<0.001).