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

Abstract

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 in utero 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.

Sonic Hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis

“…Specifically, secretion of SHH ligand from the ectoderm activates pathway activity in the adjacent cranial neural crest-derived mesenchyme (Marcucio et al, 2005;Hu and Marcucio, 2009;Hu et al, 2015;Xavier et al, 2016). Demonstrating clinical relevance, we have shown that transient in utero exposure to the Shh pathway inhibitor cyclopamine causes lateral clefts of the lip that typically extend into the primary and secondary palate, and that this model recapitulates human non-syndromic cleft lip with or without cleft palate (CL/P) (Lipinski et al, 2008b(Lipinski et al, , 2010(Lipinski et al, , 2014. Here, we utilized this pathway-specific mouse model of cleft lip to identify and investigate the mechanism of action of Shh target genes that mediate normal and abnormal upper lip morphogenesis.…”

“…Evolutionarily conserved GLI-binding sites between mouse (mm10) and human (Hg19) were identified in silico using the VISTApoint tool (Loots et al, 2002). 10 kb regions upstream and downstream of each gene coding sequence were analyzed for GLI, GLI1, GLI2 and GLI3 consensus binding sites.…”

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

“…Images were captured using a micropublisher 5.0 camera connected to a Nikon SZX-10 stereomicroscope. Linear facial measurements of formalin-fixed fetuses at GD15 were produced in Photoshop v14.1.2 as previously described (Lipinski et al, 2014). For comparison of face and brain morphology, images of Bouin's-fixed tissue were captured and converted to grayscale.…”

The Genetics of Fetal Alcohol Spectrum Disorders

“…holoprosencephaly), craniofacial and digital effects of ethanol exposure is that the timing of the ethanol exposure is just slightly prior to the onset of Shh expression. In contrast, direct inhibition of Shh signaling via cyclopamine, a potent Smo antagonist, induces holoprosencephaly only at slightly later stages of development, when Shh is actively expressed (Heyne et al, 2015; Lipinski et al, 2014). This mechanism would also explain why ethanol exposure reduces Shh expression during gastrulation, but apparently not during neurulation.…”