Molecular Etiology of Craniofacial Dysmorphology in Transition Zone Cilia Mutants

Objectives: Although craniofacial birth defects are among the most common congenital malformations, the underlying molecular etiology is poorly understood. Primary cilia are ubiquitous microtubule-based organelles that coordinate multiple signaling pathways critical for development of the craniofacial complex, including Hedgehog, Wnt and PDGF signals. Ciliary dysfunction causes a range of disorders, collectively known a ciliopathies, many of which display severe craniofacial defects including cleft lip/palate, microcephaly, micrognathia, occipital encephalocele, and tongue malformations. We previously found that the protein products of ciliopathy genes identified in human patients form a complex at the base of primary cilia to regulate ciliary membrane protein composition. This complex is termed the transition zone (TZ) complex. Our current study sets out to uncover the molecular mechanism underlying craniofacial dysmorphology in multiple TZ mutant mice.
Methods: We used geometric morphometric analysis to quantify facial shape defects in multiple TZ mutants (B9d1, Tectonic2, Cc2d2a, Tmem231, and Tmem67) and correlated facial defects with Hedgehog and Wnt pathway disruption using genetic reporters. Histochemical approaches were employed to interrogate the spatial organization of a facial signaling center critical for growth of the midface, the frontonasal ectodermal zone (FEZ). Finally, we used Cre-loxP technology to uncover tissue-specific requirements of the TZ complex in the various tissues comprising the craniofacial complex (neurectoderm, neural crest, and facial ectoderm).
Results: TZ mutants displayed hypotelorism at E11.5 and disorganization of the FEZ leading to facial clefting at E12.5. Aberrant neural tube patterning at E9.5 leading to decreased forebrain size presaged these facial defects. Decreased Hedgehog pathway activation in the face also coincided with midface narrowing. Interestingly, decreasing Ptch1 gene dosage partially rescued the hypotelorism. Finally, TZ function in the neural crest is specifically required for postnatal craniofacial development with mutants exhibiting hydrocephalus, craniosynostosis and basicranium expansion.
Conclusions: Our findings uncover the molecular underpinnings of craniofacial facial defects in a group of previously poorly understood ciliopathies.