Craniofacial Ciliopathies Reveal Specific Requirements for GLI Proteins during Development of the Facial Midline

Ciliopathies represent a broad class of disorders that affect multiple organ systems. The craniofacial complex is among those most severely affected when primary cilia are not functional. We previously reported that loss of primary cilia on cranial neural crest cells, via a conditional knockout of the intraflagellar transport protein KIF3a, resulted in midfacial widening due to a gain of Hedgehog (HH) activity. Here, we examine the molecular mechanism of how a loss of primary cilia can produce facial phenotypes associated with a gain of HH function. We show that loss of intraflagellar transport proteins (KIF3a or IFT88) caused aberrant GLI processing such that the amount of GLI3FL and GLI2FL was increased, thus skewing the ratio of GLIFL to GLIR in favor of the FL isoform. Genetic addition of GLI3R partially rescued the ciliopathic midfacial widening. Interestingly, despite several previous studies suggesting midfacial development relies heavily on GLI3R activity, the conditional loss of GLI3 alone did not reproduce the ciliopathic phenotype. Only the combined loss of both GLI2 and GLI3 was able to phenocopy the ciliopathic midfacial appearance. Our findings suggest that ciliopathic facial phenotypes are generated via loss of both GLI3R and GLI2R and that this pathology occurs via a de-repression mechanism. Furthermore, these studies suggest a novel role for GLI2R in craniofacial development.