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

Abstract

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.

Neural crest cells utilize primary cilia to regulate ventral forebrain morphogenesis via Hedgehog-dependent regulation of oriented cell division

“…We found that the transcriptional effectors of the HH pathway, the GLI proteins, did not undergo proper post-translational processing in the frontonasal prominence. We specifically found increased GLI2/3 full-length isoforms were produced at the expense of the truncated repressor isoforms (Chang et al, 2016). These data suggested that the transcriptional targets of the HH pathway in NCCs might not be appropriately regulated in the midface.…”

“…1A, B), a duplicated nasal septum, and aglossia (Brugmann et al, 2010; Chang et al, 2016; Millington et al, 2017). While each of these phenotypes was striking and severe, they were not necessarily surprising, as neural crest cells (NCCs) contribute significantly to the development of each of these structures (Chai et al, 2000; Danielian et al, 1998; Noden and Trainor, 2005).…”

“…Our prior work examined the facial phenotypes that arise when NCCs lack primary cilia ( Wnt1-Cre;Kif3a fl/fl ). We previously reported that Wnt1-Cre;Kif3a fl/fl mutants possessed multiple craniofacial phenotypes, including: severe midfacial widening, micrognathia, and aglossia (Brugmann et al, 2010; Chang et al, 2016; Chang et al, 2015; Millington et al, 2017). Furthermore, we found that these phenotypes were the result of aberrant Hedgehog signal transduction in NCCs.…”

Craniofacial Ciliopathies and the Interpretation of Hedgehog Signal Transduction

“…In this paper, Chang et al demonstrate that disruption of cilia function leads to an increase in the amount of full-length GLI2 and GLI3 in the nucleus, thus shifting the ratio of GLIA:GLIR toward the activator form [6]. This was partially rectified by increasing the amounts of GLI3R.…”

“…Altogether, this suggests that the phenotypic interpretation of HH signaling changes is more complicated than a simple on/off mechanism. In this issue, Chang et al mutate the intraflagellar transport proteins Ift88 and Kif3a in the mouse neural crest, comparing the phenotypes to those associated with functional mutations in the HH effectors Gli2 and Gli3 [6]. In doing so, they clarify the molecular basis for facial widening in ciliopathies and uncover a novel in vivo requirement for GLI-mediated transcriptional repression.…”