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The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

ABSTRACT: talpid(2) is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid(3), talpid(2) has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid(2) mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translational processing of GLI2 and GLI3 was aberrant in the developing facial prominences. Although both GLI2 and GLI3 processing were disrupted in talpid(2) mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid(2) phenotype was linked to a 1.4?Mb region on GGA1q that contained the gene encoding the ciliary protein C2CD3. We cloned the avian ortholog of C2CD3 and found its expression was ubiquitous, but most robust in the developing limbs and facial prominences. Furthermore, we found that C2CD3 is localized proximal to the ciliary axoneme and is important for docking the mother centriole to the ciliary vesicle and cell membrane. Finally, we identified a 19?bp deletion in talpid(2) C2CD3 that produces a premature stop codon, and thus a truncated protein, as the likely causal allele for the phenotype. Together, these data provide insight into the cellular, molecular and genetic etiology of the talpid(2) phenotype. Our data suggest that, although the talpid(2) and talpid(3) mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

SUBMITTER: Chang CF

PROVIDER: S-EPMC4197679 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

Chang Ching-Fang CF Schock Elizabeth N EN O'Hare Elizabeth A EA Dodgson Jerry J Cheng Hans H HH Muir William M WM Edelmann Richard E RE Delany Mary E ME Brugmann Samantha A SA

Development (Cambridge, England) 20140801 15

talpid(2) is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid(3), talpid(2) has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid(2) mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translati ...[more]

PMID: 25053433

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Project description:The primary cilium is a ubiquitous, microtubule-based organelle that cells utilize to transduce molecular signals. Ciliopathies are a group of diseases that are caused by a disruption in the structure or function of the primary cilium. Over 30% of all ciliopathies are primarily defined by their craniofacial phenotypes, which typically include midfacial defects, cleft lip/palate, micrognathia, aglossia, and craniosynostosis. The frequency and severity of craniofacial phenotypes in ciliopathies emphasizes the importance of the cilium during development of the craniofacial complex. Molecularly, many ciliopathic mutants, including the avian talpid2 (ta2 ), report pathologically high levels of full-length GLI3 (GLI3FL), which can go on to function as an activator (GLIA), and reduced production of truncated GLI3 (GLI3T), which can go on to function as a repressor (GLIR). These observations suggest that the craniofacial phenotypes of ciliary mutants like ta2 are caused either by excessive activity of the GLIA or reduced activity of GLIR. To decipher between these two scenarios, we examined GLI3 occupation at the regulatory regions of target genes and subsequent target gene expression. Using in silico strategies we identified consensus GLI binding regions (GBRs) in the avian genome and confirmed GLI3 binding to the regulatory regions of its targets by chromatin immunoprecipitation (ChIP). In ta2 mutants, there was a strikingly low number of GLI3 target genes that had significantly increased expression in facial prominences compared to the control embryo and GLI3 occupancy at GBRs associated with target genes was largely reduced. In vitro DNA binding assays, further supported ChIP results, indicated that the excessive GLI3FL generated in ta2 mutants did not bind to GBRs. In light of these results, we explored the possibility of GLI co-regulator proteins playing a role in regulatory mechanism of GLI-mediated transcription. Taken together our studies suggest that craniofacial ciliopathic phenotypes are produced via reduced GLIT production, allowing for target gene transcription to be mediated by the combinatorial code of GLI co-regulators.

Dataset Information

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

Publications

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

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