Abstract
talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 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 talpid2 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 talpid2 mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid2 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 talpid2 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 talpid2 phenotype. Our data suggest that, although the talpid2 and talpid3 mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.
Highlights
The chick is a classic embryological system that has been meticulously documented and described (Hamburger and Hamilton, 1951)
Our experiments indicate that, full-length GLI2 processing is impaired in talpid2 mutants, nuclear levels of GLI2 activator (GLI2A) and GLI2 repressor (GLI2R) are not altered
Post-translational processing of GLI3 is disrupted and GLI3 activator is increased in talpid2 mutants We examined the levels of GLI3 between control and talpid2 facial prominences (Fig. 5A)
Summary
The chick is a classic embryological system that has been meticulously documented and described (Hamburger and Hamilton, 1951). As ciliary defects have been known to affect various tissues and developmental domains differently, and because previous reports suggest a gain of SHH function in the limb with a loss of SHH function in the face of talpid (Buxton et al, 2004; Davey et al, 2006, frontonasal and maxillary prominences, pathway activity (as determined by PTC expression) is not. RNA-seq was performed on individual facial prominences of both control and talpid embryos (Fig. 3E) These data support both qRT-PCR and in situ data suggesting that, SHH ligand expression is increased in the. Post-translational processing of GLI3 is disrupted and GLI3 activator is increased in talpid mutants We examined the levels of GLI3 between control and talpid facial prominences (Fig. 5A). We conclude that truncated C2CD3 in talpid embryos impairs the ability of this protein to properly establish the transition fiber complex, which in turn affects intracellular ciliogenesis through failure of mother centriole/ciliary vesicle docking
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