The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia

Abstract

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

Developmental Regulation of the Growth Plate and Cranial Synchondrosis

“…The same group also demonstrated compromised bone collar formation in Evc −/− mice in the C57BL/6 homogenous background but showed that both proliferation and differentiation of chondrocytes are compromised in mutant mice (Pacheco et al 2012). EVC and EVC2 are mutually required for their ciliary localization and loss of Evc2/Limbin results in similar skeletal phenotypes found in Evc −/− mice (Caparrós-Martín et al 2013;Zhang et al 2015).…”

“…5) associated with dysregulated Ihh signaling in both the ISS and SOS at the neonatal stage, Patched1 expression is decreased in the perichondrium (Pacheco et al 2012), indicating that EVC and Kif3a/IFT88 regulate the synchondrosis with different mechanisms. Finally, EVC2 −/− mice also have impaired ISS development (Caparrós-Martín et al 2013). …”

Novel homozygous mutations in the EVC and EVC2 genes in two consanguineous families segregating autosomal recessive Ellis–van Creveld syndrome

“…Simultaneously, Evc and Evc2 appear to be needed for the activation of pathway in the absence of Sufu (Caparrós-Martín et al, 2013). EVC syndrome shares some phenotypic similarities with OFDI, such as orofacial and nail abnormalities and T T T T T T T TT T T T T T T TT T T T T T T T T T T T T T T T TT T T T T T T T Novel homozygous mutations in the EVC Aziz et al 5 cardiovascular defects.…”

Cilia and coordination of signaling networks during heart development

“…4), 21,22,142 and defective ciliary assembly is associated with defective cardiomyogenesis 21 as well as Hh-related heart defects that might be independent of nodal cilia, including ventricular and endocardial cushion-derived defects. 122,142,154 Further, 60% of patients with mutations in genes encoding the EVC proteins, EVC1/EVC2, which interact with Smo at the primary cilium to transduce Hh signaling [155][156][157][158][159] , display CHD, including AVSD and ASD. 47,51 In line with these findings, EVC proteins are coexpressed in the OFT and the mesenchymal structures of the atrial septa and AV cushions during heart development in mice 51 ; areas that are known to be ciliated during cardiogenesis.…”