Evc is a positive mediator of Ihh-regulated bone growth that localises at the base of chondrocyte cilia

Abstract

EVC is a novel protein mutated in the human chondroectodermal dysplasia Ellis-van Creveld syndrome (EvC; OMIM: 225500). We have inactivated Evc in the mouse and show that Evc-/- mice develop an EvC-like syndrome, including short ribs, short limbs and dental abnormalities. lacZ driven by the Evc promoter revealed that Evc is expressed in the developing bones and the orofacial region. Antibodies developed against Evc locate the protein at the base of the primary cilium. The growth plate of Evc-/- mice shows delayed bone collar formation and advanced maturation of chondrocytes. Indian hedgehog(Ihh) is expressed normally in the growth plates of Evc-/- mice, but expression of the Ihh downstream genes Ptch1 and Gli1 was markedly decreased. Recent studies have shown that Smo localises to primary cilia and that Gli3 processing is defective in intraflagellar transport mutants. In vitro studies using Evc-/- cells demonstrate that the defect lies downstream of Smo. Chondrocyte cilia are present in Evc-/- mice and Gli3 processing appears normal by western blot analysis. We conclude that Evc is an intracellular component of the hedgehog signal transduction pathway that is required for normal transcriptional activation of Ihh target genes.

Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2

“…In the absence of Shh, Flag-Gli2 was processed into a truncated repressor form (Gli2 R ) while treating cells with Shh-conditioned medium inhibits Gli2 R formation ( Figure 5M), resulting in a 4-fold increase in the ratio of Gli2 FL /Gli2 R ( Figure 5N). Consistent with a previous finding [37], we found that Evc/Evc2 RNAi had little if any effect on Gli2 R formation in the absence of Shh, suggesting that Evc/ Evc2 are not required for Gli processing to generate the repressor forms. However, we found that Shh did not effectively block the production of Gli2 R in the absence of Evc/Evc2 ( Figure 5M and 5N), suggesting that Evc/Evc2 are required for Hh to inhibit Gli2 R formation in cultured cells.…”

“…Previous work has implicated that Evc/Evc2 are required for Hh-and Smo agonist-induced pathway activation, suggesting that they act at the level of Smo in Hh signal transduction; however, it did not distinguish whether Evc/Evc2 are required for Smo activation or signal transduction downstream of activated forms of Smo [36,37]. Our above observations suggest that Evc/Evc2 act at a step downstream of Smo after it is phosphorylated in response to Hh.…”

“…For example, Hh-induced Smo phosphorylation promotes its ciliary localization and active conformation [9]. Evc and Evc2 form a complex localized in the primary cilia and both proteins have been implicated as positive regulators of mammalian Hh signaling [36,37]. We found that inactivation of Evc/Evc2 does not significantly affect Hh-induced Smo phosphory- Figure 6B).…”

“…However, we found that Shh did not effectively block the production of Gli2 R in the absence of Evc/Evc2 ( Figure 5M and 5N), suggesting that Evc/Evc2 are required for Hh to inhibit Gli2 R formation in cultured cells. A previous study indicated that Shh can still block Gli repressor formation in the absence of Evc function in vivo [37]. It is possible that Hh-mediated inhibition of Gli processing is more dependent on Evc/Evc2 in vitro, whereas redundant or context-dependent mechanisms may exist in vivo.…”

“…Previous studies demonstrated that Evc is essential for Ihh signaling in the cartilage growth plate, and Evc2, a close homolog of Evc, is a positive modulator of Shh signaling in cultured cells [36,37]. To define the underlying mechanism by which Evc/Evc2 modulate Hh signaling, we employed the RNAi approach to inactivate the two genes.…”