Abstract
BackgroundEvc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.ResultsBioinformatic analysis reveals that the Evc and Evc2 genes arose through a duplication event early in metazoan evolution and were subsequently lost in arthropods and nematodes. Here we demonstrate that Evc2 is essential for Hh pathway activation in response to the Smo agonist purmorphamine. A yeast two-hybrid screen using Evc as bait identified Evc2 as an Evc binding partner and we confirmed the interaction by immunoprecipitation. We developed anti-Evc2 antibodies and show that Evc2 and Evc co-localize at the basal body and also on primary cilia. In transfected cells, basal body and cilia localization is observed when Evc and Evc2 constructs are co-transfected but not when either construct is transfected individually. We show that Evc and Evc2 are cilia transmembrane proteins, the C-terminus for both being intracellular and Evc2, but not Evc, having an extracellular portion. Furthermore, Evc is absent at the basal body in Evc2 null cells. Using Western blots of cytoplasmic and nuclear protein, we also demonstrate that full length Evc2 but not Evc, is located in the nucleus.ConclusionsWe demonstrate for the first time that Evc2 is a positive regulator of the Hh signalling pathway and that it is located at the basal body of primary cilia. We show that the presence of Evc and Evc2 at the basal body and cilia membrane is co-dependent. In addition, Evc2, but not Evc, is present in the cell nucleus suggesting movement of Evc2 between the cilium and nucleus.
Highlights
Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate
Evc2 is a positive regulator of Hh signal transduction Evc is known to be a positive regulator of Hh signalling both in the cartilage growth plate and in cultured chondrocytes and mouse embryonic fibroblasts (MEFs)
The observation that EVC and EVC2 mutations are associated with the same phenotype indicates that Evc2 is likely to be a positive regulator of Hh signalling
Summary
Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome. EVC was first identified through a positional cloning approach as the gene defective in patients with the recessive disorder Ellis-van Creveld syndrome (EvC) [1]. Mice lacking Evc, like patients with Ellis-van Creveld syndrome, have short limbs, short ribs and dental abnormalities. In vitro studies treating mouse embryonic fibroblasts (MEFs) and chondrocytes with the Hedgehog (Hh) agonist purmorphamine confirmed that Hh signal transduction is defective in cells lacking Evc [7]. Evc is essential for Ihh signalling in the cartilage growth plate
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