The role of the EVC in development

Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive disease caused by the mutations in EVC or EVC2 genes. Human patients with EvC syndrome showed clinical features as short limbs, short ribs, postaxial polydactyly and orofacial defects and about 60% of affected individuals showed atrial septum defects. Evc-/- mice showed short limbs and short ribs similar with those observed in human patients. However, the cardiac phenotype of Evc-/- mice had not been studied. In this study, histological analysis was performed in both C57Bl/6J X 129Sv mixed background and inbred C57Bl/6J 25 background mouse embryos. No obvious cardiac development defects were observed. Parallel to this, the Evc mRNA and Evc protein were also analyzed by in situ hybridization and using the lacZ reporter system. X-gal positive cells were observed at the dorsal to atrial wall and primary atrial septum in E11.5 and E12.5, respectively. No Evc mRNA was detected in developmental heart by in situ hybridization by probes located at c.926 to c.1717. Previous study demonstrated that Evc and Evc2 protein are localized to the base of cilia. In this study, two different methods, deciliation and immuno-TEM, were applied to localize the Evc protein more precisely. The deciliation treatment sheared the cilia off at the distal end of transition zone and the immunofluorecent staining results indicated that the localization of the Evc protein was not beyond the distal end of transition zone. In the immuno-TEM experiment, although several factors were modified for proper staining, no informative results were obtained from this experiment. As the mutations of ciliary proteins might result in malformation of cilia, the ciliary structure of chondrocytes were also examined in this study. No structural II difference was observed in Evc-/- cells comparing to wildtype control. The Evc2 protein was undetectable at the base of cilia in MEFs, chondrocytes and osteoblasts when Evc protein was absent. The existence of Evc2 protein was detected in total lysate Evc-/- cells by western blot, indicating rather than affecting the expression of Evc2 protein, Evc plays a role in Evc2 protein correct localization. In human fibroblasts, EVC protein was localized to the base of cilia and the nucleus. The nuclear localization of Evc protein was never been observed in mouse cells by immunofluorescence staining. This inconsistence was examined through subcellular fractionation assay. The western blotting results demonstrated that the Evc protein exists in the nuclear fraction in the mouse MEFs. Several potential Evc associated proteins suggested by yeast two-hybrid study were also examined in subcellular fractionation assay. No distribution differences between wildtype and Evc-/- cells were observed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo