Spatio-temporal Expression And Role Of Vps4b In Knock Out Mouse Tooth Germ

Background and ObjectiveVacuolar protein sorting 4B?VPS4B?,a member of the AAA ATPase family,plays an important role in the lysosomes degradation pathway,intracellular protein trafficking,virus budding,and regulating the different stages of mitosis and cell division.Our research team has previously identified that VPS4B as one of the pathogenic genes related to Dentin dysplasia type I?DD-I?by DNA sequencing analysis of peripheral blood.Besides,through the preliminary study we have found that VPS4B gene regulates tooth development process by the Wnt pathway.Thus,a Vps4b knockout mouse model has been constructed to elucidate the mechanism of this gene in tooth development.In this experiment,the Vps4b knockout mouse was used to clarify the distribution of Vps4b gene in tooth germ development,and to explore the effect of the mutant gene on tooth development related genes,consequently further reveals the role of this gene in tooth teratogenesis.Materials and MethodsThe female and male Vps4b gene knockout mice which have been constructed by using the CRISPR-Cas9 technique were placed in the same cage at the stable time.The pregnancy time has been recorded.The fetal rats'head and tail were collected at 13.5,14.5 and 16.5 days after pregnancy,and the neonatal mice were sacrificed 2.5 to 7 days after birth to remove the jaw and tail.PCR was used to identify the genotypes of fetal and neonatal mice.The paraffin tissue sections of the first molar tooth germ were obtained from the head of the fetal mouse and the mandible of the neonatal mice.The development stages of tooth germs were determined by HE staining.The expressions of Vps4b,Dspp,and Col-I in the first mandibular molars tooth germs of normal and knockout rats at different developmental stages were examined and compared by immunohistochemical method.ResultsThe results of PCR showed that the genotypes of the offspring mice were Vps4b^+/+and Vps4b^+/-.HE staining showed that the first molars tooth germs of the embryos at 13.5,14.5 and 16.5 days were at bud,cap and bell-shaped stage respectively,while the 2.5 days of age was in the secretory stage and the 7 days in the mineralization period.Immunohistochemical staining of normal mice revealed that Vps4b was widely expressed in tooth germ in bud and cap stage,but no expression of Dspp and Col-I.In bell stage Vps4b was observed in inner and outer enamel epithelium,dental papilla and dental follicle,and Dspp was expressed in inner enamel epithelium and dental papilla,while Col-I was found in dental papilla and dental follicle.During secretory and mineralization stage Vps4b,Dspp,and Col-I were all expressed in ameloblasts,odontoblasts,and dental follicles,while in dental papilla only Vps4b and Col-I have been observed.After Vps4b gene mutation,Vps4b was negatively expressed in the bud stage of enamel organ,while in some tissues the expression intensity was decreased?P<0.05?.There was no expression of Dspp in bell stage of the dental papilla and secretory stage of the dental follicle.Besides,the number of positive cells in the ameloblasts,odontoblasts at secretory stage and dental follicles at mineralization period were decreased?P<0.05?.The expression sites of Col-I were no difference,however,the intensity of expression was decreased in some sites?P<0.05?.ConclusionDuring the development of Vps4b^+/+mice,Vps4b was expressed in different stages of tooth germs and the expression intensity changed with development,indicating that Vps4b may be involved in the normal development of tooth germs.In Vps4b^+/-mice,the expressions of Vps4b and tooth development-related protein were decreased.It is speculated that it may affect the development of tooth by regulating the expression of itself and related proteins.