Inhibition Of Hedgehog Signaling By Vismodegib Regulates Supernumerary Tooth Formation During Embryogenesis

Background:Supernumerary teeth(Hyperdontia)are teeth present in addition to the normal teeth set,often associated with complications(dental impaction,overcrowding)and syndromes(Gardner Syndrome,cleidocranial dysplasia).Tooth organogenesis is controlled by sequential and reciprocal inductive interactions between epithelium and mesenchyme,regulated by an intricate network of cell-cell signaling.Among many regulators,Shh signaling plays a fundamental role in mediating epithelium-mesenchyme interactions at early stage of tooth development.Objective:There is little report about supernumerary teeth model using exogenous inhibitors,here,by utilizing Vismodegib(a small-molecule hedgehog pathway inhibitor)to mouse embryos,we establish a novel mouse model of supernumerary teeth and further elucidate the mechanism of supernumerary teeth and explore the capacity for tooth replacement in mammals.Methods:Pregnant ICR mice in test group were administered 150mg/kg vismodegib(GDC-0449)by oral gavage at indicated time points,including: GD9.5,10.5,11.5,control group exposed to 0.5% methyl cellulose with 0.2% Tween at same time points.We detected whether supernumerary teeth appears in test and control group at E16.5embryos using HE staining technique.Embryos at E11.5-E16.5 were collected to observe the pattern formation in the developing supernumerary teeth.Expression of Pitx2 in the developing mouse tooth germ(E11.5,E12.5,E13.5)were detected by whole-mount in situ hybridization.Dental zone of mandible were removed from E16.5embryos and then transplanted into the subcapsular layer of nude mouse kidneys for tooth calcification.Immunohistochemical and immunofluorescence staining was performed to exam?-catenin and Osr2 protein expression.Results:E9.5 and E10.5 vismodegib-exposed mice displayed cleft palate and developed supernumerary teeth lingual to mandibular first molar,E11.5 vismodegib-exposed mice only showed cleft palate.Supernumerary tooth germ developed synchronously with first molar tooth germ.Pitx2 expression field in vismodegib-exposed mice were expanded than control group at E12.5,and divided into two germs at E13.5,supernumerary tooth germ and first molar germ connected at mesial side.After two months,molar tooth germs of control group gave rise to three mineralized molar teeth,representing the normal molars;vismodegib-exposed mice molar tooth germs gave rise to four mineralized teeth.Increased amounts of ?-catenin showed in vismodegib-exposed mice.Conclusions:Our result suggest inhibit Hedgehog signaling in early stage of tooth development induced initiation of supernumerary teeth lingual to first molar germ,may due to expansion of odontogenic field and splitting of primitive tooth germ caused by increased Wnt/?-catenin signaling.