Inhibition Of Hedgehog Signaling Pathway By Vismodegib Leads To Supernumerary Teeth In Mice

Objective:The purpose of this research is to establish a mouse model of supernumerary teeth lingual to mandibular first molar after inhibition of Hedgehog signaling in the intitial period of tooth development by vismodegib.And further explore the mechanism of supernumerary teeth formation.Methods:1.Pregnant SPF grade ICR mice in the test groups were fed with 150 mg/kg vismodegib by oral gavage at embryonic day(E)10.5,meanwhile the control groups were fed with the same amount solvent at the same time.The occurrence of lingual supernumerary teeth at E16.5 were detected by HE staining.The expression of Ptch1 and Gli1 genes were detected by RT-qPCR to analyze Hedgehog signaling activity.2.The pattern formation of lingual supernumerary teeth were analyzed by HE staining of the test and control groups from E11.5 to E16.5.Three-dimensional reconstructed image of dental epithelium of the mandibular molars at E15.5 and E16.5 were performed to analyze the three-dimension morphology of the supernumerary teeth.3.Changes in size of the dental epithelium along the antero-posterior axis at E12.5 were measured by three-dimension reconstruction.4.Cell proliferation and cell death of dental epithelium at E11.5-E13.5 was detected by immunefluorescence staining of PHH3 protein and TUNEL staining.5.Wnt/?-catenin signaling activity was detected by immunefluorescence staining of ?-catenin protein and expression of ?-catenin genes by RT-qPCR.Results:1.The test groups had a higher rate of supernumerary teeth and a lower death rate.Hedgehog signaling activity is effectively inhibited by exposed to vismodegib.2.The development of molar germ in the test groups were slower than the control groups.The lingual dental epithelium of the first mandibular molar germ became thicker at E12.5,and then separated into two independent molar germs at E13.5.The supernumerary teeth were smaller than the first molar and had no dental cord.3.The dental epithelial area of mandibular molar germ on test group conspicuously increased at E12.5.4.Cell proliferation diminished and cell death increased in dental epithelium of mandibular molar on test groups at E11.5-E13.5.5.Nuclear localization of ?-catenin in dental epithelial cells of lingual supernumerary were detected at E12.5-E13.5 and the expression of ?-catenin gene in mandibular molars of test groups were up-regulated.Conclusion:The use of Vismodegib to mice at E10.5 can effectively inhibit the activity of Hedgehog signaling in developing teeth germ and set up an available and stable mouse model of supernumerary teeth lingual to mandibular first molar.Inhibition of Hedgehog signaling by Vismodegib lead to cell proliferation diminishment and cell death increase in dental epithelial without affecting the subsequent formation of supernumerary teeth.Formation of lingual supernumerary teeth may relate to up-regulation of Wnt/?-catenin signaling activity and expansion of odontogenic field.