Role Of Shh Signaling At The Early Stage Of Amelogenesis

Sonic hedgehog(Shh)is required for normal embryonic development.It is a marker of odontogenesis amongst the tooth of vertebrates and is involved in oral epithelial-mesenchymal signal transduction.Shh promotes the morphogenetic movement of epithelial cells in the early tooth development,and influences the cell cycle and differentiation.However,the mechanism of Shh signaling in the early stage of amelogenesis is still unclear.By in situ hybridization analysis we found that Shh signal and its pathway molecules were expressed in the ameloblasts of early born mice.The expression was gradually decreased and finally disappeared in the pre-secretory ameloblasts of the early secretory phase.It suggests that Shh signaling may be required for differentiation of the pre-secretory ameloblasts.To accurately define the function of Shh signaling during amelogenesis,we constructed an inducible Amelx-ERCreER mouse line that is driven by the promoter of ameloblast-specific Amelx gene.Deletion of Shh and Smo in pre-secretory ameloblasts with the Amelx-ERCreER mouse could result in enamel hypoplasia.We therefore speculate that Shh signaling may be involved in the synthesis or secretion of enamel matrix proteins.By in situ hybridization and qPCR analysis,we found that removal of Shh gene in pre-secretory ameloblasts resulted in significant down-regulation of the coding genes of enamel matrix proteins,including Amelogenin(amelx),Ameloblastin(Ambn),Enamelin(Enam).Moreover,immunofluoresc ence and Western blot analyses showed a significant reduction of enamel matrix proteins in Shh mutants.We also used the recombinant Shh protein to activate Shh signaling pathway in P0 molar with an in vitro organ culture system.Q-PCR analysis showed that the expression of Amelx,Ambn and Enam was significantly up-regulated.The above results indicate that Shh signaling may directly participate in the regulation of gene expression of the enamel matrix proteins.To verify our hypothesis,we performed ChIP analysis using ALC cells and the molar epithelium of wild-type mouse at P2.We found that Gli1,Gli2,Gli3 proteins could directly bind to the promoter regions of Amelx,Ambn,and Enam,in which Gli2 showed the strongest binding ability.To determine the binding region of the Gli proteins with the Enam promoter,we performed luciferase reporter assay by co-transfection of Gli expression vectors and reporter plasmids driven by different length of Enam promoter.It showed that the binding region of Gli in Enam promoter was located at-600 bp to-1200 bp upstream of transcription start site.Taken together,these data manifest that Shh signaling participates in enamel formation by directly regulating the gene expression of enamel matrix proteins through Gli transcription factor.