The Role Of Sonic Hedgehog In Tooth Development

During organ development, neighboring cells coordinate their growth and differentiation in order to acquire form and function. In the case of teeth, cells from the oral ectoderm and mesenchyme interact to give rise to highly mineralized structures of specific shapes and sizes.Although several genes have been identified that show interesting spatial . and temporal regulation during the development of the mammalian tooth, the molecular framework underlying tooth development is poorly understood. Several homebox containing transcriptional regulators are involved in the patterning of the jaw and development of the teeth. In addition, secreted protein such as bone morphogenetic proteins(BMP), fibroblast growth factor(FGF), Wnts and Hedgehog(Hh) have been proposed to regulate epithelial-mesenchymal interactions. They are expressed in tooth germ at different stages and act as morphogenetic signals mediating the different steps of interactions. Tooth development is a continuous process controlled by a complexed network, of which any changes would affect its role in the process.Sonic hedgehog (Shh), a member of Hedgehog signaling protein family of vertebrates, plays a key role as signaling factorin the normal development. Shh is involved in growth and patterning of a number of organs including the hair. The Shh signalling pathway has been proposed to play an important role in mammalian tooth development.In the developing tooth, the dynamic expression pattern of Shh is suggestive of multiple functions for the protein. The expression of Shh has spatio-temporal characters, suggesting Shh as a paracrine and autocrine signaling molecule might stimulate epithelium and mesenchyme proliferation. Little is known about the role of Shh on cytodifferentiation and the later development of tooth. To better understand the role of shh in tooth development and mineralization, immunohistochemistry, gene transfection, antisense technology, tooth germ organ culture in vitro were used. The results showed that Shh play an important role in tooth development and mineralization.1. Expression of Shh during tooth developmentThe expression of Shh was examined by immunohistochemistry in mouse tooth germ and the bell stage of human tooth germ. The result showed that in the initiation of the mouse tooth development, the oral ectoderm formed a tooth bud by invaginating into the underling mesenchyme, Shh was stongly positive in oral and tooth epidermis; in the cup stage, Shh was found in inner and outer enamel epithelium and oral epithelium, in the bell stage, Shh was strongly positive in ameloblast and weak in odontoblast. The same pattern in the bell stage of human tooth and strong positive expression in ameloblast was found, positive signal was uncovered in odontoblast and no obvious expression in the dental papilla cells of stratum and intermedium. The expression of Shh has spatio-temporal characters during tooth development. The results suggested that Shh may play a role in cell differentiation and matrix mineralization besides tooth initiation.2. Efeects of Shh on dental papilla cells in vitroThe Shh gene specific primers was designed based on the datafrom GenBank. A specific segment with the length of 1.4kbp was obtained from human tooth germ by RT-PCR, the segment was inserted into pMD18-T vector and the recombinant plasmid was transformed into E.coli XL-10 Gold. The positive clones were analyzed with restriction endonuclease mapping and DNA sequencing. The result showed that the nucleotide sequence we cloned were consistent with the GenBank record, it suggests that the total lengthen of Shh was coloned from human tooth germ.We constructed eukaryotic expression plasmid by insering Shh cDNA into pCDNA3 vector and transfected the plasmids into COS7 cells with LipofectAMINE?2000. Western blot showed that Shh was synthesized and secreted by the cells. MTT method and enzyme dynamics method were used to invastigate the effects of Shh on the proliferation and the changes of ALPase in dental papilla cells in vitro. The results rev...