Effects Of Sfrp3knocking Down On Mouse Tooth Development

Wnt extracellular signaling molecules have essential roles in epithelial - mesenchymal interactions involved in tissue morphogenesis. Genetic studies in Drosophila and Caenorhabditis elegans, ectopic gene expression in Xenopus, and gene knockouts in the mouse have demonstrated the involvement of Wnts in processes as diverse as segmentation, CNS patterning, and control of asymmetric cell divisions. Wnts signal is one of the four growth factors that regulate the tooth developmemt. SFRPs (secreted Fizzled-related proteins) are receptors that directly bind to Wnts. In this research, we investigate the effects of Wnts signal on tooth development,and we focus on the sfrp3 which is one of the sfrp family members. We use RNAi and lentiviral gene transfer technique to knock down the expression of sfrp3 in tooth mesenchyme. Plasmids of pNL-EGFP-U₆-shsfrp3-I and II which expressed shRNA were constructed and combined with pVSVG and pHelper, respcectively, to constitute Lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce EGFP-shsfrp3-I and EGFP- shsfrp3-II lentivirus. Tooth mesenchymal cells were infected by both lentiviruses and identified by Realtime RT-PCR to examine inhibitory efficency. The EGFP- shsfrp3-II lentivirus which can inhibit 63% of the expression of sfrp3 was selected finally for knocking down sfrp3. Experimental results demonstrated knocking down of sfrp3 in recombinant tooth germs (the mesenchyme of E13.5+the oral epithelium of E10.5) will reduce the percentage of tooth formation. Inhibition of the expression of sfrp3 in intact El 1.5 malor tooth germ will enhance tooth development.These results indicate that Wnt signaling plays an important role in early tooth developmemt.