Sfrp5 Is A Key Factor For Maintaining Odontogenic Potential Of Mouse Dental Mesenchymal Cells

Tooth development is the result of reciprocal tissue interactions between ectoderm-derived epithelium and cranial neural crest-derived mesenchyme.During this process,the epithelium or mesenchyme must possess odontogenic potential.Tooth recombination experiments have demonstrated that the odontogenic potential of molar germs is located in the dental epithelium before E12.5,and then is transferred to the dental mesenchyme.The dental tissue with odontogenic potential can induce non-dental tissue to form tooth together.The usage of stem cell based tissue engineering for tooth regeneration also requires epithelial or mesenchymal stem cells to possess the odontogenic potential.However,no type of stem cell has been shown to possess this capability.Therefore,identification of the molecular components of odontogenic potential is the prerequisite for future realization of stem cell based tooth regeneration in humans,and the study is the hotspot and major challenge in this research field.The molar tooth germ has the ability to re-aggregate and develop into tooth after dispersion.According to these characteristics,our team has established systems for the loss of odontogenic potential in E13.5 and E14.5 molar mesenchymal cells: E13.5molar mesenchyme with the odontogenic potential no longer has the capability to induce tooth formation after dispersion and 8 hours in vitro culture,and even loses the potential after 9 days of culture.E14.5 molar mesenchyme with the same odontogenic potential significantly decreases this potential after 24 h in vitro culture,and this potential is completely lost after 48 hours of culture.Therefore,every four E13.5molar mesenchymal cell samples of 0h,8h,and 9d in vitro culture after dispersion and every three E14.5 molar mesenchymal tissues of 0h,24 h,and 48 h in vitro culture were collected and performed RNA-Seq to screen for the critical molecules that maintain the odontogenic potential.It is known that critical genes can be parsed from a variety of analytical methods although different bioinformatics analysis methods generate many different results for the same data.Therefore,using Fold-change(FC)?2 and FDR?0.001 as thresholds,we employed the Tophat2-Cufflinks-Cuffdiff and HISAT2-FeatureCount-DESeq2 to analyze the RNA-Seq data of E13.5 and E14.5molar mesenchymal cells,and screened 34 and 23 differentially expressed genes that were significantly expressed in both E13.5 and E14.5 molar mesenchymal cells respectively.We further found that 15 genes were identical in the differential genes obtained by the two bioinformatics methods,and Sfrp5(Secreted frizzled-related protein5,Sfrp5)was closely related to tooth development.After detection,Sfrp5 was faintly expressed in the E12.5 molar and sorely expressed in E13.5 and E14.5 molars,while its expression level decreased as the molar continued to develop.In addition,we verified that Sfrp5 was significantly down-regulated in E13.5 molar mesenchymal cells after 8 hours in vitro cultured.In order to check the biological function of Sfrp5 in maintaining the odontogenic potential of mouse molar mesenchymal cells,exogenous SFRP5 protein was added to the cultured E13.5 molar mesenchymal cells for 8h and found that SFRP5 could effectively maintain the expression of key odontogenic genes in mouse molar mesenchymal cells,including Msx1 and Lhx6.Furthermore,E13.5 molar mesenchymal cells co-cultured with SFRP5 protein for 8h were recombined with the second branchial arch epithelium(non-odontogenic epithelium),and found that SFRP5 could effectively induce the second branchial arch epithelium to differentiate into mature ameloblasts(The chimera of the control group was 0%,and the chimera of the SFRP5 treatment group was as high as 43.5%).These results demonstrate that the function of Sfrp5 is closely related to the maintenance of the odontogenic potential in mouse molar mesenchymal cells.Further study showed that SFRP5 protein could dramatically increase the Wnt/?-catenin and Wnt/PKC activity in E13.5molar mesenchymal cells after 8 hours in vitro culture,which hold Wnt activity at a certain threshold steady state level.Our results demonstrate that Sfrp5 could be one of the critical factors for the maintenance of odontogenic potential in dental mesenchyme.In general,members of the Sfrps family act as antagonists of the Wnt signaling pathway and inhibit its activity.However,it has also been reported that Sfrps can interact with Wnt's Frizzled receptor to activate Wnt signaling.Our study reveals that,unlike the function of other Sfrps during tooth development(Sfrp1,Sfrp2,and Sfrp3 always serve as antagonists of the Wnt pathway to regulate tooth morphogenesis and eruption),Sfrp5 mayfunction in activating or maintaining the Wnt activity to keep the homeostasis of Wnt signaling in dental mesenchymal cells and plays an important role in maintaining odontogenic potential.This study lays a foundation for further exploring the molecular composition of odontogenic potential.