| As an important part of the craniofacial,teeth not only mainly bear the chewing function but also play an important role in maintaining the appearance as well as the beauty of the face and helping pronunciation.Tooth development relies on reciprocal tissue interactions between ectoderm-derived dental epithelium and cranial neural crest-derived mesenchyme.During this process,the epithelium or mesenchyme must possess odontogenic potential.In the future,to achieve human tooth regeneration by stem cell-based bioengineering,it should follow the principles of tooth development and requires epithelial and mesenchymal cells and one of them possessing the tooth-inducing capability.So far,neither dental mesenchymal stem cells nor substitution of dental-derived epithelial cells have the tooth-inducing capability,and they can only differentiate into dental tissue in response to tooth-inducing signals.Thus,the major challenge and extremely critical step of tooth regeneration engineering is to identify the molecular compositions of odontogenic potential to confer the stem cells with odontogenic potential.Considering that it is easier to identify the key molecular compositions of odontogenic potential in the dental mesenchyme to confer tooth-inducing capability with mesenchymal stem cells but not the ones in the dental epithelium to confer tooth-inducing capability with non-odontogenic epithelial cells,we aim to identify the molecular compositions of odontogenic potential in the dental mesenchyme.On previously researches of our team,using mice as research model,we established the time node when the E13.5 and E14.5 mouse molar mesenchymal cells(m MMC)possess or lose the odontogenic potential during in vitro cultured.We have screened Sfrp5 that play a key role in maintenance of odontogenic potential in m MMC by RNA-Seq.First of all,to explore whether Sfrp5 can restore the odontogenic potential when m MMC have lost this capacity,we attempted two different approaches to add exogenous SFRP5 protein.One is to supplement SFRP5 protein beads in the recombinants of m MMC(lost odontogenic potential)and the second arch epithelium.The other is to add exogenous SFRP5 protein to co-culture with m MMC(lost odontogenic potential)for a certain period of time during in vitro cultured,and then m MMC are recombined with the second arch epithelium.Additionally,we detected the expression levels of critical genes for odontogenesis in the two approaches.We provide evidence that the addition of exogenous SFRP5 protein effectively increases the expression level of key genes of odontogenesis in m MMC and thereby to rescue the odontogenic potential of m MMC.Meanwhile,the rescue ability of SFRP5 protein decreased when the in vitro culture time of m MMC is prolonged and supplemented with SFRP5 protein during in vitro recombination had a better rescue effect on m MMC’s odontogenic potential when compared with adding the SFRP5 protein co-cultured in vitro.Together,our results demonstrate that Sfrp5 is one of the key factors of odontogenic potential in m MMC.Although the regulation of Sfrp5 on mesenchymal odontogenic potential may be related to multiple signaling pathways,previous data have provided evidence that Sfrp5 can act as an activator in m MMC to promote Wnt/β-catenin signaling in m MMC.Here,we made an in-depth study on the mechanism of Sfrp5 in Wnt/β-catenin signaling pathway in m MMC.We show that,after m MMC was cultured in vitro for 8h,the supplement of exogenous SFRP5 protein in the recombination significantly increase the activity of Wnt/β-catenin signaling pathway.We can see that Wnt/β-catenin signaling activity was also improved to a certain extent when adding exogenous SFRP5 protein in vitro cultured,although the overall Wnt/β-catenin signaling activity level is low.These data fit the tooth formation ratio and indicate that Sfrp5 has an activation effect rather than an antagonistic effect on the Wnt/β-catenin signaling pathway in m MMC,which is consistent with the results of previous research.To explore the regulatory mechanism of Sfrp5 on Wnt/β-catenin signaling pathway,we examined the expression patterns of endogenous Sfrp5 andβ-catenin during tooth development and find that the expression positions of endogenous SFRP5 and β-catenin overlapped.The changes of co-localization of SFRP5 and β-catenin in m MMC after the addition of exogenous SFRP5 protein were further analyzed.After 8 hours in vitro,although the expression of SFRP5 andβ-catenin in cultured m MMC overlapped to some extent,they were weak in both the cytoplasm and nucleus.However,the content and colocalization of SFRP5 andβ-catenin in both cytoplasm and nucleus of m MMC were effectively increased after the addition of exogenous SFRP5 protein.Furthermore,using in situ protein proximity ligation assay,it shows that endogenous SFRP5 and β-catenin have an interaction to some different extent in temporal and spatial during tooth development.Thus,we suggest that the odontogenic potential is closely related to the levels of SFRP5 and β-catenin in both cytoplasm and nucleus of m MMC,which may interact to increase Wnt/β-catenin signaling activity and maintain Wnt activity to a certain level.Finally,in order to identify the key downstream factors regulated by Sfrp5,m MMC samples co-cultured with SFRP5 protein(experiment group)or BSA(control group)for 5h were collected and RNA-Seq was performed to screened downstream key genes regulated by Sfrp5.51 significantly differentially expressed genes were obtained at last.The result of GO enrichment analysis shows that most of these genes involved in the modification and phosphorylation of tyrosine as well as the regulation of the frequency,rate or degree of phosphorylation of tyrosine and epidermal growth factor receptor(EGFR)signaling pathway and ERBB signaling pathway.By reviewing these genes function,we find that most of the down-regulated genes are related to the promotion of apoptosis while most of the up-regulated genes are extracellular matrix components and transcription factors acting in the nucleus.Based on the data of GO enrichment analysis and gene function,two downstream genes were selected: Pik3r2 and Sstr2.It shows that Pik3r2 encodes the regulatory subunit of PI3 K to promote the activation of PI3 K pathway;Sstr2 is not only related to the activity of tyrosine phosphokinase but also inhibits the activity of PI3K-mediated signaling pathway.It is known that many downstream reactions involved in PI3 K signaling pathway associated with β-catenin’s phosphorylation state.nucleus translocation,and stability.Therefore,in m MMC,we speculate that Pik3r2 and Sstr2 may be closely related to promote the translocation of β-catenin into nucleus,promote the interaction of β-catenin with other transcription factors,and maintain the stability and localization of β-catenin.This study provides evidences to understand the molecular constitutions of odontogenic potential in the dental mesenchyme as well as the mechanism of Sfrp5 in the Wnt/β-catenin signaling pathway in m MMC,and lays an important theoretical foundation for the realization of human tooth regeneration in the future. |
PDF Full Text Request