Overexpression Of Sfrp5 Maintains The Odontogenic Potential Of Molars In Mice

As an important organ in the craniofacial region,tooth organogenesis depends on the complex interactions between ectoderm epithelium and cranial neural crest-derived mesenchyme.During the process of odontogenesis,either epithelium or mesenchyme must own the odontogenic potential,and the other must respond to the signal of odontogenic potential to form a tooth.In reality,teeth are easily damaged or fall off due to external or genetic factors,which seriously affect the quality of human life.Based on tissue engineering,to realize tooth regeneration through epithelial-mesenchymal induction is a hotspot.In our previous researches,we had confirmed that exogenous SFRP5 protein could maintain and rescue the odontogenic potential of molar mesenchymal cells.However,in vivo,whether overexpressing Sfrp5 has the same function is still unknown.Therefore,we further constructed transgenic mice to explore the effect of Sfrp5 overexpressed in cells on the odontogenic potential of molars.To construct Sfrp5 conditionally overexpressing mice,the c DNA of E13.5 mouse molar mesenchyme was used as a template,and primers was designed with fragments homologous to the plasmid.After amplification and purification,the CDS sequence of the Sfrp5 gene was ligated with the linearized pMes-STOP-IRES-e GFP plasmids to construct the pMes-Sfrp5-IRES-e GFP recombinant plasmid.After linearization by Bci VⅠ,it was co-transfected with the TC-Cre plasmids into NIH3T3 cells to verify the effectiveness of at the cellular level.Finally,the linearized pMes-Sfrp5-IRES-e GFP plasmids were subjected to construct pMes-Sfrp5 conditionally overexpressed mice via prokaryotic microinjection.In order to verify the effectiveness of pMes-Sfrp5 mice,PCR was used to identify the pMes-Sfrp5 mice.Moreover,we crossed Wnt1-Cre mice with pMes-Sfrp5mice and identified Wnt1-Cre;pMes-Sfrp5 mice by PCR.It was found that the tissues derived from the cranial neural crest of the mutant embryos,including telencephalon,first branchial arch,second branchial arch,dorsal root neural crest,expressing GFP reporter.Furthermore,the expression of Sfrp5 in Wnt1-Cre;pMes-Sfrp5 mouse dental mesenchyme is proved to raise by quantitative real-time PCR.It confirmed the effectiveness of pMes-Sfrp5 mice.To investigate the effect of overexpression of Sfrp5 on tooth development in vivo,E13.5,E14.5,E16.5 and E18.5 Wnt1-Cre;pMes-Sfrp5 embryos were collected.The tooth morphology in mutant mice did not show differences with wild-type ones at the same stages.Considering that the five members in Sfrp family,including Sfrp1,Sfrp2,Sfrp3,Sfrp4,and Sfrp5,perform redundancy roles during organ development,q PCR was used to detect the expression of Sfrp1,Sfrp2,Sfrp3,and Sfrp4 in molar mesenchymal cells to verify whether these genes play redundancy roles with Sfrp5 in odontogenesis.It showed that the expression of Sfrp1 and Sfrp3 were significantly reduced whereas the expression of Sfrp4 was significantly increased,suggesting that Sfrps are functionally redundant,which permits normal tooth development,in mutant mice during odontogenesis.Moreover,the expression of another Wnt antagonists,including Sost,Dkk1 and Apcdd1 were also detected in the dental mesenchyme of Wnt1-Cre;pMes-Sfrp5 mice.Our data show that the expression of Dkk1 rather than others was significantly up-regulated,indicating that Dkk1 participate in regulation of Wnt signaling in Wnt1-Cre;pMes-Sfrp5 mice.Given that tooth is regulated by the complex interaction network of epithelium and mesenchyme and we had proved that exogenous SFRP5 effectively maintain the odontogenic potential of mouse molar mesenchyme cells（m MMCs）,we evaluated the effect of endogenous overexpression of Sfrp5 on the key odontogenic factors in m MMCs in vitro.Immunofluorescence and q PCR were used to detect the expression of four key odontogenic factors,including LEF1,LHX6,MSX1,PAX9 after E13.5Wnt1-Cre;pMes-Sfrp5 m MMCs were cultured for 8 hours.The results show that overexpression of Sfrp5 effectively maintained the expression of LHX6,MSX1,and PAX9 in m MMCs after cultural in vitro for 8 hours.For the reason of that the exogenous SFRP5 gradually inactivated with the extension of the culture time while the overexpression of Sfrp5 could express continuously and stably,we evaluated the effect of Sfrp5 overexpression on the expression of key dental factors,when the culture time was extended to 24h in vitro.The results reveal that overexpression of Sfrp5 could also effectively maintain the expression of LHX6,MSX1,and Lef1 in m MMCs.It has been proved that exogenous SFRP5 could maintain the odontogenic potential of m MMCs by maintaining the expression of MSX1 and LHX6.Our data have shown that overexpression of Sfrp5 could also maintain these key odontogenic factors in vitro.Therefore,we further evaluated the effect of Sfrp5 overexpression on the odontogenic potential of m MMCs through recombination ex vivo.The E13.5Wnt1-Cre;pMes-Sfrp5 m MMCs were cultured in vitro for 8 hours and recombined with the second branchial arch epithelium of E10.5 wild-type mice.It reveals that the rate of tooth formation increased from 0 to 30%.This indicates that the overexpression of Sfrp5 could maintain the odontogenic potential of m MMCs by maintaining the expression of MSX1,PAX9,and LHX6.Finally,in order to investigate the regulation of Sfrp5 overexpression on Wnt signaling in m MMCs during in vitro culture,E13.5 Wnt1-Cre;pMes-Sfrp5 dental mesenchyme were dispersed and cultured for 8 hours in vitro and the activity of Wnt signaling was examined by immunostaining.The results show that Sfrp5overexpression promotes Wnt/β-Catenin and Wnt/PCP signal activity while inhibit Wnt/Ca²⁺signaling when m MMCs were cultured in vitro for 8 hours.Considering that the overexpression is a continuous process,we extended the culture time to 24h and found that Sfrp5 overexpression also promote Wnt/β-Catenin,Wnt/PCP,and Wnt/Ca²⁺signaling.In this study,we found that both the redundant function of SFRPs and its coordination with other Wnt antagonists regulate tooth development in Sfrp5overexpression mice,and thus the Wnt activity is maintained within a stable threshold.In addition,ex vivo,the Sfrp5 overexpression could maintain the odontogenic potential via maintaining the expression of MSX1,PAX9,and LHX6 in m MMCs.Moreover,the stable and persistent Sfrp5 overexpression mice constructed in this study provides a model to analyze the molecular mechanism of odontogenic potential in m MMCs,laying a theoretical foundation for achieving tooth regeneration.