The Exploration Of Persistent Wnt/?-catenin Signaling Deprived Dental Mesenchyme Of Odontogenic Fate

Background:Tooth development is result of the interaction between cranial neural crest-derived mesenchymal cells and ectodermal-derived epithelial cells.Signaling pathways including BMP,FGF,Shh and Wnt transmit signals between epithelium and mesenchyme,regulate the initiation,morphogenesis and cell differentiation of tooth germ coordinately.In humans,19 Wnt genes have already been known.According to the signal transduction pathways in the cytoplasm,Wnt signaling pathways are divided into canonical pathway?Wnt/?-catenin pathway?and non-canonical pathway(the planar cell polarity pathway and the Wnt/Ca²⁺pathway).During tooth initiation and morphogenesis,Wnt/?-catenin signaling is only detected in dental epithelium,but absent from dental mesenchyme.However,when deleting?-catenin to inactivated Wnt/?-catenin signaling in dental mesenchyme gave rise to the development of dental germs was arrested at the bud stage or early cap stages,reminding that dental mesenchymal Wnt/?-catenin signaling is indispensable during the tooth morphogenesis,but the function of Wnt/?-catenin signaling in dental mesenchymal during the morphogenesis of tooth is still unclear.Objective:To determine the role of stabilized?-catenin in dental mesenchyme in mice,the Osr2-cre^KI;Ctnnb1^ex3f?a mutant?-catenin allele whose exon3 was sandwiched by lox P sequences?mice were used in our study to explore how mesenchymal Wnt/?-catenin signaling suppressed the odontogenic fate in vivo.Materials and Methods:?1?Morphological changes of Osr2-cre^KI;Ctnnb1^ex3fmice tooth germs were observed by tissue sections and masson staining.?2?As an inhibitor and effector of Wnt/?-catenin signaling,Axin2 and Lef1 was detected in dental mesenchyme of Osr2-cre^KI;Ctnnb1^ex3fmice by immunohistochemistry.?3?BrdU staining and TUNEL were used to detcet the proliferation and apoptosis of Osr2-cre^KI;Ctnnb1^ex3fmesenchymal cells.?4?Mesenchymal cells from wildtype mice incisor and molar were subjected to osteogenic induction for 1 week respectively and their osteogenic mineralization was analyzed by alizarin red staining.?5?In vitro tissue recombination was conducted to observe the odontogenic capability of Osr2-cre^KI;Ctnnb1^ex3fmolar mesenchyme.?6?Mesenchymal cells from EGFP mice and WT mice were cultured together in vitro to obsereved the different mineralization ability between incisor mesenchymal cells and molar mesenchymal cells.?7?The expression of mesenchymal odontogenic markers in Osr2-cre^KI;Ctnnb1^ex3fdental mesenchymal was tested by in situ hybridization.Results:?1?Immunohistochemistry with the antibody against Axin2 exhibited that the expression of Axin2 in Osr2-cre^KI;Ctnnb1^ex3fmice was extended to incisor and molar mesenchyme and even extended into the palatal and mandibular mesenchyme compared with WT mice.The immunohistochemistry with the antibody against Lef1 showed the distribution of Lef1 was almost no difference as that of Axin2 at the same time.The results above reminding us that Wnt/?-catenin signaling was activated in Osr2-cre^KI;Ctnnb1^ex3fdental mesenchyme indeed.?2?The mandibular incisor germs of Osr2-cre^KI;Ctnnb1^ex3fmice retarded at bud stage at E14.5.At E16.5,the maxillary incisors of Osr2-cre^KI;Ctnnb1^ex3fwere regressed and lack of typical bell form,the mandibular incisor germs of Osr2-cre^KI;Ctnnb1^ex3fmice even diminished completely.About 50%?15/31?of E16.5 Osr2-cre^KI;Ctnnb1^ex3fmice lost their maxillary and mandibular molars,the part of remaining Osr2-cre^KI;Ctnnb1^ex3fmolar germs were smaller than those in WT control.?3?BrdU labeling experiments show that:The BrdU positive cells of E13.5Osr2-cre^KI;Ctnnb1^ex3fmaxillary incisor mesenchyme were decreased remarkably than those in WT maxillary incisor mesenchyme?p<0.05?.Similarly,the numbers of BrdU positive cells in the Osr2-cre^KI;Ctnnb1^ex3fmandibular incisor and molar mesecnchyme were decreased compared with those in WT dental mesecnchyme?p<0.01?.In contrast,the cell proliferation of Osr2-cre^KI;Ctnnb1^ex3fmaxillary molar mesecnchyme was comparable with those in WT maxillary molar mesecnchyme?p>0.05?.?4?TUNEL assay revealed that the cell death in Osr2-cre^KI;Ctnnb1^ex3fmaxillary and mandibular incisor mesenchyme was reduced than those in WT maxillary.However,there was little difference of apoptosis number between Osr2-cre^KI;Ctnnb1^ex3fmolar germs and WT molar germs,which reminding us the morphological defects of Osr2-cre^KI;Ctnnb1^ex3fdental germs are results of reduced cell proliferation.?5?The results of Alizarin red staining showed that after osteogenic induced in vitro for 1 week,the number of mineralization nodes in WT incisor mesecnchyme was significantly higher than that in WT molar mesenchyme,which indicated that the stabilization ability of incisor mesenchyme was stronger than molar mesenchyme.?6?In situ hybridization revealed that at E14.5,the Fgf3 expression in Osr2-cre^KI;Ctnnb1^ex3fmolar germs was significantly weaker than those in WT molar germs,and the expression of Fgf3 was vanished in Osr2-cre^KI;Ctnnb1^ex3fincisors germs.The expression of Runx2 was disappeare in E14.5 Osr2-cre^KI;Ctnnb1^ex3fincisor mesenchyme,whereas expressed in WT maxillary incisor mesenchyme and silenced in mandibular incisor mesenchyme.In the E14.5 Osr2-cre^KI;Ctnnb1^ex3fmolar mesenchyme,the expression of Runx2 was mildly stronger than that in the WT molar germs.Msx1 transcription was reduced in the Osr2-cre^KI;Ctnnb1^ex3fmaxillary incisor mesenchyme and molar mesenchyme compared with that in WT dental germs,and even diminished in the mandibular incisor of the E14.5 Osr2-cre^KI;Ctnnb1^ex3fmice.These results suggested that persistent Wnt/?-catenin signaling in dental mesenchyme suppressed the expression of odontogenic markers and enhanced the expression of osteogenic markers in dental mesenchyme.?7?Tissue recombination showed neither E10.5 nor E13.5 WT molar epithelium was able to form tooth with E13.5 Osr2-cre^KI;Ctnnb1^ex3fmolar mesenchyme,indicating that the active Wnt/?-catenin signaling in Osr2-cre^KI;Ctnnb1^ex3fmolar mesenchyme was not only depress the mesenchymal odontogenic capability,but also resist the induction from the odontogenic epithelium.Conclusion:Persistent Wnt/?-catenin signaling in tooth mesenchyme not only deprive the odontogenic capability in mesenchyme by inhibit the expression of odontogenic genes,but also resist the induction from the odontogenic epithelium.