Wnt10a is Critical for Root Furcation Formation During Tooth Development

Objectives: In human, WNT10A gene mutation is the main cause of tooth agenesis. However, the functional significance of Wnt10a in regulating tooth development remains unclear.
Methods: We generated EIIa-Cre;Wnt10a^flox/flox （CKO）mice. Control and CKO mice were harvested at different time points. Tooth morphology was analyzed by micro-CT and HE staining. Immunohistochemistry were conducted to analyze the proliferation of dental mesenchymal cells and the differentiation of odontoblast. Real-time PCR and immunohistochemistry were performed to analyze the Wnt10a downstream gene expression profile that involved in tooth development.
Results: We isolated the mandibular first molars of control and CKO mice at P1, P7, P11 and P14. CKO mice showed smaller crowns with thinner dentin, lower position of tooth furcation and larger pulp cavity which was like taurodontism. We performed PCNA stainings to assess dental mensenchymal cell proliferation changes. The number of PCNA-positive cells nearby HERS in the elongation region of CKO mice was significantly reduced compared to which of control, whereas the number of PCNA-positive cells nearby the putative root furcation region was significantly upregulated. We further performed immunohistochemical staining of Dspp. The results showed a significantly reduced expression of Dspp in the coronal region of CKO mice than which of control, indicating that the odontoblast cells differentiation and the dentin formation in CKO mice were affected. Furthermore, in control mice at P1, Shh was clearly localized at the dental epithelium, whereas the expression of Shh was hardly detectable in CKO mice. Besides, the expression of Gli1, the key transcription factor activated by Shh, was significantly reduced in the mesenchymal cells adhere to HERS.
Conclusions: Our data suggest that Wnt10a is critical for tooth root morphologenesis via regulating hedgehog pathway activity. Our findings provide a new insight into understanding the tooth root developmental regulating mechanisms and the pathological mechanisms of root anomalies.