Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis

[ackground]Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. [Results]We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/− mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1−/− mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/− was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1−/−Bmp7+/− mice was significantly larger than that ofUsag-1−/−Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/− embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1−/− embryos was significantly the highest of all genotypes examined in dental papilla. [Conclusions]Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb⁻/⁻Runx2⁺/⁻ Murine Incisors

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors

Additional file 1: Figure S1. of Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis

X-gal staining in mandibular molars of Usag-1+/− (C57BL/6) mice at E14 and E15. Tissue sections from mandibular molars of Usag-1+/− (C57BL/6) mice at E14 and E15 were stained with X-gal. Scale bar: 100 μm. (A, B) Sagittal sections. (C) Coronal sections. (B, C) Usag-1 was expressed (blue) in a small portion of epithelia, except for the enamel knot and the mesenchyme near the tooth germ. (TIF 16106 kb

Additional file 2: Figure S2. of Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis

Malocclusion observed in Usag-1−/− (C57BL/6) mice and Usag-1−/− Bmp7+/− (F2 generation) mice. (A) Normal occlusion. A Usag-1−/− female mouse in the C57BL/6 background at 3 months after birth. These data were added in the analysis of the lower incisors. Scale bar: 1 mm. (B) Normal occlusion. A Usag-1−/−Bmp7+/− male mouse in the F2 generation at 4 months after birth. These data were added in the analysis of the lower incisors. (C) Malocclusion. A Usag-1−/− female mouse in C57BL/6 background at 3 months after birth. The mouse was excluded from the analysis of the lower incisors. (D) Malocclusion. A Usag-1−/−Bmp7+/− female mouse in the F2 generation at 4 months after birth. The mouse was excluded from the analysis of the lower incisors. (TIF 14671 kb