Wnt signaling in the murine diastema

Abstract

The correct number and shape of teeth are critical factors for an aesthetic and functional dentition. Understanding the molecular mechanisms regulating tooth number and shape are therefore important in orthodontics. Mice have only one incisor and three molars in each jaw quadrant that are divided by a tooth-less region, the diastema. Although mice lost teeth in the diastema during evolution, the remnants of the evolutionary lost teeth are observed as transient epithelial buds in the wild-type diastema during early stages of development. Shh and Fgf signaling pathways that are essential for tooth development have been shown to be repressed in the diastema. It remains unclear however how Wnt signaling, that is also required for tooth development, is regulated in the diastema. In this study we found that in the embryonic diastema, Wnt5a expression was observed in mesenchyme, whereas Wnt4 and Wnt10b were expressed in epithelium. The expression of Wnt6 and Wnt11 was found in both tissues. The Wnt co-receptor, Lrp6, was weakly expressed in the diastema overlapping with weak Lrp4 expression, a co-receptor that inhibits Wnt signaling. Secreted Wnt inihibitors Dkk1, Dkk2, and Dkk3 were also expressed in the diastema. Lrp4 mutant mice develop supernumerary teeth in the diastema that is accompanied by upregulation of Wnt signaling and Lrp6 expression. Wnt signaling is thus usually attenuated in the diastema by these secreted and membrane bound Wnt inhibitors.

Figure 1

Schematic diagrams showing tooth buds in rodent maxilla. Schematic representations of maxilla from wild-type mouse at adult (A), E14.5 (B), and E15.5 (C). Right diagrams of B and C showing sagittal view of tooth germs. In, incisor; M1, first molar; M2, second molar; M3, third molar; Di, rudimentary tooth bud.

Figure 2

Tooth -related genes in the diastema. Dark field (A, B, C, D, E, and F) and light field (A’, B’, C’, D’, E’, and F’) images of radioactive in situ hybridization on sagittal sections in wild-type mice at E14.5. The expression of p63 (A and A’), Msx2 (B and B’), Pitx1 (C and C’), and Notch1 (D and D’) were found in upper diastema epithelium. Runx2 (E and E’) and Msx1 (F and F’) expression were found in upper diastema mesenchyme. Both black and white arrows indicate rudimentary tooth bud.

Figure 3

Wnt ligands in the diastema. Dark field (A, B, C, D, and E) and light field (A’, B’, C’, D’, and E’) images of radioactive in situ hybridization on sagittal sections in wild-type mice at E14.5. Weak expression of Wnt4 and Wnt10b (A, A’, D, and D’) was detected in diastema epithelium. Wnt5a expression (B and B’) was observed in diastema mesenchyme. Wnt6 and Wnt11 (C, C’, E, and E’) were expressed in both diastema epithelium and mesenchyme. Arrows indicate rudimentary tooth bud.

Figure 4

Wnt signaling in the diastema. Dark field (A, B, and C) and light field (A’, B’, and C’) images of radioactive in situ hybridization on sagittal sections in wild-type mice at E14.5. Weak expression of Lrp4 (A and A’) and Lrp6 (B and B’) was observed in both upper diastema epithelium and mesenchyme. Upregulation of Lrp6 expression was detected in Lrp4^Hypo/Hypo mutants (C and C’). Supernumerary tooth in the diastema was found in Lrp4^Null/Null mutants at E18.5 (arrowhead in E). The first and the second molars were present in wild-type at E18.5 (D). Upregulation of ß-gal activity in the diastema was shown in Lrp4^Hypo/Hypo; BAT-gal mice at E14.5 (arrow in G) compared with β-gal activity in the diastema of wild-type at E14.5 (arrow in F). Both black and white arrows indicate rudimentary tooth bud.

Figure 5

Dkk expression in the diastema. Dark field (A, B, C, D, and E) and light field (A’, B’, C’, D’, and E’) images of radioactive in situ hybridization on sagittal sections in wild-type mice at E14.5. The expression of Dkk1 (A and A’) was found in presumptive maxillary bone adjacent to the diastema in wild-type. Normal expression of Dkk1 (B and B’) was detected in Lrp4^Hypo/Hypo mutant. Dkk2 (C and C’) and Dkk3 (D and D’) were expressed in upper diastema mesenchyme. Dkk4 expression (E and E’) could not be detected in the diastema. Arrows indicate rudimentary tooth bud.