Spatiotemporal Expression of Wnt/β-catenin Signaling during Morphogenesis and Odontogenesis of Deciduous Molar in Miniature Pig

Abstract

The canonical Wnt/β-catenin signaling pathway has been shown to play essential roles in tooth initiation and early tooth development. However, the role of Wnt/β-catenin signaling in cusp patterning and crown calcification in large mammals are largely unknown. In our previous study, miniature pigs were used as the animal model due to the similarity of tooth anatomy and replacement pattern between miniature pig and human. Dynamic gene expression of third deciduous molar (DM3) in miniature pig at early stages was profiled using microarray method and expression of Wnt genes was significantly correlate with odontogenesis. In the present study, dynamic expression patterns of Wnt/β-catenin signaling genes of DM3 at cap, early bell and late bell (secretory) stage were identified. We found that Lef1 and Axin2 were expressed in the enamel knot and underlying mesenchyme regions. Meanwhile, Dkk1 was expressed in the peripheral and lower parts of dental papilla, thus forming the potential Wnt signaling gradient. We also found that β-Catenin, Axin2 and Lef1 were expressed strongly in undifferentiated cells of the inner enamel epithelium (IEE), but weakly in differentiated ameloblasts. Furthermore, we found that both Wnt signaling read-out gene Lef1 and the inhibitor Dkk1 were co-expressed in the pre-odontoblasts. In conclusion, the spatiotemporal distribution and potential gradient of Wnt signaling may contribute to cusp patterning and crown calcification. These data may yield insight into future study of precise control of crown morphogenesis and regeneration in large mammals.

Keywords: Wnt signaling; development; miniature pig; tooth.

Figure 1

Characteristics of developmental stages of deciduous molar 3 of miniature pig. (A-C') H&E staining of different stages during tooth germ morphogenesis; Boxed regions in A,B,C were magnified in A',B',C'. (A,A') The tooth germ developed into cap stage at embryonic day 40 (E40). The epithelium folded into the bud and the inner enamel epithelium (IEE) and outer enamel epithelium (OEE) were separated. The stellate reticulum (SR) was located between IEE and OEE. The mesenchymal cells concentrated and became dental papilla (DP). (B,B') At E50, the tooth bud reached bell stage and the secondary and third enamel knots began to form. The out layer of dental papilla cells attached to the basement membrane of IEE. (C,C') At E60, parts of IEE and dental papilla cells at the cusp tip had differentiated into ameloblasts and odontoblasts (O). Enamel (E) and pre-dentin (pD) were secreted. Scale bars represent 50μm (A,A',B,B') and 100μm(C,C').

Figure 2

Dynamic expression of Wnt ligands (Wnt3a, Wnt5a) and inhibitor (Dkk1) during morphogenesis of DM3. (A-I') In situ hybridization of Wnt3a, Wnt5a and Dkk1 during tooth germ morphogenesis; Boxed regions in C,F,I, were magnified in C',F',I'. (A-C,C') Wnt3a was mainly expressed in dental epithelium from E40-E60. (D-E) Wnt5a was expressed in primary enamel knot at E40, and expressed in both epithelium and dental papilla at E50. (F-F') At E60, Wnt5a was expressed both in IEE and odontoblasts. (G-H) Dkk1 was expressed in lower and peripheral regions of dental mesenchyme from E40 to E50 (red arrows). (I-I') At E60, Dkk1 was mainly expressed in lower regions of dental papilla (red arrow) and in pre-odontoblasts (yellow arrow). Scale bars represent 50μm (A,D,G), 100μm (B,C',E,F',H,I') and 200μm (C,F,I).

Figure 3

Dynamic expression of Wnt read-out genes (β-Catenin, Axin2, Lef1, Tcf4) during morphogenesis of DM3. (A-L') In situ hybridization of β-Catenin, Axin2, Lef1 and Tcf4 during tooth germ morphogenesis; Boxed regions in C,F,I,L were magnified in C',F',I',L'. (A) β-Catenin was expressed both in epithelium and mesenchyme at E40. (B) β-Catenin was mainly located in the epithelium at E50. (C,C') At E60, β-Catenin was mainly expressed in the lower parts of IEE (red arrows) rather than the tip of the cone. (D) Axin2 was expressed in the epithelium and the beneath mesenchyme region at E40. (E) Axin2 was mainly expressed in the enamel knots and the underlying mesenchyme regions at E50. (F,F') At E60, Axin2 was expressed mainly in the lower parts of IEE. (G-H) Lef1 was expressed in enamel knots and the underlying mesenchyme at E40-E50. (I,I') At E60, Lef1 was expressed in lower parts of IEE and pre-odontoblasts (yellow arrows). (J) Tcf4 was mainly expressed at the mesenchyme region at E40. (K) At E50, Tcf4 was expressed both in epithelium and dental papilla. (L,L') Tcf4 was expressed in IEE, dental papilla and odontoblasts at E60. Scale bars represent 50μm (A,D,G,J), 100μm (B,C',E,F',H,I',K,L') and 200μm (C,F,I,L).

Figure 4

Dynamic protein expression of β-Catenin and Axin2 during morphogenesis of DM3. (A-F') Immunohistochemistry of β-Catenin and Axin2 during morphogenesis; Boxed regions in A,B,C,D,E,F were magnified in A',B',C',D',E',F'. (A-A') β-Catenin was expressed both in epithelium and mesenchyme at E40. (B-B') β-Catenin was mainly located in the epithelium at E50. The transition from E40 to E50 was similar with in situ hybridization. (C,C') At E60, β-Catenin was mainly expressed in IEE and odontoblasts. (D-D') Axin2 was expressed in the epithelium and the underlying mesenchyme region at E40. (E-E') Axin2 was mainly expressed in the IEE, OEE and dental papilla at E50. (F,F') At E60, Axin2 was mainly expressed in the IEE. Scale bars represent 50μm (A',B',D',E',F'), 100μm (A,B,C',D,E,F) and 200μm (C).

Figure 5

Quantity of mRNA expression dynamics of Wnt pathway genes during morphogenesis of DM3. mRNA levels were quantified with real-time RT-PCR and the differences were analyzed among three stages of each gene. (A) Wnt3a was expressed at relatively high level at E40 and E60, compared to E50. (B) Wnt5a expression level gradually increased from E40 to E60. (C) Dkk1 expression level gradually increased from E40 to E60. (D) β-Catenin was expressed at relatively high level at E40 and E60, compared to E50. (E) Axin2 was expressed at relatively high level at E40 and E60, compared to E50. (F,G) There were no significant differences of expression level of Lef1 and Tcf4 among different stages. Data presented are mean ± SD. *P< 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Schema of expression dynamics of Wnt/β-catenin signaling during cap to secretory stages of DM3. Wnt read out genes (β-Catenin, Axin2, Lef1) were marked with red color, while Wnt inhibitor (Dkk1) was marked with blue color. The transition from light to dark colors corresponds to low to high expression levels. (A) Tooth development stages from dental lamina to root development: I thickening dental lamina; II bud stage; III cap stage; IV bell stage; V secretory stage; VI root development; (B) At E40, Wnt read out genes were localized in the primary enamel knot, while Dkk1 was localized in the periphery regions of dental papilla. (C) At E50, Wnt read out genes were localized in the secondary enamel knots and underlying mesenchyme, while Dkk1 was localized in the lower parts of dental papilla. (D) At E60, Wnt read out genes were mainly localized in the undifferentiated IEE, odontoblasts, and pre-odontoblasts, while Dkk1 was localized in the lower undifferentiated parts of dental papilla and pre-odontoblasts.