Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

Abstract

TGF-beta subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-beta signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-beta signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2(fl/fl);Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-beta2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-beta signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.

Fig. 1. Abnormal odontoblastic and ameloblastic differentiation in lower 1^st molar teeth of E18.5 Tgfbr2^fl/fl;Wnt1-Cre mice

In situ hybridization of Dspp, Col1a1 and Amelogenin using wild type (A,C,E) and Tgfbr2^fl/fl;Wnt1-Cre (B,D,F) samples at E18.5. (A,B) Col1a1 is expressed in osteoblasts (arrowhead) in both wild type and Tgfbr2;wnt1-Cre mutant mice, but expression in Tgfbr2; Wnt1-Cre mice is dramatically reduced compared to wild type. (C,D) Dspp is expressed in odontoblasts and ameloblasts in wild type (arrow). In contrast, Dspp expression is not observed in Tgfbr2;wnt1-Cre mice. (E,F) Amelogenin expression is detected in ameloblasts in wild type mice (arrow), but is not detectable in Tgfbr2; wnt1-Cre mice. (G,H) In situ hybridization of Tgfbr2 using wild type 1^st lower molar at E16.5 (G) and E18.5 (H). Tgfbr2 is expressed in dental mesenchymal cells at E16.5 (arrow) (G), and odontoblast cells at E18.5 (arrow) (H). Scale bars: 100 μm in A-F, 30 μm in G and F.

Fig. 2. Tooth development defect in Tgfbr2^fl/fl;Wnt1-Cre mice after kidney capsule transplantation

Wild type and Tgfbr2^fl/fl;Wnt1-Cre lower 1^st molar tooth germ were cultivated for four weeks under host kidney capsules. Normal cusp patterning is detectable in both wild type (left side) and Tgfbr2^fl/fl;Wnt1-Cre (right side) from a lateral (A) and top view (B). However, the shape of the crown and roots in the mutant sample is different than wild type (A), and the mutant sample appears translucent (B). (C-H) H&E and Von Kossa staining of wild type and Tgfbr2^fl/fl;Wnt1-Cre tooth germ sections. In the wild type samples, the enamel and dentin are well organized (C) and well mineralized (G). The dentin is composed of two layers (E). In the Tgfbr2^fl/fl;Wnt1-Cre samples, the enamel is well formed (D), but the dentin consist of a thin monolayer (F). Both the enamel and dentin are well mineralized, although predentin is rarely observed (H). Dotted-line boxes in C and D are enlarged and shown in E and F, respectively. e, enamel; d, dentin; pd, predentin; scale bars: 1 mm in A and B; 100 μm in C and D; 50 μm in E-H.

Fig. 3. Expression of odontoblastic and ameloblastic differentiation markers in Tgfbr2^fl/fl;Wnt1-Cre tooth germ cultured under a kidney capsule

H&E staining and in situ hybridization of Dspp, Col1a1 and Amelogenin in wild type (A,C,E,G) and Tgfbr2^fl/fl;Wnt1-Cre (B,D,F,H) samples after 14 days cultivation under a kidney capsule. (C,D) Expression of Col1a1 (arrows) in Tgfbr2^fl/fl;Wnt1-Cre transplants is detectable in a similar pattern to wild type, although the level of the expression is reduced. (E,F) Expression of Dspp (arrows) is detectable in wild type and Tgfbr2^fl/fl;Wnt1-Cre odontoblasts, but the level of expression is reduced in the Tgfbr2^fl/fl;Wnt1-Cre sample. (G,H) The level and pattern of Amelogenin expression (arrows) in the Tgfbr2^fl/fl;Wnt1-Cre sample are indistinguishable from wild type. (I,J) In situ hybridization of Col1a1 (I) and Dspp (J) in wild type samples after 5 days cultivation under a kidney capsule. Both Col1a1 and Dspp are already expressed in odontoblasts at the tip of the cusp at this stage (arrow). Scale bars: 100 μm in A-H; 50 μm in I and J.

Fig. 4. Loss of dentinal tubules in Tgfbr2^fl/fl;Wnt1-Cre tooth germ

(A,B) SEM analysis of wild type and Tgfbr2^fl/fl;Wnt1-Cre teeth after 28 days cultivation under a kidney capsule. Dentinal tubules are clearly visible in the wild type dentin layer (arrowheads in A), but not in the Tgfbr2^fl/fl;Wnt1-Cre dentin (B). The structure of the Tgfbr2^fl/fl;Wnt1-Cre enamel is indistinguishable from wild type. Broken line; dentin-enamel junction, d; dentin, e; enamel. (C-J) Nestin immunohistochemistry in wild type and Tgfbr2^fl/fl;Wnt1-Cre tooth germ at E18.5 (C-F) and after 14 days cultivation under kidney capsules (G-J). E, F, I, and J are enlarged views of the yellow dashed boxes in C, D, G, and H, respectively. Nestin expression is detectable in wild type odontoblasts at the tip of the cusp (E, arrow), but not in Tgfbr2^fl/fl;Wnt1-Cre odontoblasts (F). After cultivation under the kidney capsule, nestin is highly expressed in odontoblasts and in the dentin matrix area in wild type transplants (G,I). In Tgfbr2^fl/fl;Wnt1-Cre transplants, nestin was barely detectable in odontoblasts (H,J). Scale bars: 20 μm in A, B; 100 μm in C, D, G and H; 25 μm in E, F, I and H.

Fig. 5. TGF-β induces nestin expression in odontoblasts

Nestin and Dspp expression in wild type and Tgfbr2^fl/fl;Wnt1-Cre E18.5 tooth germ implanted with BSA or TGF-β2 beads. Nestin expression is analyzed by immunohistochemistry (A-F) and Dspp expression is analyzed by in situ hybridization (G,H). Enlarged areas of A, C, E and G are shown in B, D, F and H, respectively. (A,B) Nestin expression is detectable in odontoblasts adjacent to the dentin-enamel junction in wild type tooth germ (arrow), but BSA beads do not induce nestin expression in dental papilla cells. (C,D) There is no expression of nestin in odontoblasts adjacent to the dentin-enamel junction in Tgfbr2^fl/fl;Wnt1-Cre mutant tooth germ, and nestin is not induced in dental papilla cells around TGF-β2 beads. (E,F) In wild type tooth germ, nestin expression is again visible in odontoblasts adjacent to the dentin-enamel junction (arrow in E) and is induced in dental papilla cells around TGF-β2 beads (arrowhead). Cells expressing nestin around beads appear differentiated cytologically with an enlargement of the cytoplasm and the production of processes (arrow in F). (G,H) Dspp mRNA expression in wild type tooth germ from an adjacent section to E and F shows the same pattern as nestin. Arrow indicates the Dspp expression in odontoblasts adjacent to the dentin-enamel junction, and the arrowhead highlights that in dental papilla cells around the TGF-β2 bead. (I) Double immunofluorescence for nestin and NF-M in E18.5 tooth germ implanted with TGF-β2 bead. Nestin is expressed spontaneously in odontoblasts adjacent to epithelial cells (arrow), and its expression is induced in dental papilla cells by TGF-β2 (arrowhead). NF-M expression is not observed in any area of tooth germ. (J) Antibodies against NF-M can react with neurons in trigeminal ganglia. E, dental epithelium; P, dental papilla. Scale bars: 100 μm in A, C, E, G, I and J; 20 μm in B, D, F, and H.