Cessation of epithelial Bmp signaling switches the differentiation of crown epithelia to the root lineage in a β-catenin-dependent manner

Abstract

The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial β-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial β-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/β-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/β-catenin pathways during cell fate decisions.

Fig 1

Radiological and histological analyses of enamel defects and ectopic cellular cementum-like structures in AKO teeth. Krt5-rtTA/tetO-Cre/Alk3^fl/fl (Alk3 KO [AKO]) mice and wild-type (WT) littermates were induced with Dox starting on E14.5. (A and B) Tooth samples were collected on P42 for μCT imaging (A) to examine molar surfaces or sagittal tooth sections or for backscatter SEM to show ultrastructures (B) with the green-outlined insert depicting the cellular cementum in a WT molar. (C and D) Sections of decalcified teeth were analyzed with TRI staining (C) or Krt5 IHC (D).

Fig 2

In situ hybridization for markers of ameloblasts and cementoblasts in AKO teeth. (A to P) Tooth samples were collected from WT and AKO mice induced from E14.5 on P7 (A to L) or P14 (M to P) for ISH of Alk3 (A and B), the ameloblast marker Amelx (C and D), the Bmp target gene Msx2 (E to H), and cementoblast markers Col1a1 (I to L) and Bsp (M to P). Panels E to P have the same magnification as panels A and B.

Fig 3

Morphology and differentiation of dental epithelia in AKO teeth. (A to G) P7 and P14 tooth samples were collected from WT and AKO mice induced from E14.5 for ISH or IHC of Krt14 (A and B) or double immunofluorescence staining of Krt14 and N-cadherin, vimentin, E-cadherin, collagen I or Bsp (C to G). The nucleus was counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue) in panels C to G.

Fig 4

Effects of Alk3 depletion on expression of differentiation markers and EMT-related genes in cultured dental epithelia. (A to N) Dental epithelial stem cells (DEpSCs) were isolated from the apical ends of incisors of P7 AKO and WT mice that were not exposed to Dox, expanded as spheres, and induced to differentiate in the presence of Dox for 7 days (A) for qRT-PCR (C to F), IF staining of Krt14 (B), Alk3 (G and H), or phospho-Smad1/5/8 (pSmad1/5/8) (I and J), or double IF staining for Krt14 and vimentin (K and L) or collagen I (M and N). The error bars in panels C to E show standard errors of the means (error bars) (n = 3 and P < 0.05 for all genes examined). 2D, two-dimensional.

Fig 5

Depletion of Alk3 in dental epithelial upregulated activity of Wnt/β-catenin signaling but not that of TGF-β signaling. (A to C) AKO and WT DEpSCs were induced as described in the legend to Fig. 4 and analyzed by Western blotting of nuclear proteins (A), qRT-PCR (B), or IF (C) to examine the expression of genes related to the Wnt/β-catenin pathway or the TGF-β pathway (n = 3 in each assay) (n.s., not significant; ∗, P < 0.05). (D) P7 WT and AKO teeth were examined with double IF for β-catenin and Krt14.

Fig 6

Effects of epithelial β-catenin (βcat) depletion on amelogenesis, EMT, and cementogenesis. P42 WT or BKO teeth were examined with μCT scanning (A and F), backscatter SEM (B), or IHC for Krt5 (E). P7 WT or BKO teeth were examined with ISH for Amelx (C), Col1a1 (D), and Krt14 (E). (G to I) WT and BKO DEpSCs were induced as described in the legend to Fig. 4 and analyzed with qRT-PCR for expression of genes related to amelogenesis or cementogenesis (G), EMT (H), and the Bmp pathway (I) (n = 3 and P < 0.05 for all genes except E-cad).

Fig 7

The EMT and ectopic cementogenesis caused by Alk3 depletion is dependent on Wnt/β-catenin signaling. Krt5-rtTA/tetO-Cre/Alk3^fl/fl/Ctnnb1^fl/fl compound knockout (CKO) mice were induced with Dox from E14.5. (A to D) P42 CKO tooth samples were analyzed with μCT imaging (A, C, and D) and TRI staining (B). (E to L) P7 CKO tooth samples were analyzed with ISH for Krt14 (E and F), Msx2 (G and H), Amelx (I and J), and Col1a1 (K and L). Panels F to L are the same magnification as in panel E. (M to O) CKO and WT DEpSCs were induced as described in the legend to Fig. 4 and analyzed by qRT-PCR for the expression of genes related to differentiation (M), the EMT (N), and the Bmp pathway (O) (n = 3 and P < 0.05 for all genes not labeled with n.s.).

Fig 8

Schematic summary and hypothesis of roles of Bmp and Wnt signaling in the differentiation of dental epithelia. (A) Schematic summary of tooth phenotypes in WT, AKO, BKO, and CKO mice. (B) Penetrance of enamel defects or ectopic cellular cementum-like structures in these strains (n = 16 [P7] or 24 [P42]). ND, not detected. (C) Hypothesis on roles of Bmp and Wnt signaling in the differentiation of dental epithelia. Strong Bmp signaling in crown/labial dental epithelia collaborates with weak Wnt signaling to trigger amelogenesis. The balance of strong Wnt signaling and weak Bmp signaling in apical/lingual dental epithelia promotes formation of HERS/ERM via changes in adhesion properties. Cessation of Bmp signaling in dental epithelia promotes precocious formation of HERS/ERM and epithelium-originated cementogenesis via EMT mediated by upregulation of Wnt signaling. Basal TGF-β signaling activity is required for both HERS/ERM formation and EMT.