Anat Cell Biol.  2016 Sep;49(3):199-205. 10.5115/acb.2016.49.3.199.

Requirement of Smad4-mediated signaling in odontoblast differentiation and dentin matrix formation

• Yun CY
• Choi H
• You YJ
• Yang JY
• Baek JA
• Cho ES

• KMID: 2353345
• DOI: http://doi.org/10.5115/acb.2016.49.3.199

Abstract

Dentin is the major part of tooth and formed by odontoblasts. Under the influence of the inner enamel epithelium, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and secrete pre-dentin which then undergo mineralization into dentin. Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) signaling is essential for dentinogenesis; however, the precise molecular mechanisms remain unclear. To understand the role of TGF-β/BMP signaling in odontoblast differentiation and dentin formation, we generated mice with conditional ablation of Smad4, a key intracellular mediator of TGF-β/BMP signaling, using Osr2 or OC-Cre mice. Here we found the molars of Osr2(Cre)Smad4 mutant mice exhibited impaired odontoblast differentiation, and normal dentin was replaced by ectopic bone-like structure. In Osr2(Cre)Smad4 mutant mice, cell polarity of odontoblast was lost, and the thickness of crown dentin was decreased in later stage compared to wild type. Moreover, the root dentin was also impaired and showed ectopic bone-like structure similar to Osr2(Cre)Smad4 mutant mice. Taken together, our results suggest that Smad4-dependent TGF-β/BMP signaling plays a critical role in odontoblast differentiation and dentin formation during tooth development.

Keyword

TGF-β/BMP signaling; Smad4; Odontoblasts; Dentin

MeSH Terms

Animals
Cell Polarity
Crowns
Dental Enamel
Dental Papilla
Dentin*
Dentinogenesis
Epithelium
Mice
Miners
Molar
Odontoblasts*
Tooth

Figure

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  Fig. 1 Localization of Cre recombinase for conditional inactivation of Smad4 in dental mesenchyme. β-Galactosidase activities are shown in the dental mesenchyme of Osr2CreR26R at embryo 14.5 (E14.5) (A) and in coronal odontoblasts of OCCreR26R mice at postnatal 8 (P8) (B), respectively. (C) Enlarged white-boxed area in panel B is shown. d, dentin; DM, dental mesenchyme; Od, odontoblasts; OE, oral epithelium. Scale bars=100 µm (A), 200 µm (B), 25 µm (C).

• 

  Fig. 2 Impairment of odontoblast differentiation and dentin formation by Smad4 inactivation during dentinogenesis. H&E staining of lower molar tooth germs of control and Osr2CreSmad4 mice at newborn stage (A, D). Immunohistochemical staining for Osx (B, E), Phex (C, F) with mandibular molar tooth germs of control and Osr2CreSmad4 mice at postnatal 0 (P0). Osx and Phex were highly expressed in coronal odontoblasts in control mice (B, C) while lower expression of Osx and Phex were detected in mesenchymal cells (black arrows) in Osr2CreSmad4 mice (E, F). Root forming area of mandibular molar tooth germs from control and Osr2CreSmad4 mice after kidney capsule transplantation was histologically analyzed at embryo (E) 14.5+1+2 wk (G–L). Immunohistochemical staining for Osx (H, K), Phex (I, L) with mandibular molar tooth germs of control and Osr2CreSmad4 mice. The same area of mandibular molar tooth germs from control and OCCreSmad4 mice at P21 was analyzed by H&E staining (M, N). Osx and Phex were highly expressed in differentiating odontoblasts at cervical dentin in control mice (H, I) while the lower expression of Osx was observed in entrapped cells in a bone-like tissue of Osr2CreSmad4 mice (K). The expression of Phex were not detected in the entrapped cells in Osr2CreSmad4 mice (L). The asterisks in panels J and N indicate bone-like structure of ectopic hard tissue formation. Am, ameloblasts; H&E, hematoxylin and eosin; Od, odontoblasts; Osx, osterix; Phex, phosphate regulating endopeptidase homology on the X chromosome. Scale bar=50 µm.

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  Fig. 3 Reduced dentin matrix apposition in OCCreSmad4 mice. Hematoxylin and eosin staining of crown dentin of control (A–D) and OCCreSmad4 mice (E–H) at postnal (P) 10, P14, P21, and P28. Dentin thickness was compared after the measurement of crown dentin thickness with control and OCCreSmad4 mice at P10, P14, P21, and P28 (n=5, each genotype for stage, respectively) (I). d, dentin; Od, odontoblasts. **P<0.01. Scale bar=50 µm.

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  Fig. 4 Molecular changes in odontoblasts and dentin matrix with disruption of Smad4 in odontoblasts. Immunohistochemical staining of mandibular molar crown dentin in control (A–C) and OCCreSmad4 mice (D–F) at P10. Immunohistochemical staining for Dspp (A, D), Dmp1 (B, E), and Bgn (C, F) with mandibular molar of control and OCCreSmad4 mice. The expression of Dspp was detected in odontoblast and dentinal tubules in control mice (A) while broad expression in pre-dentin and pulp of OCCreSmad4 mice (D). The expression of Dmp1 was increased in OCCreSmad4 mice (E) compared to control (B). The expression of Bgn was not changed (C, F). Bgn, biglycan; d, dentin; Dmp1, dentin matrix protein 1; Dspp, dentin sialophosphoprotein; Od, odontoblasts. Scale bar=50 µm.

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