Inactivation of Tgfbr2 in Osterix-Cre expressing dental mesenchyme disrupts molar root formation

Abstract

It has been difficult to examine the role of TGF-ß in post-natal tooth development due to perinatal lethality in many of the signaling deficient mouse models. To address the role of Tgfbr2 in postnatal tooth development, we generated a mouse in which Tgfbr2 was deleted in odontoblast-and bone-producing mesenchyme. Osx-Cre;Tgfbr2fl/fl mice were generated (Tgfbr2cko) and postnatal tooth development was compared in Tgfbr2cko and control littermates. X-ray and μCT analysis showed that in Tgfbr2cko mice radicular dentin matrix density was reduced in the molars. Molar shape was abnormal and molar eruption was delayed in the mutant mice. Most significantly, defects in root formation, including failure of the root to elongate, were observed by postnatal day 10. Immunostaining for Keratin-14 (K14) was used to delineate Hertwig's epithelial root sheath (HERS). The results showed a delay in elongation and disorganization of the HERS in Tgfbr2cko mice. In addition, the HERS was maintained and the break up into epithelial rests was attenuated suggesting that Tgfbr2 acts on dental mesenchyme to indirectly regulate the formation and maintenance of the HERS. Altered odontoblast organization and reduced Dspp expression indicated that odontoblast differentiation was disrupted in the mutant mice likely contributing to the defect in root formation. Nevertheless, expression of Nfic, a key mesenchymal regulator of root development, was similar in Tgfbr2cko mice and controls. The number of osteoclasts in the bone surrounding the tooth was reduced and osteoblast differentiation was disrupted likely contributing to both root and eruption defects. We conclude that Tgfbr2 in dental mesenchyme and bone is required for tooth development particularly root formation.

An Nfic-hedgehog signaling cascade regulates tooth root development

“…Some models, such as Osr2-Cre;Smad4 fl/fl and K14-Cre;Smad4 fl/fl (Huang et al, 2010), show phenotypes in early stages of tooth development, but it is hard to determine whether the root defect is secondary to the crown defect. Other models, including OC-Cre (Bae et al, 2013b;Gao et al, 2009;Kim et al, 2013;Zhang et al, 2013), Col1a1-Cre (Kim et al, 2012) and Sp7-Cre (Rakian et al, 2013;Wang et al, 2013), take effect too late, only targeting differentiated cells. Here, we used the Gli1-CreERT/loxP system to target the early progenitor cells for root development.…”

“…Although several reports have described root defect phenotypes following targeted gene mutation, there has not been a systematic analysis of postnatal root development (Bae et al, 2013b;Kim et al, 2013;Wang et al, 2013). Towards this end, we analyzed serial sagittal sections of the first mandibular molar in mice from PN0 to PN21 using HE staining.…”

Cellular and molecular mechanisms of tooth root development

“…For example, Bmp, Tgfβ, and their mediator Smad4, as well as Shh, Msx2 and Dlx2, are expressed in HERS cells (Åberg et al, 1997;Huang et al, 2010;Lezot et al, 2000;Nakatomi et al, 2006;Yamashiro et al, 2003). In the CNC-derived dental mesenchyme adjacent to the HERS, there is expression of Gli1, Nfic, Fgf, Tgfβ, Bmp, Wnt and its inhibitors, as well as PTHrP/PPR (Pthlh/Pth1r) (Huang et al, 2010;Ono et al, 2016;Steele-Perkins et al, 2003;Wang et al, 2013). In addition, some signaling molecules are expressed in both the HERS and the CNC-derived dental mesenchyme .…”

<b>Hertwig’s epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by </b><b>TGF-β </b>

“…In vivo, several gene mutated mice models of TGF-β signaling have reported defects in tooth root formation. Mice in which tgfbr2 was conditionally deleted in odontoblasts and osteoblasts of alveolar bone using Osterix-Cre (Osx-Cre;Tgfbr2 fl/fl ) lacked proper root formation (25), and cellular cementum was reduced when tgfbr2 was conditionally inactivated of cementoblasts using Osteocalcin-Cre (OC-Cre;Tgfbr2 fl/fl ) (6). Interestingly, mice with conditional deletion of both TGF-β/BMP signaling in odontoblasts (OC-Cre;Smad4 fl/fl ) resulted in a high rate of keratocystic odontogenic tumors in the jaw (11).…”