Parathyroid hormone receptor signalling in osterix-expressing mesenchymal progenitors is essential for tooth root formation
- Wanida Ono, Naoko Sakagami, Shigeki Nishimori, Noriaki Ono & Henry M. Kronenberg
7, Article number: 11277 (2016) | Download Citation
Dental root formation is a dynamic process in which mesenchymal cells migrate toward the site of the future root, differentiate and secrete dentin and cementum. However, the identities of dental mesenchymal progenitors are largely unknown. Here we show that cells expressing osterix are mesenchymal progenitors contributing to all relevant cell types during morphogenesis. The majority of cells expressing parathyroid hormone-related peptide (PTHrP) are in the dental follicle and on the root surface, and deletion of its receptor (PPR) in these progenitors leads to failure of eruption and significantly truncated roots lacking periodontal ligaments. The PPR-deficient progenitors exhibit accelerated cementoblast differentiation with upregulation of nuclear factor I/C (Nfic). Deletion of histone deacetylase-4 (HDAC4) partially recapitulates the PPR deletion root phenotype. These findings indicate that PPR signalling in dental mesenchymal progenitors is essential for tooth root formation, underscoring importance of the PTHrP–PPR system during root morphogenesis and tooth eruption.
Tooth morphogenesis, characterized by the two distinct stages of crown and root formation, is a prime example of organogenesis involving sequential steps of reciprocal epithelial–mesenchymal interactions1. During crown formation, the invaginated dental epithelial cells differentiate into ameloblasts that form the enamel, and then their adjacent dental papilla mesenchymal cells differentiate into odontoblasts that form the dentin. Subsequent root formation is initiated by formation of a bilayered tissue termed as Hertwig’s epithelial root sheath (HERS)2. The epithelial root sheath continues to grow downward to shape the future root of the tooth, and dental papilla and follicle mesenchymal cells in its vicinity differentiate into matrix-producing odontoblasts and cementoblasts to form dentin (inside) and cementum (outside), respectively (Fig. 1a). The dental root is a critical component of the tooth, anchored to surrounding alveolar bones by the periodontal ligament (PDL). Both human and rodent molars have multiple roots and are formed through identical developmental sequences, and formation of the tooth root and its surrounding structure, including the PDL and alveolar bone, is considered to be important for tooth eruption3.
Figure 1: A lineage-tracing model to study tooth root formation.
(a) Shown is the diagram of a developing molar during root morphogenesis. Enamel is produced by ameloblasts derived from inner enamel epithelium (IEE). Dentin is produced by odontoblasts derived from dental papilla mesenchyme. Cementum is produced by cementoblasts derived from dental follicle mesenchyme. HERS is formed by fusion of outer and inner enamel epithelia, and becomes a physical demarcation between dental papilla and follicle. HERS eventually breaks down and becomes epithelial rests of Malassez (ERM). (b) Shown is the diagram of a tamoxifen-inducible creERT2 system for lineage tracing. CreERT2 recombinase is expressed by a named promoter. It excises the stop codons in the Rosa26 locus only in the presence of tamoxifen. Once the stop codons are removed, the targeted cells permanently express tdTomato in a ubiquitously active CAG promoter-dependent manner. A low-dose tamoxifen (0.1 mg) is administered intraperitoneally, when murine molar root morphogenesis starts around postnatal day 3 (P3). Tamoxifen stays active upto 48 h after injection.
Several lines of evidence suggest that a genetic programme similar to that used during osteoblast differentiation, such as transcription factors Runx2 and osterix (Osx), governs the process of dental mesenchymal cell differentiation during root morphogenesis. Runx2- and Osx-deficient mice exhibit failure of tooth mineralization, although the role of these transcription factors in root formation is unknown because the knockout mice die before the onset of root formation4,5. Osx is expressed in odontoblasts, alveolar bone osteoblasts and dental follicle cells during tooth development6, and regulates cementum formation7. In the early foetal perichondrium of endochondral bones, Osx-expressing osteoblast precursors translocate to bone marrow and become osteoblasts and stromal cells inside the developing bone, while mature osteoblasts already expressing type I collagen (Col1) contributed only to osteoblasts in the bone collar8. Whether cells expressing Osx behave as odontoblast and cementoblast progenitors during dental root development is unknown.
The parathyroid hormone-related protein (PTHrP), a locally acting autocrine/paracrine ligand, and its receptor, the PTH/PTHrP receptor (PPR), mediate a number of important biological actions such as endochondral bone development9,10. PTHrP binds to the PPR and activates multiple heterotrimeric G proteins and triggers the intracellular signalling pathways. In chondrocytes, the PTHrP–PPR system suppresses hypertrophy through a protein kinase A (PKA) pathway-activated nuclear localization of histone deacetylace-4 (HDAC4) that inhibits MEF2 function11. In addition, the Gsα signal downstream of the PPR in cells of the osteoblast lineage facilitates the commitment of mesenchymal progenitors to the osteoblast lineage and restrains the differentiation of committed osteoblasts12. Previous studies indicate that the PTHrP–PPR system also plays important roles in tooth eruption. In humans, loss-of-function mutations of the PPR segregate with familial non-syndromic primary failure of tooth eruption13,14. The PTHrP-deficient mice whose lethal skeletal defect is rescued by expressing PTHrP in chondrocytes show failure of tooth eruption, and rescuing the mice further by expressing PTHrP in epithelial cells corrects tooth eruption15. Furthermore, PTHrP regulates extracellular matrix gene expression in cementoblastic cell lines and inhibits mineralization in vitro16. However, the underlying mechanisms whereby the PTHrP–PPR system regulates root formation and tooth eruption in vivo are largely unknown.
In this study, we hypothesize that the PPR signalling regulates proliferation and differentiation of osterix-expressing progenitors, and plays an essential role in dental root formation and tooth eruption. Our data reveal that the PPR signalling in dental mesenchymal progenitors is essential for orchestrated differentiation of cementoblasts and PDL cells, underscoring the importance of the PTHrP–PPR system in dental root formation and tooth eruption.
Progenitors of tooth root-forming cells express osterix
To understand how osterix-expressing cells participate in dental root morphogenesis of murine molars, we first mapped cell fates using a constitutively active Osx-cre and an R26R-tomato reporter system. In this system, cells expressing Osx and their descendants become permanently red. The mandibular first molar (M1) and its surrounding area were observed at postnatal day 5 (P5), shortly after the time that root morphogenesis of mandibular murine molars had been initiated17. At P5, a great majority of dental mesenchymal cells in the dental follicle and papilla were red (Fig. 2a, white arrows: dental follicle, asterisks: dental papilla, sharps: alveolar bones), whereas dental epithelial cells such as ameloblasts and the epithelial root sheath were not labelled (arrowheads), indicating a dental mesenchyme-specific activity of Osx-cre. Some distinct non-red mesenchymal populations were also observed, such as in the outer dental follicle between the apex and the alveolar bone (Fig. 2a, yellow arrows), around the furcation area (blue arrow) and in the dental papilla (green arrow). In higher magnifications, dental mesenchymal cells surround the nascent epithelial root sheath (Fig. 2a, lower panel), indicating that Osx-expressing cells and their descendants actively participate in root formation.
Figure 2: Mesenchymal progenitors of dental root-forming cells express osterix (Osx).
(a–c) P5 mandibular first molar (M1) sections of (a) Osx-cre; R26RTomato, (b) Osx-creER; R26RTomato (tamoxifen at P3) and (c) Col1-creER; R26RTomato (tamoxifen at P3) mice were stained for nuclei. Upper panels: confocal images of the distal root, lower panels: magnified views of the dotted areas. Red: tdTomato, grey: DAPI. In (a), white arrows: dental follicle, white arrowheads: ameloblasts and epithelial root sheath, asterisks: dental papilla, sharps: alveolar bone, yellow/blue arrows: Tomato− dental follicle, green arrows: Tomato− dental papilla. DP: dental papilla, DF: dental follicle, Od: odontoblasts, Am: ameloblasts. In (b), arrows: dental papilla and follicle cells surrounding HERS. Scale bars: 200 μm (upper panel) and 50 μm (lower panel). (d,e) Osx-creER; R26RTomato mice received 0.1 mg tamoxifen at P3 and were analyzed at P7 (d) and P14 (e). EdU was administered twice (6 and 3 h) prior to analysis. M1 sections were stained for nuclei and EdU. Left panels: confocal images of the distal root, right panels: magnified views of the dotted areas. Red: tdTomato, grey: DAPI, blue: EdU-Alexa647. In (d), white arrows: red cells surrounding HERS, white arrowhead: red cells in the interradicular area, yellow dotted line: HERS, yellow arrowheads: EdU+ red cells. In (e), arrowheads: cementoblasts, asterisks: odontoblasts, arrows: PDL cells. Scale bars: 200 μm (left panel) and 50 μm (right panel). (f) Oc-GFP; Osx-creER; R26RTomato mice received 0.1 mg tamoxifen at P3 and were analyzed at P25. M1 sections were stained for nuclei. Left panel: a confocal image of the distal root, right panel: a magnified view of the dotted area. Green: EGFP, red: tdTomato, grey: DAPI. Arrowheads: Oc-GFP+ cementoblasts, asterisks: odontoblasts, arrows: PDL cells. Scale bars: 200 μm (left panel) and 50 μm (right panel). (g–i) Col1-creER; R26RTomato mice received tamoxifen at P3 and were analyzed at P7 (g), P14 (h) and P25 (i). M1 sections were stained for nuclei. Red: tdTomato, grey: DAPI. Asterisks: odontoblasts. Scale bars: 200 μm.
To understand how osterix-expressing progenitors at a specific time point contribute to further root morphogenesis, a pulse-chase experiment was conducted using a tamoxifen-inducible Osx-creER and an R26R-tomato reporter system. Osx-creER; R26R-tomato mice received tamoxifen at P3 when root morphogenesis was initiated. In this paradigm, only cells actively expressing Osx at P3 undergo recombination in the presence of tamoxifen and become permanently red. Forty-eight hours after a single tamoxifen injection, osterix-expressing red cells were predominantly found among odontoblasts, alveolar bone osteoblasts/cytes and sparsely among dental papilla and follicle cells (Fig. 2b). In higher magnifications, cells in the dental papilla and follicle surrounding the epithelial root sheath were red (Fig. 2b, lower panel, white arrows). We thereafter chased the descendants of these osterix-expressing cells (Osx-P3 cells) for various days to see how these cells contribute to further dental root formation. After 4 days of chase at P7, Osx-P3 cells contributed to an increasing number of dental papilla and follicle cells surrounding the epithelial root sheath (Fig. 2d, arrows), as well as cells in the incipient interradicular area between the roots (arrowhead). Some of Osx-P3 cells were proliferating both in the dental papilla and follicle around the HERS, as they incorporated 5-ethynyl-2′-deoxyuridine (EdU) administered shortly before analysis (Fig. 2d, right panel, yellow arrowheads). In addition, Osx-P3 cells became odontoblasts of the elongating portion of the root structure (Fig. 2d, right panel, asterisks: odontoblasts). After 11 days of chase at P14, Osx-P3 cells participated in root formation by differentiating into a majority of the odontoblasts, dental pulp cells, cementoblasts on the acellular cementum (essential for establishing periodontal attachment) and some PDL cells, especially on the interalveolar septum side (Fig. 2e, arrowheads: cementoblasts, asterisks: odontoblasts, arrows: PDL cells). This trend continued when chased until P25, and Osx-P3 cells substantially contributed to most of aforementioned cell types and cementoblasts on the cellular cementum and their adjacent PDL cells (Fig. 2f, arrowheads: osteocalcin (Oc)-GFP+ (green fluorescent protein) cementoblasts, arrows: PDL cells). Osx-creER; R26R-tomato mice without tamoxifen injection did not show any accumulation of red cells (Supplementary Fig. 1a–c), suggesting that tamoxifen-independent activity of Osx-creER was negligible. Therefore, these data from the lineage-tracing experiments suggest that osterix-expressing cells at P3 include dental mesenchymal progenitors that contribute to all cell types involved in further dental root development.
Type I collagen (Col1) is a most abundant matrix protein in mineralizing tissues produced by differentiated cells, such as osteoblasts, cementoblasts and odontoblasts. To test if Col1-expressing cells contribute to dental root morphogenesis, we undertook a pulse-chase experiment using a tamoxifen-inducible Col1(3.2 kb)-creER and an R26R-tomato reporter system. Forty-eight hours after injection, Col1-expressing red cells were predominantly found among odontoblasts of the dental crown and alveolar osteoblasts/cytes, but not among dental papilla and follicle mesenchymal cells (Fig. 2c). We sought to chase the descendants of these Col1-expressing cells at P3 (Col1-P3 cells). Unfortunately, we observed substantial tamoxifen-independent activities after P14, especially among odontoblasts and alveolar bone osteoblasts, but not in PDL cells (Supplementary Fig. 1d–f). Therefore, we could not ascertain whether red odontoblasts and alveolar bone osteoblasts represented the descendants of Col1-P3 cells or ongoing 3.2 kb Col1a1 promoter activities in these cells. Nonetheless, none of dental papilla and follicle mesenchymal cells surrounding the epithelial root sheath was red at P7 (Fig. 2g), and only odontoblasts and alveolar osteoblasts/cytes, but not cementoblasts or PDL cells, were red at P14 and P25 (Fig. 2h,i, asterisks: odontoblasts). Therefore, Col1-expressing cells did not become cementoblasts or PDL cells during further dental root development.
These findings from lineage-tracing experiments suggest that osterix-expressing progenitors, but not mature matrix-producing cells such as odontoblasts and osteoblasts, differentiate into cementoblasts and their adjacent PDL cells during further dental root development.
Cells in dental follicle and on root surface express PTHrP
To delineate the pattern of PTHrP expression during dental root morphogenesis, we took advantage of a PTHrP-LacZ knock-in mouse, in which expression of β-galactosidase is regulated by the endogenous PTHrP locus18. When root morphogenesis started at P3, PTHrP-expressing blue cells were predominantly found in the dental follicle in a pattern surrounding the tooth (Fig. 2a, blue cells in the surrounding alveolar bone are LacZ-independent activities), but not within the dental papilla and dental pulp. A group of non-blue cells in the dental papilla in proximity to the incipient epithelial root sheath were proliferating as they incorporated EdU administered shortly before analysis (Fig. 3a, arrowheads). In the dental follicle, both blue and non-blue cells were proliferating (Fig. 3a, right panel, white arrows: PTHrP−, yellow arrows: PTHrP+), indicating that both PTHrP-expressing and non-expressing cells proliferated at the time of the initiation of root formation. When root morphogenesis was well in progress at P7, many dental follicle cells, but many fewer dental papilla cells, were blue at the root formation front (Fig. 3b, yellow arrowheads), suggesting that PTHrP expression during root morphogenesis was rather specific to the portion of the dental follicle that becomes the dental root in the future. In higher magnification, intense PTHrP-LacZ activity was found in the dental follicle immediately outside the epithelial root sheath and beyond (Fig. 3b, right panel, blue arrowheads), while its activity was rather weaker in the epithelial root sheath and dental papilla (see inset, Fig. 3b). When the distinct bifurcated roots were formed at P14, PTHrP was still expressed in a pattern surrounding the molar, most evidently in two locations: the dental follicle/sac on the top of the dental crown (Fig. 3c, yellow arrows) and on the root surface (yellow arrowheads). In higher magnification, PTHrP-LacZ activities were found in cementoblasts and their adjacent PDL cells (Fig. 3c, right panel, blue arrowheads). When the dental root was completely formed and the first molar completely erupted into the oral cavity at P25 (data not shown) and weeks after that at P49, the root surface-specific pattern of PTHrP expression was maintained (Fig. 3d, yellow arrowheads).
Figure 3: Cells in the dental follicle and the root surface express PTHrP.
(a) PTHrPLacZ/+ mandibular first molars (M1) sections at P3 were stained for β-galactosidase activity and EdU, which was administered twice (6 and 3 h) prior to analysis. Right panel: a magnified view of the dotted area. Blue: LacZ, green: EdU-Alexa488. White arrowheads: LacZ−EdU+ dental papilla cells, white arrows: LacZ−EdU+ dental follicle cells, yellow arrows: LacZ+EdU+ dental follicle cells. Scale bars: 500 μm (left panel) and 100 μm (right panel). (b–d) PTHrPLacZ/+ M1 sections were stained for β-galactosidase activity at P7 (b), P14 (c) and P49 (d). Right panels: magnified views of the dotted areas counterstained with eosin. The inset of the right panel shows a further magnified view of the dotted area. Yellow and blue arrowheads: LacZ+ dental follicle cells at root formation front, immediately outside HERS (b) and on root surface (c,d). DP: dental papilla, DF: dental follicle. Scale bars: 500 μm (left panels), 50 μm (right panels) and 20 μm (inset). (e,f) P7 M1 sections were stained for nuclei, anti-PPR antibody (G220) (e) or its isotype control (sc-3888) (f), both at 5 μg ml−1. Yellow arrows: PPR+ odontoblasts, yellow arrowheads: PPR+ pericytes, yellow asterisks: PPR+ dental pulp cells, blue arrows: PPR+ dental follicle cells. Red: PPR-Alexa546, blue: DAPI. Scale bar: 200 μm.
These findings suggest that the mesenchymal cells in the dental follicle and on the root surface robustly express PTHrP during root formation and after tooth eruption, suggesting that this PTHrP may have a role as an important cytokine regulating root morphogenesis and maintenance of periodontal attachment.
Tooth root formation requires PPR in mesenchymal progenitors
During active root formation, the expression pattern of PTHrP and the distribution of osterix-expressing cells and their descendants (Osx-lineage cells) overlap, particularly in the dental follicle immediately outside the epithelial root sheath (Figs 2a and 3a–d). As the biological range of action for PTHrP is limited, we hypothesize that these Osx-lineage cells in the dental follicle are the principle targets of PTHrP. PPR immunoreactivity was observed broadly among dental mesenchymal cells, such as odontoblasts, coronal dental pulp cells, pericytes and dental follicle cells, but not in dental epithelial cells such as ameloblasts or epithelial root sheath cells, confirming a mesenchyme-specific expression pattern of the PPR in developing molars (Fig. 3e). Unlike in humans, heterozygous inactivation of the PPR in mice did not cause a defect in tooth eruption (Supplementary Fig. 2a,b). To understand the roles of the PPR in dental root-forming mesenchymal cells, we conditionally deleted the PPR using Osx-cre::GFP19 and the PPR-floxed allele20. PPRfl/+, fl/fl (Control), Osx-cre::GFP; PPRfl/+ (Osx-PPR cHet) and Osx-cre::GFP; PPRfl/fl (Osx-PPR conditional knockout (cKO)) littermate mice were analyzed at P3, P7, P18 and P25.
At P3, the time that root formation was initiated, the dental follicle surrounding apical ameloblasts was comparable among the three genotypes (Fig. 4a–c, lower panels, yellow asterisks), although the mesial portion of the crown was malformed in the Osx-PPR conditional knockout mice (Fig. 4c, sharp). When root formation was in progress at P7, mesenchymal cells of the dental papilla and follicle were densely recruited around the epithelial root sheath in wild-type (WT) control teeth (Fig. 4d, yellow arrowheads). In the Osx-PPR conditional Het, the width of the dental follicle was comparable to control, although dental follicle cells adjacent to the epithelial root sheath appeared to be sparse (Fig. 4e). In the Osx-PPR conditional knockout, the dental follicle was narrower than in controls, and dental follicle cells around the epithelial root sheath were far fewer than in the conditional het (Fig. 4f). Cell proliferation was evaluated by administering EdU to these mice shortly before analysis. In both WT control and the Osx-PPR conditional Het, EdU+ dental papilla and follicle mesenchymal cells were observed surrounding the epithelial root sheath (Fig. 4g,h, yellow dotted line: epithelial root sheath). However in the Osx-PPR conditional knockout, the number of EdU+ cells was significantly reduced both in the dental papilla and follicle (Fig. 4i, quantification of EdU+ dental papilla and follicle cells is shown in Fig. 4p,q, Mann–Whitney’s U-test), suggesting that complete deletion of the PPR led to impaired proliferation of these mesenchymal cells.
Figure 4: Tooth root formation requires PPR in osterix-lineage cells.
(a–f) Mandibular first molar (M1) sections of (a,d) Control (PPRfl/+fl/fl), (b,e) Osx-PPR conditional Het (Osx-cre; PPRfl/+) and (c,f) Osx-PPR conditional KO (Osx-cre; PPRfl/fl) mice at P3 (a–c) and P7 (d–f) were stained for hematoxylin and eosin. Lower panels: magnified views of the dotted areas. Asterisks: dental follicle, sharp: malformed crown on the mesial portion. Am: ameloblasts, arrowheads: dental papilla and follicle cells around HERS. Scale bars: 500 μm (upper panels) and 50 μm (lower panels). (g–i) Sections of (g) Control, (h) Osx-PPR conditional Het and (i) Osx-PPR conditional KO molars at P7 were stained for nuclei, EdU and Osx-GFP. EdU was administered twice (6 and 3 h) prior to analysis. Green: GFP-Alexa488, red: EdU-Alexa555, grey: DAPI. Yellow dotted lines: HERS, blue arrowheads: GFP+EdU+ dental follicle cells. Scale bars: 50 μm. (j–l) Stereoscopic images of (j) Control, (k) Osx-PPR conditional Het and (l) Osx-PPR conditional KO molars at P18 are shown. Red arrowheads: crowns of M1 and M2. Scale bars: 1 mm. (m–o) M1 sections of (m) Control, (n) Osx-PPR conditional Het and (o) Osx-PPR conditional KO molars at P18 were stained for hematoxylin and eosin. Lower panels: magnified views of the dotted areas. Arrows: irregular cementum, arrowheads: ankylosis of cementum to bone. Scale bars: 500 μm (upper panels) and 50 μm (lower panels). (p,q) Shown is quantification of EdU+ cells in dental papilla (p) and dental follicle (q) around HERS at P7. Blue bars: Control, green bars: Osx-PPR conditional Het, red bars: Osx-PPR conditional KO, n=5 per group, *P<0.05, Mann–Whitney’s U-test. (r) Shown is quantification of M1 distal root length at P18. Blue bars: Control, green bars: Osx-PPR conditional Het, red bars: Osx-PPR conditional KO. n=4 per group, *P<0.05, Mann–Whitney’s U-test All data are represented as mean±s.d.
To ascertain whether the defect in proliferation occurs in osterix-expressing progenitors, we conducted anti-GFP staining in addition to EdU detection. Cells actively expressing Osx-cre::GFP are detected as green, while the nucleus of proliferating cells become red. The GFP signal was not observed in WT control sections, confirming the specificity of the antibody (Fig. 4g). In the Osx-PPR conditional Het, a number of dental mesenchymal cells surrounding the epithelial root sheath were green, and proliferating Osx+ cells were especially observed in the dental follicle (Fig. 4h, blue arrowheads). In the PPR conditional knockout, proliferation of Osx+ cells in the dental follicle was abrogated (Fig. 4i).
At P18, mandibular molars of WT control and the Osx-PPR conditional Het erupted normally into the oral cavity (Fig. 4j,k). However, in the Osx-PPR conditional knockout mice, molars did not erupt and were covered by the dental sac (Fig. 4l). In WT control mice, the dental root was normally formed with its length longer than the crown, and the PDL was highly organized with oblique fibres extending into the acellular cementum on the root surface (Fig. 4m). In the Osx-PPR conditional Het, the dental root was slightly shorter than in the control, accompanied with thinner and less organized PDL as well as irregular cementum (Fig. 4n, lower panel, arrows). In the Osx-PPR conditional knockout, the dental root was significantly truncated, making it virtually rootless (Fig. 4o, quantification of the root length of M1D is shown in Fig. 4r, Mann–Whitney’s U-test). These molars lacked the PDL, with the root cementum appearing to be ankylosed to the surrounding hypomorphic bones (Fig. 4o). Most of the Osx-PPR conditional knockout mice died after weaning for uncertain reasons. Osx-cre::GFP; PPR+/+ molars did not show an overt phenotype (Supplementary Fig. 2c,d), indicating that the presence of Osx-cre::GFP was not responsible for the root truncation. We further performed tartrate-resistant acid phosphatase (TRAP) staining to ascertain if alterations in osteoclasts accounted for the phenotype. No apparent difference was observed in terms of the location and the number of TRAP+ cells around molars before and after root formation and eruption (Supplementary Fig. 2e–m), suggesting that defect in osteoclasts was not the principal cause of these phenotypes.
Subsequently, we performed a 3D microcomputed tomography (microCT) imaging analysis of littermates with three corresponding genotypes at P18. Consistent with macroscopic findings, mandibular molars of the WT Control and the Osx-PPR conditional Het erupted into the oral cavity (Fig. 5a,b), whereas those of the Osx-PPR conditional KO did not erupt and were covered by the cortical shell (Fig. 5c). Individual slices of microCT images at an interval of 6 μm revealed that the dental root was normally formed in the WT Control and the Osx-PPR cHet (Fig. 5d,e), whereas it was truncated in the Osx-PPR cKO (Fig. 5f). These data confirm the reproducibility of our 2D histological analyses in 3D microCT imaging analyses.
Figure 5: Tooth eruption and periostin expression requires PPR in osterix-lineage cells.
(a–f) 3D microCT images of (a,d) Control, (b,e) Osx-PPR conditional Het and (c,f) Osx-PPR conditional KO molars at P18 are shown. Upper panels: 3D reconstructed microCT images of the mandible, lower panels: individual slices of microCT images at an interval of 6 μm. Scale bars: 1 mm. (g,h) Sections of (g) Control, (h) Osx-PPR conditional Het and (i,j) Osx-PPR conditional KO molars at P18 were stained for nuclei and anti-periostin (ABT280) at 0.5 μg ml−1. Osx-PPR conditional KO mice with long roots (Mouse #1) (i) and short roots (Mouse #2) (j) are shown. Red: Periostin-Alexa546, blue: DAPI, grey: DIC (differential interference contrast). Yellow arrowheads: periostin+ PDL cells in the middle portion of the dental root. Scale bars: 200 μm. (k) Body weight measurement of Control (blue line), Osx-PPR conditional Het (green line) and Osx-PPR conditional KO (red line) mice between P0 and P25. n=4–16 per group, data are represented as mean±s.d.
We also performed immunohistochemical staining for periostin as a marker for PDLs at P18. As previously reported21, strong immunoreactivity for periostin was particularly observed in the PDL, most notably in the middle portion of the dental root among periodontal fibroblasts and cementoblasts, (Fig. 5g,h, yellow arrowheads). No immunoreactivity was noted within the dental pulp or alveolar bones. In contrast, in the Osx-PPR conditional KO, no periostin immunoreactivity was observed in areas surrounding the dental roots, regardless of the length of the roots (Fig. 5i,j, see #1 long vs #2 short roots). These findings indicate that PPR signalling in Osx-expressing dental mesenchymal progenitors is essential for their differentiation into periostin-expressing PDL cells.
We also measured the body weight of three corresponding genotypes beginning the time of birth (P0) until P25 (Fig. 5k). The Osx-PPR cKO mice consistently showed lower body weight than the WT Control and the Osx-PPR cHet mice throughout postnatal growth. In addition, the Osx-PPR cHet consistently showed slightly lower body weight than the WT Control. After weaning at P21, the discrepancy of the body weight among three genotypes was enhanced, probably due to the tooth phenotype.
These data demonstrate that deletion of the PPR in Osx-lineage cells causes a defect in proliferation of dental mesenchymal cells around the epithelial root sheath, in association with agenesis of the PDL, ankylosis of the dental root to the alveolar bone and truncation of the dental root. Although underlying alveolar bones of the Osx-PPR cKO mice were underdeveloped compared to those in the other genotypes, we think this represents an independent consequence of the genetic mutation. However, as dental root formation is closely coupled with alveolar bone formation, these two biological events cannot be effectively segregated. Therefore, we cannot eliminate the possibility that abnormalities of the dental root and the mandible influence each other.
Tooth root formation does not require PPR in mature cells
The Osx-PPR conditional knockout mice showed significantly shorter dental roots than WT controls at P25 (Fig. 6b, quantification of the root length of M1D is shown in Fig. 6e, Mann–Whitney’s U-test). To determine the roles of the PPR specifically in differentiated mineralizing cells during dental root formation, we conditionally deleted the PPR using osteocalcin (Oc)-cre22 and Dmp1-cre23. PPRfl/fl (Control) and Oc-cre; PPRfl/fl (Oc-PPR cKO) or Dmp1-cre; PPRfl/fl (Dmp-PPR cKO) littermate mice were analyzed at P25.
Figure 6: PPR in differentiated matrix-producing cells is dispensable for tooth root formation.
(a–d) Mandibular first molar (M1) sections of (a) Control (PPRfl/fl), (b) Osx-PPR conditional KO (Osx-cre; PPRfl/fl), (c) Oc-PPR conditional KO (Oc-cre; PPRfl/fl) and (d) Dmp1-PPR conditional KO (Dmp1-cre; PPRfl/fl) mice at P25 were stained for hematoxylin and eosin. Lower panels: magnified views of the dotted areas. Yellow arrowhead: embedded cementocyte. Scale bars: 500 μm (upper panels) and 50 μm (lower panels). (e–g) Shown is quantification of M1 distal root length at P25. Blue bars: Control, red bars: (e) Osx-PPR conditional KO, (f) Oc-PPR conditional KO and (g) Dmp1-PPR conditional KO mice. Control: n=3–6 per group, conditional KO: n=6–7 per group, *P<0.05, **P<0.01, Mann–Whitney’s U-test. (h–m) M1 sections of (h) PPRfl/+, (i) Osx-cre; PPRfl/+, (j) Osx-cre; PPRfl/fl, (k) Col1-PPR*; PPR+/+, (l) Col1-PPR*; Osx-cre; PPRfl/+ and (m) Col1-PPR*; Osx-cre; PPRfl/fl molars at P17 were stained for hematoxylin and eosin. Lower panels: magnified views of the dotted areas. Arrowheads: cementoblasts, asterisks: PDL/dental follicle. Scale bars: 500 μm (upper panels) and 50 μm (lower panels). (n) Shown is quantification of M1 tooth height at P17. Blue bars: Col1-PPR* Control, green bars: Col1-PPR*; Osx-PPR conditional Het, red bars: Col1-PPR*; Osx-PPR conditional KO. n=4–5 per group, *P<0.05, Mann–Whitney’s U-test. All data are represented as mean±s.d.
Interestingly, fate mapping using an R26R-tomato revealed that Oc-cre and Dmp1-cre marked different mineralizing cell types in molar roots. While both cre lines similarly targeted a majority of alveolar osteoblasts/cytes, Oc-cre targeted more cementoblasts (arrowheads, Supplementary Fig. 3d,e) than odontoblasts (asterisks, Supplementary Fig. 3d,e), whereas Dmp1-cre targeted more odontoblasts than cementoblasts. Unlike Osx-cre, both Oc-cre and Dmp1-cre did not target dental pulp cells and PDL cells (Supplementary Fig. 3b,c). The Oc-PPR conditional knockout mice exhibited slightly shorter roots than controls (Fig. 6c,f). The cementum of the Oc-PPR conditional knockout appeared to be irregular and thicker, with occasional embedded cementocytes in the portion of the root not observed in WT controls (Fig. 6c, lower panel, arrowhead). Dmp1-PPR conditional knockout mice did not show any significant alteration of cell proliferation around the HERS (Supplementary Fig. 3h–k, Mann–Whitney’s U-test) or any significant root truncation, and showed no overt alteration in the cementum (Fig. 6d,g, Mann–Whitney’s U-test). Therefore, these data revealed that deletion of the PPR in differentiated matrix-producing cells leads to only minor to moderate truncation of dental roots and abnormal cementum, suggesting that the PPR in matrix-producing cells is not essential for dental root formation and may have effects directly in cementoblasts that affect cementum.
To investigate further whether an activated PPR expressed specifically in differentiated matrix-producing cells can rescue the root truncation phenotype of the Osx-PPR conditional knockout mice, we took advantage of Col1a1-caPPR transgenic mice24 in which a constitutively activated PPR with the Jansen-type constitutively active mutation is expressed in Col1+ matrix-producing cells including odontoblasts, cementoblasts and alveolar bone osteoblasts (Supplementary Fig. 3f,g). Littermates with six genotypes, PPR+/+, PPRfl/+,fl/fl (Control), Osx-cre; PPRfl/+ (Osx-PPR cHet), Osx-cre; PPRfl/fl (Osx-PPR cKO), PPR+/+,fl/+,fl/fl; Col1a1-caPPR (Col1-PPR*), Osx-cre; PPRfl/+; Col1a1-caPPR (Osx-PPR cHet; Col1-PPR*) and Osx-cre; PPRfl/fl; Col1a1-caPPR (Osx-PPR cKO; Col1-PPR*) were analyzed at P17.
Mandibular molars of the Col1-PPR* exhibited a widened dental pulp associated with thin and undermineralized enamel and dentin25, and did not erupt into the oral cavity at P17 (Fig. 6k). The roots of these molars were not bifurcated, and were morphologically different from WT controls. Nonetheless, an immature PDL and cementum were formed on the root surface of these transgenic molars (Fig. 6k, lower panel, arrowheads: cementoblasts, asterisks: PDL). In the Osx-PPR cHet; Col1-PPR*, although the height of mandibular molars was shorter than those in the Col1-PPR*, an immature PDL and cementum were still formed on the root surface, particularly on the mesial side (Fig. 6l, lower panel). In the Osx-PPR cKO; Col1-PPR*, the height of mandibular molars was even shorter than the Osx-PPR cHet; Col1-PPR*, and the root was completely absent without any PDL or cementum (Fig. 6m), while the crown morphology was comparable to that of the Col1-PPR*. The outer dentin surface of the Osx-PPR cKO; Col1-PPR* molars was covered by the dental epithelium consists of ameloblastic cells, and its adjacent dental follicle was underdeveloped (Fig. 6m, asterisk: dental follicle, quantification of the tooth height of M1 is shown in Fig. 6n).
These findings suggest that constitutive activation of the PPR in differentiated matrix-producing cells rescues the crown phenotype, but not the root phenotype of the Osx-PPR conditional knockout mice. In fact, constitutive activation of the PPR in differentiated matrix-producing cells appears to interfere with the function of root-generating cells. These observations support the idea that the PPR signalling in Osx-expressing progenitors before their differentiation into Col1-expressing cells is essential for initiation of dental root formation.
PPR orchestrates cementoblast differentiation of progenitors
To further understand how the defective PPR signalling affects osterix-expressing root-forming progenitors in a cell-autonomous manner, we conducted a lineage-tracing experiment of the PPR-deficient cells using a tamoxifen-inducible Osx-creER, an R26R-tomato reporter locus and the PPR-floxed alleles. The triple mutant mice (Osx-creER; R26R-tomato; PPRfl/fl) and their creER-negative control mice received tamoxifen at P3 when root morphogenesis was initiated. In this system, only cells actively expressing Osx at P3 undergo recombination in the presence of tamoxifen; in these cells, cre recombinase removes the “stop” sequences in the Rosa26 locus (allowing tomato expression) and the floxed exon E1 in the PPR locus (eliminating PPR function). As a result, osterix-expressing cells at P3 and their descendants become red and deficient for the receptor (PPR temporal conditional KO (Osx-PPR tcKO)), making it possible to trace the fate of PPR-deficient cells as the root develops. In addition, we also conducted a similar experiment using the triple mutant mice (Col1-creER; R26R-tomato; PPRfl/fl), in which Col1-expressing cells at P3 and their descendants become red and deficient for the receptor (Col1-PPR tcKO).
After 11 days of chase at P14 when the root was half-formed, the cementum of WT controls and the Col1-PPR tcKO was thin, acellular and organized on the root surface (Fig. 7a,c, see also Supplementary Fig. 4c,d). In contrast, the cementum was significantly thicker in the Osx-PPR tcKO, with a number of cementocytes embedded within the matrix (Fig. 7b, quantification of the cementum thickness and cementocyte number are shown in Fig. 7d,e, Mann–Whitney’s U-test). No overt phenotype was observed in the dentin and the dental pulp of the Osx-PPR tcKO, although odontoblasts appeared to be sparse on the dentin surface (Fig. 7b). Dark field images revealed that the Osx-P3 WT red cells contributed to cementoblasts on the root surface all the way from the cementoenamel junction (Fig. 7f, white arrowheads) to the apex (white arrows), and to some adjacent PDL cells (green arrows). The Osx-P3 PPR-deficient red cells also became cementoblasts along with the entire root surface and, occasionally, embedded in the cementum matrix (Fig. 7g, yellow arrowheads). Proliferation of the Osx-P3 PPR-deficient red cells was comparable to that of Osx-P3 PPR WT cells in the dental papilla, but exhibited a trend toward a decrease of proliferation in the dental follicle around the HERS after 4 days of chase at P7 (Fig. 7h, see also Supplementary Fig. 4a,b for images), suggesting that PPR-deficiency moderately affects proliferation of dental follicle cells in a cell-autonomous manner. Osx-PPR tcKO and Col1-PPR tcKO molars erupted normally into the oral cavity, and the root length of their mandibular first molar of the Osx-PPR tcKO appeared to be slightly shorter than that of controls (Supplementary Fig. 4e–h).