Shedding new light on the mysteries of tooth eruption

In PNAS, Takahashi et al. (1) shed light on the mysteries of tooth eruption, identify putative stem cells that surround the tooth, and provide insight into the function of parathyroid hormone-related peptide [PTHrP; or parathyroid hormone-like hormone (PTHLH)]. These researchers at the University of Michigan, Showa University, and Harvard Medical School wanted to solve the puzzle that surrounds the translocation of teeth from within the bone into the oral cavity. Many hypotheses were initially proposed, including biophysical traction forces exerted by the periodontal ligament (PDL) or pushing from the tooth root against the surrounding bone. However, classic developmental biology experiments that were carried out in the 1980s narrowed down the required tissue for the eruption of teeth to the dental follicle (2, 3). The dental follicle is derived from neural crest cells and is a connective-tissue capsule around the tooth (4). Despite knowing that PTHrP was required for tooth eruption (5) and that the dental follicle and the recruitment of osteoclasts were involved, there has been little progress in determining the factors that mediate this important phase of tooth development. One of the reasons that so few people in the past have studied tooth eruption is that the appearance of the teeth in the oral cavity occurs postnatally. Takahashi et al. (1) used a finely tuned genetic approach that included lineage-tracing capabilities to determine that a loss of Pth1r leads to a specific failure of eruption (Fig. 1).

Fig. 1.

Three phases of tooth eruption, preeruptive, intraosseous, and supraosseous, require signaling in the PTHrP/PTH1R pathway. (A) In the preeruptive phase, teeth are fully surrounded by a bony crypt. The enamel organ produces PTHrP and CSF1 (arrows) which feed back to the dental follicle. (B) The intraosseous phase is characterized by the spatially restricted removal of bone on the coronal side of the tooth due to signals from the dental follicle (arrows). Osteoclasts invade and resorb the bone to create an eruption pathway (ep). Cementum starts to form on the root surfaces (c). (C) First molars in the present study (1) create an eruption pathway but do not move into the occlusal plane (red X). Excess cementum forms (c), perhaps forming transient fusions with bone, preventing eruption. (D) Normally, teeth move through the eruption pathway and form an attachment to the surrounding bone via the periodontal ligament. A full deletion of Pthrp followed by restoration of expression in the enamel organ using K14-Pthrp rescues eruption (5).

Takahashi et al. (1) started by performing lineage tracing of cells that express Pthrp. Using a knockin reporter, they followed expression up to 6 mo of age (1). Initially, mCherry is expressed in the dental follicle but then becomes restricted to cementoblasts, PDL, and bony crypt cells between the roots. These fates are consistent with those found using in vitro cell culture of human dental follicle cells: Mulitpotent mesenchymal stem cells were isolated from the follicles of wisdom teeth and, depending on the culture conditions, they could form mineralized matrix, cementum-like tissue, neuronal-like cells, and adipocytes (6, 7).

Takahashi et al. (1) then removed the receptor in Pthrp-expressing cells by introducing an inducible Cre driver that contains the full regulatory region of Pthrp [bacterial artificial chromosome (BAC) containing a large region up- and downstream of the Pthrp gene]. Cre-recombinase activity was induced at one time point to target the receptor Pth1r. A check on the recombinase activity showed that a subset of cells in the follicle was labeled, but the surrounding bone and dental cells were unlabeled. Using this strategy, the authors found that loss of Pth1r within Pthrp-expressing cells produced a dramatic periodontal and root phenotype. A deletion of signaling starting 3 d after birth, which is the start of the intraosseous phase of eruption (Fig. 1A), results in a severely underdeveloped PDL, as shown by the loss of the periostin marker. The acellular cementum, the layer of mineralized tissue that covers most of the root, was replaced with cellular cementum. The ultimate phenotype was a failure of the molars to emerge into the oral cavity (68% of first molars in knockout mice failed to erupt) (Fig. 1C). Interestingly the eruption pathway formed in the conditionally deleted teeth (Fig. 1 B and C). The reason the eruption phenotype was partially penetrant is likely due to variability in the timing of peak tamoxifen activity. Indeed, the second molars were not affected because they are slightly delayed compared with the first molars. It is important to point out that even though the PDL is poorly developed, this fibrous attachment between tooth and bone is not required for intraosseous tooth eruption. Many previous studies have shown convincingly that teeth can erupt without roots and, therefore, without PDLs (8). Thus, neither the lack of PDL nor the shorter roots in the present study (1) could explain the failure of tooth eruption.

Although the failure of molars to emerge into the oral cavity was anticipated based on previous germline knockouts of Pthrp (5), the exact mechanisms are not obvious. In the original study, Philbrick et al. (5) restored Pthrp expression in cartilage, but not in the teeth, to rescue early lethality. Philbrick et al. noticed that Pthrp was normally expressed in the enamel organ at high levels, complementary to the transcripts for Pth1r in the mesenchyme. In the surviving animals that lacked Pthrp in the dental epithelium, the molars failed to erupt and were covered by bone. However, there are important differences compared with the present paper by Takahashi et al. (1). In Takashi et al., there is minimal expression of the BAC transgene in the enamel organ. Instead, there is complete overlap of expression of Pthrp and Pth1r in the cells within the follicle, so the loss of signaling is primarily mesenchymal rather than epithelial.

The differences in whether epithelial or mesenchymal cells were deficient in PTHrP/PTH1R signaling could explain the presence or absence of an eruption pathway (present in the conditional knockout of Pth1r, absent in the global knockout of Pthrp). At first glance, there is no major difference in the qualitative appearance of tartrate-resistant acid phosphatase (TRAP)⁺ cells (osteoclasts) required to form the eruption pathway in the Philbrick et al. (5) study as compared to the Takahashi et al. (1) study. However, Takahashi et al. examined TRAP staining only at P25 (postnatal day 25) and will have missed the important first wave of osteoclast invasion required to create the eruption pathway. Indeed, this original population of osteoclasts would have been present before the tamoxifen injection. The early wave of functional osteoclasts was able to create a clear path into the oral cavity in Pth1r-deficient animals (1). Despite the lack of obstruction, the first molars did not move toward the oral cavity (Fig. 1C). There may have been some transient merging of the cellular cementum and adjacent bone that prevented eruption. By 6 mo, the mutant first molars were covered by bone—a secondary rather than primary defect (1). In contrast, in the Philbrick et al. (5) study, the germline targeting of Pthrp prevented formation of the eruption pathway. Only when Pthrp was purposely expressed in the epithelium did an eruption pathway form (Fig. 1D). This study (5) indicates that PTHRP from the dental epithelium plays a key role in tooth eruption. Because an eruption pathway formed in the Takahashi et al. study (1), one must presume that residual PTHrP/PTH1R signaling remains in the coronal portion of the enamel epithelium, allowing the formation of the eruption pathway. It is interesting that in humans with a genetic basis for primary failure of eruption (PTH1R variants), an eruption pathway can often be seen on the radiograph (9). However, these teeth do not respond to orthodontic force and cannot be moved up to the occlusal plane (9). It seems likely that the human mutations, similar to the conditional knockouts in ref. 1, do not affect signaling within the enamel organ but may result in a loss of function specifically in the dental follicle. Functional analysis of some of the human PTH1R heterozygous variants suggests that they may act in a dominant-negative fashion to inhibit the wild-type receptor (10). Some variants result in a loss of function or haploinsufficiency in the G protein-coupled receptor (10). In either case, the result of most human variants is a decrease in signal transduction mediated by PTH1R.

The most likely reason for lack of eruption in the study by Takahashi et al. (1) is an abnormality in the dental follicle itself. We know that the dental follicle is absolutely required for eruption because prosthetic teeth can erupt as long as they are implanted into an intact dental follicle (2). The molecular abnormalities in the conditional KO of Pth1r were identified using bulk RNA sequencing (RNA-seq) on tdTomato⁺ cells isolated from 8-d-old mice [5 d after tamoxifen injection; Gene Expression Omnibus (GEO) database accession no. GSE117936]. A set of up-regulated genes specifically in the conditionally deleted RNA was detected. The PTHrP/PTHLH signaling loop normally represses expression of the transcription factor Mef2c and the cell-surface protein CD200. Neither gene was associated with a dental phenotype in functional studies carried out by others (11, 12). Mef2c is required for correct differentiation of cranial neural crest cells into intramembranous bone (12). The CD200 gene is downstream of RANK [coded for by TNF-receptor superfamily member 11a (Tnfrsf11a)], which is the receptor for the RANK ligand (RANKL) Tnfsf11. RANK also binds the decoy receptor, osteoprotegerin (OPG; gene symbol Tnfrsf11b) (13, 14). The CSF1-RANK-RANKL-OPG pathway is crucial for tooth eruption (15, 16). Loss of Cd200 in mouse knockouts resulted in fewer osteoclasts (11). Thus, overexpression of CD200 could somehow dysregulate osteoclast formation in the present study, even though the osteoclasts still clear an eruption pathway. Another aspect of osteoclast function may be affected, contributing to the lack of eruption.

In addition to the detailed molecular profiling of mutant cells, Takahashi et al. (1) create valuable reference data on the normal expression profile of the 6-day dental follicle cells. Single-cell RNA-seq analysis on the Pthrp-mCherry⁺ cells revealed a surprising variety of cell types (GEO database accession no. GSE120108). The authors were careful to add the mCherry sequence to the mouse reference genome to filter out any nonlabeled cells that may have been captured accidentally. The unbiased clustering algorithm (K-means clustering) identified groups of odontoblast-like, fibroblast, dental follicle, and epithelial cells. The appearance of epithelial cells is surprising. It is possible that single-cell RNA-seq is more sensitive than visualization of mCherry protein in sections. The presence of epithelial cells in the mix suggests that the regulatory regions of Pthrp may drive low levels of Cre recombinase expression in the enamel organ, thus disrupting epithelial–mesenchymal interactions. Takahashi et al. (1) identify the dental follicle cells due to relatively higher expression of genes that have been shown to be expressed in the PDL, such as Spondin1 (F-Spondin) (17), Mkx (Mohawk homeobox) (18), and Acta2 (19). It is interesting that some of the well-documented follicle genes, such as CSF1, OPG, and RANKL, were not listed as part of the expression profile (14, 15). This could be due to enrichment of a specific cell type identified by Pthrp expression.

The translational relevance of the Takahashi et al. (1) work is that the single-cell RNA-seq data can be used to identify specific cell populations in human dental follicles. These groups of cells can then be expanded in culture and used for the regeneration of the periodontium lost to disease in patients. Perhaps regulating the levels of PTHrP protein in vitro will help to direct follicular progenitor cells toward a cementoblast fate. It is important to consider that dental follicles can be harvested only from young adults. Because periodontal disease is typically a disease of older adults, other sources of mesenchymal progenitor cells need to be used. The data generated by Takahashi et al. (1) could be used to identify cell populations with similar signatures in the dental pulp, PDL, or connective tissue in the oral cavity. Autologous grafting of progenitor cells with the desired fate will one day be used in therapeutic approaches for periodontal disease.