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Home , Dental follicle, Tooth eruption

COMMENTARY

Shedding new light on the mysteries of eruption COMMENTARY Joy M. Richmana,1

In PNAS, Takahashi et al. (1) shed light on the myster- Takahashi et al. (1) then removed the receptor in ies of , identify putative stem cells that Pthrp-expressing cells by introducing an inducible Cre surround the tooth, and provide insight into the func- driver that contains the full regulatory region of Pthrp tion of parathyroid hormone-related peptide [PTHrP; [bacterial artificial chromosome (BAC) containing a or parathyroid hormone-like hormone (PTHLH)]. These large region up- and downstream of the Pthrp gene]. researchers at the University of Michigan, Showa Uni- Cre-recombinase activity was induced at one time versity, and Harvard Medical School wanted to solve point to target the receptor Pth1r. A check on the the puzzle that surrounds the translocation of teeth recombinase activity showed that a subset of cells from within the bone into the oral cavity. Many hy- in the follicle was labeled, but the surrounding bone potheses were initially proposed, including biophysi- and dental cells were unlabeled. Using this strategy, cal traction forces exerted by the periodontal ligament the authors found that loss of Pth1r within Pthrp- (PDL) or pushing from the tooth root against the sur- expressing cells produced a dramatic periodontal rounding bone. However, classic developmental biol- and root phenotype. A deletion of signaling starting ogy experiments that were carried out in the 1980s 3 d after birth, which is the start of the intraosseous narrowed down the required tissue for the eruption phase of eruption (Fig. 1A), results in a severely un- of teeth to the (2, 3). The dental folli- derdeveloped PDL, as shown by the loss of the peri- cle is derived from neural crest cells and is a ostin marker. The acellular , the layer of connective-tissue capsule around the tooth (4). De- mineralized tissue that covers most of the root, was spite knowing that PTHrP was required for tooth replaced with cellular cementum. The ultimate phe- eruption (5) and that the dental follicle and the recruit- notype was a failure of the molars to emerge into the ment of osteoclasts were involved, there has been oral cavity (68% of first molars in knockout mice failed little progress in determining the factors that mediate to erupt) (Fig. 1C). Interestingly the eruption path- this important phase of tooth development. One of way formed in the conditionally deleted teeth (Fig. the reasons that so few people in the past have stud- 1 B and C). The reason the eruption phenotype was ied tooth eruption is that the appearance of the teeth partially penetrant is likely due to variability in the in the oral cavity occurs postnatally. Takahashi et al. (1) timing of peak tamoxifen activity. Indeed, the sec- used a finely tuned genetic approach that included ond molars were not affected because they are lineage-tracing capabilities to determine that a loss slightly delayed compared with the first molars. It of Pth1r leads to a specific failure of eruption (Fig. 1). is important to point out that even though the PDL is Takahashi et al. (1) started by performing lineage poorly developed, this fibrous attachment between tracing of cells that express Pthrp. Using a knockin tooth and bone is not required for intraosseous reporter, they followed expression up to 6 mo of tooth eruption. Many previous studies have shown age (1). Initially, mCherry is expressed in the dental convincingly that teeth can erupt without roots and, follicle but then becomes restricted to cemento- therefore, without PDLs (8). Thus, neither the lack of blasts, PDL, and bony crypt cells between the roots. PDL nor the shorter roots in the present study (1) These fates are consistent with those found using could explain the failure of tooth eruption. in vitro of human dental follicle cells: Although the failure of molars to emerge into the Mulitpotent mesenchymal stem cells were isolated oral cavity was anticipated based on previous germ- from the follicles of wisdom teeth and, depending line knockouts of Pthrp (5), the exact mechanisms are on the culture conditions, they could form miner- not obvious. In the original study, Philbrick et al. (5) alized matrix, cementum-like tissue, neuronal-like restored Pthrp expression in cartilage, but not in the cells, and adipocytes (6, 7). teeth, to rescue early lethality. Philbrick et al. noticed

aLife Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Author contributions: J.M.R. wrote the paper. The author declares no conflict of interest. Published under the PNAS license. See companion article 10.1073/pnas.1810200115. 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1819412116 PNAS Latest Articles | 1of3 Downloaded by guest on October 1, 2021 Pthrp). At first glance, there is no major difference in the qualita- + tive 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 path- way. 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 obstruc- tion, the first molars did not move toward the oral cavity (Fig. 1C). There may have been some transient merging of the cellular ce- mentum 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 erup- tion 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 intootheruption.Becauseaneruptionpathwayformedinthe 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 but may result in a loss of function specifically in the Fig. 1. Three phases of tooth eruption, preeruptive, intraosseous, dental follicle. Functional analysis of some of the human PTH1R and supraosseous, require signaling in the PTHrP/PTH1R heterozygous variants suggests that they may act in a dominant- pathway. (A) In the preeruptive phase, teeth are fully surrounded by a bony crypt. The enamel organ produces PTHrP and negative fashion to inhibit the wild-type receptor (10). Some var- CSF1 (arrows) which feed back to the dental follicle. (B) iants result in a loss of function or haploinsufficiency in the G The intraosseous phase is characterized by the spatially protein-coupled receptor (10). In either case, the result of most restricted removal of bone on the coronal side of the tooth due to human variants is a decrease in signal transduction mediated signals from the dental follicle (arrows). Osteoclasts invade and resorb the bone to create an eruption pathway (ep). Cementum by PTH1R. starts to form on the root surfaces (c). (C) First molars in the The most likely reason for lack of eruption in the study by presentstudy(1)createaneruptionpathwaybutdonotmove Takahashi et al. (1) is an abnormality in the dental follicle itself. We into the occlusal plane (red X). Excess cementum forms (c), know that the dental follicle is absolutely required for eruption perhaps forming transient fusions with bone, preventing because prosthetic teeth can erupt as long as they are implanted eruption. (D) Normally, teeth move through the eruption pathway and form an attachment to the surrounding bone via the into an intact dental follicle (2). The molecular abnormalities in the periodontal ligament. A full deletion of Pthrp followed by conditional KO of Pth1r were identified using bulk RNA sequenc- + restoration of expression in the enamel organ using K14-Pthrp ing (RNA-seq) on tdTomato cells isolated from 8-d-old mice rescues eruption (5). [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 that Pthrp was normally expressed in the enamel organ at high PTHrP/PTHLH signaling loop normally represses expression of the levels, complementary to the transcripts for Pth1r in the mesen- Mef2c and the cell-surface protein CD200. chyme. In the surviving animals that lacked Pthrp in the dental Neither gene was associated with a dental phenotype in func- epithelium, the molars failed to erupt and were covered by bone. tional studies carried out by others (11, 12). Mef2c is required However, there are important differences compared with the pre- for correct differentiation of cranial neural crest cells into intra- sent paper by Takahashi et al. (1). In Takashi et al., there is minimal membranous bone (12). The CD200 gene is downstream of expression of the BAC transgene in the enamel organ. Instead, RANK [coded for by TNF-receptor superfamily member 11a there is complete overlap of expression of Pthrp and Pth1r in the (Tnfrsf11a)], which is the receptor for the RANK ligand (RANKL) cells within the follicle, so the loss of signaling is primarily mesen- Tnfsf11. RANK also binds the decoy receptor, osteoprotegerin chymal rather than epithelial. (OPG; gene symbol Tnfrsf11b) (13, 14). The CSF1-RANK-RANKL-OPG The differences in whether epithelial or mesenchymal cells pathway is crucial for tooth eruption (15, 16). Loss of Cd200 in were deficient in PTHrP/PTH1R signaling could explain the mouse knockouts resulted in fewer osteoclasts (11). Thus, over- presence or absence of an eruption pathway (present in the expression of CD200 could somehow dysregulate osteoclast conditional knockout of Pth1r, absent in the global knockout of formation in the present study, even though the osteoclasts still

2of3 | www.pnas.org/cgi/doi/10.1073/pnas.1819412116 Richman Downloaded by guest on October 1, 2021 clear an eruption pathway. Another aspect of osteoclast function (18), and Acta2 (19). It is interesting that some of the well- may be affected, contributing to the lack of eruption. documented follicle genes, such as CSF1, OPG,andRANKL, In addition to the detailed molecular profiling of mutant cells, were not listed as part of the expression profile (14, 15). This Takahashi et al. (1) create valuable reference data on the normal could be due to enrichment of a specific cell type identified by expression profile of the 6-day dental follicle cells. Single-cell Pthrp expression. + RNA-seq analysis on the Pthrp-mCherry cells revealed a sur- The translational relevance of the Takahashi et al. (1) work is prising variety of cell types (GEO database accession no. that the single-cell RNA-seq data can be used to identify specific GSE120108). The authors were careful to add the mCherry se- cell populations in human dental follicles. These groups of cells quence to the mouse reference genome to filter out any nonla- can then be expanded in culture and used for the regeneration of beled cells that may have been captured accidentally. The the lost to disease in patients. Perhaps regulating unbiased clustering algorithm (K-means clustering) identified the levels of PTHrP protein in vitro will help to direct follicular groups of -like, fibroblast, dental follicle, and epithe- progenitor cells toward a fate. It is important to lial cells. The appearance of epithelial cells is surprising. It is pos- consider that dental follicles can be harvested only from young sible that single-cell RNA-seq is more sensitive than visualization adults. Because is typically a disease of of mCherry protein in sections. The presence of epithelial cells in older adults, other sources of mesenchymal progenitor cells the mix suggests that the regulatory regions of Pthrp may drive need to be used. The data generated by Takahashi et al. (1) low levels of Cre recombinase expression in the enamel organ, could be used to identify cell populations with similar signatures thus disrupting epithelial–mesenchymal interactions. Takahashi in the dental , PDL, or connective tissue in the oral cavity. et al. (1) identify the dental follicle cells due to relatively higher Autologous grafting of progenitor cells with the desired fate will expression of genes that have been shown to be expressed in the one day be used in therapeutic approaches for periodontal PDL, such as Spondin1 (F-Spondin) (17), Mkx (Mohawk homeobox) disease.

1 Takahashi A, et al. (2019) Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption. Proc Natl Acad Sci USA, 10.1073/ pnas.1810200115. 2 Cahill DR, Marks SC, Jr (1980) Tooth eruption: Evidence for the central role of the dental follicle. J Oral Pathol 9:189–200. 3 Marks SC, Jr, Cahill DR (1984) Experimental study in the dog of the non-active role of the tooth in the eruptive process. Arch Oral Biol 29:311–322. 4 Chai Y, et al. (2000) Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127:1671–1679. 5 Philbrick WM, Dreyer BE, Nakchbandi IA, Karaplis AC (1998) Parathyroid hormone-related protein is required for tooth eruption. Proc Natl Acad Sci USA 95:11846–11851. 6 Yao S, Pan F, Prpic V, Wise GE (2008) Differentiation of stem cells in the dental follicle. J Dent Res 87:767–771. 7 Bai Y, et al. (2011) Cementum- and periodontal ligament-like tissue formation by dental follicle cell sheets co-cultured with Hertwig’s cells. Bone 48:1417–1426. 8 Wang XP (2013) Tooth eruption without roots. J Dent Res 92:212–214. 9 Frazier-Bowers SA, Puranik CP, Mahaney MC (2010) The etiology of eruption disorders—Further evidence of a ‘genetic paradigm’ Semin Orthod 16:180–185. 10 Subramanian H, et al. (2016) PTH1R mutants found in patients with primary failure of tooth eruption disrupt G-protein signaling. PLoS One 11:e0167033. 11 Cui W, et al. (2007) CD200 and its receptor, CD200R, modulate bone mass via the differentiation of osteoclasts. Proc Natl Acad Sci USA 104:14436–14441. 12 Verzi MP, et al. (2007) The transcription factor MEF2C is required for craniofacial development. Dev Cell 12:645–652. 13 Sakakura Y, et al. (2005) Immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in Meckel’s cartilage compared with developing endochondral bones in mice. J Anat 207:325–337. 14 Yao S, Ring S, Henk WG, Wise GE (2004) In vivo expression of RANKL in the rat dental follicle as determined by laser capture microdissection. Arch Oral Biol 49:451–456. 15 Wise GE, King GJ (2008) Mechanisms of tooth eruption and orthodontic tooth movement. J Dent Res 87:414–434. 16 Fleischmannova J, Matalova E, Sharpe PT, Misek I, Radlanski RJ (2010) Formation of the tooth-bone interface. J Dent Res 89:108–115. 17 Nishida E, et al. (2007) Transcriptome database KK-Periome for periodontal ligament development: Expression profiles of the extracellular matrix genes. Gene 404:70–79. 18 Koda N, et al. (2017) The transcription factor mohawk homeobox regulates homeostasis of the periodontal ligament. Development 144:313–320. 19 Beertsen W, McCulloch CA, Sodek J (1997) The periodontal ligament: A unique, multifunctional connective tissue. Periodontol 2000 13:20–40.

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