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J Dent Res 83(3): 241-244, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Opg, Rank, and Rankl in Tooth Development: Co-ordination of Odontogenesis and Osteogenesis

A. Ohazama, J.-M. Courtney, and P.T. Sharpe*

Department of Craniofacial Development, Floor 28, Guy’s Tower, GKT Dental Institute, King’s College London, Guy’s Hospital, London Bridge, London SE1 9RT, UK;

* corresponding author, paul.sharpe{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoprotegerin (OPG), receptor activator of nuclear factor-{kappa}B (RANK), and RANK ligand (RANKL) are mediators of various cellular interactions, including bone metabolism. We analyzed expression of these three genes during murine odontogenesis from epithelial thickening to cytodifferentiation stages. Opg showed expression in the thickening and bud epithelium. Expression of Opg and Rank was observed in both the internal and the external enamel epithelium as well as in the dental papilla mesenchyme. Although Rankl expression was not detected in tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells close to developing tooth germs. All three genes were detected in developing dentary bone at P0. The addition of exogenous OPG to explant cultures of tooth primordia produced a delay in tooth development that resulted in reduced mineralization. We propose that the spatiotemporal expression of these molecules in early tooth and bone primordia cells has a role in co-ordinating bone and tooth development.

KEY WORDS: Opg/Rank/Rankl • tooth development • epithelium • mesenchyme • bone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamily members, receptor activator of nuclear factor-{kappa}B (RANK) and its ligand, RANK ligand (RANKL; also known as ODF, OPGL, and TRANCE), were originally characterized as playing important roles in lymphocyte and osteoclast differentiation and activation (Kong et al., 1999a, b). Another TNF superfamily member, Osteoprotegerin (OPG), acts as a soluble decoy receptor and competes with RANK for binding to RANKL (Lacey et al., 1998). It has been shown that abnormalities in the OPG/RANK/RANKL system lead to dysfunction of several tissues (Simonet et al., 1997; Kong et al., 1999b; Honore et al., 2000). Teeth are organs that develop as a result of sequential and reciprocal interactions between the oral ectoderm and neural-crest-derived mesenchyme. Some components of the TNF pathway, such as ectodysplasin and its receptor, are required for morphogenesis of teeth (Pispa et al., 1999; Tucker et al., 2000; Laurikkala et al., 2001; Ohazama et al., 2003a,b). Furthermore, as its name indicates, RANK is an efficient NF-{kappa}B activator (Anderson et al., 1997). It has also been shown that some components of the NF-{kappa}B pathway are involved in tooth development, although there are no data linking tooth development with OPG, RANK, and RANKL (Makris et al., 2000; Schmidt-Supprian et al., 2000; Zonana et al., 2000; Döffinger et al., 2001; Sharpe lab. [unpublished data]).

Teeth must develop in the correct positions in relation to the forming jaw bones and attach to the jaw bones by tooth-associated bone (alveolar bone) via periodontal ligament. To begin to understand how these processes might be co-cordinated, we examined the expression and role of Opg, Rank, and Rankl in tooth and dentary bone development. The expression patterns of Opg, Rank, and Rankl were mapped by in situ hybridization in mouse embryonic mandibular first molar tooth germs between E11.5 and P0. This period encompasses tooth development from the earliest formation of the epithelial thickening to cytodifferentiation stages and for dentary bone development, from the earliest formation of osteogenic centers to hard tissue formation.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In situ Hybridization
Mice of the CD-1 strain were used. Embryo heads were fixed in 4% buffered paraformaldehyde (PFA), embedded in paraffin wax, and sectioned in a frontal plane. Radioactive section in situ hybridization with the use of 35S-UTP radiolabeled riboprobes was carried out according to Wilkinson (1992), with modifications as described by Tucker et al.(1998).

Explants Cultured with OPG Protein
Mandibles from embryos at E12 were dissected in D-MEM containing glutamax-1. For OPG protein (R&D systems; 50, 100, 200, 400 ng/mL) and BSA control protein, Affi-Gel-blue beads (Bio Rad) were used. Beads were washed and dried before being placed in a solution of the protein for 1 hr at 37°C. The explants were cultured as previously described on membrane filters supported by metal grids (Trowell, 1959; Saxén, 1966). Explants were cultured for 3 days in D-MEM including 10% FBS and respective concentrations of protein. After 3 days in culture, molar tooth germs were dissected from two-thirds of the explants and transferred to kidney capsules. The remaining explants were fixed and prepared for histology. The grafted explant tissues were cultured in host kidneys for 12 days. The resulting teeth were dissected from the kidneys and then fixed for 24 hrs in 4% PFA. The explants were decalcified by 0.5 M EDTA before being embedded for sectioning. Sections were stained with Alcian blue/Chlorotine fast red. All animal experiments were carried out in accordance with Home Office licences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression Patterns of Opg, Rank, and Rankl in Tooth Development
The first morphological sign of tooth development is an epithelial thickening which forms at E11-E12 (initiation). At E12.5, Rank (Fig. 1BGo) and Rankl (Fig. 1CGo) showed no expression in the mandibular epithelium and presumptive tooth mesenchyme, whereas Opg (Fig. 1AGo) was weakly expressed in the thickening tooth epithelium. Expression of Rankl was also observed in mesenchymal cells that correspond to the region of presumptive dentary bone formation (Fig. 1CGo), although no histological signs of bone formation were found.



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  		Figure 1. In situ hybridization of Opg, Rank, and Rankl expression on frontal head sections at the position of first molar tooth development. Tooth epithelium outlined in red. Scale bars: 125 µm (A-F); 300 µm (G-L); 400 µm (M-O).

 
By E13.5, the tooth epithelium has invaginated into underlying mesenchyme to form the epithelial bud (bud stage). Opg showed expression in the outer edges of the invaginated tooth epithelium (Fig. 1DGo). Opg was also weakly expressed around the mesenchyme of the bud (Fig. 1DGo). Weak expression of Rank was detected in the outer portion of the budding epithelium (Fig. 1EGo). At this stage, early osteogenic centers for dentary bone development were forming from mesenchymal cells. Rankl expression was observed in the presumptive dentary bone region but was not present in either tooth mesenchyme (including presumptive alveolar bone) or epithelium (Fig. 1FGo).

By E14.5, the bud epithelium progressively takes the form of the cap configuration and develops into the internal and the external (outer) enamel epithelium, while the mesenchyme develops into the dental papilla and follicle (cap stage). Opg and Rank were expressed strongly in both the internal and the external enamel epithelium and weakly expressed throughout the remaining dental epithelium (Figs. 1GGo, 1HGo). Opg and Rank were also expressed in dental papilla mesenchyme (Figs. 1GGo, 1HGo), and Rankl expression was retained in presumptive dentary bone mesenchyme (Fig. 1IGo).

At E15.5, the molars have started to reach the bell stage (early bell stage). Opg showed expression in the internal enamel epithelium as well as expression in the dental papilla and weak expression in the external enamel epithelium (Fig. 1JGo). Weak expression of Rank (Fig. 1KGo) and strong expression of Rankl (Fig. 1LGo) were observed in condensing mesenchyme that forms the dentary bone.

The terminal differentiation of dentin-forming odontoblasts from dental papilla cells and the enamel-forming ameloblasts from the internal epithelium are initiated between E18 and P0. Weak expression of Opg was observed in pre-ameloblasts (Fig. 1MGo), whereas Rank (Fig. 1NGo) and Rankl (Fig. 1OGo) showed no expression in the pre-ameloblasts or pre-odontoblasts. Expression of Rankl and Opg was evident in the developing alveolar bone forming from the dental follicle cells (Figs. 1MGo, 1OGo). Expression of Rank was not evident in alveolar bone but remained expressed in the forming dentary bone (Fig. 1NGo). Expression of Rank, Rankl, and Opg during murine tooth development is summarized diagrammatically in Fig. 2AGo.



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  		Figure 2. Diagrammatic representation of Opg, Rank, and Rankl expression in early tooth development and proposed interaction between RANK and RANKL. (A) Expression of Opg, Rank, and Rankl in epithelium shown in blue and in mesenchyme in red. (B) Relationship between RANKL in dentary bone mesenchymal cells and RANK and OPG in tooth germ.

 
Effects of OPG on Tooth Development
To investigate the interaction between RANK and RANKL in tooth development, we transiently inhibited RANKL/RANK signaling using exogenous OPG. Mandible primordia (E12) were cultured for 3 days in the presence of beads soaked in OPG protein (50 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL). After being cultured, tooth germs were observed in explants treated with OPG beads (Fig. 3Go), although these were retarded compared with controls (BSA) such that controls were at the cap stage, whereas explants with beads of OPG (200–400 ng/mL) were at the bud stage (Figs. 3DGo, 3EGo). Similar concentrations of OPG have been previously used in cell culture experiments (Kotake et al., 2001). To determine if tooth buds in treated explants could develop into teeth, we transplanted the explants under kidney capsules to allow for full development of teeth. Although teeth were recovered from all explants (Figs. 4AGo, 4BGo), a proportion of the teeth from explants treated with OPG (approximately 60%) showed a distinct reduction in mineralization and an abnormal pulp (Figs. 4CGo, 4DGo). In histological sections, these OPG-treated teeth showed thinner dentin and enamel, and sparse pulp tissue (Figs. 4E–GGo), although bone formation seemed to be normal (data not shown).



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  		Figure 3. Cultured mandibular primordia explants treated with protein beads. BSA control beads (A). OPG beads, 50 ng/mL (B); 100 ng/mL (C); 200 ng/mL (D), 400 ng/mL (E). Retardation of tooth epithelium invagination observed in explants treated with 200 ng/mL (D) and 400 ng/mL (E) of OPG protein. Tooth epithelium outlined in red. Scale bar: 90 µm.

 


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  		Figure 4. Teeth were recovered from renal capsules after 12 days. Teeth recovered from renal capsules (A,B). (C,D) Teeth following decalcification. Arrows show high translucency in comparison with others. (E-G) Sections of teeth. E, BSA controls; F and G, examples of teeth treated with OPG. Scale bars: 100 µm (A-D); 25 µm (E-G).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During odontogenesis and osteogenesis, Rank, Rankl, and Opg showed dynamic spatiotemporal expression patterns. In the presumptive dentary bone region, a critical regulator of osteoblast differentiation, runt-related gene 2 (Cbfa1/Runx2), is expressed in the same region as Rankl from E12 (D’Souza et al., 1999), although Cbfa1/Runx2 is additionally expressed in tooth mesenchyme. These expression patterns show that Rankl is expressed in the early ossification centers for dentary bone formation and that Rank and Opg are expressed in tooth bud epithelium and mesenchyme. It has been shown that interaction between RANK and RANKL is involved in lobulo-alveolar development of mammary glands during pregnancy (Cao et al., 2001). Teeth develop through similar epithelial-mesenchymal mechanisms to embryonic mammary gland development. It is likely that lobulo-alveolar development of mammary glands is regulated by pathways similar to the early stages of tooth development. Consistent with Rank expression in mammary gland epithelium, Rank expression was observed in tooth epithelium (Figs. 1EGo, 1HGo) (Fata et al., 2000; Cao et al., 2001). The well-established interactions among Rank, Rankl, and Opg suggested a link between the formation of ossification centers for dentary bone and tooth development at early stages of development. Such interactions may be important in co-ordinating the temporal spatial development of bone and teeth (Fig. 2BGo).

To investigate the interaction between RANK and RANKL in tooth development, we transiently inhibited RANKL/RANK signaling using exogenous OPG. The addition of exogenous OPG to explant cultures of E12 tooth primordia produced a delay in tooth development and resulted in thinner dentin and enamel, and sparse pulp tissue. This suggested that exogenous OPG did not completely inhibit tooth development, but rather retarded development and caused defective mineralization and pulp formation.

The retardation of tooth development following the addition of exogenous OPG suggests that disruption of the communication between Rank and Rankl in early tooth germs and pre-osteogenic mesenchymal condensations affects the temporal program of odontogenesis such that tooth formation is not co-ordinated with underlying bone formation. Clearly this temporal delay does not result in complete arrest of tooth development, although since the effect of exogenous OPG in these experiments is transient, it is possible that tooth germs recover as a result of degradation of OPG, and any possible later effects of exogenous OPG cannot be assessed. However, the long-term effects of this early temporal retardation appear to affect mineralization and pulp formation. A variety of mouse knockouts has revealed that bone formation is not required for tooth development, and vice versa (Satokata and Maas, 1994; Thomas et al., 1997; Depew et al., 2002). The results presented here suggest that although teeth and bone are not required for each other’s development, there is early communication between tooth germs and bone-forming cells, and that this is important for synchronizing the two processes, perhaps to ensure correct spatial positioning of teeth in the jaws.


   ACKNOWLEDGMENTS
 
We thank A. Grigoriadis for the Opg and Rankl probes and for critically reading the manuscript, Amgen Inc. for the Rank probe, and R. Schmidt-Ullrich for critically reading the manuscript. This work was supported by grants from the Wellcome Trust, MRC, BBSRC, and by Showa University, Tokyo, Japan (AO).

Received August 5, 2003; Last revision December 11, 2003; Accepted December 12, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. (1997). A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179.[Medline]

Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, et al. (2001). IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 107:763–775.[ISI][Medline]

D’Souza RN, Aberg T, Gaikwad J, Cavender A, Owen M, Karsenty G, et al. (1999). Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice. Development 126:2911–2920.[Abstract]

Depew MJ, Lufkin T, Rubenstein JL (2002). Specification of jaw subdivisions by Dlx genes. Science 298:381–385.[Abstract/Free Full Text]

Doffinger R, Smahi A, Bessia C, Geissmann F, Feinberg J, Durandy A, et al. (2001). X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nat Genet 27:277–285.[ISI][Medline]

Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al. (2000). The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103:41–50.[ISI][Medline]

Honore P, Luger NM, Sabino MA, Schwei MJ, Rogers SD, Mach DB, et al. (2000). Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat Med 6:521–528.[ISI][Medline]

Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. (1999a). OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315–323.[Medline]

Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al. (1999b). Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402:304–309.[Medline]

Kotake S, Udagawa N, Hakoda M, Mogi M, Yano K, Tsuda E, et al. (2001). Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum 44:1003–1012.[ISI][Medline]

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. (1998). Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176.[ISI][Medline]

Laurikkala J, Mikkola M, Mustonen T, Aberg T, Koppinen P, Pispa J, et al. (2001). TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Dev Biol 229:443–455.[ISI][Medline]

Makris C, Godfrey VL, Krahn-Senftleben G, Takahashi T, Roberts JL, Schwarz T, et al. (2000). Female mice heterozygous for IKK gamma/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 5:969–979.[ISI][Medline]

Ohazama A, Courtney JM, Sharpe PT (2003a). Expression of TNF-receptor-associated factor genes in murine tooth development. Gene Expr Patterns 3:127–129.[Medline]

Ohazama A, Courtney JM, Tucker AS, Naito A, Tanaka S, Inoue J, et al. (2003b). Traf6 is essential for murine tooth cusp morphogenesis. Dev Dyn 229:131–135.

Pispa J, Jung HS, Jernvall J, Kettunen P, Mustonen T, Tabata MJ, et al. (1999). Cusp patterning defect in Tabby mouse teeth and its partial rescue by FGF. Dev Biol 216:521–534.[ISI][Medline]

Satokata I, Maas R (1994). Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 6:348–356.[ISI][Medline]

Saxén L (1966). The effect of tetracyclin on osteogenesis in vitro. J Exp Zool 162:269–294.

Schmidt-Supprian M, Bloch W, Courtois G, Addicks K, Israel A, Rajewsky K, et al. (2000). NEMO/IKK gamma-deficient mice model incontinentia pigmenti. Mol Cell 5:981–992.[ISI][Medline]

Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, et al. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319.[ISI][Medline]

Thomas BL, Tucker AS, Qui M, Ferguson CA, Hardcastle Z, Rubenstein JL, et al. (1997). Role of Dlx-1 and Dlx-2 genes in patterning of the murine dentition. Development 124:4811–4818.[Abstract]

Trowell OA (1959). The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147.[ISI][Medline]

Tucker AS, Al Khamis A, Sharpe PT (1998). Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme. Dev Dyn 212:533–539.[ISI][Medline]

Tucker AS, Headon DJ, Schneider P, Ferguson BM, Overbeek P, Tschopp J, et al. (2000). Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis. Development 127:4691–4700.[Abstract]

Wilkinson DG (1992). In situ hybridisation: a practical approach. Oxford: Oxford University Press.

Zonana J, Elder ME, Schneider LC, Orlow SJ, Moss C, Golabi M, et al. (2000). A novel X-linked disorder of immune deficiency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in IKK-gamma (NEMO). Am J Hum Genet 67:1555–1562.[ISI][Medline]




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