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RESEARCH REPORTS |
1 Developmental Biology Programme, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FIN-00014, University of Helsinki, Finland; and
2 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel;
* corresponding author, irma.thesleff{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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KEY WORDS: tooth morphogenesis • permanent teeth • deciduous teeth • tooth replacement • cleidocranial dysplasia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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While the molecular mechanisms governing the morphogenesis of primary teeth have been elucidated in great detail (Thesleff, 2003), less is known of the generation of new cycles of teeth. Lower vertebrates, such as fish, replace their teeth throughout life, whereas some rodents, like mice, have only one dentition. Most mammals, including humans, replace their teeth once, and have a deciduous (primary) and permanent (secondary) dentition. The permanent teeth arise from the extension of dental lamina on the lingual aspect of the deciduous teeth or from posterior growth of the molar dental lamina (Nanci, 2003). Once the crown of the permanent tooth has formed, the dental lamina gradually degenerates. Interestingly, in the patients with cleidocranial dysplasia syndrome (CCD), multiple supernumerary teeth form, which derive from the permanent teeth and represent successional teeth, i.e., a third dentition (Jensen and Kreiborg, 1990). Histological studies have shown that there is incomplete resorption of the dental lamina of the secondary teeth (Lukinmaa et al., 1995). CCD is an autosomal-dominant disorder caused by the lack of function of one allele of the RUNX2 gene (previously named CBFA1; Mundlos et al., 1997).
Runx2 is a transcription factor, and the deletion of its function in knockout mice results in the lack of bone formation (Ducy et al., 1997). In addition, their molar development is arrested at the late bud stage, which correlates with the intense expression of Runx2 in the dental mesenchyme during the bud and cap stages (D’Souza et al., 1999). Our previous studies indicated that Runx2 mediates FGF signaling between the dental epithelium and mesenchyme (Åberg et al., 2004b). We also observed differences between the Runx2 mutant mouse upper and lower molars, with the lower molars affected more severely than the upper ones. Enamel knot marker genes, including p21, Fgf4, Edar, and Bmp4, were down-regulated in Runx2 mutant lower molars, but were expressed normally in the upper molars. Shh was completely absent in Runx2 mutant lower molars, while weak signals remained at the tip of the tooth bud in the upper molars. In addition, a prominent lingual epithelial bud was regularly observed in the mutant upper molars, but not in the lower ones (Åberg et al., 2004a). Interestingly, Runx3, another member of the Runx family, was intensely up-regulated in the Runx2 mutant upper molar mesenchyme, but was not expressed in the mutant lower molars (Åberg et al., 2004b).
In the present study, we tested whether the up-regulation of Runx3 in Runx2 mutant upper molars compensates for some functions of Runx2 and is responsible for the phenotypic differences between the molars. We analyzed the differences between upper and lower molars of Runx2 mutant mice, and compared the tooth phenotypes between Runx2 mutant and Runx2/Runx3 double-mutant mice. Since human CCD patients with heterozygous mutations in the RUNX2 gene often exhibit multiple supernumerary teeth, we also studied in detail the lingual epithelial buds as putative signs of secondary tooth initiation, and included in our analysis Runx2 heterozygote mice, representing the mouse model for CCD patients.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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Cell Proliferation Assay
E14 pregnant mice were intraperitoneally injected with 5-bromo-2'-deoxyuridine (BrdU) solution (1.5 mL/100 g body weight). After 2 hrs, the heads of embryos were harvested, processed for paraffin sections (7 µm), and immuno-stained with the use of a BrdU staining kit (Zymed, San Francisco, CA, USA). We took images of every second section from the center of the enamel knot region to the periphery of the tooth germ. Five sections for each molar were counted, and the statistical significance between wild-type and Runx2 mutant embryos was evaluated by Student’s t test.
[Details of the reverse transcription and quantitative real-time PCR can be found in supplemental information on the Web site.]
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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, 1J). In Runx2 mutant upper molars, Runx3 expression is intensely up-regulated (Fig. 1K; Åberg et al., 2004b). To examine whether Runx3 can compensate for the function of Runx2 in Runx2 mutant upper molars, we compared the tooth phenotype of Runx2/Runx3 double-knockout mice with that of Runx2 knockout mice. Both p21 and Fgf4 were expressed similarly in these mutants (Figs. 1A–1H). The development of the lingual bud in the upper molars and the expression of Shh pathway genes were also similar in the double-mutants as compared with the Runx2 mutants (see below, Figs. 2–4).
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–2C). Ptc1 and Gli1 were co-expressed in the epithelium and mesenchyme, and Ptc2 was mostly expressed in the dental epithelium (Figs. 2F, 2G, 2J, 2K, 2N, 2O; Gritli-Linde et al., 2002). In Runx2 mutants, Shh expression was absent in the lower molars, but weak signals were present in the upper molars (Fig. 2D; Åberg et al., 2004b). Ptc1, Gli1, and Ptc2 were down-regulated in Runx2 mutant lower molars, but in the upper molars, their expression was similar to that in wild-types (Figs. 2H, 2L, 2P). In Runx2/Runx3 double-mutants, the expression patterns of Shh, Ptc1, Ptc2, and Gli1 were all similar to those in the Runx2 mutants (Figs. 2E, 2I, 2M, 2Q). Smo, Gli2, and Gli3 were not affected in either the Runx2 or the Runx2/Runx3 double mutants (data not shown). Quantitative RT-PCR confirmed the reduction of the transcripts of Shh, Ptc1, Ptc2, and Gli1 in Runx2 mutant lower molars (Fig. 2R).
We examined the responsiveness of Runx2 mutant molars to the Shh signal by culturing isolated dental mesenchyme with Shh-releasing beads. Shh induced intense expression of Ptc1 and Gli1 in both upper and lower molar mesenchyme of Runx2 mutants (Figs. 3Ab,d,f,h). The induced signal was comparable with that of the wild-types (Figs. 3Aa,c,e,g). No signals were detected around the BSA control beads (Figs. 3Ai–l and data not shown).
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; p < 0.001). There was also significant reduction in cell proliferation in the mutant upper molars (Figs. 3Ba,b; p < 0.001). The reduction was more marked in the lower molars. In Runx2 mutant upper molars, there was a cluster of cells at the tip of the tooth bud devoid of BrdU, indicating that the enamel knot had been initiated (Fig. 3Bb). No such area was evident in the mutant lower molar.
A Prominent Lingual Epithelial Bud Expressing Shh Develops in Front of the Upper Molars in Runx2 Mutants, Runx2/Runx3 Double-mutants, and Runx2 Heterozygotes
E13 wild-type upper molars were at the bud stage and appeared thin and short, without any lingual buds (Fig. 4A). At E14, they were at the cap stage and appeared wider and longer. In some E14 teeth, a very small lingual bud was seen anterior to the molar epithelium (Fig. 4B). The E14 Runx2 mutant upper molars were much smaller than the wild-types, but epithelial buds were apparent on the lingual aspect of all teeth analyzed, and they were more prominent than in the wild-types (Figs. 4C, 4F). Runx2/Runx3 double-knockout mice had similar prominent lingual buds in all upper molars (Fig. 4G). Lingual buds were not detected in the lower molars.
No Shh expression was detected in the occasionally observed small lingual buds of wild-type mice (Fig. 4H; 8 samples examined), but some transcripts of Ptc1, Ptc2, and Gli1 were evident (Figs. 4K, 4N, and data not shown), suggesting that Shh signaling was present (Cobourne et al., 2004). In Runx2 mutants and Runx2/Runx3 double-mutants, Shh expression was remarkably strong in the tips of the prominent lingual buds in the upper molars (Fig. 4J), and the expression of Ptc1, Ptc2, and Gli1 was very intense (Figs. 4I, 4L, 4M, 4O, 4P, and data not shown).
The dentition of the Runx2 heterozygotes is apparently normal, and supernumerary teeth have not been observed (our unpublished data). However, we detected prominent Shh-expressing lingual buds in E14 Runx2 heterozygotes (Figs. 4Q, 4R, 4S). These buds were located anterior to the first molars, which had reached the cap stage as in the wild-type teeth (Fig. 4T). Analysis of these results suggested that reduced Runx2 activity stimulated both growth and Shh expression in the lingual epithelial buds.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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Earlier, we proposed that the different effects of Runx2 deficiency in the upper and lower molars could be due to compensation by Runx3 in the mutant upper molars (Åberg et al., 2004b). The results of our present study did not support this hypothesis, since the ablation of Runx3 function in the Runx2 knockouts did not affect the phenotype of upper molars. The third member of the Runx family, Runx1, is expressed only in the buccal dental epithelium, and its expression is not affected in Runx2 mutant teeth (Åberg et al., 2004b). Therefore, redundancy with other Runx genes does not account for the differences between Runx2 mutant upper and lower molars. Differences between the upper and lower molar phenotypes have been observed previously in transgenic mice, such as Dlx1/Dlx2 double-knockouts in which the development of upper molars is arrested, whereas the lower molars and incisors develop normally. This phenotype was explained by compensation by other Dlx family genes (Thomas et al., 1997). In contrast, the tooth phenotype of activin ßA mutant mice, which is opposite that of Dlx1/Dlx2 double-mutants, has been proposed to result from the lack of activin requirement in the maxillary molars (Ferguson et al., 1998, 2001). The different origins of the neural crest cells populating the maxillary and mandibular primordia may explain their different behavior, and hence Runx2 may have different downstream target genes in the upper and lower molars.
Cell proliferation was significantly reduced in the Runx2 mutant molars. Although the down-regulation of Fgf3 expression in the dental mesenchyme and Fgf4 in the enamel knot can affect cell proliferation (Kettunen et al., 1998; Åberg et al., 2004b), the absence of Shh signaling may also contribute to the reduced proliferation, since Shh stimulates proliferation and budding of the dental epithelium (Dassule and McMahon, 1998; Cobourne et al., 2001). Reduced proliferation in the mutant dental epithelium conceivably caused the failure of cervical loop formation and the arrest of tooth development at the bud stage.
Runx2 Inhibits the Formation of Lingual Epithelial Outgrowths and Their Shh Signaling
Although the mice do not replace their teeth, they may still have some potential to develop a secondary dentition. Small lingual epithelial buds have been reported anterior to the developing mouse molars (Peterkova et al., 2002). Based on their location, it was suggested that they develop from premolar vestigial tooth buds and represent abortive development of the secondary teeth. This would be reasonable, since, in mammals, the premolars, but not the molars, are replaced by successional teeth. In our wild-type mouse strain, a tiny bud was seen only occasionally in this region. However, lingual buds were regularly present in front of the upper molars in Runx2 knockouts and Runx2/Runx3 double-knockouts, as well as in the Runx2 heterozygotes, and they were much more prominent than in the wild-type mice. It is therefore tempting to speculate that Runx2 inhibits secondary tooth formation in wild-type mice. The formation of supernumerary teeth in human CCD patients lacking the function of one copy of the Runx2 gene indicates that Runx2 normally acts as an inhibitor of tooth cycling. It may appear contradictory that the inhibition of the Runx2 function arrests primary tooth development, but stimulates the formation of secondary teeth. However, it is not unusual, during embryogenesis, that the same gene has different effects at different developmental windows. For example, activin inhibits hair follicle development, but promotes hair cycling (Nakamura et al., 2003).
The intense expression of Shh and its target genes Ptc1 and Gli1 in the lingual buds of Runx2 mutants and Runx2/Runx3 double-mutants indicates that Shh signaling was active in these locations. Shh was expressed intensely in the lingual buds in the Runx2 heterozygotes also. These results—together with earlier evidence showing that Shh stimulates dental epithelial budding and proliferation (Cobourne et al., 2001), and that it is specifically required for the growth of lingual dental epithelium (Dassule and McMahon, 1998), where the successional teeth form—support a function for Shh as a key signal promoting tooth renewal. Further studies of Runx2 on the growth of dental lamina and successional tooth formation, and on its relationship with Shh signaling, may shed light on the molecular mechanisms of tooth cycling and may even give hints for tooth regeneration in humans.
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APPENDIX REVERSE TRANSCRIPTION AND QUANTITATIVE REAL-TIME PCR |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received July 16, 2004; Last revision November 3, 2004; Accepted November 9, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIX REVERSE TRANSCRIPTION...REFERENCES |
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Åberg T, Wang XP, Kim JH, Yamashiro T, Bei M, Rice R, et al. (2004b). Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis. Dev Biol 270:76–93.[ISI][Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
ct) compared with wild-types. Brackets represent highest and lowest 
