- Email: [email protected]
Molecular regulation of tooth development
Bone Vol. 25, No. 1 July 1999:123–125
Molecular Regulation of Tooth Development I. THESLEFF and T. ÅBERG Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
Introduction Teeth are organs that are only found in the oral cavity of vertebrates. Although they are composed of mineralized tissues and they are attached to bone, they do not form as outgrowths of bone. In fact, tooth development starts in the embryonic oral epithelium well before bone formation, and osteogenesis of the alveolar bone is later regulated by the teeth rather than vice versa. The mineralizing extracellular matrices of teeth, the enamel, dentine, and cementum, are formed by the ameloblasts, odontoblasts, and cementoblasts, respectively, which are unique dental cell types differentiating during specific stages of tooth morphogenesis. Teeth are typical examples of epithelial appendages, i.e., organs that develop from surface epithelium and underlying mesenchymal tissue. Interactions between the epithelial and mesenchymal tissues regulate the development of all epithelial appendages. These interactions, originally discovered in classic tissue recombination studies, are reciprocal and occur sequentially, constituting a kind of discussion between the neighboring tissues. The molecules of the signaling networks that mediate these interactions started to be elucidated in the late 1980s, and it has become apparent that same signaling molecules regulate the development in all epithelial appendages.19 Teeth belong to those organs in which the signaling mechanisms have been actively analyzed in the 1990s.
Figure 1. Schematic presentation of molecules in the signaling networks regulating advancing tooth morphogenesis. Arrows indicate the signals coming from the epithelium (above) or mesenchyme (below). Transcription factors in the networks are indicated in the frames.
the epithelium (Figure 1). The dental mesenchyme condenses around the invaginating epithelium and expresses signals that reciprocally act on the epithelium, which then folds and develops to the cap and bell stages. We have discovered a signaling center, the enamel knot, that appears at the tip of the epithelial bud as it transforms from the bud to cap stage. In this transient epithelial structure, several signal molecules are coexpressed, and we have suggested that the enamel knot is involved in the regulation of tooth shape.23 In the multicusped molar teeth, secondary enamel knots expressing Fgf-4 appear at the sites of future cusps, and they presumably regulate the initiation and growth of cusps (reviewed in ref. 20) Experimental studies in which the signaling proteins have been applied locally on the proposed target tissue with agarose or heparin acrylic beads have shown that the signals can, in fact, mimic the effects of the inducing tissues. This was first demonstrated in our laboratory for BMP-2 and -4, which stimulated the expression of the homeobox-containg transcription factors Msx1 and Msx2 in the presumptive dental mesenchyme.24 We have also shown that FGF-8, -9, and -4 stimulate the expression of Msx1 but not Msx2.10 Studies by others have shown that the epithelial BMPs and FGFs also stimulate the expression of several other transcription factors, including Lef1, Pax 9, Dlx1,2, Lhx6,7, and Barx1 in mesenchyme.2,6,12,14,18,22 Furthermore, Shh and Wnt proteins, which are also expressed in the budding dental epithelium, stimulate mesenchymal expression of their transcriptional targets Gli1 and Lef1.3,7 During budding of the dental epithelium, when the dental mesenchyme has acquired instructive potential, mesenchymally expressed signal molecules, including BMP-4 and FGF-10, act on dental epithelium. We have applied BMP-4 with beads on epithelium cultured on Matrigel substrate, and shown that it induces the expression of Msx2 and p21, a cyclin-dependent kinase inhibitor, which are expressed in the epithelial enamel
Signaling Networks Mediating Epithelial-Mesenchymal Interactions The most studied signaling molecules mediating epithelial-mesenchymal interactions in the tooth, as well as in other organs, belong to four major families: bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (Hh), and Wnt. Several members of these families have been implicated in tooth morphogenesis (reviewed in ref. 21). It is apparent that the same signaling molecules are used repeatedly during tooth morphogenesis. FGF-8 and BMP-4 are early signals in the oral ectoderm, which act on the underlying neural crest-derived mesenchyme and induce the capacity to form teeth, and determine tooth identity, respectively.6,22 The oral ectoderm subsequently thickens and starts to bud, and several signal molecules belonging to all four signal families (BMP, FGF, Wnt, Shh) are expressed in
Key Words: Morphogenesis; Epithelial-mesenchymal interactions; osf2; cbfa1; Odontogenesis. Address for correspondence and reprints: Irma Thesleff, Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki, Helsinki, Finland. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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may reflect a function of Cbfa1 in stabilization of the epithelial sheet. Interestingly, the human syndrome cleidocranial dysplasia, a condition mainly affecting membranous bone formation, is caused by heterozygous loss of function of the CBFA1 gene,13 and these patients have a unique tooth phenotype involving multiple extra teeth. We do not know at present which genes are regulated by Cbfa1 in the dental mesenchyme. They may be signals affecting directly epithelial cell behavior or they may be molecules regulating the composition of the extracellular matrix between the epithelium and mesenchyme. Further studies on the functions of Cbfa1 can be expected to shed more light on the molecular regulation of tooth morphogenesis, and they may also provide the first information on the virtually unknown mechanisms of the replacement of deciduous teeth by permanent teeth. Figure 2. Expression and regulation of osf2/cbfa1 expression in a cap stage mouse molar tooth germ. A) In situ hybridization analysis of the lower jaw of an E14 mouse embryo. Expression is intense in the dental mesenchyme as well as in mandibular bone. B) A bead-releasing FGF4 protein has stimulated osf2/cbfa 1 expression in the isolated dental papilla mesenchyme (whole mount in situ hybridization analysis) C) No expression is seen around the control BSA bead.
knot.8 FGF-10 stimulates cell proliferation in the dental epithelium (our unpublished observations). The distribution of the different FGF receptors is in line with a hypothesis that FGFs importantly regulate epithelial morphogenesis by stimulating locally cell proliferation.9 Transgenic mouse experiments have shown that the deficient function of several transcription factors that are parts of the signaling networks results in arrested tooth morphogenesis. These include Msx1,2, Pax9, Lef1, Dlx1,2, and Gli1,2.1,12,17,18 We have recently discovered that tooth development is arrested also in knockouts of the transcription factor Cbfa1, a runt domain factor that regulates osteoblast function and is necessary for bone development.5,11,16 The Role of Cbfa1 in Tooth Morphogenesis Our in situ hybridization analysis of Cbfa1 expression, as well as experimental studies on its regulation, suggested that Cbfa1 is part of the signaling networks regulating tooth development.4 It shows a unique expression pattern unlike any other gene reported so far (gene expression patterns in developing teeth can be viewed in the Internet data base: http://honeybee.helsinki.fi/ toothexp15). It is intensely expressed in the dental mesenchyme during active morphogenesis and is downregulated in the dental papilla during the bell stage. Expression continues in the dental follicle and is transient in ameloblasts, whereas terminally differentiated odontoblasts do not express it. Bead implantation assays showed that expression of Cbfa1 in the dental papilla is maintained by epithelial signals including FGF but not BMP (Figure 2). Analysis of the newborn Cbfa12/2 mice indicated that tooth development had been arrested at the cap/bell stage. The tooth germs were hypoplastic but, interestingly, the epithelium showed extra buddings protruding both to dental papilla and follicle.4 This phenotype is different from other mouse mutants, in which morphogenesis is arrested before or at the bud stage (see above). In the Cbfa2/2 mice, morphogenesis continues further and the teeth reach an aberrant bell stage and are severy hypoplastic. Hence, the mutant dental mesenchyme is apparently capable of inducing the onset of epithelial folding morphogenesis during bud stage, but it does not support epithelial growth and proper epithelial morphogenesis. The extra buddings in the epithelium
References 1. Bei, M. and Maas, R. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125:4325– 4333; 1998. 2. Chen, Y. and Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87:553–563; 1996. 3. Dassule, H. R. and McMahon, A. P. Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the mammalian tooth. Dev Biol 202:215–227; 1998. 4. D’Souza, R., Aberg, T. M., Gaikwad, J., et al. Cbfa1 is required for epithelialmesenchymal interactions regulating tooth morphogenesis in mice. Development. In press. 5. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754; 1997. 6. Grigoriou, M., Tucker, A. S., Sharpe, P. T., and Pachnis, V. Expression and regulation of Lhx6 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head development. Development 125:2063–2074; 1998. 7. Hardcastle, Z., Mo, R., Hui, C.-c., and Sharpe, P. T. The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants. Development 125:2803–2811; 1998. 8. Jernvall, J., Aberg, T., Kettunen, P., Keranen, S., and Thesleff, I. The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125:161–169; 1998. 9. Kettunen, P., Karavanova, I., and Thesleff, I. Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9. Dev Genet 22:374 –385; 1998. 10. Kettunen, P. and Thesleff, I. Expression and function of FGFs-4, -8, and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev Dyn 211:256 –268; 1998. 11. Komori, T., Yagi, H., Nomura, S., et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755–764; 1997. 12. Kratochwil, K., Dull, M., Farin˜as, I., Galceran, J., and Grosschedl, R. Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev 10:1382–1394; 1996. 13. Mundlos, S., Otto, F., Mundlos, C., et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89:773–779; 1997. 14. Neubu¨ser, A., Peters, H., Balling, R., and Martin, G. R. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90:247–255; 1997. 15. Nieminen, P., Pekkanen, M., Åberg, T., and Thesleff, I. A graphical WWWdatabase on gene expression in tooth. Eur J Oral Sci 106:7–11; 1998. 16. Otto, F., Thornell, A. P., Cromptom, T., et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentation and bone development. Cell 89:765–771; 1997. 17. Peters, H., Neubu¨ser, A., Kratochwil, K., and Balling, R. Pax9 deficient mice lack pharyngeal pouch derivates and teeth and exhibit craniofacial and limb abnormalities. Genes Dev 12:2735–2747; 1998. 18. Qiu, M., Bulfone, A., Ghattas, I., et al. Role of Dlx-1 and -2 in proximodistal patterning of the branchial arches: mutations alter morphogenesis of proximal skeletal elements derived from the first and second arches. Dev Biol 185:165– 184; 1997.
Bone Vol. 25, No. 1 July 1999:123–125 19. Thesleff, I. and Pispa, J. The teeth as models for studies on the molecular basis of the development and evolution of organs. In: Chuong, C.-M., Ed. Molecular Basis of Epithelial Appendage Morphogenesis. Georgetown, TX: R. G. Landes Company; 1998; 157–179. 20. Thesleff, I. and Jernvall, J. The enamel knot: a putative signaling center regulating tooth development. Cold Spring Harb Symp Quant Biol 62:257–67; 1997. 21. Thesleff, I. and Sharpe, P. Signalling networks regulating dental development. Mech Dev 67:111–123; 1997.
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22. Tucker, A. S., Matthews, K. L., and Sharpe, P. T. Transformation of tooth type induced by inhibition of BMP signaling. Science 282:1136 –1138; 1998. 23. Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S., and Thesleff, I. The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54:39 – 43; 1996. 24. Vainio, S., Karavanova, I., Jowett, A., and Thesleff, I. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75:45–58; 1993.
Molecular Regulation of Tooth Development I. THESLEFF and T. ÅBERG Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
Introduction Teeth are organs that are only found in the oral cavity of vertebrates. Although they are composed of mineralized tissues and they are attached to bone, they do not form as outgrowths of bone. In fact, tooth development starts in the embryonic oral epithelium well before bone formation, and osteogenesis of the alveolar bone is later regulated by the teeth rather than vice versa. The mineralizing extracellular matrices of teeth, the enamel, dentine, and cementum, are formed by the ameloblasts, odontoblasts, and cementoblasts, respectively, which are unique dental cell types differentiating during specific stages of tooth morphogenesis. Teeth are typical examples of epithelial appendages, i.e., organs that develop from surface epithelium and underlying mesenchymal tissue. Interactions between the epithelial and mesenchymal tissues regulate the development of all epithelial appendages. These interactions, originally discovered in classic tissue recombination studies, are reciprocal and occur sequentially, constituting a kind of discussion between the neighboring tissues. The molecules of the signaling networks that mediate these interactions started to be elucidated in the late 1980s, and it has become apparent that same signaling molecules regulate the development in all epithelial appendages.19 Teeth belong to those organs in which the signaling mechanisms have been actively analyzed in the 1990s.
Figure 1. Schematic presentation of molecules in the signaling networks regulating advancing tooth morphogenesis. Arrows indicate the signals coming from the epithelium (above) or mesenchyme (below). Transcription factors in the networks are indicated in the frames.
the epithelium (Figure 1). The dental mesenchyme condenses around the invaginating epithelium and expresses signals that reciprocally act on the epithelium, which then folds and develops to the cap and bell stages. We have discovered a signaling center, the enamel knot, that appears at the tip of the epithelial bud as it transforms from the bud to cap stage. In this transient epithelial structure, several signal molecules are coexpressed, and we have suggested that the enamel knot is involved in the regulation of tooth shape.23 In the multicusped molar teeth, secondary enamel knots expressing Fgf-4 appear at the sites of future cusps, and they presumably regulate the initiation and growth of cusps (reviewed in ref. 20) Experimental studies in which the signaling proteins have been applied locally on the proposed target tissue with agarose or heparin acrylic beads have shown that the signals can, in fact, mimic the effects of the inducing tissues. This was first demonstrated in our laboratory for BMP-2 and -4, which stimulated the expression of the homeobox-containg transcription factors Msx1 and Msx2 in the presumptive dental mesenchyme.24 We have also shown that FGF-8, -9, and -4 stimulate the expression of Msx1 but not Msx2.10 Studies by others have shown that the epithelial BMPs and FGFs also stimulate the expression of several other transcription factors, including Lef1, Pax 9, Dlx1,2, Lhx6,7, and Barx1 in mesenchyme.2,6,12,14,18,22 Furthermore, Shh and Wnt proteins, which are also expressed in the budding dental epithelium, stimulate mesenchymal expression of their transcriptional targets Gli1 and Lef1.3,7 During budding of the dental epithelium, when the dental mesenchyme has acquired instructive potential, mesenchymally expressed signal molecules, including BMP-4 and FGF-10, act on dental epithelium. We have applied BMP-4 with beads on epithelium cultured on Matrigel substrate, and shown that it induces the expression of Msx2 and p21, a cyclin-dependent kinase inhibitor, which are expressed in the epithelial enamel
Signaling Networks Mediating Epithelial-Mesenchymal Interactions The most studied signaling molecules mediating epithelial-mesenchymal interactions in the tooth, as well as in other organs, belong to four major families: bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (Hh), and Wnt. Several members of these families have been implicated in tooth morphogenesis (reviewed in ref. 21). It is apparent that the same signaling molecules are used repeatedly during tooth morphogenesis. FGF-8 and BMP-4 are early signals in the oral ectoderm, which act on the underlying neural crest-derived mesenchyme and induce the capacity to form teeth, and determine tooth identity, respectively.6,22 The oral ectoderm subsequently thickens and starts to bud, and several signal molecules belonging to all four signal families (BMP, FGF, Wnt, Shh) are expressed in
Key Words: Morphogenesis; Epithelial-mesenchymal interactions; osf2; cbfa1; Odontogenesis. Address for correspondence and reprints: Irma Thesleff, Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki, Helsinki, Finland. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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may reflect a function of Cbfa1 in stabilization of the epithelial sheet. Interestingly, the human syndrome cleidocranial dysplasia, a condition mainly affecting membranous bone formation, is caused by heterozygous loss of function of the CBFA1 gene,13 and these patients have a unique tooth phenotype involving multiple extra teeth. We do not know at present which genes are regulated by Cbfa1 in the dental mesenchyme. They may be signals affecting directly epithelial cell behavior or they may be molecules regulating the composition of the extracellular matrix between the epithelium and mesenchyme. Further studies on the functions of Cbfa1 can be expected to shed more light on the molecular regulation of tooth morphogenesis, and they may also provide the first information on the virtually unknown mechanisms of the replacement of deciduous teeth by permanent teeth. Figure 2. Expression and regulation of osf2/cbfa1 expression in a cap stage mouse molar tooth germ. A) In situ hybridization analysis of the lower jaw of an E14 mouse embryo. Expression is intense in the dental mesenchyme as well as in mandibular bone. B) A bead-releasing FGF4 protein has stimulated osf2/cbfa 1 expression in the isolated dental papilla mesenchyme (whole mount in situ hybridization analysis) C) No expression is seen around the control BSA bead.
knot.8 FGF-10 stimulates cell proliferation in the dental epithelium (our unpublished observations). The distribution of the different FGF receptors is in line with a hypothesis that FGFs importantly regulate epithelial morphogenesis by stimulating locally cell proliferation.9 Transgenic mouse experiments have shown that the deficient function of several transcription factors that are parts of the signaling networks results in arrested tooth morphogenesis. These include Msx1,2, Pax9, Lef1, Dlx1,2, and Gli1,2.1,12,17,18 We have recently discovered that tooth development is arrested also in knockouts of the transcription factor Cbfa1, a runt domain factor that regulates osteoblast function and is necessary for bone development.5,11,16 The Role of Cbfa1 in Tooth Morphogenesis Our in situ hybridization analysis of Cbfa1 expression, as well as experimental studies on its regulation, suggested that Cbfa1 is part of the signaling networks regulating tooth development.4 It shows a unique expression pattern unlike any other gene reported so far (gene expression patterns in developing teeth can be viewed in the Internet data base: http://honeybee.helsinki.fi/ toothexp15). It is intensely expressed in the dental mesenchyme during active morphogenesis and is downregulated in the dental papilla during the bell stage. Expression continues in the dental follicle and is transient in ameloblasts, whereas terminally differentiated odontoblasts do not express it. Bead implantation assays showed that expression of Cbfa1 in the dental papilla is maintained by epithelial signals including FGF but not BMP (Figure 2). Analysis of the newborn Cbfa12/2 mice indicated that tooth development had been arrested at the cap/bell stage. The tooth germs were hypoplastic but, interestingly, the epithelium showed extra buddings protruding both to dental papilla and follicle.4 This phenotype is different from other mouse mutants, in which morphogenesis is arrested before or at the bud stage (see above). In the Cbfa2/2 mice, morphogenesis continues further and the teeth reach an aberrant bell stage and are severy hypoplastic. Hence, the mutant dental mesenchyme is apparently capable of inducing the onset of epithelial folding morphogenesis during bud stage, but it does not support epithelial growth and proper epithelial morphogenesis. The extra buddings in the epithelium
References 1. Bei, M. and Maas, R. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125:4325– 4333; 1998. 2. Chen, Y. and Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87:553–563; 1996. 3. Dassule, H. R. and McMahon, A. P. Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the mammalian tooth. Dev Biol 202:215–227; 1998. 4. D’Souza, R., Aberg, T. M., Gaikwad, J., et al. Cbfa1 is required for epithelialmesenchymal interactions regulating tooth morphogenesis in mice. Development. In press. 5. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754; 1997. 6. Grigoriou, M., Tucker, A. S., Sharpe, P. T., and Pachnis, V. Expression and regulation of Lhx6 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head development. Development 125:2063–2074; 1998. 7. Hardcastle, Z., Mo, R., Hui, C.-c., and Sharpe, P. T. The Shh signalling pathway in tooth development: defects in Gli2 and Gli3 mutants. Development 125:2803–2811; 1998. 8. Jernvall, J., Aberg, T., Kettunen, P., Keranen, S., and Thesleff, I. The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125:161–169; 1998. 9. Kettunen, P., Karavanova, I., and Thesleff, I. Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9. Dev Genet 22:374 –385; 1998. 10. Kettunen, P. and Thesleff, I. Expression and function of FGFs-4, -8, and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev Dyn 211:256 –268; 1998. 11. Komori, T., Yagi, H., Nomura, S., et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755–764; 1997. 12. Kratochwil, K., Dull, M., Farin˜as, I., Galceran, J., and Grosschedl, R. Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev 10:1382–1394; 1996. 13. Mundlos, S., Otto, F., Mundlos, C., et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89:773–779; 1997. 14. Neubu¨ser, A., Peters, H., Balling, R., and Martin, G. R. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90:247–255; 1997. 15. Nieminen, P., Pekkanen, M., Åberg, T., and Thesleff, I. A graphical WWWdatabase on gene expression in tooth. Eur J Oral Sci 106:7–11; 1998. 16. Otto, F., Thornell, A. P., Cromptom, T., et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentation and bone development. Cell 89:765–771; 1997. 17. Peters, H., Neubu¨ser, A., Kratochwil, K., and Balling, R. Pax9 deficient mice lack pharyngeal pouch derivates and teeth and exhibit craniofacial and limb abnormalities. Genes Dev 12:2735–2747; 1998. 18. Qiu, M., Bulfone, A., Ghattas, I., et al. Role of Dlx-1 and -2 in proximodistal patterning of the branchial arches: mutations alter morphogenesis of proximal skeletal elements derived from the first and second arches. Dev Biol 185:165– 184; 1997.
Bone Vol. 25, No. 1 July 1999:123–125 19. Thesleff, I. and Pispa, J. The teeth as models for studies on the molecular basis of the development and evolution of organs. In: Chuong, C.-M., Ed. Molecular Basis of Epithelial Appendage Morphogenesis. Georgetown, TX: R. G. Landes Company; 1998; 157–179. 20. Thesleff, I. and Jernvall, J. The enamel knot: a putative signaling center regulating tooth development. Cold Spring Harb Symp Quant Biol 62:257–67; 1997. 21. Thesleff, I. and Sharpe, P. Signalling networks regulating dental development. Mech Dev 67:111–123; 1997.
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22. Tucker, A. S., Matthews, K. L., and Sharpe, P. T. Transformation of tooth type induced by inhibition of BMP signaling. Science 282:1136 –1138; 1998. 23. Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S., and Thesleff, I. The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54:39 – 43; 1996. 24. Vainio, S., Karavanova, I., Jowett, A., and Thesleff, I. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75:45–58; 1993.