Immunolocalization of BMP-2/-4, FGF-4, and WNT10b in the Developing Mouse First Lower Molar
INSERM U595, Institut de Biologie Médicale, Faculté de Médecine, Strasbourg, France
Correspondence to: A. Nadiri, INSERM U595, Institut de Biologie Médicale, Faculté de Médecine, 11, rue Humann, 67085 Strasbourg cedex, France. E-mail: Amal.Nadiri{at}odonto-ulp.u-strasbg.fr
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Summary |
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Key Words: tooth development immunohistochemistry epithelialmesenchymal interactions enamel knot BMP FGF WNT
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Introduction |
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BMPs influence apoptosis, cell proliferation, and cell differentiation (von Bubnoff and Cho 2001). Several genes encoding BMPs (Bmp-2, -4, -5, -6, and -7) are expressed during tooth development (Aberg et al. 1997
). BMP-2 and/or BMP-4 have been shown to mimic some of the signaling functions of the dental epithelium on mesenchyme during tooth initiation (Vainio et al. 1993
; Bei and Maas 1998
). Analysis of the expression patterns of different Bmps indicates that Bmp-2 and Bmp-4 show remarkable co-distribution. The corresponding proteins are involved in the mediation of epithelialmesenchymal interactions. They are expressed in the EK and may play a role in the control of tooth morphogenesis (Aberg et al. 1997
; Peterkova et al. 1998
). BMP-2 and -4 have been shown to induce the differentiation of the odontoblast (BègueKirn et al. 1994
) and ameloblast (Coin et al. 1999a
).
The FGF family in mammals is composed of at least 20 members (Ornitz and Itoh 2001). They have important regulatory functions in morphogenesis and organogenesis, as shown for limb, hair, lung, and feather. Several members of the FGF family (FGF-1, -2, -3, -4, -8, -9 and -10) have been implicated in dental morphogenesis (Kettunen and Thesleff 1998
; Kettunen et al. 2000
; Kratochwil et al. 2002
).
Wnt genes encode a large family of secreted cysteine-rich proteins that play key roles in intercellular signaling during development (Parr and McMahon 1994; Christian 2000
). WNTs have essential roles as regulators of cell proliferation, migration, differentiation, and in epithelialmesenchymal interactions during morphogenesis. Wnt3, 4, 5a, 6, 7b, 10a, and 10b are differentially expressed in the molar from the lamina to the early bell stages (Dassule and McMahon 1998
; Sarkar and Sharpe 1999
).
Most data on the expression of these signaling molecules during odontogenesis was obtained by in situ hybridization. They showed temporal and spatial expression patterns consistent with paracrine signaling mechanisms (for review see Thesleff and Sharpe 1997). Because BMPs, FGFs and WNTs are diffusible molecules, the purpose of the present study was to immunolocalize BMP-2, -4, FGF-4, and WNT10b, proteins that play major roles during tooth development. The stages investigated in the first lower molar were from the bud to the late bell, during which odontoblast and ameloblast differentiations are initiated.
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Materials and Methods |
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Western Blotting
First lower molars from mouse embryos at E14, 15, 17, and 19 were homogenized in lysis buffer (1% NP-40, 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 4 mg/ml NaF, 8.8 mg/ml sodium pyrophosphate decahydrate, 1 mM PMSF, 10 µg/ml aprotinin, and 20 µM leupeptin). After centrifugation at 16,000 x g for 10 min at 4C (Kwon et al. 1997), the protein concentration in the supernatant was determined by the Bradford method. Proteins (20 µg of each sample) were separated by SDS-PAGE (13% acrylamide) under reducing conditions and transferred to nitrocellulose for 3 hr at 36 V. Membranes were saturated with 1% BSA in Tris-buffered saline (TBS) pH 7.4, containing 0.1% Tween-20, incubated overnight with antibodies directed against BMP-2, BMP-4, FGF-4, and WNT10b (all from Santa Cruz; dilution 1:250) at 4C. After washing with TBS/Tween-20, the membranes were incubated for 1 hr at RT with a horseradish peroxidase-conjugated donkey anti-goat IgG (Sigma, St Louis, MO; dilution 1:40000), rinsed with TBS/Tween-20, and finally developed using the ECL Western Blotting Analysis System (Amersham Biosciences; Poole, UK).
For antigen quantitation (BMP-2, BMP-4, FGF-4, and WNT10b) during molar development, nitrocellulose membranes were then treated for 30 min at 60C with Tris 62.5 mM, pH 6.8, containing 2% SDS to detach antibodies. After two rinses with TBS, membranes were saturated as previously described and incubated for 2 hr with a primary antibody (Promega, Madison, WI; dilution 1:5000) which specifically recognized two isoforms (p44/ERK1 and p42/ERK2) of the Mitogen Activated Protein Kinase used as a reference (Seger and Krebs 1995). After washing, the membranes were incubated with an anti-rabbit IgGhorseradish peroxidase (Promega; dilution 1:5000) and developed as previously described. All protein bands were scanned and the density of each band was determined using the NIH Image 1.63 software (NIH; Bethesda, MD). Experiments were performed in triplicate. For each stage, the data were normalized against MAPK by calculating the protein:MAPK density ratio.
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Results |
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In the molar, only one protein band of 16 kD was detected with the antibody directed against BMP-2 (Figure 5). Compared to MAPK, which was used as a reference, the amount of BMP-2 was maximal at E14 and then decreased until E17 and remained almost constant at E19 (Table 1). Molars from E13 could not be assessed because of significant peridental mesenchyme contamination.
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FGF-4
At E13, FGF-4 was localized in the epithelium and mesenchyme (Figure 3A)
. From the bud to the bell stage, FGF-4 was associated with the basement membrane (Figures 3A3C) except for the basement membrane underlying cells of the primary EK at E14 and E15 (Figures 3B and 3C). At the cap stage (E14 and E15), the staining for FGF-4 was stronger in the enamel organ than in the mesenchyme (Figures 3B and 3C). At E17, the staining was very strong in the IDE and the SI (Figure 3D). In the mesenchyme, weak staining for FGF-4 was detected at the tips of cusps (Figure 3D). At the late bell stage (E19), the labeling significantly decreased (Figure 3E) except for the SI (Figure 3F).
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WNT10b
At E13, positive immunoreactivity with the anti-WNT10b antibody was observed in both the dental epithelium, mainly at the tip of the bud, and the condensing mesenchyme (Figure 4C)
. At the cap stage (E14E15), the staining in the enamel organ was maintained but showed a buccallingual asymmetry: the staining was more intense on the buccal aspect (Figures 4A, 4D, and 4E). During this period, the cells of the primary EK remained negative (Figures 4D and 4E). From E13 to E15, the staining in the mesenchyme decreased (compare Figures 4C, 4D, and 4E). From E14 to E17, the staining of the cervical loop increased (Figures 4D4F). At E17, the secondary EKs were negative (Figure 4F). At E19, WNT10b was detected in the preameloblasts and in SI cells (Figure 4G). At this stage, WNT10b staining disappeared from IDE cells at the tip of the main cusp except for the basal pole of these cells (Figure 4G).
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Discussion |
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The Bmp-2 gene is intensely expressed in the dental epithelium at the tip of the bud (Aberg et al. 1997). However, the corresponding protein was detected in the epithelium as well as in the mesenchyme, where the staining was more pronounced. This discrepancy between the in situ hybridization and immunostaining patterns is due to the ability of BMP-2 to diffuse. At the bud stage, however, the staining still remained very weak in the developing tooth compared to that observed in the cartilage or the olfactory epithelium. BMP-2 modulates chondrogenic differentiation (Zhang et al. 2002
). BMP-2 is also involved in the morphogenesis of the olfactory system (Fishell 1999
; Peretto et al. 2002
). In the developing tooth, the immunostaining for BMP-2 markedly increased in the mesenchyme at the cap stage.
At the bud stage, Bmp-4 was expressed in the condensing mesenchyme (Aberg et al. 1997). Similarly to BMP-2, BMP-4 was immunolocalized both in the epithelium and mesenchyme at the bud stage. According to Hall and Miyake (1995)
, BMP-4 might regulate mesenchymal condensation. At the cap stage, the Bmp-4 gene was strongly expressed in the dental pulp and in the distal part of the primary EK (Jernvall et al. 1998
; Keranen et al. 1998
). The corresponding protein was initially present in the dental epithelium and mesenchyme. BMP-4 from the mesenchyme may also regulate epithelial morphogenesis at the cap stage (Jernvall et al. 1998
). At E15, the staining for BMP-4 decreased in the mesenchyme but persisted in the enamel organ. Strong staining for BMP-4 was observed in the inner part of the primary EK, where apoptosis is intense (Viriot et al. 1997
; Lesot et al. 1999
). BMP-2 and -4 are known to play a role in the regulation of apoptosis during development (Jernvall et al. 1998
; Peterkova et al. 1998
; Guha et al. 2002
). The relative amounts of BMP-4 strongly increased from E14 to E15, which is in agreement with the immunohistochemical findings and with the simultaneous increase in apoptosis within the primary EK. Furthermore, the differential effects of BMPs in the epithelium (apoptosis) and mesenchyme (condensation) suggest that different signaling pathways are stimulated in each tissue.
At E13, FGF-4 was detected in the epithelium and mesenchyme and strong staining was observed at the epithelialmesenchymal junction. FGF-4 has a high affinity for heparan sulfate (Aviezer et al. 1999). Heparan sulfate, a component of the dental basement membrane, can modulate FGF-4 receptor recognition and regulate FGF-4 signaling activity (Aviezer et al. 1999
; Allen et al. 2001
). A specific CD44 splice variant binds both FGF-4 and FGF-8 and is involved in limb outgrowth (Sherman et al. 1998
). Several heparan sulfate proteoglycans have been detected in the tooth, including syndecan isoforms (Bai et al. 1994
) and the membrane-associated CD44 (Yu and Toole 1997
). At E14, Fgf-4 was expressed only in the primary EK (Kettunen and Thesleff 1998
). As a result of diffusion, the localization of the protein was again different from that of the corresponding transcripts observed by in situ hybridization. FGF-4 was present mainly in the dental epithelium and the staining was more intense in the ODE than in the IDE. FGF-4 stimulates cell proliferation in both the epithelium and mesenchyme and has been suggested to inhibit apoptosis in the dental mesenchyme (Vaahtokari et al. 1996
). Only very limited apoptosis can be detected in the mesenchyme at E14 and E15 (Viriot et al. 1997
). The relative amount of FGF-4 decreased from E14 to E15. During this period the mitotic index remained unchanged in the IDE-SI while it decreased in the mesenchyme from the central part of the tooth (Lesot et al. 1999
). After E15, the weak staining for FGF-4 in the mesenchyme progressively disappeared when tooth development progressed.
At the bud stage, Wnt10b is expressed at the tip of the epithelial bud (Dassule and McMahon 1998), while WNT10b was detected in the epithelium and in the condensing mesenchyme. At the cap stage, Wnt10b was expressed in the primary EK (Dassule and McMahon 1998
) and throughout the IDE (Sarkar and Sharpe 1999
). The corresponding protein at E14 was present in the dental and peridental mesenchyme and also in the enamel organ except for the primary EK. The relative amount of WNT10b decreased from E14 to E15. At both the bud and cap stages, the locations of Wnt10b transcripts were different from that of the corresponding protein. Depending on the context, Wnts can act either as local inducers (Zecca et al. 1996
) or as long-range morphogens to control tissue organization and growth (Zecca et al. 1996
; Neumann and Cohen 1997
). In the developing tooth, WNT10b showed significant diffusion and would therefore act as a long-range morphogen. Heparan sulfate has been proposed to be involved in the extracellular distribution of WNT (Baeg et al. 2001
).
Cap to Bell Stage
During this transition from E15 to E17, the cervical loop elongates to surround the dental mesenchyme. The segregation of the non-dividing or slowly dividing IDE cells of the EK leads to the formation of the secondary EKs (Coin et al. 1999b; Lisi et al. 2003
). The epithelialmesenchymal junction changes its shape when cusps develop.
At E14, the expression of Bmp-2 became restricted to the primary EK (Keranen et al. 1998). Bmp-2 has been suggested to play a role in the determination of the secondary EKs (Aberg et al. 1997
). Indeed, BMP-2 was mainly detected in the dental mesenchyme at E14. From E15 to E17, the staining for BMP-2 did not show any major change and remained positive in the mesenchyme only. The cap stage mesenchyme determines tooth morphogenesis (Kollar and Baird 1969
), probably by controlling the pattern of segregation of the BrdU-negative cells from the primary EK (Schmitt et al. 1999
). At E15-E17, Bmp-4 was strongly expressed in the pulp, including the preodontoblasts, when the cusps formed (Aberg et al. 1997
). From E15 to E17, the corresponding protein showed an increased staining in the mesenchyme. The analysis of E13.5 molars cultured in the presence of antisense oligodeoxynucleotides suggested that BMP-4 was necessary for cusp formation at the late bud and cap stage (Tabata et al. 2002
).
At E15, Fgf-4 was expressed only in the primary EK and later in the secondary EKs (Kettunen and Thesleff 1998; Kettunen et al. 1998
). From E15 to E17, the immunostaining for FGF-4 strongly increased in the enamel organ. Conversely, there was a decrease in the staining of the mesenchyme except for the cusp tips. This localization was in accord with the possible role of FGF-4 in tooth growth and cusp formation (Jernvall and Thesleff 2000
). During the cap to bell stage transition, the size of the molar significantly increased. The increase in the surface covered by the IDE resulted from both a histological reorganization of the IDE controlled by Shh (Lesot et al. 2002
) and cell proliferation in the IDE, which could be stimulated by FGF-4.
The staining for WNT10b at E15 was strong in the cervical loop and negative in the primary EK, where the mitotic activity is very low. At E17, WNT10b was present in the mesenchyme and the enamel organ. From E15 to E17, the staining for WNT10b became negative in the ODE. The staining in the enamel organ decreased from E15 to E17 except for the cervical loop area. This staining pattern was maintained at E19, supporting the potential role for WNT10b in the growth of the cervical loop.
Late Bell Stage and Cytodifferentiation
At E18, the first odontoblasts differentiate at the main cusp tips and their differentiation progresses towards the basal region of each cusp with a specific timespace pattern, different from the pattern of cusp formation (Lisi et al. 2003). The terminal differentiation of ameloblasts follows 24 hr later.
Bmp-2 is expressed by odontoblasts and preameloblasts when predentin secretion has started (BègueKirn et al. 1994; Aberg et al. 1997
). Similarly, Bmp-4 is expressed by odontoblasts (BègueKirn et al. 1994
) and in the IDE, where it appears to persist longer than Bmp-2 (Aberg et al. 1997
). Using in vitro functional approaches, it has been shown that TGFß1 and -3, BMP-2, and BMP-4 can induce odontoblast differentiation (Bègue-Kirn et al. 1992
; Ruch 1998
; Unda et al. 2000
). The relative amount of BMP-2 and, to a lesser extent, BMP-4 increased from E17 to E19. Immunostaining showed that TGFß1 accumulated at the epithelialmesenchymal junction where odontoblasts differentiated (Cam et al. 1997
; Kikuchi et al. 2001
). However, immunostaining for BMP-2 and -4 did not suggest any storage of these proteins at the epithelialmesenchymal junction in regions where odontoblasts had started to differentiate. This is in agreement with the fact that TGFßs could be extracted from dentin fractions, whereas there is no evidence for the presence of BMP-2 and -4 in dentin (Smith and Lesot 2001
). However, BMP-2 and BMP-4 were detected at the basal pole of preameloblasts at the tip of the main cusp at E19, just before they differentiated. BMP-2 has been shown to induce ameloblast differentiation in vitro (Coin et al. 1999a
). The molecule may therefore be involved in ameloblast differentiation in vivo.
From E17 to E19, the relative amount of FGF-4 decreased by 31% and the immunostaining for FGF-4 was very weak in all dental tissues at E19. The decrease in FGF-4 at E19 might therefore correspond to the withdrawal from the cell cycle for more and more odontoblasts and for the first ameloblasts.
At the late bell stage, there was weak staining for WNT10b in the mesenchyme except for the tip of the main cusp, which was negative. WNT10b was mainly detected in the IDE. There was an intense staining of preameloblasts and in the cervical loop area. The staining decreased towards the tip of the main cusp. Both FGF-4 and WNT10b appear to be involved in cell proliferation. However, the staining for WNT10b persisted for a longer time in the enamel organ. It can be expected that the target cells for FGF-4 and WNT10b are different. To test this hypothesis and extend this work, study of the receptors is in progress. Functional experiments will be necessary to analyze the signaling effects of these molecules after the cap stage, for which little information is available.
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Acknowledgments |
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We wish to thank Prof A.J. Smith for critical reading of the manuscript.
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aberg T, Wozney J, Thesleff I (1997) Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation. Dev Dyn 210:383396[Medline]
Aviezer D, Safran M, Yayon A (1999) Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2. Biochem Biophys Res Commun 263:621626[Medline]
Allen BL, Fill MS, Rapraeger AC (2001) Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition. J Cell Biol 155:845858
Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N (2001) Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128:8794
Bai XM, Van der Schueren B, Cassiman JJ, Van den Berghe H, David G (1994) Differential expression of multiple cell-surface heparan sulfate proteoglycans during embryonic tooth development. J Histochem Cytochem 42:10431054
BègueKirn C, Smith AJ, Loriot M, Kupferle C, Ruch JV, Lesot H (1994) Comparative analysis of TGF betas, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation. Int J Dev Biol 38:405420[Medline]
BègueKirn C, Smith AJ, Ruch JV, Wozney JM, Purchio A, Hartmann D, Lesot H (1992) Effects of dentin proteins, transforming growth factor beta1 (TGF beta1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int J Dev Biol 36:491503[Medline]
Bei M, Maas R (1998) FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125:43254333
Cam Y, Lesot H, Colosetti P, Ruch JV (1997) Distribution of transforming growth factor beta1-binding proteins and low-affinity receptors during odontoblast differentiation in the mouse. Arch Oral Biol 42:385391[Medline]
Christian JL (2000) BMP, Wnt and Hedgehog signals: how far can they go? Curr Opin Cell Biol 12:244249[Medline]
Coin R, Haikel Y, Ruch JV (1999a) Effects of apatite, transforming growth factor beta-1, bone morphogenetic protein-2 and interleukin-7 on ameloblast differentiation in vitro. Eur J Oral Sci 107:487495[Medline]
Coin R, Lesot H, Vonesch JL, Haikel Y, Ruch JV (1999b) Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area. Int J Dev Biol 43:261267[Medline]
Dassule HR, McMahon AP (1998) Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the mammalian tooth. Dev Biol 202:215227[Medline]
Fishell G (1999) BMPs: time to murder and create? Nature Neurosci 2:301303[Medline]
Guha U, Gomes WA, Kobayashi T, Pestell RG, Kessler JA (2002) In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev Biol 249:108120[Medline]
Hall BK, Miyake T (1995) Divide, accumulate, differentiate: cell condensation in skeletal development revisited. Int J Dev Biol 39:881893[Medline]
Jernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I (1998) The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125:161169
Jernvall J, Thesleff I (2000) Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech Dev 92:1929[Medline]
Keranen SV, Aberg T, Kettunen P, Thesleff I, Jernvall J (1998) Association of developmental regulatory genes with the development of different molar tooth shapes in two species of rodents. Dev Genes Evol 208:477486[Medline]
Kettunen P, Karavanova I, Thesleff I (1998) 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 22:374385[Medline]
Kettunen P, Laurikkala J, Itaranta P, Vainio S, Itoh N, Thesleff I (2000) Associations of FGF-3 and FGF-10 with signalling networks regulating tooth morphogenesis. Dev Dyn 219:322332[Medline]
Kettunen P, Thesleff I (1998) Expression and function of FGFs-4, -8 and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev Dyn 211:256268[Medline]
Kikuchi H, Amano H, Yamada S (2001) Putative role of basement membrane for dentinogenesis in the mesenchyme of murine dental papillae in vitro. Cell Tissue Res 303:93107[Medline]
Kollar EJ, Baird GR (1969) The influence of the dental papilla on the development of tooth shape in embryonic mouse tooth germs. J Embryol Exp Morphol 21:131148[Medline]
Kratochwil K, Galceran J, Tontsch S, Roth W, Grosschedl R (2002) FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(-/-) mice. Genes Dev 16:31733185
Kwon YK, Bhattacharyya A, Alberta JA, Giannobile WV, Cheon K, Stiles CD, Pomeroy SL (1997) Activation of ErbB2 during wallerian degeneration of sciatic nerve. J Neurosci 17:82938299
Lesot H, KiefferCombeau S, Fausser JL, Meyer JM, Perrin-Schmitt F, Peterkova R, Peterka M, et al. (2002) Cell-cell and cell-matrix interactions during initial enamel organ histomorphogenesis in the mouse. Connect Tissue Res 43:191200[Medline]
Lesot H, Peterkova R, Schmitt R, Meyer JM, Viriot L, Vonesch JL, Senger B (1999) Initial features of the inner dental epithelium histo-morphogenesis in the first lower molar in mouse. Int J Dev Biol 43:245254[Medline]
Lisi S, Peterkova R, Peterka M, Vonesch JL, Ruch JV, Lesot H (2003) Tooth morphogenesis and pattern of odontoblast differentiation. Connect Tissue Res 44:167170
Maas R, Bei M (1997) The genetic control of early tooth development. Crit Rev Oral Biol Med 8:439[Abstract]
Neumann CJ, Cohen SM (1997) Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124:871880
Ornitz DM, Itoh N (2001) Fibroblast growth factors. Genome Biol 2:112
Parr BA, McMahon AP (1994) Wnt genes and vertebrate development. Curr Opin Genet Dev 4:523528[Medline]
Peretto P, Cummings D, Modena C, Behrens M, Venkatraman G, Fasolo A, Margolis FL (2002) BMP mRNA and protein expression in the developing mouse olfactory system. J Comp Neurol 451:267278[Medline]
Peterkova R, Peterka M, Vonesch JL, Tureckova J, Viriot L, Ruch JV, Lesot H (1998) Correlation between apoptosis distribution and BMP-2 and BMP-4 expression in vestigial tooth primordia in mice. Eur J Oral Sci 106:667670[Medline]
Peters H, Balling R (1999) Teeth. Where and how to make them. Trends Genet 15:5965[Medline]
Ruch JV (1998) Odontoblast commitment and differentiation. Biochem Cell Biol 76:923938[Medline]
Sarkar L, Sharpe PT (1999) Expression of Wnt signalling pathway genes during tooth development. Mech Dev 85:197220[Medline]
Schmitt R, Lesot H, Vonesch JL, Ruch JV (1999) Mouse odontogenesis in vitro: the cap-stage mesenchyme controls individual molar crown morphogenesis. Int J Dev Biol 43:255260[Medline]
Seger R, Krebs EG (1995) The MAPK signaling cascade. FASEB J 9:726735
Sherman L, Wainwright D, Ponta H, Herrlich P (1998) A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth. Genes Dev 12:10581071
Smith AJ, Lesot H (2001) Induction and regulation of crown dentinogenesis: embryonic events as a template for dental tissue repair? Crit Rev Oral Biol Med 12:425437[Abstract]
Tabata MJ, Fujii T, Liu JG, Ohmori T, Abe M, Wakisaka S, Iwamoto M, et al. (2002) Bone morphogenetic protein 4 is involved in cusp formation in molar tooth germ of mice. Eur J Oral Sci 110:114120[Medline]
Thesleff I, Mikkola M (2002) The role of growth factors in tooth development. Int Rev Cytol 217:93135[Medline]
Thesleff I, Sharpe PT (1997) Signalling networks regulating dental development. Mech Dev 67:111123[Medline]
Tucker AS, Sharpe PT (1999) Molecular genetics of tooth morphogenesis and patterning: the right shape in the right place. J Dent Res 78:826834[Abstract]
Unda FJ, Martin A, Hilario E, BègueKirn C, Ruch JV, Arechaga J (2000) Dissection of the odontoblast differentiation process in vitro by a combination of FGF1, FGF2 and TGFbeta1. Dev Dyn 218:480489[Medline]
Vaahtokari A, Aberg T, Thesleff I (1996) Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development 122:121129
Vainio S, Karavanova I, Jowett A, Thesleff I (1993) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75:4558[Medline]
Viriot L, Peterkova R, Vonesch JL, Peterka M, Ruch JV, Lesot H (1997) Mouse molar morphogenesis revisited by three-dimensional reconstruction. III. Spatial distribution of mitoses and apoptoses up to bell-stages first molar teeth. Int J Dev Biol 41:679690[Medline]
von Bubnoff A, Cho KW (2001) Intracellular BMP signaling regulation in vertebrates: pathway or network? Dev Biol 239:114[Medline]
Yu Q, Toole BP (1997) Common pattern of CD44 isoforms is expressed in morphogenetically active epithelia. Dev Dyn 208:110[Medline]
Zecca M, Basler K, Struhl G (1996) Direct and long-range action of a Wingless morphogen gradient. Cell 87:833844[Medline]
Zhang W, Green C, Stott NS (2002) Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication. J Cell Physiol 193:233243[Medline]