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Published online on November 22, 2006, 10.1073/pnas.0607289103
PNAS | December 5, 2006 | vol. 103 | no. 49 | 18627-18632


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BIOLOGICAL SCIENCE / DEVELOPMENTAL BIOLOGY
Continuous tooth generation in mouse is induced by activated epithelial Wnt/beta-catenin signaling

Elina Järvinen*, Isaac Salazar-Ciudad*, Walter Birchmeier{dagger}, Makoto M. Taketo{ddagger}, Jukka Jernvall*, and Irma Thesleff*,§

*Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, University of Helsinki, FIN-00014, Helsinki, Finland; {dagger}Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13092 Berlin, Germany; and {ddagger}Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 3, 2006 (received for review August 22, 2006)


   Abstract
Top
 Abstract
Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized beta-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of beta-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes.

organ renewal | regeneration | tooth development | activator-inhibitor model

The number of teeth and their replacement patterns show remarkable variation in vertebrates. In nonmammalian vertebrates, such as fish and reptiles, continuous renewal of teeth is common, whereas in most mammals, renewal of teeth has been reduced to the formation of only two dentitions or reduced altogether as in mice having only one dentition, consisting of a single incisor and three molars in each jaw quadrant. In large part because of the lack of replacement teeth in mouse, the molecular mechanisms guiding tooth renewal have been difficult to identify. Vestigial or rudimentary tooth buds have been reported in the diastema region of mouse and other rodents (1, 2), and extra teeth appear to develop from these rudimentary buds in several transgenic or mutant mice (3–7). Despite the appearance, or reappearance, of teeth in diastema, tooth renewal does not appear to be affected in any of the reported cases. One possibility for the lack of tooth renewal in mouse experiments could be the relative antiquity of reduction in mammalian tooth replacement. By the Cenozoic, 65 million years ago, most mammalian lineages were diphyodont, replacing teeth only once, or monophyodont, having no replacement teeth. The preceeding reduction in tooth renewal during the Mesozoic was associated with increasing complexity of tooth shapes and interlocking occlusion of opposing teeth and cusps (8, 9).

Teeth develop as epithelial appendages from surface ectoderm, and the molecular mechanisms regulating tooth morphogenesis are shared with other ectodermal organs such as hairs, feathers, and scales. Conserved signaling molecules in the Wnt, fibroblast growth factor (FGF), bone morphogenetic protein, and hedgehog families mediate reciprocal interactions between the epithelial and mesenchymal tissues and regulate tooth initiation and morphogenesis (10, 11). There is evidence from animal models and human syndromes that Wnt signaling plays a role in tooth development. In mice the inhibition of canonical Wnt signaling either by deleting Lef1 function or overexpressing the Wnt inhibitor Dkk1 arrests tooth morphogenesis at an early stage (12, 13). The activation of Wnt signaling in oral epithelium induces the formation of tooth-like buds in chick embryos (14). The consequences of increased Wnt signaling have been examined in mice as well, but although a dramatic stimulation of the renewal of hairs was observed as well as formation of hair tumors, no dental phenotypes were reported (15–17). However, in humans Wnt signaling has been associated with tooth renewal. Supernumerary teeth together with odontomas (tumor-like malformations consisting of multiple small teeth) and impacted teeth occur as extracolonic symptoms in 10–20% of patients with familial adenomatosis coli, resulting from inactivating mutations of adenomatous polyposis coli, a negative regulator of Wnt signaling (18, 19). On the other hand, inactivating familial mutations in another Wnt inhibitor, Axin2, causes decreased tooth number because of the lack of tooth renewal (20). These partly contradictory observations based on two different Wnt inhibitors implicate a delicate role for Wnt signaling in tooth development, but also leave its exact role in tooth renewal unknown. To resolve the role of Wnt signaling in tooth renewal, we stimulated directly the intracellular pathway of Wnt signaling by targeting the expression of a stabilized form of beta-catenin to embryonic surface epithelium. beta-catenin is an intracellular mediator of canonical Wnt signaling, which is phosphorylated and stabilized as a result of activation of Wnt receptor and transported to the nucleus where it, as part of a transcriptional complex, regulates gene expression (21). Hence, the introduction of stabilized beta-catenin causes sustained Wnt signaling.


   Results and Discussion
Top
 Abstract
 Results and Discussion
Materials and Methods
 Acknowledgements
 References
 
Mice with a conditional beta-catenin allele (22) were crossed with K14Cre mice (16) and the resulting K14cre/+;beta-catenin{Delta}ex3fl/+ mice, hereafter called beta-cat{Delta}ex3K14/+, thus expressed one WT allele of beta-catenin and one mutated allele in oral and dental epithelium. Activity of the Cre recombinase was confirmed by crossing the K14cre mice with LacZ reporter mice. Cre recombinase was weakly active in the mandibular ectoderm already before dental placode formation in embryonic day (E) 11-E12 embryos and at E12–15 Cre activity was very strong [supporting information (SI) Fig. 6].

The mutant mice died perinatally, and we therefore focused first on the embryonic stages of tooth development. The first signs of abnormal tooth morphogenesis were detected in histological sections of E13 molar tooth buds. The beta-cat{Delta}ex3K14/+ buds were normal in size but had irregular contours (Fig. 1a). At E14 regular morphogenesis into cap stage was not detected. Instead the bud had become more irregular and small epithelial buds had formed at the surface (Fig. 1b). The extra buds grew larger and at E17 when the WT teeth had reached the bell stage of crown development, numerous long epithelial structures protruded into the mesenchyme in the region of molar development (Fig. 1 c and d). The mandibular and maxillary teeth showed similar phenotypes (Fig. 1, SI Fig. 7, and data not shown).


Figure 1
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  		Fig. 1. The development of molars is disturbed in beta-cat{Delta}ex3K14/+ embryos. (a and b) E13 (a) and E14 (b) frontal histological sections of the first molars show that mutant tooth buds have irregular shapes without clear morphogenesis into the cap stage. (c) At E16 the WT tooth is at the bell stage and the mutant shows multiple irregular and long ectopic buds protruding into the mesenchyme (arrows). White lines in a–c mark the border between dental epithelium and mesenchyme. (d) Keratin14 antibody staining of sagittal sections at E17 shows the epithelial morphology of the first and second molars in WT embryos and the malformed epithelium in the mutant with extensive epithelial budding. M1, first molar; M2, second molar. (e and f) Detection of Wnt activity in E14 mandibles by BAT-gal expression. Wnt activity is seen in molar epithelium and tongue papillae in both WT and beta-cat{Delta}ex3K14/+ mutant embryos. In the mutant, Wnt activity is detected additionally in numerous ectopic buds along the oral epithelia. Dotted line in e indicates the level of vibratome section. T, tongue. The vibratome sections (f) show that Wnt activity in mutant molar epithelium is localized to multiple separate spots. (Scale bars: 200 µm.)

As the first step to resolve the mechanism whereby activated Wnt signaling caused abnormal morphogenesis we determined the localization of Wnt signal activity by crossing the mutant mice with BAT-gal reporter mice, expressing the LacZ gene under the control of beta-catenin/Tcf responsive elements (23). In E14 WT mandibles only weak activity was observed in the molar bud epithelium although intense Wnt reporter activity was present, e.g., in tongue papillae. In the beta-cat{Delta}ex3K14/+ mandibles the reporter activity was more intense and was localized to discrete spots in the dental and oral epithelium (Fig. 1 e and f). Because high Wnt signaling activity in the dental epithelium has been localized to enamel knots (24, 25), important signaling centers regulating tooth morphogenesis (10), we next analyzed the expression of some known enamel knot marker genes. In WT embryos the first, primary enamel knot appears at the tip of the epithelial bud (Fig. 2a) and mediates the transition to cap stage, and at the bell stage secondary enamel knots determine the positions of the future cusps (26). In the mandibular and maxillary tooth buds of E13 and E14 beta-cat{Delta}ex3K14/+ embryos the enamel knot marker genes including Shh, Fgf4, Edar, Wnt10b (11), and Epiprofin (27) all were expressed in a similar pattern in dental epithelium as BAT-gal activity (Fig. 2 b and c, SI Fig. 7, and data not shown), indicating that the spots were supernumerary enamel knots. The number of supernumerary enamel knots increased markedly with advancing development in the mutant tooth germs; however, the intensity of the expression of most enamel knot genes appeared quite normal. Ectodin, a bone morphogenetic protein antagonist with Wnt modulating activity (5, 28, 29), showed a similar inverse expression pattern with the enamel knot markers as in the WT teeth (Fig. 2d). In addition, Fgf3, which is a specific marker for dental papilla identity (30), was strongly expressed below the supernumerary enamel knots in the mesenchyme (Fig. 2e) but it was not detected around the numerous buds outside the tooth region nor in the buds of the tongue. This finding implies that the odontogenic region had retained the competence to form teeth.


Figure 2
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  		Fig. 2. Supernumerary enamel knots form in beta-cat{Delta}ex3K14/+embryonic molars. (a and b) In frontal sections of WT E14 molar (a), the enamel knot is defined by Shh expression, which then spreads to the secondary enamel knots and, at E17 (b), throughout the inner enamel epithelium. In beta-cat{Delta}ex3K14/+mutants Shh is expressed in multiple patches in the tooth epithelium and the surrounding oral epithelium. (c) Epiprofin, another enamel knot marker, is expressed in spots in the dental epithelium but not in other oral epithelia of the mutant. (d) Ectodin shows inverse expression pattern to the enamel knot markers. (e) Fgf3 is expressed in the mesenchyme in the dental area but not elsewhere in oral mesenchyme. White lines in a–e mark the border between dental epithelium and mesenchyme. (f) 3D reconstructions showing epithelium from the mesenchymal side (Left) and mesenchyme from the epithelial side (Right) together with Shh expression domains. (Upper) In the E16 WT Shh expression marks the typical pattern of secondary enamel knots (arrows) in the first molar and primary enamel knot in the second molar (arrow). M1, first molar; M2, second molar. (Lower) In the E17 mutant multiple small domains expressing Shh are present, often associated with a mesenchymal structure resembling a small dental papilla (arrows). (Scale bars: 200 µm.)

We analyzed the patterning of the supernumerary enamel knots in E17 molars by a 3D reconstruction of Shh expression domains, which revealed an increased number of knots throughout the dental epithelium (Fig. 2f). Furthermore, whereas WT tooth shows secondary enamel knots forming within the first molar epithelium surrounding a large dental papilla, the mutant showed multiple small knots, some of which appeared to colocalize with a small mesenchymal papilla, possibly indicating the formation of an individual tooth (Fig. 2f). The perinatal death of the mutant mice, however, left open the fate of the supernumerary knots.

To test whether teeth actually would form in the mutant mice, we transplanted tooth germs dissected from E14 WT and mutant embryos under the kidney capsules of immunodeficient nude mice. After 3 weeks of kidney capsule culture, one WT molar tooth germ gave rise to three molars with normal shape, size, and mineralized tissues similarly as shown earlier in intraocular grafts (31). In contrast, the transplanted molar of a beta-cat{Delta}ex3K14/+ embryo had formed a large cyst with a uniform ring pattern on the surface (Fig. 3a and SI Fig. 8). When the cyst was opened, these rings on the surface were observed to be ends of the growing roots. Inside, numerous tooth crowns were present, mostly pointing internally and making the structure resemble a geode (Fig. 3 b–e). We counted 42 teeth with well mineralized crowns (Fig. 3f). The largest individual tooth crowns were close to the same height as the first molars of adult WT mice (0.70/0.85 mm) and roughly equal in height to the second WT molars. However, whereas some teeth were large and multicusped, the majority (86%) were unicusped and conical in shape. We confirmed this by using histological sections, which revealed that the more advanced teeth, both unicusped and multicusped, also had normal looking roots indicative of molar tooth identity (Fig. 3 c–e). The paucity of multicusped mutant molars may be explained by the small size of the supernumerary enamel knots (Fig. 2h), as in mouse molar the cusp number correlates with the size of the primary enamel knot (4, 32). When two molar buds were transplanted together under the kidney capsule, >80 teeth were collected after 3 weeks. When the kidney capsule culture was repeated with incisor tooth germs of E14 mutant embryos, multiple incisor-type teeth characterized by sharp and elongated crowns formed. Especially in incisor experiments, often one large incisor and multiple smaller-sized incisors were detected (SI Fig. 9).


Figure 3
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  		Fig. 3. One molar tooth bud of a beta-cat{Delta}ex3K14/+ embryo gives rise to dozens of teeth. When grown as a transplant under the kidney capsule an E14 molar tooth bud developed into a geode-resembling outgrowth containing dozens of individual teeth. (a) The ends of the roots of the teeth are seen as rings on the surface of the outgrowth. (b) Histological sections of the outgrowth show numerous individual teeth. (c and d) The teeth are in various developmental stages, including teeth with initiated root formation (arrows) (c) and crowns that have not yet begun to mineralize (arrows) (d). (e) Although most teeth appear to be simple cones, some show development of multiple cusps (magnification of the region in the black rectangle in d). (f) Forty-two mineralized teeth of various sizes were collected from one geode. (Scale bars: a–e, 200 µm; f, 1 mm.)

It was not possible to conclude from the morphology and histology of the teeth formed in the kidney transplants whether they had formed successionally, or whether the teeth had been initiated by subdivision of the dental epithelium at an early stage. To analyze the dynamics of supernumerary tooth formation we followed the development of dissected E14 and E15 molar tooth buds of beta-cat{Delta}ex3K14/+ mutant embryos and their WT littermates in organ cultures. Daily observation under the dissecting microscope revealed that the mutant tooth bud gave rise to several conical-looking teeth with enamel and dentin matrices, which formed successionally during 11 days of culture. The first forming tooth was usually clearly larger than the successively forming teeth. The WT tooth bud developed to a typical multicusped first molar followed by a smaller and multicusped second molar (Fig. 4a–e). The developmental capacity of the in vitro-formed successional tooth buds of beta-cat{Delta}ex3K14/+ embryos was tested by subculturing two tooth buds dissected from a mutant explant after 6 days of culture. During 19 days of continued culture, five additional teeth developed successionally with a concomitant increase in the tissue mass (Fig. 4g). The histological sections of the explants revealed small tooth buds interspersed between forming teeth (Fig. 4f), indicating, together with the in vitro results, that the supernumerary teeth developed by a continuing renewal process where new teeth bud off from the existing dental epithelium.


Figure 4
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  		Fig. 4. The generation of new teeth from a beta-cat{Delta}ex3K14/+ molar is continuous and occurs in all directions. (a–c) WT E14 tooth germ grown in organ culture gives rise to the first and second molar, whereas the mutant tooth germ develops a circle of tooth germs. (d) After 11 days of culture the crowns and cusps of WT molars can be readily seen under a stereomicroscope. In the mutant 10–15 structures resembling unicusped crowns have developed. M1, fist molar; M2, second molar. (e) Ameloblastin expression indicates differentiated ameloblasts, the enamel forming cells. (f) Histological sections indicate that tooth germs have started to form between the teeth (arrows, magnification of the region in the black rectangle on the left). (g) Two tooth germs were dissected from an explant of an E15 mutant molar cultured for 6 days and subcultured for 19 days. Five new teeth have formed on the sides and between the two original buds with no apparent directionality and the tissue mass have increased markedly. (Scale bars: 200 µm.)

The restricted and high Wnt reporter activity detected in the supernumerary enamel knots in the mutants indicates a direct role for Wnt signaling in the formation of supernumerary enamel knots. However, despite the beta-catenin stabilization throughout the mutant epithelium, only enamel knots and the ectodermal buds showed Wnt reporter activity. This finding suggests that besides Wnt signals additional components are required for the activation of the Wnt pathway and for the formation of enamel knots. Gat et al. (15) showed that stabilized beta-catenin could activate Wnt signaling in hairs only after induction of localized Lef1 expression. Interestingly, these mice survived to adulthood and no tooth phenotype was reported. Compared with our mutants, the unaffected tooth development may be caused by the different strategy of introducing the transgene where strongly expressing founder mouse lines may have been lost by prenatal death. Until now, mice with fully formed supernumerary teeth have been limited to cases with one extra molariform or incisiform tooth adjacent to molars or incisors, respectively. Developmentally, these individual extra teeth result from enlarged enamel knots (5, 4) and, at the level of signaling, at least from changes in the induction and inhibition of enamel knots by the bone morphogenetic protein and FGF pathways (3, 5).

The dynamics of tooth development, including basic enamel knot induction and inhibition, has been reproduced by computer model simulations (33). Based on the dynamics of activator–inhibitor systems, an increase in the production of activator and a consequent induction of enamel knots might, in principle, be expected to produce supernumerary teeth. The extensive modification of tooth development in beta-cat{Delta}ex3K14/+ mice, however, raises questions about whether a single developmental factor, in this case beta-catenin stabilization, could explain directly all of the observed changes in patterning or whether multiple, indirect effects are likely to be involved. To test the underlying principle of beta-catenin function using computer simulations, we used the published computer model (33) to simulate WT and beta-cat{Delta}ex3K14/+ molar development. The mathematical model is a morphodynamic derivative of activator–inhibitor models where, in addition to affecting the production of each other, the activator and inhibitor molecules also affect cell division and differentiation (34). Activator–inhibitor models have been previously used successfully to simulated patterns of multiple organs, including teeth and feathers (34), but they have not yet been used in connection to extensively altered mutant morphologies. Because all of the epithelial cells of the beta-cat{Delta}ex3K14/+ mice have stabilized beta-catenin we increased in the model the intrinsic production rate of activator (parameter k3), most closely matching the hypothetical effect of beta-catenin stabilization in vivo.

Whereas continuing increase in activator levels will ultimately result in uniform differentiation, the simulations show that at intermediate k3 levels activator-induced inhibitor is able to prevent knot formation laterally and an iterative knot formation dynamics is obtained (Fig. 5). Furthermore, the results show that initially large enamel knots are followed by numerous small knots (Fig. 5), which suggests that the observed beta-cat{Delta}ex3K14/+ tooth formation patterns, where initially few larger teeth are followed by multiple smaller teeth, could be a direct result of elevated levels of intrinsic enamel knot activator production.


Figure 5
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  		Fig. 5. Computer simulations of WT (Left) and beta-cat{Delta}ex3K14/+ (Right) molars. The three different time points (ages given based on morphological similarity to real teeth) of the simulations show epithelial shape and positions of the enamel knots (red). Note in the beta-cat{Delta}ex3K14/+ simulations the fast rate of knot formation and appearance of progressively smaller knots in the border of growing tissue. Because of the limits of cell number and lack of details of physical properties of tissue in the model, the simulation cannot resolve whether individual knots in beta-cat{Delta}ex3K14/+ simulation would form separate teeth later in development. The parameter values for the illustrated WT mouse simulation are k1 = 1.5; k2 = 111; k3 = 0.001; DA = 0.3; DI = 0.4; Re = 0.0005722; Rm = 0.000465; Ba = 0.0004508; Bp = 0.000756; Bb = 0.000240; Bl = 0.0009932 and the same for the beta-cat{Delta}ex3K14/+ simulation except for k3 = 8.6. The iteration values for the three different time points are 2,000, 4,000, and 7,000 for both the WT and beta-cat{Delta}ex3K14/+ simulations.

Additionally, agreeing with the computer simulations, we did not detect clear directionality in the formation of the successional teeth. In mammals, the dentition is replaced by budding of new teeth from the lingual aspect of the previous, primary, or milk teeth. On the other hand, molars develop successionally to the back of the developing tooth row and are initiated from the posterior end of the last molar (8). Both of these modes are more open-ended in most other vertebrates where dentition is replaced continuously and successional teeth are added posteriorly as the jaws grow larger (8). Our experimental and simulation results indicate that shared molecular mechanisms, in particular Wnt signaling, could, in principle, be involved in these different modes of tooth renewal. Furthermore, the possibility that the two modes of successional tooth formation, i.e., tooth replacement and addition along the tooth row, involve a similar molecular mechanism is supported by the phenotype of the human syndrome cleidocranial dysplasia (CCD) characterized by multiple supernumerary teeth, which form both as a partial third dentition and as additional posterior molars (35). CCD is caused by mutations in the Runx2 gene (35, 36), and it remains to be tested whether activated Wnt signaling may underlie tooth renewal in this human syndrome.

The composition of the geode-like outgrowths that developed from the transplanted mutant tooth buds was reminiscent of that in human compound odontomas, where multiple small teeth, sometimes >100 teeth, are found within the jaws (37). Because in some cases odontomas are caused by activated Wnt signaling due to loss-of-function mutations in the Wnt signal modulator adenomatous polyposis coli (18, 19) it is possible that supernumerary teeth are formed by the same mechanism in odontomas and our mutants. Furthermore, the odontomas are considered to be congenital anomalies rather than tumors, and it is possible that the teeth in odontomas also form successionally as we showed in our mutants, thus the mechanism of their pathogenesis involves unlocking the potential of tooth renewal.

In conclusion, we have shown that the stabilization of beta-catenin in the dental epithelium leads to continuous tooth generation and that the mechanism involves the iterative formation of ectopic enamel knot signaling centers where enamel knot activation and lateral inhibition is the underlying key mechanism. Intriguingly, a trend in mammalian evolution has been a reduction of number and renewal of teeth, concomitant with the evolution of progressively more complex, multicusped teeth (8, 9). It remains to be tested whether Wnt signaling may have played a role in the loss of tooth renewal and increase in crown complexity during evolution. Taken together, our results may have implications for organ regeneration and bioengineering of teeth and the understanding of the genetic basis of the evolution of teeth.


   Materials and Methods
Top
 Abstract
 Results and Discussion
 Materials and Methods
Acknowledgements
 References
 
Animals and Preparation of Embryonic Tissues. K14cre transgenic mice (16) and beta-catenin-flox-ex3 mice (22) were crossed into NMRI background. The K14cre/+;beta-cat{Delta}ex3fl/+ mice were generated by crossing K14cre and beta-cat-flox-ex3 mice. The BAT-gal reporter mice were kindly provided by Stefano Piccolo, University of Padova, Padova, Italy. Immunodeficient HsdCpb:NMRI-Foxn1nu (Nude) mice were from Harlan (Horst, The Netherlands), and B6;129S-Gt(ROSA)26Sor/J (Rosa 26) mice were from The Jackson Laboratory (Bar Harbor, ME). Embryos were staged according to morphological criteria, plug day was 0. Developing teeth were dissected from beta-cat{Delta}ex3K14/+ and WT littermate embryos. Teeth were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Serial sections were taken in frontal and sagittal plane at 7 µm and processed for in situ hybridization and immunohistochemistry or stained in hematoxylin-eosin for histological analysis. Serial frontal sections were taken at 10 µm for 3D analysis. Jaws were dissected and dehydrated in methanol and stored in –20°C for whole-mount in situ hybridization.

Cre Recombinase and Wnt Activity Detection. For the detection of Cre recombinase activity K14cre mice were crossed with Rosa26 mice, and for the detection of Wnt activity K14Cre mice were first crossed with BAT-gal reporter mice (23) and then with beta-cat-flox-ex3mice to achieve beta-cat{Delta}ex3K14/+;BAT-gal embryos. Embryonic tissues were dissected and fixed in 2% paraformaldehyde in 0.2% glutaraldehyde in PBS for 30 min at 4°C. Explants were incubated in X-gal staining solution [1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, and 0.02% Nonidet P-40] overnight at room temperature.

In Situ Hybridization and Immunohistochemistry. Radioactive in situ hybridization for paraffin sections was performed as described (38). Probes were labeled with 35S-UTP (Amersham, Buckinghamshire, UK); exposure time was 14 days. The probes used were Ameloblastin, Ectodin, Edar, Epiprofin, Dspp, Fgf3, Fgf4, p21, Runx2, Lef1, and Shh. Deparaffinized sections were immunostained with the Vectastain Elite ABC Kit (Vector, Burlingame, CA). Anti-Keratin14 antibody (Dako, Glostrup, Denmark) was used with the appropriate secondary antibody (Vector) and blocked with goat serum in 1% BSA.

3D Reconstruction. In situ hybridization analysis of Shh was performed on frontal serial sections of E17 beta-cat{Delta}ex3K14/+ and WT embryos. Pictures were taken at x4 magnification with an AX70 microscope Olympus (Melville, NY) and reconstructed for 3D by the NIH image and NIH 3D programs (Apple, Cupertino, CA) as described (5, 39). Because of the complex morphology of the beta-cat{Delta}ex3K14/+ teeth, the whole dental epithelium was rendered, and projections were prepared separately from both the direction of the epithelium and the mesenchyme.

Tissue Culture. E14 and E15 beta-cat{Delta}ex3K14/+ and WT littermate molars were dissected in Dulbecco's PBS (pH 7.4) under a stereomicroscope. Tooth explants were cultured on Nuclepore filters at 37°C in 5% CO2 in a Trowell type organ culture containing DMEM supplemented with 10% FCS (PAA Laboratories, Pasching, Austria). After 2 days the medium was changed to one containing 50% of F12 medium, and ascorbic acid was added to a final concentration of 0.075 g/liter. Culture medium was changed every 2 days. After culture the explants were treated with 100% methanol for 5 min, fixed in 4% paraformaldehyde, and processed for in situ hybridization analysis.

Kidney Capsule Transplantation. E14 beta-cat{Delta}ex3K14/+ molars and incisors were dissected in PBS (pH 7.4) and transplanted by a glass pipette under the kidney capsule of anesthetized nude mice. The cut was sutured. After 3 weeks the mouse was killed, and the kidney was carefully removed.

Computer Simulations. We used the morphodynamic model on tooth development (33), increased the intrinsic production rate of activator (k3), and ran the simulation with the same number of iterations as the WT mouse simulation. We examined the simulated teeth every 1,000 iterations up to 7,000 iterations and increased the k3 until the simulated patterns produced iterative formation of multiple knots.


   Acknowledgements
Top
 Abstract
 Results and Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Stefano Piccolo for the BAT-gal mice; Natalia Soshnikova (Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany) for the first samples of beta-cat{Delta}ex3K14/+ mice; and Riikka Santalahti, Merja Mäkinen, and Heidi Kettunen for excellent technical help. This work was supported by the Academy of Finland (I.T. and J.J.) and the Sigrid Juselius Foundation (I.T.).


   Footnotes
 

Abbreviations: En, embryonic day n.

§To whom correspondence should be addressed. E-mail: irma.thesleff{at}helsinki.fi

Author contributions: E.J., J.J., and I.T. designed research; E.J. and I.S.-C. performed research; W.B. and M.M.T. contributed new reagents/analytic tools; E.J., J.J., and I.T. analyzed data; and E.J., J.J., and I.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0607289103/DC1.

© 2006 by The National Academy of Sciences of the USA


   References
Top
 Abstract
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Peterkova, R, Peterka, M, Viriot, L & Lesot, H. (2002) Connect Tissue Res 43, 120–128.[ISI][Medline]
  2. Keranen, SV, Kettunen, P, Aberg, T, Thesleff, I & Jernvall, J. (1999) Dev Genes Evol 209, 495–506.[CrossRef][ISI][Medline]
  3. Klein, OD, Minowada, G, Peterkova, R, Kangas, A, Yu, BD, Lesot, H, Peterka, M, Jernvall, J & Martin, GR. (2006) Dev Cell 11, 181–190.[CrossRef][ISI][Medline]
  4. Kangas, A, Evans, AR, Thesleff, I & Jernvall, J. (2004) Nature 432, 211–214.[CrossRef][Medline]
  5. Kassai, Y, Munne, P, Hotta, Y, Penttila, E, Kavanagh, K, Ohbayashi, N, Takada, S, Thesleff, I, Jernvall, J & Itoh, N. (2005) Science 309, 2067–2070.[Abstract/Free Full Text]
  6. Tucker, AS, Headon, DJ, Courtney, JM, Overbeek, P & Sharpe, PT. (2004) Dev Biol 268, 185–194.[CrossRef][ISI][Medline]
  7. Mustonen, T, Pispa, J, Mikkola, ML, Pummila, M, Kangas, AT, Pakkasjarvi, L, Jaatinen, R & Thesleff, I. (2003) Dev Biol 259, 123–136.[CrossRef][ISI][Medline]
  8. Osborn, JW. (1973) Am Scientist 61, 548–559.[ISI][Medline]
  9. Kielan-Jaworowska, Z, Cifelli, RL & Luo, ZX. (2004) Mammals from the Age of Dinosaurs: Origins, Evolution, and Structure (Columbia Univ Press, New York,).
  10. Jernvall, J & Thesleff, I. (2000) Mech Dev 92, 19–29.[CrossRef][ISI][Medline]
  11. Wang, X-P & Thesleff, I. (2005) in Cell Signaling and Growth Factors in Development eds. Unsicker, K & Krieglstein, K. (Wiley, Weinheim, Germany,) pp. 719–754.
  12. van Genderen, C, Okamura, RM, Farinas, I, Quo, RG, Parslow, TG, Bruhn, L & Grosschedl, R. (1994) Genes Dev 8, 2691–2703.[Abstract/Free Full Text]
  13. Andl, T, Reddy, ST, Gaddapara, T & Millar, SE. (2002) Dev Cell 2, 643–653.[CrossRef][ISI][Medline]
  14. Harris, MP, Hasso, SM, Ferguso, MWJ & Fallon, J-F. (2006) Curr Biol 16, 371–377.[CrossRef][ISI][Medline]
  15. Gat, U, DasGupta, R, Degenstein, L & Fuchs, E. (1998) Cell 95, 605–614.[CrossRef][ISI][Medline]
  16. Huelsken, J, Vogel, R, Erdmann, B, Cotsarelis, G & Birchmeier, W. (2001) Cell 105, 533–545.[CrossRef][ISI][Medline]
  17. Lo Celso, CL, Prowse, DM & Watt, FM. (2004) Development (Cambridge, UK) 131, 1787–1799.[Abstract/Free Full Text]
  18. Fader, M, Kline, SN, Spatz, SS & Zubrow, HJ. (1962) Oral Surg Oral Med Oral Pathol 15, 153–172.[CrossRef][Medline]
  19. Wolf, J, Järvinen, J & Hietanen, J. (1986) Br J Oral Maxillofac Surg 24, 410–416.[CrossRef][ISI][Medline]
  20. Lammi, L, Arte, S, Somer, M, Järvinen, H, Lahermo, P, Thesleff, I, Pirinen, S & Nieminen, P. (2004) Am J Hum Genet 74, 1043–1050.[CrossRef][ISI][Medline]
  21. Logan, CY & Nusse, R. (2004) Annu Rev Cell Dev Biol 20, 781–810.[CrossRef][ISI][Medline]
  22. Harada, N, Tamai, Y, Ishikawa, T, Sauer, B, Takatu, K, Oshima, M & Taketo, MM. (1999) EMBO 18, 5931–5942.[CrossRef][ISI][Medline]
  23. Maretto, S, Cordenonsi, M, Dupont, S, Braghetta, P, Broccoli, V, Hassan, AB, Volpin, D, Bressan, GM & Piccolo, S. (2003) Proc Natl Acad Sci USA 100, 3299–3304.[Abstract/Free Full Text]
  24. Kratochwil, K, Galceran, J, Tontsch, S, Roth, W & Grosschedl, R. (2002) Genes Dev 16, 3173–3185.[Abstract/Free Full Text]
  25. Obara, N & Lesot, H. (2004) Histochem Cell Biol 121, 351–358.[CrossRef][ISI][Medline]
  26. Jernvall, J, Keränen, SVE & Thesleff, I. (2000) Proc Natl Acad Sci USA 97, 14444–14448.[Abstract/Free Full Text]
  27. Nakamura, T, Unda, F, Vilaxa, A, Fukumoto, S, Yamada, KM & Yamada, Y. (2004) J Biol Chem 279, 626–634.[Abstract/Free Full Text]
  28. Laurikkala, J, Kassai, Y, Pakkasjärvi, L, Thesleff, I & Itoh, N. (2003) Dev Biol 264, 91–105.[CrossRef][ISI][Medline]
  29. Itasaki, N, Jones, CM, Mercurio, S, Rowe, A, Domingos, PM, Smith, JC & Krumlauf, R. (2003) Development (Cambridge, UK) 130, 4295–4305.[Abstract/Free Full Text]
  30. Kettunen, P, Laurikkala, J, Itäranta, P, Vaino, S, Itoh, N & Thesleff, I. (2000) Dev Dyn 219, 322–332.[CrossRef][ISI][Medline]
  31. Lumsden, AG. (1979) J Biol Bucc 7, 77–103.
  32. Pispa, J, Jung, HS, Jernvall, J, Kettunen, P, Mustonen, T, Tabata, MJ, Kere, J & Thesleff, I. (1999) Dev Biol 216, 521–534.[CrossRef][ISI][Medline]
  33. Salazar-Ciudad, I & Jernvall, J. (2002) Proc Natl Acad Sci USA 99, 8116–8120.[Abstract/Free Full Text]
  34. Harris, MP, Williamson, S, Fallon, JF, Meinhardt, H & Prum, RO. (2005) Proc Natl Acad Sci USA 102, 11734–11739.[Abstract/Free Full Text]
  35. Jensen, BL & Kreiborg, S. (1990) J Oral Pathol Med 19, 89–93.[CrossRef][ISI][Medline]
  36. Mundlos, S, Otto, F, Mundlos, C, Mulliken, JB, Aylsworth, AS, Albright, S, Lindhout, D, Cole, WG, Henn, W & Knoll, JH, et al. (1997) Cell 89, 773–779.[CrossRef][ISI][Medline]
  37. Praetorius, F & Piatelli, A. (2005) in World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours eds. Barnes, L, Eveson, JW, Reichart, P& Sidransky, D. (IARC, Lyon, France,) pp. 311.
  38. Wilkinson, D & Green, J. (1990) in Postimplantation Mammalian Embryos eds. Copp, AJ & Cole, DE. (Oxford Univ. Press, London,) pp. 155–171.
  39. Jernvall, J, Lberg, T, Kettunen, S, Keränen, S & Thesleff, I. (1998) Development (Cambridge, UK) 125, 161–169.[Abstract]

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