Signaling Networks Regulating Tooth Organogenesis and ...cshperspectives.cshlp.org/content/early/2012/03/13/...2012/03/13 · of the development of avestigial tooth rudiment found

Signaling Networks Regulating ToothOrganogenesis and Regeneration, and theSpecification of Dental Mesenchymal andEpithelial Cell Lineages

Maria Jussila and Irma Thesleff

Developmental Biology Program Institute of Biotechnology, Biokeskus 1, P.O. Box 56, University of Helsinki,Helsinki FIN-00014, Finland

Correspondence: [email protected]

SUMMARY

Teeth develop as ectodermal appendages from epithelial and mesenchymal tissues. Toothorganogenesis is regulated by an intricate network of cell–cell signaling during all steps ofdevelopment. The dental hard tissues, dentin, enamel, and cementum, are formed by uniquecell types whose differentiation is intimately linked with morphogenesis. During evolution thecapacity for tooth replacement has been reduced in mammals, whereas teeth have acquiredmore complex shapes. Mammalian teeth contain stem cells but they may not provide a sourcefor bioengineering of human teeth. Therefore it is likely that nondental cells will have to bereprogrammed for the purpose of clinical tooth regeneration. Obviously this will requireunderstanding of the mechanisms of normal development. The signaling networks mediatingthe epithelial-mesenchymal interactions during morphogenesis are well characterized but themolecular signatures of the odontogenic tissues remain to be uncovered.

Outline

1 Morphogenesis and celldifferentiation during tooth development

2 Signal networks and signaling centers

3 Regulation of the identity anddifferentiation of odontogenicmesenchymal and epithelial cell lineages

4 Regulation of tooth replacement, continuousgrowth, and stem cells in teeth

5 Future challenges: stem cell-basedbioengineering of teeth

6 Concluding remarks

References

Editors: Patrick P.L. Tam, W. James Nelson, and Janet Rossant

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Teeth are one of the most diverse organs in vertebrates bothmorphologically and functionally. Mammalian teeth be-long to four tooth families: incisors, canine, premolars,and molars, and they are replaced either once or not atall. Humans have teeth from all four tooth families and ex-cluding molars, the teeth are replaced once (Fig. 1A). Thelaboratory mouse (Mus musculus), which is the most com-mon model animal in tooth development studies, has amuch derived dentition. It lacks the canine and premolarsand has only one incisor and three molars separated by atoothless diastema in each half of the jaw (Fig. 1B). Further-more, the mouse incisors grow continuously but the teethare not replaced. In contrast, reptiles, fish, and amphibianscan replace their teeth multiple times during the life of theanimal. Teeth of these nonmammalian species are usually

simpler in shape. Thus, during evolution the complexityof tooth shape has increased, whereas the replacement ca-pacity has been reduced.

The same conserved signaling pathways that regulatemost aspects of embryonic development are required fortooth development, and the core regulatory network seemsto have been in place already when teeth appeared in evolu-tion (Fraser et al. 2009; Tummers and Thesleff 2009). It isnoteworthy that teeth develop as epithelial appendagesand share the same regulatory molecules during the firststeps of initiation and morphogenesis with other ectoder-mal organs. However, unlike many other human epithelialappendages, human teeth have no regenerative capacity.The adult human teeth contain stem cells that are capableof differentiating to cells producing the extracellular matrix

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Figure 1. The dental formula of human and mouse, and a schematic representation of tooth development. The per-manent dentition of human consists of two incisors, a canine, two premolars, and three molars in each half of the jaw(A). Mice have one incisor and three molars separated by a toothless diastema in each half of the jaw (B). Tooth de-velopment starts from the dental lamina, a thickening of the epithelium. Individual placodes form within the dentallamina. The growing epithelium forms a bud and the dental mesenchyme condenses around the epithelium. Duringmorphogenesis, the epithelial tissue folds to cap and bell shapes. Primary and secondary enamel knots in the enamelorgan regulate the growth and shape of the tooth. During cell differentiation, enamel-secreting ameloblasts anddentin-secreting odontoblasts mature from the epithelial and mesenchymal cell compartments. The permanenttooth develops lingually to the deciduous tooth from an extension of the dental lamina (C).

M. Jussila and I. Thesleff

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of tooth-specific mineralized tissues, but so far they havenot been shown to have morphogenetic potential. The re-placement of adult teeth in humans by tissue engineeringappears still a distant goal and it is obvious that moreresearch on stem cell regulation and the molecular controlof early tooth development is required. In this article wereview the current knowledge about the mechanisms in-volved in tooth morphogenesis and replacement, and howthe epithelial and mesenchymal cell lineages acquire odon-togenic competence and differentiate into tooth-specificcells depositing the dental hard tissues, and discuss the fu-ture challenges and scenarios of tooth bioengineering.

1 MORPHOGENESIS AND CELLDIFFERENTIATION DURING TOOTHDEVELOPMENT

Teeth are initiated from two tissue components: the surfaceepithelium and the underlying mesenchyme. The dentalmesenchyme derives from cranial neural crest cells thatmigrate into the frontonasal process and first branchialarch. In mammalsthe epithelium originates from ectoderm,whereas in fish and some amphibians, pharyngeal teeth de-rive from the endoderm.

The first sign of tooth development is the formationof the dental lamina, a horseshoe-shaped epithelial stripealong the mandible and maxilla (Fig. 3B). The teeth formwithin the dental lamina, where their development startsfrom placodes, local thickenings of the epithelium (Figs.1C and 3B,C). Probably all teeth in one tooth family are in-itiated sequentially from a single placode. For instance, themouse molars develop successionally, starting from the firstmolar and followed by initiation of the second and thirdmolars from a posterior extension of the dental epithelium.

The individual teeth develop from an epithelial budthat grows down to the underlying mesenchyme. The neu-ral crest-derived mesenchyme becomes specified as the den-tal mesenchyme condenses around the bud and gives rise toall the dental tissues except enamel. The epithelial bud in-vaginates at its tip and its cervical loops grow to encompassthe dental papilla mesenchyme, which gives rise to the den-tal pulp and odontoblasts forming dentin (Fig. 1C). Themesenchyme surrounding the epithelium and dental pa-pilla becomes the dental follicle and gives rise to the peri-odontal tissues and cementoblasts forming the cementum.

During morphogenesis the epithelium acquires cap andbell shapes and is called enamel organ. It consists of severalcell types: the inner enamel epithelium facing the dentalpapilla and differentiating to enamel producing amelo-blasts, the outer enamel epithelium facing the dental fol-licle, and the stellate reticulum and stratum intermediumcells in between. The growth and folding of the inner

enamel epithelium during the bell stage determine thesize and shape of the tooth crown. The shape becomes fixedwhen the organic matrices of dentin and enamel mineralizebecause no remodeling of either dentin or enamel takesplace later.

Root formation is initiated after crown developmentwhen ameloblast differentiation reaches the future crown-root boundary, and the cells of the inner enamel epitheliumno longer differentiate into ameloblasts. Instead they formthe Hertwig’s epithelial root sheath (HERS) with the outerenamel epithelium. HERS proliferates and migrates down-ward guiding root formation, and it also induces the differ-entiation of odontoblasts forming root dentin. HERS has alimited growth potential, which determines the length ofthe root. The disintegration of HERS results in the forma-tion of an epithelial network called epithelial rests of Malas-sez (ERM) and this allows the cells of dental follicle to comein contact with root dentin and their differentiation intocementoblasts depositing cementum on the root surface.The periodontal ligament that connects the tooth to thebone is formed by fibroblasts differentiating from the den-tal follicle cells. In addition, the dental follicle gives rise toosteoblasts that form the alveolar bone where the fibers ofthe periodontal ligament are embedded (Nanci 2008).The dental follicle has an important function later in tootheruption as it regulates bone remodeling around the tooth(Marks and Cahill 1987).

2 SIGNAL NETWORKS AND SIGNALING CENTERS

All aspects of tooth morphogenesis are regulated byepithelial-mesenchymal interactions, which are mediatedby the conserved signaling pathways including Hedgehog(Hh), Wnt, Fibroblast growth factor (FGF), Transforminggrowth factor b (Tgfb), Bone morphogenic protein (Bmp),and Ectodysplasin (Eda) (Fig. 2). Their interactions, targets,and expression patterns have been elucidated in considerabledetail in teeth (http://bite-it.helsinki.fi; Bei 2009b; Tum-mers and Thesleff 2009). Epithelial signaling centers playa pivotal role regulating the different steps of tooth devel-opment. There are three sets of such centers: the placodes,the primary enamel knots, and the secondary enamelknots. Their formation is regulated by epithelial-mesen-chymal interactions and they all express largely the same ar-ray of multiple growth factors.

All ectodermal organs begin to develop from a placode,and the molecular mechanisms of tooth placode formationand signaling are shared to a great extent with placodes ofother organs such as hairs (Mikkola 2009b). One of theimportant genes regulating placode formation is the tran-scription factor p63 that is expressed throughout the sur-face ectoderm. When p63 function is deleted in mice, the

Signaling Networks Regulating Tooth Development

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placodes of teeth and other ectodermal appendages do notdevelop, but the dental lamina forms (Laurikkala et al.2006). Key signaling pathways including Bmp, Fgf, Notch,and Eda are impaired in the absence of p63 (Laurikkalaet al. 2006). The importance of the signaling that takes

place at the placode stage is further highlighted by the phe-notype of several mouse mutants where tooth developmentstops before epithelial budding (Bei 2009b).

Ectodysplasin (Eda) is a signal of the tumor necrosisfactor family and signals through its receptor Edar that islocally expressed in the placodes of all ectodermal appen-dages as well as in primary and secondary enamel knots(Mikkola 2009b). Mutations in the Eda pathway genescause the human syndrome hypohidrotic ectodermal dys-plasia (HED) manifesting multiple missing teeth as wellas defects in other ectodermal organs, e.g., sparse hairand reduced sweating (Mikkola 2009b). Mice lacking func-tional Eda often lack third molars or incisors and the cusppatterning of molars is abnormal, indicating a requirementof Eda in the function of placodes and enamel knots (Pispaet al. 1999). Mice that overexpress Eda in epithelium (underkeratin14-promotor) develop an extra tooth in front ofthe molars as well as supernumerary hairs and mammaryglands (Fig. 5A,B) (Mustonen et al. 2003). The targets ofEda signaling include molecules from all the other impor-tant signaling pathways (e.g., Shh, Fgf20, Dkk4, ctgf, Folli-statin) making Eda a key regulator of ectodermal organdevelopment (Mikkola 2009b).

The primary enamel knot appears in the dental epithe-lium at the transition from bud to cap stage. In addition todirecting crown formation, in molars it determines the po-sitions of the secondary enamel knots which in turn markthe positions of the cusp tips in the molar crown (Fig. 2B)(Jernvall et al. 2000). Wnts are important upstream regula-tors of enamel knots as shown by the requirement of Lef1for Fgf4 expression in the enamel knot (Kratochwil et al.2002) and the induction of new enamel knots and placodesby forced activation of Wnt/b-catenin signaling in oralepithelium (Järvinen et al. 2006; Wang et al. 2009). Morethan a dozen different signal molecules belonging to allfour conserved signal families are locally expressed in theprimary and secondary enamel knots. The enamel knotsinitiate and regulate the folding of the epithelium by stim-ulating the surrounding epithelium to proliferate throughFgfs (Fgfs 3, 4, 9, and 20) and remaining nonproliferativethemselves. They express the cyclin-dependent kinase in-hibitor p21 and lack Fgf receptors making them insensitiveto the proliferative signals (Jernvall et al. 1998; Kettunenet al. 1998). The Fgfs also signal to dental mesenchyme andinduce e.g., Runx2, and Fgf3, which signals back to epithe-lium illustrating the bidirectional Fgf signaling betweenepithelium and mesenchyme regulating tooth morpho-genesis (Klein et al. 2006). Shh from the enamel knotstimulates epithelial morphogenesis indirectly via the mes-enchyme (Gritli-Linde et al. 2002).

Important aspects of enamel knot signaling are themodulation and fine-tuning, which affect the patterning

Shh

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Bmp4

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Enamel knot signals: Fgf3,4,9,20 Shh Wnt3,6,10a,10b Bmp2,4,7

Mesenchymal signals: Fgf3,10 Bmp4 Wnt5a,5b

Cusp patterning and cell differentiation

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Figure 2. Cross talk between epithelium and mesenchyme throughthe conserved signaling pathways regulates all aspects of tooth devel-opment. When tooth development is initiated, signals from the epi-thelium activate a set of transcription factors in the mesenchyme,leading to condensation of the mesenchyme and formation of the ep-ithelial placode (A). The enamel knot is a signaling center expressingmultiple signaling molecules that induce reciprocal signals from themesenchyme. Enamel knots determine the position of the cusps andinitiate differentiation of odontoblasts (B). Tgfb, Bmp, and Shh sig-naling regulate epithelial-mesenchymal interactions in the cervicalloop of the mouse incisor. They support the maintenance and prolif-eration of the stem cells as well as ameloblast differentiation and en-amel production (C).





of the secondary enamel knots and thereby the patternof molar cusps via lateral inhibition and reaction diffusionmechanisms. Different shapes of molars can be generatedby mathematical modeling using parameters of activatingand inhibiting enamel knot signals, and it has been sug-gested that changes in signaling during evolution are re-sponsible for the species-specific cusp patterns (Salazar-Ciudad and Jernvall 2002). This hypothesis has gained ex-perimental support from phenotypes of transgenic micewhere signal modulation has resulted in phenotypes resem-bling teeth of other species. Examples include molarsof K14-Eda resembling kangaroo teeth, and of Sostdc1knockout (inhibitor of Wnt and Bmp signaling) resem-bling rhino teeth (Kangas et al. 2004; Kassai et al. 2005).Furthermore, epithelial deletion of Dicer, which is requiredfor processing of microRNA (miRNA), results in the mod-ulation of molar cusp pattern (Michon et al. 2010).

Fine-tuning of the activity of the conserved signalingpathways controls many other aspects of tooth formationas well. For example, a supernumerary tooth forms in frontof the first molar in several mutant mouse lines when sig-naling activity is modulated. These teeth do not representde novo tooth induction. Instead they form from activationof the development of a vestigial tooth rudiment found inwild-type mice in the diastema and represent premolarslost during the evolution of rodents. Examples are theK14-Eda mouse (Fig. 5A,B) (Mustonen et al. 2003) andthe Sprouty mutants (Klein et al. 2006). In the Osr2 knock-out an extra tooth develops lingually to the first molar(Zhang et al. 2009). This is accompanied by spreading ofBmp4 expression to the lingual mesenchyme and resultsprobably from a subsequent broadening of the dental field(Mikkola 2009a). The relative sizes of the mouse molars are

influenced by activation and inhibition between succes-sionally developing teeth (Kavanagh et al. 2007), the sizeand number of mouse incisors is affected by fine-tuningBmp signaling in the placodes (Munne et al. 2010), andthe continuous growth and enamel deposition in incisorscan be modulated by the levels of Fgf, Activin, and Bmp sig-naling in the epithelial stem cell niche (Fig. 2C) (Wang et al.2007).

3 REGULATION OF THE IDENTITY ANDDIFFERENTIATION OF ODONTOGENICMESENCHYMAL AND EPITHELIAL CELLLINEAGES

Classical recombination experiments have shown that theodontogenic potential shifts from the epithelium to mes-enchyme in mouse teeth between embryonic days 11 and12, i.e., around the time of placode formation (Fig. 3).When epithelium of the first branchial arch from an E9-11 mouse embryo was recombined with second arch mes-enchyme, a tooth formed (Fig. 3A) (Mina and Kollar 1987).Similarly, first arch epithelium from an E9-10 embryo in-duced tooth formation when recombined with cranial neu-ral crest cells that normally form the dental mesenchymeand, interestingly, also when combined with premigratorytrunk neural crest cells (Lumsden 1988). At E12 the epithe-lium no longer has inductive potential and now the firstarch mesenchyme can induce tooth formationwhen recom-bined with second arch epithelium (Fig. 3A). The mesen-chyme from E13 or older tooth germs has the informationon the tooth identity as the shape of the tooth in the recip-rocal recombinations between incisor and molar epithe-lium and mesenchyme will form according to origin of

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Figure 3. Shift of the odontogenic potential from epithelium to mesenchyme between dental lamina and placodestages as shown by reciprocal tissue recombinations (Mina and Kollar 1987). Epithelium is capable of inducing toothdevelopment when recombined with nondental mesenchyme until E11 stage of mouse development. At E12 theodontogenic potential has shifted to mesenchyme, and it can induce tooth development when recombined with non-dental epithelium (A). Pitx2 is expressed in the dental lamina of the mouse lower jaw at E11 (t ¼ tongue) (B). At E12.5the Pitx2 expression is restricted to the placode epithelium of the incisors (arrows) and molars (asterisks) (C).





the mesenchyme (Kollar and Baird 1969). In addition, thedental papilla can induce tooth formation when recom-bined with limb epithelium (Kollar and Baird 1970). Ithas been proposed, based on in vitro experiments, thatthe incisor versus molar identity of teeth is determined bythe level of Bmp signaling (Tucker et al. 1998). However,this conclusion was challenged recently by the observationthat inhibition of Bmp signaling caused partial splittingof the incisor placode resulting in the formation of twofused incisors rather than incisor to molar transformation(Munne et al. 2010).

The molecular basis of odontogenic competence inearly jaw epithelium and later in the condensed dental mes-enchyme remains elusive. As all the genes that are knownto regulate tooth development are also expressed in otherdeveloping organs, it seems that there is no single tooth-specific gene that defines the odontogenic tissues. Cur-rently only few genes such as Sonic hedgehog (Shh) andthe transcription factor Pitx2 are known to be restrictedto the dental lamina (Fig. 3B,C) (Keränen et al. 1999). Itis not known how the dental lamina becomes established,and to date there is no mouse mutant reported where thedental lamina would be missing. All teeth develop withinthe dental lamina, even in micewhere extra teeth are induced.Activation of Wnt signaling in the epithelium induces super-numerary placodes throughout the surface epithelium andthey give rise to various epithelial appendages. Yet extra teethform only in the region of dental arches and mostly in con-nection with other teeth (Järvinen et al. 2006; Wang et al.2009). These observations indicate that the odontogeniccompetence is present only in the oral region.

It is likely that spatiotemporal patterns of the epithelialsignals are involved in the shift of competence to mesen-chyme (Fig. 2A). In addition to Shh, which is restrictedto the dental lamina, many Wnt ligands are expressed inthe oral epithelium (Sarkar and Sharpe 1999). Wnt/b-cat-enin signaling regulates Fgf8 expression in the early epithe-lium (Wang et al. 2009), and placodes do not form in miceoverexpressing the Wnt inhibitor Dkk1 (Andl et al. 2002).Bmp4 and Fgf8 are expressed in the jaw epithelium in over-lapping patterns before any morphological sign of toothdevelopment. Bmp4 is expressed more distally at the sitewhere molars will develop and Fgf8 more proximally inthe incisor region (Neubüser et al. 1997). Epithelial Bmpsinduce the expression of Bmp4 in the mesenchyme beforebud stage correlating with the shift in the odontogenic po-tential (Vainio et al. 1993). Also, Wnt/b-catenin signalingin the incisor mesenchyme stimulates the expression ofBmp4 that in turn regulates Shh in the epithelium (Fujimoriet al. 2010). Furthermore, Wnt signaling was shown to berequired in the molar mesenchyme for the bud to cap stagetransition and primary enamel knot formation (Chen

et al. 2009). The signals induced in the mesenchyme byepithelial Fgfs and acting reciprocally to the epithelium in-clude Activin, Fgf3, and Fgf10 (Ferguson et al. 1998; Ket-tunen et al. 2000). These signals regulate the subsequentepithelial morphogenesis and the enamel knot formation(Fig. 2A).

The shift of the odontogenic competence from epithe-lium to mesenchyme is accompanied by the induction ofimportant transcription factors in the dental mesenchyme(Fig. 2A). The deletion of the function of several of theseeither alone or together with another transcription factorin the same family results in tooth arrest at placode orbud stage. All four signal pathways have been shown tobe involved in the regulation of these transcription factors(Bei 2009b). For example, Bmp4 induces the expression ofMsx1 and Fgf8 induces the expression of Pax9 (Vainio et al.1993, Neubüser et al. 1997). Other targets of Bmp and Fgfsignaling in mesenchyme at this stage include Lhx6,7,Barx1, Dlx1,2, and Runx2 (Bei 2009b, Tummers and Thes-leff 2009). The Shh mediators Gli2 and Gli3 are expressed inthe mesenchyme and are required for tooth formation(Hardcastle et al. 1998). In addition, the expression ofLef1, a Wnt effector, shifts from the epithelium to the mes-enchyme together with the shift in the odontogenic poten-tial and is regulated by Bmp4 in mesenchyme (Kratochwilet al. 1996). Perhaps a combination of these transcriptionfactors constitutes the code for the odontogenic identityof the mesenchyme.

The differentiation of the tooth-specific cell types is in-timately linked with epithelial morphogenesis. Odonto-blasts and cementoblasts differentiate from the lineage ofdental mesenchyme, the odontoblasts from the dental pa-pilla, and cementoblasts from the dental follicle, whereasameloblasts differentiate from the epithelial lineage. Theyare responsible for the formation and the deposition ofthe extracellular matrices of the tooth-specific mineralizedtissues, dentin, cementum, and enamel, respectively. It isnot known exactly at which stage of tooth formation thecells become committed, but the final steps of odontoblastand ameloblast differentiation have been analyzed in detailduring the bell stage of tooth formation.

The mesenchyme is first induced to differentiate intoodontoblasts by the inner enamel epithelium. The differen-tiation starts from the cusp tips and proceeds downward tocervical and intercuspal directions. Signals in Tgfb/Bmpfamilies have been implicated in odontoblast induction(Ruch et al. 1995), and it was shown recently that the condi-tional loss of Smad4, a mediator of Tgfb/Bmp signaling,from the dental papilla prevents the terminal differen-tiation of odontoblasts and dentin deposition (Li et al.2011a). As the formation of enamel knots is temporallyassociated with the initiation of odontoblast differentiation





at the cusp tips, the enamel knot signals have been sug-gested to play a role (Fig. 2B) (Thesleff et al. 2001). Oneof these signals, Wnt10b, was suggested to regulate the ex-pression of dentin sialophosphoprotein (Dspp) and odonto-blast differentiation (Yamashiro et al. 2007). The localizationof Wnt reporter activity in odontoblasts is also in line withthe role of Wnts in the process (Suomalainen and Thesleff2010). In addition, the basement membrane is importantfor the polarization and differentiation of the odontoblastsand serves presumably as a reservoir of signal molecules(Thesleff and Hurmerinta 1981; Ruch et al. 1995). Dentinis composed mainly of type I collagen, dentin phosphopro-tein, and Dspp, and mutations in these genes cause dentino-genesis imperfecta in humans (Shields et al. 1973).

After the odontoblasts have been induced to differenti-ate, they signal back to the epithelium. The signals fromthe mesenchyme involved in the ameloblast inductioninclude Bmp2, Bmp4, and Tgfb1 (Fig. 4) (Coin et al.1999; Wang et al. 2004). In addition, Shh from the epithe-lial stratum intermedium cells is required to support ame-loblast differentiation and maturation (Dassule et al. 2000;Gritli-Linde et al. 2002). Other epithelial growth factorsregulating ameloblasts are TFGb1, Wnt3, Eda, and Follista-tin (Bei 2009a). Ameloblasts express transcription factorssuch as Sp6 and Msx2 that have been shown to play a

role in amelogenesis in mice (Bei 2009a). Mutations inameloblast-specific genes including ameloblastin, ameloge-nin, enamelin, and Mmp20 cause human amelogenesisimperfecta (Bei 2009a). Very little is known about the mo-lecular regulation of cementoblast development. Bmp sig-naling was reported to induce cementoblast differentiationfrom dental follicle cells, whereas Wnt signaling promotestheir proliferation (Zhao et al. 2002; Nemoto et al. 2009).

4 REGULATION OF TOOTH REPLACEMENT,CONTINUOUS GROWTH, AND STEM CELLSIN TEETH

As the mouse teeth are not replaced, relatively little isknown about the mechanisms of tooth replacement in +mammals. Histological observations in nonmodel animalsindicate that replacement teeth develop from the dentallamina associated with their predecessors (Luckett 1993;Järvinen et al. 2009). The ferret (Mustela putorius furo) re-places its incisors, canines, and premolars, and it was shownthat the deciduous teeth are connected to each other by acontinuous dental lamina, and the permanent teeth startto grow from the lingual side of each deciduous tooth asan extension of the dental lamina (Fig. 5C–E) (Järvinenet al. 2009). Similarly, in the reptiles the replacement tooth

Pulp

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Figure 4. Bmp4 is one of the signals regulating ameloblast induction. A schematic view of the postnatal mouse in-cisor shows the asymmetrical deposition of enamel only on the labial side of the tooth and the cervical loop stem cellniche (A). Amelogenin protein is present in the ameloblasts (a) and in the first enamel matrix on the labial side ofnewborn (NB) incisor (arrow) but not on the lingual side [asterisk; o, odontoblasts]) (B). Bmp4 is expressed in themesenchyme and is intense in the odontoblasts (arrows) of the developing incisor at E16. The white line surroundsthe epithelium (C). A bead soaked in Bmp4 protein induces ameloblastin expression in E16 incisors (D). (B and Dreprinted, with permission, from Wang et al. 2004.)





arises from an outgrowth of the dental lamina each time theprevious tooth has grown to a certain size (Richman andHandrigan 2011). In contrast, in the fish species studied,there seems to be no successional dental lamina, and thenew teeth are initiated directly from the epithelium of the pre-vious tooth or from the oral epithelium (Smith et al. 2009).

Wnt signaling has been associated with tooth replace-ment both in mammals and reptiles and may be a key factorregulating tooth renewal across vertebrates (Järvinen et al.2009; Richman and Handrigan 2011). In the ferret, the ex-pression of Sostdc1, an inhibitor of Wnt and Bmp signaling,marks the border between the deciduous tooth and thedental lamina that gives rise to the permanent tooth (Fig.5E) (Järvinen et al. 2009). The expression of Axin2, a feed-back inhibitor of Wnt signaling, was also detected in themesenchyme between the tooth and the growing dentallamina (Järvinen et al. 2009). During snake tooth replace-ment, there is Wnt activity in the tip of the dental lamina

and it is promoted by Shh and Bmp signaling from the mes-enchyme (Richman and Handrigan 2011).

The phenotypes of some human syndromes and theirmouse models support the role of Wnt signaling in toothreplacement. Mutations in the human AXIN2 gene causeoligodontia, which specifically affects permanent teeth(Lammi et al. 2004). On the other hand, supernumeraryteeth are common in familial adenomatous polyposis(FAP), which is caused by mutations in APC, an inhibitorycomponent of the Wnt pathway, and the patients also de-velop odontomas, benign tumors composed of numeroussmall teeth (Wang and Fan 2011). A similar phenotype isseen in mice when Wnt signaling is activated in the epithe-lium either by deletion of APC or stabilization of b-catenin(Fig. 5F–H) (Järvinen et al. 2006; Liu et al. 2008; Wanget al. 2009). Sp62/2 (Epiprofin) mutants have a similarphenotype but this gene has not been associated with hu-man conditions (Nakamura et al. 2008; Wang and Fan

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Figure 5. Supernumerary teeth, tooth replacement, and continuous tooth renewal. Overexpression of ectodysplasinin the surface epithelium results in development of a supernumerary tooth (arrow) in front of the molars (m1-3) inthe K14-Eda mice (A). The rudiment of the supernumerary tooth (blue arrow) in front of the first molar (red arrow)can be visualized by Shh expression in E14.5 lower jaw of K14-Eda embryo (B). The permanent canine (C) of theferret develops as an extension of the dental lamina (dl) on the lingual side of the deciduous canine (dC) at E33 (C).Both deciduous and permanent canine express Pitx2 in the epithelium (D). Sostdc1 is expressed in the intersectionbetween the dental lamina and the deciduous third premolar (dP3) at the time when permanent P3 is initiated in theE35 ferret embryo (arrow) (E). Stimulation of Wnt signaling by stabilized b-catenin in mouse oral epithelium leadsto the development of multiple small teeth from a single E14 mutant tooth germ cultured under the kidney capsule(F). Fgf20 is expressed in the enamel knots of upper and lower molars of E14.5 wild-type mouse embryos (G). Multi-ple enamel knots expressing Fgf20 have been induced in the dental epithelium of b-catDex3K14/+ embryos (H ).





2011). The teeth in the Wnt gain-of-function mouse modelswere shown to develop successionally from previous teeth re-sembling the continuous generation of the simple-shapedreplacement teeth in fish and reptiles. This led to a sugges-tion, that Wnt signaling may have been involved in the re-duction in the replacement capacity and in the gain intooth complexity during evolution (Järvinen et al. 2006).

Interestingly, the phenotypes of two syndromes suggestthat the capacity for continued tooth replacement can beunlocked in humans. The supernumerary teeth were sug-gested to represent a third dentition in cleidocranial dys-plasia (CCD) and in a novel craniosynostosis syndrome,caused by mutations in the transcription factor RUNX2and interleukin receptor IL11RA, respectively (Jensen andKreiborg 1990; Nieminen et al. 2011). Unfortunately, themouse models of these syndromes do not show supernum-erary teeth, likely because the mouse teeth are not normallyreplaced, and they are therefore not suitable for studies ontooth replacement (D’Souza et al. 1999; Nieminen et al.2011). Runx2 has been associated with Fgf as well as Wntsignaling in tooth development as the induction of theWnt inhibitor Dkk1 in dental mesenchyme by epithelialFgf4 requires Runx2 (James et al. 2006).

Putative epithelial stem cells have been identified duringtooth replacement in gecko, a reptile (Handrigan et al.2010). These cells reside in the lingual side of dental laminaand express some known stem cell marker genes. It is possi-ble that the reduction of tooth replacement capacity inmammals to maximally one replacement has involved deple-tion of such stem cells and that they are maintained in thecleidocranial dysplasia and craniosynostosis syndromes.

Although the human teeth do not regenerate and theirdevelopment is completed already during adolescence, thereare stem cells in the adult teeth. Human dental mesenchymalstem cells were first isolated from the dental pulp and whentransplanted they formed dentin (Gronthos et al. 2000).Stem cells in periodontal ligament were shown to producecementum and periodontal ligament-like structures (Seoet al. 2004). Similar stem cells were also characterized in ex-foliated deciduous teeth and third molars (Rodriguez-Loza-no et al. 2011). Epithelial stem cells may reside within theepithelial cell rests of Malassez as these cells can be inducedto ameloblastlike cells (Shinmura et al. 2008).

Some mammals have teeth that grow continuouslythroughout life and thus have stem cells. The most studiedsuch tooth is the mouse incisor, which harbors epithelialstem cells in a niche situated in the cervical loop at its prox-imal end (Fig. 2C) (Harada et al. 1999). The mouse incisorhas an asymmetric structure with enamel deposited only onthe labial side, whereas the lingual side is covered by dentin(Fig. 4A). The asymmetry arises from differences betweenthe lingual and labial cervical loops as only the larger labial

cervical loop contains label-retaining stem cells and transi-ent amplifying (TA) cells (Harada et al. 1999; Seidel et al.2010). The stem cells reside within the stellate reticulumcells in the core of cervical loop, which is surroundedby dental mesenchyme. The progeny of stem cells invadesthe basal epithelium and proliferates as TA cells before dif-ferentiating into ameloblasts (Figs. 2C and 4A,B).

Mesenchymal signals play key roles in the regulation ofthe epithelial stem cells and their progeny (Fig. 2C). Fgf10is the key mesenchymal signal required for epithelial stemcell maintenance and proliferation, and Fgf3 has a partlyredundant function as stimulator of TA cell proliferation(Wang et al. 2007). Mesenchymal Fgfs act in a regulatoryloop with epithelial Fgfs, notably Fgf9, and stimulation ofFgf signaling by deleting the function of Sprouty genes resultsin extensive growth of the incisors and ectopic deposition ofenamel on the lingual surface (Klein et al. 2008). Lingualenamel also forms in Follistatin knockout mice, whereasenhanced Follistatin expression results in complete absenceof enamel from labial side as well as in growth inhibition(Wang et al. 2004). Follistatin is expressed in the lingual epi-thelium and it antagonizes Activin function in the cervicalloop epithelium while, interestingly, inhibiting Bmp4 func-tion in the zone of differentiation, which prevents enamelformation. It was shown that Bmp signaling is required forameloblast differentiation (Fig. 4) (Wang et al. 2004). Ac-cordingly, when the Bmp inhibitor Noggin is overexpressedin epithelium, the mouse incisors grow extensively and lackenamel (Plikus et al. 2005). Bmps and Activin function in aregulatory network with Fgf3, which is inhibited by Bmp4,which is in turn repressed by Activin that is strongly ex-pressed in the labial dental mesenchyme but not on the lin-gual side. This contributes to the asymmetric production ofTA cells only in the labial cervical loop (Wang et al. 2007).

Fgf and Shh signaling play a role in the postnatal ho-meostasis of the TA cell production in the mouse incisorsbut not in stem cell survival (Parsa et al. 2010; Seidelet al. 2010). On the other hand, Wnt signaling activity isnot detected in the stem cells in the cervical loops (Suoma-lainen and Thesleff 2010). Some stem cell marker genes,such as Lgr5, Bmi1, Oct3/4, and Yap, have been localizedin the incisor stem cells (Suomalainen and Thesleff 2010;Li et al. 2011b). Despite the intense investigations on thesignal pathways regulating the incisor stem cell niche, thecharacterization of these stem cells is still on its way.

5 FUTURE CHALLENGES: STEM CELL-BASEDBIOENGINEERING OF TEETH

Different scenarios have been proposed for bioengineeringof human teeth. One possibility could be the direct induc-tion of tooth development in the jaws with activators such





as Wnt and Eda. However, although supernumerary teethare induced in mouse models and in human syndromesby modulation of signal pathways, it is not likely that thisapproach would function in the adult jaws. The main rea-son is that the supernumerary teeth in mice—as well as inthe rare human syndromes—form from the tissue associ-ated with developing teeth and such teeth would not bepresent in adult jaws anymore. The stem cells discoveredin adult teeth described above, including the mesenchymalstem cells in dental papilla, pulp, and periodontal ligamenthave the capacity to generate cells forming dentin, cementum,and periodontal ligament, but it is unlikely that they havemorphogenetic potential, and even less likely that the cellscould be targeted in vivo to undergo tooth morphogenesis.

A more realistic approach would be to engineer a toothin vitro and implant it to patient’s mouth. It has been pro-posed that such teeth could be generated by growing cells intooth-shaped scaffolds. However, taking into account thecomplex structure and organization of the hard and softtissues of teeth and the fact that tooth size and shapeemerge during the multistep process of morphogenesis to-gether with the periodontal tissue attaching the tooth to thebone, it is difficult to imagine how a functional tooth couldbe developed within a scaffold. Therefore, the preferableway would be to trigger the initiation of tooth developmentprogram in progenitor cells and let the tooth develop itself.

The classical tooth bud transplantation and tissue re-combination experiments have shown that the programfor tooth morphogenesis is present very early in the jawsand that a tooth bud can form a complete tooth whentransplanted to various ectopic sites. The proof of principleexperiments in mouse have already shown that even disso-ciated embryonic dental epithelial and mesenchymal cellscan regenerate a tooth germ in vitro and that this forms afunctional tooth when implanted to the jaw of an adultmouse (Nakao et al. 2007; Oshima et al. 2011).

To use such an approach in human therapy, one wouldneed to replace the embryonic dental cells with adult cellspreferably with tooth forming capacity. Obviously, bothepithelial and mesenchymal cell lineages are needed, butbased on the classical recombination experiments onlyone cell type needs to have the odontogenic potential.Although there is some evidence that adult mouse bonemarrow stem cells can form a tooth together with embry-onic branchial arch epithelium (Ohazama et al. 2004), itis probable that in the recombinations the nonodontogenicmesenchyme should have properties of cranial neural crestand the epithelium should be ectodermal. The inductivepotential of the current human dental stem cell lines hasnot been explored. However, although dental stem cellsfrom adult teeth could perhaps be used for tooth bioengin-eering because they are likely to share characteristics with

embryonic dental tissue, collecting dental stem cells fromadults is challenging as it would imply sacrificing a toothfrom the patient needing a new tooth.

Odontogenic cells might be produced from adult so-matic cells by iPS cell technology or direct reprogramming(Hanna et al. 2010). Thus they could be first reprogram-med to embryonic stem cells by iPS technology and pro-grammed further to dental epithelial or mesenchymal cellfates. Oral mucosal epithelium and stromal cells could befeasible sources for reprogramming because their develop-mental history is likely more similar to dental tissues. Alter-natively, the oral mucosal cells or other adult somatic cellscould perhaps be directly converted to tooth epitheliumand mesenchyme as recently shown in other tissues (Zhouet al. 2008; Hanna et al. 2010).

The knowledge lacking at the moment is the molecularsignatures of the epithelial and mesenchymal lineages thatcould be used in reprogramming. The key genes likelyinclude transcription factors expressed by the early embry-onic tissues such as Pitx2 in the branchial arch epitheliumand Msx1,2, Dlx1,2,5, Runx2, Pax9, Lhx6,7,8, and Prx1,2in the odontogenic mesenchyme (Fig. 2A) (Thesleff andTummers 2008). So far these are onlyeducated guesses basedon expression patterns and mutant phenotypes (http://bite-it.helsinki.fi; Bei 2009b).

Finally, it should be noted that it is unrealistic to aim atgenerating a perfect tooth crown, because it is rather ob-vious that the right shape and size of the crown as well asthe color and proper structure of enamel cannot be gener-ated by bioengineering. Therefore, the crown needs to becompleted prosthetically. The most important aspect ofthe bioengineered tooth would be a functional root pro-viding physiological anchorage of the tooth to jaw bone.However, initiation of root formation, without a precedingcrown appears impossible, at least by mimicking normaldevelopmental mechanisms. The physiological anchorageis lacking in the titanium implants that otherwise are suc-cessfully used for tooth replacement. To this end interestingexperiments have been performed in minipigs, where stemcells from the root apical papilla and periodontal ligamentstem cells were used. When the cells were seeded on a root-shaped cylindrical hydroxyapatite/tricalcium phosphatescaffold, and implanted in the jaw, dentin, cementum, andperiodontal ligament were generated and a structure resem-bling root developed (Sonoyama et al. 2006). It is not yetknown whether the bioengineered root has adequate phys-iological properties to be used in clinical tooth replacement.

6 CONCLUDING REMARKS

Studies on the laboratory mouse have given a great deal ofknowledge on the molecular regulation of tooth initiation,





morphogenesis, and stem cell maintenance. However, be-fore the building of teeth by tissue engineering becomes areality, more detailed understanding of the process of toothdevelopment and regeneration is required. In particular,the gene regulatory networks during cell lineage specifi-cation in dental epithelium and mesenchyme need to beunderstood more thoroughly and the origins of the twotypes of progenitor cells to be used for tooth bioengineer-ing should be determined.

ACKNOWLEDGMENTS

We thank Emma Juuri, Otso Häärä, Elina Järvinen, andAapo Kangas for providing illustrations.

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published online March 13, 2012Cold Spring Harb Perspect Biol Maria Jussila and Irma Thesleff Epithelial Cell LineagesRegeneration, and the Specification of Dental Mesenchymal and Signaling Networks Regulating Tooth Organogenesis and

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P.L. TamRobert O. Stephenson, Janet Rossant and Patrick

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Signaling Networks Regulating Tooth Organogenesis and ...cshperspectives.cshlp.org/content/early/2012/03/13/...2012/03/13 · of the development of avestigial tooth rudiment found