Signaling Networks Regulating Tooth Organogenesis and Regeneration, and the Speciﬁcation of Dental Mesenchymal and Epithelial Cell Lineages

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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: maria.jussila@helsinki.ﬁ

SUMMARY

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

Outline

1 Morphogenesis and cell 4 Regulation of tooth replacement, continuous differentiation during tooth development growth, and stem cells in teeth 2 Signal networks and signaling centers 5 Future challenges: stem cell-based bioengineering of teeth 3 Regulation of the identity and 6 Concluding remarks differentiation of odontogenic mesenchymal and epithelial cell lineages References

Editors: Patrick P.L. Tam, W. James Nelson, and Janet Rossant Additional Perspectives on Mammalian Development available at www.cshperspectives.org

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Teethare one of the most diverse organs in vertebrates both simpler in shape. Thus, during evolution the complexity morphologically and functionally. Mammalian teeth be- of tooth shape has increased, whereas the replacement ca- long to four tooth families: incisors, canine, premolars, pacity has been reduced. and molars, and they are replaced either once or not at The same conserved signaling pathways that regulate all. Humans have teeth from all four tooth families and ex- most aspects of embryonic development are required for cluding molars, the teeth are replaced once (Fig. 1A). The tooth development, and the core regulatory network seems laboratory mouse (Mus musculus), which is the most com- to have been in place already when teeth appeared in evolu- mon model animal in tooth development studies, has a tion (Fraser et al. 2009; Tummers and Thesleff 2009). It is much derived dentition. It lacks the canine and premolars noteworthy that teeth develop as epithelial appendages and has only one incisor and three molars separated by a and share the same regulatory molecules during the ﬁrst toothless diastema in each half of the jaw (Fig. 1B). Further- steps of initiation and morphogenesis with other ectoder- more, the mouse incisors grow continuously but the teeth mal organs. However, unlike many other human epithelial are not replaced. In contrast, reptiles, ﬁsh, and amphibians appendages, human teeth have no regenerative capacity. can replace their teeth multiple times during the life of the The adult human teeth contain stem cells that are capable animal. Teeth of these nonmammalian species are usually of differentiating to cells producing the extracellular matrix

AB Incisors Incisors Canine

Premolars Diastema

Molars Molars

C Dental lamina Placode Bud Cap Bell Eruption Epithelium Placode Enamel knot Secondary Enamel Stellate enamel knot Dentin reticulum ri Juu E. E E. Dental Ameloblasts Developing Dental pulp Mesen- Mesen- mesen- Dental permanent chyme chyme chyme papilla Odontoblasts tooth Alveolar bone

Cell Initiation Morphogenesis differentiation Matrix secretion

Figure 1. The dental formula of human and mouse, and a schematic representation of tooth development. The permanent 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 development starts from the dental lamina, a thickening of the epithelium. Individual placodes form within the dental lamina. The growing epithelium forms a bud and the dental mesenchyme condenses around the epithelium. During morphogenesis, the epithelial tissue folds to cap and bell shapes. Primary and secondary enamel knots in the enamel organ regulate the growth and shape of the tooth. During cell differentiation, enamel-secreting ameloblasts and dentin-secreting odontoblasts mature from the epithelial and mesenchymal cell compartments. The permanent tooth develops lingually to the deciduous tooth from an extension of the dental lamina (C).

2 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Signaling Networks Regulating Tooth Development of tooth-specific mineralized tissues, but so far they have enamel epithelium during the bell stage determine the not been shown to have morphogenetic potential. The re- size and shape of the tooth crown. The shape becomes fixed placement of adult teeth in humans by tissue engineering when the organic matrices of dentin and enamel mineralize appears still a distant goal and it is obvious that more because no remodeling of either dentin or enamel takes research on stem cell regulation and the molecular control place later. of early tooth development is required. In this article we Root formation is initiated after crown development review the current knowledge about the mechanisms in- when ameloblast differentiation reaches the future crown- volved in tooth morphogenesis and replacement, and how root boundary, and the cells of the inner enamel epithelium the epithelial and mesenchymal cell lineages acquire odon- no longer differentiate into ameloblasts. Instead they form togenic competence and differentiate into tooth-specific the Hertwig’s epithelial root sheath (HERS) with the outer cells depositing the dental hard tissues, and discuss the fu- enamel epithelium. HERS proliferates and migrates down- ture challenges and scenarios of tooth bioengineering. ward guiding root formation, and it also induces the differentiation of odontoblasts forming root dentin. HERS has a limited growth potential, which determines the length of 1 MORPHOGENESIS AND CELL the root. The disintegration of HERS results in the forma- DIFFERENTIATION DURING TOOTH tion of an epithelial network called epithelial rests of Malas- DEVELOPMENT sez (ERM) and this allows the cells of dental follicle to come Teethare initiated from two tissue components: the surface in contact with root dentin and their differentiation into epithelium and the underlying mesenchyme. The dental cementoblasts depositing cementum on the root surface. mesenchyme derives from cranial neural crest cells that The periodontal ligament that connects the tooth to the migrate into the frontonasal process and first branchial bone is formed by fibroblasts differentiating from the den- arch.Inmammalstheepitheliumoriginatesfromectoderm, tal follicle cells. In addition, the dental follicle gives rise to whereas in fish and some amphibians, pharyngeal teeth de- osteoblasts that form the alveolar bone where the fibers of rive from the endoderm. the periodontal ligament are embedded (Nanci 2008). The first sign of tooth development is the formation The dental follicle has an important function later in tooth of the dental lamina, a horseshoe-shaped epithelial stripe eruption as it regulates bone remodeling around the tooth along the mandible and maxilla (Fig. 3B). The teeth form (Marks and Cahill 1987). within the dental lamina, where their development starts from placodes, local thickenings of the epithelium (Figs. 2 SIGNAL NETWORKS AND SIGNALING CENTERS 1C and 3B,C). Probably all teeth in one tooth family are initiated sequentially from a single placode. For instance, the All aspects of tooth morphogenesis are regulated by mouse molars develop successionally, starting from the first epithelial-mesenchymal interactions, which are mediated molar and followed by initiation of the second and third by the conserved signaling pathways including Hedgehog molars from a posterior extension of the dental epithelium. (Hh), Wnt, Fibroblast growth factor (FGF), Transforming The individual teeth develop from an epithelial bud growth factor b (Tgfb), Bone morphogenic protein (Bmp), that grows down to the underlying mesenchyme. The neu- and Ectodysplasin (Eda) (Fig. 2). Their interactions, targets, ral crest-derived mesenchyme becomes specified asthe den- and expression patterns have been elucidated in considerable tal mesenchyme condenses around the bud and gives rise to detail in teeth (http://bite-it.helsinki.fi; Bei 2009b; Tum- all the dental tissues except enamel. The epithelial bud in- mers and Thesleff 2009). Epithelial signaling centers play vaginates at its tip and its cervical loops grow to encompass a pivotal role regulating the different steps of tooth devel- the dental papilla mesenchyme, which gives rise to the den- opment. There are three sets of such centers: the placodes, tal pulp and odontoblasts forming dentin (Fig. 1C). The the primary enamel knots, and the secondary enamel mesenchyme surrounding the epithelium and dental pa- knots. Their formation is regulated by epithelial-mesen- pilla becomes the dental follicle and gives rise to the peri- chymal interactions and they all express largely the same ar- odontal tissues and cementoblasts forming the cementum. ray of multiple growth factors. During morphogenesis the epithelium acquires cap and All ectodermal organs begin to develop from a placode, bell shapes and is called enamel organ. It consists of several and the molecular mechanisms of tooth placode formation cell types: the inner enamel epithelium facing the dental and signaling are shared to a great extent with placodes of papilla and differentiating to enamel producing amelo- other organs such as hairs (Mikkola 2009b). One of the blasts, the outer enamel epithelium facing the dental fol- important genes regulating placode formation is the tran- licle, and the stellate reticulum and stratum intermedium scription factor p63 that is expressed throughout the sur- cells in between. The growth and folding of the inner face ectoderm. When p63 function is deleted in mice, the

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A place at the placode stage is further highlighted by the phenotype of several mouse mutants where tooth development

Fgf8Bmp4 Shh Wnts stops before epithelial budding (Bei 2009b). Ectodysplasin (Eda) is a signal of the tumor necrosis factor family and signals through its receptor Edar that is Bmp4 Activin Fgfs locally expressed in the placodes of all ectodermal appen- Pax9, Msx1,2, Runx2, Barx1, Lhx6,7 Dlx1,2,5 dages as well as in primary and secondary enamel knots (Mikkola 2009b). Mutations in the Eda pathway genes Mesenchymal condensation cause the human syndrome hypohidrotic ectodermal dys- and placode formation plasia (HED) manifesting multiple missing teeth as well B as defects in other ectodermal organs, e.g., sparse hair and reduced sweating (Mikkola 2009b). Mice lacking functional Eda often lack third molars or incisors and the cusp patterning of molars is abnormal, indicating a requirement Enamel knot signals: Fgf3,4,9,20 of Eda in the function of placodes and enamel knots (Pispa Shh Wnt3,6,10a,10b et al. 1999). Mice that overexpress Eda in epithelium (under Bmp2,4,7 keratin14-promotor) develop an extra tooth in front of the molars as well as supernumerary hairs and mammary glands (Fig. 5A,B) (Mustonen et al. 2003). The targets of Mesenchymal signals: Eda signaling include molecules from all the other impor- Fgf3,10 Bmp4 Cusp patterning tant signaling pathways (e.g., Shh, Fgf20, Dkk4, ctgf, Folli- Wnt5a,5b and cell differentiation statin) making Eda a key regulator of ectodermal organ C development (Mikkola 2009b). Renewal The primary enamel knot appears in the dental epithelium at the transition from bud to cap stage. In addition to Fgf3 Bmp4 Fgf10 Activin directing crown formation, in molars it determines the positions of the secondary enamel knots which in turn mark the positions of the cusp tips in the molar crown (Fig. 2B) Bmp4 Fgf9 (Jernvall et al. 2000). Wnts are important upstream regula-

Shh tors of enamel knots as shown by the requirement of Lef1 for Fgf4 expression in the enamel knot (Kratochwil et al. 2002) and the induction of new enamel knots and placodes

Maintenance of stem cells by forced activation of Wnt/b-catenin signaling in oral and ameloblast production epithelium (Ja¨rvinen et al. 2006; Wang et al. 2009). More than a dozen different signal molecules belonging to all Figure 2. Cross talk between epithelium and mesenchyme through four conserved signal families are locally expressed in the the conserved signaling pathways regulates all aspects of tooth devel- primary and secondary enamel knots. The enamel knots opment. When tooth development is initiated, signals from the epi- initiate and regulate the folding of the epithelium by stim- thelium activate a set of transcription factors in the mesenchyme, leading to condensation of the mesenchyme and formation of the ep- ulating the surrounding epithelium to proliferate through ithelial placode (A). The enamel knot is a signaling center expressing Fgfs (Fgfs 3, 4, 9, and 20) and remaining nonproliferative multiple signaling molecules that induce reciprocal signals from the themselves. They express the cyclin-dependent kinase in- mesenchyme. Enamel knots determine the position of the cusps and hibitor p21 and lack Fgf receptors making them insensitive initiate differentiation of odontoblasts (B). Tgfb, Bmp, and Shh sig- to the proliferative signals (Jernvall et al. 1998; Kettunen naling regulate epithelial-mesenchymal interactions in the cervical loop of the mouse incisor. They support the maintenance and prolif- et al. 1998). The Fgfs also signal to dental mesenchyme and eration of the stem cells as well as ameloblast differentiation and en- induce e.g., Runx2, and Fgf3, which signals back to epithe- amel production (C). lium illustrating the bidirectional Fgf signaling between epithelium and mesenchyme regulating tooth morpho- placodes of teeth and other ectodermal appendages do not genesis (Klein et al. 2006). Shh from the enamel knot develop, but the dental lamina forms (Laurikkala et al. stimulates epithelial morphogenesis indirectly via the mes- 2006). Key signaling pathways including Bmp, Fgf, Notch, enchyme (Gritli-Linde et al. 2002). and Eda are impaired in the absence of p63 (Laurikkala Important aspects of enamel knot signaling are the et al. 2006). The importance of the signaling that takes modulation and ﬁne-tuning, which affect the patterning

4 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Signaling Networks Regulating Tooth Development of the secondary enamel knots and thereby the pattern influenced by activation and inhibition between succes- of molar cusps via lateral inhibition and reaction diffusion sionally developing teeth (Kavanagh et al. 2007), the size mechanisms. Different shapes of molars can be generated and number of mouse incisors is affected by fine-tuning by mathematical modeling using parameters of activating Bmp signaling in the placodes (Munne et al. 2010), and and inhibiting enamel knot signals, and it has been sug- the continuous growth and enamel deposition in incisors gested that changes in signaling during evolution are re- can be modulated by the levels of Fgf, Activin, and Bmp sig- sponsible for the species-specific cusp patterns (Salazar- naling in the epithelial stem cell niche (Fig. 2C) (Wanget al. Ciudad and Jernvall 2002). This hypothesis has gained ex- 2007). perimental support from phenotypes of transgenic mice where signal modulation has resulted in phenotypes resem- 3 REGULATION OF THE IDENTITY AND bling teeth of other species. Examples include molars DIFFERENTIATION OF ODONTOGENIC of K14-Eda resembling kangaroo teeth, and of Sostdc1 MESENCHYMAL AND EPITHELIAL CELL knockout (inhibitor of Wnt and Bmp signaling) resem- LINEAGES bling rhino teeth (Kangas et al. 2004; Kassai et al. 2005). Furthermore, epithelial deletion of Dicer, which is required Classical recombination experiments have shown that the for processing of microRNA (miRNA), results in the mod- odontogenic potential shifts from the epithelium to mes- ulation of molar cusp pattern (Michon et al. 2010). enchyme in mouse teeth between embryonic days 11 and Fine-tuning of the activity of the conserved signaling 12, i.e., around the time of placode formation (Fig. 3). pathways controls many other aspects of tooth formation When epithelium of the first branchial arch from an E9- as well. For example, a supernumerary tooth forms in front 11 mouse embryo was recombined with second arch mes- of the first molar in several mutant mouse lines when sig- enchyme, a tooth formed (Fig. 3A) (Mina and Kollar 1987). naling activity is modulated. These teeth do not represent Similarly, first arch epithelium from an E9-10 embryo in- de novo tooth induction. Instead they form from activation duced tooth formation when recombined with cranial neu- of the development of a vestigial tooth rudiment found in ral crest cells that normally form the dental mesenchyme wild-type mice in the diastema and represent premolars and, interestingly, also when combined with premigratory lost during the evolution of rodents. Examples are the trunk neural crest cells (Lumsden 1988). At E12 the epithe- K14-Eda mouse (Fig. 5A,B) (Mustonen et al. 2003) and lium no longer has inductive potential and now the first the Sprouty mutants (Klein et al. 2006). In the Osr2 knock- archmesenchymecaninducetooth formationwhenrecom- out an extra tooth develops lingually to the first molar bined with second arch epithelium (Fig. 3A). The mesen- (Zhang et al. 2009). This is accompanied by spreading of chyme from E13 or older tooth germs has the information Bmp4 expression to the lingual mesenchyme and results on the tooth identity as the shape of the tooth in the recip- probably from a subsequent broadening of the dental field rocal recombinations between incisor and molar epithe- (Mikkola 2009a). The relative sizes of the mouse molars are lium and mesenchyme will form according to origin of

A 100 B Epithelium Mesenchyme t

Pitx2 E11 50 C * * t odontogenic potential (%)

0 Pitx2 E12.5 E10 E11E12 E13 Age

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

Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 5 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press M. Jussila and I. Thesleff the mesenchyme (Kollar and Baird 1969). In addition, the et al. 2009). The signals induced in the mesenchyme by dental papilla can induce tooth formation when recom- epithelial Fgfs and acting reciprocally to the epithelium in- bined with limb epithelium (Kollar and Baird 1970). It clude Activin, Fgf3, and Fgf10 (Ferguson et al. 1998; Ket- has been proposed, based on in vitro experiments, that tunen et al. 2000). These signals regulate the subsequent the incisor versus molar identity of teeth is determined by epithelial morphogenesis and the enamel knot formation the level of Bmp signaling (Tucker et al. 1998). However, (Fig. 2A). this conclusion was challenged recently by the observation The shift of the odontogenic competence from epithe- that inhibition of Bmp signaling caused partial splitting lium to mesenchyme is accompanied by the induction of of the incisor placode resulting in the formation of two important transcription factors in the dental mesenchyme fused incisors rather than incisor to molar transformation (Fig. 2A). The deletion of the function of several of these (Munne et al. 2010). either alone or together with another transcription factor The molecular basis of odontogenic competence in in the same family results in tooth arrest at placode or early jaw epithelium and later in the condensed dental mes- bud stage. All four signal pathways have been shown to enchyme remains elusive. As all the genes that are known be involved in the regulation of these transcription factors to regulate tooth development are also expressed in other (Bei 2009b). For example, Bmp4 induces the expression of developing organs, it seems that there is no single tooth- Msx1 and Fgf8 induces the expression of Pax9 (Vainio et al. specific gene that defines the odontogenic tissues. Cur- 1993, Neubu¨ser et al. 1997). Other targets of Bmp and Fgf rently only few genes such as Sonic hedgehog (Shh) and signaling in mesenchyme at this stage include Lhx6,7, the transcription factor Pitx2 are known to be restricted Barx1, Dlx1,2, and Runx2 (Bei 2009b, Tummers and Thes- to the dental lamina (Fig. 3B,C) (Kera¨nen et al. 1999). It leff 2009). The Shh mediators Gli2 and Gli3 are expressed in is not known how the dental lamina becomes established, the mesenchyme and are required for tooth formation and to date there is no mouse mutant reported where the (Hardcastle et al. 1998). In addition, the expression of dental lamina would be missing. All teeth develop within Lef1, a Wnt effector, shifts from the epithelium to the mes- the dental lamina, evenin micewhere extrateethare induced. enchyme together with the shift in the odontogenic poten- Activation of Wnt signaling in the epithelium induces super- tial and is regulated by Bmp4 in mesenchyme (Kratochwil numerary placodes throughout the surface epithelium and et al. 1996). Perhaps a combination of these transcription they give rise tovarious epithelial appendages. Yet extra teeth factors constitutes the code for the odontogenic identity form only in the region of dental arches and mostly in con- of the mesenchyme. nection with other teeth (Ja¨rvinen et al. 2006; Wang et al. The differentiation of the tooth-specific cell types is in- 2009). These observations indicate that the odontogenic timately linked with epithelial morphogenesis. Odonto- competence is present only in the oral region. blasts and cementoblasts differentiate from the lineage of It is likely that spatiotemporal patterns of the epithelial dental mesenchyme, the odontoblasts from the dental pa- signals are involved in the shift of competence to mesen- pilla, and cementoblasts from the dental follicle, whereas chyme (Fig. 2A). In addition to Shh, which is restricted ameloblasts differentiate from the epithelial lineage. They to the dental lamina, many Wnt ligands are expressed in are responsible for the formation and the deposition of the oral epithelium (Sarkar and Sharpe 1999). Wnt/b-cat- the extracellular matrices of the tooth-specific mineralized enin signaling regulates Fgf8 expression in the early epithe- tissues, dentin, cementum, and enamel, respectively. It is lium (Wang et al. 2009), and placodes do not form in mice not known exactly at which stage of tooth formation the overexpressing the Wnt inhibitor Dkk1 (Andl et al. 2002). cells become committed, but the final steps of odontoblast Bmp4 and Fgf8 are expressed in the jaw epithelium in over- and ameloblast differentiation have been analyzed in detail lapping patterns before any morphological sign of tooth during the bell stage of tooth formation. development. Bmp4 is expressed more distally at the site The mesenchyme is first induced to differentiate into where molars will develop and Fgf8 more proximally in odontoblasts by the inner enamel epithelium. The differen- the incisor region (Neubu¨ser et al. 1997). Epithelial Bmps tiation starts from the cusp tips and proceeds downward to induce the expression of Bmp4 in the mesenchyme before cervical and intercuspal directions. Signals in Tgfb/Bmp bud stage correlating with the shift in the odontogenic po- families have been implicated in odontoblast induction tential (Vainio et al. 1993). Also, Wnt/b-catenin signaling (Ruch et al. 1995), and it was shown recently that the condi- in the incisor mesenchyme stimulates the expression of tional loss of Smad4, a mediator of Tgfb/Bmp signaling, Bmp4 that in turn regulates Shh in the epithelium (Fujimori from the dental papilla prevents the terminal differen- et al. 2010). Furthermore, Wnt signaling was shown to be tiation of odontoblasts and dentin deposition (Li et al. required in the molar mesenchyme for the bud to cap stage 2011a). As the formation of enamel knots is temporally transition and primary enamel knot formation (Chen associated with the initiation of odontoblast differentiation

6 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Signaling Networks Regulating Tooth Development at the cusp tips, the enamel knot signals have been sug- role in amelogenesis in mice (Bei 2009a). Mutations in gested to play a role (Fig. 2B) (Thesleff et al. 2001). One ameloblast-speciﬁc genes including ameloblastin, ameloge- of these signals, Wnt10b, was suggested to regulate the ex- nin, enamelin, and Mmp20 cause human amelogenesis pression of dentin sialophosphoprotein (Dspp) and odonto- imperfecta (Bei 2009a). Very little is known about the mo- blast differentiation (Yamashiro et al. 2007). The localization lecular regulation of cementoblast development. Bmp sig- of Wnt reporter activity in odontoblasts is also in line with naling was reported to induce cementoblast differentiation the role of Wnts in the process (Suomalainen and Thesleff from dental follicle cells, whereas Wnt signaling promotes 2010). In addition, the basement membrane is important their proliferation (Zhao et al. 2002; Nemoto et al. 2009). for the polarization and differentiation of the odontoblasts and serves presumably as a reservoir of signal molecules (Thesleff and Hurmerinta 1981; Ruch et al. 1995). Dentin 4 REGULATION OF TOOTH REPLACEMENT, is composed mainly of type I collagen, dentin phosphopro- CONTINUOUS GROWTH, AND STEM CELLS tein, and Dspp, and mutations in these genes cause dentino- IN TEETH genesis imperfecta in humans (Shields et al. 1973). As the mouse teeth are not replaced, relatively little is After the odontoblasts have been induced to differenti- known about the mechanisms of tooth replacement in + ate, they signal back to the epithelium. The signals from mammals. Histological observations in nonmodel animals the mesenchyme involved in the ameloblast induction indicate that replacement teeth develop from the dental include Bmp2, Bmp4, and Tgfb1 (Fig. 4) (Coin et al. lamina associated with their predecessors (Luckett 1993; 1999; Wang et al. 2004). In addition, Shh from the epithe- Ja¨rvinen et al. 2009). The ferret (Mustela putorius furo)re- lial stratum intermedium cells is required to support ame- places its incisors, canines, and premolars, and it was shown loblast differentiation and maturation (Dassule et al. 2000; that the deciduous teeth are connected to each other by a Gritli-Linde et al. 2002). Other epithelial growth factors continuous dental lamina, and the permanent teeth start regulating ameloblasts are TFGb1, Wnt3, Eda, and Follista- to grow from the lingual side of each deciduous tooth as tin (Bei 2009a). Ameloblasts express transcription factors an extension of the dental lamina (Fig. 5C–E) (Ja¨rvinen such as Sp6 and Msx2 that have been shown to play a etal.2009).Similarly,inthereptilesthereplacementtooth

A B NB Lingual - enamel free *

Dentin Pulp

Enamel o Cervical Labial - enamel loop a Amelogenin

C E16 D Bmp4 bead

Bmp4 Ameloblastin

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

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A K14-Eda B K14-Eda

m1 m2 m3

Shh E14.5

C D E

dl dl dl dC dC P3 dP3 C C E33 Pitx2 E33Sostdc1 E35

Δ F -cat Δx3K14/+ G wt H -cat ex3K14/+

Fgf20 E14.5 Fgf20 E14.5

Figure 5. Supernumerary teeth, tooth replacement, and continuous tooth renewal. Overexpression of ectodysplasin in the surface epithelium results in development of a supernumerary tooth (arrow) in front of the molars (m1-3) in the 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 the ferret 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 intersection between the dental lamina and the deciduous third premolar (dP3) at the time when permanent P3 is initiated in the E35 ferret embryo (arrow) (E). Stimulation of Wnt signaling by stabilized b-catenin in mouse oral epithelium leads to 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 ). arises from an outgrowth of the dental lamina each time the and it is promoted by Shh and Bmp signaling from the mes- previous tooth has grown to a certain size (Richman and enchyme (Richman and Handrigan 2011). Handrigan 2011). In contrast, in the fish species studied, The phenotypes of some human syndromes and their thereseemstobenosuccessionaldentallamina,andthe mouse models support the role of Wnt signaling in tooth new teeth are initiated directly from the epithelium of the pre- replacement. Mutations in the human AXIN2 gene cause vious tooth or from the oral epithelium (Smith et al. 2009). oligodontia, which specifically affects permanent teeth Wnt signaling has been associated with tooth replace- (Lammi et al. 2004). On the other hand, supernumerary ment both in mammals and reptiles and may be a key factor teeth are common in familial adenomatous polyposis regulating tooth renewal across vertebrates (Ja¨rvinen et al. (FAP), which is caused by mutations in APC, an inhibitory 2009; Richman and Handrigan 2011). In the ferret, the ex- component of the Wnt pathway, and the patients also de- pression of Sostdc1, an inhibitor of Wnt and Bmp signaling, velop odontomas, benign tumors composed of numerous marks the border between the deciduous tooth and the small teeth (Wang and Fan 2011). A similar phenotype is dental lamina that gives rise to the permanent tooth (Fig. seen in mice when Wnt signaling is activated in the epithe- 5E) (Ja¨rvinen et al. 2009). The expression of Axin2, a feed- lium either by deletion of APC or stabilization of b-catenin back inhibitor of Wnt signaling, was also detected in the (Fig. 5F–H) (Ja¨rvinen et al. 2006; Liu et al. 2008; Wang mesenchyme between the tooth and the growing dental et al. 2009). Sp62/2 (Epiprofin) mutants have a similar lamina (Ja¨rvinen et al. 2009). During snake tooth replace- phenotype but this gene has not been associated with hu- ment, there is Wnt activity in the tip of the dental lamina man conditions (Nakamura et al. 2008; Wang and Fan

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2011). The teeth in the Wnt gain-of-function mouse models cervical loop contains label-retaining stem cells and transi- were shown to develop successionally from previous teeth re- ent amplifying (TA) cells (Harada et al. 1999; Seidel et al. sembling the continuous generation of the simple-shaped 2010). The stem cells reside within the stellate reticulum replacement teeth in fish and reptiles. This led to a sugges- cells in the core of cervical loop, which is surrounded tion, that Wnt signaling may have been involved in the re- by dental mesenchyme. The progeny of stem cells invades duction in the replacement capacity and in the gain in the basal epithelium and proliferates as TA cells before dif- tooth complexity during evolution (Ja¨rvinen et al. 2006). ferentiating into ameloblasts (Figs. 2C and 4A,B). Interestingly, the phenotypes of two syndromes suggest Mesenchymal signals play key roles in the regulation of that the capacity for continued tooth replacement can be the epithelial stem cells and their progeny (Fig. 2C). Fgf10 unlocked in humans. The supernumerary teeth were sug- is the key mesenchymal signal required for epithelial stem gested to represent a third dentition in cleidocranial dys- cell maintenance and proliferation, and Fgf3 has a partly plasia (CCD) and in a novel craniosynostosis syndrome, redundant function as stimulator of TA cell proliferation caused by mutations in the transcription factor RUNX2 (Wang et al. 2007). Mesenchymal Fgfs act in a regulatory and interleukin receptor IL11RA, respectively (Jensen and loop with epithelial Fgfs, notably Fgf9, and stimulation of Kreiborg 1990; Nieminen et al. 2011). Unfortunately, the Fgf signaling by deleting the function of Sprouty genes results mouse models of these syndromes do not show supernum- in extensive growth of the incisors and ectopic deposition of erary teeth, likely because the mouse teeth are not normally enamel on the lingual surface (Klein et al. 2008). Lingual replaced, and they are therefore not suitable for studies on enamel also forms in Follistatin knockout mice, whereas tooth replacement (D’Souza et al. 1999; Nieminen et al. enhanced Follistatin expression results in complete absence 2011). Runx2 has been associated with Fgf as well as Wnt of enamel from labial side as well as in growth inhibition signaling in tooth development as the induction of the (Wang et al. 2004). Follistatin is expressed in the lingual epi- Wnt inhibitor Dkk1 in dental mesenchyme by epithelial thelium and it antagonizes Activin function in the cervical Fgf4 requires Runx2 (James et al. 2006). loop epithelium while, interestingly, inhibiting Bmp4 func- Putative epithelial stem cells have been identified during tion in the zone of differentiation, which prevents enamel tooth replacement in gecko, a reptile (Handrigan et al. formation. It was shown that Bmp signaling is required for 2010). These cells reside in the lingual side of dental lamina ameloblast differentiation (Fig. 4) (Wang et al. 2004). Ac- and express some known stem cell marker genes. It is possi- cordingly, when the Bmp inhibitor Noggin is overexpressed ble that the reduction of tooth replacement capacity in in epithelium, the mouse incisors grow extensively and lack mammals to maximallyone replacement has involved deple- enamel (Plikus et al. 2005). Bmps and Activin function in a tion of such stem cells and that they are maintained in the regulatory network with Fgf3, which is inhibited by Bmp4, cleidocranial dysplasia and craniosynostosis syndromes. which is in turn repressed by Activin that is strongly ex- Although the human teeth do not regenerate and their pressed in the labial dental mesenchyme but not on the lin- development is completed already during adolescence, there gual side. This contributes to the asymmetric production of are stem cells in the adult teeth. Human dental mesenchymal TA cells only in the labial cervical loop (Wang et al. 2007). stem cells were first isolated from the dental pulp and when Fgf and Shh signaling play a role in the postnatal ho- transplanted they formed dentin (Gronthos et al. 2000). meostasis of the TA cell production in the mouse incisors Stem cells in periodontal ligament were shown to produce but not in stem cell survival (Parsa et al. 2010; Seidel cementum and periodontal ligament-like structures (Seo et al. 2010). On the other hand, Wnt signaling activity is et al. 2004). Similar stem cells were also characterized in ex- not detected in the stem cells in the cervical loops (Suoma- foliated deciduous teeth and third molars (Rodriguez-Loza- lainen and Thesleff 2010). Some stem cell marker genes, no et al. 2011). Epithelial stem cells may reside within the such as Lgr5, Bmi1, Oct3/4, and Yap, have been localized epithelial cell rests of Malassez as these cells can be induced in the incisor stem cells (Suomalainen and Thesleff 2010; to ameloblastlike cells (Shinmura et al. 2008). Li et al. 2011b). Despite the intense investigations on the Some mammals have teeth that grow continuously signal pathways regulating the incisor stem cell niche, the throughout life and thus have stem cells. The most studied characterization of these stem cells is still on its way. such tooth is the mouse incisor, which harbors epithelial stem cells in a niche situated in the cervical loop at its prox- imal end (Fig. 2C) (Harada et al. 1999). The mouse incisor 5 FUTURE CHALLENGES: STEM CELL-BASED has an asymmetric structure with enamel deposited only on BIOENGINEERING OF TEETH the labial side, whereas the lingual side is covered by dentin Different scenarios have been proposed for bioengineering (Fig. 4A). The asymmetry arises from differences between of human teeth. One possibility could be the direct induc- the lingual and labial cervical loops as only the larger labial tion of tooth development in the jaws with activators such

Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 9 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press M. Jussila and I. Thesleff as Wnt and Eda. However, although supernumerary teeth embryonic dental tissue, collecting dental stem cells from are induced in mouse models and in human syndromes adults is challenging as it would imply sacrificing a tooth by modulation of signal pathways, it is not likely that this from the patient needing a new tooth. approach would function in the adult jaws. The main rea- Odontogenic cells might be produced from adult so- son is that the supernumerary teeth in mice—as well as in matic cells by iPS cell technology or direct reprogramming the rare human syndromes—form from the tissue associ- (Hanna et al. 2010). Thus they could be first reprogram- ated with developing teeth and such teeth would not be med to embryonic stem cells by iPS technology and pro- present in adult jaws anymore. The stem cells discovered grammed further to dental epithelial or mesenchymal cell in adult teeth described above, including the mesenchymal fates. Oral mucosal epithelium and stromal cells could be stem cells in dental papilla, pulp, and periodontal ligament feasible sources for reprogramming because their develop- have the capacity to generate cells forming dentin, cementum, mental history is likely more similar to dental tissues. Alter- and periodontal ligament, but it is unlikely that they have natively, the oral mucosal cells or other adult somatic cells morphogenetic potential, and even less likely that the cells could perhaps be directly converted to tooth epithelium couldbetargetedinvivotoundergotoothmorphogenesis. and mesenchyme as recently shown in other tissues (Zhou A more realistic approach would be to engineer a tooth et al. 2008; Hanna et al. 2010). in vitro and implant it to patient’s mouth. It has been pro- The knowledge lacking at the moment is the molecular posed that such teeth could be generated by growing cells in signatures of the epithelial and mesenchymal lineages that tooth-shaped scaffolds. However, taking into account the could be used in reprogramming. The key genes likely complex structure and organization of the hard and soft include transcription factors expressed by the early embry- tissues of teeth and the fact that tooth size and shape onic tissues such as Pitx2 in the branchial arch epithelium emerge during the multistep process of morphogenesis to- and Msx1,2, Dlx1,2,5, Runx2, Pax9, Lhx6,7,8, and Prx1,2 gether with the periodontal tissue attaching the tooth to the in the odontogenic mesenchyme (Fig. 2A) (Thesleff and bone, it is difficult to imagine how a functional tooth could Tummers2008).Sofartheseareonlyeducatedguessesbased be developed within a scaffold. Therefore, the preferable on expression patterns and mutant phenotypes (http:// way would be to trigger the initiation of tooth development bite-it.helsinki.fi; Bei 2009b). program in progenitor cells and let the tooth develop itself. Finally, it should be noted that it is unrealistic to aim at The classical tooth bud transplantation and tissue re- generating a perfect tooth crown, because it is rather ob- combination experiments have shown that the program vious that the right shape and size of the crown as well as for tooth morphogenesis is present very early in the jaws the color and proper structure of enamel cannot be gener- and that a tooth bud can form a complete tooth when ated by bioengineering. Therefore, the crown needs to be transplanted to various ectopic sites. The proof of principle completed prosthetically. The most important aspect of experiments in mouse have already shown that even disso- the bioengineered tooth would be a functional root pro- ciated embryonic dental epithelial and mesenchymal cells viding physiological anchorage of the tooth to jaw bone. can regenerate a tooth germ in vitro and that this forms a However, initiation of root formation, without a preceding functional tooth when implanted to the jaw of an adult crown appears impossible, at least by mimicking normal mouse (Nakao et al. 2007; Oshima et al. 2011). developmental mechanisms. The physiological anchorage To use such an approach in human therapy, one would is lacking in the titanium implants that otherwise are suc- need to replace the embryonic dental cells with adult cells cessfully used for tooth replacement. Tothis end interesting preferably with tooth forming capacity. Obviously, both experiments have been performed in minipigs, where stem epithelial and mesenchymal cell lineages are needed, but cells from the root apical papilla and periodontal ligament based on the classical recombination experiments only stem cells were used. When the cells were seeded on a root- one cell type needs to have the odontogenic potential. shaped cylindrical hydroxyapatite/tricalcium phosphate Although there is some evidence that adult mouse bone scaffold, and implanted in the jaw, dentin, cementum, and marrow stem cells can form a tooth together with embry- periodontal ligament were generated and a structure resem- onic branchial arch epithelium (Ohazama et al. 2004), it bling root developed (Sonoyama et al. 2006). It is not yet is probable that in the recombinations the nonodontogenic known whether the bioengineered root has adequate phys- mesenchyme should have properties of cranial neural crest iological properties to be used in clinical tooth replacement. and the epithelium should be ectodermal. The inductive potential of the current human dental stem cell lines has not been explored. However, although dental stem cells 6 CONCLUDING REMARKS from adult teeth could perhaps be used for tooth bioengin- Studies on the laboratory mouse have given a great deal of eering because they are likely to share characteristics with knowledge on the molecular regulation of tooth initiation,

10 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008425 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Signaling Networks Regulating Tooth Development morphogenesis, and stem cell maintenance. However, be- Hardcastle Z, Mo R, Hui C-C, Sharpe PT. 1998. The Shh signalling path- fore the building of teeth by tissue engineering becomes a way in tooth development: Defects in Gli2 and Gli3 mutants. Develop- ment 125: 2803–2811. reality, more detailed understanding of the process of tooth James MJ, Ja¨rvinen E, WangXP,Thesleff I. 2006. Different roles of Runx2 development and regeneration is required. In particular, during early neural crest-derived bone and tooth development. J Bone the gene regulatory networks during cell lineage specifi- Miner Res 21: 1034–1044. cation in dental epithelium and mesenchyme need to be Ja¨rvinen E, Salazar-Ciudad I, Birchmeier W, Taketo MM, Jernvall J, The- sleff I. 2006. Continuous tooth generation in mouse is induced by ac- understood more thoroughly and the origins of the two tivated epithelial Wnt/b-catenin signaling. Proc Natl Acad Sci 103: types of progenitor cells to be used for tooth bioengineer- 18627–18632. ing should be determined. Ja¨rvinen E, Tummers M, Thesleff I. 2009. The role of the dental lamina in mammalian tooth replacement. J Exp Zool B Mol Dev Evol 312B: 281–291. ACKNOWLEDGMENTS Jensen BL, Kreiborg S. 1990. Development of the dentition in cleidocranial dysplasia. J Oral Pathol Med 19: 89–93. We thank Emma Juuri, Otso Haä¨ra¨, Elina Ja¨rvinen, and Jernvall J, A˚ berg T,Kettunen P,Kera¨nen S, Thesleff I. 1998. The life history Aapo Kangas for providing illustrations. of an embryonic signaling center: BMP-4 induces P21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125: 161–169. REFERENCES Jernvall J, Kera¨nen SV,Thesleff I. 2000. 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Signaling Networks Regulating Tooth Organogenesis and Regeneration, and the Specification of Dental Mesenchymal and Epithelial Cell Lineages

Maria Jussila and Irma Thesleff

Cold Spring Harb Perspect Biol 2012; doi: 10.1101/cshperspect.a008425 originally published online March 13, 2012

Subject Collection Mammalian Development

The Dynamics of Morphogenesis in the Early Cell Division Modes and Cleavage Planes of Mouse Embryo Neural Progenitors during Mammalian Cortical Jaime A. Rivera-Pérez and Anna-Katerina Development Hadjantonakis Fumio Matsuzaki and Atsunori Shitamukai microRNAs as Developmental Regulators Blood and Lymphatic Vessel Formation Kathryn N. Ivey and Deepak Srivastava Victoria L. Bautch and Kathleen M. Caron Development of the Endochondral Skeleton Transcriptional Networks in Liver and Intestinal Fanxin Long and David M. Ornitz Development Karyn L. Sheaffer and Klaus H. Kaestner Adipogenesis Pluripotency in the Embryo and in Culture Kelesha Sarjeant and Jacqueline M. Stephens Jennifer Nichols and Austin Smith Molecular Mechanisms of Inner Ear Development Signaling and Transcriptional Networks in Heart Doris K. Wu and Matthew W. Kelley Development and Regeneration Benoit G. Bruneau Polarity in Mammalian Epithelial Morphogenesis Signals and Switches in Mammalian Neural Crest Julie Roignot, Xiao Peng and Keith Mostov Cell Differentiation Shachi Bhatt, Raul Diaz and Paul A. Trainor Eye Development and Retinogenesis Hematopoiesis Whitney Heavner and Larysa Pevny Michael A. Rieger and Timm Schroeder Primordial Germ Cells in Mice Intercellular Interactions, Position, and Polarity in Mitinori Saitou and Masashi Yamaji Establishing Blastocyst Cell Lineages and Embryonic Axes Robert O. Stephenson, Janet Rossant and Patrick P.L. Tam For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/