Observations on Continuously Growing Roots of the Sloth and the K14-Eda

Home , Cementoblast, Cervical loop, Epithelial root sheath, Stellate reticulum

EVOLUTION & DEVELOPMENT 10:2, 187–195 (2008)

Observations on continuously growing roots of the sloth and the K14-Eda transgenic mice indicate that epithelial stem cells can give rise to both the ameloblast and root epithelium cell lineage creating distinct tooth patterns

Mark TummersÃ and Irma Thesleff1 Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland ÃAuthor for correspondence (email: [email protected]) 1Current address and address at time of work for both authors: Institute of Biotechnology, P.O. Box 56, FIN-00014 University of Helsinki, Finland.

SUMMARY Root development is traditionally associated that it acts as a functional stem cell niche. Similarly we show with the formation of Hertwig’s epithelial root sheath (HERS), that continuously growing roots represented by the sloth molar whose fragments give rise to the epithelial cell rests of and K14-Eda transgenic incisor maintain a cervical loop with a Malassez (ERM). The HERS is formed by depletion of the small core of stellate reticulum cells around the entire core of stellate reticulum cells, the putative stem cells, in the circumference of the tooth and do not form a HERS, and still cervical loop, leaving only a double layer of the basal give rise to ERM. We propose that HERS is not a necessary epithelium with limited growth capacity. The continuously structure to initiate root formation. Moreover, we conclude that growing incisor of the rodent is subdivided into a crown analog crown vs. root formation, i.e. the production of enamel vs. half on the labial side, with a cervical loop containing a large cementum, and the differentiation of the epithelial cells into core of stellate reticulum, and its progeny gives rise to enamel ameloblasts vs. ERM, can be regulated independently from producing. The lingual side is known as the root analog and the regulation of stem cell maintenance. This developmental gives rise to ERM. We show that the lingual cervical loop flexibility may underlie the developmental and evolutionary contains a small core of stellate reticulum cells and suggest diversity in tooth patterning.

INTRODUCTION abrasive diets. For instance, a significant increase in the prev- alence of hypsodonty in commonly found mammals of many The teeth can be roughly subdivided into three groups. The taxonomic groups occurred during the Neogene due to an first group consists of brachydont or low-crowned teeth where increased aridity in the environment of Europe (Jernvall and the root is relatively long compared with the crown. This is Fortelius 2002). Hypselodonty can be seen as an extreme form the tooth type usually described in textbooks when describing of hypsodonty. The crown never stops growing and root for- root formation (Nanci 2003). The second group consists of mation is postponed indefinitely, but often with small tracts of hypsodont, or high–crowned, teeth, where the crown is high dentin covered with cementum acting as regions attaching the compared with the root. The third group consists of the tooth to the jaw bone with a periodontal ligament. hypselodont teeth, ever-growing or open-rooted teeth that Within closely related species there can be a variation be- grow continuously during the lifetime of the animal. Open- tween brachydont, hypsodont, and hypselodont teeth, indi- rooted refers solely to the large apical opening present in all cating that the regulation of crown height is rather flexible. continuously growing teeth and does not imply that the tooth For instance in closely related rodent species, the Mouse (Mus actually needs to have a root in a classical sense as described musculus) molar is brachydont, the molars of the Bank vole in the textbooks. and the Southern Red-backed vole (Clethrionomys glareolus, It is thought that brachydonty is the ancestral state Clethrionomys gapperi) are hypsodont, and the molars of the of mammalian teeth. During evolution the shift from Sibling vole and Meadow vole (Microtus rossiaemeridionalis brachydont to hypsodont teeth is a common phenomenon and Microtus clethrionomys) are hypselodont (Phillips and (Macfadden 2000). This trend is often initiated by environ- Oxberry 1972; Tummers and Thesleff 2003). It has been mental pressures. Teeth with higher crowns last longer with proposed that the switch from hypsodont to hypselodont

& 2008 The Author(s) 187 Journal compilation & 2008 Blackwell Publishing Ltd. 188 EVOLUTION & DEVELOPMENT Vol. 10, No. 2, MarchÂpril 2008 between the microtine genera of Clethrionomys and Microtus basal epithelium is left that is known as Hertwig’s epithelial is caused by the maintenance of a regenerative unit, possibly root sheath (HERS) (Thomas 1995). As HERS proliferates, due to a simple mutation (Phillips and Oxberry 1972), or that the growing epithelial sheet becomes discontinuous and forms increased crown height results from delayed termination/ a fenestrated network lining the root surface known as the cytodifferentiation and that hypselodonty is an extreme out- epithelial cell rests of Malassez (ERM) (Ten Cate 1996). come of such a delay (von Koenigswald 1982). We have more Through this network the follicular mesenchyme cells can recently proposed that the increase in crown height is a result migrate to the dentin surface and differentiate into cemento- of prolonging the period during which the epithelial stem cell blasts depositing the cementum. The ERM functions in the niche is maintained (Tummers and Thesleff 2003). induction of cementoblast differentiation and regulation of During classic root formation the dental epithelium of their function (Thomas 1995; Bosshardt and Schroeder 1996; the cervical loop undergoes some major structural changes Kagayama et al. 1998). Fibers of the periodontal ligament are (Fig. 1). The cervical loop is created during crown morpho- embedded in the cementum and connect the root to the jaw genesis and with root initiation loses the central core of bone. HERS forms in brachydont and hypsodont teeth when stellate reticulum and stratum intermedium cells, including the root formation is initiated and crown formation ends, and its putative epithelial stem cells (Ten Cate 1961; Starkey 1963; transition to ERM is generally regarded as a typical feature of Harada et al. 1999; Harada et al. 2002). A double layer of root formation. Interestingly, the continuously growing rodent incisor is subdivided into two halves. The labial side is called crown analog because it produces ameloblasts and enamel, whereas the lingual half is called root analog, because its epithelium fragments and forms ERM and cementum is produced. Both root and crown analogs are generated continuously by the apical end of the incisor. It has been suggested that the labial cervical loop of the crown analog in the incisor is a specialized stem cell niche providing the epithelial progeny for the entire incisor and that lingually HERS is formed (Ohshima et al. 2005). This last notion is questioned by the existence of a special type of continuously growing or hypselodont teeth as is represented by the sloth molar. The dentition of the contemporary sloth species is heavily modified, lacking both incisors and ca- nines. Sloth teeth are open-rooted, grow continuously, and at the same time lack enamel (Naples, 1982). In juvenile speci- Fig. 1. Formation of the cervical loop, the putative epithelial stem mens the tooth erupts as a simple cone. In adult specimens the cell niche, during early development and its fate in the mouse molar cap of the dentin is worn off, leaving a hard shell of dentin with and incisor. During early stages of morphogenesis all teeth go a soft pulp in the center. The edges of the dentin get sharper through the same developmental stages (initiation, bud stage, cap stage, and the bell stage) generating the crown. During bud stage a with age due to wear (Naples 1982). Similarly, the dentition of core of loosely arranged epithelium is formed in the center of the the mouse, as a representative of the rodents, is also heavily bud. During the cap stage the cervical loop is formed, a protrusion modified during evolution, with only two incisors in each jaw, from the bud that envelopes the condensed dental papilla me- followed by a diastema region lacking teeth, and three molars. senchyme. The cervical loop is extended during the bell stage and The transgenic K14-Eda mouse has ectodysplasin (Eda) the inner enamel epithelium starts to differentiate into ameloblasts. During the late bell stage crown morphology is established and expressed under the keratin 14 promoter leading to an ex- cells producing mineralized tissues differentiate terminally. Cell cessive production of Eda throughout the ectoderm from E10 differentiation starts from the cusp tips and extends toward the onwards, including the oral and dental epithelium (Mustonen base. Enamel is deposited by ameloblasts and dentin by odonto- et al. 2003). The constitutive expression of Eda in the dental blasts. In the mouse molar the cervical loop loses its core of stellate epithelium leads to the formation of supernumerary molars reticulum, the putative stem cells, and forms the HERS, which fragments into ERM, typical of a root. On the labial side of the and the loss of enamel on crown analog of the incisors. This mouse incisor the cervical loop is maintained as a stem cell niche transgenic mouse line therefore has possibly transformed its and it keeps giving rise to ameloblasts. On the lingual side no incisor into a continuously growing root, and serves in this ameloblasts differentiate and instead ERM is formed. The fate of paper as a model system for continuously growing roots. If the lingual cervical loop is unclear, although it has been suggested HERS is required for the production of root epithelium, these that HERS is present (Ohshima et al. 2005). ERM, epithelial cell rests of Malassez; HERS, Hertwig’s epithelial root sheath; iee, teeth would not have a cervical loop and a stem cell niche. inner enamel epithelium; oee, outer enamel epithelium; sr, stellate Here we investigate the structure of the cervical loop area reticulum. of continuously growing roots of a sloth molar that has Tummers and Thesle¡ Root growth and epithelial stem cells 189 erupted into the oral cavity and shows the typical cone-shaped has erupted into the oral cavity, similar to the juvenile stage morphology of a juvenile stage, and in the incisors of the wild- (Naples 1982). This molar is characterized by a prominent type and the K14-Eda transgenic mouse to investigate if root thick cap of dentin at the tip. This cap was not covered by formation is truly linked to HERS formation characterized by enamel typical of the crown of brachydont and hypsodont the loss of the stellate reticulum containing the putative stem teeth. Also the side surface of the tooth seemed to lack enamel cells. Furthermore, we analyzed molecular markers of the in- and we confirmed this with a close-up of a representative area cisor stem cell niche and differentiation in the K14-Eda incisor (Fig. 2B). The sloth molar lacked enamel-producing amelo- in order to check the state of the stem cell niche and the fate of blasts and the surface of this molar was entirely covered with the epithelial progeny. We show that stem cells exist in con- dentin and cementum, with occasional cementoblasts visible tinuously growing roots in the sloth molar, in the K14-Eda within the cementum, all typical features of a root surface incisor, and in the root analog side of the wild-type incisor. (Fig. 2C). The sloth molar therefore lacked any enamel from This indicates that the crown vs. root formation, i.e., the the tip to the base of the tooth and instead had acquired a production of enamel vs. cementum, and the differentiation of root surface. the epithelial cells into ameloblasts vs. ERM, can be regulated The general overview (Fig. 2A) showed a thin epithelial independently from the maintenance of the stem cells, and structure at the base of the tooth, where normally the HERS that the maintenance of stem cells does not indicate an im- is found in brachydont roots. However, a close-up of this area plicit ameloblast fate for the progeny. showed that the typical structure of the HERS, consisting only of inner and outer enamel epithelium, was not found in the sloth. Instead we found that the cervical loop was main- MATERIAL AND METHODS tained and it contained a core of cells surrounded by inner and outer enamel epithelium (Fig. 2C). The sloth histological sections are of a Bradypus tridactylus spec- imen. The teeth have started to erupt and resemble the juvenile stage (Naples 1982). The sections were collected and processed by Histological structure of the cervical loop of the the Dutch researcher Van den Broek in 1913 and are part of the wild-type incisor historical collection of the Hubrecht Laboratory in Utrecht. The The mouse incisor is subdivided into two domains, the labial K14-Eda mouse is a transgenic mouse that overexpresses the signal crown analog and the lingual root analog. The crown analog molecule Ectodysplasin-A1 under the Keratin14 promoter and has is characterized by an enamel surface whereas the lingual side been previously described (Mustonen et al. 2003). Wild-type tissue has a cementum surface. An overview of the apical end of the was used from 1, 4, and 12 days, and 4-week post-natal NMRI mice. K14-Eda tissue was from 4-week-old specimens. incisor was dominated by the presence of the prominent labial Radioactive in situ hybridization with 35 S labeled RNA probes cervical loop and a reduced epithelial structure on the lingual was performed on serial paraffin sections as described previously side. It has recently been suggested that the lingual aspect (Tummers and Thesleff 2003). Immunohistochemistry was per- of the incisor consists of HERS instead of a cervical loop formed on 7-mm-thick paraffin sections. After deparaffination the (Ohshima et al. 2005) and therefore we closely examined the sections were microwaved for 10 min in 10 mM natrium citrate structural organization of the epithelium on the lingual side buffer, pH 6.0, and then treated for 20 min in Proteinase K 7 mg/ml (Fig. 3B). We observed that there are indeed two epithelial cell in phosphate-buffered saline (PBS). After washes in PBS the sec- layers, apparently representing the inner and outer enamel tions were incubated for 1 h in 3% BSA in PBS and then with epithelium, but also that a small core of stellate reticulum is polyclonal rabbit anti-human keratin (Dako, Glostrup, Denmark, retained between these layers. We checked this at older stages A0575) 1:250 overnight at 41C. The Vectastain ABC kit was used as well, and this phenotype did not change from 1 day post- for detection and the sections were stained with DAB (Vector Laboratories, Burlingame, CA). natal to 4 weeks post-natal. In HERS this core is lost; hence For histology the tissues were sectioned at 4 and 7 mm thickness, the lingual side of the mouse incisor has maintained the cer- deparaffinized and stained with hematoxylin–eosin. The histolog- vical loop structure although diminished in size. The labial ical structures were identified based on definitions and examples in cervical loop is much enlarged as described previously due to Ten Cate’s Oral Histology (Nanci 2003). a large amount of stellate reticulum in the core of the cervical loop and here the inner enamel epithelium proliferates actively and subsequently differentiates into ameloblasts (Fig. 3C). RESULTS The sloth molar Histological structure of the cervical loop in the Figure 2A shows the general histology of a frontal section of K14-Eda incisor the sloth molar (Bradypus tridactylus) from an unspecified Previously we have shown that the K14-Eda incisor stage, showing a conical-shaped molar of which the tip lacks enamel on its labial aspect (Mustonen et al. 2003). We 190 EVOLUTION & DEVELOPMENT Vol. 10, No. 2, MarchÂpril 2008

Fig. 2. Histological structure of continuously growing sloth molar. (A) The frontal section shows the general structure of the sloth molar with open root and a massive core of dental mesenchyme topped off with a thick cap of dentin. B and C are higher magnifications of the boxes in A. (B) A continuous layer of polarized odontoblasts is evident as well as thick layers of dentin and cementum. Neither ameloblasts nor enamel is observed. The arrow shows a cementoblast inside the cementum. (C) At the apex of the root a cervical loop, i.e. the putative epithelial stem cell niche, is present. Some stellate reticulum cells are visible in the core. This cervical loop is magnified in D and a schematic representation shows the structure of the cervical loop and the basal lamina that separates the epithelium from the mesenchyme. Scale bars are 1 mm in A and 200 mminBandC.

therefore investigated here the fate of the cervical loop area of ameloblasts on the labial side but instead the epithelium frag- the K14-Eda incisor to determine whether HERS was formed mented and generated ERM typical of root surface. or the cervical loop was maintained. Eda is highly expressed throughout the dental epithelium in the K14-Eda incisor at 4 days and 5 weeks post-natal (data not shown). We observed Three-dimensional (3D) reconstruction of the that the lingual and labial aspects of this incisor looked strik- apical end of the K14-Eda incisor ingly similar (Fig. 3D) and resembled the lingual aspect of the To determine whether the stem cell niche is localized to a wild incisor. A few cells of stellate reticulum were present in certain region or is a continuous structure in the K14-Eda the cervical loop between the inner and outer enamel epithe- incisor, we analyzed the spatial location of the cervical loop lium (Fig. 3E). Moreover, we also observed that the progeny area at the tooth base. 3D reconstructions of serial sections of this cervical loop did not diﬀerentiate into elongated revealed that the cervical loop was not limited to the most Tummers and Thesle¡ Root growth and epithelial stem cells 191

Fig. 3. Fate of the epithelial stem cell niche in the wild-type mouse incisor and K14-Eda transgenic mouse incisor. (A) The apical end of a 1 day post-natal wild-type incisor with the large labial cervical loop and the smaller lingual cervical loop. (B) Magnification of the lingual cervical loop showing a small core of stellate reticulum cells surrounded by inner and outer enamel epithelium. (C) The specialized enlarged structure of the labial cervical loop with a large core of stellate reticulum cells surrounded by outer and inner enamel epithelium. The inner enamel epithelium is starting to differentiate into preameloblasts. (D) The apical end of the K14-Eda incisor. (E) Higher magnification of the labial cervical loop shows a signifi- cantly reduced core of stellate reticulum and lack of preameloblasts. (F) A pan-keratin antibody immunohistochemistry of the frontal sections of the K14- Eda incisor shows that from posterior to anterior the lateral cervical loops appeared first, with the labial cervical loop (asterix) closing more anteriorly, and the lingual loop did not close in a small area (arrowhead). (G) Three-dimensional reconstructions of the images in F confirmed lateral cervical loops protruding. The arrowheads show the start of epithelial fragmenta- tion. Scale bars are 200 mminA–C and 100 mm in D and E. labial or lingual aspects in the wild-type incisor but runs aspect of the cervical loop however never fully closed in the around the entire base of the tooth. In the following we refer K14-Eda incisor. We confirmed our findings by making a 3D to the cervical loop area situated between the lingual and reconstruction of the apical end of the incisor (Fig. 3G). We labial aspect as the lateral cervical loop. In the wild-type in- confirmed that the lateral cervical loops protruded posteriorly cisor the labial cervical loop appeared first in frontal sections and that the labial cervical loop closes more anteriorly com- that go from posterior to anterior because the lingual loop is pared with the sections containing the lateral cervical loops. located more toward the tip (Fig. 3A). Frontal sections of the Also the transition was clearly visible from cervical loop K14-Eda incisor where the epithelium was labeled with a pan- epithelium to fragmented epithelium, i.e., ERM in this re- keratin antibody showed a different picture with the lateral construction. Interestingly, on the lingual side, a small area of cervical loops appearing first on the most posterior sections a few cells width did not see closure of the cervical loop. (Fig. 3F). The most labial aspect remained open for a very Instead this area remained free from epithelium and imme- long time but eventually closed (Fig. 3F – asterix). The lingual diately undergoes the transition into ERM. 192 EVOLUTION&DEVELOPMENT Vol. 10, No. 2, MarchÂpril 2008

Molecular markers of the stem cell niche in the central cells of the lingual cervical loop of the wild-type In the wild-type incisor, notch1 has been shown to be speciﬁ- incisor (Fig. 4C) as well as in the lateral cervical loop area cally expressed in the stellate reticulum and stratum interme- (data not shown). Also in the K14-Eda incisor notch1 was dium cells of the labial cervical loop (Harada et al. 1999) expressed in the central epithelial cells of the cervical loop (Fig. 4A, and B). We showed that Notch1 was also expressed (Fig. 4D, and E) resembling the lingual wild-type pattern Tummers and Thesle¡ Root growth and epithelial stem cells 193

Fig. 4. Molecular regulation of the epithelial stem cell niche. (A) Notch1 expression in the wild-type incisor (4 dpn) is confined to the stellate reticulum and stratum intermedium of the labial as well as the lingual aspects. (B) Magnification of the labial cervical loop with a large core of stellate reticulum. (C) Magnification of the lingual cervical loop and although much smaller than the labial loop in B, stellate reticulum cells are present as indicated by notch1 expression at the core. (D) Notch1 expression in the K14-Eda incisor. (E) The K14-Eda labial cervical loop is much smaller than in the wild type resembling the wild-type lingual cervical loop B. (F–H) Other markers of the stem cell niche are normal in the K14-Eda incisor. (F) Fgf10 is expressed in the supporting mesenchyme. (H) Lfng is expressed in the inner enamel epithelium. (G) Fgf3 is only expressed on the labial side of the K14-Eda incisor similar to the wild type. In the sections of the K14-Eda incisor the lingual cervical loop is absent due to sectioning exactly through the cervical loop free zone as described in Fig. 3F and H; however, Notch1, Lfng and Fgf10 are present in neighboring and lateral sections.

(Fig. 4C). In the K14-Eda incisor Fgf10 was expressed in the aspects of the incisor (Fig. 5G) while in the wild type they mesenchyme directly surrounding the cervical loop similar to were restricted to the lingual side. the wild type (Fig. 4F). Fgf3 expression is restricted in the wild-type incisor to the labial mesenchyme and this pattern was similar in the K14-Eda incisor (Fig. 4G). Lunatic DISCUSSION AND CONCLUSIONS fringe is a marker for the transit-amplifying epithelial cells of the inner enamel epithelium (Harada et al. 1999) and it was The rodent incisor is functionally and morphologically sub- expressed in the K14-Eda incisor similar to the wild type divided into the labial crown analog and the lingual root in the inner enamel epithelium of the labial cervical loop analogue. Each side shows a typical differentiation pattern (Fig. 4H). where the crown analog is covered by enamel deposited by ameloblasts and the root analog is covered by cementum deposited by cementoblasts. It has been suggested that the large Differentiation in wild-type and K14-Eda incisor cervical loop at the labial aspect of the incisor represents the We compared cell differentiation in the K14-Eda and wild- sole epithelial stem cell niche supplying epithelial stem cells for type incisor by means of histology and markers for different the growth of all aspects, and that HERS typical of roots in cell types. The labial aspect of the K14-Eda incisor showed an molars, forms lingually and is responsible for root formation identical picture to the lingual side of the wild-type incisor there (Ohshima et al. 2005). However, we found no presence with a layer of odontoblasts, dentin, cementum, and peri- of HERS at the lingual side; instead there were stellate re- odontal ligament typical of a root. The transformation of the ticulum cells present in the core of the lingual cervical loop, as crown analog into a root analog in the K14-Eda incisor was was confirmed by the notch1 expression in these cells. More- confirmed by frontal sections labeled with a pan-keratin an- over, the cervical loop was shown to be a continuous structure tibody. The distinctive cap of tall ameloblasts was obvious in around the base of the incisor. the wild-type incisor on the labial aspect, and fragmented Similarly, no HERS typical of brachydont teeth (Ten Cate ERM covered the lingual side (Fig. 5D), whereas ERM sur- 1996) was found in the continuously growing sloth molar or rounded the entire circumference of the K14-Eda incisor the K14-Eda incisor. Both these teeth were covered by a typ- (Fig. 5E). The absence of ameloblasts was confirmed by the ical root surface consisting of dentin covered by cementum, differentiation marker jagged1, which is normally expressed in and they had cervical loops in their apical ends that had differentiating ameloblasts (Harada et al. 1999), and it was maintained stellate reticulum cells in the center. Moreover, the absent in the epithelium of the K14-Eda incisor (Fig. 5F). cervical loop was present in all sections of the apical aspect, Bsp1 is a marker for cementoblasts and odontoblast differ- indicating that it is present in the entire circumference of the entiation (Yamashiro et al. 2003), and in the K14-Eda incisor base of the tooth. A similar situation is present in the con- cementoblasts were present on both the lingual and labial tinuously growing molar of the sibling vole where the cervical

Fig. 5. Cell differentiation in the wild-type and K14-Eda incisor. (A) The labial aspect or crown analog of the wild-type incisor with the typical layer of elongated epithelial ameloblasts producing enamel and the mesenchymal odontoblasts generating dentin. (B) The root analog side has no ameloblasts or enamel. Dentin produced by odontoblasts is covered with cementum and the periodontal ligament attaches the cementum surface to the bone. (C) The labial aspect of the K14-Eda incisor is similar to the lingual root analog of the wild type in B. (D) Immunohistochemical staining with a pan-keratin antibody in the frontal sections of the wild-type incisor shows ameloblasts on the labial side and fragmented ERM epithelium on the lingual side. (E) Similar frontal section of the K14-Eda incisor shows fragmented ERM epithelium surrounding the entire tooth. (F,G) Sagittal sections of the K14-Eda incisors. (F) Jagged1 expression is missing in the epithelial compartment indicating the lack of preameloblasts, although jagged1 is still expressed normally in differentiating odontoblasts (arrowheads). (G) Bsp1 is expressed in odontoblasts (arrowheads) and cementoblasts (arrows) in a similar pattern on both lingual and labial side indicating that the labial side has adopted the lingual root phenotype. Scale bars are 200 mm. 194 EVOLUTION&DEVELOPMENT Vol. 10, No. 2, MarchÂpril 2008 loop is not restricted to a specific local area (Tummers and the progeny, because the enlarged cervical loops in the K14- Thesleff 2003). The continuously growing molar of the guinea noggin transgenic mouse form no enamel (Plikus et al. 2005). pig shows a morphology comparable to that of the vole (Hunt We propose that in the wild-type incisor the labial cervical 1958). loop is enlarged due to the functional requirement to produce The 3D reconstruction of the apical end of the K14-Eda a large amount of ameloblast progeny, whereas the lingual incisor allowed the observation of the cervical loop structures cervical loop merely provided progeny for fragmented epi- and this indicated that the labial cervical loop had a similar thelium of the ERM. sized core of stellate reticulum as the cervical loop at other We do not suggest that modification of Eda signaling is the aspects of the tooth (Fig 3F). In addition, the lateral cervical mechanism used in the sloth tooth to acquire the continuously loops were seen to protrude slightly from the apical end and growing root phenotype and lack of enamel. The acquisition the labial cervical loop was folded inwards. At the most lin- of this phenotype may occur at different regulatory levels. gual aspect the lateral loops did not meet and close. The total Recent studies on the regulation of the asymmetric develop- lack of epithelium here may indicate that the tooth is sub- ment of the mouse incisor have revealed a central role for divided into individual sections representing a lineage from follistatin, an inhibitor of TGFb signaling. The expression of stem cell to differentiated cell similar to that of the crypt of the follistatin in the lingual epithelium prevents enamel formation gut (Crosnier et al. 2006). Lack of the cervical loop in a spe- by inhibiting the inductive effect of BMPs on ameloblast cific area of the incisor therefore means local depletion of stem differentiation (Wang et al. 2004). Interestingly, follistatin also cells and eventually all epithelial structures deriving from inhibits the proliferation of the epithelial cells in the cervical those stem cells, because no replenishment takes place from loop, but this effect is due to inhibition of the positive effect of neighboring areas. The husbandry of the K14-Eda transgenic activin on stem cell proliferation (Wang et al. 2007). Recom- mice shows that the reduced cervical loop of the K14-Eda binant Eda protein induces the expression of follistatin as well incisor is indeed a functional stem cell niche because the in- as another BMP inhibitor CCN2 and prevents BMP-induced cisors need to be clipped regularly to prevent misalignment ameloblastin expression in vitro, showing that the lack of due to constant regeneration of this tooth. In conclusion, our enamel in the K14-Eda mice may result from inhibited BMP data clearly showed that the HERS is not an obligatory signaling (Pummila et al. 2007). These studies indicate that the structure for root formation, that no specialized stem cell maintenance of stem cells and their differentiation are regu- niche existed in the sloth molar or mouse incisor that is related by different molecular mechanisms supporting the find- stricted to a local area, and that a cervical loop with a reduced ings we have presented here. Taken together the different core of stellate reticulum cells can still act as a stem cell niche. models show that there are many possible ways to create a It is known that Fgf10 is important for the maintenance of sloth tooth phenotype. the stem cell niche in the incisor (Harada et al. 1999; Harada In conclusion, there appears to be regulatory flexibility in et al. 2002), and we have suggested that Fgf10 signaling is the decision between crown and root fate that is independent maintained in all continuously growing teeth to maintain the of the depletion of the stem cells in the niche. The differen- epithelial stem cell niche based to the similarities in the con- tiation compartment and stem cell compartment of the niche tinuously growing molar of the sibling vole and the rodent can be regulated independently, giving rise to multiple pat- incisor (Tummers and Thesleff 2003). It has also been pro- terns (Fig. 6): the brachydont pattern with low crown and posed that the disappearance of Fgf10 signaling leads to the high roots, the hypsodont pattern with high crown and low transition from crown to root formation due to a loss of the roots, the crown hypselodont pattern with a continuously dental epithelial stem cell compartment (Yokohama-Tamaki growing crown domain and root domain, and the exclusively et al. 2006). Based on our observations we would however like hypselodont root pattern. In the brachydont tooth, the dis- to suggest that although lack of Fgf10 can lead to a reduction appearance of the stem cells coincides with the switch to root of the stem cell niche and switch to root fate as can be seen in fate of the epithelial progeny during late development. the mouse molar (Tummers and Thesleff 2003), differentiation Hypsodonty can be seen as a simple extension of the brachy- into root can also take place in the presence of a functional dont pattern where the stem cells are maintained longer dur- epithelial stem cell niche. In the K14-Eda incisor, Fgf10 and ing crown development, and root formation is postponed Fgf3 expression was continued and although the amount of leading to a higher crown. In sharp contrast, the fate of root stellate reticulum, containing the putative stem cells, was re- and crown domains in continuously growing teeth is probably duced, it was not lost. At the same time the stem cell niches in already determined during early development, and is inde- the K14-Eda incisor and sloth molar give rise to root epithe- pendent of the maintenance of the stem cells. We propose that lium, suggesting that maintenance of the stem cells has no the diversity of tooth patterns is possible because the differ- default effect on the differentiation of the progeny, which ap- entiation of the progeny of the epithelial stem cells in the parently can differentiate into either ameloblasts or ERM. It cervical loop is not restricted to one specific fate, the amelo- does not seem that the size of the niche determines the fate of blast cell lineage, but also root epithelium can form. Tummers and Thesle¡ Root growth and epithelial stem cells 195

REFERENCES

Bosshardt,D.D.,andSchroeder,H.E.1996.Cementogenesisreviewed:a comparison between human premolars and rodent molars. Anat. Rec. 245: 267–292. Crosnier, C., Stamataki, D., and Lewis, J. 2006. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat. Rev. Genet. 7: 349–359. Harada, H., Kettunen, P., Jung, H. S., Mustonen, T., Wang, Y. A., and Thesleff, I. 1999. Localization of putative stem cells in dental epithelium andtheirassociationwithNotchandFGFsignaling.J. Cell Biol. 147: 105–120. Harada, H., et al. 2002. FGF10 maintains stem cell compartment in developing mouse incisors. Development 129: 1533–1541. Hunt, A. M. 1958. A description of the molar teeth and investing tissues of normal guinea pigs. J. Dent. Res. 38: 216–231. Jernvall, J., and Fortelius, M. 2002. Common mammals drive the evolutionary increase of hypsodonty in the Neogene. Nature 417: 538–540. Kagayama,M.,Sasano,Y.,Zhu,J.,Hirata,M.,Mizoguchi,I.,and Kamakura, S. 1998. Epithelial rests colocalize with cementoblasts forming acellular cementum but not with cementoblasts forming cellular cementum. Acta Anat. 163: 1–9. MacFadden, B. J. 2000. Cenozoic mammalian herbivores from the Amer- icas: reconstructing ancient diets and terrestrial communities. Annu. Rev. Ecol. Syst. 31: 33–59. Mustonen, T., et al. 2003. Stimulation of ectodermal organ development by Ectodysplasin-A1. Dev. Biol. 259: 123–136. Nanci, A. 2003. Development of the tooth and its supporting tissues. In A. Nanci (ed.) Ten Cate’s Oral Histology: Development, Structure, and Function. 6th ed. Mosby-Year Book, St. Louis, MO, pp. 79–110. Naples,V.L.1982.Cranialosteologyandfunctioninthetreesloths, Bradypus and Choloepus. Am Museum Novitiates 2739: 1–41. Ohshima, H., Nakasone, N., Hashimoto, E., Sakai, H., Nakaura-Ohshima, K., and Harada, H. 2005. The eternal tooth germ is formed at the apical end of continuously growing teeth. Arch. Oral Biol. 50: 153–157. Fig. 6. Diversity in tooth patterning is due to independent regu- Phillips, C. J., and Oxberry, B. 1972. Comparative histology of molar dentitions of Microtus and Clethrionomys, with comments on dental lation of the epithelial stem cell cells and differentiation of its evolution in Microtine Rodents. J. Mammal. 53: 1–20. progeny in the stem cell niche. In brachydont teeth HERS is Plikus, M. V., et al. 2005. Morphoregulation of teeth: modulating the formed after completion of crown morphogenesis and growth of number, size, shape and differentiation by tuning Bmp activity. Evol Dev the root is limited to a typical length. During crown morphogenesis 7: 440–457. the stem cell niche is formed and a signaling event leads to Pummila, M., et al. 2007. Ectodysplasin has a dual role in ectodermal the formation of HERS simultaneously with the disappearance of organogenesis: inhibition of Bmp activity and induction of Shh expres- the stem cells. In hypsodont teeth root formation is delayed and sion. Development 134: 117–125. crown formation is extended, which is accompanied by the main- Starkey, W. E. 1963. The migration and renewal of tritium labeled cells in tenance of the stem cell niche. Root initiation is accompanied the developing enamel organ of rabbits. Br. Dent. J. 115: 143–153. Ten Cate, A. R. 1961. Recruitment in the internal enamel epithelium by the loss of stem cells, which results in the formation of HERS. asafactoringrowthofthehumantoothgerm.Br. Dent. J. 110: 267–273. In continuously growing hypselodont teeth such as the sloth molar Ten Cate, A. R. 1996. The role of epithelium in the development, struc- and the rodent incisor, stem cells are present in the stellate retic- ture and function of the tissues of tooth support. Oral. Dis. 2: ulum of the cervical loop and the stem cells can give rise to either 55–62. ameloblasts or ERM. Hence, the fate of the epithelial progeny is Thomas,H.F.1995.Rootformation.Int. J. Dev. Biol. 39: 231–237. independent of the maintenance of the stem cells. The flexibility of Tummers, M., and Thesleff, I. 2003. Root or crown: a developmental choice regulation allows the diversity in tooth types and classic spatial orchestrated by the differential regulation of the epithelial stem cell niche association of root with jaw and crown with oral cavity becomes in the tooth of two rodent species. Development 130: 1049–1057. pointless. ERM, epithelial cell rests of Malassez (fragmented root Von Koenigswald, W. 1982. Zum verstandnis der Morphologie der wuhlmausmolaren. Z. Geol. Wiss. Berlin 10: 951–962. epithelium); scn, stem cell niche; HERS, Hertwig’s epithelial root Wang, X. P., et al. 2007. An integrated gene regulatory network controls sheath. stem cell proliferation in teeth. PLoS Biology 5: e159. Wang, X. P., Suomalainen, M., Jorgez, C. J., Matzuk, M. M., Werner, S., and Thesleff, I. 2004. Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast Acknowledgments differentiation. Dev. Cell. 7: 719–730. Yamashiro, T., Tummers, M., and Thesleff, I. 2003. Expression of bone We thank Raija Savolainen, Merja Ma¨kinen, and Riikka San- morphogenetic proteins and Msx genes during root formation. J. Dent. talahti for their excellent technical assistance, and we thank the Res. 82: 172–176. Hubrecht Laboratory for supplying the sloth sections. This work was Yokohama-Tamaki, T., et al. 2006. Cessation of Fgf10 signaling, result- supported by the Academy of Finland and the Sigfrid Juselius ing in a defective dental epithelial stem cell compartment, leads to the Foundation. transition from crown to root formation. Development 133: 1359–1366.