observations on continuously growing roots of the sloth...

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 Tummers� and Irma Thesleff1

Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland�Author 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 associatedwith the formation of Hertwig’s epithelial root sheath (HERS),whose fragments give rise to the epithelial cell rests ofMalassez (ERM). The HERS is formed by depletion of thecore of stellate reticulum cells, the putative stem cells, in thecervical loop, leaving only a double layer of the basalepithelium with limited growth capacity. The continuouslygrowing incisor of the rodent is subdivided into a crown analoghalf on the labial side, with a cervical loop containing a largecore of stellate reticulum, and its progeny gives rise to enamelproducing. The lingual side is known as the root analog andgives rise to ERM. We show that the lingual cervical loopcontains a small core of stellate reticulum cells and suggest

that it acts as a functional stem cell niche. Similarly we showthat continuously growing roots represented by the sloth molarand K14-Eda transgenic incisor maintain a cervical loop with asmall core of stellate reticulum cells around the entirecircumference of the tooth and do not form a HERS, and stillgive rise to ERM. We propose that HERS is not a necessarystructure to initiate root formation. Moreover, we conclude thatcrown vs. root formation, i.e. the production of enamel vs.cementum, and the differentiation of the epithelial cells intoameloblasts vs. ERM, can be regulated independently fromthe regulation of stem cell maintenance. This developmentalflexibility may underlie the developmental and evolutionarydiversity in tooth patterning.

INTRODUCTION

The teeth can be roughly subdivided into three groups. The

first group consists of brachydont or low-crowned teeth where

the root is relatively long compared with the crown. This is

the tooth type usually described in textbooks when describing

root formation (Nanci 2003). The second group consists of

hypsodont, or high–crowned, teeth, where the crown is high

compared with the root. The third group consists of the

hypselodont teeth, ever-growing or open-rooted teeth that

grow continuously during the lifetime of the animal. Open-

rooted refers solely to the large apical opening present in all

continuously growing teeth and does not imply that the tooth

actually needs to have a root in a classical sense as described

in the textbooks.

It is thought that brachydonty is the ancestral state

of mammalian teeth. During evolution the shift from

brachydont to hypsodont teeth is a common phenomenon

(Macfadden 2000). This trend is often initiated by environ-

mental pressures. Teeth with higher crowns last longer with

abrasive diets. For instance, a significant increase in the prev-

alence of hypsodonty in commonly found mammals of many

taxonomic groups occurred during the Neogene due to an

increased aridity in the environment of Europe (Jernvall and

Fortelius 2002). Hypselodonty can be seen as an extreme form

of hypsodonty. The crown never stops growing and root for-

mation is postponed indefinitely, but often with small tracts of

dentin covered with cementum acting as regions attaching the

tooth to the jaw bone with a periodontal ligament.

Within closely related species there can be a variation be-

tween brachydont, hypsodont, and hypselodont teeth, indi-

cating that the regulation of crown height is rather flexible.

For instance in closely related rodent species, the Mouse (Mus

musculus) molar is brachydont, the molars of the Bank vole

and the Southern Red-backed vole (Clethrionomys glareolus,

Clethrionomys gapperi) are hypsodont, and the molars of the

Sibling vole and Meadow vole (Microtus rossiaemeridionalis

and Microtus clethrionomys) are hypselodont (Phillips and

Oxberry 1972; Tummers and Thesleff 2003). It has been

proposed that the switch from hypsodont to hypselodont

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

& 2008 The Author(s)

Journal compilation & 2008 Blackwell Publishing Ltd.

187

between the microtine genera of Clethrionomys and Microtus

is caused by the maintenance of a regenerative unit, possibly

due to a simple mutation (Phillips and Oxberry 1972), or that

increased crown height results from delayed termination/

cytodifferentiation and that hypselodonty is an extreme out-

come of such a delay (von Koenigswald 1982). We have more

recently proposed that the increase in crown height is a result

of prolonging the period during which the epithelial stem cell

niche is maintained (Tummers and Thesleff 2003).

During classic root formation the dental epithelium of

the cervical loop undergoes some major structural changes

(Fig. 1). The cervical loop is created during crown morpho-

genesis and with root initiation loses the central core of

stellate reticulum and stratum intermedium cells, including the

putative epithelial stem cells (Ten Cate 1961; Starkey 1963;

Harada et al. 1999; Harada et al. 2002). A double layer of

basal epithelium is left that is known as Hertwig’s epithelial

root sheath (HERS) (Thomas 1995). As HERS proliferates,

the growing epithelial sheet becomes discontinuous and forms

a fenestrated network lining the root surface known as the

epithelial cell rests of Malassez (ERM) (Ten Cate 1996).

Through this network the follicular mesenchyme cells can

migrate to the dentin surface and differentiate into cemento-

blasts depositing the cementum. The ERM functions in the

induction of cementoblast differentiation and regulation of

their function (Thomas 1995; Bosshardt and Schroeder 1996;

Kagayama et al. 1998). Fibers of the periodontal ligament are

embedded in the cementum and connect the root to the jaw

bone. HERS forms in brachydont and hypsodont teeth when

root formation is initiated and crown formation ends, and its

transition to ERM is generally regarded as a typical feature of

root formation. Interestingly, the continuously growing ro-

dent 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 con-

tinuously by the apical end of the incisor. It has been sug-

gested 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 rep-

resented 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-

mens the tooth erupts as a simple cone. In adult specimens the

cap of the dentin is worn off, leaving a hard shell of dentin with

a soft pulp in the center. The edges of the dentin get sharper

with age due to wear (Naples 1982). Similarly, the dentition of

the mouse, as a representative of the rodents, is also heavily

modified during evolution, with only two incisors in each jaw,

followed by a diastema region lacking teeth, and three molars.

The transgenic K14-Eda mouse has ectodysplasin (Eda)

expressed under the keratin 14 promoter leading to an ex-

cessive production of Eda throughout the ectoderm from E10

onwards, including the oral and dental epithelium (Mustonen

et al. 2003). The constitutive expression of Eda in the dental

epithelium leads to the formation of supernumerary molars

and the loss of enamel on crown analog of the incisors. This

transgenic mouse line therefore has possibly transformed its

incisor into a continuously growing root, and serves in this

paper as a model system for continuously growing roots. If

HERS is required for the production of root epithelium, these

teeth would not have a cervical loop and a stem cell niche.

Here we investigate the structure of the cervical loop area

of continuously growing roots of a sloth molar that has

Fig. 1. Formation of the cervical loop, the putative epithelial stemcell niche, during early development and its fate in the mouse molarand incisor. During early stages of morphogenesis all teeth gothrough the same developmental stages (initiation, bud stage, capstage, and the bell stage) generating the crown. During bud stage acore of loosely arranged epithelium is formed in the center of thebud. During the cap stage the cervical loop is formed, a protrusionfrom the bud that envelopes the condensed dental papilla me-senchyme. The cervical loop is extended during the bell stage andthe inner enamel epithelium starts to differentiate into ameloblasts.During the late bell stage crown morphology is established andcells producing mineralized tissues differentiate terminally. Celldifferentiation starts from the cusp tips and extends toward thebase. Enamel is deposited by ameloblasts and dentin by odonto-blasts. In the mouse molar the cervical loop loses its core of stellatereticulum, the putative stem cells, and forms the HERS, whichfragments into ERM, typical of a root. On the labial side of themouse incisor the cervical loop is maintained as a stem cell nicheand it keeps giving rise to ameloblasts. On the lingual side noameloblasts differentiate and instead ERM is formed. The fate ofthe lingual cervical loop is unclear, although it has been suggestedthat HERS is present (Ohshima et al. 2005). ERM, epithelial cellrests of Malassez; HERS, Hertwig’s epithelial root sheath; iee,inner enamel epithelium; oee, outer enamel epithelium; sr, stellatereticulum.

188 EVOLUTION & DEVELOPMENT Vol. 10, No. 2, March^April 2008

erupted into the oral cavity and shows the typical cone-shaped

morphology of a juvenile stage, and in the incisors of the wild-

type and the K14-Eda transgenic mouse to investigate if root

formation is truly linked to HERS formation characterized by

the loss of the stellate reticulum containing the putative stem

cells. Furthermore, we analyzed molecular markers of the in-

cisor stem cell niche and differentiation in the K14-Eda incisor

in order to check the state of the stem cell niche and the fate of

the epithelial progeny. We show that stem cells exist in con-

tinuously growing roots in the sloth molar, in the K14-Eda

incisor, and in the root analog side of the wild-type incisor.

This indicates that the crown vs. root formation, i.e., the

production of enamel vs. cementum, and the differentiation of

the epithelial cells into ameloblasts vs. ERM, can be regulated

independently from the maintenance of the stem cells, and

that the maintenance of stem cells does not indicate an im-

plicit ameloblast fate for the progeny.

MATERIAL AND METHODS

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

the Dutch researcher Van den Broek in 1913 and are part of the

historical collection of the Hubrecht Laboratory in Utrecht. The

K14-Eda mouse is a transgenic mouse that overexpresses the signal

molecule Ectodysplasin-A1 under the Keratin14 promoter and has

been previously described (Mustonen et al. 2003). Wild-type tissue

was used from 1, 4, and 12 days, and 4-week post-natal NMRI

mice. K14-Eda tissue was from 4-week-old specimens.

Radioactive in situ hybridization with 35S labeled RNA probes

was performed on serial paraffin sections as described previously

(Tummers and Thesleff 2003). Immunohistochemistry was per-

formed on 7-mm-thick paraffin sections. After deparaffination the

sections were microwaved for 10min in 10mM natrium citrate

buffer, pH 6.0, and then treated for 20min in Proteinase K 7mg/ml

in phosphate-buffered saline (PBS). After washes in PBS the sec-

tions were incubated for 1h in 3% BSA in PBS and then with

polyclonal rabbit anti-human keratin (Dako, Glostrup, Denmark,

A0575) 1:250 overnight at 41C. The Vectastain ABC kit was used

for detection and the sections were stained with DAB (Vector

Laboratories, Burlingame, CA).

For histology the tissues were sectioned at 4 and 7mm thickness,

deparaffinized and stained with hematoxylin–eosin. The histolog-

ical structures were identified based on definitions and examples in

Ten Cate’s Oral Histology (Nanci 2003).

RESULTS

The sloth molar

Figure 2A shows the general histology of a frontal section of

the sloth molar (Bradypus tridactylus) from an unspecified

stage, showing a conical-shaped molar of which the tip

has erupted into the oral cavity, similar to the juvenile stage

(Naples 1982). This molar is characterized by a prominent

thick cap of dentin at the tip. This cap was not covered by

enamel typical of the crown of brachydont and hypsodont

teeth. Also the side surface of the tooth seemed to lack enamel

and we confirmed this with a close-up of a representative area

(Fig. 2B). The sloth molar lacked enamel-producing amelo-

blasts and the surface of this molar was entirely covered with

dentin and cementum, with occasional cementoblasts visible

within the cementum, all typical features of a root surface

(Fig. 2C). The sloth molar therefore lacked any enamel from

the tip to the base of the tooth and instead had acquired a

root surface.

The general overview (Fig. 2A) showed a thin epithelial

structure at the base of the tooth, where normally the HERS

is found in brachydont roots. However, a close-up of this area

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-

tained and it contained a core of cells surrounded by inner

and outer enamel epithelium (Fig. 2C).

Histological structure of the cervical loop of thewild-type incisor

The mouse incisor is subdivided into two domains, the labial

crown analog and the lingual root analog. The crown analog

is characterized by an enamel surface whereas the lingual side

has a cementum surface. An overview of the apical end of the

incisor was dominated by the presence of the prominent labial

cervical loop and a reduced epithelial structure on the lingual

side. It has recently been suggested that the lingual aspect

of the incisor consists of HERS instead of a cervical loop

(Ohshima et al. 2005) and therefore we closely examined the

structural organization of the epithelium on the lingual side

(Fig. 3B). We observed that there are indeed two epithelial cell

layers, apparently representing the inner and outer enamel

epithelium, but also that a small core of stellate reticulum is

retained between these layers. We checked this at older stages

as well, and this phenotype did not change from 1 day post-

natal to 4 weeks post-natal. In HERS this core is lost; hence

the lingual side of the mouse incisor has maintained the cer-

vical loop structure although diminished in size. The labial

cervical loop is much enlarged as described previously due to

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).

Histological structure of the cervical loop in theK14-Eda incisor

Previously we have shown that the K14-Eda incisor

lacks enamel on its labial aspect (Mustonen et al. 2003). We

Root growth and epithelial stem cells 189Tummers and Thesle¡

therefore investigated here the fate of the cervical loop area of

the K14-Eda incisor to determine whether HERS was formed

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

that the lingual and labial aspects of this incisor looked strik-

ingly similar (Fig. 3D) and resembled the lingual aspect of the

wild incisor. A few cells of stellate reticulum were present in

the cervical loop between the inner and outer enamel epithe-

lium (Fig. 3E). Moreover, we also observed that the progeny

of this cervical loop did not differentiate into elongated

ameloblasts on the labial side but instead the epithelium frag-

mented and generated ERM typical of root surface.

Three-dimensional (3D) reconstruction of theapical end of the K14-Eda incisor

To determine whether the stem cell niche is localized to a

certain region or is a continuous structure in the K14-Eda

incisor, we analyzed the spatial location of the cervical loop

area at the tooth base. 3D reconstructions of serial sections

revealed that the cervical loop was not limited to the most

Fig. 2. Histological structure of continuously growing sloth molar. (A) The frontal section shows the general structure of the sloth molarwith open root and a massive core of dental mesenchyme topped off with a thick cap of dentin. B and C are higher magnifications of theboxes in A. (B) A continuous layer of polarized odontoblasts is evident as well as thick layers of dentin and cementum. Neither ameloblastsnor enamel is observed. The arrow shows a cementoblast inside the cementum. (C) At the apex of the root a cervical loop, i.e. the putativeepithelial stem cell niche, is present. Some stellate reticulum cells are visible in the core. This cervical loop is magnified in D and a schematicrepresentation shows the structure of the cervical loop and the basal lamina that separates the epithelium from the mesenchyme. Scale barsare 1mm in A and 200mm in B and C.


labial or lingual aspects in the wild-type incisor but runs

around the entire base of the tooth. In the following we refer

to the cervical loop area situated between the lingual and

labial aspect as the lateral cervical loop. In the wild-type in-

cisor the labial cervical loop appeared first in frontal sections

that go from posterior to anterior because the lingual loop is

located more toward the tip (Fig. 3A). Frontal sections of the

K14-Eda incisor where the epithelium was labeled with a pan-

keratin antibody showed a different picture with the lateral

cervical loops appearing first on the most posterior sections

(Fig. 3F). The most labial aspect remained open for a very

long time but eventually closed (Fig. 3F – asterix). The lingual

aspect of the cervical loop however never fully closed in the

K14-Eda incisor. We confirmed our findings by making a 3D

reconstruction of the apical end of the incisor (Fig. 3G). We

confirmed that the lateral cervical loops protruded posteriorly

and that the labial cervical loop closes more anteriorly com-

pared with the sections containing the lateral cervical loops.

Also the transition was clearly visible from cervical loop

epithelium to fragmented epithelium, i.e., ERM in this re-

construction. Interestingly, on the lingual side, a small area of

a few cells width did not see closure of the cervical loop.

Instead this area remained free from epithelium and imme-

diately undergoes the transition into ERM.

Fig. 3. Fate of the epithelial stemcell niche in the wild-type mouseincisor and K14-Eda transgenicmouse incisor. (A) The apical endof a 1 day post-natal wild-type in-cisor with the large labial cervicalloop and the smaller lingual cervi-cal loop. (B) Magnification of thelingual cervical loop showing asmall core of stellate reticulumcells surrounded by inner and out-er enamel epithelium. (C) The spe-cialized enlarged structure of thelabial cervical loop with a largecore of stellate reticulum cells sur-rounded by outer and inner enam-el epithelium. The inner enamelepithelium is starting to differenti-ate into preameloblasts. (D) Theapical end of the K14-Eda incisor.(E) Higher magnification of thelabial cervical loop shows a signifi-cantly reduced core of stellatereticulum and lack of pre-ameloblasts. (F) A pan-keratin an-tibody immunohistochemistry ofthe frontal sections of the K14-Eda incisor shows that fromposterior to anterior the lateral cer-vical loops appeared first, with thelabial cervical loop (asterix) closingmore anteriorly, and the lingualloop did not close in a small area(arrowhead). (G) Three-dimension-al reconstructions of the images inF confirmed lateral cervical loopsprotruding. The arrowheads showthe start of epithelial fragmenta-tion. Scale bars are 200mm in A–Cand 100mm in D and E.


Molecular markers of the stem cell niche

In the wild-type incisor, notch1 has been shown to be specifi-

cally expressed in the stellate reticulum and stratum interme-

dium cells of the labial cervical loop (Harada et al. 1999)

(Fig. 4A, and B). We showed that Notch1 was also expressed

in the central cells of the lingual cervical loop of the wild-type

incisor (Fig. 4C) as well as in the lateral cervical loop area

(data not shown). Also in the K14-Eda incisor notch1 was

expressed in the central epithelial cells of the cervical loop

(Fig. 4D, and E) resembling the lingual wild-type pattern


(Fig. 4C). In the K14-Eda incisor Fgf10 was expressed in the

mesenchyme directly surrounding the cervical loop similar to

the wild type (Fig. 4F). Fgf3 expression is restricted in

the wild-type incisor to the labial mesenchyme and this pat-

tern was similar in the K14-Eda incisor (Fig. 4G). Lunatic

fringe is a marker for the transit-amplifying epithelial cells of

the inner enamel epithelium (Harada et al. 1999) and it was

expressed in the K14-Eda incisor similar to the wild type

in the inner enamel epithelium of the labial cervical loop

(Fig. 4H).

Differentiation in wild-type and K14-Eda incisor

We compared cell differentiation in the K14-Eda and wild-

type incisor by means of histology and markers for different

cell types. The labial aspect of the K14-Eda incisor showed an

identical picture to the lingual side of the wild-type incisor

with a layer of odontoblasts, dentin, cementum, and peri-

odontal ligament typical of a root. The transformation of the

crown analog into a root analog in the K14-Eda incisor was

confirmed by frontal sections labeled with a pan-keratin an-

tibody. The distinctive cap of tall ameloblasts was obvious in

the wild-type incisor on the labial aspect, and fragmented

ERM covered the lingual side (Fig. 5D), whereas ERM sur-

rounded the entire circumference of the K14-Eda incisor

(Fig. 5E). The absence of ameloblasts was confirmed by the

differentiation marker jagged1, which is normally expressed in

differentiating ameloblasts (Harada et al. 1999), and it was

absent in the epithelium of the K14-Eda incisor (Fig. 5F).

Bsp1 is a marker for cementoblasts and odontoblast differ-

entiation (Yamashiro et al. 2003), and in the K14-Eda incisor

cementoblasts were present on both the lingual and labial

aspects of the incisor (Fig. 5G) while in the wild type they

were restricted to the lingual side.

DISCUSSION AND CONCLUSIONS

The rodent incisor is functionally and morphologically sub-

divided into the labial crown analog and the lingual root

analogue. Each side shows a typical differentiation pattern

where the crown analog is covered by enamel deposited by

ameloblasts and the root analog is covered by cementum de-

posited by cementoblasts. It has been suggested that the large

cervical loop at the labial aspect of the incisor represents the

sole epithelial stem cell niche supplying epithelial stem cells for

the growth of all aspects, and that HERS typical of roots in

molars, forms lingually and is responsible for root formation

there (Ohshima et al. 2005). However, we found no presence

of HERS at the lingual side; instead there were stellate re-

ticulum cells present in the core of the lingual cervical loop, as

was confirmed by the notch1 expression in these cells. More-

over, the cervical loop was shown to be a continuous structure

around the base of the incisor.

Similarly, no HERS typical of brachydont teeth (Ten Cate

1996) was found in the continuously growing sloth molar or

the K14-Eda incisor. Both these teeth were covered by a typ-

ical root surface consisting of dentin covered by cementum,

and they had cervical loops in their apical ends that had

maintained stellate reticulum cells in the center. Moreover, the

cervical loop was present in all sections of the apical aspect,

indicating that it is present in the entire circumference of the

base of the tooth. A similar situation is present in the con-

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 thetypical layer of elongated epithelial ameloblasts producing enamel and the mesenchymal odontoblasts generating dentin. (B) The rootanalog side has no ameloblasts or enamel. Dentin produced by odontoblasts is covered with cementum and the periodontal ligamentattaches 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 typein B. (D) Immunohistochemical staining with a pan-keratin antibody in the frontal sections of the wild-type incisor shows ameloblasts onthe labial side and fragmented ERM epithelium on the lingual side. (E) Similar frontal section of the K14-Eda incisor shows fragmentedERM epithelium surrounding the entire tooth. (F,G) Sagittal sections of the K14-Eda incisors. (F) Jagged1 expression is missing in theepithelial 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 labialside indicating that the labial side has adopted the lingual root phenotype. Scale bars are 200mm.

Fig. 4. Molecular regulation of the epithelial stem cell niche. (A) Notch1 expression in the wild-type incisor (4 dpn) is confined to the stellatereticulum and stratum intermedium of the labial as well as the lingual aspects. (B) Magnification of the labial cervical loop with a large coreof stellate reticulum. (C) Magnification of the lingual cervical loop and although much smaller than the labial loop in B, stellate reticulumcells are present as indicated by notch1 expression at the core. (D)Notch1 expression in the K14-Eda incisor. (E) The K14-Eda labial cervicalloop 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 arenormal 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 thelingual 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.


loop is not restricted to a specific local area (Tummers and

Thesleff 2003). The continuously growing molar of the guinea

pig shows a morphology comparable to that of the vole (Hunt

1958).

The 3D reconstruction of the apical end of the K14-Eda

incisor allowed the observation of the cervical loop structures

and this indicated that the labial cervical loop had a similar

sized core of stellate reticulum as the cervical loop at other

aspects of the tooth (Fig 3F). In addition, the lateral cervical

loops were seen to protrude slightly from the apical end and

the labial cervical loop was folded inwards. At the most lin-

gual aspect the lateral loops did not meet and close. The total

lack of epithelium here may indicate that the tooth is sub-

divided into individual sections representing a lineage from

stem cell to differentiated cell similar to that of the crypt of the

gut (Crosnier et al. 2006). Lack of the cervical loop in a spe-

cific area of the incisor therefore means local depletion of stem

cells and eventually all epithelial structures deriving from

those stem cells, because no replenishment takes place from

neighboring areas. The husbandry of the K14-Eda transgenic

mice shows that the reduced cervical loop of the K14-Eda

incisor is indeed a functional stem cell niche because the in-

cisors need to be clipped regularly to prevent misalignment

due to constant regeneration of this tooth. In conclusion, our

data clearly showed that the HERS is not an obligatory

structure for root formation, that no specialized stem cell

niche existed in the sloth molar or mouse incisor that is re-

stricted to a local area, and that a cervical loop with a reduced

core of stellate reticulum cells can still act as a stem cell niche.

It is known that Fgf10 is important for the maintenance of

the stem cell niche in the incisor (Harada et al. 1999; Harada

et al. 2002), and we have suggested that Fgf10 signaling is

maintained in all continuously growing teeth to maintain the

epithelial stem cell niche based to the similarities in the con-

tinuously growing molar of the sibling vole and the rodent

incisor (Tummers and Thesleff 2003). It has also been pro-

posed that the disappearance of Fgf10 signaling leads to the

transition from crown to root formation due to a loss of the

dental epithelial stem cell compartment (Yokohama-Tamaki

et al. 2006). Based on our observations we would however like

to suggest that although lack of Fgf10 can lead to a reduction

of the stem cell niche and switch to root fate as can be seen in

the mouse molar (Tummers and Thesleff 2003), differentiation

into root can also take place in the presence of a functional

epithelial stem cell niche. In the K14-Eda incisor, Fgf10 and

Fgf3 expression was continued and although the amount of

stellate reticulum, containing the putative stem cells, was re-

duced, it was not lost. At the same time the stem cell niches in

the K14-Eda incisor and sloth molar give rise to root epithe-

lium, suggesting that maintenance of the stem cells has no

default effect on the differentiation of the progeny, which ap-

parently can differentiate into either ameloblasts or ERM. It

does not seem that the size of the niche determines the fate of

the progeny, because the enlarged cervical loops in the K14-

noggin transgenic mouse form no enamel (Plikus et al. 2005).

We propose that in the wild-type incisor the labial cervical

loop is enlarged due to the functional requirement to produce

a large amount of ameloblast progeny, whereas the lingual

cervical loop merely provided progeny for fragmented epi-

thelium of the ERM.

We do not suggest that modification of Eda signaling is the

mechanism used in the sloth tooth to acquire the continuously

growing root phenotype and lack of enamel. The acquisition

of this phenotype may occur at different regulatory levels.

Recent studies on the regulation of the asymmetric develop-

ment of the mouse incisor have revealed a central role for

follistatin, an inhibitor of TGFb signaling. The expression of

follistatin in the lingual epithelium prevents enamel formation

by inhibiting the inductive effect of BMPs on ameloblast

differentiation (Wang et al. 2004). Interestingly, follistatin also

inhibits the proliferation of the epithelial cells in the cervical

loop, but this effect is due to inhibition of the positive effect of

activin on stem cell proliferation (Wang et al. 2007). Recom-

binant Eda protein induces the expression of follistatin as well

as another BMP inhibitor CCN2 and prevents BMP-induced

ameloblastin expression in vitro, showing that the lack of

enamel in the K14-Eda mice may result from inhibited BMP

signaling (Pummila et al. 2007). These studies indicate that the

maintenance of stem cells and their differentiation are regu-

lated by different molecular mechanisms supporting the find-

ings we have presented here. Taken together the different

models show that there are many possible ways to create a

sloth tooth phenotype.

In conclusion, there appears to be regulatory flexibility in

the decision between crown and root fate that is independent

of the depletion of the stem cells in the niche. The differen-

tiation compartment and stem cell compartment of the niche

can be regulated independently, giving rise to multiple pat-

terns (Fig. 6): the brachydont pattern with low crown and

high roots, the hypsodont pattern with high crown and low

roots, the crown hypselodont pattern with a continuously

growing crown domain and root domain, and the exclusively

hypselodont root pattern. In the brachydont tooth, the dis-

appearance of the stem cells coincides with the switch to root

fate of the epithelial progeny during late development.

Hypsodonty can be seen as a simple extension of the brachy-

dont pattern where the stem cells are maintained longer dur-

ing crown development, and root formation is postponed

leading to a higher crown. In sharp contrast, the fate of root

and crown domains in continuously growing teeth is probably

already determined during early development, and is inde-

pendent of the maintenance of the stem cells. We propose that

the diversity of tooth patterns is possible because the differ-

entiation of the progeny of the epithelial stem cells in the

cervical loop is not restricted to one specific fate, the amelo-

blast cell lineage, but also root epithelium can form.


AcknowledgmentsWe thank Raija Savolainen, Merja Makinen, and Riikka San-talahti for their excellent technical assistance, and we thank theHubrecht Laboratory for supplying the sloth sections. This work wassupported by the Academy of Finland and the Sigfrid JuseliusFoundation.

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Fig. 6. Diversity in tooth patterning is due to independent regu-lation of the epithelial stem cell cells and differentiation of itsprogeny in the stem cell niche. In brachydont teeth HERS isformed after completion of crown morphogenesis and growth ofthe root is limited to a typical length. During crown morphogenesisthe stem cell niche is formed and a signaling event leads tothe formation of HERS simultaneously with the disappearance ofthe stem cells. In hypsodont teeth root formation is delayed andcrown formation is extended, which is accompanied by the main-tenance of the stem cell niche. Root initiation is accompaniedby the loss of stem cells, which results in the formation of HERS.In continuously growing hypselodont teeth such as the sloth molarand the rodent incisor, stem cells are present in the stellate retic-ulum of the cervical loop and the stem cells can give rise to eitherameloblasts or ERM. Hence, the fate of the epithelial progeny isindependent of the maintenance of the stem cells. The flexibility ofregulation allows the diversity in tooth types and classic spatialassociation of root with jaw and crown with oral cavity becomespointless. ERM, epithelial cell rests of Malassez (fragmented rootepithelium); scn, stem cell niche; HERS, Hertwig’s epithelial rootsheath.


observations on continuously growing roots of the sloth...

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