Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki, Finland
* Author for correspondence (e-mail: mark.tummers{at}helsinki.fi)
Accepted 27 November 2002
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SUMMARY |
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Key words: Morphogenesis, Evolution, Tooth, Stem Cell, Root Formation, Crown formation, Sibling vole, Microtus rossiaemeridionalis, Mouse, Mus musculus, FGF, BMP, Notch, Hypselodont, Hypsodont
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INTRODUCTION |
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The incisor of rodents represents a special tooth type since it grows
continuously throughout the lifetime of the animal. Therefore, it must possess
adult mesenchymal and epithelial stem cells. The exact location of the
mesenchymal stem cells in teeth is not clear, although dental mesenchymal
cells have been isolated from the pulp of adult human teeth
(Gronthos et al., 2000). The
cervical loop area located opposite of the distal tip of the tooth, and more
specifically the epithelial tissue named stellate reticulum, has been put
forward as the putative site of the epithelial stem cell compartment in the
mouse incisor (Harada et al.,
1999
). It was also suggested that Notch signaling and FGF10 are
involved in the regulation of this epithelial stem cell compartment
(Harada et al., 2002
). In the
incisor the stem cells migrate from the stellate reticulum to the inner enamel
epithelium and contribute to a pool of proliferating cells, also known as
transit-amplifying cells. These cells then move towards the distal tip and
show increasingly higher states of differentiation of the ameloblast cell
lineage. The ameloblasts produce and deposit the enamel matrix responsible for
the hardness of the tooth. The enamel is then constantly worn down at the
distal tip of the incisor. In contrast, in mouse molars and all human teeth
for instance, the stellate reticulum is lost after crown formation and a
double layer of root sheath epithelium is left that directs root formation
(Fig. 1).
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The rodent incisor is not the only tooth type that shows continuous growth.
Also molars of certain species grow continuously. One such species is the
rabbit. Tritium labeling studies suggested that also in the rabbit molar the
cervical loop area is the origin of the epithelial cell lineage
(Starkey, 1963). It was
concluded that the pool of transit-amplifying cells in the inner enamel
epithelium cannot be sustained by itself, but probably originates from the
stratum intermedium, which is the denser layer of the stellate reticulum
closest to the inner enamel epithelium. A cell that is recruited into the
basal layer of proliferating cells must therefore change epithelial
compartments and actually re-laminate itself into the inner enamel epithelium,
since the opposite side of the inner enamel epithelium faces the dental
mesenchyme and therefore also borders with a basal lamina
(Ten Cate, 1961
).
Here we focus on a less well-known tooth system; the continuously growing
molar of a vole species known as the sibling vole (Microtus
rossiaemeridionalis). The vole and mouse are both rodents and are closely
related species. Previously, the earlier stages of the vole and mouse molar
were compared until the late bell stage of development (E17), and the basic
aspects of morphogenesis and the distribution of important developmental
regulatory molecules were found to be almost identical
(Keränen et al., 1998).
Nature, therefore, provided for us the experimental setup. We have two tooth
systems, the molar of the vole and the molar of the mouse, whose early
development and morphogenesis are remarkably similar, but later venture on
different developmental paths. The mouse molar develops roots and stops
growing, the vole molar maintains the crown fate and will grow continuously.
Therefore, a regulatory difference must be present and through comparison of
the two phylogenetically closely related systems the developmental mechanism
responsible for this divergence might be discovered.
From the incisor it is known that FGF10 and Notch signaling is important
for the maintenance of the stem cell niche and the continuous growth of the
mouse incisor (Harada et al.,
1999; Harada et al.,
2002
). We compared the expression patterns of these genes between
mouse and vole molar. Similarities in this regulatory system between the vole
molar and mouse incisor suggested that both share a common regulatory
mechanism that maintains the epithelial stem cell niche and epithelial cell
lineage. In the mouse molar this regulatory system disappears and root
development takes place. The analysis of the morphology of the vole molar
additionally revealed the presence of three small domains lacking ameloblasts
and enamel. These areas represented functional root structures and can be
compared to the lingual side of the rodent incisor. This area in the incisor
looks and functions as a root, but no classic root formation takes place, and
hence is known as the root analogue. The switch between root and crown
epithelium can therefore occur locally during development and is flexible in
nature.
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MATERIALS AND METHODS |
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In situ hybridization
The in situ hybridization with 35S-UTP (Amersham) labeled
riboprobes was performed as described previously
(Wilkinson and Green, 1990).
The slides from the radioactive in situ hybridization were photographed with
an Olympus Provis microscope equipped with CCD camera (Photometric Ltd).
Figures were processed using Adobe Photoshop (Adobe Systems, CA; the
dark-field images were inverted and artificially stained red and combined with
the bright-field image) and Micrographx Designer software. Every radioactive
in situ hybridization was carried out at least two times, and during each
experiment each individual probe was tested on several different sections.
Most genes were examined more than twice and also in sections cut at different
angles.
Probes
The following plasmids were used for the 35S-UTP probes. All
probes originated from mouse sequences except for FGF10, which originated from
rat. The Lfng probe was a kind gift from Alan Wang
(Harada et al., 1999), jagged1
(Jag1)- and delta1 (Dll1)-containing plasmids were
a kind gift from Domingos Henrique
(Mitsiadis et al., 1997
;
Bettenhausen et al., 1995
),
Notch1, 2, 3 probes from Urban Lendahl
(Lardelli et al., 1994
;
Larsson et al., 1994
) and
Hes1 and Hes5 from Royuchiro Kageyama
(Sasai et al., 1992
). The rat
Fgf10 and murine Fgf3 probes have been described previously
(Kettunen et al., 2000
). It
has been shown that probes that work in the mouse generally also work in the
sibling vole (Keränen et al.,
1998
).
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RESULTS |
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Notch, BMP and FGF signaling identifies the epithelial stem cell
niche in the vole molar
In the mouse incisor the Notch signaling pathway genes and Fgf10
are expressed in a distinct pattern and are thought to be important for the
regulation of the epithelial stem cell niche. In the vole molar we found a
similar pattern and this pattern did not change from 2 dpn to 14 dpn in the
crown area (14 dpn not shown). Here Notch1, 2 and 3 were
expressed most strongly in the stellate reticulum compartment of the cervical
and intercuspal loop, with especially strong expression in the stratum
intermedium (Fig. 3B,D,F,
Fig. 5B,D). Notch2 and
3 were also expressed throughout the mesenchyme at lower levels and
Notch1 and 3 were associated with blood vessels.
Lfng expression was present in the inner and outer enamel epithelium
corresponding roughly to the regions containing the transit-amplifying cells
(Fig. 3H,
Fig. 5F). Above this
Lfng domain Jag1 was expressed in the inner enamel
epithelium in a restricted domain corresponding to differentiating ameloblasts
(Fig. 3J, Fig. 5H). The mesenchymal
Dll1 expression domain was found at the same height as the epithelial
Jag1 domain and was restricted to preodontoblasts
(Fig. 4A,B). This indicated
that odontoblast and ameloblast differentiation take place at the same crown
level in the vole molar. Hes5 expression could not be detected (data
not shown), but Hes1 was present throughout the stellate reticulum
and mesenchyme with the highest levels of expression in the stratum
intermedium, similar to the combined Notch expression patterns
(Fig. 4D,
Fig. 6D). Bsp1 (black
spleen) was used as a differentiation marker and its expression domain was
exactly above that of Jag1 in the inner and outer enamel epithelium
(Fig. 4H,
Fig. 6J). Near the cusps,
Bsp1 was also expressed in the coronal layer of the mesenchyme of the
pulp chamber indicating a function in the closure of the pulp chambers
(Fig. 4I).
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Previously it has been shown that mesenchymal Fgf10 supports the
epithelial stem cell compartment in the mouse incisor
(Harada et al., 2002;
Harada et al., 1999
). In the
vole molar, Fgf10 expression was also seen in the mesenchyme with the
strongest expression near the base of the tooth, and was especially strong
around the intercuspal and cervical loop
(Fig. 4E,F,
Fig. 6E,F). These are the sites
where the proliferating cells and the putative stem cells are located.
Bmp4 showed an identical mesenchymal pattern as Fgf10
(Fig. 6G,H). Hes1 and
Lfng expression could both be seen as a broad band running from
anterior to posterior (Fig.
3G,H, Fig. 4C,D,
Fig. 5E,F,
Fig. 6C,D). The band of
Lfng expression was bordering that of Hes1 but was located
higher, nearer the cusps. Inside the Hes1 domain, gaps were present
surrounding the intercuspal folds and cervical loop, which coincided with the
expression domains of Fgf10 and Bmp4
(Fig. 4C,D,
Fig. 6C,D).
Three root analogue domains in the vole molar
Almost the entire area of cervical loop epithelium of the vole molar
maintains the crown fate throughout the life of the animal and continues to
produce ameloblasts. Coronal sections revealed the presence of three small
functional root domains, similar in appearance to the root of the mouse molar
(Fig. 2D-G). A larger domain
was present at the anterior and two smaller ones at the very ends of the
crescent-shaped posterior section. None of these root domains were present at
2 days after birth, but their development had visibly started 5 days after
birth. As can be seen in Fig.
2C the root domain reached only halfway up the tooth at 5 dpn.
Above it there are still ameloblasts with enamel. At 14 dpn the root zone was
continuous from apex to the cusps. Histological analysis showed that this
enamel-free zone contained all the structures of a root, such as dentin,
cementum, cementoblasts, cementocytes and periodontal ligament
(Fig. 2C). Also, the apical
epithelium was seen as a double layer, identical to Hertwig's epithelial root
sheath, which became fragmented further away from the apex. The cementoblasts
showed a strong Bsp1 signal in the radioactive in situ hybridization
(Fig. 6I).
Differential gene expression in the root and crown domain of the vole
molar
In the vole molar two distinct domains are present, the crown and root
domain. All the Notch signaling pathway gene expression patterns were similar
at the anterior and posterior side of the vole molar in the 2 dpn tissue when
the root domains are still visibly absent (Figs
3,
4). Fgf10 was
expressed at the anterior root side similarly to the posterior crown side with
strong expression near the anterior cervical loop. At 5 dpn, however, the
formation of the root analogue in their restricted domains is well underway
and now the general trend is the absence of the Notch pathway genes in the
epithelial compartments of the anterior root analogue. Notch1 was no
longer expressed in the anterior cervical loop epithelium
(Fig. 5A). Epithelial
Hes1 expression was missing here, although the mesenchymal band of
Hes1 expression still ran across the entire tooth base
(Fig. 6C,D). No anterior
epithelial Lfng expression was visible and only a slight expression
of mesenchymal Lfng was detected in the subodontoblastic layer
(Fig. 5E). Anterior
Jag1 expression was weak and lacked clear boundaries
(Fig. 5G). Mesenchymal
Fgf10 was still present, albeit lacking the intensive regions of
expression normally found around the intercuspal folds and posterior cervical
loop (Fig. 6E). Delta1
expression was found in the differentiating odontoblast similarly to the
posterior side, indicating that odontoblast differentiation is not affected
(Fig. 6A).
Notch and FGF10 signaling in the mouse molar root
Unlike the vole molar, the crown of the mouse molar stops growing
completely and the entire epithelium switches to the root fate. On a molecular
level the most striking difference is the absence of some of the key genes
involved in the regulation of the epithelial stem cell compartment.
Fgf10 and Fgf3 are absent in 10 and 14 dpn mouse molars
(Fig. 7K,L: 10 dpn and
Fgf3 data not shown). Notch1, 3, Hes1 and 5 also
showed no expression in or near the cervical loop area at these stages
(Fig. 7A,B,E,F,I,J:
Hes5 data not shown). Patches of Notch1 and 3 were
associated with blood vessels in the dental mesenchyme. Only Notch2
and Lfng were expressed in the cervical loop area. Notch2
was expressed throughout the dental mesenchyme, with the exception of the
odontoblasts (Fig. 7C,D). Strong expression was also found in Hertwig's epithelial root sheath, the
outer enamel epithelium and the epithelium covering the crown. It was absent
in the ameloblasts. Lfng was also expressed in Hertwig's epithelial
root sheath, albeit not with the same intensity as in the transit-amplifying
cells in the vole molar (Fig.
7G,H). Lfng expression was lacking in the remainder of
the tooth, except for some light expression in the mesenchyme near the root
tips, likely representing the differentiating odontoblasts. Hes1 was
expressed in preodontoblasts and odontoblasts, and in mature ameloblasts.
Hes5 expression was not found. Jagged1 only showed
expression in ameloblasts. Delta1 expression was mesenchymal and not
very strong and showed a slight gradient, with intensity increasing towards
the cusps (data not shown).
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DISCUSSION |
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If the cervical loop changes its developmental fate into a root the
stellate reticulum degenerates leaving just the inner and outer enamel
epithelium as a double layer of epithelium and is known as Hertwig's
epithelial root sheath (HERS) (Fig.
1). This structure continues to proliferate for a limited time and
directs root formation (Thomas,
1995). As the root lengthens the HERS is fragmented and invaded by
cementoblast precursors and is known as the epithelial cell rests of Malassez.
The limited proliferative capacity of both structures suggests that both these
epithelial structures lack stem cells.
What makes a continuously growing tooth: morphology
We propose that for the hypselodont (continuously growing) teeth the
cervical loop is an essential structure. We showed that the continuously
growing molar of the vole maintains its cervical loop during late postnatal
development, and that it is present in the entire circumference of the tooth
base. It has been suggested that the bulging nature caused by the large
stellate reticulum compartment of the cervical loop typical of the rodent
incisor is a requirement for a functional stem cell niche
(Harada et al., 2002). However,
the entire cervical loop area of the vole molar is rather flat
(Fig. 2B). We therefore propose
that the functional essence of the cervical loop of a continuously growing
tooth during development is not size, but merely the presence of the proper
structural components: the inner and/or outer enamel epithelium and within it
the stem cell containing stellate reticulum and/or stratum intermedium
cells.
What makes a continuously growing tooth: molecular regulation
In order to keep producing crown, the original structure of the cervical
loop, and therefore the stem cell niche, has to be maintained. Epithelial
Notch and mesenchymal FGF10 signaling are part of the molecular regulation of
the epithelial stem cell niche in the mouse incisor and the subsequent
differentiation of the progeny into functional ameloblasts
(Harada et al., 1999;
Harada et al., 2002
). We found
a similar regulatory set-up in the vole molar. Based on the similar
distribution of expression patterns, the epithelial cell lineage can be
subdivided into several domains with increasing levels of differentiation
(Fig. 8). There is the stem
cell compartment characterized by Notch expression, a proliferation
compartment characterized by Lfng expression and then a subset of
differentiation domains each representative of a different degree of
differentiation and each with their own specific marker, such as Jag1
and Bsp1. It is not known how Notch regulates the stem cells, but
previously Notch has been associated with stem cell differentiation in many
different tissues, such as neurons and glia
(Wang and Barres, 2000
;
Lütolf et al., 2002
),
lymphocytes (Anderson et al.,
2001
), pancreas (Apelqvist et
al., 1999
) and epidermis
(Lowell et al., 2000
). One
possible function could be the regulation of stem cell division
(Chenn and McConnell, 1995
).
Hes1 is one of the downstream targets of Notch signaling, but its
expression was almost identical to that of Notch1, 2 and 3,
with the highest levels of expression in the stratum intermedium.
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We showed that Fgf10 was expressed in the dental mesenchyme near
the cervical loop and around the base of the intercuspal folds
(Fig. 4E,F,
Fig. 6E,F) similar to the
incisor. It has been postulated that FGF10 stimulates the division of both
stem cells and transit-amplifying cells in the cervical loop
(Harada et al., 1999;
Harada et al., 2002
). Harada
and co-workers also showed that the FGF receptors FGFR1b and 2b are expressed
throughout the cervical loop epithelium. The identical expression domain of
Bmp4 (Fig. 6G,H) and
Fgf10 suggests that BMP signaling could be involved in stem cell
regulation, perhaps in cooperation with FGF10. We showed earlier that FGF and
BMP are involved in the regulation of Lfng expression in the
progenitors of cervical loop epithelium during early tooth development, where
they have an antagonistic effect on Lfng and a synergistic effect on
Hes1 expression (Mustonen et al.,
2002
). A similar system could remain in place in the continuously
growing tooth.
The intercuspal loop: a stem cell niche similar to the cervical
loop
The most striking difference between mouse incisor and vole molar is the
presence of the complex cusp pattern in the vole molar
(Fig. 1), whereas the incisor
ends in a simple cone-shaped tip. The cusp pattern is a result of the
epithelial folding mediated by the actions of the enamel knots earlier during
development (Jernvall et al.,
2000; Vaahtokari et al.,
1996
). The folding leads to the formation of intercuspal loops
(Fig. 1,
Fig. 2A,B). These loops consist
of a basal layer of inner enamel epithelium which envelopes first a layer of
stratum intermedium and more to the inside the stellate reticulum. The
intercuspal loop is therefore structurally comparable to the cervical loop.
Moreover, Notch signaling pathway genes, Fgf10 and Bmp4 were
present in the intercuspal loop in a similar pattern to that in the cervical
loop. The intercuspal loop could therefore function in the same way as the
cervical loop, including acting as a stem cell niche.
Root analogue in the vole molar
We have shown that the vole molar possessed three small root-like domains
(Fig. 2C-G). The root domains
in the vole molar contained all the histological features of a root and a
typical expression of Bsp1 in the cementoblasts
(D'Errico et al., 1997). We
therefore propose that continuously growing teeth in general solve the problem
of lack of anchorage, resulting from the absence of a classic root system, by
transforming several small domains into the root fate. The presence of the
anterior root analogue in the vole molar confirmed the lack of involvement of
the Notch signaling pathway in root differentiation. In the anterior part of
the vole molar, all Notch pathway genes were absent in the dental epithelium
once root formation had been initiated (Figs
5,
6). The situation seems to be
different for the dental mesenchyme since odontoblast formation is still an
ongoing process, whereas the epithelial ameloblast differentiation is halted
when root formation starts.
Continuous growth in vole molar versus growth arrest in mouse
molar
Although the early development of the mouse molar is almost identical to
that of the vole (Keränen et al.,
1998), later in development an important developmental decision is
made differently. The mouse molar arrests its crown development and the
epithelium switches to a root fate. Therefore, it is to be expected that the
molecular regulation of the cervical loop area is different in vole and mouse
molars. From the viewpoint of functional morphology the vole molar resembles
the mouse molar more than the mouse molar resembles the incisor. Relatively
recently on the evolutionary time scale, a change occurred in the molecular
regulation of the vole molar, which resulted in the extended growth period of
the crown epithelium. The Notch pathway genes were not expressed in the root
epithelium of the mouse molar at 10 or 14 dpn with the exception of
Notch2 and Lfng (Fig.
7: 10 dpn not shown). These two mRNAs were found in Herwig's
epithelial root sheath. Since the HERS is the site of epithelial proliferation
in the root (Kaneko et al.,
1999
), Lfng could be associated with proliferation
similar to that in the cervical loop area. The Notch pathway was, however,
mostly active in the mesenchyme of the crown area instead of the cervical loop
area (Fig. 7).
It has been reported earlier that Fgf10 and Fgf3
expression is diminished in mouse molars soon after birth
(Kettunen et al., 2000). We
have shown that Fgf10 and Fgf3 are completely absent at 10
and 14 dpn when root growth still continues
(Fig. 7K,L). In Fgf10
knockout mice the early morphogenesis of the incisor is normal, but at later
stages the cervical loop is greatly diminished in size and the incisor stops
growing (Harada et al., 2002
).
Also in these mice antibodies against FGF10 halted the growth of the incisor
and human FGF10 protein could rescue this phenotype. Although FGF10 controls
the survival of the cervical loop area, it is unknown if FGF10 is needed for
the stem cells or just the proliferation of the transit-amplifying cell pool.
It is also unclear how the downregulation of Notch and FGF signaling is
regulated.
Evolutionary flexibility of the regulation of the adult stem cell
compartment in teeth
Not only is the regulation of the epithelial stem cell compartment
conserved between hypselodont molars and incisors, it also seems that this
regulation is flexible. Not all vole species have continuously growing molars.
Some do develop roots, but only after an extended period of crown growth when
compared to the mouse. This results in hypsodont teeth with a typical high
crown. Hypsodont teeth are quite common in the animal kingdom
(Janis and Fortelius, 1988),
suggesting that the prolongation of crown growth and subsequent delayed switch
to root fate is a flexible regulatory process. The continuously growing vole
molar could therefore be seen as an extreme modification of this regulatory
switch, i.e. the growth period of the crown is extended for so long that the
switch is never used. We suggest that all continuously growing teeth use the
same molecular pathways, FGF, Notch and perhaps BMP for the regulation of the
epithelial stem cell niche (Fig.
8). These pathways can be altered spatially and temporally to
fulfil the different functional requirements of teeth, be it continuous
growth, root development, restricted root development, or postponement of root
development.
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ACKNOWLEDGMENTS |
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