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First published online January 13, 2009
doi: 10.1242/10.1242/dev.025064
Developmental Biology Program, Institute of Biotechnology, PO Box 56, University of Helsinki, FIN-00014, Helsinki, Finland.
* Author for correspondence (e-mail: jernvall{at}fastmail.fm)
Accepted 20 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Wise, Usag1, Vestigial organs, Mouse, Sostdc1 (ectodin)
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), long-lost structures are exceedingly rare to reappear. Even
long-lost features, however, may still be retained as vestigial structures
before disappearing altogether. For example, the developing mouse dentition
contains potential to make more teeth than are present in the adult dental
pattern, which is often attributed to be due to the presence of vestigial
teeth (Peterkova et al., 2002;
Keränen et al., 1999).
Vestigial tooth germs have been reported in the mouse toothless diastema
region between the incisors and the first molar
(Peterkova et al., 2002), and
also in the mouse incisor region
(Peterkova et al., 2002).
These tooth rudiments, which would have been retained for over 45 million
years, do not develop beyond early invagination of the epithelium, or the
early bud stage, and they degenerate apoptotically during later development
(Keränen et al., 1999;
Peterkova et al., 2002). In
addition to these missing tooth loci, rodents have lost the ability for tooth
replacement from deciduous to permanent incisors. The loss of successional
replacement of molars is evolutionarily basal for all living mammals.
Developmentally, vestigial mouse tooth rudiments disappear after the
inductive potential of tooth formation has shifted from the epithelium to the
mesenchyme (Vainio et al.,
1993; Mina and Kollar,
1987; Kollar and Baird,
1969). The neural crest-derived dental mesenchyme can induce tooth
formation when combined with non-dental epithelium, raising the question of
what mechanism prevents the full development of rudimentary teeth? Several
mutant and transgenic mouse strains have supernumerary molars, predominantly
anterior to the first molar, where previous reports have identified vestigial
tooth buds (Mustonen et al.,
2003; Klein et al.,
2006; Kassai et al.,
2005). The analysis of the developmental origins of these
supernumerary molars has indicated that they develop from the vestigial buds,
and thus appear to `rescue' the teeth lost during evolution
(Mustonen et al., 2003;
Klein et al., 2006;
Kassai et al., 2005). At the
molecular level, these works suggest a role for multiple feedback loops
activating and inhibiting tooth development and number, but it remains unknown
how these interactions may modulate the inductive potential of the dental
mesenchyme.
A signaling pathway affecting both incisor and diastema vestigial teeth in
mice is the bone morphogenetic protein (BMP) pathway. BMP4 is required for the
shift of the inductive potential from the presumptive dental epithelium to the
dental mesenchyme after embryonic day 12.5 (E12.5)
(Vainio et al., 1993;
Chen et al., 1996). Epithelial
BMP4 induces the expression of the homeobox gene Msx1, which is
required for advancing tooth development and for the shift of Bmp4
expression to mesenchyme (Vainio et al.,
1993; Chen et al.,
1996). In Msx1 mutants, development of all teeth is
arrested at the early bud stage, and mesenchymal Bmp4 expression is
lacking (Chen et al., 1996).
This phenotype can be rescued by forced Bmp4 expression in the
mesenchyme (Zhao et al., 2000;
Bei et al., 2000). Tooth
development also ceases at the bud stage in mice that lack the functional BMP
receptor type 1A in the epithelium (Andl et
al., 2004).
Whereas partial downregulation of BMP signaling decreases tooth number
(Plikus et al., 2005),
deletion of the BMP antagonist Sostdc1 rescues the vestigial molar
tooth buds (Kassai et al.,
2005). Sostdc1, which was discovered three times
independently and has also been called ectodin, Usag1 and
Wise (Laurikkala et al.,
2003; Yanagita et al.,
2004; Itasaki et al.,
2003), is a secreted molecule identified as a BMP inhibitor
binding to BMPs with high affinity
(Laurikkala et al., 2003;
Yanagita et al., 2004). BMPs
themselves induce the expression of Sostdc1
(Laurikkala et al., 2003).
Sostdc1 has also been shown to regulate the Wnt pathway in a context-dependent
manner. It is able to compete with Wnts for binding to the Wnt co-receptor
Lrp6 (Itasaki et al.,
2003).
We have previously shown that Sostdc1 affects the morphology of mouse
molars by restricting the size and the placement of epithelial signaling
centers: the enamel knots (Kassai et al.,
2005). In addition, in Sostdc1 knockout mice, an extra
tooth develops in the location of the vestigial premolar primordium
(Kassai et al., 2005) and in
the incisor region (Murashima-Suginami et
al., 2007) (Fig.
1). In this study, we used Sostdc1 null mutant mice
(Mus musculus Linnaeus) to explore the role of the mesenchyme in
modulating tooth number. We focused on the incisor because previous reports
have suggested the presence of multiple vestigial incisor buds in mice
(Peterkova et al., 2002).
Compared with living rodents, ancestors of rodents had both higher number of
incisors and replacement of incisors from deciduous to permanent teeth. This
raises the issue of whether the extra incisors in Sostdc1-deficient
mice represent rescued teeth or rescued tooth replacement. Our results show
that the extra incisors in Sostdc1-deficient mice are likely to be
the latter: successional incisors. Furthermore, we show the amount of
mesenchymal tissue alone may influence the outcome between induction and
inhibition of extra teeth and that Sostdc1 is a central modulator of
the inductive potential of dental mesenchyme.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and the heterozygous littermates were used as controls.
Wild-type embryos were from the NMRI strain and from heterozygous
Shh-GFP mice, which express a reporter gene [green fluorescence
protein (GFP)] under a Shh-promoter
(Harfe et al., 2004).
Sostdc1-deficient mice were crossed with the Shh-GFP reporter line.
The Catenb
ex3k14/+ mice have been described
previously (Järvinen et al.,
2006). Two different reporter mouse lines were crossed with
Sostdc1-deficient mice to study the localization of Wnt activity:
TOPgal (Jackson laboratory) and BATgal
(Maretto et al., 2003). The
reporter constructs consist of multiple TCF/Lef-binding sites and a WNT target
gene minimal promoter driving the expression of lacZ reporter gene
(DasGupta and Fuchs, 1999;
Maretto et al., 2003). The age
of the embryos was estimated from the appearance of the vaginal plug (E0) and
from their exterior features.
Organ culture
The teeth with some surrounding tissue were dissected from the lower jaw of
E12 to E14 embryos and cultured using a Trowell type organ culture system
(Sahlberg et al., 2002). The
medium was changed every second day and contained DMEM and F12 (Ham's Nutrient
Mix: Life Technologies) (1:1) supplemented with 10% fetal calf serum (PAA
laboratories, Pasching, Austria), 150 mg/ml ascorbic acid, glutamine and
penicillin-streptomycin. The explants were photographed every day using light
(Olympus SZX9) or fluorescent (Leica MZFLIII) microscopy.
Recombinant BMP4 (0.5 ng/µl R&D), Noggin (1 ng/µl R&D), Dkk1 (1 ng/µl R&D) and Sostdc1 (1 ng/µl, a kind gift from Dr N. Itoh, Kyoto, Japan) proteins were added into the medium. Canonical Wnt signaling was activated using a specific Gsk3beta inhibitor BIO (2 mM Calbiochem). In bead experiments, Affi-gel agarose beads (Biorad) were soaked in recombinant BMP4 (100 ng/µl R&D) or BSA, and they were placed on top of the tooth explants.
Processing of tissues for histology and in situ hybridization
Heads from wild-type and Sostdc1 knockout mouse embryos (E12-E17)
were dissected and fixed with 4% paraformaldehyde (PFA) at +4°C overnight.
They were dehydrated paraffin-embedded and serially sectioned at 7 µm.
Sections were counterstained with Hematoxylin and Eosin. Jaws for whole-mount
in situ hybridization were fixed similarly. The tissue culture samples were
treated with 100% ice-cold methanol for 5 minutes before fixing with 4% PFA.
Sample preparation was same for the TUNEL staining.
Three dimensional (3D) reconstructions
Digital pictures were taken of frontal serial sections of the incisor. For
the shape 3D reconstruction, the pictures were imported into a stack with
Scion Image software (version 4) and the individual slices were aligned with
the register function using the midline of the jaw as reference. The
epithelial shape was manually traced in Adobe Photoshop CS2 (PS) with the
brush function. The distance between the sections (7 µm) was translated to
a corresponding pixel distance based on a reference scale bar. The stack with
the modified slices was re-sliced at a perpendicular angle in order to create
a new stack with only one pixel distance between individual slices. The jagged
epithelial shape in the resulting slices were smoothened in PS with a batch
command: select white, expand five pixels, smooth 15 pixels, contract four
pixels, fill white (shape), invert selection, fill black (background). The E16
Sostdc1 -/- stack was smoothened entirely manually using the brush
function in PS. A projection was made of the stack and the sagittal view
selected for presentation.
For the 3D reconstruction of expression patterns, first the shape 3D reconstruction was created according to the previous protocol. Subsequently, the blue X-Gal staining was isolated by applying the high contrast red filter with the `black&white' command in PS, the levels adjusted and inverted. The epithelial expression of interest was isolated and treated in the same sequence of steps as the shape and projected on the corresponding slice of the shape projection.
In situ hybridization, TUNEL labeling and detection of lacZ
Whole-mount in situ hybridization was performed by using the InSituPro
robot (Intavis AG, Germany) as described earlier
(Laurikkala et al., 2003). BM
Purple AP Substrate Precipitating Solution (Boehringer Mannheim Gmbh, Germany)
was used to visualize the digoxigenin-labeled probes. Radioactive in situ
hybridization for paraffin sections was carried out according to standard
protocols using 35S-UTP labeling (Amersham). The following probes
were used: murine Shh (Vaahtokari
et al., 1996), p21
(Jernvall et al., 1998),
Msx2 (Jowett et al.,
1993), Sostdc1
(Laurikkala et al., 2003;
Kassai et al., 2005) and
Bmp4 (Vainio et al.,
1993).
Apoptotic cells were visualized by detecting DNA fragmentation in whole-mount samples and sections with the ApoTag Apoptosis Detection Kit.
Localization of Wnt activity through β-galactosidase in the embryonic tissue was revealed using X-Gal staining. E13-14 lower jaws were collected in Dulbecco's 1xPBS and fixed in 2% PFA, 0.2% glutaraldehyde for 30 minutes at 4°C. Tissues were incubated for 3x10 minutes in X-gal washing buffer (2 mM MgCl2, 0.02% NP-40 in PBS) and stained with X-gal staining solution (1 mg/ml X-Gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 in X-gal washing buffer) in room temperature. Serial sections were counterstained with Nuclear Fast Red.
DiI-labeling
Microinjections were performed by injecting fluorescent DiI [1,1'-di-
octadecyl-6,6-di(4-sulfophenyl)-3,3,3',3'-tetramethylindocarbocyanine]
(Invitrogen) to the in vitro cultured incisor explants. The color (diluted in
2 µg/ml DMSO) was injected to the cells that will later form the extra
incisor.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kassai et al., 2005). As in
molars, the incisor Sostdc1 expression correlates with Bmp4
expression (Fig. 1F), except
that in incisors, these genes are strongly expressed in the lingual side
(Fig. 1E,F), whereas in the
molars they are expressed in the buccal side
(Laurikkala et al., 2003;
Kassai et al., 2005).
Furthermore, compared with Sostdc1, Bmp4 expression is located in
closer proximity to the dental epithelium, including in the enamel knot cells
themselves and throughout the underlying mesenchyme.
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From E12, the expression of Shh, an epithelial placode and enamel
knot marker in Sostdc1-deficient jaws is stronger and broader in
incisors in comparison with the controls
(Fig. 1I-L; see Fig. S1 in the
supplementary material). The expanded expression of Shh in the
epithelium correlates with the expanded and stronger expression of
p21 expression (see Fig. S1 in the supplementary material), a
differentiation marker of the enamel knots
(Jernvall et al., 1998).
Moreover, the expanded Shh expression in the
Sostdc1-deficient epithelium corresponds to stages when
Sostdc1 expression is limited to the mesenchyme in the wild-type
incisors (Fig. 1C).
Specific lack of epithelial apoptosis in Sostdc1-deficient mice
Because rudimentary tooth germs and enamel knots are known to disappear
through apoptosis (Keränen et al.,
1999; Peterkova et al.,
2002), we used TUNEL-staining to study effects of Sostdc1
on apoptosis (Fig. 2A,B).
Almost no apoptosis was detected in the incisor buds at E12 and E13, and no
difference was seen between wild-type and mutant incisors (not shown).
However, at E14, the cap stage Sostdc1-deficient incisor epithelium
shows a marked lack of apoptosis (n=6/6) in the enamel knot, and
especially in the area closer to the oral epithelium
(Fig. 2A,B). There was only
limited apoptosis in the mesenchyme and it appeared similar in wild type and
the Sostdc1-deficient mice.
In order to examine whether the area of apoptosis in the epithelium close
to oral surface of E14 wild-type incisor is associated with the removal of a
vestigial incisor rudiment, we localized enamel knot markers at an earlier
stage - E13-E13.5 (Fig. 2C,D).
Because the Wnt pathway is another potential Sostdc1 target, and enamel knots
are sites of high Wnt signaling activity
(Järvinen et al., 2006),
we examined activity of Wnt pathway using TOPgal and BATgal reporter mice
crossed with the Sostdc1-deficient mice. Epithelial Wnt activity was
detected in the incisor enamel knot both in Sostdc1-deficient mice
and controls at E13-E13.5 (Fig.
2C,D; data not shown). In addition, we detected a small domain of
Wnt activity in the area of intense apoptosis in the E14 wild-type incisors
(Fig. 2A).
3D analysis reveals the origin of extra incisor in Sostdc1-deficient mice
We used 3D analysis of serial sections to determine the relationship
between enamel knot dynamics and extra incisor formation. The results show
that the small domain of Wnt activity located orally to the incisor enamel
knot is larger and longer in duration in the Sostdc1-deficient
incisors compared with the wild-type incisors
(Fig. 3A). Furthermore, 3D
analysis of later developmental stages
(Fig. 3B) show that the extra
incisor develops at the location previously showing Wnt activity and intense
apoptosis in the wild-type incisors. The illustrated 3D renderings are
projections from the medial side, and thus the location of the extra incisor
closely matches the final position of this tooth in the jaw
(Fig. 1H). Taken together, the
extra incisor appears not to originate from a separate bud along the dental
lamina, but rather to be developmentally part of the main incisor, much like
replacement teeth in mammals with more complete tooth replacements.
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Additionally, we tested whether the removal of the surrounding mesenchyme
from wild-type molar buds would also rescue the development of the vestigial
bud anterior to the first molar, which develops to an extra tooth in
Sostdc1 null mutants (Kassai et
al., 2005). We dissected the mandibular molar tooth germs at E12
stage and removed most of the surrounding mesenchyme. In a small number of
cases (n=4/20) supernumerary teeth formed anterior to the first
molars and their development resembled the phenotype of
Sostdc1-deficient mice (see Fig. S2 in the supplementary
material).
Removal of surrounding mesenchyme from Sostdc1-deficient incisors initiates the development of de novo incisors from the lingual dental epithelium
When the surrounding mesenchyme was trimmed from the
Sostdc1-deficient incisor explants, the extra incisor that forms in
vivo developed also in vitro (Fig.
5). However, the formation of extra incisors appears to continue,
and additional de novo incisors appeared after a few days of culture from the
lingual cervical loop area (Fig.
5A-D) (n=48/109). Because the de novo incisors develop in
immediate proximity to the other incisors, we examined the origins of these
teeth by microinjecting DiI-label in Sostdc1-deficient and control
explants expressing GFP in the Shh locus
(Fig. 6A-C). DiI was injected
after 4 days of culture to the lingual epithelium of E13 incisor at the site
where the `normal' extra incisor form. The DiI-label spreads during subsequent
culture and labels the extra incisor as it grows in both
Sostdc1-deficient and control explants
(Fig. 6A-C; see Fig. S3 in the
supplementary material). However, the de novo incisor in the trimmed
Sostdc1-deficient explant appears as a new DiI-negative outgrowth
from, or close to, the cervical loop of the main incisor
(Fig. 6A-C).
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We introduced exogenous BMP4 to dissected E13 control and Sostdc1-deficient incisor explants at the site where Bmp4 is normally co-expressed with Sostdc1 in the mesenchyme. BMP4-soaked beads were placed on the lingual side of incisor buds (owing to the flattening of in vitro cultured teeth, lingual position is an approximation) and the day when extra incisors appeared was tabulated (Fig. 7C). In control samples, extra incisors did not develop either in BMP4-treated or in the control explants (Fig. 7F). By contrast, in homozygous Sostdc1-deficient explants, BMP4 beads accelerated the formation of extra teeth (Fig. 7F). They had already started to form at the lingual aspect of the incisors during the second culture day, whereas in the absence of BMP bead, the formation of extra incisors was first seen after 4 days of culture (Fig. 7C).
When the timing of the initiation of extra incisor development was compared between the different explants, the Sostdc1-deficient teeth cultured with BMP4 beads showed the fastest initiation of extra incisors (Fig. 7C). However, the wild-type incisors with trimmed mesenchyme showed faster initiation of extra incisors than did Sostdc1-deficient incisors cultured with mesenchyme, suggesting the presence of other mesenchymal inhibitory factors in addition to Sostdc1.
Inhibition of either BMP or Wnt signal activity prevents the formation of the extra incisor in Sostdc1-deficient embryos
We tested whether known inhibitors of BMP and Wnt signaling could mimic the
function of Sostdc1 and prevent the formation of extra incisors in
Sostdc1-deficient embryos. Dissected E13 incisor explants were
cultured in the presence of Noggin and Dkk1, antagonists of BMP and Wnt
signaling, respectively. In addition, recombinant Sostdc1 was used as a
control to rescue Sostdc1 deficiency. All three inhibitors prevented
the formation of the extra incisors in Sostdc1-deficient and control
explants (see Fig. S4 in the supplementary material).
Activation of epithelial Wnt signaling mimics the effect of Sostdc1 deficiency in inducing de novo incisor formation from the lingual epithelium
Stimulation of Wnt signaling in dental epithelium by forced activation of
β-catenin causes dramatic stimulation of both molars and incisors in mice
(Järvinen et al., 2006).
Although the formation of extra molars in these mice was analyzed in detail
and it was demonstrated to occur continuously from previously formed teeth,
the details of the generation of the extra incisors were not studied. In order
to examine whether there were similarities with the formation of the extra
teeth in Sostdc1 knockout mice, we observed the development of
Catenbex3k14/+ incisors in organ culture. E13-E15
incisor tooth germs were dissected from the lower jaws of
Catenbex3k14/+ embryos and cultured for 1 week (see
Fig. S5 in the supplementary material). The formation of the supernumerary
teeth was progressive, and they formed successively, especially from the
lingual incisor epithelium of previously formed incisors (see Fig. S5A in the
supplementary material). These supernumerary incisors started to form earlier
in vitro than in vivo, which was similar to our observations in
Sostdc1-deficient explants. The de novo formation of incisors was
morphologically very similar in the two mouse mutants and indicates that the
lingual dental epithelium has the capacity for tooth formation. Additionally,
we administered BIO, a specific inhibitor of GSK3b, into the culture medium,
thus activating the canonical Wnt signaling pathways throughout the explants.
No extra incisor formed in these explants (not shown), but as BIO also had
inhibitory effects on the main incisor of the mutant, as well as on control
teeth, this may indicate inhibitory effects of enhanced mesenchymal Wnt
signaling on tooth development.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). After
this, the neural crest-derived dental mesenchyme can induce tooth formation
when combined with non-dental epithelium. Furthermore, several mutants are
known that interfere with the mesenchymal induction of the epithelial
signaling centers, enamel knots, causing the tooth development to arrest at
E13 bud stage (Kratochwil et al.,
1996; Bei and Maas,
1998; Peters and Balling,
1999; Åberg et al.,
2004). Whereas the inductive role of mesenchyme in tooth
development is well established (Vainio et
al., 1993; Mina and Kollar,
1987), our results show that induction of additional teeth can be
achieved by reducing the amount of dental mesenchyme, perhaps uncovering a
role for the mesenchyme in limiting excess tooth induction.
Mesenchymal Sostdc1 expression inhibits development of extra teeth
In the Sostdc1-deficient embryos, markers of tooth formation have
broader expression domains, encompassing the areas giving rise to the extra
teeth (Fig. 1). In themselves,
these kind of broader expression domains are to be expected because similar
changes have been reported in the molar region of genetically altered mouse
strains with extra molars (Kangas et al.,
2004; Mustonen et al.,
2003; Klein et al.,
2006; Zhang et al.,
2003). Our results also agree with previous reports on enlarged
expression domains of enamel knot markers, such as p21, in
Sostdc1-deficient molars and incisors
(Kassai et al., 2005;
Murashima-Suginami et al.,
2007; Murashima-Suginami et
al., 2008). These patterns are intriguing because, until E14,
Sostdc1 expression is predominantly limited to the mesenchyme
(Fig. 1), and the E13 broadened
expression patterns in the epithelium are thus suggestive of a role for
Sostdc1 in interfering with epithelio-mesenchymal induction.
The in vitro culture system allowed us to test how reducing the amount of
surrounding mesenchyme, and consequently decreasing Sostdc1
expression, would affect the incisor formation in the wild-type teeth
(Fig. 4). Our result, i.e. the
in vitro formation of the extra incisor, essentially phenocopies the
Sostdc1-deficient phenotype. The peak effect of mesenchymal trimming
was reached at E13, well within the stage of development when the inductive
potential for tooth formation resides in the mesenchyme
(Mina and Kollar, 1987). Our
work thus appears to have uncovered a complex role for the mesenchyme in the
regulation of tooth induction. Initially, the mesenchyme is obviously required
for normal tooth morphogenesis and the induction of epithelial enamel knots
(Kollar and Baird, 1970a;
Kollar and Baird, 1970b;
Jernvall et al., 1998), but it
might acquire inhibitory roles as the development proceeds and the
successional incisor starts to develop. One possibility for the observed
activation of the successional tooth after mesenchymal tissue reduction could
be slightly different expression domains of genes that induce and inhibit
tooth formation. For example, Sostdc1 appears to have broad
expression domains that expand laterally around developing teeth
(Fig. 1). Furthermore, peak
Sostdc1 expression intensity in E14 cap stage is further away from
the center of the tooth than Bmp4 expression
(Fig. 1), which has been
implicated in the induction of enamel knots and the regulation of
Sostdc1 itself (Laurikkala et
al., 2003; Kassai et al.,
2005; Bei et al.,
2000). Interestingly, the bioengineering of tooth germs from
dissociated dental cells in vitro has been shown to lead to the formation of
multiple incisors (Nakao et al.,
2007); it is plausible that these in vitro procedures disrupt the
inhibitory mechanisms of in vivo mesenchyme.
Both Bmp4 and Sostdc1 are intensely expressed in the
mesenchyme at the lingual side of incisor, corresponding to the location of
extra incisors in the Sostdc1-deficient mouse. This contrasts with
the buccal expression bias of Sostdc1 and Bmp4 in molars
(Jernvall et al., 1998;
Laurikkala et al., 2003;
Kassai et al., 2005). However,
in molars of Sostdc1-deficient mice, the cuspal morphology is
substantially altered in the buccal side. We interpret these results as
further supporting the role of Sostdc1 as a feedback inhibitor of
BMP4 in tooth development, and the role of BMP4 in regulating the lateral bias
in tooth patterning (Kassai et al.,
2005).
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) and the
mesenchyme (Yamamoto et al.,
2005; Yuan et al.,
2008) have been proposed to inhibit diastema tooth development.
Recently Yuan et al. (Yuan et al.,
2008) have shown that, whereas post E12 diastema mesenchyme
retains the capacity to form teeth, it cannot initiate tooth formation by
inducing epithelium. Because Sostdc1 expression domains partially
cover the diastemal regions, we propose that Sostdc1 may be one of
the genes suppressing diastemal tooth formation.
Normal removal of incipient extra incisors involves epithelial apoptosis
In our in vitro cultures of wild-type teeth, the experimental phenocopying
of the Sostdc1-deficient extra incisors was possible until the
incisors reached E14 cap-stage. By this developmental stage, Sostdc1
expression is upregulated also in the epithelium
(Fig. 1). This alone suggests
that trimming the E14 dental mesenchyme in vitro may not reduce
Sostdc1 expression enough to allow the development of extra incisors.
Another possibility for the decreased tooth making potential of E14 explants
may be apoptotic removal of the epithelial area that has potential to form
extra incisors. Increased apoptosis has been reported in multiple rudimentary
teeth (Tureckova et al., 1996;
Peterkova et al., 2002;
Keränen et al., 1999) and
we detected a distinct population of apoptotic cells in the dental epithelium
of the wild-type incisors at the site where the extra incisors of
Sostdc1 mutants develop. There was a dramatic lack of apoptotic
bodies in the dental epithelium of Sostdc1-deficient teeth at E14,
both in the enamel knot and especially in the region of the epithelium giving
rise to the extra incisors (Fig.
2). Unlike Murashima-Suginami
(Murashima-Suginami, 2007;
Murashima-Suginami, 2008), we
did not detect marked changes in mesenchymal apoptosis in the
Sostdc1-deficient teeth. Taken together, because earlier removal of
mesenchyme expressing Sostdc1 causes the development of the extra
incisors in the wild-type explants and because this ability is lost with the
upregulation of Sostdc1 expression and apoptosis in the epithelium,
we suggest that apoptosis may be a relatively downstream event in the in vivo
suppression of extra tooth formation.
Sostdc1 may not be the only mesenchymal factor inhibiting development of extra teeth
Because the trimming of the mesenchyme in the wild-type explants
consistently produced the extra incisor found in Sostdc1-deficent
mice in vivo and in vitro, we tested whether the trimming of mesenchyme in the
mutants had any effect. The results showed that, in addition to the one extra
incisor, additional teeth were initiated later from the lingual cervical loop
epithelium of the previously initiated incisors (Figs
5 and
6). This de novo incisor
formation in the trimmed Sostdc1 null explants suggests that there
are additional genes expressed in the mesenchyme that suppress extra teeth.
The phenotype of these de novo incisors resembles the supernumerary teeth that
form when epithelial β-catenin is constitutively stabilized in transgenic
mice (Catenbex3k14/+) (see Fig. S5 in the
supplementary material). Normally, the stabilization of epithelial
β-catenin requires canonical Wnt signaling and the additional mesenchymal
factors may thus involve modulators of Wnt signaling pathway. As
Sostdc1 has been implicated in the inhibition of both BMP and Wnt
signaling (Laurikkala et al.,
2003; Itasaki et al.,
2003; Yanagita et al.,
2004), there may be partly redundant signaling pathways that
restrict extra tooth formation.
Inhibition of both BMP and Wnt signaling contribute to the inhibitory role of the dental mesenchyme
We have previously linked Sostdc1 to BMP signaling in tooth
development (Laurikkala et al.,
2003; Kassai et al.,
2005). BMP4 can induce the expression of Sostdc1, which
in turn antagonizes the induction of enamel knot marker p21 by BMPs
in tissue cultures (Laurikkala et al.,
2003; Kassai et al.,
2005). The role of BMP4 as mesenchymally expressed enamel knot
inducer was strengthened in the absence of Sostdc1, as shown by our
experiments where we placed BMP4-releasing beads on the lingual side of the in
vitro mutant explants (Fig. 7).
BMP4 accelerated extra incisor formation even further than did the trimming of
the mesenchyme. However, we observed acceleration of extra incisor formation
in only the Sostdc1-deficient explants, perhaps further supporting
the role of Sostdc1 as a feedback inhibitor of BMP signaling
(Kassai et al., 2005).
Additionally, we have previously shown a relatively subtle acceleration of
normal molar initiation by BMP4 (Kavanagh
et al., 2007), and it is conceivable that the `rescue' of extra
teeth may require additional factors.
|
). This link is indirectly supported by the resemblance of the
de novo supernumerary tooth formation in Sostdc1-deficient mice with
the phenotype of Catenbex3k14/+ mice (see Fig. S5
in the supplementary material). In addition, Dkk1, a potent Wnt signal
inhibitor inhibited extra incisor formation in vitro. The BMP antagonist
Noggin had a similar inhibitory effect, as also shown recently by
Murashima-Suginami et al.
(Murashima-Suginami et al.,
2008) (see Fig. S4 in the supplementary material). These
inhibitory effects agree with previous works implicating both Wnt and BMP as
activators of tooth initiation
(Järvinen et al., 2006;
Bei et al., 2000;
Wise and Stock, 2006). It is
noteworthy that the de novo incisors formed only in the absence of epithelial
Sostdc1 expression. Whereas mesenchymal Sostdc1 expression
may predominantly antagonize mesenchymally expressed Bmp4, the
epithelially expressed Sostdc1 may also interfere with epithelial Wnt
signaling crucial for tooth induction, a possibility supported by the
increased Wnt activity in the Sostdc1-deficient incisors
(Fig. 3). In developing hair
placode epithelia, Bmp expression is upregulated as a result of
β-catenin stabilization (Närhi
et al., 2008), and it is conceivable that Wnt and BMP signaling
may function both up- and downstream from each other at different stages of
development.
Conclusions and a hypothesis on the identity of the extra incisors
Our results are indicative of a role for mesenchyme, through
Sostdc1 and at least one additional factor, in inhibiting extra tooth
formation. The exact developmental origin of the extra incisor
(Fig. 3) is reminiscent of the
location from which normal replacement teeth are initiated in many mammals
(e.g. Luckett, 1985;
Järvinen et al., 2009).
Therefore, we propose that the most parsimonious interpretation of the extra
incisor in Sostdc1-deficient mice is to consider it as a replacement
tooth. This fits with the views that, evolutionarily, rodent incisors are
interpreted to be second, and sometimes first, deciduous incisors
(Luckett 1985;
Meng et al., 2003;
Asher et al., 2005). The
permanent replacement incisor was lost early in the evolutionary history of
rodents, together with the reduction of the number of incisors to the one pair
found in all rodents. This scenario for the role of Sostdc1 may be
further supported by the de novo supernumerary incisors formed in the
Sostdc1 mutants with trimmed mesenchyme. These teeth resemble the
continuous tooth budding found in the
Catenbex3k14/+ mice. Hence, a central role of
Sostdc1 in normal tooth development may be to modulate BMP and Wnt
signaling in limiting tooth replacement. In addition to tooth replacement, the
Sostdc1-Wnt-BMP signaling may be part of the developmental program limiting
the induction of teeth spatially. Interestingly, the region-specific absence
of dentition in teleost fish seems to correlate with the lack of Bmp
expression (Wise and Stock,
2006), and it remains to be tested whether Sostdc1-like
inhibition may also be involved in spatial delineation of tooth-forming areas
in other vertebrates. Considering the role of mesenchyme in tooth induction
and the design of tissue engineering protocols, our work may have uncovered
how delicate control of tissue quantities alone may influence the outcome
between induction and inhibition.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/393/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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