The extensive variation in tooth number and morphology in
vertebrates raises important questions about how patterning of
dentition is controlled during evolution and development. In
mammals, teeth develop sequentially in an anterior-to-posterior
direction. The initiation of tooth development is characterized by
thickening of the oral ectoderm and subsequent condensation of
neural-crest-derived mesenchyme around the invaginating
epithelium to form tooth buds (Tucker and Sharpe, 2004).
Signaling between the dental epithelium and mesenchyme
modulates the survival and growth of tooth buds. This signaling is
crucial for determining tooth number, as rudimentary or vestigial
buds initially form in the toothless diastema region between the
incisors and molars but degenerate without reaching the cap stage
(Peterkova et al., 2006). At the beginning of the cap stage, a
transient epithelial signaling center, called the enamel knot, is
induced at the tip of the tooth bud and it regulates tooth growth and
morphogenesis (Tucker and Sharpe, 2004).
Mutations in a number of genes encoding components of major
signaling pathways have been shown to influence tooth number.
This is consistent with the idea that crosstalk between several major
signaling pathways, such as Fgf, Shh, Wnt and Bmp, regulates
tissue interactions and modulates tooth formation (Tummers and
Thesleff, 2009). Many of these pathways are reiteratively used at
different stages of tooth development. In the case of Wnt signaling,
tooth development is arrested at the early bud stage when Wnt
signaling is inactivated, either by conditional knockout of b-catenin
or by overexpression of the Wnt antagonist dickkopf 1 (Dkk1) in
the dental epithelium (Andl et al., 2002; Liu et al., 2008).
Conversely, ectopic Wnt activation leads to supernumerary teeth as
well as abnormal cusp patterning (Jarvinen et al., 2006; Wang et
al., 2009). Therefore, tight control of Wnt signaling activity is
essential for normal tooth development, yet it is unclear how this
control is achieved and how Wnt signaling interacts with other
signaling pathways during tooth development.
Wise (also known as Sostdc1, ectodin and USAG-1) was
identified in a functional screen as a gene encoding a conserved
secreted protein capable of modulating canonical Wnt signaling
(Itasaki et al., 2003). In vitro assays revealed that Wise and the
closely related Sost protein bind to the extracellular domain of the
Wnt co-receptors Lrp5 and Lrp6 and inhibit Wnt signaling (Ellies
and Krumlauf, 2006; Itasaki et al., 2003; Li et al., 2005; Lintern et
al., 2009; Semenov et al., 2005). In vitro assays have also revealed
that Wise can bind to the extracellular domain of the related Lrp4
receptor (Ohazama et al., 2008). Based on mutations of Lrp4 in
humans and mice, it has been postulated that Lrp4 can modulate
Wnt signaling mediated by Lrp5 and Lrp6 (Choi et al., 2009; Li et
al., 2010; Ohazama et al., 2008; Weatherbee et al., 2006). In
addition, Wise is phylogenetically related to several subgroups of
Bmp antagonists within the cystine-knot superfamily, and Wise has
been shown to bind to a subset of Bmps in vitro and to influence
Bmp signaling (Laurikkala et al., 2003). Therefore, Wise has the
potential to provide multiple regulatory inputs into Lrp5/6, Bmp-
and Lrp4-dependent pathways.
Wise loss-of-function mutants display defects in many aspects of
tooth development including tooth number, size and cusp pattern
(Kassai et al., 2005; Yanagita et al., 2006). Loss of Wise can increase
the sensitivity to excess Bmp in cultured teeth, suggesting that Wise
might have a function as a Bmp antagonist in teeth (Kassai et al.,
2005). Mice homozygous for a hypomorphic allele of Lrp4
displayed tooth defects similar to those of Wise-null mice,
Development 137, 3221-3231 (2010) doi:10.1242/dev.054668
© 2010. Published by The Company of Biologists Ltd
1Stowers Institute for Medical Research, Kansas City, MO 64110, USA. 2Departments
of Orofacial Sciences and Pediatrics, Program in Craniofacial and Mesenchymal
Biology, University of California at San Francisco, San Francisco, CA 94143-0442,
USA. 3Department of Anatomy and Cell Biology, University of Kansas Medical
Center, Kansas City, KS 66160, USA.
*Author for correspondence (firstname.lastname@example.org)
Accepted 14 July 2010
Mice carrying mutations in Wise (Sostdc1) display defects in many aspects of tooth development, including tooth number, size
and cusp pattern. To understand the basis of these defects, we have investigated the pathways modulated by Wise in tooth
development. We present evidence that, in tooth development, Wise suppresses survival of the diastema or incisor vestigial buds
by serving as an inhibitor of Lrp5- and Lrp6-dependent Wnt signaling. Reducing the dosage of the Wnt co-receptor genes Lrp5
and Lrp6 rescues the Wise-null tooth phenotypes. Inactivation of Wise leads to elevated Wnt signaling and, as a consequence,
vestigial tooth buds in the normally toothless diastema region display increased proliferation and continuous development to
form supernumerary teeth. Conversely, gain-of-function studies show that ectopic Wise reduces Wnt signaling and tooth number.
Our analyses demonstrate that the Fgf and Shh pathways are major downstream targets of Wise-regulated Wnt signaling.
Furthermore, our experiments revealed that Shh acts as a negative-feedback regulator of Wnt signaling and thus determines the
fate of the vestigial buds and later tooth patterning. These data provide insight into the mechanisms that control Wnt signaling
in tooth development and into how crosstalk among signaling pathways controls tooth number and morphogenesis.
KEY WORDS: Wnt signaling, Shh, Fgf, Wnt antagonists, Feedback regulation, Tooth development, Mouse
Inhibition of Wnt signaling by Wise (Sostdc1) and negative
feedback from Shh controls tooth number and patterning
Youngwook Ahn1, Brian W. Sanderson1, Ophir D. Klein2and Robb Krumlauf1,3,*
suggesting that they might cooperate in regulating Wnt signaling
(Ohazama et al., 2008). Furthermore, a recent study with cultured
Wise-deficient incisors suggested that Wise from the dental
mesenchyme can limit tooth formation via inhibition of both Bmp
and Wnt signaling (Munne et al., 2009). As signaling pathways play
diverse roles at multiple stages of tooth development it is important
to understand the stage- and process-specific mechanisms through
which Wise exerts its in vivo regulatory activity.
In this study, we have used Wise-null mutants and a transgenic
gain-of-function system to investigate crosstalk between signaling
pathways and the molecular and cellular mechanisms that regulate
the development and ultimately the number of teeth. We present
genetic evidence that Wise inhibits Wnt signaling dependent upon
the Wnt co-receptors Lrp5 and Lrp6 to suppress survival of
vestigial buds in the incisor and molar regions. We found that the
Fgf and Shh pathways are major downstream targets of Wise-
regulated Wnt signaling and that Shh acts as a negative-feedback
regulator of Wnt signaling during the bud-to-cap transition. Our
data provide insight into how signaling pathways interact with each
other to regulate cellular processes which govern key aspects of
MATERIALS AND METHODS
Lrp5, Lrp6, Top-Gal, Ctnnb1(ex3)fx, ShhGFPcre, ShhcreERT, Shhneo, Shhfx,
Ptch1LacZ, R26R, Fgfr1, Fgfr2, Fgf10, Bmpr1a and K14-Cre mice were
described previously (Chiang et al., 1996; DasGupta and Fuchs, 1999;
Dassule et al., 2000; Harada et al., 1999; Harfe et al., 2004; Kato et al.,
2002; Milenkovic et al., 1999; Min et al., 1998; Mishina et al., 2002;
Pinson et al., 2000; Soriano, 1999; Trokovic et al., 2003; Yu et al., 2003).
All experiments involving mice were approved by the Institutional Animal
Care and Use Committee of the Stowers Institute for Medical Research
Generation of transgenic mice
To produce a Wise-LacZ BAC reporter, a LacZ-SV40polyA sequence was
inserted in-frame into the first coding exon of Wise in a mouse BAC clone,
RP23-166E23, which contains an 191 kb genomic region. BAC DNA was
prepared using Qiagen Maxi-Prep Kit, linearized with PI-SceI and used for
pronuclear injection into C57B6/JxCBA-F1 embryos.
A 2.2 kb promoter of the human keratin 14 gene was amplified from a
BAC clone (RP11-434D2) using the primers 5?-AAGATCTAGG -
TGCGTGGGGTTGGGATG-3? and 5?-GAAGCTTGAGCGAGCAG -
TTGGCTGAGTG-3? and subcloned into the pCMS-EGFP vector
(Clontech) replacing the CMV promoter. The mouse Wise cDNA was
inserted downstream of the promoter. The 3.5 kb insert was gel-purified
and injected into either C57B6/JxCBA-F1 or Top-Gal one-cell embryos.
b b-gal staining, in situ hybridization and BrdU analysis
For b-gal staining, embryonic jaws were fixed in either 0.1%
paraformaldehyde with 0.2% glutaraldehyde or 4% paraformaldehyde
(PFA) for one hour on ice. After washing in phosphate-buffered saline,
samples were stained in X-gal for 6-20 hours at room temperature.
Wholemount in situ hybridization was performed with jaws fixed in 4%
PFA according to standard protocols using DIG-labeled riboprobes. Cell
proliferation was measured by injecting BrdU into pregnant females 2
hours before embryo harvest. Histological samples were paraffin-
embedded, sectioned at 8 m and stained with a rabbit caspase 3 antibody
(Cell Signaling) or a mouse anti-BrdU antibody (Amersham). To induce
Cre-recombination in Shh+/CreERTembryos, Tamoxifen was injected into
pregnant females at the dose of 3-6 mg/40 g body weight.
Embryonic day (E) 13.5 tooth germs were dissected from 14 mandibles of
Wise-null embryos and Wise heterozygous littermates. Total RNA was
isolated and used for qPCR according to the manufacturer’s protocol
(SABiosciences). Four replicas were run for each signaling pathway array
and the statistical analysis was performed using the software provided by
the manufacturer. To compare gene expression between Wise+/–and
Wise+/–;Shh+/–tooth germs at E13.5, total RNA was extracted from six
mandibular tooth germs for each genotype and four replicas were run with
a TaqMan Array (Applied Biosystems).
Tooth phenotypes in Wise-null mice
We have used Wise-null mutants, which exhibit a variety of tooth
defects, to investigate the genetic mechanisms that regulate the
number and shape of teeth. Our Wise-null mice were generated by
knock-in of a selection cassette into the first exon of the gene.
Homozygous mutants displayed all the tooth abnormalities
previously reported by other groups, including supernumerary
incisors and molars, fused molars and cusp defects (Fig. 1; see Fig.
S1A-F in the supplementary material) (Kassai et al., 2005; Yanagita
et al., 2006). The maxillary molar region of Wise-null mice
displayed extensive fusion of anterior teeth with full penetrance,
resulting in two teeth in each jaw quadrant (Fig. 1B). In the
mandibular molar region, there were two general phenotypes
observed. In two-thirds of the animals, in place of the three molars
(M1-M3) seen in control mice, four teeth (T1-T4) were observed in
each jaw quadrant. In the remaining animals, we observed T1-T2
and/or T2-T3fusions (Fig. 1B,H). We also observed a small extra
tooth lateral to T2on the lingual side and a varying number of
small teeth in the T4region (Fig. 1B). The frequency of the fusions
and lateral teeth varied with strain backgrounds and was higher in
Genetic interactions between Wise and the Fgf
and Bmp pathways
It is known that mutations in Spry2 or Spry4, which are Fgf
antagonists, result in formation of a premolar-like tooth due to
elevated Fgf signaling and survival of a diastema bud (Klein et al.,
2006; Peterkova et al., 2009). As the supernumerary teeth in the
sprouty mutants were rescued by reducing dosage of genes
encoding Fgf receptors (Fgfr1 or Fgfr2) or ligands (Fgf10) (Klein
et al., 2006), we attempted to rescue Wise-null tooth phenotypes
through a similar strategy. However, the appearance of four molar
teeth or supernumerary maxillary incisors in Wise-null mice was
not affected in any of the compound mutant mice (see Fig. S1 in
the supplementary material). Thus, modest reductions in Fgf
signaling were not sufficient to compensate for the loss of Wise
with respect to tooth number. As Wise has been implicated as a
Bmp antagonist, we also attempted to rescue the Wise tooth
phenotypes by reducing levels of Bmp signaling through removal
of a copy of the Bmpr1a type I receptor gene. There were no
significant changes in any of the phenotypes in Wise–/–;Bmpr1+/–
mice (see Fig. S1J,N in the supplementary material).
Dosage-dependent rescue of Wise tooth
phenotypes by Lrp5 and Lrp6
Wise has been implicated in modulation of the canonical Wnt
signaling pathway through its ability to interact with the
extracellular domain of the Lrp5 and Lrp6 co-receptors (Itasaki et
al., 2003). We hypothesized that elevated Wnt signaling might
account for abnormal tooth development in Wise-null mice.
Therefore, we attempted to lower levels of Wnt signaling by
reducing the dosage of the Lrp5 and Lrp6 co-receptor genes.
Reduction in copies of Lrp5 and/or Lrp6 themselves did not result
in tooth abnormalities (data not shown). However, in Wise-null
Development 137 (19)
mice, we observed strong genetic interactions between Wise and
Lrp5 and Lrp6, as evidenced by rescue of all incisor and molar
Removal of only a single copy of Lrp6 in Wise-null mice
rescued both the supernumerary maxillary (90%) and mandibular
(100%) incisor phenotypes (Fig. 1C,H; data not shown). Lrp6–/–
mutants are embryonic lethal and hence we could not test this
combination. Removing one copy of Lrp5 had no effect (Fig.
1D,H), but in Wise–/–;Lrp5–/–mice, supernumerary incisors were
rescued in 22% of cases (Fig. 1E,H). When mice carried one or
two Lrp5-null alleles in addition to an Lrp6-null allele, the
supernumerary incisor phenotypes were completely rescued (Fig.
With respect to the abnormalities in the mandibular molars of
Wise-null mice, removing one copy of either Lrp5 or Lrp6 rescued
some aspects of the phenotype, as evidenced by the absence of a
lateral supernumerary molar in 98% of cases (Fig. 1C,D). Although
molar fusions completely disappeared in Wise–/–;Lrp5+/–mice, only
T2-T3fusions were rescued in Wise–/–;Lrp6+/–mice (Fig. 1H).
Removing two of the four copies of the co-receptors resulted in a
smaller T1, but four molars were still present (Fig. 1E,F). Finally,
in Wise–/–;Lrp5–/–;Lrp6+/–mice, the normal pattern of three molars
in each jaw quadrant was restored in the majority of cases (5/6),
including a fairly normal cusp pattern (Fig. 1G).
The maxillary molar region of Wise-null mice displayed two
teeth in each jaw quadrant owing to extensive fusion of anterior
teeth (Fig. 1B). Removing one copy of Lrp6 had no effect on this
fusion phenotype (Fig. 1C,H). However, the fusion phenotype
was impacted by dosage of Lrp5, as three teeth were observed in
25% of Wise–/–;Lrp5+/–mice and three or four teeth were
observed in 92% of Wise–/–;Lrp5–/–mice (Fig. 1D,E,H). In the
majority of animals (5/6), removing three of the four copies of
the co-receptors (Wise–/–;Lrp5–/–;Lrp6+/–) also restored the
normal tooth number, size and cusp pattern in the maxilla (Fig.
The dosage-dependent rescue by decreases in Lrp5 and Lrp6
demonstrates that most, if not all, of the diverse tooth defects of
Wise-null mice are mediated by Lrp5/6-dependent processes.
Although these experiments show that the Wnt co-receptors have
overlapping and additive roles in tooth development, they also
illustrate that the individual Lrp5 and Lrp6 genes contribute
differently to regional aspects of tooth development. To probe this
issue, we examined the expression pattern of Lrp5 by in situ
hybridization and of Lrp6 by detection of LacZ expression from the
Lrp6-null allele. We found that both genes are broadly expressed
in dental epithelium and mesenchyme (data not shown). Hence,
differential patterns of expression do not appear to account for
specific roles for each co-receptor in tooth development. With
respect to Wnt ligands, expression analyses have showed that
multiple Wnt ligands are differentially expressed in early tooth
germs (Sarkar and Sharpe, 1999).
Elevated Wnt signaling leads to continuous
development of R2
The dependence of the Wise phenotypes on Lrp5 and Lrp6 suggests
that elevated Wnt signaling in the absence of Wise causes the tooth
abnormalities both in incisors and molars. Previously, Munne et al.
reported an additional epithelial Wnt activity in Wise-null incisors
utilizing the Top-Gal transgenic line (DasGupta and Fuchs, 1999;
Munne et al., 2009). To monitor Wnt signaling during molar
development, we also used the Top-Gal reporter and observed
dynamic changes in the relative levels, number and spatial
distribution of sites of Top-Gal expression during early stages of
tooth development (E12.5-E15.5) in Wise mutants (Fig. 2A-H).
Therefore, we investigated how these early alterations in Wnt
activity might account for tooth abnormalities observed in adult
In mouse tooth development, premolars do not form,
contributing to a toothless diastema region between the incisors and
molars (Fig. 3A). Tooth buds are initiated in the diastema but they
regress (Peterkova et al., 2006; Viriot et al., 2000). Two diastema
buds, called ‘MS’ and ‘R2’ for historical reasons, form in a
progressive anterior-posterior (AP) manner (Fig. 3A). MS is the
first to form (E12.5), followed by R2 in the adjacent posterior
territory (E13.5). At E14.5, as MS and R2 continue to regress, the
first molar (M1) begins to develop posterior to R2. The lack of
markers for such transient structures has made it difficult to follow
the fate of the diastema buds.
Control of Wnt signaling in tooth development
Fig. 1. Dosage-dependent rescue of Wise tooth phenotypes in
Lrp5 and Lrp6 mutants. (A-G)Tooth phenotypes in Wise-null mutants
carrying varying doses of the Lrp5 and Lrp6 Wnt-coreceptor genes. The
genotypes are listed on the left and the respective incisor or molar
regions noted at the top. M, molar; Mn, mandibular; Mx, maxillary;
T, tooth. Asterisks mark the supernumerary incisor and the black
arrowhead marks the supernumerary lateral molar. (H)Summary of the
genetic interaction with the number of scored jaw quadrants shown as
n. All phenotypes were scored with littermates in a mixed background
of C57/BL6 and 129Sv/Ev. SN, supernumerary.
We generated parasagittal sections to precisely map the domains
of reporter staining (Fig. 2A?-H?). Top-Gal expression is
colocalized with the markers of the epithelial signaling center in
early tooth buds as well as the enamel knot of the cap stage molars
(Fig. 2; see Fig. S2 in the supplementary material). In control mice,
the MS and R2 vestigial buds were marked by Top-Gal at E12.5
and E13.5, respectively (Fig. 2A?,B?). At E14.5, Top-Gal staining
was maintained in R2 and a new domain appeared in M1(Fig.
2C?). By E15.5, reporter staining in R2 was almost undetectable,
whereas it significantly increased and expanded in M1(Fig. 2D?).
The transient Top-Gal expression in MS and R2 is consistent with
the regression of these vestigial buds (Fig. 3A).
In Wise-null mandibles, Top-Gal expression was slightly
increased in MS at E12.5 (Fig. 2E?). We detected elevated and
sustained reporter staining in R2 from E13.5-E15.5 (Fig. 2F?-H?).
R2 displayed characteristics of an advanced cap stage tooth germ
at E14.5, suggesting that it continued to develop rather than regress.
Reporter staining in the mutant M1region was delayed by one day,
being first detected at E15.5 (Fig. 2H?).
To determine whether activation of Wnt signaling is sufficient for
survival of R2, we genetically elevated Wnt signaling utilizing a
conditional gain-of-function allele (Ctnnb1(ex3)fx) of the b-catenin
gene (Harada et al., 1999). By combining it with a Tamoxifen-
inducible ShhCreERTallele (Hayashi and McMahon, 2002), Wnt
signaling was activated in the Shh-expressing cells of R2 and M1as
shown by elevated Top-Gal expression (Fig. 2I-J?). Sustained Wnt
activation led to continuous development of R2, as evidenced by
invagination and morphogenesis (Fig. 2J?). These data underscore
the crucial role of Wnt signaling in determining the fate of R2.
A previous study on the root pattern of maxillary molars
suggested that the large molar of Wise-null mice was formed by
fusion of multiple molars (Ohazama et al., 2008). To further
explore the impact of sustained growth of R2 in Wise-null mice, the
root pattern of the mandible was examined. In wild-type mandibles,
each quadrant contains five roots: the first and second roots in M1,
the third and fourth roots in M2and the fifth root in M3(Fig. 3B).
The same total number of roots was observed in Wise-null and
Wise–/–;Lrp5–/–mice (Fig. 3C,D). However, in both mutants, T1
only contained a single enlarged root and T2contained the second
and third roots, although the second and third roots were often
fused in Wise-null mice. T3and T4each had a single root. These
changes demonstrated that the tooth field was repartitioned in the
mutants as a consequence of the survival of R2.
The temporal changes in Wnt signaling together with alterations
in the root pattern suggest that, in Wise mutants, R2 overcame
developmental arrest and continued to grow, eventually forming T1.
Correlated with these changes in R2, M1 displayed delayed
development and subsequently gave rise to the second tooth (T2;
Wise expression in tooth development
To monitor Wise expression during tooth development, we
generated transgenic mice harboring a Wise-LacZ BAC reporter
construct. The Wise-LacZ expression mimicked endogenous gene
expression in the tooth germ (Fig. 2K; see Fig. S3D,E in the
supplementary material) (Laurikkala et al., 2003). At E12.5, Wise
expression was dynamic and appeared in the epithelium
immediately surrounding the signaling center and in the underlying
mesenchyme of MS (see Fig. S2D? in the supplementary material).
At E14.5, the Wise-LacZ reporter was strongly expressed in the
condensing mesenchymal cells adjacent to R2 (Fig. 2K,K?).
Weaker expression was also detected in the outermost layer of
mesenchymal cells in the M1region (Fig. 2K?). Wise-LacZ reporter
expression was strongly upregulated in the developing R2 region
of Wise-null tooth germ (Fig. 2L,L?), suggesting the presence of a
Development 137 (19)
Fig. 2. Elevated Wnt signaling and continuous
development of the R2 vestigial bud.
(A-H)Wholemount Top-Gal expression in tooth
germs of E12.5-E15.5 mandibles. (A? ?-H? ?) Parasagittal
sections (anterior to the left) of the tooth germs from
panels A-H show Top-Gal expression in the epithelial
signaling center of the vestigial buds MS and R2, and
in M1. (I-J? ?) Tamoxifen (Tmx)-inducible mutation of
b-catenin leads to ectopic Wnt activation in Shh-
expressing cells of R2 and M1and continued
development of R2. Tmx was injected at E13.5.
(K-L? ?) Wise-LacZ BAC reporter expression in E14.5
tooth germs. Parasagittal (K,L) and frontal (K?,L?)
sections showing reporter expression in mesenchymal
cells. The dotted lines indicate the boundary between
the dental epithelium and mesenchyme.
Overexpression of Wise disrupts tooth
We used gain-of-function to investigate the inhibitory potential of
Wise on the Wnt pathway in tooth development. Transgenic mice
overexpressing Wise in epidermal tissues using the human keratin
14 promoter (K14-Wise) displayed a variety of tooth abnormalities
including reduced size, loss of M3in the maxilla and cusp defects
(Fig. 4A-E). To determine how Wnt signaling is affected by Wise
overexpression, we also monitored Top-Gal expression in K14-
Wise transgenic embryos. We found that tooth germs were growth-
retarded and displayed reduced levels of Top-Gal expression (Fig.
4F-I). The data indicate that ectopic Wise can disrupt tooth
development by inhibiting Wnt signaling.
Sustained proliferation and survival of the R2 bud
in the Wise-null mice
The loss of Wise resulted in elevated epithelial Wnt activity, which
promoted the continuous development of R2. To investigate the
basis of altered R2 development, we examined rates of cell
proliferation and cell death (Fig. 5A-H). BrdU incorporation assays
showed that the non-proliferating epithelial signaling center of R2
was surrounded by proliferating epithelial and mesenchymal cells
in both control and Wise-null embryos at E13.5 (Fig. 5A,B). No
obvious differences were observed between control and Wise-null
tooth germs at this stage. However, in Wise-null tooth germs at
E14.5, unlike control mice, robust proliferation was detected in the
underlying mesenchyme of R2 (Fig. 5C,D, asterisk). In addition,
the non-proliferating epithelial signaling center of R2 was
maintained only in the mutants. At this stage, a new epithelial
signaling center (M1) emerged posterior to R2 in control mice, but
evidence for initiation of M1was not observed in the posterior
region of Wise-null mice, providing further evidence for delayed
development of M1(Fig. 5C,D).
To examine the rate of cell death, cells undergoing apoptosis
were labeled by immunostaining against activated caspase 3
(Shigemura et al., 2001). A small group of apoptotic cells marked
the primary enamel knot from E14.5-E15.5, but no significant
difference was observed between control and mutant tooth germs
over these stages (Fig. 5E-H). This indicated that apoptosis was
unlikely to play a crucial role in determining the fate of R2. This
is in agreement with recent genetic studies that suggested apoptosis
is largely dispensable for tooth development (Matalova et al., 2006;
Setkova et al., 2007).
The fate and contribution of cells from MS and R2 to M1has
been difficult to determine because these structures are normally
transient in nature and undergo degeneration. We first examined the
expression of Shh, an established enamel knot marker (Hardcastle
et al., 1998). In control animals, Shh was progressively activated in
a transient manner in MS and R2 and, by E15.5, Shh expression
was observed only in the M1enamel knot of control mice (Fig.
5I,I?). By contrast, as previously shown in Wise-null mice (Kassai
et al., 2005), Shh expression was sustained in R2 and also present
in M1at E15.5 (Fig. 5J,J?). To trace the fate of cells descended
from the MS and R2 in control and Wise-null mice, we utilized a
GFP-Cre knock-in allele of the Shh gene (Harfe et al., 2004). In
combination with an R26R-floxstop-LacZ reporter line, descendants
of cells that had expressed Shh at earlier stages were genetically
marked. At E15.5, the putative MS and R2 regions anterior to M1
were occupied by the descendants of the Shh-expressing cells in
control tooth germs (Fig. 5K,K?). To ensure that the large group of
Control of Wnt signaling in tooth development
Fig. 3. Abnormal partitioning of the tooth field in Wise-null mice.
(A)Schematic diagrams summarizing the fate of tooth buds in control
and Wise-null mice. (B-D)The root pattern indicates that the tooth field
was repartitioned in Wise-null mice. Mandibular molars (top) and
corresponding coronal sections (bottom).
Fig. 4. Overexpression of Wise disrupted tooth development.
(A)K14-Wise transgene. (B-E)Molars of transgenic mice were smaller
with an abnormal cusp pattern and M3was frequently missing in the
maxilla (E). (F-I)In the K14-Wise;Top-Gal embryo, tooth germs were
growth-retarded with reduced Top-Gal expression, as shown in
wholemount lower jaws (F,G) and frontal sections (H,I). Mn,
mandibular; Mx, maxillary.
positive cells anterior to R2 were actually descended from MS and
not from cells present in other, earlier domains of Shh expression,
we utilized the Tamoxifen-inducible ShhCreERTallele to mark cells
at a specific stage. By comparing LacZ-positive cells after
Tamoxifen injection at E12.5 and E13.5, we confirmed that
descendants of MS contribute to the dental epithelial cells anterior
to R2 at E15.5 (Fig. 5M-N?). Staining in both the MS and R2
regions was greatly expanded in Wise-null tooth germs (Fig.
5L,L?). This was consistent with continued expression of Shh in R2
at earlier stages and underscored the fact that MS was also altered
in the mutant (see Figs S2, S3 in the supplementary material).
Fgf and Shh pathways are major targets of Wise-
regulated Wnt signaling
Our analyses demonstrate that inactivation of Wise leads to
increased signaling activity in R2 as early as E13.5, whereas the
morphological differences only become apparent a day later. To
examine the degree to which signaling pathways were misregulated
in the R2 of Wise-null mice, tooth germs were dissected from
E13.5 mandibles and expression analysis was performed using
qPCR arrays designed for Wnt, Tgfb and/or Bmp, hedgehog and
growth factor pathways (SABiosciences). Differential expression
was confirmed for some of the genes by wholemount in situ
hybridization (see Fig. S3 in the supplementary material). Major
changes were observed in components of the Fgf and Shh
pathways with a large increase in several Fgf genes and Shh (Table
1), whereas some minor alterations were observed in Tgfb and/or
Bmp or other pathways (see Table S1 in the supplementary
Genetic interaction between Wise and Shh
The significant changes in gene expression observed for several
components of the Shh signaling pathway and elevated Patch1-
LacZ expression (see Fig. S3 in the supplementary material) in the
Wise-null tooth buds prompted us to investigate genetic interactions
between Wise and Shh. There were no detectable tooth phenotypes
in the Shh+/GFPcreor Wise+/–heterozygous mice (Fig. 6A). Hence,
we were surprised to discover that a supernumerary tooth anterior
to M1 was generated in Wise+/–;Shh+/GFPcremice with high
penetrance (81%, n42; Fig. 6B). The ShhGFPcreallele displayed a
dynamic pattern of Gfp expression in the diastema buds and molars
that mimicked expression of the endogenous Shh gene (Fig. 6D-L).
The tooth phenotype in double-heterozygous embryos correlated
with changes in the temporal pattern of Shh as Gfp expression was
significantly elevated in R2 and restricted to a smaller domain of
cells in M1 at E14.5 (Fig. 6H). At E15.5, double-heterozygous
embryos continued to express Gfp in R2 and expression in the M1
enamel knot appeared to be smaller and shifted posteriorly (Fig.
6K). These results indicate that, in the compound heterozygous
mutants, R2 escaped developmental arrest to form a cap-stage tooth
and initiation and/or growth of M1was delayed. Two additional
Shh-null alleles, ShhcreERTand Shhneo, also generated the same
Development 137 (19)
Fig. 5. Proliferation and fate-mapping of the
vestigial buds. (A-D)BrdU incorporation assays
measuring cell proliferation in tooth germs. Asterisks
mark mesenchymal cells underlying R2.
(E-H)Immunostaining for cleaved caspase 3 to
detect apoptotic cells. (I-J? ?) Shh was ectopically
expressed in R2 of Wise-null tooth germs at E15.5,
as shown in the whole mandibles (I,J) and on
parasagittal sections (I?,J?). (K-L? ?) Shh-expressing cells
and their descendants were marked by b-gal
staining of the Shh+/GFPcre;R26R embryos at E15.5.
(M-N? ?) Tmx-induced labeling of the descendants of
Shh-expressing cells indicates that MS contributes to
the anterior outer enamel epithelium of M1.
Table 1. Differentially expressed genes in Wise-null tooth
germs at E13.5 identified in qPCR arrays
Fold upregulation or
Genes with >1.4-fold change (P≤0.05) are shown.
supernumerary tooth phenotype when combined with the Wise
mutant allele (data not shown). A reduction in the dosage of Shh
significantly increased the severity of Wise-null molar phenotypes
(Fig. 6C,M). Gfp expression in the Wise–/–;Shh+/GFPcretooth germs
was elevated to even higher levels in R2, and it was more delayed
in M1(Fig. 6I,L).
These genetic interactions demonstrated that phenotypes in Wise
mutant backgrounds were highly sensitive to the dosage of Shh and
thus presumably to levels of Shh signaling. Therefore, we tested
the effect of reducing Ptch1, which encodes a negative regulator of
Shh signaling. Removal of one copy of Ptch1 significantly
decreased the frequency of the supernumerary tooth phenotype in
Wise+/–;Shh+/GFPcremice (33%, n18, P<0.0002; Fig. 6N). This
suggests that reduced Shh signaling was the cause of the defect in
Shh negatively regulates Wnt signaling in tooth
The effects seen upon reduction of Shh might reflect changes in
Wnt signaling. Therefore, we measured Top-Gal activity in tooth
germs with varying dosages of Wise and Shh (Fig. 7A-D). There
was no change in patterns of reporter expression in Wise+/–or
Shh+/GFPcremice compared with control animals. However, in the
significantly increased in R2 and delayed in M1 (Fig. 7D).
Furthermore, reducing the dosage of Lrp6 rescued the
supernumerary tooth formation
Wise+/–;Shh+/GFPcre;Lrp6+/–mice (20/22; Fig. 6N). The elevated
Wnt signaling and genetic rescue by Lrp6+/–in the compound
mutants closely resemble those observed in Wise-null mice,
suggesting that the reduction in Shh signaling leads to sustained
growth of R2 through an elevation in Wnt signaling.
These genetic data point to a role for Shh as an antagonist of
Wnt signaling and a suppressor of the bud-to-cap transition of R2.
In addition, enhanced tooth fusion in Wise–/–;Shh+/–mice implies
that Shh is required for separation of teeth by antagonizing Wnt
signaling as the fusion phenotype was highly sensitive to dosage of
Lrp5. To investigate this idea, we have utilized a conditional allele
of Shh (Shhfx) in combination with a K14-Cre driver to delete Shh
in the dental epithelium. In the K14-Cre;Shhneo/fxmice, Top-Gal
expression in tooth germs was highly upregulated compared with
control mice (Fig. 7I-L,I?,J?). Elevation of Wnt signaling was also
observed in the maxilla where a single extended domain of Top-
Gal expression was associated with tooth fusion (Fig. 7M-P). To
rule out the possibility that the elevated Wnt signaling is an indirect
effect of disruption in earlier tooth development, Shh was
tooth germs, Top-Gal expression was
in the majority of
Control of Wnt signaling in tooth development
Fig. 6. Genetic interaction between Wise, Shh, Ptch1 and Lrp6.
(A-C)Adult molars in the mandible. The asterisk marks a
supernumerary tooth. (D-L)Gfp expression from the GfpCre knock-in
Shh allele in wholemount tooth germs shown as inverted gray scale.
(M)Exacerbation of Wise-null tooth phenotypes by reducing Shh
dosage. (N)Rescue of Wise+/–;Shh+/–supernumerary tooth by reducing
dosage of Ptch1 or Lrp6. The ShhGFPcreallele in a C57/BL6 background
was used for analyses. n, number of mandibular jaw quadrants.
Fig. 7. Shh negatively regulates Wnt signaling in tooth germs.
(A-H)Wnt signaling was elevated in R2 of Wise+/–;Shh+/–mice, as
shown by Top-Gal expression in whole mandibles and parasagittal
sections. (I-L)Top-Gal expression was elevated in Shh-deficient (K14-
cre;Shhneo/fx) tooth germs. Increased Top-Gal expression was also
observed in fungiform taste papillae (I?,J?). (M-R)Top-Gal expression in
the maxillary tooth germs of Wise (M,N), K14-cre;Shhneo/fx(O,P) and
Shhfx/CreERT(Q,R; Tmx injection at E13.5) mice. (S)qPCR analysis of
selected genes in Wise+/–and Wise+/–;Shh+/–tooth germs at E13.5.
temporally reduced in R2 and M1buds using the ShhCreERTallele.
Two days after Tamoxifen injection, Top-Gal was upregulated in
the tooth buds, which appeared to be fused (Fig. 7Q,R). These data
support the idea that Shh suppresses the survival of R2 and
prevents fusion between neighboring teeth by antagonizing Wnt
signaling. We also observed elevated levels of Top-Gal staining in
taste papillae and hair follicles in these conditional Shh mutants
(Fig. 7I?,J?; data not shown). Therefore, Shh might have a related
role in modulating Wnt signaling in other tissue contexts (Iwatsuki
et al., 2007).
To identify candidate genes that might participate in the
antagonistic action of Shh on Wnt signaling, we utilized qPCR to
examine the expression of 90 selected genes relevant to tooth
development, including the differentially regulated genes in Wise-
null tooth germs (Table 1). Besides Shh, Dkk1 was downregulated
by about 50% in Wise+/–;Shh+/–tooth germs at E13.5 compared
with Wise+/–tooth germs (Fig. 7S). Dkk1 was the only gene that
showed more than 1.6-fold change (P<0.05), suggesting that it is
an early downstream target of Shh (data now shown). This raises
the possibility that simultaneous reduction of the two Wnt
antagonists, Wise and Dkk1, might account for the elevation of
Wnt signaling above a threshold level and lead to survival of R2 in
In this study, we have demonstrated that precise control of the
level of Wnt signaling plays a crucial role in determining whether
diastema buds survive and go on to develop into mature teeth. We
have also shown that survival of a diastema bud results in delayed
development of the adjacent posterior molar buds, suggesting that
reduced inhibitory signals resulting from the regression of R2
might be a prerequisite for the timely initiation of M1. These tissue
interactions between developing buds are consistent with a
temporal model for tooth formation in which pre-existing tooth
buds inhibit initiation of new posterior teeth and form a
progressive inhibitory cascade affecting the timing of tooth
formation (Kavanagh et al., 2007). We have shown that, in tooth
development, Wise suppresses survival of the diastema and incisor
vestigial buds by serving as an inhibitor of Lrp5- and Lrp6-
dependent Wnt signaling. Our genetic and expression analyses
reveal that the Fgf and Shh pathways are major downstream
targets of the Wnt signaling regulated by Wise. Furthermore,
through genetic interaction studies, we have discovered that Shh
acts as a negative-feedback regulator of Wnt signaling during the
bud-to-cap transition. Our data provide insight into the
mechanisms that control the levels of Wnt signaling and crosstalk
between the Wnt, Shh and Fgf pathways that regulate the timing
and number of teeth.
Wise as a Wnt antagonist
In Wise mutants, our qPCR array data indicated that expression of
Wnt pathway components was only moderately affected and the Wnt
pathway activity as a whole was highly elevated, as detected by Top-
Gal. Conversely, overexpression of Wise resulted in reduced Wnt
activity in the dental epithelium and inhibition of tooth growth. The
phenotypes of our K14-Wise mice (Fig. 4; data not shown) were also
strikingly similar to those of K14-Dkk1 mice, with abnormal
development of multiple tissues including hair follicles, mammary
glands, taste buds and teeth (Andl et al., 2002; Chu et al., 2004; Liu
et al., 2008). The upregulation of Wise-LacZ reporter expression in
Wise-null tooth germs implies that Wise might be activated by
signaling molecules from the enamel knot through a negative-
feedback loop. This is consistent with the elevated Wise expression
observed in tooth germs with constitutively active Wnt signaling
(Jarvinen et al., 2006; Liu et al., 2008).
The genetic interaction data revealed that, in the absence of
Wise, multiple aspects of abnormal tooth development are highly
sensitive to the dosage of Lrp5 and Lrp6. Complete rescue of the
Wise-null molar and incisor phenotypes was only observed in
Lrp5–/–;Lrp6+/–mice, indicating that both genes contribute to Wnt
signaling in tooth development. This supports a model whereby
Wise acts as a Wnt antagonist through its direct interaction with
these Wnt co-receptors. In the absence of inhibition by Wise, there
is an elevation of Lrp5/6-dependent Wnt signaling, which induces
tooth phenotypes. Decreasing copies of the Lrp5/6 co-receptors
rescues the Wise-null phenotypes presumably through restoration
of normal levels of Wnt activity. There were no obvious tooth
abnormalities in Lrp5–/–;Lrp6+/–mice in the presence of Wise. This
indicates that one copy of Lrp6 is sufficient to provide levels of
Wnt signaling able to potentiate normal tooth development. This
could be a consequence of feedback mechanisms that would
compensate for reduced levels of the co-receptor (Fig. 8).
Lrp4 might provide an additional means through which Wise
mediates antagonistic action on Wnt signaling. Lrp4 can antagonize
canonical Wnt signaling when overexpressed in cultured cells (Li
et al., 2010). Although in vitro Wise can bind to Lrp4 (Ohazama et
al., 2008), it is unknown whether interactions with Wise can
influence Lrp4 function. Developmental abnormalities associated
with inactivation of Lrp4 were similar to Wnt loss-of-function
phenotypes in certain tissues rather than phenotypes caused by
excess Wnt signaling (Choi et al., 2009; Weatherbee et al., 2006).
This suggests that the function of Lrp4 can be context-dependent.
As mice homozygous for a hypomorphic allele of Lrp4 displayed
tooth defects similar to those of Wise-null mice, it was proposed
that Wise binds to Lrp4 to initiate intracellular events leading to
inhibition of Wnt signaling (Ohazama et al., 2008). Overgrowth
and fusion of molars in these Lrp4 mutant mice was associated
with elevated Wnt signaling, as assayed by BAT-gal staining in
bell-stage tooth germs (Ohazama et al., 2008), but the early
changes in Wnt, Shh and other signaling activities have not been
examined in Lrp4 mutant mice. Preliminary analyses of mice
homozygous for a null allele of Lrp4 (Weatherbee et al., 2006)
have shown that loss of Lrp4 does not phenocopy loss of Wise
during R2 development. This raises the possibility that Lrp4 and
Wise play distinct roles in the diastema buds and that multiple
molecular mechanisms underlie tooth defects in these two mouse
Signaling network in diastema tooth
In the diastema region of the mouse, it has been proposed that
phylogenetic memory of odontogenesis is manifested by the
formation of vestigial tooth buds that undergo degeneration without
reaching the cap stage. In mouse models with genetic disruptions
of major signaling pathways, a supernumerary tooth forms in front
of M1, indicating that crosstalk between many pathways is involved
in controlling diastema tooth development. In this regard, our
analyses with Wise mutants have shed light on the important role
of Wnt signaling in these events.
Data with the Top-Gal reporter in control and Wise-null mice
demonstrated that Wnt signaling is sequentially activated in
epithelia of the two vestigial buds, MS and R2, and then
subsequently in molar buds M1, M2 and M3. However, during
normal development, Top-Gal expression is rapidly downregulated
Development 137 (19)
in MS and R2 in contrast to the M1-M3buds, coincident with the
inability of the rudimentary buds to make the bud-to-cap transition.
Reporter analysis showed that Wise is highly expressed in the
mesenchymal cells that surround the arrested buds. The loss of
Wise leads to elevated and sustained levels of Wnt signaling in the
epithelium and continuous development of R2.
Together with the fate mapping data that shows the persistence
of descendants of Shh-positive cells, the results from cell
proliferation and cell death analyses suggest that the transition is
controlled largely by proliferative signals from the epithelial
signaling center to the surrounding mesenchyme. Fgf4 is a strong
candidate for the signals, as it is an epithelial target of Wnt
signaling and can rescue the developmental arrest of cultured Lef1
mutant tooth germs with its ability to activate mesenchymal Fgf3
expression (Kratochwil et al., 2002). Our quantitative expression
analyses showed that Fgf4, as well as Fgf3, are highly upregulated
in Wise-null tooth germs, further supporting the notion that Fgf
signaling is a major downstream target of epithelial Wnt signaling.
There are similarities and differences in the roles for Wise in
molar and incisor development. In both molars (Fig. 2) and incisors
(Munne et al., 2009), Wnt signaling is significantly elevated, in
agreement with the idea that Wise can inhibit the Wnt pathway.
Furthermore, reducing the dosage of the Lrp co-receptors rescues
supernumerary tooth phenotypes in molars and incisors (Fig. 1).
With respect to cell death in R2 and incisors, there were a limited
number of apoptotic cells at early stages (Fig. 5) (Munne et al.,
2009). However, reduction in the number of apoptotic cells was
reported in later stage incisors (Munne et al., 2009; Murashima-
Suginami et al., 2007), suggesting that apoptosis might play a
different role in incisors compared with molars.
Interaction between Shh and Wnt signaling in
diastema tooth development
An important aspect of the regulatory network uncovered by our
studies was the nature of the genetic interactions between Wise
(Wnt signaling) and Shh in tooth development. In normal tooth
development, there was a tight spatial and temporal correlation
between Wnt activity and Shh expression in the epithelial signaling
center of tooth buds. The elevated Wnt signaling in Wise-null mice
led to increased Shh expression in the R2 bud. Ectopic activation
of Wnt signaling in the dental epithelium has been shown to induce
Shh expression in tooth buds (Jarvinen et al., 2006; Wang et al.,
2009). Inactivation of Wnt signaling results in loss of Shh
expression (Liu et al., 2008). These data indicate that the level of
Shh expression in the developing tooth bud is dependent upon the
relative level of Wnt signaling. Conversely, we have found that Shh
modulates levels of Wnt signaling. In Wise+/–;Shh+/–mice, Wnt
signaling was significantly elevated, leading to survival of R2 and
supernumerary tooth formation. This suggests that Shh signaling
normally participates in a negative-feedback loop that controls the
level of Wnt signaling in R2 (Fig. 8).
A key aspect of this regulatory model is that maintaining a
proper balance between Wnt and Shh signaling is more important
than the absolute level of either signaling activity. For example,
during normal development of R2, levels of Wnt signaling are
relatively low and transient. Therefore, low levels of Shh are
sufficient to repress Wnt activity and suppress the bud-to-cap
transition of R2. Phenotypes arise when either of the pathways is
disrupted and normal regulatory feedback modulations are unable
to restore a proper balance at the appropriate time. Fig. 8 presents
a model for the regulatory interactions between signaling
components in R2 of wild-type and genetic mutant backgrounds.
This hypothesis was further supported by the observation that
reducing the dosage of Ptch1 (elevating Shh signaling) or Lrp6
(reducing Wnt signaling) rescues the supernumerary tooth
phenotype of Wise+/–;Shh+/–mice. We also observed elevated Wnt
signaling in Shh-deficient tooth buds. This inhibitory role of Shh
on Wnt signaling in the bud-to-cap transition is consistent with the
recent finding that ectopic Shh activity in K14-Shh mice arrests
tooth development at the bud stage (Cobourne et al., 2009). In
addition to the early role in R2, Shh signaling is required for proper
separation of teeth (Gritli-Linde et al., 2002; Ohazama et al., 2008),
consistent with the exacerbated fusion in Wise–/–;Shh+/–mice. The
elevated Wnt signaling in the Shh-deficient tooth germs suggests
that Shh prevents tooth fusion by antagonizing Wnt signaling.
Recently, ectopic tooth formation was reported in mice deficient
for polaris and Gas1 (Ohazama et al., 2009). Polaris is a component
of primary cilia which is required for Shh signaling (Huangfu et al.,
2003). Gas1 acts as a facilitator of Shh signaling in different
developmental contexts (Allen et al., 2007; Martinelli and Fan, 2007;
Seppala et al., 2007). Our genetic data on interactions between Shh
and Wnt signaling suggest that the supernumerary tooth in polaris
and Gas1 mutant mice results from disruption of the Wnt-Shh
feedback loop in R2, in which temporal reduction in Shh signaling
results in elevated Wnt signaling and hence survival of R2.
In conclusion, our findings highlight how the processes of tooth
development are highly sensitive to spatiotemporal changes in Wnt
signaling activity. Even a small disruption in the signaling network,
through loss or gain of Wnt antagonists (Wise) or feedback
regulators such as Shh, is sufficient to change the fate of tooth
buds, leading to abnormal tooth number and size. Changes in
Control of Wnt signaling in tooth development
Fig. 8. Model for signaling network in diastema tooth
development. Schematic diagrams of the signaling network regulating
development of the diastema R2 bud at E13.5-E14.5 in wild-type and
genetic mutant backgrounds. In wild-type teeth, Wnt signaling is
required for the bud-to-cap transition of R2 by inducing Fgfs. Wise and
Spry2/4 antagonize Wnt and Fgf signaling, respectively, to suppress the
transition. Shh is a downstream target of Wnt signaling and acts as a
negative-feedback regulator of Wnt signaling via Dkk1 and other
targets. Elevated Wnt signaling feeds back to stimulate Wise
expression. The thickness of the lines represents relative levels of
activity. Red indicates repressive input and black indicates positive input
expression of signaling modulators such as Wise might represent
an important mechanism that underlies the evolutionary diversity
in mammalian dentition.
We thank S. Miura for analysis of the Bmp pathway, G. R. Martin for
discussion and providing the Fgf10, Fgfr1 and Fgfr2 mutants and the following
for providing mutant mouse lines: L. Chan, Lrp5; W. C. Skarnes, Lrp6; M.
Taketo, Ctnnb1(ex3)fx; C. Tabin and B. Harfe, ShhGFPcreand ShhcreERT; P. Beachy,
Shhneo; A. P. McMahon, Shhfx; M. P. Scott, Ptch1LacZ; and Y. Mishina, Bmpr1a.
We thank M. L. Johnson, D. L. Ellies and Krumlauf laboratory members for
discussions, the Stowers Institute histology facility, K. Westpfahl for animal
support and S. D. Weatherbee for sharing unpublished data on Lrp4 mutants.
Y.A., B.W.S. and R.K. were supported by funds from the Stowers Institute.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
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Control of Wnt signaling in tooth development