|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 8, 2009
doi: 10.1242/10.1242/dev.033803
1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, MA 02115, USA.
2 Division of The Channing Laboratory, Department of Medicine, Brigham and
Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
3 Children's Hospital Informatics Program and Department of Pediatrics, Boston
Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
4 Department of Oral Anatomy, School of Dentistry, Iwate Medical University,
Iwate 020-8505, Japan.
5 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Author for correspondence (e-mail:
maas{at}genetics.med.harvard.edu)
Accepted 31 March 2009
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Apc, Wnt, β-catenin, Fgf8, Msx1, Stem cells, Tooth regeneration
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). For
example, in sharks, 
200 teeth can develop successively in one location,
and one primary tooth may be replaced by two successors
(Butler, 1995). During
evolution, tooth replacement becomes progressively restricted or absent
(Jarvinen et al., 2008).
Humans have only two sets of dentition and mice have just one. In lower
vertebrates, successional teeth, like primary teeth, form directly from the
epidermal or oral epithelium. However, in all jawed vertebrates, successional
teeth derive from the dental lamina of primary teeth, and the dental lamina is
thought to provide the genetic information that controls the timing, position
and shape of replacement teeth (Smith,
2003).
Tooth germs develop through sequential and reciprocal interactions between
epithelium and mesenchyme that are common to many ectoderm organs
(Pispa and Thesleff, 2003). In
mouse molar tooth germ, morphological initiation begins at embryonic day 11
(E11), when oral ectodermal placode thickens to form the dental lamina, which
then develops through the bud, cap and bell stages. A unique feature of rodent
incisors that makes it an especially compelling system with which to
investigate regenerative mechanisms is the existence of an endogenous adult
stem cell niche that allows the teeth to grow continuously throughout life
(Harada et al., 1999a).
Humans have only two sets of dentition; after the crown of a permanent
tooth forms, the dental lamina degenerates, ending the tooth cycle and further
tooth development. However, supernumerary teeth do form in certain human
disease states, including Gardner's syndrome, a variant of familial
adenomatous polyposis (FAP), which is caused by loss-of-function germline
mutations in the adenomatous polyposis coli (APC) gene
(Madani and Madani, 2007). Apc
inhibits the activity of the Wnt/β-catenin signaling pathway that plays a
pivotal role during development and in the maintenance of homeostasis in
adults (Clevers, 2006;
Nusse, 2008;
Rajagopal et al., 2008). In
the absence of Wnt ligand, Apc together with Axin and Gsk3β form an
inhibitory complex that phosphorylates β-catenin, targeting it for
degradation. Binding of Wnt ligand to its co-receptors frizzled and Lrp
triggers a signaling cascade that involves inhibition of Gsk3β and the
cytosolic stabilization and nuclear translocation of β-catenin, which
then interacts with Tcf/Lef family members to regulate Wnt target genes. Thus,
Apc loss-of-function is associated with the nuclear accumulation of
β-catenin and mimics the constitutive activation of Wnt signaling.
We previously showed that genetic deletion of Apc in embryonic
mouse oral epithelium (K14-Cre;Apccko/cko) results in
supernumerary tooth formation, suggesting that Wnt signaling and the level of
Apc protein are crucial determinants of tooth initiation
(Kuraguchi et al., 2006).
Consistent with this, constitutive activation of β-catenin in mouse
embryonic oral epithelium
(K14-Cre;β-cat
ex3f/+)
also initiates supernumerary tooth formation
(Jarvinen et al., 2006;
Liu et al., 2008). In the
present study, we further analyzed the tooth phenotype in
K14-Cre;Apccko/cko mice. We find that epithelial deletion
of Apc in mouse embryos and in young mice results in continuous
supernumerary tooth formation from multiple regions of the jaw. Surprisingly,
the genetic deletion of Apc or activation of β-catenin
(Ctnnb1) in the oral epithelium of old adult mice also produced
multiple supernumerary teeth in the incisor region. The formation of
supernumerary teeth is Apc non-cell-autonomous and, in contrast to
endogenous tooth formation, can occur in the absence of Msx1. We also
identify Fgf8, an early tooth initiation marker, as a direct target
of Wnt/β-catenin signaling. These studies provide key insights into the
mechanisms of supernumerary tooth formation, and highlight similarities and
differences between endogenous and supernumerary tooth formation.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Chen et al.,
1996; Harada et al.,
1999b; Kuraguchi et al.,
2006). K14-Cre8Brn mice were from Mouse
Repository (Jonkers et al.,
2001), K14-Cre1Amc mice, R26R
reporter mice and K14-CreERTM mice were from Jackson
Laboratories (Dassule et al.,
2000; Soriano et al., 1999;
Vasioukhin et al., 1999). The
CreER transgene was activated by intraperitoneal injection of
4-hydroxytamoxifen (Sigma-Aldrich) at 50 mg/kg body weight once a day for 3-5
days. Mice were sacrificed 2-3 weeks after the last injection. All animal work
was carried out under an animal protocol approved by Harvard Medical Area
IACUC.
In situ hybridization and immunohistochemistry
Riboprobes used for in situ hybridization: mouse amelogenin
(Luo et al., 1991), activin
βA, Fgf3, Fgf4, Pitx2, dentin sialophosphoprotein, p21,
Fgf8, Ctnnb1 and rat Shh
(Aberg et al., 2004;
Wang et al., 2005). Antibodies
used in immunohistochemistry: anti-β-galactosidase (Immunology
Consultants Laboratory, USA), anti-β-catenin (BD Transduction), anti-Ki67
(BD Pharmingen), anti-neurofilament (Abcam), and anti-Cd31 (BD Pharmingen).
Mouse anti-dentin sialoprotein (DSP) antibody was a kind gift from Dr Chunlin
Qin (Baylor College of Dentistry).
Cell proliferation assays
Staged mice were intraperitoneally injected with
5-bromo-2'-deoxyuridine (BrdU) solution (Invitrogen) at 1.5 ml/100 g
body weight, and sacrificed 2 hours after injection. Mouse heads were
harvested and processed for paraffin sections (7 µm). BrdU-incorporated
cells were detected using a BrdU staining kit (Invitrogen).
Tissue culture and bead implantation assay
The culture of E9.5 or E13.5 CD1 mouse lower mandibles on Nuclepore filters
using a Trowell-type culture system, and the bead implantation assay, were as
described previously (Vainio et al.,
1993; Wang et al.,
2004). Affi-Gel agarose beads (Bio-Rad) were incubated in Dkk1
(200 ng/ml; R&D), and heparin beads (Bio-Rad) were soaked in Fgf8 (100
ng/ml; R&D) or BSA (1 µg/µl; Sigma) at 37°C for 45 minutes.
Cell culture and transfection
LS8 cells were a kind gift of Dr Malcolm Snead (USC, Los Angeles). HAT-7
cells were generated as described (Kawano
et al., 2002). LS8 cells were transfected with constitutively
activated β-catenin (S33Y) or control pBabe plasmid, and with either
pTopflash (wild type) or pFopflash (mutant) luciferase reporter plasmids using
Qiagen SuperFect (301307). To knockdown endogenous β-catenin, we used two
different Ctnnb1 stealthTM select RNAi vectors (Invitrogen,
RSS331356 and RSS331358), with scrambled stealthTM RNAi as negative
control (Invitrogen, 12935-300). Western blot analyses were performed using
anti-β-catenin mouse monoclonal antibody (BD Transduction, 1:500) and
anti-β-actin mouse antibody (Sigma, 1:2000).
Real-time PCR
Fgf8 (Rn00590996_m1, Mm00438921_m1) and control (β-actin,
Rn00667869_ml; 
-tubulin, Mm00506159_m1) transcripts were quantitated
using TaqMan Gene Expression Assays (Applied Biosystems). Fold change was
calculated using
2Ct(Fgf8)-Ct(Control),
where Ct is the difference in threshold cycle between samples.
Chromatin immunoprecipitation (ChIP) and transactivation assay
ChIP analyses were performed as previously described
(Lee et al., 2006;
Yochum et al., 2007) using
anti-Lef1 antibody (Santa Cruz), anti-β-catenin antibody (BD
Transduction), and rabbit and mouse IgG (Jackson ImmunoResearch). ChIP PCR
primers: Fgf8 intron 3F, 5'-CTGGCCAGGCAGTTTACAGA-3';
Fgf8 intron 3R, 5'-CCTCTTCTCGAGCCAGTTTG-3'; 3.5 kb
Control F, 5'-TGGCACAACCTTCCACAATA-3'; 3.5 kb Control R,
5'-AACCCCTCCAAATTCTGCTT 3'. C57BL/6 mouse genomic DNA was used to
PCR amplify the 1 kb conserved element of Fgf8 intron 3 containing
the Lef1 binding site identified by ChIP. Primers used in PCR for subsequent
ligation into the pGL3-promoter vector (Promega, E1761): 1 kb Fgf8
intron 3 F, 5'-GAAGATCTCAAGGATGCTAGGCCATTTG-3'; 1 kb Fgf8
intron 3 R, 5'-GGGGTACCAGGGGCTGAGAACTGATTGA-3'. The mutant
construct contained a 3 bp deletion (CTTTGA to CTxxxA) in the core of the Lef1
binding site, and was prepared using the QuikChange Site-Directed Mutagenesis
Kit (Stratagene, 200518). Primers for site-directed mutagenesis: del385-387F,
5'-TGTCTCCTCCTCTAGCTAAGGGAAGTCAGCTATG-3'; del385-387R,
5'-CATAGCTGACTTCCCTTAGCTAGAGGAGGAGACA-3'.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
Based on analyses employing Rosa26R (R26R) reporter mice,
K14-Cre1Amc mice exhibited uniform Cre recombinase
activity throughout the oral and dental epithelium, whereas
K14-Cre8Brn mice exhibited a mosaic pattern of recombinase
activity in 20-60% of oral and dental epithelial cells
(Fig. 1A,B; see Fig. S1A in the
supplementary material) (Dassule et al.,
2000; Jonkers et al.,
2001). In our experiments, we used the complementary features of
these two Cre alleles, focusing initially on the
K14-Cre8Brn allele that generated a postnatal viable
phenotype. K14-Cre8Brn;Apccko/cko mice survived until postnatal day 18 (P18) and the intrinsic development of their principal teeth was not obviously affected. However, the principal teeth were surrounded by numerous supernumerary teeth on both their labial and lingual sides (Fig. 1C-G). Some supernumerary teeth were contiguous with the oral epithelium and vestibular lamina, which is the invagination of proliferating epithelial cells responsible for the formation of the vestibule, i.e. the space between the gingiva and the inner cheek (Fig. 1H,J,K). With increasing age of K14-Cre8Brn;Apccko/cko mice, from E14 to P18, increasing numbers of teeth were observed, and these formed from both the principal teeth and from pre-existing supernumerary teeth (Fig. 1I). BrdU labeling revealed cell proliferation in the cervical loop and dental mesenchyme of supernumerary teeth (Fig. 1J,K). Although the majority of supernumerary teeth were simple unicuspid cones, some multicuspid teeth formed in both the molar and incisor regions (see Fig. S2 in the supplementary material). The supernumerary teeth in the proximal molar region, both unicuspid and multicuspid, expressed Barx1 in their dental mesenchyme, whereas no Barx1 was expressed in the teeth at the distal incisor region of the jaw (see Fig. S2C-J in the supplementary material; data not shown). More-mature supernumerary teeth possessed well-differentiated ameloblasts and odontoblasts with enamel and dentin matrix deposition and also root development, with Hertwig's epithelial root sheath (HERS) extending internally and apically (Fig. 1L,M). Immunofluorescence revealed the presence of neurofilament in the dental pulp and dental tubules, and Cd31 (Pecam1) in the dental pulp, indicating that supernumerary teeth contained nerves and blood vessels, respectively (Fig. 1N-Q). Thus, the supernumerary teeth may function as natural teeth.
Msx1 is dispensable for supernumerary tooth development
In wild-type mice, Shh is expressed at the tip of the tooth bud in
the enamel knot region, a signaling center that regulates growth of the enamel
organ and differentiation of the underlying mesenchyme
(Jernvall et al., 1994;
Vaahtokari et al., 1996). By
the bell stage, Shh is expressed along the inner dental epithelium
and thus also serves as a marker for differentiating ameloblasts
(Fig. 2A,E)
(Gritli-Linde et al., 2002).
In K14-Cre8Brn;Apccko/cko mice, ectopic
Shh and Fgf8 expression was present in the dental lamina
epithelium, the oral epithelium and the vestibular lamina
(Fig. 2B,D,F, arrows).
Supernumerary tooth buds from incisor epithelium developed on both the labial
side, which contains differentiating ameloblasts, and on the lingual side,
which in wild-type mice does not undergo differentiation
(Fig. 2B). Identical sections
revealed that these supernumerary tooth buds also expressed activin βA
and Fgf3 in their dental mesenchyme
(Fig. 2C; data not shown). At
later time points, supernumerary teeth expressed the same differentiation
marker genes as endogenous teeth, including amelogenin (Amelx - Mouse
Genome Informatics) and dentin sialophosphoprotein (Dspp)
(Fig. 2G,H; data not
shown).
|
). In
Msx1-null knockout mice, Bmp4 and Fgf3 are
downregulated and tooth development arrests at the bud stage, without
progression to the cap stage or expression of enamel knot markers such as
Shh (Fig. 2J,N,R,V).
When we introduced the Msx1-null mutation into
K14-Cre8Brn;Apccko/cko mice, however, we
observed numerous supernumerary tooth germs that protruded from the oral
epithelium into the mesenchyme and that expressed Shh in the
epithelium, as in
K14-Cre8Brn;Apccko/cko;Msx1+/- mice
(Fig. 2K,O,L,P). Moreover,
Bmp4 was expressed in both the epithelium and mesenchyme of these
supernumerary tooth germs (Fig.
2S,T), and Fgf3 was expressed in their mesenchyme
(Fig. 2U-X). Msx2 was
expressed in dental papilla mesenchyme of supernumerary teeth and was
expressed in differentiating ameloblasts (data not shown). These results
indicate that although supernumerary and endogenous tooth development exhibit
similarities, they exhibit distinct differences with respect to Msx1
dependency.
Apc-deficient cells can recruit wild-type epithelial and mesenchymal cells to an odontogenic fate
The mosaic expression of the K14-Cre8Brn allele allowed
us to examine whether the formation of supernumerary teeth proceeds by a
cell-autonomous or non-cell-autonomous mechanism. We crossed the R26R
reporter allele into K14-Cre8Brn;Apccko/cko
mice and assayed for β-galactosidase (β-gal) expression as a proxy
for Apc-deficient cells. Immunohistochemistry for β-gal in
K14-Cre8Brn;Apccko/cko;R26R mice revealed that
only a subset of epithelial cells within the supernumerary tooth buds were
β-gal positive (Fig.
3Aa-d). Consistent with the fact that Apc
loss-of-function results in an obligate upregulation of β-catenin, double
immunofluorescence detection revealed elevated levels of β-catenin
protein in both the nucleus and cytoplasm of β-gal-positive cells
(Fig. 3Ba-d). In most cases,
β-gal-positive cells or cells exhibiting elevated levels of
β-catenin comprised only a subset of dental epithelial cells in
supernumerary tooth germs, and wild-type epithelial and mesenchymal cells thus
participated in supernumerary tooth formation.
|
|
).
In E14.5 wild-type cap-stage tooth germs, β-catenin was expressed in both
dental epithelial and mesenchymal cells, with strong nuclear staining in the
enamel knot signaling center (see Fig. S3 in the supplementary material)
(Liu et al., 2008). Most cells
within the enamel knot region were Ki67 negative, indicating mitotic
inactivity. In K14-Cre8Brn;Apccko/cko mice, the
β-catenin-expressing cells in the supernumerary tooth buds were mostly
Ki67 positive (Fig. 3Da-d).
However, some cells with elevated β-catenin were Ki67 negative
(Fig. 3De-l, arrows); these
cells were often concentrated at the tips of presumptive supernumerary tooth
buds, and are likely to represent incipient enamel knot signaling centers.
Induction of supernumerary teeth by Apc loss-of-function occurs via β-catenin signaling
K14-Cre1Amc;Apccko/cko mice, which express
Cre recombinase uniformly throughout skin ectoderm and oral and dental
epithelium, died at birth. Although their tooth germs appeared normal at E13.5
(data not shown), by E14.5 the mutant teeth were severely disrupted, with
numerous irregular epithelial buds protruding from the oral epithelium into
jaw mesenchyme, and intense expression of Fgf8, Shh, Pitx2, p21
(Cdkn1a - Mouse Genome Informatics) and Fgf4 transcripts and
elevated β-catenin levels were observed (see Figs S1 and S4 in the
supplementary material). Expression of β-catenin transcripts
(Ctnnb1) was also upregulated (see Fig. S1 in the supplementary
material). The odontogenic phenotype in these mice is similar to that reported
in mice with constitutive activation of β-catenin in embryonic oral
epithelium (Jarvinen et al.,
2006; Liu et al.,
2008).
During early stages of tooth initiation (E9-12), Fgf8 is expressed
in oral and dental epithelium, but is downregulated after E13. By contrast,
Fgf4 begins expression at E13 in the prospective enamel knot at the
tip of the tooth bud, and becomes intensely expressed at E14 in the enamel
knot (Kettunen and Thesleff,
1998). There is no overlap in Fgf8 and Fgf4
expression in endogenous tooth development in wild-type mice. However, in
E14.5 K14-Cre1Amc;Apccko/cko mice, we detected
many oral epithelial cells in the invaginating ectoderm that co-expressed
Fgf8 and Fgf4 (see Fig. S1N-Q in the supplementary
material), suggesting that their expression is not mutually exclusive, and
that the temporal process of tooth development is compressed.
Apc has other functions besides that in Wnt/β-catenin signaling,
including the regulation of cell migration and maintenance of chromosomal
stability during mitosis (Clevers,
2006). To test whether the induction of supernumerary teeth by
Apc loss-of-function was mediated by β-catenin, we bred the
K14-Cre1Amc;Apccko/cko mice with a
β-catenin loss-of-function allele (Ctnnb1cko/cko).
K14-Cre1Amc;Ctnnb1cko/cko mouse tooth germs
arrest at the bud stage (see Fig. S4G-I in the supplementary material)
(Liu et al., 2008). When we
genetically compounded Ctnnb1cko/cko with
K14-Cre1Amc;Apccko/cko mice, the formation of
supernumerary teeth was suppressed. Only rare, small patches of epithelial
cells could be found that expressed low levels of Shh and
Fgf8 (see Fig. S4J-O in the supplementary material). These data prove
that signaling via the Wnt/β-catenin pathway is responsible for
supernumerary tooth formation in Apc loss-of-function mice.
|
). We
systematically administered 4-OHT to mice at different ages, ranging from P5
to 10 months. Prior to 4-OHT injection,
K14-CreERTM;Apccko/cko mice were indistinguishable
from control wild-type mice. Administration of 4-OHT had no effect on
K14-CreERTM-negative littermates, and corn oil alone had no
effect on K14-CreERTM;Apccko/cko mice. However,
administration of 4-OHT to P5 to 10-month-old
K14-CreERTM;Apccko/cko mice induced large numbers of
supernumerary teeth on both the labial and lingual sides of incisors
(Fig. 4A-G).
K14-CreERTM;Apccko/cko mice treated with 4-OHT also
showed variation in the developmental stages of the supernumerary teeth,
ranging from initiation stages to those with well-differentiated ameloblasts
and odontoblasts and enamel and dentin matrix deposition. Most supernumerary
teeth were unicuspid, but some multicuspid supernumerary teeth were also
observed surrounding the principal teeth
(Fig. 4B,D, yellow asterisks).
The development of principal teeth was not grossly affected; however, in some
cases, supernumerary teeth destroyed the continuity of the principal teeth
(Fig. 4C, arrow).
|
In contrast to the incisors, we did not observe supernumerary tooth formation in molar regions of old adult mice. Mice have only a single dentition, and unlike the incisors, their molar teeth do not grow continuously. Once the molar teeth erupt into the oral cavity, the dental lamina epithelial cells degenerate. We therefore focused on young mice in which the molar teeth have not yet erupted into the oral cavity and the associated dental epithelial cells persist. We injected 4-OHT into K14-CreERTM;Apccko/cko mice at P5 for 3 consecutive days and analyzed their teeth 12 days after the last injection. Upon histological examination, the principal molars in control wild-type mice had erupted into the oral cavity (Fig. 4K), whereas K14-CreERTM;Apccko/cko mice treated with 4-OHT at P5 did not show any molar eruption. However, these mice exhibited many supernumerary teeth forming from multiple regions of the jaw, including the dental lamina and outer dental epithelium in the crown region of the molar teeth (Fig. 4L,M). Immunohistochemical analysis of Dspp confirmed that these supernumerary tooth germs had undergone odontoblast differentiation (Fig. 4L). In addition, we also observed supernumerary tooth budding directly from the oral epithelium of the jaw (Fig. 4N). Supernumerary tooth buds emanating from the dental lamina also expressed Shh and amelogenin, as did the crown region of the molars (Fig. 4O,P). Remarkably, we even observed supernumerary tooth formation in HERS under the cemento-enamel junction (CEJ) in the developing root, with intense amelogenin expression (Fig. 4Q). Similarly, injection of 4-OHT into K14-CreERTM;Ctnnb1(ex3)fl/+ mice at P5 also induced supernumerary tooth formation in the crown region of the molar, as well as in HERS and the furcation area in the developing root (Fig. 4R-T). These results indicate that young mice retain odontogenic potential in multiple regions of the jaw in the vicinity of incisor and molar teeth.
Wnt/β-catenin signaling regulates and maintains Fgf8 expression in oral epithelium
Fgf8 is one of the earliest molecules expressed during the
initiation stage of tooth development
(Kettunen and Thesleff, 1998).
To analyze the effect of Wnt/β-catenin signaling on Fgf8, we
utilized HAT-7 rat dental epithelial progenitor cells and LS8 mouse
ameloblast-like cells (Kawano et al.,
2002; Zhou and Snead,
2000). We first assayed Fgf8 expression in the presence
of LiCl, a Gsk3β inhibitor that blocks β-catenin degradation
(Hedgepeth et al., 1997). In
the presence of 10 or 50 mM LiCl for 6-18 hours, Fgf8 was upregulated
1.5-fold after 6 hours and 2.5-fold after 18 hours in LS8 cells
(Fig. 6A). In HAT-7 cells,
Fgf8 was upregulated 2.5-fold after 6 hours and 5.2-fold after
18 hours (see Fig. S6 in the supplementary material). Transfection of plasmid
encoding constitutively activated β-catenin into LS8 cells activated
canonical Wnt signaling and also upregulated Fgf8 transcripts
2-fold (Fig. 5B).
Previous studies identified a Tcf4 binding site 3 kb upstream of the
Fgf8 start site (Hatzis et al.,
2008). We identified four additional putative Tcf/Lef binding
sites around the mouse Fgf8 genomic locus using TRANSFAC
(Matys et al., 2006) (see
Table S1 in the supplementary material). In this study, we focused on a Lef1
binding site in Fgf8 intron 3 that is conserved in mouse, rat and
human, and performed chromatin immunoprecipitation (ChIP) experiments for Lef1
and β-catenin in HAT-7 cells treated for 18 hours with 50 mM LiCl. PCR
comparison of immunoprecipitated DNA using an IgG control antibody and
antibodies against either Lef1 or β-catenin revealed increased recovery
of the genomic fragment specifically containing the Lef1 binding site in
Fgf8 intron 3 (Fig.
5C). To test whether this binding site is functional, we subcloned
a conserved 1 kb sequence surrounding the Lef1 binding site into a
pGL3-promoter luciferase reporter vector. When this reporter construct was
co-transfected with constitutively activated β-catenin (S33Y) plasmid
into LS8 cells, luciferase activity was activated 1.7-fold compared with
the pGL3-promoter vector alone (Fig.
5D). This activation was abolished when 3 bp of the Lef1 binding
site core was deleted from the 1 kb construct, indicating that the Lef1
binding site in intron 3 of Fgf8 is functional. These results suggest
that Fgf8 is a direct target gene of Wnt/β-catenin signaling,
and that Wnt/β-catenin signaling is sufficient to stimulate Fgf8
expression.
|
To determine whether Wnt/β-catenin signaling is also necessary for Fgf8 expression, we cultured HAT-7 cells with two different Ctnnb1 RNAi constructs to knockdown endogenous β-catenin mRNA. Scrambled RNAi constructs with a similar GC content were used as negative controls. RNAi-mediated knockdown of β-catenin dramatically reduced β-catenin protein levels and significantly downregulated Fgf8 expression (Fig. 6A). To determine whether direct antagonism of Wnt ligand could also affect endogenous Fgf8 expression, we applied Dkk1-soaked beads to isolated E9.5 mouse mandibles. After 12-18 hours of in vitro culture, explants were fixed and Fgf8 expression detected by whole-mount in situ hybridization. BSA-soaked beads were used as negative controls, and expression was also compared with the contralateral side of the explant (0/12). In contrast to BSA-soaked beads, which had no effect on Fgf8 expression, Dkk1-soaked beads dramatically downregulated Fgf8 (8/13) (Fig. 6B). These results support an obligatory role for Wnt/β-catenin signaling in maintaining Fgf8 expression in the prospective tooth-forming oral ectoderm.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Soukup et al., 2008). During
evolution, teeth gradually became confined to the oral region. The most
primitive arrangement of oral teeth is a general distribution of denticles
throughout the oral cavity that are replaced continuously throughout life. For
example, in bony fishes, teeth are attached to bones surrounding the mouth
cavity, including the palate and medial surface of the lower jaw
(Johanson and Smith, 2003). By
contrast, in mice and humans, teeth are confined to U-shaped areas in the
upper and lower jaws, and these organisms exhibit just one and two sets of
dentition, respectively. Strikingly, however, we demonstrate here that
extensive regions of the mouse jaw exhibit odontogenic potential. By
modulation of just one gene, in this case deletion of Apc, or by
activation of Wnt/β-catenin signaling, supernumerary teeth can be induced
to form in embryonic and young mice in multiple regions of the jaw, including
oral and dental epithelia, the vestibular lamina and HERS of the developing
root.
Previous studies have shown that in tooth development, the initial
odontogenic signal comes from oral ectoderm, which instructs the underlying
mesenchyme of the first branchial arch to form teeth; after E12, the
tooth-forming potential shifts to the mesenchyme
(Lumsden, 1988;
Mina and Kollar, 1987).
Ablation of Apc or activation of Wnt/β-catenin signaling may
reprogram or reactivate the oral and dental epithelia, which then interact
with adjacent mesenchyme to form new teeth. Even mesenchymal cells along the
lingual aspect of the mouse incisor, which represents the root analog of the
molar tooth and contains periodontal ligaments
(Tummers and Thesleff, 2008),
retain responsiveness to initiation signals from the dental epithelium and are
able to participate in new tooth formation. Notably, the supernumerary teeth
form in the vicinity of the incisor and molar teeth. In this context,
depletion of Apc or activation of Wnt/β-catenin signaling in the
epidermis or in the tongue generates ectopic hair follicles or taste buds
(Kuraguchi et al., 2006;
Liu et al., 2007). Thus,
whether the identity of the supernumerary teeth is determined by the activated
oral epithelium or by the jaw mesenchyme remains to be determined.
Nevertheless, once initiated, the odontogenic program proceeds autonomously to
stages of terminal differentiation and root formation, indicating that
activation of Wnt signaling is a key switch for the entire tooth-forming
program. Collectively, these results suggest that Apc is a bona fide
endogenous inhibitor of supernumerary tooth formation during embryogenesis and
throughout adulthood. Moreover, the balance between Apc and Wnt signaling
controls the position and number of teeth, and regulates the processes of
tooth replacement and successional tooth formation.
Wnt signaling is active in developing teeth from the initiation stage, and
continues throughout tooth differentiation. At the initiation of tooth
development, Wnt4 and Wnt6 are expressed throughout oral and
dental epithelium, whereas Wnt10a and Wnt10b transcripts are
concentrated in the presumptive dental epithelium. Wnt3 and
Wnt7b are not expressed in dental epithelium, but in the flanking
oral epithelium. Wnt5a is the only Wnt signal reported to be
expressed in the mesenchyme (Dassule and
McMahon, 1998; Sarkar and
Sharpe, 1999). Apc is an inhibitor of Wnt/β-catenin
signaling, and in K14-Cre1Amc;Apccko/cko mice,
β-catenin protein levels were significantly upregulated. Not
surprisingly, therefore, the dental epithelial phenotypes in
K14-Cre1Amc;Apccko/cko mice resemble those in
mice with constitutive activation of β-catenin in the epithelium
(Jarvinen et al., 2006;
Liu et al., 2008). However,
both Apc and β-catenin have additional functions besides their
interaction in the Wnt/β-catenin signaling pathway. Nonetheless, we found
that genetic depletion of β-catenin in
K14-Cre1Amc;Apccko/cko mice blocked
supernumerary tooth formation. This result formally proves that Apc acts to
prevent supernumerary tooth formation by inhibiting Wnt/β-catenin
signaling, and that the induction of supernumerary teeth by Apc
loss-of-function is due to activation of the Wnt/β-catenin signaling
pathway.
Embryonic and young mice formed supernumerary teeth continuously from
multiple regions of the jaw, whereas in older adult mice, supernumerary teeth
were mainly observed in regions around the incisors. The lack of supernumerary
teeth in the molar regions of older adult mice might reflect the degeneration
of the dental lamina and dental epithelial cells that normally occurs
coincident with the eruption of the molar teeth, and the differentiation of
remaining jaw epithelium into stratified epithelium and oral mucosa. The mouse
incisor differs from these regions in that it contains epithelial stem cells
in the central core of the cervical loop regions, and both the labial and
lingual dental epithelia contain stem cells
(Harada et al., 1999a;
Wang et al., 2007). Indeed,
supernumerary teeth were formed from both the labial and lingual sides of the
principal incisors. However, they were not only restricted to cervical loop
regions. Some supernumerary teeth were located near the incisor tip, far from
the cervical loop region, whereas others developed from among differentiating
ameloblasts. Whether supernumerary teeth form from progenitor cells that
migrate from the cervical loops, or whether dental progenitor cells also
reside in other locations, requires further investigation.
The formation of supernumerary teeth is non-cell-autonomous
Using confocal analyses we obtained evidence that supernumerary tooth
formation is non-cell-autonomous. To obviate the concern that differential
recombination kinetics between the two floxed alleles (R26R and
Apccko) might lead to an underestimate of the number of
Apc-deficient cells, we also detected β-catenin expression in
these mice. Surprisingly, we observed that Apc-deficient cells
constituted only a modest subset of the epithelial cells in supernumerary
tooth buds. We therefore conclude that Apc-deficient epithelial cells
are able to recruit surrounding wild-type epithelial and mesenchymal cells
into supernumerary tooth formation. Further studies are required to determine
whether Apc-deficient cells directly induce surrounding wild-type
epithelial cells into the odontogenic program, or whether they do so
indirectly, via reciprocal signaling through the dental mesenchyme.
In K14-Cre8Brn;Apccko/cko mice, most of the
cells with elevated levels of β-catenin were Ki67 positive, but a subset
of these cells, located at the tips of supernumerary tooth buds in the
prospective enamel knot region, were Ki67 negative and hence in G0. This state
is similar to that in cells in the endogenous tooth enamel knot, which is
non-proliferative owing to p21 expression
(Jernvall et al., 1998) and is
therefore mostly Ki67 negative. Thus, similar to endogenous tooth development
(see Fig. S3 in the supplementary material), β-catenin-positive cells are
also involved in enamel knot formation in supernumerary tooth germs. Since
both Apc-deficient and adjacent wild-type epithelial cells can
express high levels of β-catenin, it appears that the formation of
supernumerary teeth is Apc non-cell-autonomous, and that even
wild-type epithelial cells can participate in enamel knot formation in
supernumerary teeth.
Supernumerary and endogenous tooth development exhibit mechanistic similarities and differences
The development of supernumerary teeth involves many of the same signaling
molecules that are employed in endogenous tooth development, including Shh,
activin βA and Fgf8. Fgf8 is an early epithelial marker for
tooth initiation, as its expression starts at E9 and is downregulated after
E13 (Neubuser et al., 1997;
Kettunen and Thesleff, 1998).
Fgf8 can induce the expression of many key transcription factors in the
underlying dental mesenchyme, and genetic ablation of Fgf8 in first
branchial arch ectoderm results in cell death in proximal ectomesenchyme and
in an absence of molar teeth (Grigoriou et
al., 1998; Trumpp et al.,
1999). In this study, we identified a novel, occupied Lef1 binding
site within the third intron of Fgf8, in addition to a previously
reported Tcf4 occupancy in the Fgf8 proximal promoter
(Hatzis et al., 2008), and we
confirmed its functionality by performing ChIP and site-directed mutagenesis
experiments. Importantly, beads soaked with Dkk1, a Wnt antagonist,
downregulated endogenous Fgf8 in E9.5 mandibular explants. These
results indicate that Fgf8 is a direct downstream target of
Wnt/β-catenin signaling in tooth formation, and that Wnt/β-catenin
is required for the maintenance of endogenous Fgf8 expression in oral
epithelium.
A previous study showed that Fgf4 is a direct target gene of Lef1
and Wnt signaling in the developing teeth
(Kratochwil et al., 2002).
Fgf4 is not expressed during the initiation stage of tooth
development. However, it is expressed in the enamel knot signaling center of
the tooth germ at the late bud and cap stage, as well as in the secondary
enamel knots of bell-stage tooth germs that mark the future tooth cusps
(Kettunen and Thesleff, 1998).
As a result, there is no overlap between Fgf8 and Fgf4
expression during endogenous tooth development in wild-type mice. The
observation that Fgf8 and Fgf4 are co-expressed in the
invaginating oral epithelium of E14.5
K14-Cre1Amc;Apccko/cko mice indicates that
their expression is not mutually exclusive. Furthermore, the co-expression of
Fgf8, a tooth initiation marker, and Fgf4, an enamel knot
marker (Jarvinen et al.,
2006), suggests a temporal compression of tooth development in
Wnt/β-catenin-activated supernumerary teeth.
A key difference between supernumerary and endogenous tooth formation
concerns their Msx1 dependence. In Msx1 knockout mice, tooth
development arrests at the bud stage (Chen
et al., 1996). By contrast, the development of supernumerary teeth
progressed well beyond the bud stage in
K14-Cre8Brn;Apccko/cko mice that were also
homozygous null for Msx1. It has been shown that Bmp4 in the dental
mesenchyme, which requires Msx1 for its expression, acts upon the dental
epithelium to initiate enamel knot formation and to promote the tooth bud to
cap stage transition (Chen et al.,
1996). Our results suggest that this requirement can be bypassed
through the activation of Wnt/β-catenin signaling in the epithelium. For
example, both Bmp4 and Fgf3, which are downregulated in
Msx1 knockout mice, are expressed in the mesenchyme of supernumerary
teeth in
K14-Cre8Brn;Apccko/cko;Msx1-/- mice,
and Bmp4 is also expressed in the epithelium of these supernumerary
teeth. We showed previously that the addition of Bmp4 to Msx1 mutant
tooth germs can rescue the development of Msx1 mutant arrested tooth
buds to advanced stages (Bei et al.,
2000). The upregulation of Bmp4 that we observed in
supernumerary tooth germs resembles that observed in β-catenin
gain-of-function [Ctnnb1(ex3)fl] mice with supernumerary
tooth and hair follicle development (Liu
et al., 2008; Narhi et al.,
2008). These results suggest that Apc loss-of-function
rescues Bmp4 expression and thus bypasses the mesenchymal
Msx1-Bmp4 feedback loop normally required for endogenous tooth
development (Chen et al., 1996;
Bei et al., 2000), thereby
permitting the dental epithelium to develop past the bud stage to more
advanced stages. Mutations in MSX1 in humans cause tooth agenesis
(Vastardis et al., 1996). It
would be interesting to see whether human MSX1-deficient oral tissues also
possess the ability to form supernumerary teeth. Taken together, our data
indicate that the induction of supernumerary teeth by Wnt/β-catenin
signaling involves mechanisms that resemble those used in endogenous tooth
development (e.g. induction of Fgf8), as well as those that differ
(e.g. Msx1 independence).
Supernumerary teeth and the potential for in vivo tooth regeneration
We demonstrate that supernumerary teeth resemble natural teeth, with
well-differentiated ameloblasts and odontoblasts that support
biomineralization. Enamel matrix secreted by these teeth is indistinguishable
from native enamel (X.-P.W., R.L.M. and Z. Skobe, unpublished). Similar to
natural teeth, the supernumerary teeth possess well-distributed blood vessels
within dental pulp, and neural innervation within dental pulp and dentin
tubules. These teeth also form HERS, a structure responsible for tooth root
formation and elongation.
Previous studies showed that recombination of E10 mouse embryonic oral
epithelium with embryonic stem cells, neural stem cells, or with adult bone
marrow-derived mesenchymal cells (BMSCs) induced the expression of Msx1,
Pax9 and Lhx6/7 in the stem cells, and the combination of dental
epithelium and BMSCs followed by kidney capsule culture resulted in tooth
formation (Ohazama et al.,
2004). Adult mesenchymal stem cells have been identified in human
dental pulp, periodontal ligaments and exfoliated deciduous teeth
(Mao et al., 2006). However,
potential limitations of human tooth regeneration include limiting sources of
a suitable inducing tissue - in particular, of early embryonic oral
epithelium. Our results reveal a remarkable latent odontogenic potential in
adult rodent dental tissue, and especially in the oral tissue of young mice.
In humans, permanent tooth development starts during the embryonic and fetal
periods, and then continues for many years after birth into adolescence.
Children and adolescents thus retain dental lamina epithelial cells in their
jaws. This fact, together with the identification of signaling pathways that
can initiate new tooth formation in an adult animal, offer a potentially
viable strategy for human tooth regeneration and repair and might provide
insight into the regeneration of other organs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/11/1939/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aberg, T., Wang, X. P., Kim, J. H., Yamashiro, T., Bei, M.,
Rice, R., Ryoo, H. M. and Thesleff, I. (2004). Runx2 mediates
FGF signaling from epithelium to mesenchyme during tooth morphogenesis.
Dev. Biol. 270,76
-93.[CrossRef][Medline]
Bei, M., Kratochwil, K. and Maas, R. L. (2000).
BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development.
Development 127,4711
-4718.[Abstract]
Brault, V., Moore, R., Kutsch, S., Ishibashi, M., Rowitch, D.
H., McMahon, A. P., Sommer, L., Boussadia, O. and Kemler, R.
(2001). Inactivation of the beta-catenin gene by
Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure
of craniofacial development. Development
128,1253
-1264.[Abstract]
Butler, P. M. (1995). Ontogenetic aspects of
dental evolution. Int. J. Dev. Biol.
39, 25-34.[Medline]
Byrne, C., Tainsky, M. and Fuchs, E. (1994).
Programming gene expression in developing epidermis.
Development 120,2369
-2383.
Chen, Y., Bei, M., Woo, I., Satokata, I. and Maas, R.
(1996). Msx1 controls inductive signaling in mammalian tooth
morphogenesis. Development
122,3035
-3044.[Abstract]
Clevers, H. (2006). Wnt/beta-catenin signaling
in development and disease. Cell
127,469
-480.[CrossRef][Medline]
Dassule, H. R. and McMahon, A. P. (1998).
Analysis of epithelial-mesenchymal interactions in the initial morphogenesis
of the mammalian tooth. Dev. Biol.
202,215
-227.[CrossRef][Medline]
Dassule, H. R., Lewis, P., Bei, M., Maas, R. and McMahon, A.
P. (2000). Sonic hedgehog regulates growth and morphogenesis
of the tooth. Development
127,4775
-4785.[Abstract]
Grigoriou, M., Tucker, A. S., Sharpe, P. T. and Pachnis, V.
(1998). Expression and regulation of Lhx6 and Lhx7, a novel
subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head
development. Development
125,2063
-2074.[Abstract]
Gritli-Linde, A., Bei, M., Maas, R., Zhang, X. M., Linde, A. and
McMahon, A. P. (2002). Shh signaling within the dental
epithelium is necessary for cell proliferation, growth and polarization.
Development 129,5323
-5337.
Harada, H., Kettunen, P., Jung, H. S., Mustonen, T., Wang, Y. A.
and Thesleff, I. (1999a). Localization of putative stem cells
in dental epithelium and their association with Notch and FGF signaling.
J. Cell Biol. 147,105
-120.
Harada, N., Tamai, Y., Ishikawa, T., Sauer, B., Takaku, K.,
Oshima, M. and Taketo, M. M. (1999b). Intestinal polyposis in
mice with a dominant stable mutation of the beta-catenin gene. EMBO
J. 18,5931
-5942.[CrossRef][Medline]
Hatzis, P., van der Flier, L. G., van Driel, M. A., Guryev, V.,
Nielsen, F., Denissov, S., Nijman, I. J., Koster, J., Santo, E. E., Welboren,
W. et al. (2008). Genome-wide pattern of TCF7L2/TCF4
chromatin occupancy in colorectal cancer cells. Mol. Cell.
Biol. 28,2732
-2744.
Hedgepeth, C. M., Conrad, L. J., Zhang, J., Huang, H. C., Lee,
V. M. and Klein, P. S. (1997). Activation of the Wnt
signaling pathway: a molecular mechanism for lithium action. Dev.
Biol. 185,82
-91.[CrossRef][Medline]
Jarvinen, E., Salazar-Ciudad, I., Birchmeier, W., Taketo, M. M.,
Jernvall, J. and Thesleff, I. (2006). Continuous tooth
generation in mouse is induced by activated epithelial Wnt/beta-catenin
signaling. Proc. Natl. Acad. Sci. USA
103,18627
-18632.
Jarvinen, E., Valimaki, K., Pummila, M., Thesleff, I. and
Jernvall, J. (2008). The taming of the shrew milk teeth.
Evol. Dev. 10,477
-486.[CrossRef][Medline]
Jernvall, J., Kettunen, P., Karavanova, I., Martin, L. B. and
Thesleff, I. (1994). Evidence for the role of the enamel knot
as a control center in mammalian tooth cusp formation: non-dividing cells
express growth stimulating Fgf-4 gene. Int. J. Dev.
Biol. 38,463
-469.[Medline]
Jernvall, J., Aberg, T., Kettunen, P., Keranen, S. and Thesleff,
I. (1998). The life history of an embryonic signaling center:
BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel
knot. Development 125,161
-169.[Abstract]
Johanson, Z. and Smith, M. M. (2003). Placoderm
fishes, pharyngeal denticles, and the vertebrate dentition. J.
Morphol. 257,289
-307.[CrossRef][Medline]
Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van
der Valk, M. and Berns, A. (2001). Synergistic tumor
suppressor activity of BRCA2 and p53 in a conditional mouse model for breast
cancer. Nat. Genet. 29,418
-425.[CrossRef][Medline]
Kawano, S., Morotomi, T., Toyono, T., Nakamura, N., Uchida, T.,
Ohishi, M., Toyoshima, K. and Harada, H. (2002).
Establishment of dental epithelial cell line (HAT-7) and the cell
differentiation dependent on Notch signaling pathway. Connect.
Tissue Res. 43,409
-412.[Medline]
Kettunen, P. and Thesleff, I. (1998).
Expression and function of FGFs-4, -8, and - 9 suggest functional redundancy
and repetitive use as epithelial signals during tooth morphogenesis.
Dev. Dyn. 211,256
-268.[CrossRef][Medline]
Kratochwil, K., Galceran, J., Tontsch, S., Roth, W. and
Grosschedl, R. (2002). FGF4, a direct target of LEF1 and Wnt
signaling, can rescue the arrest of tooth organogenesis in Lef1(-/-) mice.
Genes Dev. 16,3173
-3185.
Kuraguchi, M., Wang, X. P., Bronson, R. T., Rothenberg, R.,
Ohene-Baah, N. Y., Lund, J. J., Kucherlapati, M., Maas, R. L. and
Kucherlapati, R. (2006). Adenomatous polyposis coli (APC) is
required for normal development of skin and thymus. PLoS
Genet. 2,e146
.[CrossRef][Medline]
Lee, T. I., Johnstone, S. E. and Young, R. A.
(2006). Chromatin immunoprecipitation and microarray-based
analysis of protein location. Nat. Protoc.
1, 729-748.[CrossRef][Medline]
Liu, F., Thirumangalathu, S., Gallant, N. M., Yang, S. H.,
Stoick-Cooper, C. L., Reddy, S. T., Andl, T., Taketo, M. M., Dlugosz, A. A.,
Moon, R. T. et al.(2007). Wnt-beta-catenin signaling
initiates taste papilla development. Nat. Genet.
39,106
-112.[CrossRef][Medline]
Liu, F., Chu, E. Y., Watt, B., Zhang, Y., Gallant, N. M., Andl,
T., Yang, S. H., Lu, M. M., Piccolo, S., Schmidt-Ullrich, R. et al.
(2008). Wnt/beta-catenin signaling directs multiple stages of
tooth morphogenesis. Dev. Biol.
313,210
-224.[CrossRef][Medline]
Lumsden, A. G. (1988). Spatial organization of
the epithelium and the role of neural crest cells in the initiation of the
mammalian tooth germ. Development
103 Suppl,155
-169.
Luo, W., Slavkin, H. C. and Snead, M. L.
(1991). Cells from Hertwig's epithelial root sheath do not
transcribe amelogenin. J. Periodontal. Res.
26, 42-47.[CrossRef][Medline]
Madani, M. and Madani, F. (2007). Gardner's
syndrome presenting with dental complaints. Arch. Iran.
Med. 10,535
-539.[Medline]
Mao, J. J., Giannobile, W. V., Helms, J. A., Hollister, S. J.,
Krebsbach, P. H., Longaker, M. T. and Shi, S. (2006).
Craniofacial tissue engineering by stem cells. J. Dent.
Res. 85,966
-979.
Matys, V., Kel-Margoulis, O. V., Fricke, E., Liebich, I., Land,
S., Barre-Dirrie, A., Reuter, I., Chekmenev, D., Krull, M., Hornischer, K. et
al. (2006). TRANSFAC and its module TRANSCompel:
transcriptional gene regulation in eukaryotes. Nucleic Acids
Res. 34,D108
-D110.
Mina, M. and Kollar, E. J. (1987). The
induction of odontogenesis in non-dental mesenchyme combined with early murine
mandibular arch epithelium. Arch. Oral Biol.
32,123
-127.[CrossRef][Medline]
Narhi, K., Jarvinen, E., Birchmeier, W., Taketo, M. M., Mikkola,
M. L. and Thesleff, I. (2008). Sustained epithelial
beta-catenin activity induces precocious hair development but disrupts hair
follicle down-growth and hair shaft formation.
Development 135,1019
-1028.
Neubuser, A., Peters, H., Balling, R. and Martin, G. R.
(1997). Antagonistic interactions between FGF and BMP signaling
pathways: a mechanism for positioning the sites of tooth formation.
Cell 90,247
-255.[CrossRef][Medline]
Nusse, R. (2008). Wnt signaling and stem cell
control. Cell Res. 18,523
-527.[CrossRef][Medline]
Ohazama, A., Modino, S. A., Miletich, I. and Sharpe, P. T.
(2004). Stem-cell-based tissue engineering of murine teeth.
J. Dent. Res. 83,518
-522.
Pispa, J. and Thesleff, I. (2003). Mechanisms
of ectodermal organogenesis. Dev. Biol.
262,195
-205.[CrossRef][Medline]
Rajagopal, J., Carroll, T. J., Guseh, J. S., Bores, S. A.,
Blank, L. J., Anderson, W. J., Yu, J., Zhou, Q., McMahon, A. P. and Melton, D.
A. (2008). Wnt7b stimulates embryonic lung growth by
coordinately increasing the replication of epithelium and mesenchyme.
Development 135,1625
-1634.
Sarkar, L. and Sharpe, P. T. (1999). Expression
of Wnt signalling pathway genes during tooth development. Mech.
Dev. 85,197
-200.[CrossRef][Medline]
Scholzen, T. and Gerdes, J. (2000). The Ki-67
protein: from the known and the unknown. J. Cell
Physiol. 182,311
-322.[CrossRef][Medline]
Smith, M. M. (2003). Vertebrate dentitions at
the origin of jaws: when and how pattern evolved. Evol.
Dev. 5,394
-413.[CrossRef][Medline]
Smith, M. M. and Hall, B. K. (1990).
Development and evolutionary origins of vertebrate skeletogenic and
odontogenic tissues. Biol. Rev. Camb. Philos. Soc.
65,277
-373.
Soriano, P. (1999). Generalized lacZ expression
with the ROSA26 Cre reporter strain. Nat. Genet.
21, 70-71.[CrossRef][Medline]
Soukup, V., Epperlein, H. H., Horacek, I. and Cerny, R.
(2008). Dual epithelial origin of vertebrate oral teeth.
Nature 455,795
-798.[CrossRef][Medline]
Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and
Martin, G. R. (1999). Cre-mediated gene inactivation
demonstrates that FGF8 is required for cell survival and patterning of the
first branchial arch. Genes Dev.
13,3136
-3148.
Tummers, M. and Thesleff, I. (2008).
Observations on continuously growing roots of the sloth and the K14-Eda
transgenic mice indicate that epithelial stem cells can give rise to both the
ameloblast and root epithelium cell lineage creating distinct tooth patterns.
Evol. Dev. 10,187
-195.[Medline]
Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S. and
Thesleff, I. (1996). The enamel knot as a signaling center in
the developing mouse tooth. Mech. Dev.
54, 39-43.[CrossRef][Medline]
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I.
(1993). Identification of BMP-4 as a signal mediating secondary
induction between epithelial and mesenchymal tissues during early tooth
development. Cell 75,45
-58.[CrossRef][Medline]
Vasioukhin, V., Degenstein, L., Wise, B. and Fuchs, E.
(1999). The magical touch: genome targeting in epidermal stem
cells induced by tamoxifen application to mouse skin. Proc. Natl.
Acad. Sci. USA 96,8551
-8556.
Vastardis, H., Karimbux, N., Guthua, S. W., Seidman, J. G. and
Seidman, C. E. (1996). A human MSX1 homeodomain missense
mutation causes selective tooth agenesis. Nat. Genet.
13,417
-421.[CrossRef][Medline]
Wang, X. P., Suomalainen, M., Jorgez, C. J., Matzuk, M. M.,
Wankell, M., Werner, S. and Thesleff, I. (2004). Modulation
of activin/bone morphogenetic protein signaling by follistatin is required for
the morphogenesis of mouse molar teeth. Dev. Dyn.
231,98
-108.[CrossRef][Medline]
Wang, X. P., Aberg, T., James, M. J., Levanon, D., Groner, Y.
and Thesleff, I. (2005). Runx2 (Cbfa1) inhibits Shh signaling
in the lower but not upper molars of mouse embryos and prevents the budding of
putative successional teeth. J. Dent. Res.
84,138
-143.
Wang, X. P., Suomalainen, M., Felszeghy, S., Zelarayan, L. C.,
Alonso, M. T., Plikus, M. V., Maas, R. L., Chuong, C. M., Schimmang, T. and
Thesleff, I. (2007). An integrated gene regulatory network
controls stem cell proliferation in teeth. PLoS Biol.
5, e159.[CrossRef][Medline]
Yochum, G. S., McWeeney, S., Rajaraman, V., Cleland, R., Peters,
S. and Goodman, R. H. (2007). Serial analysis of chromatin
occupancy identifies beta-catenin target genes in colorectal carcinoma cells.
Proc. Natl. Acad. Sci. USA
104,3324
-3329.
Zhou, Y. L. and Snead, M. L. (2000).
Identification of CCAAT/enhancer-binding protein alpha as a transactivator of
the mouse amelogenin gene. J. Biol. Chem.
275,12273
-12280.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  S. Miyagawa, A. Moon, R. Haraguchi, C. Inoue, M. Harada, C. Nakahara, K. Suzuki, D. Matsumaru, T. Kaneko, I. Matsuo, et al. Dosage-dependent hedgehog signals integrated with Wnt/{beta}-catenin signaling regulate external genitalia formation as an appendicular program Development, December 1, 2009; 136(23): 3969 - 3978. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
