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897
Development 136, 897-903 (2009) doi:10.1242/dev.027979
Primary cilia regulate Shh activity in the control of molar
tooth number
Atsushi Ohazama1, Courtney J. Haycraft2, Maisa Seppala1, James Blackburn1, Sarah Ghafoor1,
Martyn Cobourne3, David C. Martinelli4, Chen-Ming Fan4, Renata Peterkova5, Herve Lesot6,
Bradley K. Yoder2 and Paul T. Sharpe1,*
Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a
cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the
embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is
specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth
development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice
mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth and
accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh
activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and
shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.
INTRODUCTION
Tooth number is highly regulated in mammals. Species such as mice
have a highly reduced dentition consisting of only molars and
incisors, whereas the development of other mammalian tooth types
has been lost during evolution. Evidence from three-dimensional
reconstructions of early tooth development in mouse embryos has
identified tooth primordia in the diastema, a toothless region
between the incisors and molars, which are initiated but are later
suppressed by apoptosis (Tureckova et al., 1996; Peterkova et al.,
2002). These primordia are believed to be the vestiges of teeth lost
during mouse evolution and suggest that a mechanism for
suppression of tooth formation in the diastema involves selective
apoptosis of tooth primordia (Peterkova et al., 2003). In the early
oral cavity, Shh expression is restricted to the thickenings of oral
epithelium (dental placodes), from which the tooth germs arise
(Bitgood and McMahon, 1995; Dassule and McMahon, 1998;
Hardcastle et al., 1998). A loss of Shh activity in these regions at
embryonic day (E)10 results in a lack of epithelial cell proliferation
and a failure of tooth bud formation (Cobourne et al., 2001). Once
the early tooth bud has formed, continued Shh activity is required
within discrete regions of the tooth germ epithelium and
mesenchyme for normal growth and morphogenesis of the
developing tooth (Dassule et al., 2000; Gritli-Linde et al., 2002;
Jeong et al., 2004). Significantly, Shh transcriptional activity is
lacking in the diastema and, thus, during normal development of the
1
Department of Craniofacial Development, Dental Institute, King’s College London,
London SE1 9RT, UK. 2Department of Cell Biology, University of Alabama at
Birmingham, Birmingham, AL 35294-0005, USA. 3Department of Craniofacial
Development and Orthodontics, Dental Institute, King’s College London, UK.
4
Department of Embryology, Carnegie Institution of Washington 3520 San Martin
Drive, Baltimore, MD 21218, USA. 5Department of Teratology, Institute of
Experimental Medicine, Academy of Sciences of the CR, Prague, Czech Republic.
6
INSERM UMR977, Dental School, Strasbourg University, Strasbourg, France.
*Author for correspondence (e-mail: [email protected])
Accepted 21 January 2009
murine dentition, suppression of Shh pathway activity in the
diastema may play an important role in controlling primary tooth
number (Cobourne et al., 2004).
Primary cilia have recently been shown to play a crucial role in
Shh signalling (Rohatgi et al., 2007; Caspary et al., 2007; Singla
and Reiter, 2006; Huangfu and Anderson, 2005). The Shh
receptor Ptch1, its activator Smo and the transcriptional effector
Gli1 are all found to localize in cilia, suggesting that the Shh
signalling pathway is active in cilia and that cilia are required for
Shh signal transduction (Rohatgi et al., 2007; Haycraft et al.,
2005). Intraflagellar transport (IFT) proteins are highly conserved
in all ciliated eukaryotic cells, and mutation of the proteins that
comprise the IFT process result in defects in cilia formation in all
organisms studied to date (Rosenbaum and Witman, 2002; Pan et
al., 2005; Scholey and Anderson, 2006). Mice with null mutations
in Ift88, which encodes the IFT protein polaris, lack cilia on all
cells and die mid-gestation with severe defects in neural tube
patterning and closure, polydactyly, and left-right axis
determination (Murcia et al., 2000; Zhang et al., 2003). Defects
in the neural tube have been associated with loss of Shh signalling
regulation through disruption of Gli activation, whereas the limb
patterning defects are associated with a gain of Shh activity as a
result of a loss of Gli3 repression (Huangfu and Anderson, 2005;
Haycraft et al., 2005; Michaud and Yoder, 2006). Similar defects
have also been reported for mice with severe mutations in other
IFT proteins (Huangfu and Anderson, 2005; Liu et al., 2005; May
et al., 2005). Tg737orpk is a hypomorphic allele of polaris;
homozygous Tg737orpk mice exhibit a complex pathology,
including kidney and pancreatic cysts, preaxial polydactyly, and
supernumerary teeth (Zhang et al., 2003).
We examined the development of vestigial diastema tooth
primordia in Tg737orpk mice and found ectopic tooth formation that
correlates with ectopic Shh signalling activity in the diastema
mesenchyme. Using Wnt1-Cre and keratin(K)5-Cre to conditionally
inactivate polaris in tooth mesenchyme and epithelium, respectively,
we show that this cilia-mediated Shh signalling is required only in
DEVELOPMENT
KEY WORDS: Intraflagellar transport, Cilia, Shh, Supernumerary tooth, Tooth development, Tooth number, Orpk, Tg737, Gas1, Epithelium,
Mesenchyme, Mouse
RESEARCH REPORT
the mesenchyme. We further show that mutants in the Shh
regulatory protein Gas1 also develop diastema teeth as a result of
ectopic Shh activity in diastema mesenchyme. Therefore, in the
context of control of tooth number, cilia are involved in the
repression of Shh activity in diastema mesenchyme cells.
Development 136 (6)
Epithelial three-dimension reconstruction
Epithelial three-dimension reconstruction of mandible dental and adjacent
oral epithelium were performed as previously described (Lesot et al., 1996).
Transmission electron microscopy (TEM) analysis
Tg737orpk and Tg737Δ2-3β-gal were produced as described previously (Moyer
et al., 1994). Gas1 mutant mice, polarisLoxP mice, K5-Cre and Wnt1-Cre
mice were produced as described previously (Chai et al., 2000; Lee et al.,
2001; Ramirez et al., 2004; Haycraft et al., 2007).
Heads were fixed in 2.5% glutaraldehyde (phosphate buffer) overnight at
4°C and postfixed in 2% osmium tetroxide (Millonigs buffer) after washing
by buffer. Specimens were dehydrated through a graded series of ethanols
and embedded in Epon 812-equivalent (TAAB Lab). Semithin sections
(1 μm) were stained with Toluidine Blue for light microscopy analysis.
Ultrathin sections (40-90 nm) were cut, stained with uranyl acetate and lead
citrate and examined with a Hitachi H7600 transmission electron
microscope.
In situ hybridisation
Immunohistochemistry
MATERIALS AND METHODS
Production and analysis of mice
Radioactive section in situ hybridisation using 35S-UTP radiolabelled
riboprobes was carried out as described previously (Ohazama et al., 2008).
Whole-mount in situ hybridisation was carried out as described previously
(Pownall et al., 1996).
Micro-CT analysis
Heads were scanned with Explore Locus SP (GE Pre-clinical imaging) high
resolution Micro-CT with a voxel dimension of 8 μm. Three-dimension
reconstruction was performed by the three structure analysis software
Microview (GE Pre-clinical imaging).
β-Galactosidase (β-gal) staining
Embryo heads were processed as previously described (Taulman et al.,
2001).
Sections were incubated with antibody to acetylated α-tubulin or γ-tubulin
(Sigma). Alexa488 or Alexa594 was used (Molecular probe) for detecting
primary antibody. Immunohistochemistry for Polaris or Shh were performed
as previously described (Rosenbaum and Witman 2002; Martinelli and Fan,
2007; Gritli-Linde et al., 2001).
RESULTS AND DISCUSSION
Shh activity in the diastema
Primary cilia have been shown to be important mediators of Shh
activity and, as Shh controls tooth initiation, we examined tooth
development in Tg737orpk adult mice, which contain a hypomorphic
mutation in the IFT protein polaris. We identified teeth mesial to the
Fig. 1. Lower molar tooth phenotypes and Shh signalling in Tg737orpk homozygous mutant mice. (A-D) Whole teeth (A,B) and sagittal
sections (C,D) show that supernumerary teeth develop mesial to the first molars (sn in B,D) in mutants. (E,F) Increase of Ptch1-lacZ staining in the
mandible of Tg737orpk mutants with Ptch1-lacZ allele at E13.5. Ptch1-lacZ staining was expanded into the diastema region (arrowhead). Arrows
indicate molar germs. (G-J) Radioactive in situ hybridisation on sagittal sections showing Gli1 (G,H), Gas1 (I,J) and Shh (G’,H’,I’,J’) expression in
mandibles of embryo heads at E12.5. (G,H,I,J) Yellow circles represent positions of Shh expression in adjacent sections (blue arrowheads in
G’,H’,I’,J’). Gli1 expression (H) was expanded into the diastema and Gas1 (J) was downregulated in the diastema of Tg737orpk mutants (green
arrowheads). (K-N) Sagittal sections (N) and three-dimensional reconstructions (K-M) of the dental epithelium of the mandible molar region at E15.5
(N) and E16.5 (K-M) in Tg737orpk mutants. The supernumerary tooth buds (arrows in K-M) were observed in the position of the mesial swellings
(arrowhead in N). (O-T) SEM analysis shows that the lingual cusp of maxillary supernumerary tooth (red line in T) is more prominent in comparison
with that of mandibular supernumerary tooth (red line in S). (U,V) Horizontal micro-CT sections of Tg737orpk show that maxillary supernumerary
teeth have concave roots (circle in V) and in mandibles show round roots (circle in U). m1, first molar; m2, second molar; m3, third molar; mand,
mandible jaw; max, maxillary jaw.
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first molar in all four jaw quadrants with 100% penetrance that were
not present in wild-type animals (Fig. 1A-D) (Zhang et al., 2003).
Because the development of ectopic digits in Tg737orpk mutants is
due to alternations in Shh signalling in the limb bud, we examined
the expression of Ptch1 and Gli1, which are upregulated in response
to Shh signal transduction, in the region of ectopic tooth
development in the Tg737orpk homozygous mutants at E12.5 and
E13.5. To determine the expression pattern of Ptch1, we generated
Tg737orpk homozygous mutants that were also heterozygous for a
Ptch1-lacZ allele and examined the activity of β-gal. At E13.5,
Ptch1-lacZ staining was increased in the incisor and molar areas of
Tg737orpk homozygous mutants and expanded into the diastema,
which is normally devoid of Shh signalling activity (Fig. 1E,F). Gli1
expression was also expanded into the diastema region (Fig. 1G-H⬘)
and expression of Gas1, a Shh antagonist in tooth development that
is downregulated by Shh activity, was reduced (Fig. 1I-J⬘)
(Cobourne et al., 2004; Martinelli and Fan, 2007). The expansion of
Ptch1 and Gli1 expression into the diastema correlates with the
placement of premolar-like tooth buds that develop in the Tg737orpk
mutants. These observations suggest that an absence of the IFT
protein polaris results in a Shh gain-of-function phenotype. In limb
buds, hypomorphic mutation of polaris leads to a series of changes
in Shh signalling that include disruption of Gli3 processing, resulting
in severe polydactyly similar to that found in Gli3 mutants (Haycraft
et al., 2005; Litingtung et al., 2002). The expression of Shh is
downregulated in the midline of polaris null mutants, which show
severe midline defects (Murcia et al., 2000). It has been suggested
that the loss of Gli activators, which play a major role in neural tube
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899
patterning, is consistent with the loss of ventral neural tube cells in
the IFT protein mutant mice, whereas loss of Gli3 repressor
function, which is more important in digit patterning, is consistent
with the formation of extra digits in the IFT protein mutants
(Huangfu and Anderson, 2005; Michaud and Yoder, 2006). The
absence of an IFT protein can therefore affect both positive and
negative aspects of Shh signalling, depending upon the particular
Gli activity. In the context of the control of tooth number, increased
Shh activity is thus consistent with loss of Gli3 repressor which is
prominently expressed in the wild-type diastema (see Fig. S1 in the
supplementary material) (Huangfu and Anderson, 2005; Haycraft et
al., 2005; Michaud and Yoder, 2006).
Cilia and cilia proteins in tooth development
To investigate the cells that require fully functional cilia during tooth
formation, we first determined the location of cilia in tooth germs
using a cilia marker, acetylated α-tubulin and a marker of basal
bodies of cilia, γ-tubulin. Cilia were found in tooth epithelium and
mesenchyme cells at early stages of development (Fig. 2A,B,D).
Cilia were observed on both epithelial and mesenchymal cells of
supernumerary and endogenous tooth germs in Tg737orpk mice (Fig.
2C,E). Transmission electron microscopy (TEM) was performed to
investigate the structure of the cilia on tooth cells. Cilia were found
on dental epithelial and mesenchymal cells and no obvious
morphological differences were visible between Tg737orpk and wildtype cells (Fig. 2F-I). We next examined Tg737 expression and
polaris protein localisation in tooth development to confirm that this
protein is a component of cilia on dental cells. Tg737 was expressed
Fig. 2. Cilia, and Tg737 expression and localisation in tooth germs. (A-C) Acetylated α-tubulin-positive cells (immunohistochemistry) are found
in both tooth epithelium and mesenchyme of wild type (A,B). There are no significant differences between wild-type and Tg737orpk mice, or
between first molar (m1) and supernumerary tooth (sn) in Tg737orpk mice (C). (D,E) γ-Tubulin-positive cells are observed in both tooth epithelium
and mesenchyme of wild-type (D) and Tg737orpk mice (E). (F-I) TEM analysis of molar teeth show that cilia were found in both epithelium (G) and
mesenchyme (F) of molar tooth germs in wild-type (F,G) and in molar tooth epithelium (H) and mesenchyme (I) in Tg737orpk (arrows). (J,K) β-Gal
expression in molar of Tg737Δ2-3β-gal show that Tg737 was expressed in both epithelium and mesenchyme. (L,M) Immunohistochemistry sections
also show that polaris protein is localised in both dental epithelium (DE) and dental mesenchyme (DM). Frontal sections (A,B,D-J,L,M) and sagittal
sections (C,K) of lower molars at E10.5 (A), E13.5 (B,D-J,L), E14.5 (C), E16.5 (M) and E18.5 (K) of wild-type littermates (A,B,D,F,G,J-M) and
Tg737orpk mutants (C,E,H,I). Dots represent boundaries between epithelium and mesenchyme (A,M). Tooth epithelium is outlined in red (B-E) or
green (L). Nuclei are shown in blue by DAPI (A’-E’,L,M).
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Development 136 (6)
throughout the dental epithelium and in the underlying
mesenchyme, as determined by expression of β-gal in embryos
heterozygous for the Tg737Δ2-3β-gal allele (Fig. 2J,K). In agreement
with the β-gal expression pattern, immunolocalisation of polaris
showed cilia were present on both tooth epithelial and underlying
dental mesenchyme cells (Fig. 2L,K).
Tooth phenotype in Gas1 mutant mice
In order to confirm that ectopic Shh signalling in the diastema was
the direct cause of supernumerary tooth formation, we examined
the dentition of mice with a targeted disruption in the Gas1 gene,
which we has previously identified as an inhibitor of Shh in
diastema mesenchyme. Gas1 is expressed in diastema
mesenchyme and non-dental mesenchyme, but not in tooth germs
(Fig. 4G,H) (Cobourne et al., 2004). Supernumerary teeth in the
diastema region of both the maxilla and mandible were observed
with 100% penetrance in Gas1 mutants (Fig. 4A-F). These teeth,
positioned mesial to the first molars, were identical to those
observed in Tg737orpk mice. The presence of these teeth was
associated with ectopic Shh signalling activity within the
diastema region, demonstrated by analysis of Ptch1, Gli1 and Shh
expression, and Shh protein localization, as observed in the
Tg737orpk mutants (Fig. 4I-U). Significantly, we also found that
Gas1 expression was downregulated in diastema mesenchyme of
Tg737orpk mutant embryos (Fig. 1I-J⬘). Ectopic Shh activity in the
Fig. 3. Molar teeth of K5-Cre/polarisflox/flox mice and Wnt1Cre/polarisflox/flox mice. (A,B) There are no supernumerary teeth in
K5Cre (A) and K5-Cre/polarisflox/flox (B) mice. (C) Sagittal sections
showing first molar (m1) and second molar (m2) in mandible.
(D) Supernumerary teeth (sn) were observed in Wnt1-Cre/polarisflox/flox
mice. (E-F⬘) Radioactive in situ hybridisation on sagittal sections
showing Gli1 (E,F) and Shh (E’,F’) expression in mandibles of embryo
heads. (E,F) Yellow circles represent positions of Shh expression in
adjacent sections (blue arrowheads in E’,F’). Gli1 expression (E,F) was
expanded into the diastema of Wnt1-Cre/Polarisflox/flox mice (green
arrowheads). Images show lower mandibles of E11.5 (E-F’), newborn
(C,D) and adult (A,B).
diastema mesenchyme was thus observed in two different
mutants, both resulting in ectopic tooth formation mesial to the
first molars. In Gas1 mutants, the effect on Shh signalling can be
attributed to a direct consequence of loss of an antagonist,
whereas Tg737orpk may be indirect following reduction in polaris
that results in loss of Gas1.
In the developing tooth, Shh signalling is required for initiation
and subsequent downgrowth of the dental epithelium into the
underlying mesenchyme to control development of the teeth (Sarkar
et al., 2000; Cobourne et al., 2001); in the diastema, Shh signalling
is normally specifically inhibited by Gas1 (Cobourne et al., 2004).
When Gas1 is lost either directly, or indirectly in the mesenchyme
of Tg737orpk mice, Shh signalling is increased. As a result, the
vestigial tooth rudiments mesial to the first molar do not undergo
apoptosis, but survive and develop into a functional tooth. This
suggests that modulation of Shh signalling has played an important
part in the evolution of dental patterns by regulating tooth number.
Interestingly, in the talpid2 chick mutant, ectopic activation of Shh
signalling in the developing oral cavity correlates with the formation
of reptile-like teeth [reminiscent of avian ancestors (Harris et al.,
2006)]. The requirement for Shh signalling in the mesenchyme to
regulate events in the epithelium also identifies a mesenchyme-toepithelium interaction in this process.
DEVELOPMENT
Polaris in epithelial and mesenchymal cells of
tooth germs
As cilia are present on both epithelial and mesenchymal cells of
early tooth germs, the effect of reduction in polaris in Tg737orpk
mice could be mediated in either or both cell types. Conditional
loss-of-function of polaris in epithelial cells was investigated by
crossing polaris floxed mice with K5-Cre mice. The K5Cre/polarisflox/flox mice survived but there was no evidence of
supernumerary teeth, although they did show polydactyly (Fig.
3A,B; data not shown). To test whether polaris is required in
dental mesenchyme, we crossed polaris floxed mice with Wnt1Cre mice. Wnt1-Cre/polarisflox/flox mice died at birth and showed
severe craniofacial abnormalities (data not shown).
Supernumerary tooth germs were observed mesial to first molars
in the Wnt1-Cre/polarisflox/flox mice (Fig. 3C,D). The expansion
of Gli1 expression into the diastema was seen in the Wnt1Cre/polarisflox/flox mice as also observed in Tg737orpk mice (Fig.
3E-F⬘). This confirmed that the formation of supernumerary teeth
results from loss of polaris in dental mesenchyme. Although Shh
is only expressed in the epithelium during tooth development,
Ptch1 and other Shh pathway molecules are expressed in both
epithelial and mesenchymal cells in early tooth primordia.
Moreover, a role for Shh in dental epithelial cells is confirmed by
abnormal cell differentiation in K14-Cre/Shh and K14-Cre/Smo
conditional mutant mice; however, the teeth that develop in K5Cre/polarisflox/flox mice undergo apparently normal morphogenesis
and cytodifferentiation (Dassule et al., 2000; Gritli-Linde, 2002).
This suggests that cilia-mediated Shh activity mediated by polaris
is dispensable in dental epithelial cells, whereas Shh signalling in
early dental mesenchyme requires polaris-mediated cilia function.
Despite the expectation that cilia would be absent in tooth
mesenchymal cells in Wnt1-Cre/polarisflox/flox mice, α-tubulin
immunohistochemistry did show cilia present but in reduced
numbers (see Fig. S2 in the supplementary material). This may
represent incomplete expression of Cre or a contribution of nonneural crest cells (Chai et al., 2000).
Shh and tooth number
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Fig. 4. Molar tooth phenotypes of Gas1 mutant mice.
(A-F) Sagittal sections (A-D) and micro-CT analysis (E,F)
show that supernumerary teeth develop mesial to the first
molars (sn in C,F). Micro-CT analysis showed that
supernumerary teeth were smaller than first molars and
had fewer cusps (F). At the section level of the second
molar, the supernumerary teeth were not visible (D).
(G,H) Radioactive in situ hybridisation on sagittal sections,
showing Gas1 expression in mandibles of embryo heads
at E13.5. Gas1 was expressed in the diastema region
(arrowheads), whereas it was absent in tooth regions
(incisor in G; molar in H). (I,J) Whole-mount in situ
hybridisation showing Ptch1 expression in the mandibular
process of embryo heads at E12.5. Ptch1 expression was
expanded into the diastema region in Gas1 mutants
(arrowheads in J). (K,L) Radioactive in situ hybridisation on
sagittal sections showing Gli1 expression in mandibles of
embryo heads at E14.5. Gli1 expression was expanded
into the diastema region of Gas1 mice (arrowhead in L).
(N,P,R) Radioactive in situ hybridisation on sagittal
sections showing Shh expression in maxillae of embryo
heads at E14.5. Shh expression was found in both
supernumerary (sn in R) and the first molars in Gas1
mutants (m1 in P). (M,O,Q) Haematoxylin and Eosin
stained sections adjacent to N, P and R, respectively. Shh
expression could not be seen in the first molar at the
section level of the supernumerary tooth (R).
(S-U) Immunohistochemistry on sagittal sections showing
Shh protein localisation in mandibles of embryo heads at
E12.5. Shh localisation was found in the diastema region
of Gas1 mice (between arrowheads in U), as well as
endogenous tooth germs (arrowheads in T); it was not
observed in the diastema of wild type (between
arrowheads in S). Shh protein deposition could not be
seen in the incisors and molars at the section level of the
diastema showing Shh expression (U). m1, first molar; m2,
second molar; sn, supernumerary tooth. Lingual cusps (L1L3) and extra cusp (ec). Scale bar: 500 μm.
that Shh is the direct determinant of tooth initiation and that the roles
of FGF, BMP and other signalling pathways in tooth suppression are
mediated through Shh.
Premolar formation in the diastema
Mouse first molar tooth buds form from a fusion of four
epithelial swellings, the most mesial of which is excluded from
the first molar tooth bud and undergoes apoptosis (Peterkova et
al., 2002). We used histology and 3D reconstruction of E15.5
embryos to show that the supernumerary tooth primordia were
observed in the position of the former mesial swellings,
indicating that rather than being suppressed, these swellings
continued to develop into independent teeth (Fig. 1K-N). The
cyclin-dependent kinase inhibitor p21 provides a marker for
diastema bud apoptosis and in Tg737orpk homozygous embryos
we found that p21 expression was downregulated in diastema
DEVELOPMENT
Ectopic tooth formation mesial to the first molars has been
observed in mice with mutation of the BMP/Wnt antagonist ectodin
(Wise) and following misexpression of Eda and Edar (Mustonen et
al., 2003; Kassai et al., 2005; Tucker et al., 2004). In both these
cases, development of the molars is impaired. The formation of
diastema teeth in the context of normal molar development is
observed in Sprouty2 and Sprouty4 mutant mice, and has been
interpreted as a role for suppression of FGF signalling in suppression
of diastema tooth formation. In Sprouty4 mutants, ectopic teeth are
restricted to one quadrant with low penetrance; in Sprouty2 mutants,
92% have bilateral, 5% have unilateral and 3% have no ectopic teeth
in the mandible, whereas ectopic teeth are observed in less than 5%
of the maxilla (Klein et al., 2006) (J.-M. Courtney and P.T.S,
unpublished). We show here that ectopic Shh signalling in the
diastema produces ectopic teeth with 100% frequency in all four jaw
quadrants without affecting molar development and thus we propose
RESEARCH REPORT
epithelial buds (see Fig. S3 in the supplementary material). The
premolar-like teeth in Tg737orpk thus form from the survival of
vestigial swellings.
To verify whether the supernumerary teeth developed with a
molar-like or incisor-like programme, we performed in situ
hybridisation at E15.5 for Barx1, which is specifically expressed in
developing molars, in addition to dHAND (Hand2 – Mouse Genome
Informatics) and Islet1 (Isl1– Mouse Genome Informatics), which
are expressed in incisors (Thomas et al., 1998; Tucker et al., 1998;
Mitsiadis et al., 2003). The supernumerary teeth expressed Barx1 in
a similar manner to the developing first and second molars, but did
not express Hand2 or Isl1 (see Fig. S4 in the supplementary
material). These data support the identification of these ectopic teeth
as molar-like, and confirm they develop from oral epithelium in the
molar region.
In several other mouse mutants that have been reported to develop
extra teeth, the form and position of the molars are highly abnormal,
which makes analysis of the morphology of diastema teeth difficult
(Mustonen et al., 2003; Kassai et al., 2005). Interestingly, the molar
and incisor teeth of homozygous Tg737orpk mutants showed no
major abnormalities in shape, size or position. The supernumerary
teeth were smaller than the first molars, had fewer cusps and a cusp
pattern that was consistent with a premolar-like identity. The lingual
cusps of maxillary supernumerary teeth were more prominent in
comparison with the lingual cusps of mandibular supernumerary
teeth (Fig. 1O-T). Furthermore, the supernumerary teeth in the
maxilla showed a concave-shape root, whereas those in the
mandible had a round root shape (Fig. 1U,V). These differences in
root and cusp shapes are a consistent feature of human premolars
(Ash and Nelson, 2003; Berkovitz et al., 2002).
The fact that the extra teeth that develop as a result of ectopic Shh
form a shape that is appropriate for their position indicates that
despite having lost the ability to form a premolar tooth shape over
50-100 million years ago, the mouse embryo has retained all the
genetic information necessary to make this tooth type (Meng et al.,
1994; Ji et al., 2002). This supports the concept that loss of tooth
types during evolution results from suppression of tooth initiation.
This is also consistent with the specification of tooth shape being
provided by expression of specific homeobox genes such as Dlx and
Barx1 in the mesenchyme prior to initiation (Tucker and Sharpe,
2004). These gene expression domains in the mesenchyme of the
facial primordia provide cells with positional information. This
information is used to direct cells to follow particular pathways of
hard tissue morphogenesis. In animals where tooth development is
suppressed, such as birds or the mouse diastema, this information is
still required for bone and cartilage morphogenesis. Thus, what has
been ‘lost’ in the evolution of mice is the ability to initiate and
maintain epithelial tooth buds, mesial to the first molars. When
ectopic tooth development is stimulated as a result of ectopic Shh
signalling, the tooth primordia develop from mesenchyme cells with
the information appropriate for that position.
We thank Karen Liu and Isabelle Miletich for critical reading of the manuscript,
Deepak Srivastava for dHAND plasmid, Andrew McMahon for Shh plasmid,
Ken Brady for TEM analysis, and Chris Healy for micro-CT analysis. Maisa
Seppala is a European Union Marie Curie Early Stage Fellow (grant number
MEST-CT-2004-504025). Funding for this work was provided by the Medical
Research Council, The Wellcome Trust, GACR-304/07/0223 and MSM
0021620843. Atsushi Ohazama is an Research Councils UK Fellow. Deposited
in PMC for release after 6 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/6/897/DC1
Development 136 (6)
References
Ash, M. M. and Nelson, S. J. (2003). Wheeler’s Dental Anatomy, Physiology, and
Occlusion. Philadelphia, PA: W. B. Saunders.
Berkovitz, B. K. B., Holland, G. R. and Moxham, B. J. (2002). Oral Anatomy,
Histology and Embryology. St Louis, MO: Mosby.
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are
coexpressed at many diverse sites of cell-cell interaction in the mouse embryo.
Dev. Biol. 172, 126-138.
Caspary, T., Larkins, C. E. and Anderson, K. V. (2007). The graded response to
Sonic Hedgehog depends on cilia architecture. Dev. Cell 12, 767-778.
Chai, Y., Jiang, X., Ito, Y., Bringas, P., Jr, Han, J., Rowitch, D. H., Soriano, P.,
McMahon, A. P. and Sucov, H. M. (2000). Fate of the mammalian cranial
neural crest during tooth and mandibular morphogenesis. Development 127,
1671-1679.
Cobourne, M. T., Hardcastle, Z. and Sharpe, P. T. (2001). Sonic hedgehog
regulates epithelial proliferation and cell survival in the developing tooth germ. J.
Dent. Res. 80, 1974-1979.
Cobourne, M. T., Miletich, I. and Sharpe, P. T. (2004). Restriction of sonic
hedgehog signalling during early tooth development. Development 131, 28752885.
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.
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.
Gritli-Linde, A., Lewis, P., McMahon, A. P. and Linde, A. (2001). The
whereabouts of a morphogen: direct evidence for short- and graded long-range
activity of hedgehog signaling peptides. Dev. Biol. 236, 364-386.
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.
Hardcastle, Z., Mo, R., Hui, C. C. and Sharpe, P. T. (1998). The Shh signalling
pathway in tooth development: defects in Gli2 and Gli3 mutants. Development
125, 2803-2811.
Harris, M. P., Hasso, S. M., Ferguson, M. W. and Fallon, J. F. (2006). The
development of archosaurian first-generation teeth in a chicken mutant. Curr.
Biol. 16, 371-377.
Haycraft, C. J., Banizs, B., Aydin-Son, Y., Zhang, Q., Michaud, E. J. and
Yoder, B. K. (2005). Gli2 and Gli3 localize to cilia and require the intraflagellar
transport protein polaris for processing and function. PLoS Genet. 1, e53.
Haycraft, C. J., Zhang, Q., Song, B., Jackson, W. S., Detloff, P. J., Serra, R. and
Yoder, B. K. (2007). Intraflagellar transport is essential for endochondral bone
formation. Development 134, 307-316.
Huangfu, D. and Anderson, K. V. (2005). Cilia and Hedgehog responsiveness in
the mouse. Proc. Natl. Acad. Sci. USA 102, 11325-11330.
Jeong, J., Mao, J., Tenzen, T., Kottmann, A. H. and McMahon, A. P. (2004).
Hedgehog signaling in the neural crest cells regulates the patterning and growth
of facial primordia. Genes Dev. 18, 937-951.
Ji, Q., Luo, Z. X., Yuan, C. X., Wible, J. R., Zhang, J. P. and Georgi, J. A. (2002).
The earliest known eutherian mammal. Nature 416, 816-822.
Kassai, Y., Munne, P., Hotta, Y., Penttila, E., Kavanagh, K., Ohbayashi, N.,
Takada, S., Thesleff, I., Jernvall, J. and Itoh, N. (2005). Regulation of
mammalian tooth cusp patterning by ectodin. Science 309, 2067-2070.
Klein, O. D., Minowada, G., Peterkova, R., Kangas, A., Yu, B. D., Lesot, H.,
Peterka, M., Jernvall, J. and Martin, G. R. (2006). Sprouty genes control
diastema tooth development via bidirectional antagonism of epithelialmesenchymal FGF signaling. Dev. Cell 11, 181-190.
Lee, C. S., May, N. R. and Fan, C. M. (2001). Transdifferentiation of the ventral
retinal pigmented epithelium to neural retina in the growth arrest specific gene
1 mutant. Dev. Biol. 236, 17-29.
Lesot, H., Vonesch, J. L., Peterka, M., Tureckova, J., Peterkova, R. and Ruch,
J. V. (1996). Mouse molar morphogenesis revisited by three-dimensional
reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell
staged first and second upper molar teeth. Int. J. Dev. Biol. 40, 1017-1031.
Litingtung, Y., Dahn, R. D., Li, Y., Fallon, J. F. and Chiang, C. (2002). Shh and
Gli3 are dispensable for limb skeleton formation but regulate digit number and
identity. Nature 418, 979-983.
Liu, A., Wang, B. and Niswander, L. A. (2005). Mouse intraflagellar transport
proteins regulate both the activator and repressor functions of Gli transcription
factors. Development 132, 3103-3111.
Martinelli, D. C. and Fan, C. M. (2007). Gas1 extends the range of Hedgehog
action by facilitating its signaling. Genes Dev. 21, 1231-1243.
May, S. R., Ashique, A. M., Karlen, M., Wang, B., Shen, Y., Zarbalis, K.,
Reiter, J., Ericson, J. and Peterson, A. S. (2005). Loss of the retrograde motor
for IFT disrupts localization of Smo to cilia and prevents the expression of both
activator and repressor functions of Gli. Dev. Biol. 287, 378-389.
Meng, J., Wyss, A. R., Dawson, M. R. and Zhai, R. (1994). Primitive fossil
rodent from Inner Mongolia and its implications for mammalian phylogeny.
Nature 370, 134-136.
DEVELOPMENT
902
Michaud, E. J. and Yoder, B. K. (2006). The primary cilium in cell signaling and
cancer. Cancer Res. 66, 6463-6467.
Mitsiadis, T. A., Angeli, I., James, C., Lendahl, U. and Sharpe, P. T. (2003). Role
of Islet1 in the patterning of murine dentition. Development 130, 4451-4460.
Moyer, J. H., Lee-Tischler, M. J., Kwon, H. Y., Schrick, J. J., Avner, E. D.,
Sweeney, W. E., Godfrey, V. L., Cacheiro, N. L., Wilkinson, J. E. and
Woychik, R. P. (1994). Candidate gene associated with a mutation causing
recessive polycystic kidney disease in mice. Science 264, 1329-1333.
Murcia, N. S., Richards, W. G., Yoder, B. K., Mucenski, M. L., Dunlap, J. R.
and Woychik, R. P. (2000). The Oak Ridge Polycystic Kidney (orpk) disease gene
is required for left-right axis determination. Development 127, 2347-2355.
Mustonen, T., Pispa, J., Mikkola, M. L., Pummila, M., Kangas, A. T.,
Pakkasjarvi, L., Jaatinen, R. and Thesleff, I. (2003). Stimulation of
ectodermal organ development by Ectodysplasin-A1. Dev. Biol. 259, 123-136.
Ohazama, A., Johnson, E. B., Ota, M. S., Choi, H. J., Porntaveetus, T.,
Oommen, S., Itoh, N., Eto, K., Gritli-Linde, A., Herz, J. et al. (2008). Lrp4
modulates extracellular integration of cell signaling pathways in development.
PLos One 3, e4092.
Pan, J., Wang, Q. and Snell, W. J. (2005). Cilium-generated signaling and ciliarelated disorders. Lab. Invest. 85, 452-463.
Peterkova, R., Peterka, M., Viriot, L. and Lesot, H. (2002). Development of the
vestigial tooth primordia as part of mouse odontogenesis. Connect Tissue Res.
43, 120-128.
Peterkova, R., Peterka, M. and Lesot, H. (2003). The developing mouse
dentition: a new tool for apoptosis study. Ann. NY Acad. Sci. 1010, 453-466.
Pownall, M. E., Tucker, A. S., Slack, J. M. and Isaacs, H. V. (1996). eFGF, Xcad3
and Hox genes form a molecular pathway that establishes the anteroposterior
axis in Xenopus. Development 122, 3881-3892.
Ramirez, A., Page, A., Gandarillas, A., Zanet, J., Pibre, S., Vidal, M., Tusell, L.,
Genesca, A., Whitaker, D. A., Melton, D. W. et al. (2004). A keratin K5Cre
transgenic line appropriate for tissue-specific or generalized Cre-mediated
recombination. Genesis 39, 52-57.
RESEARCH REPORT
903
Rohatgi, R., Milenkovic, L. and Scott, M. P. (2007). Patched1 regulates
hedgehog signaling at the primary cilium. Science 317, 372-376.
Rosenbaum, J. L. and Witman, G. B. (2002). Intraflagellar transport. Nat. Rev.
Mol. Cell. Biol. 3, 813-825.
Sarkar, L., Cobourne, M. T., Naylor, S., Smalley, M., Dale, T. and Sharpe, P. T.
(2000). Wnt/Shh interactions regulate ectodermal boundary formation during
mammalian tooth development. Proc. Natl. Acad. Sci. USA 97, 4520-4524.
Scholey, J. M. and Anderson, K. V. (2006). Intraflagellar transport and ciliumbased signaling. Cell 125, 439-442.
Singla, V. and Reiter, J. F. (2006). The primary cilium as the cell’s antenna:
signaling at a sensory organelle. Science 313, 629-633.
Taulman, P. D., Haycraft, C. J., Balkovetz, D. F. and Yoder, B. K. (2001). Polaris,
a protein involved in left-right axis patterning, localizes to basal bodies and cilia.
Mol. Biol. Cell 12, 589-599.
Thomas, T., Kurihara, H., Yamagishi, H., Kurihara, Y., Yazaki, Y., Olson, E. N.
and Srivastava, D. (1998). A signaling cascade involving endothelin-1, dHAND
and msx1 regulates development of neural-crest-derived branchial arch
mesenchyme. Development 125, 3005-3014.
Tucker, A. and Sharpe, P. (2004). The cutting-edge of mammalian development:
how the embryo makes teeth. Nat. Rev. Genet. 5, 499-508.
Tucker, A. S., Matthews, K. L. and Sharpe, P. T. (1998). Transformation of tooth
type induced by inhibition of BMP signaling. Science 282, 1136-1138.
Tucker, A. S., Headon, D. J., Courtney, J. M., Overbeek, P. and Sharpe, P. T.
(2004). The activation level of the TNF family receptor, Edar, determines cusp
number and tooth number during tooth development. Dev. Biol. 268, 185-194.
Tureckova, J., Lesot, H., Vonesch, J. L., Peterka, M., Peterkova, R. and Ruch,
J. V. (1996). Apoptosis is involved in the disappearance of the diastemal dental
primordia in mouse embryo. Int. J. Dev. Biol. 40, 483-489.
Zhang, Q., Murcia, N. S., Chittenden, L. R., Richards, W. G., Michaud, E. J.,
Woychik, R. P. and Yoder, B. K. (2003). Loss of the Tg737 protein results in
skeletal patterning defects. Dev. Dyn. 227, 78-90.
DEVELOPMENT
Shh and tooth number