897RESEARCH REPORT

INTRODUCTIONTooth 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

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

Primary cilia regulate Shh activity in the control of molartooth numberAtsushi 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 acilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of theembryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype isspecific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on toothdevelopment. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed micemutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth andaccompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shhactivity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size andshape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.

KEY WORDS: Intraflagellar transport, Cilia, Shh, Supernumerary tooth, Tooth development, Tooth number, Orpk, Tg737, Gas1, Epithelium,Mesenchyme, Mouse

Development 136, 897-903 (2009) doi:10.1242/dev.027979

1Department of Craniofacial Development, Dental Institute, King’s College London,London SE1 9RT, UK. 2Department of Cell Biology, University of Alabama atBirmingham, Birmingham, AL 35294-0005, USA. 3Department of CraniofacialDevelopment and Orthodontics, Dental Institute, King’s College London, UK.4Department of Embryology, Carnegie Institution of Washington 3520 San MartinDrive, Baltimore, MD 21218, USA. 5Department of Teratology, Institute ofExperimental Medicine, Academy of Sciences of the CR, Prague, Czech Republic.6INSERM UMR977, Dental School, Strasbourg University, Strasbourg, France.

*Author for correspondence (e-mail: paul.sharpe@kcl.ac.uk)

Accepted 21 January 2009 DEVELO

PMENT

898

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.

MATERIALS AND METHODSProduction and analysis of miceTg737orpk 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).

In situ hybridisationRadioactive 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 analysisHeads 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) stainingEmbryo heads were processed as previously described (Taulman et al.,

2001).

Epithelial three-dimension reconstructionEpithelial three-dimension reconstruction of mandible dental and adjacent

oral epithelium were performed as previously described (Lesot et al., 1996).

Transmission electron microscopy (TEM) analysisHeads 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.

ImmunohistochemistrySections 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 DISCUSSIONShh activity in the diastemaPrimary 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

RESEARCH REPORT Development 136 (6)

Fig. 1. Lower molar tooth phenotypes and Shh signalling in Tg737orpk homozygous mutant mice. (A-D) Whole teeth (A,B) and sagittalsections (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 themandible of Tg737orpk mutants with Ptch1-lacZ allele at E13.5. Ptch1-lacZ staining was expanded into the diastema region (arrowhead). Arrowsindicate 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 inmandibles of embryo heads at E12.5. (G,H,I,J) Yellow circles represent positions of Shh expression in adjacent sections (blue arrowheads inG’,H’,I’,J’). Gli1 expression (H) was expanded into the diastema and Gas1 (J) was downregulated in the diastema of Tg737orpk mutants (greenarrowheads). (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 comparisonwith that of mandibular supernumerary tooth (red line in S). (U,V) Horizontal micro-CT sections of Tg737orpk show that maxillary supernumeraryteeth 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. D

EVELO

PMENT

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). Gli1expression 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

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 developmentTo 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 wild-

type 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

899RESEARCH REPORTShh and tooth number

Fig. 2. Cilia, and Tg737 expression and localisation in tooth germs. (A-C) Acetylated α-tubulin-positive cells (immunohistochemistry) are foundin both tooth epithelium and mesenchyme of wild type (A,B). There are no significant differences between wild-type and Tg737orpk mice, orbetween first molar (m1) and supernumerary tooth (sn) in Tg737orpk mice (C). (D,E) γ-Tubulin-positive cells are observed in both tooth epitheliumand 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) andmesenchyme (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-Galexpression in molar of Tg737Δ2-3β-gal show that Tg737 was expressed in both epithelium and mesenchyme. (L,M) Immunohistochemistry sectionsalso show that polaris protein is localised in both dental epithelium (DE) and dental mesenchyme (DM). Frontal sections (A,B,D-J,L,M) and sagittalsections (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) andTg737orpk mutants (C,E,H,I). Dots represent boundaries between epithelium and mesenchyme (A,M). Tooth epithelium is outlined in red (B-E) orgreen (L). Nuclei are shown in blue by DAPI (A’-E’,L,M). D

EVELO

PMENT

900

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).

Polaris in epithelial and mesenchymal cells oftooth germsAs 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 K5-

Cre/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 Wnt1-

Cre 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 Wnt1-

Cre/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 Shhis 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 K5-

Cre/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 non-

neural crest cells (Chai et al., 2000).

Tooth phenotype in Gas1 mutant miceIn 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 Shhexpression, 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

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-to-

epithelium interaction in this process.

RESEARCH REPORT Development 136 (6)

Fig. 3. Molar teeth of K5-Cre/polarisflox/flox mice and Wnt1-Cre/polarisflox/flox mice. (A,B) There are no supernumerary teeth inK5Cre (A) and K5-Cre/polarisflox/flox (B) mice. (C) Sagittal sectionsshowing 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 sectionsshowing Gli1 (E,F) and Shh (E’,F’) expression in mandibles of embryoheads. (E,F) Yellow circles represent positions of Shh expression inadjacent sections (blue arrowheads in E’,F’). Gli1 expression (E,F) wasexpanded into the diastema of Wnt1-Cre/Polarisflox/flox mice (greenarrowheads). Images show lower mandibles of E11.5 (E-F’), newborn(C,D) and adult (A,B).

DEVELO

PMENT

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

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 diastemaMouse 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

901RESEARCH REPORTShh and tooth number

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 firstmolars (sn in C,F). Micro-CT analysis showed thatsupernumerary teeth were smaller than first molars andhad fewer cusps (F). At the section level of the secondmolar, the supernumerary teeth were not visible (D).(G,H) Radioactive in situ hybridisation on sagittal sections,showing Gas1 expression in mandibles of embryo headsat 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 situhybridisation showing Ptch1 expression in the mandibularprocess of embryo heads at E12.5. Ptch1 expression wasexpanded into the diastema region in Gas1 mutants(arrowheads in J). (K,L) Radioactive in situ hybridisation onsagittal sections showing Gli1 expression in mandibles ofembryo heads at E14.5. Gli1 expression was expandedinto the diastema region of Gas1 mice (arrowhead in L).(N,P,R) Radioactive in situ hybridisation on sagittalsections showing Shh expression in maxillae of embryoheads at E14.5. Shh expression was found in bothsupernumerary (sn in R) and the first molars in Gas1mutants (m1 in P). (M,O,Q) Haematoxylin and Eosinstained sections adjacent to N, P and R, respectively. Shhexpression could not be seen in the first molar at thesection level of the supernumerary tooth (R). (S-U) Immunohistochemistry on sagittal sections showingShh protein localisation in mandibles of embryo heads atE12.5. Shh localisation was found in the diastema regionof Gas1 mice (between arrowheads in U), as well asendogenous tooth germs (arrowheads in T); it was notobserved in the diastema of wild type (betweenarrowheads in S). Shh protein deposition could not beseen in the incisors and molars at the section level of thediastema showing Shh expression (U). m1, first molar; m2,second molar; sn, supernumerary tooth. Lingual cusps (L1-L3) and extra cusp (ec). Scale bar: 500μm.

DEVELO

PMENT

902

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 GenomeInformatics) 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. MaisaSeppala is a European Union Marie Curie Early Stage Fellow (grant numberMEST-CT-2004-504025). Funding for this work was provided by the MedicalResearch Council, The Wellcome Trust, GACR-304/07/0223 and MSM0021620843. Atsushi Ohazama is an Research Councils UK Fellow. Depositedin PMC for release after 6 months.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/6/897/DC1

ReferencesAsh, 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 toSonic 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 cranialneural crest during tooth and mandibular morphogenesis. Development 127,1671-1679.

Cobourne, M. T., Hardcastle, Z. and Sharpe, P. T. (2001). Sonic hedgehogregulates 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 sonichedgehog signalling during early tooth development. Development 131, 2875-2885.

Dassule, H. R. and McMahon, A. P. (1998). Analysis of epithelial-mesenchymalinteractions 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). Sonichedgehog regulates growth and morphogenesis of the tooth. Development127, 4775-4785.

Gritli-Linde, A., Lewis, P., McMahon, A. P. and Linde, A. (2001). Thewhereabouts of a morphogen: direct evidence for short- and graded long-rangeactivity 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 cellproliferation, growth and polarization. Development 129, 5323-5337.

Hardcastle, Z., Mo, R., Hui, C. C. and Sharpe, P. T. (1998). The Shh signallingpathway in tooth development: defects in Gli2 and Gli3 mutants. Development125, 2803-2811.

Harris, M. P., Hasso, S. M., Ferguson, M. W. and Fallon, J. F. (2006). Thedevelopment 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. andYoder, B. K. (2005). Gli2 and Gli3 localize to cilia and require the intraflagellartransport 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. andYoder, B. K. (2007). Intraflagellar transport is essential for endochondral boneformation. Development 134, 307-316.

Huangfu, D. and Anderson, K. V. (2005). Cilia and Hedgehog responsiveness inthe 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 growthof 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 ofmammalian 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 controldiastema tooth development via bidirectional antagonism of epithelial-mesenchymal FGF signaling. Dev. Cell 11, 181-190.

Lee, C. S., May, N. R. and Fan, C. M. (2001). Transdifferentiation of the ventralretinal pigmented epithelium to neural retina in the growth arrest specific gene1 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-dimensionalreconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bellstaged 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 andGli3 are dispensable for limb skeleton formation but regulate digit number andidentity. Nature 418, 979-983.

Liu, A., Wang, B. and Niswander, L. A. (2005). Mouse intraflagellar transportproteins regulate both the activator and repressor functions of Gli transcriptionfactors. Development 132, 3103-3111.

Martinelli, D. C. and Fan, C. M. (2007). Gas1 extends the range of Hedgehogaction 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 motorfor IFT disrupts localization of Smo to cilia and prevents the expression of bothactivator and repressor functions of Gli. Dev. Biol. 287, 378-389.

Meng, J., Wyss, A. R., Dawson, M. R. and Zhai, R. (1994). Primitive fossilrodent from Inner Mongolia and its implications for mammalian phylogeny.Nature 370, 134-136.

RESEARCH REPORT Development 136 (6)

DEVELO

PMENT

Michaud, E. J. and Yoder, B. K. (2006). The primary cilium in cell signaling andcancer. Cancer Res. 66, 6463-6467.

Mitsiadis, T. A., Angeli, I., James, C., Lendahl, U. and Sharpe, P. T. (2003). Roleof 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. andWoychik, R. P. (1994). Candidate gene associated with a mutation causingrecessive 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 geneis 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 ofectodermal 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). Lrp4modulates 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 cilia-related disorders. Lab. Invest. 85, 452-463.

Peterkova, R., Peterka, M., Viriot, L. and Lesot, H. (2002). Development of thevestigial tooth primordia as part of mouse odontogenesis. Connect Tissue Res.43, 120-128.

Peterkova, R., Peterka, M. and Lesot, H. (2003). The developing mousedentition: 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, Xcad3and Hox genes form a molecular pathway that establishes the anteroposterioraxis 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 K5Cretransgenic line appropriate for tissue-specific or generalized Cre-mediatedrecombination. Genesis 39, 52-57.

Rohatgi, R., Milenkovic, L. and Scott, M. P. (2007). Patched1 regulateshedgehog 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 duringmammalian tooth development. Proc. Natl. Acad. Sci. USA 97, 4520-4524.

Scholey, J. M. and Anderson, K. V. (2006). Intraflagellar transport and cilium-based 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, dHANDand msx1 regulates development of neural-crest-derived branchial archmesenchyme. 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 toothtype 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 cuspnumber 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 dentalprimordia 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 inskeletal patterning defects. Dev. Dyn. 227, 78-90.

903RESEARCH REPORTShh and tooth number

DEVELO

PMENT