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First published online May 8, 2009
doi: 10.1242/10.1242/dev.033803

Development 136, 1939-1949 (2009)
Published by The Company of Biologists 2009
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Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood

Xiu-Ping Wang1,*, Daniel J. O'Connell1,*, Jennifer J. Lund1, Irfan Saadi1, Mari Kuraguchi1, Annick Turbe-Doan1, Resy Cavallesco1, Hyunsoo Kim2, Peter J. Park3, Hidemitsu Harada4, Raju Kucherlapati1,5 and Richard L. Maas1,{dagger}

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.


Figure 1
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  		Fig. 1. Supernumerary teeth form in multiple regions of the jaw. (A,B) β-Gal immunostaining in K14-Cre8Brn;R26R mice. (C,D) X-ray microtomograph (micro-CT) of P13 wild-type (C) and K14-Cre8Brn;Apccko/cko (D) mice. (E) Supernumerary tooth (asterisk) next to principal molars after surface soft tissue removal in a K14-Cre8Brn;Apccko/cko lower mandible. (F-K) Supernumerary teeth (asterisks) form from lingual (F) and labial (G) sides of principal teeth, directly from oral epithelium (H), continuously from principal teeth and pre-existing supernumerary teeth (I), and from vestibular lamina (VL in J). (K) Higher magnification of J. (L) Some supernumerary teeth start to form roots. (M) Higher magnification of L. Red arrows indicate Hertwig's epithelial root sheath (HERS) in developing root. (N,O) Histology (N) and immunostaining for Cd31 (O) reveal vascularization within dental pulp of supernumerary teeth (asterisk). Arrow in O indicates non-specific background in enamel matrix. (P) Immunostaining for neurofilament reveals innervation in dental pulp and within dental tubules of supernumerary teeth. (Q) Higher magnification of P. D, dentin; E, enamel; Lab, labial; Lin, lingual; UM, upper molar; UI, upper incisor; LI, lower incisor; od, odontoblasts. Scale bars: 100 µm in A,B,L,N,O; 200 µm in F-I,K; 500 µm in J; 50 µm in M; 10 µm in P; 5 µm in Q; 1 mm in C,D.

 

Figure 2
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  		Fig. 2. Supernumerary and endogenous tooth development employ similar genes, but differ in Msx1 dependence. (A) In wild-type mice, Shh is expressed in differentiating ameloblasts along the labial side of lower incisor (LI). Red dashed lines mark the boundary between epithelium and mesenchyme. (B,C) Identical sections of K14-Cre8Brn;Apccko/cko mice reveal ectopic Shh expression in the vestibular lamina (VL; arrow), the lingual side of the lower incisor (arrow), and in a supernumerary tooth budding from differentiating ameloblasts on the labial side of the lower incisor that expresses epithelial Shh and mesenchymal activin βA (arrowhead). (D) Ectopic Fgf8 expression in K14-Cre8Brn;Apccko/cko mice (arrow). (E-H) Supernumerary teeth expressing Shh and amelogenin develop from oral epithelium and vestibular lamina (arrows). (I-X) Histology (top row) and RNA in situ hybridization for Shh, Bmp4 or Fgf3 on adjacent parasagittal sections of E18.5 mandibles in Apc and Msx1 loss-of-function genotype combinations. Scale bars: 100 µm in A-D,I-X; 200 µm in E-H.

 

Figure 3
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  		Fig. 3. Apc-deficient cells induce surrounding wild-type epithelial cells to adopt an odontogenic fate. K14-Cre8Brn;Apccko/cko;R26R mouse embryos at E16.5. (A) Supernumerary tooth bud that developed directly from the oral epithelium (a) and exhibits mesenchymal condensation (arrow in b, a higher magnification view of the boxed area from a). Only a subset of dental epithelial cells are β-Gal positive (c,d). (B) Double fluorescent label immunostaining for β-Gal and β-catenin. (C) Double label experiment using anti-β-catenin antibody and Shh mRNA riboprobe. (D) Double immunostaining for β-catenin and Ki67. White solid and dashed lines mark the boundaries between epithelium and mesenchyme. Scale bars: 50 µm in Aa-d,Ca-d; 25 µm in Ba-h,Ce-h,Da-d; 10 µm in De-l.

 

Figure 4
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  		Fig. 4. Supernumerary teeth form in adult mice following epithelial deletion of Apc or activation of β-catenin. (A) Control mice injected with 4-hydroxytamoxifen (4-OHT) at 3 weeks. (B-G) K14-CreERTM;Apccko/cko mice injected with 4-OHT at 3 weeks (B), 8 weeks (C), 6 months (D) and 10 months (E-G) of age. Some supernumerary teeth are multicuspid (yellow asterisks) with well-differentiated ameloblasts and odontoblasts (enamel was lost owing to decalcification). (G) Higher magnification of boxed region from F. (H-J) Constitutive activation of β-catenin in adult oral epithelium at 6 months of age also results in supernumerary teeth. (K) Control mice injected with 4-OHT at P5. (L-Q) K14-CreERTM;Apccko/cko mice injected with 4-OHT at P5. (R-T) K14-CreERTM;Ctnnb1(ex3)fl/+ mice injected with 4-OHT at P5. D, dentin; E, enamel; Lab, labial; Lin, lingual. Scale bars: 100 µm in G,S,T; 200 µm in A-E,K,N-R; 400 µm in F,L,M; 500 µm in H-J.

 

Figure 5
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  		Fig. 5. Fgf8 is a direct downstream target of Wnt/β-catenin signaling. (A) LiCl increased the levels of β-catenin and upregulated Fgf8 transcripts ~1.5-2.5-fold in LS8 cells. (B) Transfection of constitutively active β-catenin (S33Y) plasmid into LS8 cells activated Wnt signaling (Top/Fop) and upregulated Fgf8 ~2-fold, as compared with control plasmid (pBabe). (C) ChIP analysis of Lef1 binding site occupation in Fgf8. (Above) Location of Fgf8 primers. (Below) HAT-7 cells were treated with 50 mM LiCl for 18 hours and PCR comparison of DNA immunoprecipitated with antibodies against Lef1 or β-catenin showed enrichment of the amplicon representing the genomic region surrounding the Lef1 binding site in Fgf8 intron 3. (D) The conserved 1 kb sequence surrounding the Lef1 binding site in Fgf8 intron 3 was inserted into the pGL3-promoter luciferase reporter vector and co-transfected with S33Y or pBabe plasmid into LS8 cells. A 3 bp deletion in the Lef1 binding site core abolished luciferase activation by the β-catenin S33Y plasmid. *P<0.05; **P<0.01; error bars, s.e.m.

 

Figure 6
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  		Fig. 6. Wnt/β-catenin signaling is necessary for Fgf8 expression in oral epithelium. (A) HAT-7 cells transfected with two different Ctnnb1 RNAi vectors (RNAi 56, RNAi 58) exhibit markedly reduced β-catenin protein levels (left), and Fgf8 transcripts are downregulated to 40-60% of those of control scrambled RNAi (right). **P<0.001; error bars, s.e.m. (B) E9.5 mouse mandibular explants cultured with BSA- or Dkk1-soaked beads (blue).

 

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