DEV033803 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Supernumerary tooth bud formation in K14-Cre^1Amc;Apc^cko/cko mice. (A) Anti-β-gal immunostaining reveals uniform Cre recombinase activity throughout the oral and dental epithelia of K14-Cre^1Amc;R26R mice. (B-Q) Parasagittal sections of mouse lower mandible. (B,C) At E14.5, β-catenin protein is dramatically upregulated throughout oral ectoderm in K14-Cre^1Amc;Apc^cko/cko mice. (D-M) Expression of the endogenous β-catenin gene (Ctnnb1), Pitx2, p21, Fgf8 and Fgf4 is also upregulated throughout the oral ectoderm. (N-Q) Fgf8 and Fgf4 are both upregulated and co-localize in some oral ectodermal cells. Scale bars: 50 µm in A; 100 µm in D-K; 200 µm in B,C,L,M; 25 µm in N-Q.

• Supplemental Figure S2 -

  Fig. S2. Multicuspid supernumerary teeth develop from proximal molar and distal incisor regions, whereas Barx1 expression is confined to the proximal molar tooth region. (A-F) K14-Cre^8Brn;Apc^cko/cko mice. (A,B) Histology reveals multicuspid (asterisk) and unicuspid (arrow) supernumerary teeth in both the distal incisor region and proximal molar region. (C,D) Barx1 expression in multicuspid supernumerary teeth in the proximal molar region. (D) Higher magnification of arrowed region from C. (E,F) Barx1 expression in unicuspid supernumerary teeth in the proximal molar region. (F) Higher magnification of arrowed region from E. (G-J) K14-Cre^1Amc;Apc^cko/cko and control mice. (G,H) Barx1 expression is excluded from the distal incisor region ectomesenchyme in both control and K14-Cre^1Amc;Apc^cko/cko mice. (I,J) Barx1 is expressed in proximal molar region dental mesenchyme. Scale bars: 200 µm in A-C,E,I,J; 50 µm in D,F,G,H.

• Supplemental Figure S3 -

  Fig. S3. Ki67 and β-catenin protein expression in the developing tooth. Dental epithelial cells within the enamel knot (encircled) of E14.5 wild-type mice are mostly Ki67 negative. (A) DAPI staining of nuclei. (B) Immunostaining for Ki67 in proliferating cell nuclei. (C) Merge of A and B. (D) β-catenin protein levels are elevated in epithelial cell nuclei within the enamel knot. Scale bars: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. Loss of β-catenin blocks supernumerary tooth formation. Parasagittal sections of lower mandibles. (A-C) E17.5 control molar teeth at the bell stage with Shh expression along the inner dental epithelium. Fgf8 is undetectable at this stage. (D-F) At E17.5, numerous supernumerary teeth budding from the oral ectoderm of K14-Cre^1Amc;Apc^cko/cko mice express Fgf8 and Shh. (G-I) At P0 in K14-Cre^1Amc;Ctnnb1^cko/cko mice, tooth development arrests at the bud stage with no Fgf8 or Shh expression. (J-O) At E17.5, deletion of endogenous β-catenin (Ctnnb1) in K14-Cre^1Amc;Apc^cko/cko mice suppresses the formation of supernumerary teeth, with rare detection of ectopic Fgf8 and Shh transcripts (arrow in M-O). Scale bars: 200 µm.

• Supplemental Figure S5 -

  Fig. S5. The number of supernumerary teeth increases with age. In K14-Cre^8Brn;Apc^cko/cko mice at P3, we detected only a few supernumerary tooth buds (yellow asterisks) beyond the cap stage. At P8, some supernumerary teeth have undergone odontogenic cell differentiation, with new buds continuously forming from pre-existing supernumerary teeth. By P13, numerous supernumerary teeth are found within the jaw. Scale bars: 500 µm.

• Supplemental Figure S6 -

  Fig. S6. LiCl increases Fgf8 expression in HAT-7 cells. The glycogen synthase kinase 3 β (GSK3β) inhibitor LiCl increases β-catenin levels and upregulates Fgf8 transcripts ∼2.5- to 5.2-fold in HAT-7 cells. *P<0.04; **P<0.01; error bars, s.e.m.

• Supplemental Table S1 - Adobe PDF