DEV025064 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The expression patterns of Shh and the BMP target gene p21 are expanded in the Sostdc1 (ectodin)-deficient incisor region. (A-D) Shh-expression in wild-type and Sostdc1-deficient lower jaws at E12 (A,B) and E14 (C,D). Shh marks the potential tooth-forming regions. Arrow indicates the separate Shh expression domain, which disappears from the wild type, but not from the Sostdc1-deficient mice. (E,F) Expression of p21 in Sostdc1-deficient lower jaws indicates stronger and expanded expression domains in Sostdc1-deficient jaws compared with wild type. Scale bar: 0.5 mm in A-D; 0.25 mm in E,F.

• Supplemental Figure S2 -

  Fig. S2. Minimizing the surrounding mesenchyme from wild-type molar tooth germs rescues the development of premolar rudiment in vitro. (A-C) Molar tooth germs were dissected from the mandibles of E12 wild-type and Sostdc1-deficient embryos and cultured for 5 days in vitro. When surrounding mesenchyme was trimmed from wild-type explants before culture in four out of 20 explants, an extra tooth emerged anterior to the molar (B). This phenotype was similar to the extra tooth developing in the cultures of Sostdc1 mutant molars (C). Scale bar: 0.5 mm.

• Supplemental Figure S3 -

  Fig. S3. Fluorescent DiI-label reveals the origin of extra incisor in in vitro cultured wild-type incisor explants. DiI was injected in the in vitro cultured E13 Sostdc1-deficient (Fig. 6) and wild-type incisor explants that expressed GFP under the Shh promoter. The dye was injected at the epithelial site where the extra incisor starts to form. The dye incorporated to the forming extra incisor (asterisks), but not to the main incisor. (C) Schematic drawing summarizes the bright field picture (A) and fluorescent picture (B); green is the GFP expression and red is the injected DiI. Scale bar: 0.5 mm.

• Supplemental Figure S4 -

  Fig. S4. Inhibition of either BMP or Wnt signal activity prevents the formation of extra incisors in Sostdc1-deficient and wild-type explants. (A) Vestigial and de novo incisors (broken lines) have formed in the control culture of Sostdc1-deficient incisor after minimizing the amount of tooth surrounding mesenchyme. (E) The extra incisor has formed also to the wild-type control culture after removing most of the surrounding mesenchyme. The presence of Sostdc1 (B,F), Dkk1 (C,G) and Noggin (D,H) in the culture medium prevent the formation of extra incisors. The arrow indicates the site that should form the extra incisors. Scale bar: 0.5 mm.

• Supplemental Figure S5 -

  Fig. S5. Activation of canonical Wnt signaling in the epithelium leads to supernumerary incisor formation. (A-D) Activation of epithelial Wnt-signaling in the Catenb^Dex3k14/+ mutant leads to supernumerary incisor formation predominantly from the lingual epithelium. The incisor tooth buds were dissected from E15 embryos and cultured in vitro for several days. Asterisks indicate the supernumerary incisors. (E,F) The pattern of Sostdc1 expression in Catenb^Dex3k14/+ mouse appears to be broadly similar to that in wild-type teeth. The expression, however, seems to be wider in the epithelium in the Catenb^Dex3k14/+ mouse and weaker in dental papilla. Li, lingual cervical loop; La, labial cervical loop. Scale bar: 1 mm.