DEV02325 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of DNp63 in embryonic ectoderm. (A) Immuno-histochemical analysis using a DNp63-specific antibody revealed DNp63 protein expression throughout skin epithelium at E12. (B) Intense expression was evident in the basal layer of the epidermis and in developing hair follicles at E17. (C,D) DNp63 was also expressed in oral and dental epithelium at E13, as well as in P1 incisor epithelium.

• Supplemental Figure 2 -

  Fig. S2. Expression of Fgf8, Fgf9, Bmp4, Msx1, and activinbA in wild type and p63 mutants. (A,B) Intense Fgf8 expression was seen in the thickened wild-type and mutant epithelium at E12. (C,D) At E10, Fgf9 transcripts were expressed similarly in the oral epithelium in wild-type and p63 mutant embryos. (E,F) Bmp4 is expressed in the mesenchyme after the initiation of tooth development, and is more intense on the buccal side of the molar tooth germ. Bmp4 transcripts were localized in a similar pattern in p63^-/- embryos. (G,H) Msx1 becomes concentrated to the dental mesenchyme during the initiation of tooth development and continues during subsequent morphogenesis (E11-E12). Msx1 transcripts were reduced in p63 mutant mesenchyme underlying dental lamina. (I,J) Activin was expressed with a similar pattern and intensity in wild type and p63^-/- dental mesenchyme. (K-N) Bmp4 and ActivinbA were detected in similar patterns in the skin mesenchyme in wild-type and p63 null embryos. Scale bar: 200 mm.

• Supplemental Figure 3 -

  Fig. S3. Expression of WNT pathway genes, ectodysplasin, Tnfrsf19 and pvrl1 in wild-type and p63 mutant epithelium. (A-D) Wnt6 and Wnt10b expression was similar in wild-type and p63 mutant oral ectoderm at E12. The intense expression of Wnt10b in the dental placode was, however, not seen in the mutant. (E,F) Lef1 transcripts were expressed similarly in oral epithelium in wild type and p63 mutant, but in the mutant mesenchyme they were downregulated (E12, arrows). (G,H) At E12, Wnt3 was expressed similarly in wild-type and mutant surface ectoderm. (I-K) Ectodysplasin (Eda; I,J) and Tnfrsf19  (K,L) were expressed similarly in wild-type and p63 mutant epithelium. (M-P) pvrl1 was expressed in the epidermis, as well as in dental lamina, at E11 in wild-type embryos. At E12, expression was confined to the suprabasal cell layer. There was no difference in the expression of pvrl1 in p63 deficient oral, dental or skin epithelium when compared with wild-type embryos, indicating that the defects in ectodermally derived organs that are affected in human ectodermal dysplasia syndromes caused by mutations in either p63 or pvrl1 may have different pathogenetic mechanisms. Scale bar: 500 mm for A-D,K,L; 200 mm for E-J,M-P.

• Supplemental Figure 4 -

  Fig. S4. p63 binds to specific sequences in the Notch1 and Bmp7 genes in vivo. (A) Schematic representation of part of the Bmp7 and Notch1 genes (not in scale), showing locations of putative p63-binding sites and the control site tested in ChIP assay. The p63-binding site consists of two or more copies of a 10-bp conserved motif separated by 0 to 13 bp. Comparison of the p63-responsive elements in Bmp7 and Notch1 genes with the consensus sequence is shown. R, purine; Y, pyrimidine; W, adenosine or thymidine. (B) Freshly isolated E13 epidermal cells, i.e. cells that express DNp63, were cross-linked with formaldehyde, and DNA fragments immunoprecipitated with a p63-specific antibody (4A4) or with an isotype-specific control antibody were PCR amplified with primers flanking the putative p63-binding sites or control regions lacking the consensus site. After immunoprecipitation, part of the sample was analyzed by western blotting with an anti-DNp63 antibody, verifying the presence of DNp63 in the immune complexes (data not shown). The known binding site in the p21 promoter was used as a positive control. Input indicates PCR amplification of a portion of the sonicated chromatin before immunoprecipitation. No amplification of target sites was detected in the absence of an antibody or when an isotype-specific control antibody was used.