DEV02471 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Expression of Wnt9a in the perichondrium and joints. (A-F) β-Galactosidase activity in Wnt9a-lacZ homozygous mice at E13.5 (A-C) and newborns (P0) (D-F). (A) Elbow region; (B) higher magnification of elbow joint; (C) digits; (D) knee; (E) foot; (F) skull. There is partial fusion in the foot between the calcaneus and cuboid tarsal elements (asterisk in E) and ectopic cartilage is present in the sagittal suture (arrows) in the skull of Wnt9a-lacZ homozygous mice above the β-gal-positive cells (F).

• Supplemental Figure 2 -

  Fig. S2. Ihh expression levels are altered in proximal but not in distal appendicular skeletal elements. (A,B) Relative expression levels of Ihh in wild-type and Wnt9a^-/- humeri at E12.5 and E13.5, showing decreased Ihh expression in the mutant. (C,D) Ihh expression in the digits of wild-type and Wnt9a^−/− limbs at E12.5 (C) and E14.5 (D), showing no significant difference between wild type and mutant.

• Supplemental Figure 3 -

  Fig. S3. Alteration of Fgf-signaling in wild-type and Wnt9a mutant limbs. (A,B) Relative expression of Fgf1, Fgf4, Fgf6, Fgf15, Fgf20 and Fgf1, in wild-type and Wnt9a^−/− humeri at E12.5 (A) and E13.5 (B). Error bars represent the standard deviation of the mean of the results (n=3 for all samples at both time points). Asterisks indicate the changes in the relative expression levels, which are significant (P<0.025). (C) Expression of Ihh in wild-type and Fgfr3ach humeri at E12.5. (D) Expression of Ihh and Col10a1 in wild-type and Fgfr3ach humeri at E13.5, showing a slight increase of the expression domains in comparison with wild type. (E) Expression of Ihh in DMSO- and SU5402-treated wild-type and Wnt9a mutant E12.5 humeri. (F) Ihh expression in DMSO- and SU5402-treated wild-type and Wnt9a mutant E13.5 humeri. (G) Col10a1 expression in DMSO- and SU5402-treated wild-type and Wnt9a mutant E13.5 humeri.

• Supplemental Figure 4 -

  Fig. S4. Early joint interzone markers are expressed in limbs lacking β-catenin activity. (A-D) In situ hybridization for Gdf5, Wnt4, Gli1 and Col2a1 on sections from wild-type and β-cat^Prx/− E11.5 forelimbs, showing that early joint interzone markers, such as Gdf5, Wnt4 and Gli1, are expressed in limbs lacking β-catenin activity in the mesenchyme.

• Supplemental Figure 5 -

  Fig. S5. Wnt9a and Wnt4 act redundantly in chondrocyte maturation and upregualation of Wnt4 in Wnt9a mutant humeri. (A) Comparison of Col10a1, Ihh and Col2a1 expression between wild-type, Wnt9a^−/− and Wnt9a^−/−; Wnt4^−/− humeri at E13.5 and E15.5; showing that loss of Wnt4 further enhances the chondrocyte maturation delay of the Wnt9a mutants. (B) Reduced expression domain of Col10a1 and Ihh in Wnt4^−/− humeri, suggesting a slight delay of chondrocyte maturation. (C) Relative expression of Wnt4 in wild-type and Wnt9a^−/− humeri at E12.5 and E13.5, showing an increase in Wnt4 expression in the mutant, which is significant at E13.5 (P<0.05; asterisk). Error bars represent the standard deviation of the mean of the results (n=3 for both time points). (D) Expression of Wnt4 in the joint region and prehypertrophic chondrocytes of wild-type humeri at E12.5 and E13.5.