DEV02546 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The elbow joint interzone failed to form in Ptch1^c−;Catnb^c/−; Col2a1-Cre mutant embryo. Consecutive sections of the developing forelimbs at 12.5 dpc were examined by Alcian Blue/Nuclear Fast Red staining and in situ hybridization with the indicated ³⁵S-labelled riboprobes. Elbow joint/interzone formation is indicated by lighter Alcian Blue staining and the differentiation of fibroblast-like cells in the joint/interzone area (arrows). Col2a1 expression was lost and Gdf5, the joint/interzone marker, was strongly expressed in the joint/interzone region (arrows). In the Ptch1^c/−;Catnb^c/−; Col2a1-Cre mutant limb there was a continuous Alcian Blue staining in the presumptive elbow joint/interzone region without differentiation of fibroblast-like cells (arrowhead). Persistent strong Col2a1 expression and loss of Gdf5 expression were also observed (arrowhead). In the Ptch1^c/−;Catnb^c/−; Col2a1-Cre mutant limb examined for Gdf5 expression, skeletal elements were outlined by dotted lines.

• Supplemental Figure 2 -

  Fig. S2. Blocking Bmp signaling by noggin treatment rescued Gdf5 expression that was lost in the fused humerus-scapula joint in the Ptch1^c/−;Catnb^c/−; Col2a1-Cre mutant limb. In situ hybridization with ³⁵S-labelled riboprobes of Gdf5 was performed on sections of the forelimbs that were isolated from 12.5 dpc embryos and cultured in vitro for 4 days. Gdf5 expression in the humerus-scapula joint is indicated by an arrow. Noggin treatment increased Gdf5 expression in the wild-type limb. In the Ptch1^c/−;Catnb^c/−; Col2a1-Cre mutant limb, Gdf5 expression was lost in the fused humerus-scapula joint (arrowhead). In the Ptch1^c/−;Catnb^c/−; Col2a1-Cre mutant limb treated with noggin, Gdf5 expression was rescued significantly in the humerus-scapula joint.