DEV01252 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of Dach1 and Bmp4 in the chick limb bud. In the limb bud, Dach1 is first detected in the anterior mesenchyme at stage 17, and its expression becomes intense at stage 19 (A). It is also expressed in the neural tube and the lateral part of the somites (A). Dach1 is expressed in both the AER and the mesenchyme at stage 20, leaving the posterior part Dach1 negative (B). At stage 23, expression of Dach1 expands to the entire limb mesenchyme and the AER (C). Bmp4 is expressed in the anterior mesenchyme, the AER and the mesendermal cells beneath the AER at stage 20 to stage 23 (D-F). (G) Dach1 is not expressed in the ectoderm in the prospective AER region, but its expression is evident in the mesenchyme of the limb field at stage 13 (yellow arrowhead). A red arrowhead indicates the ventral limit of the limb field. The expression of Dach1 is confined to the prospective limb field. (H) Expression of Bmp4 is evident in the Dach1-positive limb field (yellow arrowhead) and the lateral plate mesoderm. (I) Msx2 is weakly expressed in the limb field (yellow arrowhead) and the lateral plate mesoderm beyond the ventral limit of the limb field (red arrowhead). (J) At stage 15, expression of Dach1 becomes faint transiently (red arrowheads). (K,L) Expression of Bmp4 and Msx2 becomes robust at this stage. (M) Expression of Wnt7a starts at the prospective dorsal ectoderm. (N) Dach1 expression is first detected in the AER at stage 19 (red arrowhead) and the underlying mesenchyme. (O) At this stage, Bmp-4 expression is detected in the mesenchyme and the AER. (P) Msx2 is restricted to the ventral part of the limb mesenchyme and the AER. At this stage, Dach1 expression overlaps with that of Bmp4 in both the AER and the mesenchyme (N,O). (Q) Expression of the dorsal marker Wnt7a becomes evident in the dorsal ectoderm.

• Supplemental Figure 2 -

  Fig. S2. When DD2 was misexpressed in the ectoderm, disruption of Fgf8 expression was observed at stages 22 (A) and 23 (B). (C) In the limb bud, expression of Msx2 in the AER was also disrupted with repression of its mesenchymal expression. When VP=Dach1 was misexpressed in the limb mesoderm, the AER was deformed with irregular and faint expression of Fgf8 at stage 23. (E) One week after electroporation, digit I was not formed at stage 37. (F) Expression of Meis2 was evident in the VP=Dach1-misexpressed limb bud with strong induction in the anterior region (red arrowheads). (G) When VP=Dach1 was misexpressed both in the ectoderm and the mesoderm, distal expansion of Meis2 expression was obtained. The distal limit of Meis2 expression was shifted distally with robust induction in the anterior mesenchyme (red arrowheads). (H) Higher magnification of G. Striped induction of Meis2 in the ectoderm is evident. (I) Implantation of BMP4-soaked beads (0.01 mg/ml) in the anterior part of the limb bud repressed Meis2 expression. (J) When BMP4-soaked beads (0.05 mg/ml) were implanted in the proximal part, repression of Meis2 was observed in an area surrounding the implanted bead. (K) When TGFb1-soaked beads (0.1 mg/ml) were implanted in the middle of the limb bud, Meis2 was also repressed.