Fig. 3. Additional limb formation induced by Wnt2b, Tbx5 and Tbx4 in the flank. (A) When Wnt2b-expressing cells were implanted in the flank, a mosaic limb was formed (red arrowheads). (A') Careful examination of this additional limb revealed its mosaic structures with wing-like morphology on the anterior side (W') and leg-like structure on the posterior side (L'). (B) At earlier stages, the Wnt2b-induced additional limb (green arrow) expressed Tbx5 (red arrow) and Tbx4 (blue arrow) in its anterior and posterior sides, respectively. A white circle indicates the position of cell implantation. (C to C'') In the Wnt2b-induced limb buds, leg-specific Hoxb9 (C) and Hoxc9 (C') were expressed in the posterior region, whereas wing-specific Hoxd9 (C'') in the anterior. Arrows correspond to the positions indicated in B. (D) Schematic representation of the Wnt- or Fgf-induced additional limb and its mosaic structures. (E) When CAGGS-Tbx5-EGFP plasmid was electroporated in the flank, wing-like limb was formed (red arrowhead), covered extensively by feather buds. (F) About 16 hours after electroporation, Wnt2b expression was induced (arrowhead) in the electroporated area visualized by the GFP (G). Expression of Fgf10 (H) was observed in the induced limb bud (red arrowhead). L, normal leg bud. (I) About 60 hours after electroporation, an additional limb (red arrowhead) was formed at the caudal side of the normal wing (W). This additional limb bud expressed Fgf8 in the AER (red arrowhead). (J) In the Tbx5-induced limb, expression of leg-specific Hoxb9 was observed weakly only in the posterior margin, excluded form the anterior part of limb. (K) Expression of another leg-specific Hoxc9 was very faint. (L) By contrast, wing-specific Hoxd9 was expressed. Red brackets indicate additional limbs. (M) When the CAGGS-Tbx4-EGFP plasmid was electroporated in the flank, a leg-like limb was formed (red arrowheads). Black arrowheads indicate a boundary between feather and scale domains. When expression of several marker genes was examined, Wnt8c (N, red arrowheads) was induced. Electroporation was monitored by the GFP as shown in O. (P) Likewise, expression of Fgf10 was induced in the additional limb bud (red arrowhead). (Q) In the Tbx4-induced limb bud, expression of Fgf8 (red arrowhead) was observed in the AER. In the additional limb buds, leg-specific Hoxb9 (R) and Hoxc9 (S) were expressed. (T) By contrast, expression of wing-specific Hoxd9 was suppressed, although faint expression was observed at the anterior margin. Red brackets indicate additional limbs.