Fig. 3. (A) Schematic representation of the experimental procedure. As graft tissues for transplantations, the presumptive limb region was taken from wing level (W and blue arrows) or leg level (L and red arrows), and the lateral plate mesoderm with overlying ectoderm was separated from endoderm. Then, the donor graft was inserted in the cavity of the host and placed between the dorsal medial ectoderm and the underlying neural tube and somites at the level of the forelimb or hindlimb region. (B) An example of a wing graft in the leg-level median region. Tbx5 is expressed in the grafted limb bud (white arrowhead) as well as in the host wing bud. (C) Another example of a wing graft in the leg-level median region. Tbx4 is expressed in the host leg bud but is not detected in the grafted limb bud (arrowhead). (D) Expression of Tbx5/Tbx4 in a wing graft at the leg level, analyzed by double in situ hybridization. Tbx5 (turquoise) and Tbx4 (red) are detectable in the host wing and leg bud, respectively. Only blue staining for Tbx5 is obvious in the graft (arrowhead). (E) An example of a leg graft in the wing-level median region. Tbx5 is clearly detectable in the grafted limb bud (arrow). (F) Another example of a leg graft at the wing level. Tbx4 is detectable in the grafted limb bud. Note that Tbx4 expression in the leg graft (arrow) appears weaker than that in the host leg bud. (G) Expression of Tbx5/Tbx4 in a leg graft at the wing level. Chimeric staining with blue (for Tbx5) and red (for Tbx4) is apparent in the graft (arrows). (H,I) Tbx5/Tbx4 expression in serial sections of a leg graft at the wing level. In the area to the right of the arrowheads Tbx4 expression is much weaker than on the left of arrowheads, and the expression of Tbx5 is reversed.