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Figure 1. Muscle-specific GFP expression in Car-GFP transgenic tadpole. A,B: Transgenic tadpoles expressing GFP under control of Cardiac promoter. In a tadpole at stage 47, GFP expression was detected only at differentiated muscle tissues in various areas, including tail, eyes, and jaw. C–H: GFP expression in the hindlimb buds of Car-GFP transgenic tadpoles. The GFP signal could be observed throughout the early limb bud at stage 48 (C), stage 49 (D), and stage 50 (E). At later stage 51 (F), stage 52 (G), and stage 55 (H), the fluorescent signal of GFP increases in the proximal region. I,J: Serial sections prepared from a stage-55 hindlimb bud of Car-GFP transgenic tadpole. Immunostaining for GFP (I) and with anti-MyHC antibody (J).
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Figure 3. Transplantation of the hindlimb bud at stage 51 into the lower jaw cavity. A: Schematic representation of transplantation. Stage-51 limb bud of a Car-GFP transgenic was transplanted into the cavity between lower jaw and trunk of a wild-type stage 51 tadpole. B,C: Grafted limb bud at one day after transplantation. D,E: Grafted limb bud at 2 weeks after transplantation. The inset in D shows higher magnification, demonstrating GFP-positive fibers in transplanted limb. F–L: Serial sections of the limb formed ectopically in the host cavity. F: Double-staining with alcian blue and mayer's hemotoxylin. G–L: Immunostainings for MyHC (G, J), GFP (H, K), and PAX7 (L) proteins. Both signals for MyHC and GFP can be observed in the same region, and the GFP-positive muscle tissue contains PAX7-positive cells.
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Figure 4. Transplantation of the hindlimb bud at stage 51 onto the amputated hind limb bud. A–F: A Car-GFP transgenic limb bud was used as a graft, and wild-type tadpole as a host. A,B: One day after implantation. C,D: Ten days after implantation. The chimeric limb contains GFP-positive muscle. Sections from the chimera were stained with MF20 (E) and anti-GFP (F) antibody. Many MF20-positive cells are also GFP-positive. G–L: Wild-type limb bud was used as a graft, and cmv-GFP tadpole as a host. G: Grafted limb bud just after implantation. Arrowheads trace shape of the graft. H: Grafted limb bud that developed normally for 20 days. GFP signals were observed in the overlying ectoderm, suggesting that ectoderm was exchanged. I–L: Serial sections of the chimeric limb bud in H. I,J: Double-staining for GFP (I) and MF20 (J) in a section. Signals for GFP and MF20 are not overlapping. K,L: Double-staining for GFP (K) and α-tubulin (L). GFP signals in the graft are co-localized with α-tubulin.
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