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Figure 5. Labeling of myogenic cells in Xenopus embryo. A: Schematic representation of labeling of myogenic cells in somites. A transgenic vector, pCX-DsRed2/EGFP, was introduced into the genome by a transgenic technique. When pCX-DsRed2/EGFP transgenic eggs are generated, a circular pCar-hCre plasmid is also injected. The pCar-hCre plasmid is not integrated into the genome and would exist episomally, since this plasmid is not linearized. A temporal muscle-specific expression of hCre under the control of Cardiac actin promoter makes muscle cells change in color from red (DsRed) to green (EGFP). B: A control; pCX-DsRed2/EGFP transgenic tadpole without injection of pCar-hCre plasmid. Cells are labeled in red. C,D: GFP fluorescence in a pCX-DsRed2/EGFP transgenic tadpole with an injection of pCar-hCre plasmid. Muscle cells are in green. Some leaky signals are observed in the ectoderm, but these signals disappear as development proceeds. D: Muscle-specific GFP fluorescence in stage-52 tadpole. Many muscle fibers are labeled in green, and little GFP signal can be seen in ectoderm at this stage.
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Figure 6. Location of labeled myogenic cells. A–F: Whole-mount observation of GFP expression in the stage-55 tadpole whose myogenic cells are labeled by the method in Figure 5. A,B: At the forelimb level, no signal for GFP is detected in the forelimb bud (flb), although many muscle fibers in the main body express GFP. C,D: At the abdomen level. Many abdominal muscle express GFP. E,F: At the hindlimb level, GFP fluorescence cannot be detected inside the hindlimb bud (hlb). G–Q: Immunostaining for GFP (G,J,M,N,P) and MyHC (H,I,K,O,Q) on a series of sections prepared from the tadpoles in A–F. G-L: At the forelimb level, many trunk muscles (H,I) are GFP-positive (G,J). However, GFP-positive cells are not detected in the forelimb (L), although MyHC-positive differentiated muscles are observed (K). M–Q: Sections at the hindlimb level. GFP-positive muscle is not detected inside the hindlimb bud (M). MyHC-positive muscle cells on a transverse (O) section and a cross (Q) section are GFP-negative (N,P).
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