Next, we focused on the muscle in the induced limb. Histological observation suggested that muscles were induced in the ectopic limb (Fig. 2C,D). To confirm the presence of muscle with molecular marker genes and investigate myogenesis in the ectopic blastema, the expression pattern of myogenic marker genes in the ectopic limb was tested by in situ hybridization and immunohistochemistry. We compared transverse sections of a growing flank bump (Fig. 1C) with that of the fully patterned flank limb (Fig. 1E). In the growing flank bump, there were histologically undifferentiated cells (Fig. 3A). Cells expressing cardiac actin mRNA, which serves as a marker for early myogenesis (Nicolas et al., 1998; Satoh et al., 2005), could be detected throughout the growing bump (Fig. 3C). The expression of the mRNA-encoding MHC, which serves as marker for late myogenesis, was barely detectable (Fig. 3E,G). In the fully patterned flank limb, histologically differentiated cells were observed (Fig. 3B). Cardiac actin, MHC, and MHC proteins were detected (Fig. 3D,F,H). We also investigated the expression pattern of these two genes in regular regenerating blastemas (Fig. 4). Cardiac actin was broadly expressed between the early bud (EB) and late bud (LB) stages and was detectable before MHC expression (Fig. 4A,D,D′,G). In the blastema at the LB and the pallet (PS) stage, cardiac actin expression seemed up-regulated in the dorsal and ventral regions (Fig. 4G,J). MHC mRNA and protein were still not detected at the LB stage of blastemas; however, these were detected in the proximal (nonamputated) region (Fig. 4B,C,E,F,H,I). MHC expression was slow to appear, but we confirmed that MHC was detectable in the muscle tissue of digit stage (DS) blastemas (Fig. 4N,N′,O,O′) and cardiac actin gene expression was more restricted at this stage (Fig. 4M,M′). The myogenic marker gene expression pattern in the ectopic limb on a flank was similar to that of the regenerating blastema. These data indicated that myogenesis occurs normally during flank limb formation and that muscle tissues exist in the flank limb.
Figure 3. Muscle marker genes were expressed in the growing flank limb. A,B: Transverse sections of a growing flank bump (A) and the ectopic limb (B) were stained with Alcian blue and hematoxylin–eosin stain. B was sectioned at the level of the autopod of an induced limb (Fig. 1E). C,E,G: The boxed areas of the adjacent sections to A. D,F,H: The boxed areas of the sections adjacent to B. C,D: Sections were analyzed for a maker of early myogenesis (cardiac actin) by in situ hybridization and shows that the pattern of expression of cardiac actin (blue). E,F: The pattern of expression of MHC, a maker of terminal myogenesis expression, is shown (blue). G,H: Immunohistochemical analysis of MHC expression (green). Nuclei were counterstained with Hoechst dye (blue). The arrow in G indicates cells expressing MHC. Scale bars = 1 mm in A, 5 mm in B, 500 μm in C–H.
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Figure 4. A–O′: Expression of muscle differentiation markers in the blastema. Gene expression patterns of myogenic marker genes in longitudinal (A–F,G–O) and transverse sections (D′–F′,M′–O′) of regenerating blastemas are shown. The expression of cardiac actin (A,D,D′,G,J,M,M′) and MHC (B,E,E′,H,K,N,N′) mRNAs was detected by in situ hybridization (blue). C,F,F′,I,L,O,O′: Immunohistochemistry for MHC (green), and counter-stained with Hoechst (blue). Cardiac actin was first expressed in entire blastema, and then split into the dorsal and ventral region. MHC mRNA and protein were expressed in the DS blastema. Scale bars = 5 mm.
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