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A spike, a resultant regenerate made after amputation of a Xenopus froglet limb, has no muscle tissue. This muscle-less phenotype was analyzed by molecular approaches, and the results of analysis revealed that the spike expresses no myosin heavy chain or Pax7, suggesting that neither mature muscle tissue nor satellite cells exist in the spike. The regenerating blastema in the froglet limb lacked some myogenesis-related marker genes, myoD and myf5, but allowed implanted muscle precursor cells to survive and differentiate into myofiber. Implantation of hepatocyte growth factor (HGF) -releasing cell aggregates rescued this muscle-less phenotype and induced muscle regeneration in Xenopus froglet limb regenerates. These results suggest that failure of regeneration of muscle is due to a disturbance of the early steps of myogenesis under a molecular cascade mediated by HGF/c-met. Improvement of muscle regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic tissue regeneration in amphibians and other vertebrates.
Figure 2. Myogenic marker gene expressions in the regenerating blastema. A-F: Sections of the froglet blastema that developed for 14 days after amputation. G-L: Sections of the tadpoleblastema at 5 days after amputation. A,G: Sox9 transcripts were found in the middle of the blastema. B,H: Slight myoD expression was observed only in the stump region (H) and not within the froglet blastema (B). MyoD is expressed both in the tadpoleblastema and stump region (H). C,D,I,J: Faint expressions of hgf (C)and c-met (D) were detectable in the froglet blastema, and in the tadpoleblastema, significant signals for both hgf (I) and c-met (J) were detected. E,F,K,L: Immunostainings for myosin heavy chain (MyHC) and Pax7 proteins. E,K: Pax7-positive cells were scattered in the proximal stump region (E,K), and the froglet blastema was Pax7-negative (E), although Pax7-positive cells were detected also in the tadpoleblastema (K). F,L: Anti-MyHC immunoreactivity was located only in the stump region. Scale bars = 400 mu m n A (applies to A-F), in G (applies to G-L).
Figure 5. P14 cells stimulate muscle formation in the froglet blastema. A,B: Hepatocyte growth factor (HGF) -releasing P14 cells labeled with red fluorescent dye PKH26 were implanted into the froglet blastema (A), and after 2 weeks, implanted P14 cells were detected in the froglet blastema (B). C-J: An anterior-posterior series of sections made from a specimen shown in B was stained with MF20 antibody. Arrows indicate that clusters of differentiated muscle cells existed in the manipulated spike, and higher magnifications of the regions indicated by the arrows are shown in D,F,H,J. These well-organized muscle fibers seem to consist of the bundled multinucleate fibers. K: Immunostaining with anti-Pax7 antibodies shows Pax7-expressing cells detectable at the MF20-positive region. Scale bars = 400 mu m.
Figure 1. A Xenopus froglet limb regenerates a muscle-less spike after amputation. A,B: The same individual of Xenopus froglet before and after limb regeneration. Amputation of a forelimb at wrist level (A) gives rise to a spike (B). C–H: Histological and immunohistochemical analyses of muscle formation in Xenopus limb regeneration. C: A section of the spike was stained with eosin, Mayer's hematoxylin solution, and Alcian blue. Note that no muscletissue can be observed in the spike, except in the most proximal region (arrowheads). D: Anti–myosin heavy chain immunoreactivity (fluorescein isothiocyanate [FITC] in green) was detectable in mature muscle in the stump and the most proximal region of the spike (indicated by white arrowheads). E: Pax7-positive cells (in green) were dotted in muscletissue of the stump, but no signal can be seen in the spike (see also Figs. 2E, 4A). F–H: Sections of a regenerate after amputation at the ankle level of a stage 52 hindlimb bud. F: A histological section that shows muscle fibers (arrowheads) and cartilage. G,H: Immunostaining with MF20 (G) and anti-Pax7 (H) antibodies (FITC in blue–green). MF20-positive mature muscle cells (G) and Pax7-positive muscle precursor cells (H) were detectable in the regenerate as well as in the proximal stump. Lines indicate the estimated amputation sites. Scale bars = 400 μm in C–H.
Figure 4. Froglet blastema has a permissive environment for survival and differentiation of muscle precursor cells. A,B:Xenopus limbmuscletissue in the adult frog possesses Pax7-positive cells (A, fluorescein isothiocyanate [FITC] in blue–green); a transverse section of the muscletissue was stained with anti-laminin (FITC in green) and anti-Pax7 (rhodamine in red) antibodies, and a high magnification of a section shows that a Pax7-positive in red is located inside basal laminae visualized by anti-laminin immunoreactivity in green (B). C–K: Cells isolated from muscletissue of froglet femora were cultured for 1 day and stained with anti-Pax7 antibody. C: In low cell density, many cultured cells were Pax7-positive (C, insert, diaminobenzidine staining in brown). D: After 10 days incubation, a lot of elongated multinucleate cells were MF20-positive. E: An overlap image of bright and fluorescent field embosses implanted cell aggregates that contain 1 day-cultured Pax7-positive cells from cmv–green fluorescent protein (GFP) transgenic frogs. F–J: After 14–21 days, samples were stained with anti-GFP (F,I) and MF20 (G,J) antibody; a brightfield view (H). F,I: GFP-positive cells were recognizable in the blastema (arrows and arrowheads). F,G,I,J: Some GFP-positive cells were MF20-positive (arrowheads), and other ones were MF20-negative (arrows). K: The 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining (blue) shows that some of these double-positive cells are multinucleate (arrowheads). Scale bars = 400 μm.
