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In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limbmuscle in Xenopus. Taken together, the results suggest that limbmuscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.
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).
Myogenic marker gene expression and protein distribution in the developing hindlimb bud. Sections of limb buds at stage 51(A,D,G,J,M,P,S), stage 52 (B,E,H,K,N,Q,T), and stage 53 (C,F,I,L,O,R,U). A–C: Cardiac actin expression. D–F: MyoD expression. G–I: Myogenin expression. J,L: Pax3 expression. M–O: Immunostaining for PAX7. P,R: Immnostaining for MyHC. S–U: Sox9 expression.
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 muscletissue contains PAX7-positive cells.
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.
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.
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).
