Figure 3

Muscle dedifferentiation requires metamorphosis. (a–d) Induction of metamorphosis in metamorphosis-inhibited (MI) giant larvae. (b–d) Representative set of images obtained from the same individual. For this experiment, animals were grown to ~ 6 cm (total body length) in tap water containing 0.2 g/l thiourea for 1.1 year while preserving their larval characteristics such as gills and tail fin (age and body size corresponded to those of preadolescence). MI giant larvae metamorphosed over ~ 2 months after they were transferred into plain tap water. These animals are referred to as MI-released (MIR) juveniles. (e,f) Representatives showing a digit-forming pattern during limb regeneration in MI giant larvae and MIR juveniles, respectively. These are different individuals from those shown in (b) and (d). (g–o) Contribution of muscle dedifferentiation to limb regeneration in MI giant larvae and MIR juveniles. The images in (g,h) and (i,j) are representative of blastema and almost regenerated limbs in MI giant larvae, and those in (k,l) and (m–o) show blastema and autopod-forming regenerating limbs in MIR juveniles. Dotted line: amputation plane. U ulna, R radius, H humerus. The images in (n,o) are an enlargement of a distal part of the regenerating limb in (m), which is indicated by a u-shaped right parenthesis. Note that in both MI giant larvae and MIR juveniles, the ulna and radius, which were still cartilage, regenerated quickly by growing distally. Even at the blastema stage, the distal ends of the ulna and radius were observed inside the blastema (g,h). In MI giant larvae, mono-SMFCs were not detected in the blastema (g,h; n = 8), nor in the following stages of the regenerating limb (i,j; n = 5). On the other hand, in MIR juveniles, even though mono-SMFCs were not detected in the blastema (g,h; n = 8), a large number of mesenchymal cells with N-mCherry nuclei appeared along the space under the skin of a distal part of the regenerating limb, when autopod (hand) formation started in the regenerating limb (n,o; n = 6). Arrowheads in (n) point to the N-mCherry nuclei of mesenchymal cells. At this stage, as in normal juveniles, muscle fibers with N-mCherry nuclei had already extended distally from the amputation region. Note that the animals used in (g–o) were mosaics that expressed EGFP at least in muscle. Scale bar: 2 cm (b–d); 2 mm (e,f); 200 μm (g–o).