Figure 4
Newt limb muscles are capable of dedifferentiation before metamorphosis. (a–g) Competency of swimming larvae at St. 57 to metamorphose in response to triiodothyronine (T3). Arrows in (c,f) point to the location of the gills. Numbers in (d,g) indicate number of fingers. Scale bar: 1 cm (b,e); 5 mm (c,f); 1 mm (d,g). (h,j) Explant culture of larval muscles in the presence of T3. Explants were obtained from the forelimbs of swimming larvae at St. 57. The images in (i) are representatives. Fibroblast-like mesenchymal cells started migration immediately after the explant adhered to the dish in 7 days. Scale bar: 500 μm. (j) Changes of the total number of migrating cells during culture. The number of cells counted on each day was normalized with explant volume, which was estimated on day 7. The mean values on each day were not statistically different between control (n = 4) and T3 (n = 5) conditions. (k–m) A striated muscle fiber with two N-mCherry nuclei in the explant of (i). The images represent different magnifications to show the muscle fiber (black arrowhead), nuclei (white arrowheads), and a striped pattern of the muscle fiber (for a more enlarged image, see Supplementary Fig. S9). The image in (m) shows N-mCherry fluorescence only. Scale bars: 200 μm (k); 100 μm (l); 50 μm (m). (n) Summary of iv-mono-SMFC tracking during culture. Horizontal bars indicate the period between the day of appearance and the day the cell could no longer be followed. Note that the cells, except for cell 1 of explant T37-2 [shown in (o,p)] and cell 1 of explant C2 [shown in (q)], were already a single mesenchymal cell with one nucleus when we discovered them near the explant. (o,p) Tracking of the muscle fiber of (k–m) during culture. Arrowheads indicate the nuclei. The image of day 13 is enlarged in (p). The right-hand panel is a phase-contrast image. Scale bars: 200 μm (n); 100 μm (o). (q) Tracking a protrusion of muscle fiber. Arrowheads indicate the nuclei. Insets indicate phase-contrast images. In this case, the muscle explant was cultured in the control condition. The protrusion contained two nuclei in its leading end on day 19. It gave rise to two mononucleated cells on day 20. One of those (right-hand cell) had an elongated shape and survived (see Supplementary Fig. S12). Scale bar: 200 μm.
