Fig. 6

Lineage-tracing and ultrastructural analysis of regenerating muscle. (A) Schematic representation of transgenes used for myocyte ablation and lineage tracing. (B) Triple transgenic (Z-CAT; bactin2:loxp-DsRed-STOP-loxp-EGFP) zebrafish were injected once with vehicle (left) or 4-HT. In 4-HT-injected animals, EGFP labeled the majority of spared cardiomyocytes by 7 dpi (middle) and the majority of regenerated cardiomyocytes by 30 days post-injection (dpi; right). Arrowheads indicate examples of MHC⁺ tissue expressing EGFP. (C) Quantification of EGFP⁺ myocardium as a percentage of MHC⁺ ventricular tissue. At 7 dpi, 96% of myocardium was EGFP-labeled, and 95% at 30 dpi. For each group, 4-10 animals were assessed. Insets show enlarged views of the boxed area. (D) Assessment of sarcomere structure in Z-CAT fish crossed to a transgenic strain that labels Z-lines (green). Cardiomyocytes of control animals show well-organized sarcomeres with clear Z-lines. By contrast, many myocytes of 4-HT-injected animals display disorganized sarcomeres and loss of Z-lines (arrowheads) by 7 dpi and 14 dpi. By 30 dpi, sarcomeric structure and Z-lines are typically restored. Scale bars: 50 μm. (E) Transmission electron micrographs of ventricular myocytes from vehicle- or 4-HT-injected Z-CAT animals. Control myocytes have prominent sarcomeric structure and normal mitochondria (m), whereas 7 and 14 dpi myocytes appear less organized with fewer Z-lines (arrows) and swollen mitochondria (arrowheads). Scale bar: 0.5 μm.