Myogenic Stem Cell-Dependent Regeneration in Fish Figure 3. Pax7 labels myogenic satellite cells in adult skeletal muscle and electric organ of S. macrurus. (A) Pax7 RT-PCR shows expression in skeletal muscle (SM), electric organ (EO), brain (B), spinal cord (SC), but not liver (L). Negative controls for each sample omitted reverse transcriptase (nrt). (B) Pax7 antibody detects in vitro translation products from partial Pax7 cDNA sequence (1,435 bp) and expected band (,55 kDa) in homogenates (20 mg/lane) from control muscle (M) and electric organ (EO). (C) Pax7-positive cells (red) co-label with laminin (green) in tissue cryosections (20-mm thick). (D) Electron micrograph cross-sections of adult intact tail show satellite cells (SC) unlike other nuclei (N) are found between the plasma membrane (arrow) and basal lamina in both electrocytes and muscle fibers. (E) Few Pax7 cells (green) colabel with BrdU (red) in control longitudinal sections (20-mm thick) (2 Arrow points to co-localization of Pax7 and BrdU. Abbreviations: EC, electrocyte; mm, muscle fiber; epi, epidermis. Scale bars: 50 mm. doi:10.1371/journal.pone.0036819.g003 amputation. Pax7-positive cells were detected within muscle fibers adjacent to the amputation site. However, despite the lack of quantitative assessment, the apparent number of Pax7-positive cells observed in response to tail amputation appeared higher than that observed in response to ventral muscle amputation. This difference is visibly evident in Figures 7 and 8 wherein the incidence of Pax7-positive cells within a 200 mm segment of tissue in tails after amputation (Figure 7) is higher compared to that within a 200 mm segment of tissue after ventral muscle amputation (Figure 8). BrdU incorporation was observed but mainly within the fin and not in muscle fibers adjacent to the injury sites (Figure 8). These data could be explained by an injury response that involves a differential potential in the activation of Pax7-positive cells under different injury scenarios in S. macrurus – a hypothesis that will be tested in future studies. No Evidence for Myogenic Cell Fragmentation after Tail Amputation The occurrence of cell dedifferentiation has been a major focus in urodele limb regeneration studies. Morphological and cell tracing experiments, for instance, have demonstrated that mature skeletal muscle fibers can fragment (or cellularize) into smaller nuclei-containing units, proliferate, and contribute as a source of new cells that make up the blastema [48–51]. In this study, when we stained longitudinal tissue sections with hematoxylin and eosin (Figure 6A–B) or immunolabeled them with anti-laminin (Figure 9A–B), we did not observe changes in cell morphology of muscle fibers and electrocytes proximal to the transection site that resembled cell fragmentation. Similarly, our BrdU incorpo- ration studies did not reveal labeling of nuclei inside muscle fibers or electrocytes (Figure 9C) showing that these cells did not dedifferentiate via reentry into the cell cycle. Instead, BrdU incorporation observed was in cells outside the plasma membrane of muscle fibers and electrocytes (Figure 9C, arrows). There were few intact electrocytes that were immunolabeled with the anti- sarcomeric MHC antibody MF20 (Figure 9D) manifesting an earlier stage in their differentiation from skeletal muscle fibers. However, these MF20-positive electrocytes did not show any other evidence of a dedifferentiation process beyond the re-expression of MyHC. To more rigorously test the incidence of cell fragmentation of myogenic tissues during regeneration in S. macrurus, fluorescent dextran was injected into single electrocytes and muscle fibers prior to tail amputation. Longitudinal cryosections of the distal- most region of the ventral fin (50 mm from amputation site) were processed for detection of dextran during the first two weeks after tail amputation. Our in vivo dye-injection studies showed no evidence of cell fragmentation in any of the tails analyzed up to 14 days post-amputation. Electrocytes labeled with single-cell dextran injections showed an intact morphology with no fragmentation PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36819