Myogenic Stem Cell-Dependent Regeneration in Fish Figure 2. S. macrurus Pax7 protein is highly conserved. Sequence comparison of the deduced protein encoded by S. macrurus Pax7 (GenBank accession number ACC86106) with comparable sequences from mouse (A) and other piscine species (B). Protein sequences from mouse (M. musculus, GenBank accession number NP_035169.1), zebrafish (D. rerio; accession number NP_571400), Atlantic salmon (S. salar; accession number CAF02090.1), and arctic char (S. alpinus; accession number CAG25716.1) were used. The boxed areas are labeled to indicate the highly conserved regions of members of the Pax family: the paired domain, octapeptide, and homeodomain. Asterisks represent amino acid similarities across sequences. doi:10.1371/journal.pone.0036819.g002 within developing muscle fibers and electrocytes (Figures 4C and 5A-B) clearly demonstrate the contribution of Pax7-positive cells to the formation of the same myogenic tissues they are associated with in adult intact tails. Outside of these myogenic zones in the blastema, Pax7 immunolabeling was only detected in cells along the entire antero-posterior axis of the regenerating spinal cord (Figure 5A, C). The occurrence of Pax7-positive cells exclusively in the dorsal zones of the spinal cord in blastema and adult tails of S. macrurus is consistent with expression patterns reported in developing and regenerating spinal cords in other vertebrates [37,39,45–47]. In sum, our data support a mechanism of regeneration that is mediated primarily through a population of Pax7-positive stem cells that give rise to dorsal spinal cord, muscle fibers and electrocytes. Pax7 expression increases in tissues proximal to the amputation site. Changes in Pax7 expression were determined at different time periods after amputation in the tail region proximal to the amputation site, a tail segment that included both injured and intact cells (Figure 6). Electrocytes injured by tail transection were easily distinguished from intact electrocytes based on their staining with hematoxylin and eosin (Figure 6A–B). Similar distinctions among muscle fibers before and after injury were not made due to their much smaller size and number. Injured electrocytes atrophied into long thin strips that extended into the regeneration blastema (Figure 6A–B). At seven days after amputation, these thin strips of damaged electrocytes contained Pax7-positive cells (Figure 6C–D). BrdU incorporation studies showed higher Pearson’s correlation r values for colocalization of Pax7 and BrdU (Figure 6E) in tails from both 7 and 14 days after amputation compared to tails from control uncut fish (Figure 6F), providing further support that they are the product of a recent cell division. The longitudinal cryosections shown in Figure 7 are representative of the density of Pax7-positive cells proximal to the amputation site observed in all fish studied. We observed an increase in the density of Pax7-positive cells associated with electrocytes and muscle fibers in the proximal stump seven days after tail amputation (Figure 7A) that was .86higher than that in control uncut tails (Figure 7B). The increased density of Pax7- positive cells in blastema was .1006 higher than found in control tissues (Figure 7B). By day 14 post-amputation, most Pax7-positive cells occupied the extracellular space (Figure 7C–D) a distribution not observed in control intact tails (Figure 3C). Injury-dependent responses of muscle and Pax7 expression. To further investigate the regenerative potential of skeletal muscle Pax7-positive cells in S. macrurus, a section of ventral muscle was amputated. In this procedure, electric organ, central blood vessel and spinal cord were left intact. Pax7 immunolabeling and BrdU incorporation studies were done in the same way as with studies after tail amputation described above. The response to removal of ventral muscle was similar to the process observed after tail amputation, i.e., immediate formation of a wound followed by the formation of a blastema and its growth to give rise to new ventral muscle (Figure 8). Full restoration of all ventral muscle tissue removed was generally completed 20 to 22 weeks after its removal, that is, 15 to 18 weeks longer than when a similar length of muscle was removed after complete tail PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36819