Myogenic Stem Cell-Dependent Regeneration in Fish Figure 10. Intracellular tracer dye injections into single muscle fibers and electrocytes at the level of the distal-most ventral fin reveal no cell fragmentation. Fluororuby (red) was injected into single electrocytes (A) and muscle fibers (B) and visualized 7 days after injection by retracting the overlying skin of the fish. Tail sections at the level of the distal-most region of the ventral fin were processed for immunolabeling and viewed under a fluorescent microscope. (C), region of a cryosection containing muscle fibers injected with Fluororuby dextran (red) and counterstained with DAPI (blue). (D), same image as C showing only DAPI labeling. Scale bars: A and B, 20 mm; C and D, 10 mm. doi:10.1371/journal.pone.0036819.g010 reflect their cell type of origin as has been observed with satellite cell populations that give rise to either slow or fast muscle fibers in birds and rodents [64–66] – or not, as it has been reported in humans [67]. Our present characterization of satellite cells in muscle and electric organ of S. macrurus represent a modest beginning of a large-scale project that ultimately includes understanding how the skeletal muscle phenotype is transformed to acquire new functions and how this may occur independently in multiple lineages in teleosts. We studied the response of Pax7-positive cells following amputation, blastema formation, and regeneration of muscle fibers and electrocytes. Upon tail amputation, Pax7 positive cells associated with intact electrocytes and muscle fibers proliferated profusely (Figure 7). Pax7-positive cells associated with electro- cytes and muscle fibers that were damaged by tail amputation were also observed to extend in narrow strip-like formations into the blastema (Figures 3A, 5C–D). Within the blastema, the incorporation of Pax7-positive cells into developing dorsal spinal cord, muscle fibers and electrocytes was unmistakable (Figures 4 and 5). This cellular distribution corresponds to tissues which Pax7-positive cells manifest in adult intact tails of S. macrurus (Figure 3). Hence, with regards to the incorporation of Pax7- positive cells into myogenic tissues, our results demonstrate that muscle and electrocyte regeneration in adult S. macrurus is similar to the satellite-dependent muscle repair process common in vertebrates including mammals, frogs, birds, fish and some salamanders [9,55–56,a˚68–70]. That skeletal muscle regeneration in S. macrurus shows considerable dependence on activation of myogenic Pax7-positive stem cells is further supported by results obtained from our ventral muscle amputation experiments that showed a slower regeneration process associated with consider- ably fewer changes in Pax7-positive cell number (Figure 8) compared to that observed after tail amputation (Figures 5 and 7). It has been well documented that formation of a blastema can result from the dedifferentiation of mature tissues proximal to the amputation site [6–7,71]. Muscle dedifferentiation can involve the reentry of myonuclei into the cell cycle, cellularization or fragmentation of muscle fibers, and/or the loss of muscle-specific proteins, such as, sarcomeric proteins. Our morphological analysis of muscle fibers and electrocytes did not detect evidence of fragmentation of these myogenic cells that was indisputable when compared to control muscle and electrocytes (Figure 9). Our experiments also do not support the occurrence of myonuclei reentering the cell cycle since BrdU incorporation was only detected in satellite cells (Figure 9C). We did observe a few intact electrocytes near the amputation site that re-expressed sarcomeric myosin heavy chain (Figure 9D) indicating a partial reversal in their myogenic phenotype. However, our single-cell tracer dye injection experiments provide the strongest evidence against in vivo fragmentation or cellularization of either muscle fibers or electrocytes in response to tail amputation (Figure 10). Data from these intracellular dye injection experiments together with our Pax7 expression studies suggest that skeletal muscle and muscle-derived electrocytes are regenerated in S. macrurus by a mechanism similar to the myogenic stem cell-dependent muscle repair process seen in mammals. Hence, muscle dedifferentiation is likely one regeneration strategy, but not the requirement in the myogenic lineage of the adult teleost S. macrurus. In sum, this first characterization of the cellular mechanisms involved in the regeneration of myogenic tissues of the adult tail in S. macrurus: 1) demonstrates the important implications that studying regener- ation in different animals with robust regeneration potential have on our understanding of the evolution of regeneration processes, 2) support the emergent concept in vertebrate regeneration that different tissues provide a distinct progenitor cell population to the regeneration blastema, and these progenitor cells subse- quently restore the original tissue [10], and 3) represent an exciting starting point in the comparative investigation of regeneration mechanisms underlying the restoration of myogeni- cally derived, highly specialized non-contractile tissues in other teleosts. Acknowledgments We are grateful to Vince Gutschick for live fish photography, Shannon Manuelito and Amanda Meenach for technical support in confocal microscopy, and Jung Kim for assistance in image processing. Author Contributions Conceived and designed the experiments: GAU MQM SJT. Performed the experiments: CMW MQM GAU. Analyzed the data: CMW GAU. Contributed reagents/materials/analysis tools: MQM GAU. Wrote the paper: GAU CMW. PLoS ONE | www.plosone.org 12 May 2012 | Volume 7 | Issue 5 | e36819