regeneration potential in other vertebrates is not known. In order to test this idea by studying different species, it is important to first recognize that cell dedifferentiation involves different (and likely, independent) cellular events [14]. The first involves the reentry of myonuclei into the cell cycle suggesting that the postmitotic arrest of nuclei in mature muscle fibers is reversible in urodeles [15]. The second type of dedifferentiation involves the reversal of multinu- cleated muscle fibers to mononucleated cells (aka, cellularization). The third event involves the loss of gene expression markers of the differentiated state such as sarcomeric proteins. Each of these processes has been demonstrated to occur in the dedifferentiation of skeletal muscle fibers after limb amputation in urodeles. However, the extent to which each of these events occurs in response to injury among other vertebrates has not been well studied. Investigation of the regenerative processes in vertebrates other than urodeles with extensive regenerative capacities is essential for the identification of the limiting factors that constrain the restoration of tissues in some species and development of strategies to improve their regeneration potential. The South American gymnotiform electric fishes are unique vertebrate teleosts in that they possess a muscle-derived electric organ (EO) that is specialized for the production of an electric field outside the body [16] and they also possess a robust capacity to regenerate lost tissues. Of the seventy known species of gymnoti- forms, all can regenerate their tails following amputation. Specif- ically, they can replace lost spinal cord, skin, electroreceptors, skeleton, blood vessels, skeletal muscle and the muscle-derived electric organ [17,18,19,20,21,22,23,24]. Tail regeneration in all gymnotiforms begins by epidermal cells covering the wound followed by formation of a blastema of apparently undifferentiated cells appearing as a small swelling at the end of the tail that gives rise to all newly formed adult tissues [25,18,19], a process widespread in the animal kingdom, appearing in both invertebrate and vertebrate organisms [26,27]. Moreover, some gymnotiforms can replace all tissues lost after repeated tail amputations, suggesting an inexhaust- ible regeneration capacity in the adult [28] and representing the ideal experimental system for investigation of regeneration in teleosts. Tail regeneration in gymnotiforms has been most studied in the yellow stripe knife fish S. macrurus [21,28,29]. As in all gymnoti- forms, S. macrurus responds to tail amputation by wound healing, blastema formation, and blastema elongation by cell proliferation. At the site of amputation at the end of the ventral fin, electrocytes predominate, but also present are skeletal muscle fibers found peripherally beneath the skin and vertebrae protecting the central artery and motoneurons that innervate electrocytes and muscle fibers (Figure 1). Cell differentiation during regeneration proceeds with slow-twitch fibers appearing under the skin followed by differentiation of fast-twitch fibers medial to the slow fibers [21]. A subpopulation of the most centrally-located fast-twitch fibers then fuses and subsequently downregulates many muscle genes to give rise to cells of the electric organ, i.e., electrocytes, all while the spinal cord, vertebrae and blood vessel are differentiating within the center of the tail [21,28]. Within three weeks of tail amputation, a new tail develops complete with peripheral slow and fast muscle fibers and a centrally located electric organ surrounding the vertebrae, spinal cord and central blood vessel [28]. A previous study looking at the labeling patterns of cell replication markers during tail regeneration in S. macrurus suggested that the source of the regeneration blastema consisted of local reserve cells that became activated to contribute to the restoration of all lost tissues [21]. Specifically, the authors concluded that myogenic satellite cells contributed to the blastema Myogenic Stem Cell-Dependent Regeneration in Fish and the regeneration of skeletal muscle and the myogenically derived EO reviving the argument for determining the extent to which stem cell activation and dedifferentiation are conserved across different vertebrate species. Here we tested the hypothesis that myogenic stem cells contribute to blastema formation during adult tail regeneration in S. macrurus. We detected myogenic cells associated with adult skeletal muscle and electric organ that express the mammalian satellite cell marker Pax7 [30]. After tail amputation, these Pax7-positive satellite cells proliferated and populated blastema regions that exclusively regenerate muscle fibers and electrocytes. Accompanying in vivo microinjection studies using high molecular weight lineage tracers showed that multinucleate muscle fibers and electrocytes proximal to the injury site do not undergo fragmentation (cellularization) and do not participate in the formation of regenerated myogenic cells. Our results reveal a regeneration process in S. macrurus that is dependent on the activation of myogenic stem cells for the renewal of both skeletal muscle and the muscle-derived electric organ. These data also suggest that muscle dedifferentiation is not required for a robust regenerative capacity in the myogenic lineage of the adult teleost S. macrurus. Materials and Methods Animal Surgeries and Tissue Preparation S. macrurus is a fresh-water species of knife fish native to South America and was obtained commercially from Segrest Farms (Gibsonton, FL). Adult fish, 20–35 cm in length, were housed individually in 15 to 20-gallon aerated aquaria maintained at 25– 28uC and fed three times weekly. Fish were anesthetized in 2- phenoxyethanol (1:1,500 in tank water) and a tail segment distal to the ventral fin was amputated with a sharp blade as described previously [28,29,31]. In one group the most-distal segment (50 mm) of the tail stump along with the blastema was harvested 7 (n = 4) and 14 (n = 3) days after initial tail amputation. In a second group, only the regeneration blastema was removed 1 (n = 5) or 2 (n = 5) weeks after initial tail amputation. In a third group, only the distal-most section of ventral muscle (2–3 cm) was amputated. In this procedure, electric organ, central blood vessel and spinal cord were left intact and regeneration studied until full restoration of muscle was complete. Immediately following each amputation surgery, fish were treated with a topical antibiotic (Nystatin and Triamcinolone Acetonide Ointment USP), returned to their tanks, Stress CoatH (Aquarium Pharmaceuticals, Inc., Chalfont, PA) was added to the aquarium tank as an additional anti-infection agent, and fish were monitored until they recovered fully from anesthesia. Control fish were overdosed in 2-phenoxyethanol (1:500 in tank water) and the liver, brain, spinal cord, ventral skeletal muscle and EO were excised under a dissecting microscope. All procedures used in this study followed the American Physiological Society Animal Care Guidelines and were approved by the Animal Use Committee at New Mexico State University. Excised tail segments were 1) immediately immersed in RNAlaterTM (Ambion, Austin, TX) and stored at 280uC until RNA extraction, 2) frozen directly in liquid nitrogen and stored at 280uC until protein extraction for Western blots, or 3) frozen on cork with Tissue Freezing MediumTM (TBSTM, Durham, NC) in isopentane chilled in liquid nitrogen and used for immunohistochemical analysis. RNA Isolation and Reverse-transcription and Polymerase Chain Reaction (RT-PCR) Total cellular RNA was isolated from skeletal muscle, EO, spinal cord, brain, and liver of adult control fish using a protocol adapted from the guanidinium isothiocyanate method used in PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36819