Image Analysis Images of immunolabeled tissue sections were visualized and captured on a BioRad 1024 confocal microscope (Bio-Rad Laboratories, Richmond, CA), controlled by OpenLab imaging software (Improvision, Lexington, MA). To quantify Pax7-positive cells in control versus regeneration, images from separate samples of proximal tissue at day 7 (n = 4), control (n = 3) and day 7 blastema (n = 3) were sampled using ImageJ. In each image, three different 100 mm2 areas with highest Pax7-positive density were converted to 8-bit and the threshold was modified to reflect positively labeled cells. For proximal and control samples, cells were manually counted using the cell counter plugin. Day 7 blastema was counted using the analyze particle function and the results were displayed to validate correct counts. Manual counting produced the same numbers as produced by the analyze particle plugin. Counts for each image were averaged and the average value from the set of images for each experimental condition were graphed with error bars representing the standard deviation. To quantify colocalization of Pax7 and BrdU, day 7, day 14, and control (n = 3) images were used. For each image, red and green channels were converted to 8-bit and the JACoP plugin [36] was used to calculate Pearson’s coefficient. Results S. macrurus Pax7 Protein is Highly Conserved PCR-coupled RACE generated two Pax7 cDNA sequences from S. macrurus skeletal muscle and electric organ tissues: a 2,187 bp cDNA (GenBank accession number EU624121.1) and a 1,809 bp cDNA (GenBank accession number EU624122.1). The longest cDNA contains a 507 amino acid open reading frame that is flanked by a 216 bp 59 UTR and a 447 bp 39 UTR. Multiple sequence alignments of the predicted S. macrurus Pax7 protein with mouse (accession number NP_035169.1; Figure 2A), and other teleosts including zebrafish (D. rerio; accession number NP_571400), Atlantic salmon (S. salar; accession number CAF02090.1), and arctic char (S. alpinus; accession number CAG25716.1) (Figure 2B) revealed a high degree of conservation among these Pax7 amino acid sequences. The similarity between Pax7 of S. macrurus, mouse, and other piscine species ranged from 87% (mouse) to 96% identity (D. rerio) (Figure 2). Vertebrate Pax7 contains distinct protein regions that are required for transcriptional activation of its target genes, i.e., the paired box domain, the octapeptide, and the homeodomain (Figure 2A; 37; 38). Within the paired box domain, S. macrurus Pax7 showed a 93% homology compared to the mouse protein (Figure 2A), and 98– 100% homology to that of other teleosts (Figure 2B). The octapeptide was 100% similar to both mouse and teleost regions (Figure 2), and the homeodomain region of S. macrurus Pax7 differed only at one of sixty amino acids (98%) with the other vertebrate Pax7 sequences. In sum, S. macrurus Pax7 protein exhibits a high degree of sequence conservation in the specific amino acids that are important for its transcriptional activation. The shorter Pax7 variant contains a 300 amino acid open reading frame that is flanked by a 216 bp 59 UTR and a 689 bp 39 UTR (data not shown). The sequences of the three functional protein domains, the paired box domain (100%), the octapeptide (100%), and the homeodomain (78%) were highly conserved between the two Pax7 variants in S. macrurus. Compared to the longer variant, the shorter Pax7 variant was truncated downstream of the homeodomain resulting in a shorter putative protein by 207 amino acids. Pax7 Expression in Adult Tissues We used RT-PCR and immunolabeling to compare the tissue- specific patterns of Pax7 mRNA and protein expression, Myogenic Stem Cell-Dependent Regeneration in Fish respectively, in adult S. macrurus tissues. Pax7 transcript was detected in mature skeletal muscle, electric organ, brain, and spinal cord tissues, but not in liver (Figure 3A). This tissue expression pattern in S. macrurus is consistent with that reported in other vertebrates [39,30]. In vitro translation of a partial 1,435-bp Pax7 cDNA transcript generated protein products that immunor- eacted with the mouse monoclonal Pax7 antibody obtained from the Developmental Hybridoma Bank (Figure 3B). A band corresponding to the predicted S. macrurus Pax7 protein size of ,55 kDa was strongly labelled and two smaller bands of sizes between 44–55 kDa were also detected (Figure 3B). All bands detected correspond to sizes that are within the range in Pax7 protein sizes found in mouse (,55 kDa; accession number P47239), and other teleosts including zebrafish (,56 kDa; accession number NP_571400.1), arctic char (,55 kDa; accession number Q5ZNB2), and Atlantic salmon (,56 kDa; accession number Q683Y9). Western blots using S. macrurus control skeletal muscle and electric organ tissues revealed a single band of approximately 55 kDa. These data provide support for the specificity of the mammalian Pax7 antibody against S. macrurus Pax7 protein. It was not determined whether the multiple in vitro translation products were due to internal translation initiation of the cDNA. However, proteins of approximately 54, 53, 49, and 43 kD could be explained by leaky initiation of translation at the first four internal in-frame ATG residues of the S. macrurus Pax7 mRNA. On tissue sections, Pax7 co-localized with laminin immunolabeling in both muscle fibers and electrocytes (Figure 3C). Ultrastructural analysis using electron micrographs of longitudinal and cross sections from muscle and electric organ of adult tails corroborated the location of mononuclear cells beneath the basement membrane of muscle cells and electrocytes (Figure 3D). In control tails, the incidence of Pax7-positive cells that incorporated BrdU was relatively low (Figure 3E). By virtue of their Pax7 expression, morphology, and location, these cells associated with muscle fibers and electrocytes can be considered to be homologs of satellite cells observed in mammals [40,41], salamanders [9], frogs [42], and other teleosts [43] and birds [44]. Spatial distribution of Pax7-positive cells in regeneration blastemas. Characterization of the tail regeneration process after tail amputation in S. macrurus has been described in previous studies [21,28,31]. Briefly, when the tail is amputated at the end of the ventral fin, epidermal cells at the wound margin rapidly proliferated and covered the wound within 24 hr. After one week, blastemas reached an average length of 4 mm (range: 2.2– 4.8 mm; SD: 61.1) and were composed primarily of undifferen- tiated mesenchymal cells. During the second week, blastemas grew to an average length of 7 mm and contained spinal neurons, muscle fibers and electrocytes at different stages of differentiation. Muscle fibers formed adjacent to the epithelium with some of the larger muscle cells farthest from the epithelium fusing and converting into electrocytes – data that supports previous observations in S. macrurus tail regeneration [28,29,31]. To determine whether Pax7-positive cells are associated with the restoration of myogenic cells in the blastema, we studied the expression and distribution of Pax7 at different stages of regeneration. Within the first week after amputation, most Pax7- positive cells were detected peripherally underneath the epithelium of the blastema (arrowheads, Figure 4A). This distribution was also true for 2-week blastemas, i.e., Pax7-positive cells were abundant and highly concentrated beneath the epithelium (Figure 4B). Blastema regions under the epithelium correspond to those that give rise to myogenic cells as evident by co-labeling with antibodies against muscle markers desmin (Figure 4C) and sarcomeric myosin heavy chain (Figure 5B). The co-localization of Pax7-positive cells PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36819