Myogenic Stem Cell-Dependent Regeneration in Fish Figure 4. Spatial distribution of Pax7-positive cells in regeneration blastema. Confocal images of longitudinal cryosections (20-mm thick) from 7-day (A) and 14-day (B) blastemas immunolabeled with anti-Pax7 (A, B, C), BrdU (B) and desmin (C) antibodies. Dashed line in A denotes the site of amputation with the newly regenerated blastema to the right of the amputation site. Arrows point to electrocytes damaged by amputation. Arrowheads point to Pax7-positive cells adjacent to the epithelium (Ep). Pax7-positive cells are red in A and C, and green in B. Images in C represent the region enclosed in the dotted box in B from a serial cryosection co-labelled with anti-desmin and Pax7 antibodies. Scale bars: A, 400 mm; B, 150 mm; C, 50 mm. doi:10.1371/journal.pone.0036819.g004 into smaller cellular components (Figure 10A). Similar results were obtained with dextran-injected muscle fibers (Figure 10B–C). Small fluorescent remnants of dextran labeling were at times detected around some electrocytes and muscle fibers (Figure 10C), but this fluorescence was not co-labeled with nuclear stain DAPI (Figure 10C–D) indicating these were acellular debris generated by cutting labeled cells. Hence, at least with regard to data from laminin labeling and intracellular dye injection experiments, multinucleated muscle fibers and electrocytes proximal to the injury site did not undergo fragmentation. Hence, these findings are contrary to the association between blastema formation and dedifferentiation of cells near the injury site that has been reported in urodeles [6,48–51]. Discussion Our cloning analysis revealed that specific amino acids important for transcriptional activation by Pax7 in mammals are also highly conserved in the S. macrurus ortholog (Figure 2). A common feature of the Pax7 gene is the generation of different transcripts through alternative splicing [37–39;52–53], a feature confirmed for the Pax7 gene in S. macrurus. We identified 2 alternate Pax7 isoforms (GenBank accession numbers EU624121 and EU624122), which varied in size from 300 to 507 amino acid residues, a range similar to that found among Pax7 isoforms reported within different vertebrate species. The number of Pax7 variants confirmed in S. macrurus is fewer than the number of alternate Pax7 transcripts identified in other vertebrates, which range from at least four variants in mice [38–39] and zebrafish [37] to at least 10 variants in other teleosts including Atlantic salmon [52] and the arctic charr [53]. Hence, it remains to be determined whether the Pax7 gene of S. macrurus encodes additional isoforms, an issue that was beyond the scope of the present study. In addition, establishing whether alternate tran- scripts are expressed in different cell populations in S. macrurus as it is known in mice and other teleosts would suggest different tissue- specific roles of Pax7 function that are highly conserved in vertebrates. Our RT-PCR and immunolabeling studies also suggest that expression of Pax7 mRNA and protein, respectively, is highly conserved in S. macrurus. Pax7 transcripts in S. macrurus were mainly found in mature skeletal muscle, electric organ, brain and spinal cord where Pax7 expression was restricted to a subpopu- lation of neural cells in the dorsal spinal cord. These Pax7 expression patterns in skeletal muscle, brain and spinal cord are similar to those in adult mouse [30,38], urodele amphibians [46] and other teleosts [52–53]. We also show that the commonly used mammalian antibody against Pax7 from the Developmental Hybridoma Bank labeled protein products from in vitro translated PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36819