Myogenic Stem Cell-Dependent Regeneration in Fish Figure 7. Pax7 expression increases proximal to the amputation site. A, Black and white image of a portion of a longitudinal cryosection (20-mm thick) of a 7-day tail immunolabeled with the antibody against Pax7. Proximal stump is the region below to the dashed line and regeneration blastema is the region above dashed line. B, The density of Pax7-positive cells is increased in the proximal stump (.86) (stump) and regeneration blastema (.1006) (blast) compared to control, based on images from 4 different tails taken from control fish, and 7-day amputated tails. C and D, Portions of longitudinal sections (20-mm thick) of 14-day amputated tails immunolabeled with antibodies against Pax7 (green) and BrdU (red). Dashed lines denote the site of amputation with the proximal stump to the left. Arrows point to cells that were co-labeled with Pax7 and BrdU. Abbreviations: EC, electrocyte; Ep, epithelium. Scale bars: A, 250 mm; B, 100 mm. doi:10.1371/journal.pone.0036819.g007 Pax7 antibody (Figure 3C). By combining nuclear Pax7 expression with morphological observations of satellite cells by electron microscopy we conclude that Pax7 is a reliable satellite cell marker in S. macrurus muscle and electric organ. Other molecules like c- Met and M-cadherin have been used to identify satellite cells by other investigators [54–55]. However, unlike the Pax7 antibody, antibodies against these other markers did not cross-react successfully with S. macrurus tissues. Pax7 is a commonly used marker for identifying quiescent satellite cells [30,56]. In control intact fish, Pax7-positive cells associated with electrocytes and muscle fibers rarely incorporated BrdU (Figure 3E and Figure 6E) suggesting a predominant state of quiescence. Following tail amputation, only some Pax7-positive cells proximal to the amputation site were BrdU positive, and the low incidence of Pax7-BrdU-positive co-labeling was similar regardless of whether satellite cells were associated with, or free of, electrocytes and muscle fibers (Figure 7). These data may reflect the BrdU injection paradigm used for this timepoint wherein, in all likelihood, was not incorporated into all cells that re-entered cell division in response to tail amputation. Since the first description in frog muscle by Mauro in 1961 [57], satellite cells have been identified in many vertebrate species and have been considered distinct progenitor cells with the unique ability of associating with, generating, repairing, and maintaining skeletal muscle. This muscle-specific fate has recently been challenged by studies showing that satellite cells can adopt different cell phenotypes [58–61]. In each of these cases, satellite cells have been taken away from their natural niche and cultured under conditions to promote the conversion of satellite cells into cells with adipocyte and osteocyte [58–60], and even some neuronal characteristics [61]. Yet, we are not aware of any studies wherein satellite cells show this cell fate potential in vivo and/or demonstrate the occurrence of satellite cells in mature fat, bone or neuronal tissues. In contrast, the association of satellite cells with mature electrocytes is a natural occurrence that represents an exception to the tissue-specificity of these myogenic stem cells to striated muscle fibers. A major quest of future studies is to determine whether the Pax7-positive cells in muscle and electric organ of S. macrurus form a single multipotent class of cells or whether they represent discrete satellite cells for muscle and electrocyte lineages. One way to explore this is to replicate differentiation of muscle fibers and electrocytes in culture. Our ability to isolate satellite cells from muscle fibers or electrocytes will allow us to directly test the potential of these myogenic cells under suitable differentiation conditions [62–63]. It will be interesting to find out if satellite cell populations isolated from either tissue PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e36819