Degeneration of electrocytes injured by amputation.

<p>Portions of longitudinal cryosections (14–30 µm thick) from 7-day (<b>A</b>) and 10-day (<b>B</b>) blastemas stained with hematoxyline and eosin. (<b>C</b>) 7-day blastema co-labeled with anti-Pax7 (red) and anti-laminin (green) antibodies. (<b>D</b>) 7-day blastema co-labeled with anti-Pax7 (red) and anti-BrdU (green) antibodies. Dashed line in <b>A</b>, <b>B</b> and <b>D</b> show the sites of tail amputation. Arrows in <b>A</b>, <b>B</b>, <b>C</b> and <b>D</b> point to electrocytes that were damaged by the amputation. <b>E</b>, Enlargement of the region within the dashed area in <b>D</b> to more clearly visualize the colocalization of Pax7 (red) and BrdU (green) as yellow. <b>F</b>, Correlation of Pax7 and BrdU immunolabeling in cells from control tails (Con), and tails after amputation at 7 (7 d) and 14 (14 d) days. Abbreviations: EC, electrocyte; Ep, epithelium; mm, muscle fiber. Scale bars: <b>A</b>, 2 mm; <b>B</b>, 5 mm; <b>C</b>, 500 µm; <b>D</b>, 200 µm. <b>F</b>, Range of Pearson’s correlation values for Pax7/BrdU co-localization for images from 3 different tails taken from control fish, and 7-day and 14-day amputated tails.</p

Pax7 labels myogenic satellite cells in adult skeletal muscle and electric organ of S. macrurus.

<p>(<b>A</b>) Pax7 RT-PCR shows expression in skeletal muscle (SM), electric organ (EO), brain (B), spinal cord (SC), but not liver (L). Negative controls for each sample omitted reverse transcriptase (nrt). (<b>B</b>) Pax7 antibody detects in vitro translation products from partial Pax7 cDNA sequence (1,435 bp) and expected band (∼55 kDa) in homogenates (20 µg/lane) from control muscle (M) and electric organ (EO). (<b>C</b>) Pax7-positive cells (red) co-label with laminin (green) in tissue cryosections (20-µm thick). (<b>D</b>) Electron micrograph cross-sections of adult intact tail show satellite cells (SC) unlike other nuclei (N) are found between the plasma membrane (arrow) and basal lamina in both electrocytes and muscle fibers. (<b>E</b>) Few Pax7 cells (green) colabel with BrdU (red) in control longitudinal sections (20-µm thick) (2 Arrow points to co-localization of Pax7 and BrdU. Abbreviations: EC, electrocyte; mm, muscle fiber; epi, epidermis. Scale bars: 50 µm.</p

Intracellular tracer dye injections into single muscle fibers and electrocytes at the level of the distal-most ventral fin reveal no cell fragmentation.

<p>Fluororuby (red) was injected into single electrocytes (<b>A</b>) and muscle fibers (<b>B</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. (<b>C</b>), region of a cryosection containing muscle fibers injected with Fluororuby dextran (red) and counterstained with DAPI (blue). (<b>D</b>), same image as C showing only DAPI labeling. Scale bars: <b>A</b> and <b>B</b>, 20 µm; <b>C</b> and <b>D</b>, 10 µm.</p

Spatial distribution of Pax7-positive cells in regeneration blastema.

<p>Confocal images of longitudinal cryosections (20-µm thick) from 7-day (<b>A</b>) and 14-day (<b>B</b>) blastemas immunolabeled with anti-Pax7 (<b>A</b>, <b>B</b>, <b>C</b>), BrdU (<b>B</b>) and desmin (<b>C</b>) antibodies. Dashed line in <b>A</b> 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 <b>A</b> and <b>C</b>, and green in <b>B</b>. Images in <b>C</b> represent the region enclosed in the dotted box in <b>B</b> from a serial cryosection co-labelled with anti-desmin and Pax7 antibodies. Scale bars: <b>A</b>, 400 µm; <b>B</b>, 150 µm; <b>C</b>, 50 µm.</p

S. macrurus Pax7 protein is highly conserved.

<p>Sequence comparison of the deduced protein encoded by S. macrurus Pax7 (GenBank accession number ACC86106) with comparable sequences from mouse (<b>A</b>) and other piscine species (<b>B</b>). Protein sequences from mouse (M. musculus, GenBank accession number NP_035169.1), zebrafish (D. rerio; accession number NP_571400), Atlantic salmon (S. salar; accession number CAF02090.1), and arctic char (S. alpinus; accession number CAG25716.1) were used. The boxed areas are labeled to indicate the highly conserved regions of members of the Pax family: the paired domain, octapeptide, and homeodomain. Asterisks represent amino acid similarities across sequences.</p

Pax7 expression increases proximal to the amputation site. A

<p>, Black and white image of a portion of a longitudinal cryosection (20-µm 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>B</b>, The density of Pax7-positive cells is increased in the proximal stump (>8×) (stump) and regeneration blastema (>100×) (blast) compared to control, based on images from 4 different tails taken from control fish, and 7-day amputated tails. <b>C</b> and <b>D</b>, Portions of longitudinal sections (20-µm 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: <b>A</b>, 250 µm; <b>B</b>, 100 µm.</p

Schematic illustration of <i>S. macrurus</i> and the amputation protocol used in the present study.

<p>Tails were amputated at the end of the ventral fin (scissors). At the site of amputation, vertebrae, central artery, motor neurons, electrocytes and muscle fibers are present. The spinal cord contains two distinct populations of motor neurons: electromotoneurons that innervate electrocytes and somatomotoneurons that innervate muscle fibers. Muscle fibers are located peripherally and surround a central core of electrocytes. Electrocytes are up to 3 mm in length with a cross sectional area up to 30 times that of the adjacent muscle fibers. As shown in the cross sectional view at the bottom, muscle fibers and electrocytes are separated into distinct compartments. In this study, we analyzed the most-distal segment (50 mm) of the tail stump after tail amputation to investigate effects of amputation on tissues immediately adjacent to the injury site.</p

No evidence of cell dedifferentiation.

<p>Portions of longitudinal cryosections (14-µm thick) from intact tails (<b>A</b> and <b>D</b>) and tails 4 (<b>B</b>) and 14 days (<b>C</b>) after amputation. Tissue sections were immunoreacted with anti-laminin (<b>A</b> and <b>B</b>) to study changes in basal lamina morphology that may indicate cellularization (arrows), anti-BrdU (<b>C</b>) to investigate myonuclei proliferation after tail cut (arrow), and anti-myosin heavy chain (<b>D</b>, red) to examine changes in muscle protein expression in intact mature electrocytes proximal to the tail transection site. Sections in <b>D</b> were co-immunolabeled with anti-Pax7 (green). Abbreviations: EC, electrocyte. Scale bars: 50 µm.</p

Delayed regeneration of ventral muscle associated with fewer Pax7-positive cells.

<p>An adult S. macrurus and delineation of its ventral and dorsal muscle groups. Four images show the regeneration of the most distal portion (2 to 3 cm) of the ventral muscle over a 120-day period after its ampuation. Blue arrow points to the distal ventral muscle region removed including portions of the ventral fin. Serial longitudinal cryosections of a tail 10 days after ventral muscle excision were immunolabeled with anti-Pax7 (red) antibody to show the incidence of Pax7 immunolabeling at two different depths of the tail region adjacent to the muscle excision area. Dotted lines on the top two Pax7 images represent site of ventral muscle excision. The bottom panel shows longitidunal section from the same animal immunolabeled with anti-BrdU (red) and MyHC (MF20; green) antibodies. A dotted line has been added to the bottom image (BrdU/MyHC) to clearly discern the boundary between skeletal muscle and fin adjacent to the site of excision. MyHC is not expressed in the fin. Scale bar for Pax7/BrdU immunolabeled images: 200 µm.</p

Pax7-positive cells are localized in blastema regions that give rise to muscle, electric organ and dorsal spinal cord.

<p>Serial cross-sections taken from distal half of 14-day blastemas (<b>A</b> and <b>B</b>) immunolabeled with antibodies against Pax7 (<b>A</b>, green) and MF20 (<b>B</b>, green). MF20 labels all mammalian myosin heavy chain (MyHC) isoforms present in all muscle fibers and developing electrocytes that arise from the fusion of muscle fibers. Immunolabeling by Pax7 and MF20 was detected in peripheral regions of the blastema underneath the epithelium. Pax7 immunolabel was also detected in cells within the dorsal spinal cord of regenerating blastema (<b>A</b>, arrowhead). <b>C</b>, Pax7 immunolabeling was detected along the entire regenerating dorsal spinal cord in 14-day blastemas as shown in this longitudinal section. Abbreviations: ECs, electrocytes; mm, muscle fibers; SC, spinal cord. Scale bars: <b>A</b>, 300 µm; <b>B</b>, 300 µm; <b>C</b>, 150 µm.</p