Expression of myogenic regulatory factors in the muscle derived electric organ of Sternopygus macrurus

In most groups of electric fish, the current-producing cells of electric organs (EOs) derive from striated muscle fibers but retain some phenotypic characteristics of their precursor muscle cells. Given the role of the MyoD family of myogenic regulatory factors (MRFs) in the transcriptional activation of the muscle program in vertebrates, we examined their expression in the electrocytes of the gymnotiform Sternopygus macrurus. We estimated the number of MRF genes in the S. macrurus genome and our Southern blot analyses revealed a single MyoD, myogenin, myf5 and MRF4 gene. Quantitative RT-PCR showed that muscle and EO transcribe all MRF genes. With the exception of MyoD, the endogenous levels of myogenin, myf5 and MRF4 transcripts in electrocytes were greater than those detected in muscle fibers. These data indicate that MRF expression levels are not sufficient to predict the level to which the muscle program is manifested. Qualitative expression analysis of MRF co-regulators MEF2C, Id1 and Id2 also revealed these genes not to be unique to either muscle or EO, and detected similar expression patterns in the two tissues. Therefore, the partial muscle program of the EO is not associated with a partial expression of MRFs or with apparent distinct levels of some MRF co-factors. In addition, electrical inactivation by spinal cord transection (ST) resulted in the up-regulation of some muscle proteins in electrocytes without an accompanying increase in MRF transcript levels or notable changes in the co-factors MEF2C, Id1 and Id2. These findings suggest that the neural regulation of the skeletal muscle program via MRFs in S. macrurus might differ from that of their mammalian counterparts. Together, these data further our understanding of the molecular processes involved in the plasticity of the vertebrate skeletal muscle program that brings about the muscle-like phenotype of the non-contractile electrogenic cells in S. macrurus

The myogenic electric organ of Sternopygus macrurus: a non-contractile tissue with a skeletal muscle transcriptome

In most electric fish species, the electric organ (EO) derives from striated muscle cells that suppress many muscle properties. In the gymnotiform Sternopygus macrurus, mature electrocytes, the current-producing cells of the EO, do not contain sarcomeres, yet they continue to make some cytoskeletal and sarcomeric proteins and the muscle transcription factors (MTFs) that induce their expression. In order to more comprehensively examine the transcriptional regulation of genes associated with the formation and maintenance of the contractile sarcomere complex, results from expression analysis using qRT-PCR were informed by deep RNA sequencing of transcriptomes and miRNA compositions of muscle and EO tissues from adult S. macrurus. Our data show that: (1) components associated with the homeostasis of the sarcomere and sarcomere-sarcolemma linkage were transcribed in EO at levels similar to those in muscle; (2) MTF families associated with activation of the skeletal muscle program were not differentially expressed between these tissues; and (3) a set of microRNAs that are implicated in regulation of the muscle phenotype are enriched in EO. These data support the development of a unique and highly specialized non-contractile electrogenic cell that emerges from a striated phenotype and further differentiates with little modification in its transcript composition. This comprehensive analysis of parallel mRNA and miRNA profiles is not only a foundation for functional studies aimed at identifying mechanisms underlying the transcription-independent myogenic program in S. macrurus EO, but also has important implications to many vertebrate cell types that independently activate or suppress specific features of the skeletal muscle program

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

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

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

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