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Abstract
BackgroundFish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.ResultsIn transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.ConclusionsWe produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.
Highlights
Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber
Transgene construct The fMyLC2-GFP transgene construct used during this study contained a 1 kilobase zebrafish fast myosin light chain 2 (MLC2f ) promoter at the 5' [9], the 0.8 kb GFP coding region, a 860pb fragment including the small t intron and the polyadenylation signal from SV40 [10] and a mylc 1/3 enhancer from the rat mylc gene [11] (Figure 1A)
All subcloning procedures were performed in a modified pBluescriptII SK+ carrying I-SceI sites in both ends of its polylinker to enable I-SceI meganuclease-mediated transgenesis [12]
Summary
Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. Muscle growth results from successive phases of myogenesis that involve distinct myogenic precursor populations [1]. During the final and most important phase of myogenesis (termed mosaic hyperplasia) which starts around the larval stage, new muscle fibers are continuously being added throughout the myotome, giving a typical mosaic appearance to a muscle cross section [2]. Unlike mammals, in which postnatal growth is solely dependent on the hypertrophy of muscle fibers formed during the embryonic period, fish combine muscle fiber hypertrophy and hyperplasia to generate indeterminate muscle growth throughout life [3].
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