Characterization and functional analysis of the 5' flanking region of myosin light chain-2 gene expressed in white muscle of the gilthead sea bream (Sparus aurata).

The promoter region ( approximately 1400 bp) of myosin light chain 2 gene of fast skeletal muscle from the marine fish Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box, six perfect E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors and four putative MEF2-binding sites. Three genomic fragments (truncated at their upstream region) of 244, 650 and 1400 bp showed promoter activity evidenced by muscle-specific reporter gene activity using transient expression of green fluorescent protein in microinjected zebrafish embryos and in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA. The three genomic fragments also directed luciferase activity in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA showing a 60 to 150-fold higher luciferase activity compared to that obtained with pGL3-Basic. These experiments show that the three genomic fragments are functional muscle-specific promoters which will be useful for directing myostatin and follistatin expression in fish muscle in order to study their effect on fish muscle growth.

Long-term culture of muscle explants from Sparus aurata.

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.