Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer.

BACKGROUND: Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. RESULTS: Modulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId). CONCLUSIONS: In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish.

Myostatin is a member of the TGF-beta superfamily and a potent negative regulator of muscle growth and development in mammals. Its expression is limited primarily to skeletal muscle in mammals, but occurs in many different fish tissues, although quantitative measurements of the embryonic and tissue-specific expression profiles are lacking. A recent phylogenetic analysis of all known myostatin genes identified a novel paralogue in zebrafish, zfMSTN-2, and prompted the reclassification of the entire subfamily to include MSTN-1 and -2 sister clades in the bony fishes. The differential expression profiles of both genes were therefore determined using custom RNA panels generated from pooled (100-150/sampling) embryos at different stages of development and from individual adult tissues. High levels of both transcripts were transiently present at the blastula stage, but were undetectable throughout gastrulation (7 hpf). Levels of zfMSTN-2 peaked during early somitogenesis (11 hpf), returned to basal levels during late somitogenesis and did not begin to rise again until hatching (72 hpf). By contrast, zfMSTN-1 mRNA levels peaked during late somitogenesis (15.5-19 hpf), returned to baseline at 21.5 hpf and eventually rose 25-fold by 72 hpf. In adults, both transcripts were present in a wide variety of tissues, including some not previously known to express myostatin. Expression of zfMSTN-1 was highest in brain, muscle, heart and testes and was 1-3 log orders above that in other tissues. It was also greater than zfMSTN-2 expression in most tissues, nevertheless, levels of both transcripts increased almost 600-fold in spleens of fish subjected to stocking stress. Myostatin expression was also detected in mouse spleens, suggesting that myostatin may influence immune cell development in mammals as well as fish. These studies indicate that zfMSTN-1 and -2 gene expression is differentially regulated in developing fish embryos and in adult tissues. The increased expression of both genes in spleens from stressed fish is further supportive of an immunomodulatory role and may explain increased disease susceptibility associated with stocking stress.

Coulter counter use in the enumeration of muscle and fat stem cells.

Although the manual counting chamber (hemacytometer) is the gold standard for counting cells, this method is subject to great variability due to the 'human factor'. The automated cell counter (Coulter Counter) can enumerate cells in less time and with greater accuracy than the hemacytometer by removing many of the steps in which errors are made. While the Coulter Counter (and others of its type) has been used for many years in the cell culture field, there have been few studies to validate its use with specific cell types. We conducted several experiments in which we assessed the accuracy of the Coulter Counter over counts made with a hemacytometer as well as validated its use for the counting of satellite cells and preadipocytes.

Oil red-O stains non-adipogenic cells: a precautionary note.

Bovine adipofibroblasts, 3T3-L1 cells, L-6 myogenic cells, and sheep satellite cells were allowed to proliferate for 48 h. Oil red-O (ORO) was dissolved in three different solvents isopropanol, propylene glycol and triethyl phosphate. At 48 h, the proliferative cultures were stained with the three stains. ORO stain prepared in both propylene glycol and triethyl phosphate resulted in bright red droplets appearing in all cultures, whereas ORO dissolved in isopropanol was not taken up by any of the cells. These data suggest that certain preparations of ORO may stain cells in non-adipogenic lineages as well as undifferentiated pre-adipocytes. Caution must be exercised when choosing solvents for ORO in differentiation studies using cells of the fat/adipose lineage.