The α-actin–RFP transgenic fish line was a generous gift from H.J. Tsai (Taiwan National University) and has been described previously.17
Additional experiments were done utilizing the wild-type AB strain, which was obtained from the Children's Hospital aquatics program and maintained in their aquatics facility. All animal protocols were approved by the animal resources committee of Children's Hospital.
Isolation of Zebrafish Myogenic Muscle Cells from Whole Dorsal Myotome
For each cell preparation, 15–20 adult zebrafish were euthanized in tricaine (Sigma-Aldrich) and the whole zebrafish was placed in 100% ethanol for 30 seconds as the first step for sterilization. The fish's head, tail, and fins were removed with a scalpel, and the skin and internal organs were removed with forceps. The fish's body was sterilized in 10% bleach for 30 seconds and then washed twice in sterile phosphate-buffered saline (PBS) for another 30 seconds. Fish dorsal muscle and bone were minced with a scalpel and then transferred to a pre-weighed culture plate. For every gram of fish tissue, 3.5 ml of collagenase IV (10 mg/ml stock solution) and 3.5 ml of dispase (2.4 units/ml stock solution; Worthington Chemicals) were added and mixed by pipetting (Worthington). The solution was incubated at room temperature for 45 minutes (mixed every 10 minutes by pipette) before 10 ml of growth medium (L15; Sigma-Aldrich), 3% fetal calf serum, 100 μg/ml penicillin/streptomycin, 2 mM glutamine, and 0.8 mM CaCl2 (all Sigma-Aldrich) were added to the cells to quench the activity of the collagenase and dispase proteases. Debris was removed by filtering the cells through a 70-μm filter and then through two 40-μm filters (BD Biosciences). On each occasion, the filters were washed with 5 ml of L15 medium.
The cells were isolated by centrifugation at 1000 × g for 10 minutes at 9°C, and the supernatant was aspirated. The cells were then resuspended in 3 ml of red blood cell lysis buffer (Qiagen) and incubated for 3 minutes at room temperature before neutralization with 22 ml of L15 growth medium. The cells were then pelleted at 1000 × g for 10 minutes at 9°C, the supernatant aspirated, and the cell pellet resuspended in 3 ml of cold 1× PBS and layered on top of 4 ml of Ficoll-Paque gradient (GE Healthcare) in a 15-ml tube. Samples were then centrifuged at 1400 × g for 40 minutes at 9°C. A mononuclear cell layer was then extracted by pipette and washed with 10 ml of ice-cold 1× PBS. Afterwards, the cells were resuspended in 10 ml of ice-cold L15 buffer. The cell density was determined using an automated hemocytometer (Countess; Invitrogen), and the cell suspension was diluted in L15 growth medium.
The cells were then pre-plated on uncoated plates for 1 hour in a 28°C tissue culture incubator at 5% CO2. After pre-plating, the cellular supernatant (non-adherent cells) was removed and placed on laminin-coated plates (BD Biocoat). Alternatively, 0.1% gelatin-coated (porcine) plates can be used. The medium was changed every 3 days. The zebrafish myogenic progenitor cells were able to be grown for up to seven doublings before evidence of cellular senescence, with an average of four and five doublings per myoblast isolation. On average, a yield of 5–10 million live (trypan blue–negative) cells were isolated from each preparation of between 15 and 20 adult zebrafish. Lower yields of 100,000–500,000 live cells were isolated when using 1–5 adult zebrafish.
An alternative to the L15 growth medium was later used in zebrafish myogenic progenitor cell cultures and achieved the same results. Human skeletal myoblast growth medium (Promocell) that contained 20% fetal bovine serum (Atlanta Biologicals), 1× antibiotic–antimycotic (Invitrogen), and 1× Glutamax (Invitrogen), and supplemented with 3 ng/ml recombinant human fibroblast-like growth factor (rhFGF; Promega), can be used in lieu of the L15 growth medium.
Myogenic Differentiation of Adult Zebrafish Myogenic Progenitor Cells
Approximately 300,000 cells/well were plated into six-well 0.1% gelatin-coated plates in 2 ml of growth medium and grown to 95% confluence. The medium was then changed to differentiation medium consisting of: 2% horse serum (Gibco) in Dulbecco modified Eagle medium (DMEM; Mediatech, Inc.) supplemented with 1× antibiotic–antimycotic (Invitrogen) and 1× Glutamax (Invitrogen). The differentiation medium was changed every other day, and cells were monitored for myotube fusion by phase and fluorescent microscopy. Multinucleated myotubes were observed during days 4–7.
The following primary antibodies were used for immunohistochemistry of zebrafish myogenic progenitor cells: Pax3 mouse monoclonal (1:25; Developmental Studies Hybridoma Bank); Pax7 mouse monoclonal (1:25; Developmental Studies Hybridoma Bank); anti-MyoD1 rabbit polyclonal (1:50; Santa Cruz Biotechnology); and anti-myogenin rabbit polyclonal (1:50; M-225; Santa Cruz Biotechnology). The myogenin antibody has been characterized previously in early zebrafish myogenic progenitor cells.18
The zebrafish myod1 epitope has been shown to be recognized by the myf5 antibody (Santa Cruz Biotechnology).19
Approximately, 100,000 cells were pre-plated on uncoated coverslides (Nunc, Lab-Tek) and, after a 1-hour pre-plating, the supernatant was plated onto 0.1% gelatin-collated coverslips. The following day, the zebrafish myogenic cells attached were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at 4°C for 10 minutes. To block nonspecific binding of the antibodies, slides were incubated for 30 minutes at room temperature in PBS + 10% goat serum. After blocking, the slides were incubated overnight at 4°C using the primary antibodies. Slides were washed three times in 1× PBS, and sections were incubated with Alexa 488 (anti-mouse IgG)- or 568 (anti-rabbit IgG)-conjugated goat secondary antibodies (Invitrogen) at a 1:500 dilution for 45 minutes at room temperature. The slides were then washed three times in 1× PBS before mounting in Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Slides were analyzed by microscope (E1000 Nikon Eclipse; Nikon) and OpenLab software.
RNA Isolation and Microarray Analysis
RNA was extracted directly from zebrafish myogenic progenitor cells in culture at various stages of differentiation using Tripure (Roche Applied Science), following the manufacturer's protocol. Zebrafish cDNA was hybridized to the Affymetrix GeneChip Zebrafish Genome Array (GenBank Release 36.0, June 2003) and processed following the manufacturer's protocol at the Molecular Genetics Core Facility at Children's Hospital Boston. The resulting. CEL files, which contain probe signal intensities of the samples, were preprocessed and normalized together using robust multiarray averaging (RMA), which returns the expression level of each probe set or gene as a positive real number in logarithmic base 2 scale.20
The complete microarray data are available from the NCBI Gene Expression Omnibus (GEO) as GSE19754.
Principal component analysis (PCA) was used to survey gene variation across sample (time) and space, and sample variation across transcriptome space, separately.21
Because most of the time-points had replicate sample measurements, we computed the linear correlation between the unlogged replicate time profiles (A, B) for each probe set to assess the reproducibility of their time profile. We selected the probe set with the maximum replicate time profile correlation as the unique representative for genes with more than one probe set representative. The fold change of a probe set for days 10–14 vs. days 0–1 was computed as the average RMA signal of days 10–14 minus the average RMA signal of days 0–1. This fold change is in log base 2 scale, because the RMA signal is in log base 2 scale. Gene ontology (GO) enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID 6.7; http://david.abcc.ncifcrf.gov) on the mouse homologs of zebrafish genes, because the ontological characterization of genes is currently richer for the mouse than for the zebrafish.22
We used the mouse C2C12 myogenic differentiation microarray dataset (GEO, GSE19968) for comparative genomic analysis.23
Quantitative Real-Time Polymerase Chain Reaction
Total RNA (1 μg) was extracted from the zebrafish muscle myogenic progenitor cells in culture at various time-points during differentiation and subjected to reverse transcriptase using the First Strand Synthesis Kit (Invitrogen). cDNA was then diluted in sterile water into tenfold serial dilutions, and real-time polymerase chain reaction (PCR) was performed (SYBR Green Master Mix; Applied Biosystems). Gene-specific primers that overlapped introns were used (refer to Supplementary Material, Table S5). All samples were amplified on a light cycler (Model 7900HT; ABI). Cycle time (CT) values were normalized to a zebrafish ef1α loading control. All significant values were determined using Student t-tests (two-tailed).