Musclebow2 fish allow genetic marking of muscle fibres and their precursor cells. Transgenic Musclebow2 fish were selected from an enhancer trap screen with larvae at the indicated stages shown in lateral views with anterior to left, dorsal up. (A) The BB3 and HS:Cre.cryaa:RFP DNA constructs used for making transgenic fish. (B) Dissecting scope view of BB3mus7kg309 yielding tdTomato detectable in a subset of muscle fibres from 1 dpf and HS:Cre.cryaa:RFPkg310 yields RFP in lens from 2 dpf (arrow, top). Germline activity of HS:Cre.cryaa:RFPkg310 yielded lines BB3mus7:EYFPkg311 (EYFP, middle) or BB3mus7:mCeruleankg321 (mCer, bottom) expressing throughout muscle tissue. (C) Maximum intensity projection confocal stack (mip) after heat shock at 30% epiboly yielding subsets of fibres expressing one or two recombined and unrecombined alleles, presumably reflecting mosaic Cre action followed by fusion of mpcs expressing BB3mus7 in different recombination states. Note that recombination yielded EYFP fibres more frequently than mCer fibres and that some fibres remain unlabelled. (D) Three-channel confocal mip stack showing BB3mus7 expression at 3 dpf in morphological slow (cyan arrows) and fast (green arrows) fibres and in mncs (arrowheads) in somites. Note the clustered mncs (green box, short stack shown magnified beneath). (E) Single cluster of mCer-marked mncs (arrowheads) in somite 9. Yellow crosshairs indicate planes in XY, YZ and XZ projections. Inset shows location of cluster (white box). Note absence of mCer mncs in rest of plane. (F) BB3mus7 expression in pectoral fin muscles. (G) Time-lapse analysis of short 20 µm stack mips showing the persistent set of marked fibres (numbered) in the deep region of somites 15 and 16 at 3, 6 and 13 dpf. The 6 dpf stack extends further superficially within the myotome than the 3 and 13 dpf stacks. A cluster of mncs (bracket) appears in the deep myotome between 3 and 6 dpf and forms a nascent fibre (arrowhead) by 13 dpf. Note the loss of tdT in dual labelled fibres (asterisks) and the clonal growth of gut-associated cells (hashes). (H) Time-lapse analysis of somite 11 shows maintenance of overall pattern of fibre labelling but changes in colour of some fibres (arrowheads). Note dramatic increase in EYFP-marked superficial mncs (arrows) in this somite which correlates with accumulation of extra EYFP-marked fibres (upper arrowheads). (I) Confocal maximum intensity projection of 4 dpf BB3mus7:EYFPkg311 larva with strong near-uniform signal in myotomal fibres and stronger signal in some mncs on myosepta (arrows) or within the myotome (arrowheads). Note the strongly marked nascent fibres (magenta arrows). Scale bars: 50 µm.
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