BB3mus7 recombination reveals dynamics of mpc clones. Live confocal microscopy of BB3mus7kg309/+; HS:Cre.cryaa:RFPkg310/+ embryos after heat-shock at 24 hpf. (A) Three-channel confocal slice showing BB3mus7 expression in somites 12-15 at 3 dpf in morphological slow (white arrowheads) and fast (other arrowheads) fibres and in mncs (white arrows) in somites. Note the clustered mncs. Examples of individual fibres marked by tdT-only (white arrowheads), EYFP-only (yellow arrowheads), mCer-only (cyan arrowheads), EFYP and tdT (orange arrowheads) and mCer and tdT (magenta arrowheads). Asterisk indicates autofluorescence from xanthophores in the mCer channel. Faint breakthrough of bright EYFP signal into the mCer detection channel that can be seen, for example, in the mncs and the fibre indicated by the orange arrowhead is readily distinguished from genuine mCer. (B) Short stack (22 µm) mip of somites 12-14 at 3 dpf showing recombined mncs (arrows), superficial slow fibres (white arrowheads), muscle pioneers (magenta arrowheads), fast fibres (yellow arrowheads) and unrecombined slow fibres (cyan arrowheads). Note that some fibres contain both tdT and mCer or EYFP proteins, indicating perdurance of tdT after Cre-driven recombination. (C) Quantification of numbers of marked cells in individual somites of a single fish at 3 and 6 dpf, determined from high-resolution confocal stacks. Only somites 11-16 were scored at both timepoints. SSF, superficial slow fibre; MP, muscle pioneer slow fibre; mnc, mononuclear non-fibre cell; Fast, fast fibre with T-tubules; Nascent, thin nascent fibre spanning anteroposterior extent of somite. (D) XYZ slice projections (indicated on mip at top left) of somites 10-13 at 6 dpf showing reduction in mncs and increase in elongated cells spanning the myotome marked with EYFP (white arrowheads) or mCer (blue arrowheads) within the hypaxial myotome. Coloured crosshairs indicate the plane of the orthogonal boxed optical sections. Scale bars: 50 µm.
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