Single and double, fluorescent and non-fluorescent, in situ hybridization and immunohistochemical stainings were performed using standard protocols. To analyze the ltbp3 loss of function phenotype, we injected anti-sense ltbp3 morpholinos into one-cell stage WT and transgenic embryos. For genetic lineage tracing, a transgenic driver strain expressing Cre recombinase in ltbp3+ cells and four Cre-responsive “color switching” reporter strains were generated using standard methods. The driver strain was crossed individually to each of the reporter strains and their double transgenic progenies were analyzed for ZsYellow protein fluorescence using confocal microscopy. To follow the migration of zebrafish SHF cells, a tracking dye was injected into the ZsYellow+, AmCyan− region of Tg(nkx2.5::ZsYello); Tg(cmlc2::CSY) embryos at 24hpf, the embryos were imaged immediately, and then again at 48 and/or 72hpf. A transgenic strain carrying a cDNA encoding a constitutively active human TGFβ type I receptor (caALK5) under control of the zebrafish heat shock promoter was generated and used to rescue the myocardial defect in ltbp3 morphant embryos.
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