Microinjection of mRNA into Ascidians and Zebrafish

Microinjections of mRNA in ascidians were carried out as previously described [25] (link). All synthetic mRNA were transcribed with mMachine kit (Ambion). Approximately 30pl of solution at 20 ng/µl were injected per ascidian egg. Fertilized zebrafish eggs were injected manually at the one cell stage, with injection solutions containing 1% phenol red and the specified plasmids at 25 ng/µl. Injected embryos were kept at 28°C and collected for expression analysis at 30 h post fertilization. GFP constructs injected embryos were analysed live under an epifluorescence microscope. lacZ construct injected embryos were fixed and X-gal stained as described previously [45] (link).