Sp6 mmessage mmachine system

pCS2-Lifeact-GFP and pCS2-MRLC-GFP [gifts from Drs Noriyuki Kinoshita (National Institute for Basic Biology, Japan) and Yasuyuki Fujita (The University of Hokkaido, Japan), respectively] were used as templates for mRNA synthesis. Lifeact-GFP and MRLC-GFP mRNAs were synthesized using the SP6 mMessage mMachine System (Thermo Fisher Scientific). Lifeact-GFP mRNA (100 pg) or Myosin II regulatory light chain-GFP (MRLC-GFP) mRNA (200 pg) were injected into the yolk of one-cell-stage zebrafish embryos, as described previously (Matsui et al., 2005 (link)).

The Erk biosensor Eevee-ERKnls comprises an enhanced cyan-emitting mutant of GFP (ECFP), a WW domain, an EV linker, an Erk substrate, a yellow fluorescent protein for energy transfer (Ypet), and a nuclear localization signal (NLS)^{22 (link)}. When Erk phosphorylates the Erk substrate, the WW domain binds to the Erk substrate, thereby bringing ECFP closer to Ypet and inducing FRET from ECFP to Ypet. The pCS2-Erk biosensor subcloned from pPBbsr2-3594NLS^{22 (link)} was used as a template for mRNA synthesis. Erk biosensor mRNAs were synthesized using the SP6 mMessage mMachine system (Thermo Fisher Scientific).

We injected 200 pg ytip-mCherry mRNA (Fukui et al., 2014 (link)), 100 or 200 pg zebrafish-smad7 mRNA, 1.2 ng lats1-atg MO (5′-CCTCGGGTTTCTCGGCCCTCCTCAT-3′) (Chen et al., 2009a (link)), 1.2 ng lats2-atg MO (5′-CATGAGTGAACTTGGCCTGTTTTCT-3′) (Chen et al., 2009a (link)), 8 ng ajuba-atg MO (5′-TGAGTTTGATGCCAAGTCGATCCAT-3′) (Witzel et al., 2012 (link)), and 5 ng control MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) as previously reported (Fukui et al., 2014 (link)). These morpholinos were purchased from Gene Tools (Philomath, OR). Capped mRNAs were synthesized using the SP6 mMessage mMachine system (Thermo Fisher Scientific). Microinjection was performed using FemtoJet (Eppendorf, Hamburg, Germany). MOs, mRNA, and Tol2 plasmids were injected into blastomeres at the one- to two-cell stage.