The following constructs were used in this study: pGF-CMV-GCaMP6s (Chen et al., 2013 (link)), pBH-R4/R2 (Heim et al., 2014 (link)), p5E-bactin2, pCS2FA-transposase (Kwan et al., 2007 ), p5E-ubi (Mosimann et al., 2011 (link)), pCS2+wnt5b (Lin et al., 2010 (link)), and pCS2+cyc/ndr2 (Rebagliati et al., 1998 (link)). The GCaMP6s open reading frame (ORF) was amplified from pGF-CMV-GCaMP6s using Phusion High-Fidelity DNA polymerase (Invitrogen) with GCaMP6s-Forward and -Reverse primers (Table S1 ). The amplified GCaMP6s ORF was cloned into pENTR/D-TOPO (Invitrogen) and sequenced. To generate Tol2 destination constructs, p5E-βactin2 or p5E-ubi promoter sequences were recombined with pENTR/D-GCaMP6s into pBH-R4/R2 using the Gateway LR Clonase II Plus enzyme (Invitrogen).
To generate cyc/ndr2 RNA, pCS2+cyc/ndr2 plasmid was linearized with Asp718I, purified (Qiagen), and used as a template for RNA synthesis with the mMessage mMachine SP6 kit (Ambion). The resulting RNA was purified with the Micro Bio-Spin P-30 columns (Bio-Rad). 50 pg cyc/ndr-2 RNA mixed with 0.1% Texas Red was injected at 64-cell stage to 1–2 marginal blastomeres, or 400 pg was injected at 512-cell stage to the yolk cell. pCS2+wnt5b was linearized with ApaI, and transcribed and purified as described above. 50–100 pg wnt5b synthetic RNA was injected at 1-cell stage.
To generate cyc/ndr2 RNA, pCS2+cyc/ndr2 plasmid was linearized with Asp718I, purified (Qiagen), and used as a template for RNA synthesis with the mMessage mMachine SP6 kit (Ambion). The resulting RNA was purified with the Micro Bio-Spin P-30 columns (Bio-Rad). 50 pg cyc/ndr-2 RNA mixed with 0.1% Texas Red was injected at 64-cell stage to 1–2 marginal blastomeres, or 400 pg was injected at 512-cell stage to the yolk cell. pCS2+wnt5b was linearized with ApaI, and transcribed and purified as described above. 50–100 pg wnt5b synthetic RNA was injected at 1-cell stage.