Morpholino injection

To knockdown zebrafish Greb1 expression, we injected 2 nl of the following splicing-blocking morpholinos into 1–2-cell embryos at 2–8 ng/µl in 0.4 mM MgSO₄, 0.6 mM CaCl₂, 0.7 mM KCl, 58 mM NaCl, 5 mM HEPES pH 7.6; 0.05% Phenol Red: (M1) 5′-GGAAGACTGTAAAAGCTCACCCTCA-3′, (M2) 5′AATACTGAAATCACACCTCTCCTCC-3′ (Fig. S1; Gene Tools, Philomath, OR, USA). Control injections used a mutated M2 oligo (MM) with five nucleotide mismatches: 5′-AATAGTCAAATCAGACCTGTGCTCC-3′. To test for non-specific toxicity, 4 ng/µl of blocking or control morpholino was co-injected with 6 ng/µl p53 antisense morpholino (Robu et al., 2007). Efficacy and specificity were tested by sizing and sequencing RT-PCR products of total RNA from morpholino-injected embryos, SuperScript III One-Step RT-PCR mix (Invitrogen, #12574035).