G5SP MO creates GATA5 protein lacking the C-terminal Zn finger and causes heart and liver defects. A: G5SP MO causes dose-dependent splicing out of exon 4 in both Xenopus laevis and Xenopus tropicalis. The injected dose is indicated (in ng). 3–4–5, cDNA that contains exon 4 and regions of exons 3 and 5 determined by target sites of the primers; 3–5, cDNA without exon 4. Below are shown the sequences of the wt 3–4–5 and 3–5 cDNAs showing in-frame splicing in both species. B: G5SP MO causes a dose-dependent reduction in the level of the wt full-length mRNA (including exon 4; 3–4–5) and concomitant increase in the level of the mRNA that lacks exon 4 (3–5), as revealed by RT-PCR with primers based in exons 2 and 4. Injection of 9 ng of G5SP MO causes partial loss (~50%) of wt GATA5 mRNA. The dose in ng used per embryo is given for each MO. -PCR, control with no cDNA input. M-DNA marker. ODC- Orhithine Decarboxylase loading control. Embryos were collected for RNA analysis at st. 15. C: Injection of 9 ng of G5SP MO into the same group of embryos analysed in (B) causes severe reduction of heart and liver st. 37 (C1,2). Injection of 50 ng of C1 MO has no effect on heart and liver development (C3). D: The dGATA5 protein can neither activate transcription nor can it significantly affect the ability of GATA5 or GATA4 to activate a firefly luciferase reporter driven by 2 GATA sites in animal cap explants. Dual luciferase assays were performed 3 hours after excision of explants, and firefly luciferase activity was normalised to renilla luciferase activity resulting from TK-RL DNA. A representative experiment (out of 3) is shown; whilst the levels of induction varied between experiments, the trend (activation by GATA4 or GATA5 and lack of substantial effect by dGATA5) remained consistent. E: Schematic representation of the effect of G4/5SP MOs (exon-specific part shown as red line) on the domain structure of their targets. TAD-Trans Activation Domain; NLS-Nuclear Localisation Signal; N, C-Zn fingers. Below-Western blot showing efficient translation of the dGATA5 protein in embryos, detected with anti-HA antibody. E-uninjected embryos. d, wt-embryos injected with the d- or wtGATA5-GR.HA mRNA, respectively.