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Figure 4. GATA4 MOs cause defects in heart and liver development. A: Translation of injected GATA4-GR.HA mRNA, detected by Western blotting with anti-HA antibody, is blocked by G4 but not by other MOs indicated. 1 ng of mRNA was injected into 1- or 2-cell embryos and 105 minutes later the embryos were injected with 50 ng of indicated MOs. E-Uninjected embryos. B: Injection of 50 ng/embryo of G4 MO leads to a reduction in heart and liver precursors and to cardiac morphogenesis defects such as cardia bifida, highlighted by arrows in B3 (B1-5). The same dose (50 ng/embryo) of the C2 MO has no effect on heart and liver development (B6,7). B1-3,5,7: ventral view. B4,6: lateral view. Heart and liver precursors were revealed by MLC2 and Hex probes (BM purple and BCIP, respectively). C: 10 ng/embryo of C2 MO has no effect on heart in X. tropicalis embryos, but the same dose of G4 MO causes heart defects. D: G4SP MO causes dose-dependent splicing out of exon 4 in both Xenopus laevis and Xenopus tropicalis. The injected dose is indicated (in ng). 3, cDNA that contains exon 4 and regions of exons 3 and 5 determined by target sites of the primers; 3, cDNA without exon 4. Below are shown the sequences of the wt 3 and 3 cDNAs showing in-frame splicing in both species. E (top): Injection of 80 or 100 ng/embryo of G4SP MO, but not of 80 ng of C1 MO, causes splicing out of exon 4 with ~90% efficiency, as detected by RT-PCR analyses of mRNA from st. 15 embryos. +/- RT-indicates presence or absence of Reverse Transcriptase in samples that were analysed by PCR.E (bottom): 80 ng of C1 MO has no effect on heart and liver development (E1), whereas the same amount of the G4SP MO causes cardia bifida and liver defects (E2-4). In E2 remnants of cardiac tissue detected by weak expression of cardiac marker in severely affected embryo with cardia bifida are shown by arrows. Ventral views are shown. Heart was labelled by cTnI (purple) and liver with Hex (light blue/turquoise) probes. |
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Figure 2. 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, cDNA that contains exon 4 and regions of exons 3 and 5 determined by target sites of the primers; 3, cDNA without exon 4. Below are shown the sequences of the wt 3 and 3 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) and concomitant increase in the level of the mRNA that lacks exon 4 (3), 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. |
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Figure 3. Summary of effects of GATA4 and GATA5 MOs in Xenopus embryos. A: Examples of phenotypic classes caused by GATA5 and GATA4 MOs. Ventral views of embryos injected with G5UTR MO or G4 MO are shown (50 ng/embryo). Heart and liver precursors have been revealed by MLC2 (BCIP) and FOR1 (BM purple) probes, respectively. FOR1 was developed first. Ventral views are shown, with anterior at the top. B, C: Summary of frequencies with which the heart and liver phenotypes were observed for GATA5 MOs (B) and for GATA4 MOs (C). The doses of splicing MOs are indicated and for other MOs are as in Figs. 1 and 4. |
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Figure 7. Interaction between GATA4 and GATA6 in heart and liver development. Suboptimal doses of G4 (A, 25 ng) or G6 (B, 5 ng) MOs have only minor effects on heart and liver development at st. 37. 50 ng of G4 MO (C, D) or 10 ng of G6 MO cause heart and liver defects. Similar defects are seen in embryos coinjected with 25 ng of G4 MO and 5 ng of G6 MOs (F). G, H: 50 ng/embryo of C1 MO has no effect on heart and liver. I-frequencies of the phenotypes observed. Heart and liver precursor have been analysed as in Fig. 1 with MLC2 and Hex markers. |
