GATA4 and GATA5 are essential for heart and liver development in Xenopus embryos

Background: GATA factors 4/5/6 have been implicated in the development of the heart and endodermal derivatives in vertebrates. Work in zebrafish has indicated that GATA5 is required for normal development earlier than GATA4/6. However, the GATA5 knockout mouse has no apparent embryonic phenotype, thereby questioning the importance of the gene for vertebrate development. Results: In this study we show that in Xenopus embryos GATA5 is essential for early development of heart and liver precursors. In addition, we have found that in Xenopus embryos GATA4 is important for development of heart and liver primordia following their specification, and that in this role it might interact with GATA6. Conclusion: Our results suggest that GATA5 acts earlier than GATA4 to regulate development of heart and liver precursors, and indicate that one early direct target of GATA5 is homeobox gene Hex

Cyclin D2 is a GATA4 cofactor in cardiogenesis

Cyclin D2 is a cell cycle regulator with spatially restricted expression. Loss and gain of function in animal models also revealed a role in cell differentiation, but the mechanisms underlying this are incompletely understood. The cardiogenic transcription factor GATA4 is an upstream regulator of cyclin D2. We show that GATA4 and cyclinD2 are part of a forward reinforcing loop in which cyclin D2 feeds back to enhance GATA4 activity through direct interaction. Mutations in GATA4 that abrogate cyclin D2 interactions are associated with human congenital heart disease. The results unravel a unique transcriptional role of cyclin D2 that may underlie its cell specificity. The finding that cyclin D2 is a cardiogenic GATA4 cofactor may be exploitable therapeutically for heart repair

NKCC1 (SLC12a2) induces a secondary axis in Xenopus laevis embryos independently of its co-transporter function

NKCC1 is a broadly expressed Na(+)-K(+)-Cl(-) co-transporter involved in regulation of ion flux across the cell membrane and in regulating cell volume. Whilst much is known about the co-transporter activity of NKCC1 and its regulation by protein kinases and phosphatases, little is known about the activities of NKCC1 that are co-transporter independent. In this report we show that over-expression of NKCC1 in embryos of Xenopus laevis induces secondary axes, independently of its co-transporter activity. In addition, over-expression of NKCC1 results in the formation of neural tissue in ectodermal explants. We also show that NKCC1 is expressed broadly but non-uniformly in embryos of Xenopus laevis and Xenopus tropicalis, with prominent expression in the notochord, nervous system and stomach. These results provide insights into an additional, previously unreported activity of NKCCl.</p

GATA4 and GATA5 are essential for heart and liver development in embryos-3

H 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 10–15 minutes later the embryos were injected with 50 ng of indicated MOs. E-Uninjected embryos. 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). 10 ng/embryo of C2 MO has no effect on heart in embryos, but the same dose of G4 MO causes heart defects. G4SP MO causes dose-dependent splicing out of exon 4 in both and . 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. 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.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.<p><b>Copyright information:</b></p><p>Taken from "GATA4 and GATA5 are essential for heart and liver development in embryos"</p><p>http://www.biomedcentral.com/1471-213X/8/74</p><p>BMC Developmental Biology 2008;8():74-74.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2526999.</p><p></p

GATA4 and GATA5 are essential for heart and liver development in embryos-7

H anti-HA antibody, is blocked by G5 but not by other MOs indicated. G5UTR MO ("G5U") does not affect translation of GATA5-GR.HA, as this constructs lacks the 5'UTR sequence. 1 ng of mRNA was injected into 1- or 2-cell embryos and 10–15 minutes later the embryos were injected with 10 ng of G5, or 50 ng of G5UTR or G4 MOs. E-Uninjected embryos. Injection of 5 ng of G5 MO results in a loss (B1,2) or severe reduction (B3-5) of cardiac and liver precursors, as revealed by whole-mount in situ hybridisation for MLC2 (purple) and Hex (turquoise). Posterior injection of G5 MO (5ng) has no obvious effects (B6). Control MOs 1 or 2 (C1 or C2, 50 ng/embryo) have no effect on normal development of heart and liver precursors (B7-9). B1-4, 8,9: ventral view; B5-7: lateral view (anterior to the left). In B3-5 arrows point to the remnants of the heart. G5 MO (1 ng/embryo) causes heart defects in () embryos (arrow points to the heart remnant), and C1 MO (10 ng) has no effect on normal heart development (arrowhead in C2). Complete bleaching of embryos has removed the morphological landmarks, and cement gland (cg) and eyes (e) have been indicated to add visualisation.<p><b>Copyright information:</b></p><p>Taken from "GATA4 and GATA5 are essential for heart and liver development in embryos"</p><p>http://www.biomedcentral.com/1471-213X/8/74</p><p>BMC Developmental Biology 2008;8():74-74.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2526999.</p><p></p

GATA4 and GATA5 are essential for heart and liver development in embryos-2

S 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. 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.<p><b>Copyright information:</b></p><p>Taken from "GATA4 and GATA5 are essential for heart and liver development in embryos"</p><p>http://www.biomedcentral.com/1471-213X/8/74</p><p>BMC Developmental Biology 2008;8():74-74.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2526999.</p><p></p

GATA4 and GATA5 are essential for heart and liver development in embryos-8

Ected 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. 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. 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). : 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. 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.<p><b>Copyright information:</b></p><p>Taken from "GATA4 and GATA5 are essential for heart and liver development in embryos"</p><p>http://www.biomedcentral.com/1471-213X/8/74</p><p>BMC Developmental Biology 2008;8():74-74.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2526999.</p><p></p

GATA4 and GATA5 are essential for heart and liver development in embryos-5

Xtran (red fluorescence). The drawing of ventral view of Nieuwkoop and Faber stage 41 embryo [] is from . Cardiac actin/GFP embryos were injected at 32-cell stage with indicated MOs and the fate of injected cells was recorded at st. 41 by rhodamine fluorescence . GFP fluorescence was used to observe heart development (cardiac actin/GFP is expressed in all striated muscles, and head muscles are visible in all images), and GFP images were merged with rhodamine images to highlight injected areas. Heart is indicated by arrows, and in specimens where heart was partially injected , uninjected area is highlighted by an arrowhead. All properly targeted embryos were similarly affected by G4SP and G5SP MOs (n = 8, 10, respectively), but not by C1 MO (n = 7). Ventral views are shown, with anterior at the top.<p><b>Copyright information:</b></p><p>Taken from "GATA4 and GATA5 are essential for heart and liver development in embryos"</p><p>http://www.biomedcentral.com/1471-213X/8/74</p><p>BMC Developmental Biology 2008;8():74-74.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2526999.</p><p></p