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

Haworth KE, Kotecha S, Mohun TJ, Latinkic BV - BMC Dev. Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, Wales, UK. haworthk@cardiff.ac.uk

ABSTRACT

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.

Show MeSH
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.
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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). 3–4–5, 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.

Mentions: Our attempts to rescue GATA5 morphants by injection of a wide range of GATA5 mRNA amounts proved inconclusive (data not shown), most likely because overexpression of even small amounts of GATA5 mRNA causes a severe phenotype [24]; data not shown). It is likely that a successful rescue requires the precise regulation of the amount, place and time of exogenous GATA5 expression. We therefore wished to provide an additional line of evidence for the specificity of action of GATA5 MOs by downregulating GATA5 in a way that would enable assessment of the status of endogenous GATA5. To achieve this, a splice site-blocking MO was used and its action was monitored by RT-PCR analyses of mRNA. Using genomic sequence available for X. tropicalis, a splice junction blocking MO (G5SP) was designed which was predicted to cause in-frame skipping of exon 4. This exon encodes the C-terminal Zn finger (Fig. 2). Injection of G5SP MO caused an efficient and dose-dependent reduction in the level of the full-length GATA5 mRNA in X. tropicalis embryos (Fig. 2A). This MO also caused exon skipping in X. laevis (Fig. 2A, B), indicating conservation of intronic sequence at the exon/intron and intron/exon boundaries (currently X. laevis GATA5 genomic sequence is not available to confirm this assumption).


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

Haworth KE, Kotecha S, Mohun TJ, Latinkic BV - BMC Dev. Biol. (2008)

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.
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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). 3–4–5, 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.
Mentions: Our attempts to rescue GATA5 morphants by injection of a wide range of GATA5 mRNA amounts proved inconclusive (data not shown), most likely because overexpression of even small amounts of GATA5 mRNA causes a severe phenotype [24]; data not shown). It is likely that a successful rescue requires the precise regulation of the amount, place and time of exogenous GATA5 expression. We therefore wished to provide an additional line of evidence for the specificity of action of GATA5 MOs by downregulating GATA5 in a way that would enable assessment of the status of endogenous GATA5. To achieve this, a splice site-blocking MO was used and its action was monitored by RT-PCR analyses of mRNA. Using genomic sequence available for X. tropicalis, a splice junction blocking MO (G5SP) was designed which was predicted to cause in-frame skipping of exon 4. This exon encodes the C-terminal Zn finger (Fig. 2). Injection of G5SP MO caused an efficient and dose-dependent reduction in the level of the full-length GATA5 mRNA in X. tropicalis embryos (Fig. 2A). This MO also caused exon skipping in X. laevis (Fig. 2A, B), indicating conservation of intronic sequence at the exon/intron and intron/exon boundaries (currently X. laevis GATA5 genomic sequence is not available to confirm this assumption).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, Wales, UK. haworthk@cardiff.ac.uk

ABSTRACT

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.

Show MeSH