Limits...
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

Related in: MedlinePlus

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 10–15 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). 3–4–5, 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. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2526999&req=5

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 10–15 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). 3–4–5, 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. 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.

Mentions: GATA4 was shown in both zebrafish [12] and mouse [11,26,27] to be essential for heart and liver development following specification, but its role in Xenopus has not been investigated in detail. To address this question we designed a MO targeted against both X. laevis alleles and X tropicalis GATA4 (Additional File 1). This MO, G4, causes specific blocking of translation of injected mRNA (Fig. 4A) and results in heart and liver defects (Fig. 4B and also Fig. 3). The majority of G4 morphants had heart morphogenesis defects but had detectable heart and liver gene expression (Figs 3, 4). However, in these experiments the status of endogenous GATA4 in G4 morphants was unknown. Therefore, we additionally tested the role of GATA4 by using a splice-blocking MO, G4SP, which was designed like the G5SP MO to cause in-frame removal of exon 4 in its target. As shown in Fig. 4D, G4SP MO caused a dose-dependent splicing out of exon 4 in both X. tropicalis and X. laevis, resulting in heart and liver defects. G4SP morphants closely resembled G4 morphants (Fig. 4). However, the maximum activity of the G4SP MO (~90% efficiency in inducing splicing) was reached at 80 ng/embryo (Fig. 4D, E), unlike G5SP MO, which was effective in causing complete splicing above 18 ng/embryo. Further increase in the dose of the G4SP MO to 100 and 120 ng/embryo did not improve the efficiency of splicing (Fig. 4E and data not shown). This result was observed in both X. laevis and X. tropicalis (Fig. 4C) and therefore likely represents inherent property of the G4SP MO, rather then inefficient action caused by sequence divergence. G4SP MO did not cause splicing out of exon 4 in GATA5 mRNA (Fig. 4D).


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)

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 10–15 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). 3–4–5, 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. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2526999&req=5

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 10–15 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). 3–4–5, 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. 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.
Mentions: GATA4 was shown in both zebrafish [12] and mouse [11,26,27] to be essential for heart and liver development following specification, but its role in Xenopus has not been investigated in detail. To address this question we designed a MO targeted against both X. laevis alleles and X tropicalis GATA4 (Additional File 1). This MO, G4, causes specific blocking of translation of injected mRNA (Fig. 4A) and results in heart and liver defects (Fig. 4B and also Fig. 3). The majority of G4 morphants had heart morphogenesis defects but had detectable heart and liver gene expression (Figs 3, 4). However, in these experiments the status of endogenous GATA4 in G4 morphants was unknown. Therefore, we additionally tested the role of GATA4 by using a splice-blocking MO, G4SP, which was designed like the G5SP MO to cause in-frame removal of exon 4 in its target. As shown in Fig. 4D, G4SP MO caused a dose-dependent splicing out of exon 4 in both X. tropicalis and X. laevis, resulting in heart and liver defects. G4SP morphants closely resembled G4 morphants (Fig. 4). However, the maximum activity of the G4SP MO (~90% efficiency in inducing splicing) was reached at 80 ng/embryo (Fig. 4D, E), unlike G5SP MO, which was effective in causing complete splicing above 18 ng/embryo. Further increase in the dose of the G4SP MO to 100 and 120 ng/embryo did not improve the efficiency of splicing (Fig. 4E and data not shown). This result was observed in both X. laevis and X. tropicalis (Fig. 4C) and therefore likely represents inherent property of the G4SP MO, rather then inefficient action caused by sequence divergence. G4SP MO did not cause splicing out of exon 4 in GATA5 mRNA (Fig. 4D).

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
Related in: MedlinePlus