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The transcription factors, GATA4, 5 and 6, recognize the same DNA sequence and are all expressed in the developing myocardium. However, knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium. Here we present evidence for redundancy in this family for the first time. Using morpholinos in both Xenopus and zebrafish embryos, we show that GATA4 knockdown, for example, only affects cardiac marker expression in the absence of either GATA5 or GATA6. A similar situation pertains for GATA5 in Xenopus whereas, in zebrafish, GATA5 (faust) plays a major role in driving the myocardial programme. This requirement for GATA5 in zebrafish is for induction of the myocardium, in contrast to the GATA6 requirement in both species, which is for differentiation. This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling, suggesting that a mutual maintenance loop for GATA, BMP and Nkx expression is the evolutionarily conserved entity.
Fig. 1. Cardia bifida is evident in GATA4, 5 and 6 morphants, but cardiac gene expression is only affected in GATA6 depleted Xenopus embryos. (A) Cardiac tissue stained for MLC fails to migrate to the midline in embryos injected singly with GATA4, 5 or 6 morpholinos. (B, C) Expression levels of MLC, Nkx2.5, Tbx5 and CA remain unchanged in GATA4 and 5 morphants compared with control uninjected embryos at stage 28. (D) Expression levels of MLC, Nkx2.5, Tbx5 and CA are substantially decreased in GATA6 morphants at stage 28.
Fig. 2. Functional redundancy between GATA4, 5 and 6. (A) Xenopus embryos were injected singly and in combination with GATA4, 5 and/or 6 morpholinos, harvested at stage 28 and analysed by whole mount in situ hybridisation for MLC and Nkx2.5 expression. Morphant embryos were classified into four classes: wild type (type +, light blue bar), mild down regulation of MLC or Nkx2.5 (type −, mid blue bar), strong down regulation (type −−, purple bar) and no expression (type −−−, dark blue bar). (B, C) Graphical representations of the proportion of embryos in each class.
Fig. 3. Induction of cardiac precursor gene expression is unaffected in morphant Xenopus neurulae. (A) Nkx2.5 was expressed normally at stage 16/17 when GATA4, 5 or 6 were depleted individually or in combination. (B) Cryostat sections confirmed that expression of Nkx2.5 in the cardiac mesoderm (delineated by red dashed lines) was not affected. (C) Expression of Nkx2.3, which unlike Nkx2.5 is restricted to the cardiac mesoderm (D) also remained unchanged in the triple morphants, as does eHAND (E). (Red dashed lines mark the cardiac precursors, remaining stain reflects expression in the blood island mesoderm).
tbx5 (T-box 5) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28, lateral view, anteriorright, dorsal up.
Supplementary Fig. 1. GATA4 MO specifically inhibits the translation of GATA4 RNA and GATA5 MO inhibits the production of endogenous GATA5 RNA. (A) GATA4 MO sequence and the ATG region to which it binds. (B) A Western blot to show the inhibition by GATA4 MO of translation of exogenous HA-tagged GATA4 RNA injected into Xenopus animal caps. (C) A Western blot to show that GATA4 MO specifically inhibits translation of GATA4 RNA injected into Xenopus oocytes, and has no effect on GATA5 or GATA6 translation. (D) Genomic structure of the GATA5 gene. Exons (ex) depicted as boxes, intron sizes not to scale. Splice MO binding site (red line) and primer binding sites for RTâPCR analysis (black lines) are shown. (E) The amplified 260Â bp GATA5 fragment is present at a much lower level in the RNA extracted from embryos injected with 25Â ng MO, indicating that there is a reduced level of correctly spliced GATA5 in the injected embryos. (F) Analysis of vmhc expression at 26Â hpf (dorsal anterior view) and nkx2.5 expression at 10 somites (flat mount, dorsal view) shows that 25Â ng GATA5 MO effectively phenocopues the fausttm236a mutant phenotype, black brackets identify cardiac expression.
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