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Comparative Study
. 2008 May 15;317(2):614-9.
doi: 10.1016/j.ydbio.2008.03.013. Epub 2008 Mar 20.

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice

Roong Zhao  1 , Alistair J Watt, Michele A Battle, Jixuan Li, Benjamin J Bondow, Stephen A Duncan
Affiliations
Comparative Study

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice

Roong Zhao et al. Dev Biol. .

Abstract

Despite significant advances in identifying signaling molecules that induce cardiogenesis in mammals, the transcription factors that control the onset of cardiac myocyte gene expression have remained elusive. Candidates include the zinc finger transcription factors GATA binding proteins 4 and 6 (GATA4, GATA6). The individual loss of either protein in mice results in lethality prior to the onset of heart development due to defects in the extra-embryonic endoderm; however, when this extra-embryonic deficiency is circumvented using tetraploid embryo complementation, cardiac myocyte differentiation initiates normally. Here we show that these factors have redundant roles in controlling the onset of cardiac myocyte differentiation. As a consequence, Gata4(-/-)Gata6(-/-) embryos completely lack hearts, although second heart field progenitor cells are still generated. Our data support a model whereby GATA4 or GATA6 are essential for expression of the network of transcription factors that regulate the onset of cardiac myocyte gene expression during mammalian development.

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Figures

Figure 1
Figure 1. Loss of both GATA4 and GATA6 disrupts cardiac myocyte gene expression in ES cell embryoid bodies
a,b, Schematic overview of the strategy used to generate Gata4−/− Gata6−/− ES cells. Genomic structure of wild type (WT) and mutated (MUT) GATA4 (a) and GATA6 (b) alleles with exons presented as open boxes, the loxP site as a closed circle, and the Pgk-Neo cassette as a shaded box. Position of Southern blot probes and sizes of relevant genomic DNA fragments following digestion with EcoRI(E) or BamHI(B) are indicated. c, Southern blot confirming the genotype of ES cell lines with DNA fragment sizes shown in kb. d, Graph showing percentage of contractile embryoid bodies generated from control (R1) and experimental Gata4−/−Gata6+/+, Gata4+/+ Gata6−/−, Gata4−/− Gata6+/−, Gata4−/− Gata6−/− (clones 1 and 2) ES cells in three independent experiments (error bars). e, RT-PCR analysis of steady-state cardiac mRNA levels in control and GATA knockout embryoid bodies. Polr2a; RNA Polymerase II, Actc1; cardiac alpha actin, Cnn1;Calponin, Myh6;cardiac alpha myosin heavy chain, Myh7;cardiac beta myosin heavy chain, Myl2;cardiac myosin light chain 2v, Myl7;myosin light chain 2a. f, Table showing changes, determined by real-time quantitative RT-PCR analyses, in abundance of mRNAs encoding cardiac transcription factors.
Figure 2
Figure 2. Loss of both GATA4 and GATA6 precludes development of the heart
a, Distribution of Gata4 and Gata6 mRNAs detected in E7.5, early headfold stage, and E8.5, 6–8 somite stage, embryos by in situ hybridization. b, Micrograph showing typical E8.5 embryos generated from either control (G4+/+ G6+/+) or GATA4/6 doubly null (G4−/− G6−/−) ES cells from which the yolk sac has been removed. c, Sections of control or GATA4/6 null embryos stained with hematoxylin and eosin (H&E), by immunohistochemistry for expression of FoxA1, HNF4α, or Pecam (CD31), and by in situ hybridization for Foxa2 mRNA. The presence of cardiac tissue in control embryos is indicated with an arrowhead, the expected location of the heart in mutant embryos with an arrow, and the anterior intestinal portal by an asterisk (*). c, cardiac crescent; 1°,presumptive primary heart field; 2° presumptive second heart field; en, definitive endoderm; ht, primary heart tube; fp, neural tube floorplate; ec, endocardium; ExE, extra embryonic endoderm; v, blood vasculature. The positions of the proximal-distal (prox-dist), anterior-posterior (A–P) and dorsal-ventral (D–V) axes are shown where appropriate.
Figure 3
Figure 3. Differentiated cardiac myocytes are undetectable in GATA4/GATA6 null embryos
Micrographs showing control (G4+/+ G6+/+) and mutant (G4−/− G6−/−) whole embryos or sections of embryos at E8.5 (8–10 somites) or E7.5 (1–2 somites) stained by immunohistochemistry (brown staining) to reveal expression of smooth muscle actin (SMA), myosin heavy chain (MHC), sarcomeric actin (αSrcAct), or by in situ hybridization to identify Smarcd3 mRNA. Cardiac myocytes are indicated with an arrowhead in control embryos and the expected position of such cells with an arrow in mutant embryos. Anterior-posterior axis (A-P) and extra-embryonic endoderm (ExE) is shown where appropriate.
Figure 4
Figure 4. GATA4 and GATA6 are dispensable for formation of second heart field cardiac progenitor cells
Micrographs showing the expression of Isl1 in E8.5 (8–10 somites) and E7.5 (early headfold) embryos, and Nkx2.5 and Tbx5 in E8.5 embryos (8–10 somites) by in situ hybridization. Arrowheads and arrows show the presence of presumptive cardiac progenitor cells in control and mutant embryos, respectively. Sections through Isl1 and Nkx2.5–stained embryos confirm the presence of these mRNAs in the mesoderm. All panels show ventral views of embryos with the proximal-distal axis positioned from top to bottom.
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