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. 2001 Apr 1;15(7):839-44.
doi: 10.1101/gad.875201.

Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors

J D Crispino  1 , M B Lodish, B L Thurberg, S H Litovsky, T Collins, J D Molkentin, S H Orkin
Affiliations

Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors

J D Crispino et al. Genes Dev. .

Abstract

GATA-family transcription factors are critical to the development of diverse tissues. In particular, GATA-4 has been implicated in formation of the vertebrate heart. As the mouse Gata-4 knock-out is early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, we created mice harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2. Gata4(ki/ki) mice die just after embryonic day (E) 12.5 exhibiting features in common with Fog2(-/-) embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. We also infer that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation.

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Figures

Figure 1
Figure 1
Targeting the GATA4–FOG-2 interaction in mice. (A) Structure of the N finger of chicken GATA-1 modeled with DNA. The essential valine is highlighted in red in both the illustration and in the sequence alignment of the murine factors, GATA-1 (V205) and GATA-4 (V217), shown below. Note the high conservation of the residues within the N finger of the two GATA proteins. (B) Partial restriction map of the murine Gata4 wild-type locus (top), the Gata4 knock-in targeting vector (middle), and targeted homologous recombination before excision of the selection cassette (bottom). The targeting construct contains the HSV-tk and neomycin resistance (neoR) genes under the control of the mouse phosphoglycerate kinase (PGK) promoter. Homologous recombination results in replacement of wild-type Gata4 with genomic DNA harboring a substitution of valine to glycine at position 217 in the N finger of GATA-4, as well as the incorporation of neomycin cassette. Gata4 coding exons are shown as empty boxes, whereas the exon used as a probe used for Southern blot analysis is highlighted by a black box. S, SacI; E, EcoRV; B, BglII; N, NotI. (C) Southern blot analysis of ES cell DNA and mouse tail DNA (left panel) showing the presence of heterozygous mutant animals (ki/+). Analysis of E12.5 embryos resulting from an intercross of Gata4 knock-in heterozygotes (ki/+), demonstrating the presence of all expected genotypes (right panel). The wild-type allele (WT) generated a 3.8-kb band after digestion of genomic DNA with BglII. In contrast, the knock-in mutated allele (Ki) generated a much larger fragment because of the replacement of the intronic BglII site with NotI.
Figure 1
Figure 1
Targeting the GATA4–FOG-2 interaction in mice. (A) Structure of the N finger of chicken GATA-1 modeled with DNA. The essential valine is highlighted in red in both the illustration and in the sequence alignment of the murine factors, GATA-1 (V205) and GATA-4 (V217), shown below. Note the high conservation of the residues within the N finger of the two GATA proteins. (B) Partial restriction map of the murine Gata4 wild-type locus (top), the Gata4 knock-in targeting vector (middle), and targeted homologous recombination before excision of the selection cassette (bottom). The targeting construct contains the HSV-tk and neomycin resistance (neoR) genes under the control of the mouse phosphoglycerate kinase (PGK) promoter. Homologous recombination results in replacement of wild-type Gata4 with genomic DNA harboring a substitution of valine to glycine at position 217 in the N finger of GATA-4, as well as the incorporation of neomycin cassette. Gata4 coding exons are shown as empty boxes, whereas the exon used as a probe used for Southern blot analysis is highlighted by a black box. S, SacI; E, EcoRV; B, BglII; N, NotI. (C) Southern blot analysis of ES cell DNA and mouse tail DNA (left panel) showing the presence of heterozygous mutant animals (ki/+). Analysis of E12.5 embryos resulting from an intercross of Gata4 knock-in heterozygotes (ki/+), demonstrating the presence of all expected genotypes (right panel). The wild-type allele (WT) generated a 3.8-kb band after digestion of genomic DNA with BglII. In contrast, the knock-in mutated allele (Ki) generated a much larger fragment because of the replacement of the intronic BglII site with NotI.
Figure 1
Figure 1
Targeting the GATA4–FOG-2 interaction in mice. (A) Structure of the N finger of chicken GATA-1 modeled with DNA. The essential valine is highlighted in red in both the illustration and in the sequence alignment of the murine factors, GATA-1 (V205) and GATA-4 (V217), shown below. Note the high conservation of the residues within the N finger of the two GATA proteins. (B) Partial restriction map of the murine Gata4 wild-type locus (top), the Gata4 knock-in targeting vector (middle), and targeted homologous recombination before excision of the selection cassette (bottom). The targeting construct contains the HSV-tk and neomycin resistance (neoR) genes under the control of the mouse phosphoglycerate kinase (PGK) promoter. Homologous recombination results in replacement of wild-type Gata4 with genomic DNA harboring a substitution of valine to glycine at position 217 in the N finger of GATA-4, as well as the incorporation of neomycin cassette. Gata4 coding exons are shown as empty boxes, whereas the exon used as a probe used for Southern blot analysis is highlighted by a black box. S, SacI; E, EcoRV; B, BglII; N, NotI. (C) Southern blot analysis of ES cell DNA and mouse tail DNA (left panel) showing the presence of heterozygous mutant animals (ki/+). Analysis of E12.5 embryos resulting from an intercross of Gata4 knock-in heterozygotes (ki/+), demonstrating the presence of all expected genotypes (right panel). The wild-type allele (WT) generated a 3.8-kb band after digestion of genomic DNA with BglII. In contrast, the knock-in mutated allele (Ki) generated a much larger fragment because of the replacement of the intronic BglII site with NotI.
Figure 2
Figure 2
Heart defects in Gata4 mutant (ki/ki) embryos. (A,B) Wild-type (A) and mutant (B) embryos at E13.5 showing edema and peripheral hemorrhaging in a mutant. (C,D) Transverse sections through wild-type (C) and mutant (D) hearts at E13.5 at the level of the atrioventricular (AV) junction show enlarged atria, thin myocardium, and the absence of a ventricular septum. Original magnification, 40×. (E,F) Transverse sections of wild-type (E) and mutant (F) hearts at the level of the aortic and pulmonary outflow tracts. Gata4ki/ki hearts have a double outlet right ventricle, in which all blood exits the heart into both great arteries, the pulmonary artery and the aorta. The left ventricle, which normally delivers blood to the aorta, fails to communicate with an artery in the mutant. Also note the apparent increase in cellularity of both outflow tracts and semilunar valves in the mutant. Original magnification, 400×. (G,H) Transverse sections of wild-type (G) and mutant (H) hearts at the level of the AV junction. Gata4ki/ki hearts form a common AV valve that is situated between the left and right ventricles. For comparison, the mitral (MV) and tricuspid (TV) valves of the wild-type heart are indicated by arrowheads. Original magnification, 100×.
Figure 3
Figure 3
Expression of Gata4 in the heart. Sagittal sections of wild-type (A–D) and Gata4 ki/ki (E–H) embryos at E12.5 were stained with an α-GATA-4 antibody. Both wild-type and mutant hearts display similar staining within the semilunar and AV valve cells. Note the staining of outflow tracts in both the wild-type and the mutant heart. At, atrium; AV, atrioventricular; V, ventricle. Original magnification, A,E,G, 40×; B,D,F,H, 400×; C, 100×.
Figure 4
Figure 4
Aberrant expression of coronary vessel and myocardial transcripts. (A) Staining of E12.5 wild-type (+/+) and mutant (ki/ki) hearts with an α-Flk-1 antibody (dorsal view). Staining of the mutant is vastly reduced within the heart, but lung tissue stained with equal intensity. (B) Immunostaining of wild-type and mutant hearts at E12.5 using the α-ICAM-2 antibody (dorsal view). Note the absence of a well-developed vascular tree in the mutant heart. (C) Whole-mount RNA in situ staining of eHand in E11.5 hearts. eHand expression is down-regulated in the outer myocardial layer (white arrows), whereas there is more intense staining in the outflow tract of the mutant (dark arrows). Note that the direction of the outflow tract relative to the heart is altered in the mutant, consistent with the pathological findings.
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