Jiang Y and Evans T (1996), The Xenopus GATA-4/5/6 genes are associated wit...

XB-ART-18423

Dev Biol 1996 Mar 15;1742:258-70. doi: 10.1006/dbio.1996.0071.

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The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis.

Jiang Y , Evans T .

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The GATA family of nuclear factors has been implicated in the regulation of cell type-specific transcription. We report the isolation of the Xenopus GATA-4 and GATA-6 cDNA clones and characterize the expression patterns of the xGATA-4/5/6 genes. By comparing the sequence of the cDNAs with those previously reported from chick and mammalian sources, we conclude that each is conserved across vertebrate evolution as a distinct gene product. Each gene is expressed in differentiated adult heart and gut, but maintains distinct transcript patterns in various other adult tissues. During embryogenesis, each gene displays a similar overlapping distribution of transcripts localized throughout the developing cardiogenic region. The xGATA-4 gene can be detected in dorsal cardiac progenitor rudiments prior to migration. Axis disruption experiments were used to demonstrate that transcription of these genes is intimately associated with the specification of cardiac progenitors. Ectopic expression of each gene is specifically capable of activating during embryogenesis the transcription of the cardiac genes encoding actin and myosin heavy chain alpha. The data are consistent with a primary role for the GATA-4/5/6 genes in regulating heart development.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a gata4 gata5 gata6 gcg myh6 tbx2

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	FIG. 1. The GATA-4, GATA-5, and GATA-6 sequences have been conserved during vertebrate evolution as distinct genes. (A) The predicted amino acid sequence of xGATA-4 and xGATA-6 is shown aligned with that of the previously reported (Kelley et al., 1993) xGATA-5a gene. Arrows indicate the sequences used to design PCR primers used for isolating finger region sequences (1 and 2) or for isolating probes specific for xGATA-4 (3 and 5) or xGATA-6 (4 and 5). The highly conserved GATA factor DNA-binding domain containing two similar but distinct zinc fingers is in bold. (B) The amino acid sequences of various GATA factors were compared using the GAP program (GCG, Inc.). The data is given as (% similarity allowing conservative changes)/(% identity). For each set of alignments, the most similar pair is boxed and confirms that genes are conserved across species. The genes are abbreviated, for example: xG4 Xenopus GATA-4, cG5 chicken GATA-5. (C) The highly conserved DNA-binding domain for all reported GATA-4/5/6 genes is shown. The chicken sequences are from Laverriere et al. (1994), mGATA-4 is from Arceci et al. (1993), xGATA-5 is from Kelley et al. (1993), and rGATA-6 is from Tamura et al. (1993). Amino acids that are diagnostic for each individual gene across species are in bold.
	FIG. 2. Transcription patterns for the xGATA-4/5/6 genes. (A) A semiquantitative RT/PCR assay was used to analyze transcript levels in various tissues of the adult frog. Each gene is most highly expressed in heart and small intestine; note that levels cannot be analyzed between the genes, but only among tissues for a given gene. Lanes are abbreviated: H, heart; SK, skin; LU, lung; K, kidney; SP, spleen; P, pancreas; SI, small intestine; C, colon; ST, stomach; M, skeletal muscle; O, ovary; LI, liver. Additional reactions used primers to detect levels of EF-1a, to demonstrate the presence of cDNA in each reaction. (B) RNA from the indicated stages of development was analyzed by RT/PCR for the relative abundance of each gene product. (C) Northern blots were performed using RNA isolated from adult blood (B), gut (G), heart (H), liver (Li), or lung (Lu). Blots were analyzed using gene-specific probes derived from the xGATA-4/5/6 genes, or the EF- 1a gene, as indicated. Transcript sizes (GATA-4: 5.7 and 4.3 kb, GATA-5: 1.9 kb, GATA-6: 4.4 and 3.8 kb) were estimated using RNA molecular weight standards (not shown). The GATA-4 and GATA-5 blots were exposed 4 days, the GATA-6 blot was exposed for 2 days, and the EF-1a blot was exposed for 12 hr.
	FIG. 3. The xGATA-4/5/6 genes are expressed specifically in the developing cardiogenic region during embryogenesis. Whole mount in situ hybridization assays were performed on albino embryos to analyze the early transcript patterns. (A) Stage 22 embryos show transcripts localized to the developing cardiovascular region at the ventralmidline. (B) Bystage 35/36, transcripts are localized to the beating heart. High-magnification views (right) show that staining is diffuse and throughout the heart region, including endocardium,myocardium, and great vessel endothelium. (C) Early staining pattern detecting xGATA-4 transcripts. At stage 10 (left) RNA is detected along one half of the marginal zone. At stage 12 (center) two distinct rudiments located on opposite sides of the blastopore (bottom) express xGATA-4 transcripts. By stage 30 (right), the cardiac progenitors have migrated and transcripts are localized to the developing heart region at the ventral midline.
	FIG. 4. LiCl treatment results in hyperdorsalized embryos and a concomitant expansion of GATA-4/5/6 expression reflecting the radial distribution of cardiac progenitors. (A) Embryos were treated with LiCl at the 32-cell stage and allowed to develop until untreated siblings had reached stage 22, before being analyzed for GATA-4/5/6 transcripts by whole mount in situ hybridization. Arrows indicate the width (along the A/P axis) of the band of cells expressing the genes around the radius of the embryo. (B) The analysis was performed on similarly treated and staged embryos, using probes specific for cardiac actin (top) or MHCa (bottom). Note that, compared to (A), a relatively ‘‘thin’’ band of cells (most similar to the xGATA-6 pattern) marks the presumptive cardiac skeletal muscle progenitors (arrows), while both of these genes are in addition expressed in a much broader region reflecting presumptive embryonic dorsal mesoderm (left side of each embryo).
	FIG. 5. Ectopic expression of xGATA-4/5/6 genes specifically activates the transcription of cardiac-specific genes but does not disrupt heart development. (A) Quantitative RT/PCR was used to measure transcript levels of the MHCa gene, in RNA derived fromstage 9 embryos. Embryos were injected at the one-cell stage with RNA encoding xGATA-1 (G1), a nonfunctional mutant form of xGATA-4 (mG4), xGATA-4 (G4), xGATA-5 (G5), xGATA-6 (G6), or the anti-sense strand of xGATA-5 (not shown; this gave identical results to mG4). (B) In the same manner, transcript levels were analyzed for the cardiac actin gene. Although these data do not show a significant activation by GATA-6, in multiple experiments we have not detected a significant difference in the ability of each GATA-4/5/6 gene to activate transcription of the target genes. (C) Whole mount in situ hybridization experiments were used to analyze expression patterns of cardiac actin and MHCa in untreated and injected embryos. Top row (c-actin, stage 32) left to right: uninjected, xGATA-4 injected, xGATA-5 injected. Middle row (c-actin, stage 25) left to right: uninjected, xGATA-4 injected. Bottom row (MHCa, stage 32) left to right: uninjected, xGATA-4 injected, xGATA-5 injected. Similar results were obtained for xGATA-6 injected embryos (not shown). Embryos were either wild-type (pigmented) or albino.