Kelley C et al. (1993), GATA-4 is a novel transcription factor expresse...

XB-ART-22460

Development 1993 Jul 01;1183:817-27.

Show Gene links Show Anatomy links

GATA-4 is a novel transcription factor expressed in endocardium of the developing heart.

Kelley C , Blumberg H , Zon LI , Evans T .

???displayArticle.abstract???
We have isolated and characterized Xenopus cDNA clones for a new transcription factor that represents an early marker for the developing heart. The cDNAs encode a protein that we have designated GATA-4; it contains the highly conserved DNA-binding domain that characterizes this family of cell-type restricted transcriptional activators. Whole-embryo in situ analysis of Xenopus embryos demonstrates that the GATA-4 gene is transcribed in presumptive cardiac ventral mesoderm at the time that bilateral progenitors fuse and form the cardiac tube. GATA-4 is therefore the earliest molecular marker of cardiogenesis yet characterized. By stage 30, the GATA-4 mRNA is expressed in the developing atria and ventricles; at stage 38, cross-sections reveal that the gene is active in the endocardial layer, but not in myocardium. By stage 40, GATA-4 message is detected in the great vessels. In the adult frog, the GATA-4 gene is highly transcribed in heart and gut; lower levels of message are detected in various endoderm-derived tissues and gonads. Expression in the stomach is largely confined to the epithelium. The GATA-4 gene is first activated at stage 11; mRNA is initially present throughout the marginal zone of explants and later partially localized to the ventral marginal zone. GATA-4 mRNA is also detected at high levels in cultured endodermal explants derived from the vegetal region of early embryos. In mesoderm induction experiments, GATA-4 transcription is not induced in animal caps treated with activin or bFGF. The GATA-4 gene may provide a new early marker for studying the inductive processes that lead to the formation of the cardiovascular system and for the specification of the endocardial lineage.

???displayArticle.pubmedLink??? 8076520

???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: fgf2 fubp1 gata4 gata5 gnl3 odc1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. The amino acid sequence of Xenopus GATA-4 as predicted from the cDNA clones is shown compared to the other known Xenopus GATA factors. Numbering refers to amino acids and is relative to xGATA-4a. Brackets indicate the highly conserved DNA-binding domain containing the two characteristic related fingers. Genbank nucleotide accession numbers are xGATA-4a: L13701, xGATA-4b: L13702.
	Fig. 2. GATA-4 binds specifically to a GATA cis-element. Gel mobility-shift assays were performed using a labeled oligomer probe (P) containing a GATA cis-element from the chicken aDglobin promoter [TE72/73 (Evans and Felsenfeld, 1991)]. The probe was incubated in the absence of added extract (-) or with 1 or 2 ml (left to right) of nuclear extract prepared from chicken erythroid cells containing cGATA-1 (RBC), COS cells transfected with the pXM expression vector without an insert (COS), or COS cells transfected with the xGATA-4 expression plasmid (xG4). All other samples included 2 ml of the xG4 nuclear extract in addition to competitor. Competitors were a 30- or 60-fold excess (left to right) of unlabeled TE72/73 oligomer as a specific DNA competitor (Sp), the same amounts of unlabeled TE78/79 (Evans and Felsenfeld, 1991), which is identical to TE72/73 except for a mutation of the GATA motif, as a non-specific DNA competitor (Ns), 1 or 2 ml of pre-immunized rabbit serum (pb), 1 or 2 ml of polyclonal rabbit anti-GATA-4 serum (pAb), 1, 2 or 4 ml of protein A affinity-purified monoclonal antibody directed against GATA-4 (mAb). Note that the complex formed by xGATA-4 (arrow) is of slightly higher mobility compared to that formed with cGATA-1 (arrowhead), consistent with the larger molecular weight of xGATA-4. The asterisk indicates a complex formed to a partial degradation product of GATA-4, which retains binding activity.
	Fig. 3. The GATA-4 gene is transcribed in heart, gonads and endodermal derivatives. Northern blots containing 10 mg of total cellular RNA were hybridized first to a GATA-4 cDNA probe. After exposure, the blots were stripped and reprobed using an EF- 1a cDNA probe to demonstrate RNA integrity. RNA standards (not shown) were used to determine an approximate size of the GATA-4 message as 1.8 kb. The arrow indicates a faint but reproducible slightly larger message found in ovary and testis. Additional minor higher molecular weight RNA species are evident in tissues that express high levels of GATA-4. Small intestine was divided into duodenum (f, female; m, male) and a distal or proximal (prox) segment of the ileum. Additional tissues were derived from 2-week-old tadpoles (tp). In the experiment shown, transcription in the lung was unusually high. Also, although not obvious in the reproduction, the GATA-4 message was consistently detected in liver.
	Fig. 4. GATA-4 is highly transcribed in gut epithelium. Sections of adult Xenopus stomach were hybridized in situ to antisense (A-D) or control sense (E,F) 35S-labeled RNA probes derived from the GATA-4a cDNA. (A,C,E) Bright-field photographs; (B,D,F) corresponding dark-field views. Indicated in A are the lumen (l), epithelium (e), submucosa (s) and smooth muscle (m). C is a higher magnification view of the section shown in A. The arrows in C indicate regions of the lumenal crypts with particularly high levels of GATA-4 RNA.
	Fig. 5. Activation of the GATA-4 gene during development. Northern blots were used to detect RNA from whole embryos taken at the indicated stage of development. Each lane contained 20 mg of total RNA. The blot was reprobed for EF-1a message. Note that zygotic EF-1a begins to accumulate at stage 8; GATA-4 is first detected around stage 11.
	Fig. 6. GATA-4 is an early marker for the developing heart. Embryos were analyzed by whole-mount in situ hybridization for GATA-4 RNA. Stages are as follows: 18 (A,B); 26 (C,D); 34 (E,F); 40 (G,H). The photographs shown in B,D,F and H are high magnification views of the embryos shown in A,C,E and G, respectively. Arrows indicate detection of GATA-4 message by alkaline phosphatase staining in the region of the developing heart; non-specific staining around the residual blastocoel (for example, in A) is seen with control sense-strand probes. Note that by stage 40, GATA-4 message is detected in other organs where transcription is high in adults, such as the stomach. In all cases, dorsal is towards the top. In C and D, anterior is to the left, otherwise anterior is to the right. Large arrows in F and H indicate staining in the developing heart (h). For reference, cg indicates the position of the cement gland.
	Fig. 7. The GATA-4 gene is activated in the endocardium of the developing heart. Shown is a sagittal section of a stained stage 38 embryo which has undergone the whole-mount in situ hybridization procedure. Ventral is towards the bottom and anterior is to the left. Note that only the inner layer (endocardium) of the developing heart is stained. In this sample, the endocardial cells have separated from the overlying primitive myocardium in the region consisting of cardiac jelly (arrow).
	Fig. 8. Quantitative RT/PCR analysis of GATA-4 RNA in embryo explants. (A) The GATA-4 gene is activated in the presumptive mesoderm and vegetal explants. Embryos were dissected at stage 8 and explants were incubated in 0.1´ MBS until 30 hours postfertilization. RNA was then prepared and analyzed as described in Materials and Methods. Multiplex PCR employed primers specific for either GATA-4 cDNA or ornithine decarboxylase (ODC) cDNA. The latter transcripts were co-analyzed as an internal control. A, animal caps; V, vegetal explants (three independent experiments are shown); M, presumptive mesoderm; E, whole embryo; NT, no RT included control. (B) The GATA-4 gene is activated in both the ventral marginal zone (VMZ) and the dorsal marginal zone (DMZ). Marginal zones were dissected at stage 12.25 and incubated in buffer alone until control embryos reached the indicated stages. RNA was analyzed as above. Two independent experiments are shown for each VMZ and DMZ explant. In the control lane reaction, RT was omitted.