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EVOLUTIONARY HOMOLOGS

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General considerations of the GATA-4/5/6 subfamily

GATA-4 is one of the earliest developmental markers of the precardiac mesoderm, heart, and gut; it has been shown to activate regulatory elements controlling transcription of genes encoding cardiac-specific proteins. To elucidate the molecular mechanisms underlying the transcriptional activity of the GATA-4 protein, structure-function analyses were performed; they reveal that the C-terminal zinc finger and adjacent basic domain of GATA-4 is bifunctional, modulating both DNA-binding and nuclear localization activities. The N terminus of the protein encodes two independent transcriptional Activation Domains (amino acids 1-74 and amino acids 130-177). Amino acid residues were identified within each domain that are required for transcriptional activation. Regions of Xenopus GATA-5 and -6 corresponding to Activation Domains I and II, respectively, function as potent transcriptional activators. The identification and functional characterization of two evolutionarily conserved transcriptional Activation Domains within the GATA-4/5/6 subfamily suggests that each of these domains modulates critical functions in the transcriptional regulatory program(s) encoded by GATA-4, -5, and -6 during vertebrate development. As such these data provide novel insights into the molecular mechanisms that control development of the heart (Morrisey, 1997a).

The GATA-4/5/6 genes encode transcription factors implicated previously in the regulation of cardiac-specific differentiation programs. However, recent analyses of mouse GATA-4 null mutations found evidence for function in endoderm development (in vitro) and embryonic morphogenesis (in vivo). Whether each of the three cardiac-associated GATA factors function within distinct or common developmental programs has been previously untested; past studies defined specific and distinct roles for each of the GATA-1/2/3 genes in embryonic hematopoiesis. In this study, a comparison was made of cGATA-4/5/6 transcript patterns during chick embryogenesis. Each of the three GATA factors is expressed in a similar pattern within gastrulating cells of the primitive streak, prior to determination of the cardiomyocyte progenitors, and later within the lateral plate mesoderm and associated endoderm layer. The patterns overlap but extend beyond the presumptive cardiomyocyte population expressing cNkx-2.5. Later in development, cGATA-4/5/6 are all transcribed throughout the differentiating heart, in similar but not identical patterns, within the endocardium, myocardium, and great vessels. In order to test the function of GATA factors during chick cardiogenesis, embryos were cultured in vitro in the presence of antisense oligomers designed to specifically deplete transcripts encoding cGATA-4/5/6, beginning around stage 7. When oligomers are used to target transcripts for all three genes, a high percentage of the embryos develop abnormal hearts related to the failure to form a normal primitive heart tube. In the most severe phenotype, cardiac bifida results in two bilateral beating hearts. In some embryos, the paired heart primordia undergo partial fusion but fail to form a single looping heart tube. In all cases, cellular differentiation is not obviously affected, as the abnormal hearts form beating tissue. Depletion of transcripts encoding any single GATA factor, or any combination of two GATA factors, does not affect development. The partial depletion of all three genes in chick results in a remarkably similar phenotype compared to the null GATA-4 mutation in mouse. Therefore, in the chick, each of the GATA-4/5/6 genes functions in a common pathway, at the time of cardiac crescent formation, for regulating early embryonic cardiac morphogenesis, apparently associated with embryonic folding or the migration of primordia to form a primitive tube (Jiang, 1998).

Based on conserved expression patterns, three members of the GATA family of transcriptional regulatory proteins, GATA-4, -5, and -6, are thought to be involved in the regulation of cardiogenesis and gut development. Functions for these factors are known in the heart, but relatively little is understood regarding their possible roles in the regulation of gut-specific gene expression. In this study, the expression and function of GATA-4, -5, and -6 was analyzed using three separate but complementary vertebrate systems; the results support a function for these proteins in regulating the terminal-differentiation program of intestinal epithelial cells. xGATA-4, -5, and -6 from Xenopus can directly stimulate promoter activity for the gene coding for the intestinal fatty acid-binding protein (xIFABP), which is a marker for differentiated enterocytes. This is the first direct demonstration of a target for GATA factors in the vertebrate intestinal epithelium. Transactivation by xGATA-4, -5, and -6 is mediated (at least in part) by a defined proximal IFABP promoter element. The expression patterns for chicken cGATA-4, -5, and -6 are markedly distinct along the proximal-distal villus axis. Transcript levels for cGATA-4 increase along the axis toward the villus tip; likewise, cGATA-5 transcripts are largely restricted to the distal tip containing differentiated cells. In contrast, the pattern of cGATA-6 transcripts is complementary to cGATA-5, with highest levels detected in the region of proliferating progenitor cells. Undifferentiated and proliferating human HT-29 cells express hGATA-6 but not hGATA-4 or hGATA-5. When stimulated to differentiate, the transcript levels for hGATA-5 increase: this occurs prior to increased transcription of the terminal differentiation marker intestinal alkaline phosphatase. At the same time, hGATA-6 steady-state transcript levels decline appreciably. All of the data are consistent with evolutionarily conserved but distinct roles for these factors in regulating the differentiation program of intestinal epithelium. Based on this data, it is suggested that GATA-6 might function primarily within the proliferating progenitor population, while GATA-4 and GATA-5 function during differentiation to activate terminal-differentiation genes including IFABP (Gao, 1998).

Interactions between the key regulatory genes of the cardiogenic pathway, including those from the GATA and Nkx2 transcription factor families, are not well defined. Treating neurula-stage Xenopus embryos with retinoic acid (RA) causes a specific block in cardiomyocyte development that correlates with a progressive reduction in the region of the presumptive heart-forming region expressing Nkx2.5. In contrast, RA does not block expression of the GATA-4/5/6 genes, which are transcribed normally in an overlapping pattern with Nkx2.5 throughout cardiogenesis. Instead, GATA-4/5/6 transcription levels are increased, including an expansion of the expression domain corresponding to lateral plate mesoderm that is part of the early heart field, but that normally is progressively restricted in its ability to contribute to the myocardium. GATA-dependent regulatory sequences of the Nkx2.5 gene implicate GATA-4/5/6 as upstream positive regulators. However, experiments indicate that GATA factors might normally antagonize transcription of Nkx2.5. To test this hypothesis, a dominant negative isoform of GATA-4 (SRG4) capable of inhibiting transcription of GATA-dependent target genes was generated. Ectopic expression of SRG4 results in a transient expansion of the Nkx2.5 transcript pattern, indicating that a normal function of GATA factors is to limit the boundary of the Nkx2.5 expression domain to the most anterior ventral region of the heart field. Regulatory mechanisms altered by excess RA must function normally to limit GATA-4/5/6 expression levels, to define the region of Nkx2.5 expression and regulate myocardial differentiation (Jiang, 1999).

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND (Deciduum, Heart, Autonomic Nervous System and Neural Crest Derivatives-Expressed Protein) is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, it has been shown that cardiac and neural crest expression of dHAND are each controlled by separate upstream enhancers: a composite cardiac-specific enhancer is described that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis has reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence has revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolishes transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart (McFadden, 2000).

To test whether GATA4 was uniquely required for enhancer activity in vivo, a 5.5 kb enhancer transgenic line was crossed into a GATA4 mutant background. Analysis of lacZ expression in GATA4 null embryos showed that transgene expression is maintained in the absence of GATA4. GATA4 mutant embryos exhibit cardia bifida due to a defect in ventral morphogenesis of the embryo. Intriguingly, dHAND-lacZ expression is much stronger in the right compared to the left cardiac tube in the mutant, suggesting that the mechanisms that restrict dHAND expression to the right ventricular chamber at the onset of cardiac looping are at least partially intact in these embryos. The finding that the GATA-dependent dHAND enhancer is active in GATA4 mutant embryos suggests that GATA5 or GATA6, which are expressed in an overlapping pattern with GATA4 during cardiac development, can substitute for GATA4 to activate the dHAND enhancer (McFadden, 2000).

The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, nucleosome arrays containing albumin enhancer sequences were assembled and they were compacted with linker histone. HNF3 and GATA-4 but not NF-1, C/EBP, and GAL4-AH, bind their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events (Cirillo, 2002).

How might HNF3 access its binding sites in vivo? Under physiological salt conditions, nucleosome arrays exist in a dynamic equilibrium between folded and compacted states. Additionally, linker histone is rapidly exhanged on chromatin in living cells. HNF3 could exploit both of these properties to initially bind its sites in compacted chromatin. Previously published data indicate that the essential HNF3 binding sites eG and eH flank the dyad axis of the nucleosome particle to which HNF3 binds. This would place HNF3 on the bound particle in the vicinity of where X-ray crystallography data places histones H3 and H4 in the nucleosome. Histones H3 and H4 make internucleosomal contacts which have been implicated in the formation of nucleosomal arrays, and nucleosome arrays lacking the H3/H4 amino-terminal tails fail to fold into a fully compacted state or undergo mitotic chromosome condensation. It is suggested that HNF3 disrupts internucleosomal interactions promoted by H3/H4 tetramers, thereby decompacting the array locally and making it accessible to other proteins. In addition, HNF3 binding helps stabilize the position of an underlying nucleosome, which could affect the length of linker DNA on either side of the N1 particle. Small variations in linker length can have dramatic effects on the compaction of nucleosomes in chromatin. It is therefore suggested that HNF3 disrupts local chromatin structure by a combination of core histone interactions and by inducing changes in the position or orientation of nearby linker regions (Cirillo, 2002).

The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. This study details the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3ß by all three GATA factors is abolished when protein synthesis is inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17alpha and HNF1ß by GATA4 and 5 is substantially reduced when protein synthesis is inhibited, induction by GATA6 is minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induces GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, block induction of Sox17alpha and HNF1ß by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors are induced by activin, although GATA4 and 6 require lower concentrations. GATA MOs inhibit Sox17alpha and HNF1ß induction by activin at low and high concentrations in the order: GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFß/Nodal signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, reveals GATA5 and 6 as the most critical for the development of the gut and the liver (Afouda. 2005).

A loss-of-function model for Gata4 has been examined in zebrafish in order to examine broadly its requirement for organogenesis. The function of Gata4 in zebrafish heart development is well conserved with that in mouse, and, in addition, Gata4 is required for development of the intestine, liver, pancreas and swim bladder. Therefore, a single transcription factor regulates the formation of many organs. Gata6 is a closely related transcription factor with an overlapping expression pattern. Zebrafish depleted of Gata6 show defects in liver bud growth similar to mouse Gata6 mutants and zebrafish Gata4 morphants, and zebrafish embryos depleted of both Gata4 and Gata6 display an earlier block in liver development, and thus completely lack liver buds. Therefore, Gata4 and Gata6 have distinct non-redundant functions in cardiac morphogenesis, but are redundant for an early step of liver development. In addition, both Gata4 and Gata6 are essential and non-redundant for liver growth following initial budding (Holtzinger, 2005).

GATA-4 and heart morphogenesis (part 1/2)

Homozygous GATA4-deficient (GATA4-/-) mice die between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos display severe defects in both rostral-to-caudal and lateral-to-ventral folding, which are reflected in a generalized disruption of the ventral body pattern. This results in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrates that these embryos develop splanchnic mesoderm, which differentiates into primitive cardiac myocytes that express contractile proteins. Therefore GATA4 is not required either for the specification of committed cardiac myocyte progenitors within the splanchnic mesoderm or for the differentiation of atrial or ventricular myocytes and endocardial cells. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes fail to migrate to the ventral midline to form a linear heart tube and instead form aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes is not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populate the hearts of GATA4-/--C57BL/6 chimeric mice. Taken together, these results demonstrate that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis. In this role, GATA 4 is distinct from Nkx2.5, dHAND, and eHAND, any of which are required for the later looping stages of cardiac morphogenesis. Nkx2.5 is expressed normally in GATA4 knockouts, suggesting that it is not regulated directly by GATA4 (Kuo, 1997).

To determine the role of GATA4 in embryogenesis, mice homozygous for a GATA4 null allele were generated. Homozygous GATA4 null mice arrest in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lack a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia fail to migrate ventrally but instead remained lateral and generate two independent heart tubes that contained differentiated cardiomyocytes. These deformities result from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. It is proposed that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the anterior intestinal portal. Approximately one-third of the GATA4 null embryos arrest at the egg cylinder stage and fail to gastrulate. GATA4 is expressed in visceral endoderm, suggesting that it may be required for the proper development of this cell layer. The finding that approximately two-thirds of the GATA4 null embryos progress through gastrulation suggests that there is a critical point in development in which visceral endoderm function can be rescued. Obvious candidates for factors that might compensate for loss of GATA4 are GATA5 and GATA6. GATA6 is up-regulated in GATA4 null embryos (Molkentin, 1997).

The earliest step in heart formation in vertebrates occurs during gastrulation, when cardiac tissue is specified. Dorsoanterior endoderm is thought to provide a signal that induces adjacent mesodermal cells to adopt a cardiac fate. However, the nature of this signalling and the precise role of endoderm are unknown because of the close proximity and interdependence of mesoderm and endoderm during gastrulation. To better define the molecular events that underlie cardiac induction, attempts were made to develop a simple means of inducing cardiac tissue. The transcription factor GATA4, which has been implicated in regulating cardiac gene expression, is sufficient to induce cardiac differentiation in Xenopus embryonic ectoderm (animal pole) explants, frequently resulting in beating tissue. Lineage labelling experiments demonstrate that GATA4 can trigger cardiac differentiation not only in cells in which it is present, but also in neighboring cells. Surprisingly, cardiac differentiation can occur without any stable differentiation of anterior endoderm and is in fact enhanced under conditions in which endoderm formation is inhibited. Remarkably, cardiac tissue is formed even when GATA4 activity is delayed until long after explants have commenced differentiation into epidermal tissue. Thus, cardiomyocytes can be induced in explants of presumptive ectodermal tissue from Xenopus embryos by ectopic expression of GATA4. Expression of cardiomyocyte markers is neither precocious nor selective, indicating that their expression reflects genuine induction of cardiac muscle tissue rather than the selective transactivation of myocardial genes containing GATA-factor binding sites. GATA4 apparently triggers the formation of several mesendodermal tissues, including cardiac, but not skeletal, muscle. These findings provide a simple assay system for cardiac induction that may allow elucidation of pathways leading to cardiac differentiation. Better knowledge of the pathways governing this process may help develop procedures for efficient generation of cardiomyocytes from pluripotent stem cells (Latinkic, 2003).

In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. Cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 is a member of a multigene family containing four members: NF-ATc, FF-ATp, NF-AT3 and NF-AT4. These factors bind the consensus DNA sequence GGAAAAT as monomers or dimers through a Rel homology domain (see Drosophila Dorsal). NF-AT3 interacts with the cardiac zinc finger transcription factor GATA4, resulting in synergistic activation of cardiac transcription. The region of NF-AT3 that interacts with GATA4 extends from amino acid 522, which is near the middle of the Rel homology domain, to the carboxy terminus. Residues 181-328 of GATA4, which encompass the two zinc fingers and the nuclear localization sequence, interact with NF-AT3 as efficiently as full-length GATA4. GATA4 and NF-AT3 interact synergistically in the activation of b-type natriuretic peptide, which decreases blood pressure by vasodilation and natriuresis and is rapidly up-regulated in the heart in response to hypertrophic signals. Transgenic mice that express activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure that mimic human heart disease. Pharmacologic inhibition of calcineurin activity by immunosuppressant drugs cyclosporin A and FK506 blocks hypertrophy in vivo and in vitro. These results define a novel hypertrophic signaling pathway and suggest pharmacologic approaches to prevent cardiac hypertrophy and heart failure (Molkentin, 1998).

GATA-4 knockout mice die by 9.5 days postcoitum and exhibit profound defects in ventral morphogenesis, including abnormal foregut formation and a failure of fusion of the bilateral myocardial primordia. During early mouse development, GATA-4 is expressed in cardiogenic splanchnic mesoderm and associated endoderm, suggesting that the presence of this transcription factor in one or both of these tissue types is essential for ventral development. To distinguish whether GATA-4 expression in mesoderm or endoderm accounts for the phenotype in knockout mice, chimeric mice were prepared by injecting Gata4-/- ES cells into 8-cell stage ROSA26(Gata4+/+) embryos. A series of high percentage null chimeras (8-10 days postcoitum) were found in which Gata4+/+ cells are restricted to visceral yolk sac endoderm and small portions of the foregut/hindgut endoderm. Despite an absence of GATA-4 in all other cells of these embryos, there is normal development of the heart, foregut, and surrounding tissues. It is concluded that expression of GATA-4 in endoderm rather than cardiogenic mesoderm is required for ventral morphogenesis (Narita, 1997a).

In situ hybridization studies, promoter analyses and antisense RNA experiments have all implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. Gata4-/- embryonic stem (ES) cells were used to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. The ability of Gata4-/- ES cells to form cardiomyocytes was assessed during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes are seen in both wild-type and Gata4-/- embryoid bodies, although cardiomyocytes are observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4-/- embryoid bodies reveal the presence of sarcomeres and junctional complexes, while immunofluorescence confirms the presence of cardiac myosin. To assess the capacity of Gata4-/- ES cells to differentiate into cardiomyocytes in vivo, chimeric mice were prepared and analyzed. Gata4-/- ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4-/- ES cells contribute to endocardium, myocardium and epicardium. In situ hybridization shows that myocardium derived from Gata4-/- ES cells expresses several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4 (Narita, 1997b).

Studies have shown that GATA-4 is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. Consistent with a role for GATA-4 in cardiomyocyte formation, inhibition of GATA-4 expression by antisense transcripts interferes with expression of cardiac muscle genes and blocks development of beating cardiomyocytes in P19 embryonic stem cells. In order to better define the function of GATA-4 in cardiogenesis, molecular analysis of early stages of cardiomyocyte differentiation has been carried out in GATA-4-deficient P19 cell lines and in P19 cells stably overexpressing GATA-4. The results indicate that GATA-4 is not required for either endodermal or mesodermal commitment or for initiation of the cardiac pathway. However, in the absence of GATA-4, differentiation is blocked at the precardiac (cardioblasts) stage and cells are lost through extensive apoptosis. In contrast, ectopic expression of GATA-4 in P19 cells accelerates cardiogenesis and markedly increases (over 10-fold) the number of terminally differentiated beating cardiomyocytes following cell aggregation. Together, these findings suggest that in addition to its role in activation of the cardiac genetic program, GATA-4 may be the nuclear target of inductive and/or survival factors for precardiac cells (Grepin, 1997).

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein (Drosophila homolog: Tinman) are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of either GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. Whether or not GATA-4 and Nkx2-5 are cofactors for one another was examined by using transcription and binding assays with the the only known target for Nkx2-5, the cardiac atrial natriuretic factor (ANF) promoter. Co-expression of GATA-4 and Nkx2-5 results in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 in an interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis (Durocher, 1997).

GATA-4 has been implicated in formation of the vertebrate heart. Since 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, mice were created harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2, a homolog of Drosophila U-shaped. Gata4^ki/ki mice die just after embryonic day 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. It is inferred that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation (Crispino, 2001).

The power of this analysis rests on the exquisite specificity of the knock-in mutation within the N finger of GATA-4. The residue that was modified is required for physical interaction with FOG-like proteins and does not influence the DNA-binding specificity of the GATA-protein. Although displaying many similar features, Gata4^ki/ki hearts are distinguished from Fog2-/- hearts, however, by the presence of a double-outlet right ventricle and defects in the semilunar valves and outflow tracts. Since immunostaining confirms that GATA-4 is expressed at wild-type levels in the semilunar valves of the Gata4 mutant heart, it is likely that another FOG, or FOG-like protein, that functions as a cofactor for GATA-4 in transcription, is expressed in these valve cells. Though high-level expression of the only other known vertebrate FOG-like factor, FOG-1, has not previously been observed by in situ RNA hybridization, FOG-1 transcripts are present at low levels in Northern blots of total heart RNA. Thus, it is quite possible that disruption of the physical interaction between GATA-4 and FOG-1, or a novel, undefined FOG protein, is responsible for impaired development of the semilunar valves and the appearance of a double-outlet right ventricle (Crispino, 2001).

Given the profound effects of mutation of either GATA-4 or FOG-2 proteins on heart morphogenesis in mice, it is worth considering their potential relevance to human congenital heart defects, such as the Tetralogy of Fallot or the double-outlet right ventricle. Whereas mutation of several genes, such as Jmj (Jumanji), Sox4, and Egfr/Shp2, give rise to the double-outlet right ventricle defect in mice, and defects in other genes for transcription factors, such as FOG-2, NF1, neurotrophin 3, and RXR, result in all or a subset of the Tetralogy of Fallot, the consistent and combined phenotype seen in the Gata4^ki/ki mice is unique. Although it is sometimes difficult to distinguish between double-outlet right ventricle with associated pulmonary stenosis and the Tetralogy of Fallot, it is clear that the defects in the Gata4^ki/ki hearts are different in the outflow tracts than those observed in Fog2-deficient embryos (Crispino, 2001).

Through the use of an altered specificity mutant, it has been demonstrated that GATA-4 very likely requires both FOG-2 and an additional FOG, or FOG-like protein, as cofactors for distinct aspects of heart development. Interaction with FOG-2 is essential for the initiation of coronary vasculature and for some morphogenetic events, whereas interaction with a distinct FOG protein appears to be required for formation of cardiac valves. The results are surprising in that two other GATA-factors, GATA-5 and GATA-6, are also expressed in myocardium, and indirect data have suggested that they might compensate for the absence of GATA-4. For example, previous studies show that GATA-4 is dispensable for terminal differentiation of cardiomyocytes and that Gata4-/- ES cells contribute to all layers of the heart. In these experiments, it has been suggested that GATA-5 or GATA-6 functionally replace GATA-4. It is possible that proper expression of the GATA-4^ki/ki protein, as distinguished from the absence of GATA-4 in the knock-out situation, precludes compensation by other GATA factors. Indeed, immunostaining with an alpha-GATA-6 antibody has demonstrated that GATA-6 expression, though similar to that of GATA-4, is not up-regulated in the Gata4^ki/ki hearts. In addition, staining with alpha-GATA-5 antibody reveals a normal pattern in Gata4^ki/ki hearts. Since GATA-5 is no longer expressed within the ventricles of the heart at E12.5, it is unlikely that it would compensate for the absence of functional GATA-4 (Crispino, 2001).

These findings implicate GATA-4 as the principal GATA factor relevant to heart morphogenesis and coronary vasculature development and as the primary partner for FOG proteins in the heart. This represents the second example of transcriptional regulation involving GATA-FOG protein complexes and argues for their broad involvement as key regulators of multiple developmental pathways (Crispino, 2001).

An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activate expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the alpha-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy is suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND is associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacts with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. These results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart (Dai, 2002).

The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. The Xenopus ANF promoter was isolated in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. It is proposed that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene (Small, 2003).

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