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GATA-4 and GATA-6 function in early mammalian development: GATA factors are master genes for endodermal differentiation functioning dowstream of FGF as revealed by studies of embryonic stem cells

GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, embryoid bodies derived from mouse embryonic stem (ES) cells were studied in which both copies of the Gata-4 gene are disrupted. Embryoid bodies formed from wild-type and heterozygous deficient ES cells are covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm is evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines display this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, are absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. It is concluded that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm (Soudais, 1995).

GATA-4, a transcription factor implicated in lineage determination, is expressed in both parietal and visceral endoderm of the early mouse embryo. In embryonic stem cell-derived embryoid bodies, GATA-4 mRNA is first detectable at 4-5 days of differentiation and is confined to visceral endoderm cells on the surface of the bodies. Targeted mutagenesis of the Gata4 gene in embryonic stem cells results in a block in visceral endoderm differentiation in vitro. In an attempt to elucidate the role of GATA-4 in the formation of visceral endoderm, Gata4 -/- and wild type embryoid bodies were differentiated in the presence of retinoic acid with and without dbcAMP, known inducers of endoderm formation. Differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid results in formation of visceral endoderm, while differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid plus dbcAMP causes parietal endoderm formation. The presence of these yolk sac endoderm layers was confirmed by light microscopy and analysis of biochemical markers including alpha-fetoprotein, type IV collagen, laminin, and binding sites for Dolichos biflorus agglutinin. Treatment of Gata4 -/- embryoid bodies with retinoic acid induces expression of another GATA-binding protein, GATA-6, in both visceral and parietal endoderm cells. The fact that another GATA-binding protein is induced in the absence of GATA-4 suggests that this family of transcription factors plays an important role in yolk sac differentiation (Bielinska, 1997).

GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient [GATA6(-/-)] ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6(-/-) ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, alpha-fetoprotein (AFP), and HNF3beta. Homozygous GATA6(-/-) mice die between embryonic day (E) 6.5 and E7. 5 and exhibit a specific defect in endoderm differentiation, including severely down-regulated expression of GATA4 and the absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activates the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6(-/-)-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6(-/-) ES cells contribute to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium (Morrisey, 1998).

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. This is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells (Fujikura, 2002).

These data have suggested that the repression of this GATA network is an important part of the molecular mechanism to maintain the ES cells in an undifferentiated state, a state that was released by withdrawal of LIF or up-regulation of Oct-3/4. However, down-regulation of Oct-3/4 induces TE transcription factors such as Hand-1 (see Drosophila Hand) and Cdx-2 but not Gata-4 and Gata-6, indicating that Oct-3/4 does not repress the GATA network directly. It is hypothesized that unidentified transcription factor(s) activated by the cooperation of Oct-3/4 and its cofactor(s) acts as a repressor of Gata-6. During ES self-renewal, expression of Gata-6 is repressed by the hypothetical repressor X, at a point below the threshold level necessary to trigger differentiation; however, once expression of the repressor X is down-regulated, Gata-6 expression is up-regulated initially, then Gata-6 and Gata-4 trans- and self-activate themselves. Once the GATA protein expression level is increased beyond the threshold, the ExE differentiation process can progress. Because COUP-TF can act as a repressor on the Oct-3/4 promoter, COUP-TF induced by GATA expression might play some role to block stem cell self-renewal. Indeed, overexpression of Coup-tf I in ES cells represses endogenous Oct-3/4 expression. This results in differentiation to epithelial cells that express the TE marker gene Hand-1, a marker gene that is never induced in GATA transfectants. However, the morphology of these epithelial cells is different from that of the TE-like cells induced by the down-regulation of Oct-3/4. These data also suggest that COUP-TF alone is not sufficient to mimic the effect of GATA factors, and that the cooperative function of the GATA factors and their target genes, including Coup-tf, might be important to trigger the proper differentiation program toward ExE, and the down-regulation of stem-cell-specific genes (Fujikura, 2002).

Transcription factors GATA-4, -5, and -6 constitute an evolutionary conserved subfamily of vertebrate zinc finger regulators highly expressed in the developing heart and gut. Genetic evidence suggests that each protein is essential for embryonic development, but their exact functions are not fully elucidated. Moreover, because all three proteins share similar transcriptional properties in vitro, and because transcripts for two or more GATA genes are present in similar tissues, the molecular basis underlying in vivo specificity of GATA factors remains undefined. Knowledge of the exact cell types expressing each protein and identification of downstream targets would greatly help define their function. High-resolution immunohistochemistry has been used to precisely determine the cellular distribution of the GATA-4, -5, and -6 proteins in murine embryogenesis. The results reveal novel sites of expression in mesodermal and ectodermal cells. In particular, GATA-4 and -6 expression are closely associated with yolk sac vasculogenesis and early endoderm-mesoderm signaling. Additionally, GATA-6 is strongly expressed in the embryonic ectoderm, neural tube, and neural crest-derived cells. This pattern of expression closely parallels that of BMP-4, and the BMP-4 gene has been identified as a direct downstream target for GATA-4 and -6. These findings offer new insight into the function of GATA-4 and -6 during early stages of embryogenesis and reveal the existence of a positive cross-regulatory loop between BMP-4 and GATA-4. They also raise the possibility that part of the early defects in GATA-4 and/or GATA-6 null embryos may be due to impaired BMP-4 signaling (Nemer, 2003).

The establishment of alternative cell fates during embryoid body differentiation has been investigated, when embryonic stem (ES) cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. Endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, it was demonstrated that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. Pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is stem cell flow through two cell layers connected by laminin 1 and stem cell involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase (Li, 2004).

The inner cell mass (ICM) of preimplantation and early postimplantation mammalian embryos contain cells ancestral to the entire individual, that undergo extensive morphological change prior to gastrulation. In the blastocyst and early egg cylinder the ICM consists of an aggregate of non-polar stem cells, which before gastrulation undergo epithelialization and cavitation, creating a pseudostratified columnar epithelium that surrounds a central cavity similar to the proamniotic canal of the early embryo. The pseudostratified columnar epithelium or epiblast attaches to the sub-endodermal basement membrane (BM). This polarized epithelium allows intermingling of clonal derivatives and is thought to be necessary for gastrulation. Much is known about the role of endoderm to ectoderm signalling in anteroposterior patterning of the early embryo. The establishment of major elements of the amniote body plan during gastrulation has been also studied in detail. However, the mechanism that precedes these changes and transforms the non-polar primitive ectoderm into the columnar polar epiblast is little understood (Li, 2004).

Embryonic stem cell derived embryoid bodies (EBs) are similar to the egg cylinder embryo, but, in contrast to it, they can be grown in large quantities, providing a useful model for early embryogenesis. The mechanism of EB differentiation has been set out as a model for pregastrulation development and tube formation by cavitation. EBs have an external endoderm that is similar to the primitive or visceral endoderm of the embryo and is separated from the inner columnar ectoderm by a basement membrane (BM). Using a genetically undefined spontaneous mutation, which fails to form the columnar ectoderm layer, it was proposed that cavitation is regulated by two signals: one emanating from the outer endoderm layer was thought to be responsible for the apoptotic signal/s of cavitation; the second, originating in the BM, was considered necessary for the maintenance and survival of the columnar ectoderm (Li, 2004 and references therein).

The work carried out in this study started as a study of the role of FGF signalling in EB differentiation and led to questions regarding BM assembly that were investigated using ES cells that express truncated Fgfr2 cDNA as a dominant-negative mutation. ES cells expressing dnFgfr fail to develop the two characteristic cell layers of the EB. They display a homogenous aggregate of non-polar cells and form no endoderm or ectoderm-like elements, but survive for weeks during cultivation. EBs formed by dnFgfr ES cells fail to synthesize laminin and collagen IV isotypes, which supply the protein network of the BM. Co-cultivating wild-type and dnFgfr ES cells rescued EB differentiation, suggesting that an FGF-controlled extracellular substance, subsequently identified as laminin 1, is required for epiblast differentiation. Exogenously added laminin 1 partially rescues the EB phenotype and induces epithelial transformation, demonstrating that laminin 1 produced by the endoderm is necessary and sufficient to induce epiblast polarization (Li, 2004).

Laminin 1 has been shown to be required for EB differentiation. Targeted disruption of ß1-integrin, which inhibits laminin alpha1 synthesis, interferes with epiblast differentiation. Disruption of Lamc1 encoding laminin gamma1, one of the three polypeptides of the laminin 1 heterotrimer, leads to a similar phenotype. Significantly, defective epiblast differentiation caused by loss of either gene was rescued by exogenously added laminin 1, which in turn could be inhibited by the E3 fragment of laminin alpha1 containing the heparin and sulfatide binding site of the LG4 globular domain of the laminin alpha1-chain. Recognising the potential importance of these findings for understanding epithelial differentiation and early development, it would help their analysis if the succession and main intermediates of EB differentiation were defined (Li, 2004).

In the present study, attempts were made to obtain a comprehensive view of the developmental interactions that precede gastrulation. To achieve this, several specific questions had to be answered. Is FGF signalling required for the differentiation of both epithelia and the pattern of their arrangement in the EB, or for only an initial step that is necessary for later events? Defective FGF signalling could be partially restored by exogenous laminin 1. The next question is can the same effect be obtained by laminin 1 presented by the BM in a physiological cell-matrix interaction? It was also important to determine whether laminin affects the stem cell directly, or whether it activates precursors after they reached a specific stage of FGF dependent differentiation. To answer these questions, mutant and wild-type ES cell lines were used , and their behaviour was studied as an effect of chemical inhibitors and co-cultivation experiments between mutant and wild-type cells (Li, 2004).

As an experimental system to elucidate interactions between the endoderm and primitive ectoderm GATA4- or GATA6-transformed endoderm-like cells co-cultivated with mutant ES cell lines were used. This system demonstrated that GATA4 and GATA6 transform ES cells into functional extra-embryonic endoderm that deposits a BM, which in turn mediates epiblast polarization. GATA transformed cells synthesize and later secrete laminin 1 and collagen IV into the culture supernatant, which could be used to rescue epiblast differentiation. Genetic evidence of laminin gamma1 null ES cells has demonstrated the specificity of mutant rescue. This experimental system thus recreated the physiological BM-mediated interaction and allowed the separation of endoderm and epiblast differentiation according to their respective FGF/GATA6 and laminin/Rho kinase-dependent mechanisms (Li, 2004).

Endoderm differentiation depends on FGF signalling, as demonstrated by the targeted disruption of Fgf4. Fgf4 is expressed in the ICM and contributes to the maintenance of the endoderm, where the multiple FGF receptors that read its signals are localized. Expression of GATA4 and GATA6, where GATA4 is regulated by GATA6, is controlled by FGF signalling. Nevertheless, the immediate downstream elements of FGF signalling are insufficiently understood in EB differentiation. In vitro evidence suggests that most FGF dependent signals go through Frs2a, a docking protein, which communicates with the Grb2 adaptor. Interestingly although null mutants of Fgf4 die with defective endoderm development shortly after implantation, Frs2a null embryos survive until advanced gastrulation, indicating that FGF signalling may exhibit unique characteristics in the early embryo. Analysis of signal transduction in dnFgfr ES cells revealed that PI3K-Akt/PKB rather than MAPK-ERK signalling is affected by defective FGF activity. In agreement, this study found that constitutively active Akt/PKB enhances endoderm development and the synthesis of laminin and collagen IV isotypes, indicating that the PI3K-Akt/PKB pathway predominates in FGF-dependent endoderm differentiation (Li, 2004).

GATA6 is an intermediary of FGF signalling. GATA6, which is transcribed already in the ICM, behaves as a master gene for endoderm differentiation. GATA6 activates the synthesis of all three polypeptide chains of laminin 1, which together with collagen IV, nidogen and perlecan assemble into the sub-endodermal BM. GATA factors induce endoderm differentiation and BM assembly even in dnFgfr ES cells, indicating that once activated, these transcription factors induce endoderm differentiation independently from FGF signalling. Because endoderm differentiation requires GATA6 and because cysts of GATA6 transformed cells contain only endoderm-like elements, it is concluded that GATA factors are required and sufficient to induce endoderm development and deposition of the subendodermal BM (Li, 2004).

Additional elements of this pathway are the transcription factors COUP-TFs I and II, which are upregulated by GATA4/6 during endoderm development and induce Lamc1 and Lamb1 expression. It follows that minimal elements of this interaction are, sequentially, Fgf4, multiple Fgfr, PI3K and AKT/PKB, GATA6 and GATA4, COUP-TFs I and II, as well as the genes encoding the three polypeptide chains of laminin 1 (Li, 2004).

Evidence demonstrates that E-cadherin is also required for early EB differentiation. E-cadherin-null ES cells fail to aggregate, do not form a normal ectoderm and do not undergo EB differentiation. Therefore, E-cadherin-dependent ES cell aggregation may be a prerequisite for the restriction of FGF signalling to the outer cells of the developing EB. E-cadherin is connected to the ß-catenin-GSK3-wnt pathway. Patterning events involving cadherin-Wnt/ß-catenin interactions have been shown to be controlled by FGF signalling (Li, 2004).

There is strong evidence for the epithelialization of ES cells by exogenous laminin 1. Laminin 1 can induce epiblast differentiation as part of the BM that mediates the physiological interaction of the endoderm with the epiblast. While laminin 1 binds to ES cells and their ectodermal derivatives, it does not associate with the primitive endoderm. Thus, the cell-binding domains of the laminin alpha1 chain determine the location of the subendodermal BM by interacting with their receptors displayed by the stem cells localized below the endoderm layer. This therefore defines the direction of laminin-mediated signalling, thereby determining the topographical relationship of endoderm and ectoderm (Li, 2004).

Besides inducing epiblast polarization, the BM affects the simple two-cell layer pattern of the EB and egg cylinder embryo. Since cell-to-matrix interactions take place through direct contact, epithelialization of residual stem cells is precluded, and a single epiblast monolayer develops from cells immediately adjacent to the BM. It has been proposed that the residual stem cells are removed by programmed cell death induced by factors derived from the endoderm, to form a central cavity. Investigation of the role of BMP signalling in cavitation indicates that BMP2 synthesizes in the endoderm, and BMP4 in the primitive ectoderm can both contribute to cavitation, although BMP4 is expressed only for a short period. The data indicate that cavitation and columnar ectoderm differentiation do not require the endoderm, provided that exogenous laminin 1 is presented. It is therefore possible that the developing ectoderm itself secretes the necessary apoptotic factors, such as BMP4, although inhibition of ROCK activity uncouples cavitation from full epithelialization of the primitive ectoderm and argues that cavitation may be either not different from necrosis, or it might be due to mechanical separation of the columnar ectoderm from the residual stem cells. This issue requires further study (Li, 2004).

Dominant-negative ROCK abolishes epiblast polarization without affecting endoderm differentiation, suggesting that it may be regulated separately in the two cell lineages. This assumption was supported by observing that ROCK expression and epiblast polarization does not require the endoderm for the laminin-induced differentiation of dnFgfr ES cells. Although ROCK is required for the epithelialization of the primitive ectoderm, it is not sufficient to induce this process, as suggested by the observation that dominant-active ROCK does not rescue dnFgfr differentiation. Although in the epiblast ROCK activity may be induced by laminin, in the endoderm it appears to be under FGF control and the resistance of endodermal differentiation to ROCK inhibition is consistent with the possibility that RAC1 or Cdc42, which are co-expressed in the endoderm, may have a role in endodermal differentiation (Li, 2004).

Separation of endoderm and epiblast differentiation has been repeatedly observed in this study. FGF signalling is shown to be required for endoderm differentiation but not for epiblast polarization, which is independently induced by laminin 1 of the sub-endodermal BM. The two lineages are also distinguished by laminin binding. ES cells and their ectodermal derivatives bind laminin, while the primitive and visceral endoderm do not, which defines the direction of laminin-induced differentiation. It follows that the extra-embryonic and embryonic epithelium of the EB and egg cylinder embryo develop by distinct mechanisms, which are connected by the inductive activity of the laminin component of their common BM. Future research will have to clarify whether other epithelial transitions are also controlled by laminin-dependent mechanisms (Li, 2004).

GATA6 Levels Modulate Primitive Endoderm Cell Fate Choice and Timing in the Mouse Blastocyst

Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, an unbiased, quantitative, single-cell-resolution image analysis pipeline was applied to analyze embryos lacking or exhibiting reduced levels of GATA6. Gata6 mutants were found to exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, it was shown that GATA6 is necessary for PrE specification by FGF signaling, and a model is proposed where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification (Schrode, 2014).

Other GATA-4 functions

Gene inactivation studies have shown that members of the GATA family of transcription factors are critical for endoderm differentiation in mice, flies and worms, yet how these proteins function in such a conserved developmental context has not been understood. In vivo footprinting of mouse embryonic endoderm cells was used to show that a DNA-binding site for GATA factors is occupied on a liver-specific, transcriptional enhancer of the serum albumin gene. The albumin enhancer is co-occupied with an adjacent binding site for HNF3 in the embryonic gut endoderm, embryonic hepatocytes and adult liver. Increasing amounts of HNF3 leads to increasing amounts of a complex with both factors bound and a depletion of the GATA-4/DNA complex. The appearance of GATA-4 and HNF3 bound to the same DNA in the presence of excess enhancer probe indicates that the factors bind cooperatively. GATA site occupancy occurs in gut endoderm cells at their pluripotent stage: the cells have the potential to initiate tissue development but they have not yet been committed to express albumin or other tissue-specific genes. The GATA-4 isoform accounts for about half of the nuclear GATA-factor-binding activity in the endoderm. GATA site occupancy persists during hepatic development and is necessary for the activity of albumin gene enhancer. Thus, GATA factors in the endoderm are among the first to bind essential regulatory sites in chromatin. Binding occurs prior to activation of gene expression, changes in cell morphology or functional commitment that would indicate differentiation. It is suggested that GATA factors at target sites in chromatin may generally help potentiate gene expression and tissue specification in metazoan endoderm development (Bossard, 1998).

Mammalian gonadal development and sexual differentiation are complex processes that require the coordinated expression of a specific set of genes in a strict spatiotemporal manner. Although some of these genes have been identified, the molecular pathways, including transcription factors, that are critical for the early events of lineage commitment and sexual dimorphism, remain poorly understood. GATA-4, a member of the GATA family of transcription factors, is present in the gonads and may be a regulator of gonadal gene expression. The ontogeny of gonadal GATA-4 expression has been analyzed by immunohistochemistry. GATA-4 protein is detected as early as embryonic day 11.5 in the primitive gonads of both XX and XY mouse embryos. In both sexes, GATA-4 specifically marks the developing somatic cell lineages (Sertoli in testis and granulosa in ovary) but not primordial germ cells. Interestingly, abundant GATA-4 expression is maintained in Sertoli cells throughout embryonic development but is markedly down-regulated shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual dimorphism. Consistent with this hypothesis, it is found that the Mullerian inhibiting substance promoter, which harbors a conserved GATA element, is a downstream target for GATA-4. Thus, transcription factor GATA-4 may be a new factor in the cascade of regulators that control gonadal development and sex differentiation in mammals (Viger, 1998).

Friend of GATA-1 (FOG: Drosophila homolog U-shaped) is a multitype zinc finger protein that interacts with GATA-1 and serves as a cofactor for GATA-1-mediated transcription. FOG is coexpressed with GATA-1 in developing erythroid and megakaryocyte cell lineages and FOG cooperates with GATA-1 to control erythropoiesis. A novel FOG-related factor, FOG-2, is described that is expressed predominantly in the developing and adult heart, brain, and testis. FOG-2 interacts with GATA factors, and interaction of GATA-4 and FOG-2 results in either synergistic activation or repression of GATA-dependent cardiac promoters, depending on the specific promoter and the cell type in which they are tested. The properties of FOG-2 suggest its involvement in the control of cardiac and neural gene expression by GATA transcription factors. In contrast to FOG, which is restricted to developing hematopoietic cell lineages, FOG-2 is expressed predominantly in developing heart and brain during embryogenesis. FOG-2 expression is first detected in the developing cardiac tube at E9.0, and expression is maintained throughout the myocardium during embryogenesis and postnatal development. Considering the importance of GATA-4 for cardiac morphogenesis and myogenesis, it is likely that FOG-2 plays an important role in GATA-dependent transcriptional activation in the developing heart. FOG-2 expression becomes detectable in neurons within the brain and neural tube beginning at about E10.5. Several members of the GATA family have been shown to be expressed in the brain and to activate various brain-specific promoters, which also suggests a potential role for FOG-2 in the regulation of neural genes (Lu, 1999).

Endothelins are a family of biologically active peptides that are critical for development and function of neural crest-derived and cardiovascular cells. These effects are mediated by two G-protein-coupled receptors and involve transcriptional regulation of growth-responsive and/or tissue-specific genes. The cardiac ANF promoter, which represents the best-studied tissue-specific endothelin target, has been used to elucidate the nuclear pathways responsible for the transcriptional effects of endothelins. Cardiac-specific response to endothelin 1 (ET-1) requires the combined action of the serum response factor (SRF) and the tissue-restricted GATA proteins that bind over their adjacent sites, within a 30-bp ET-1 response element. SRF and GATA proteins form a novel ternary complex reminiscent of the well-characterized SRF-ternary complex factor interaction required for transcriptional induction of c-fos in response to growth factors. In transient cotransfections, GATA factors and SRF synergistically activate atrial natriuretic factor and other ET-1-inducible promoters that contain both GATA and SRF binding sites. Thus, GATA factors may represent a new class of tissue-specific SRF accessory factors that account for muscle- and other cell-specific SRF actions (Morin, 2001).

SRF, initially isolated as the nuclear protein that mediates transcriptional response of c-fos and other immediate-early genes to growth factors, has been one of the most extensively characterized transcription factors. It is now well established that many SRF-dependent responses to growth factor stimulation are mediated by an SRF-containing ternary complex in which the TCF is the target of several MAPK cascades. At least three different but related TCFs have been identified; functional as well as structural analyses of the TCF-SRF-DNA ternary complex suggest that TCFs act as growth-regulated SRF cofactors. Unexpectedly, while mutations that abolish TCF binding render the c-fos promoter unresponsive to some growth factors, they do not abolish serum regulation or endothelin stimulation. This has led to the speculation that an unidentified SRF cofactor that would interact with the DNA-binding SRF domain and form a ternary complex with SRF and DNA must exist. These results suggest that GATA factors may fulfill these criteria. Indeed, GATA-4 and -6 interact with the DNA-binding domain of SRF and form a stable ternary complex, as evidenced by gel shift analysis and supported by molecular modeling. Remarkably, it was found that the well-studied c-fos SRE contains two inverted GATA motifs flanking the SRF binding sequences that bind recombinant GATA factors, albeit with lower affinity than the ANF GATA sites. Moreover, the c-fos promoter as well as a c-fos SRE heterologous promoter are synergistically activated by SRF and GATA factors in many cell types. Whether a GATA-SRF ternary complex can substitute for the TCF-SRF complex over the c-fos promoter and mediate cell-specific serum or growth-differentiation responses in GATA-expressing cells deserves to be investigated (Morin, 2001).

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse have indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, it has been shown that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, it has been found that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources (Rossi, 2001).

During mouse embryogenesis GATA-4 is expressed first in primitive endoderm and then in definitive endoderm derivatives, including glandular stomach and intestine. To explore the role of GATA-4 in specification of definitive gastric endoderm, chimeric mice were generated by introducing Gata4 minus ES cells into ROSA26 morulae or blastocysts. In E14.5 chimeras, Gata4 minus cells were represented in endoderm lining the proximal and distal stomach. These cells express early cytodifferentiation markers, including GATA-6 and ApoJ. However, by E18.5, only rare patches of Gata4 minus epithelium are evident in the distal stomach. This heterotypic epithelium has a squamous morphology and does not express markers associated with differentiation of gastric epithelial cell lineages. Sonic Hedgehog, an endoderm-derived signaling molecule normally down-regulated in the distal stomach, is overexpressed in Gata4 minus cells. It is concluded that GATA-4-deficient cells have an intrinsic defect in their ability to differentiate. Similarities in the phenotypes of Gata4 minus chimeras and mice with other genetically engineered mutations that affect gut development suggest that GATA-4 may be involved in the gastric epithelial response to members of the TGF-beta superfamily (Jacobsen, 2002).

Sox is a large family of genes related to the sex-determining region Y gene (designated as the SRY gene), In mammals, Sry expression in the bipotential, undifferentiated gonad directs the support cell precursors to differentiate as Sertoli cells, thus initiating the testis differentiation pathway. In the absence of Sry, or if Sry is expressed at insufficient levels, the support cell precursors differentiate as granulosa cells, thus initiating the ovarian pathway. The molecular mechanisms upstream and downstream of Sry are not well understood. The transcription factor GATA4 and its co-factor FOG2 are required for gonadal differentiation. Mouse fetuses homozygous for a null allele of Fog2 or homozygous for a targeted mutation in Gata4 (Gata4ki) that abrogates the interaction of GATA4 with FOG co-factors exhibit abnormalities in gonadogenesis. Sry transcript levels are significantly reduced in XY Fog2–/– gonads at E11.5, which is the time when Sry expression normally reaches its peak. In addition, three genes crucial for normal Sertoli cell function (Sox9, Mis and Dhh) and three Leydig cell steroid biosynthetic enzymes (p450scc, 3ßHSD and p450c17) are not expressed in XY Fog2–/– and Gataki/ki gonads, whereas Wnt4, a gene required for normal ovarian development, is expressed ectopically. By contrast, Wt1 and Sf1, which are expressed prior to Sry and necessary for gonad development in both sexes, are expressed normally in both types of mutant XY gonads. These results indicate that GATA4 and FOG2 and their physical interaction are required for normal gonadal development (Tevosian, 2002).

Mammalian sexual differentiation requires both the GATA4 and FOG2 transcriptional regulators to assemble the functioning testis. The sexual development of female mice is profoundly affected by the loss of GATA4-FOG2 interaction. The Dkk1 gene, which encodes a secreted inhibitor of canonical β-catenin signaling, has been identified as a target of GATA4-FOG2 repression in the developing ovary. The tissue-specific ablation of the β-catenin gene in the gonads disrupts female development. In Gata4ki/ki; Dkk1-/- or Fog2-/-; Dkk1-/- embryos, the normal ovarian gene expression pattern is partially restored. Control of ovarian development by the GATA4-FOG2 complex presents a novel insight into the cross-talk between transcriptional regulation and extracellular signaling that occurs in ovarian development (Manuylov, 2009).

GATA-5 and GATA-6

The amino terminus of the mouse GATA-5 protein shares high level amino acid sequence identity with the murine GATA-4 and -6 proteins, but not with other members of the GATA family. GATA-5 binds to the functionally important CEF-1 nuclear protein binding site in the cardiac-specific slow/cardiac troponin C (cTnC) transcriptional enhancer and overexpression of GATA-5 transactivates the cTnC enhancer in noncardiac muscle cell lines. During embryonic and postnatal development, the pattern of GATA-5 gene expression differs significantly from that of other GATA family members. In the primitive streak embryo, GATA-5 mRNA is detectable in the precardiac mesoderm. Within the embryonic heart, the GATA-5 gene is expressed within the atrial and ventricular chambers (ED 9.5), becomes restricted to the atrial endocardium (ED 12.5), and is subsequently not expressed in the heart during late fetal and postnatal development. Coincident with the earliest steps in lung development, only the GATA-5 gene is expressed within the pulmonary mesenchyme. The GATA-5 gene is also expressed in tissue-restricted subsets of smooth muscle cells (SMCs), including bronchial SMCs and SMCs in the bladder wall. These data are consistent with a model in which GATA-5 performs a unique temporally and spatially restricted function in the embryonic heart and lung. Moreover, these data suggest that GATA-5 may play an important role in the transcriptional program(s) that underlies smooth muscle cell diversity (Morrisey, 1997b).

Two members of the GATA family of transcription factors correspond to chicken cDNAs for cGATA-4 and cGATA-5. Another new member of this family corresponds to a third factor designated cGATA-6. Each of these mRNAs displays a differential expression pattern. The cGATA-5 gene is initially transcribed in the cardiac crescent prior to formation of the primordial heart tube. Following formation of the primitive heart, cGATA-5 transcripts are evident in both endocardium and myocardium as well as in other lateral plate derivatives. The cGATA-5 gene is also transcribed in the primitive embryonic gut and in late stage embryos is sequentially up-regulated in distinct segments of gastrointestinal epithelia as they undergo terminal differentiation. These studies thus provide novel insights into tissue-specific regulation by GATA-5, as well as into possibly overlapping regulatory functions for these three family members (Laverriere, 1994).

In the adult mouse and adult chicken, GATA-4 genes are transcribed predominantly in the heart, small intestine, and gonads. GATA-5 transcripts are abundant in the heart throughout the gut, and in gut derivatives. While chicken GATA-6 is also transcribed in heart, stomach, and small intestine, transcripts are also relatively abundant in lung, liver and ovary. These genes are each expressed in differentiated heart tissue, while mRNA is absent from skeletal muscle. High-level expression of GATA-5 in the gut is specific to epithelium and has been shown to correlate with its terminal differentiation. Xenopus GATA-4, -5 and -6 genes are expressed in differentiated adult heart and gut, but maintain 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. During embryogenesis, ectopic expression of each gene is specifically capable of activating 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 (Jiang, 1996).

The endoderm gives rise to the gut and tissues that develop as outgrowths of the gut tube, including the lungs, liver and pancreas. GATA5 is expressed in the yolk-rich vegetal cells of Xenopus embryos from the early gastrula stage onwards, when these cells become committed to form endoderm. At mid-gastrula stages, GATA5 is restricted to the sub-blastoporal endoderm and is the first molecular marker for this subset of endodermal cells so far identified. GATA4 and GATA5 are potent inducers of endodermal marker genes in animal cap assays, while other GATA factors induce these genes only weakly, if at all. When injected into the dorsal marginal zone, GATA5 respecifies prospective mesoderm towards an endodermal fate, thereby disrupting the convergence and extension movements normally undergone by the dorsal mesoderm. The resulting phenotype is very similar to those seen after injection of dominant negative versions of the FGF-receptor or the T-box transcription factor, Xbra and can be rescued by eFGF. The ability of GATA5 to respecify ectodermal and mesodermal cells toward endoderm suggests an important role for GATA5 in the formation of this germlayer. In animal cap assays, GATA5 is induced by concentrations of activin above those known to induce dorsal mesoderm and heart, in an FGF-independent manner. These data indicate that the emerging view for endodermal induction in general, namely that it is specified by high levels of TGF-beta in the absence of FGF signaling, is specifically true for sub-blastoporal endoderm (Weber, 2000).

gata5, coding for a zinc-finger transcription factor, is required for the development of the zebrafish gut tube. gata5 mutants also display defects in the development of other endodermal organs such as the liver, pancreas, thyroid and thymus. gata5 is expressed in the endodermal progenitors from late blastula stages, suggesting that it functions early during endoderm development. During gastrulation stages, gata5 mutants form fewer endodermal cells than their wild-type siblings. In addition, the endodermal cells that form in gata5 mutants appear to express lower than wild-type levels of endodermal genes such as sox17 and axial/foxA2. Conversely, overexpression of gata5 leads to expanded endodermal gene expression. These data indicate that Gata5 is involved both in the generation of endodermal cells at late blastula stages and in the maintenance of endodermal sox17 expression during gastrulation. The relationship of Gata5 to other factors involved in endoderm formation has been examined. Using complementary mutant and overexpression analyses, Gata5 has been shown to regulate endoderm formation in cooperation with the Mix-type transcription factor Bon. Gata5 and Bon function downstream of Nodal signaling, and casanova (cas) function is usually required for the activity of Gata5 in endoderm formation. fau/gata5, bon and cas exhibit dominant genetic interactions providing additional support that they function in the same pathway. Together, these data demonstrate that Gata5 plays multiple roles in endoderm development in zebrafish, and position Gata5 relative to other regulators of endoderm formation (Reiter, 2001).

A model of zebrafish endoderm formation is presented. The Nodal-related proteins Cyc and Sqt act through TGFbeta-type receptors. Oep is also essential for Nodal signaling and is thought to act upstream of TGFbeta-type receptors. Nodal signaling induces the expression of bon and gata5. Other Nodal- and Oep-dependent factors may also be required for endoderm formation. Bon and Gata5 cooperatively regulate the expression of sox17 and foxA2, but do not regulate each other’s expression. Although Cas is required for sox17 expression and appears to function downstream of, or in parallel to, Bon and Gata5, it is not yet clear how it interacts with other members of the pathway. cas may encode an obligate downstream effector of Bon and Gata5, or the cas gene product may antagonize a repressor of foxA2 and sox17 expression (Reiter, 2001).

Members of both the bone morphogenetic protein (Bmp) and EGF-CFC families have been implicated in vertebrate myocardial development. Zebrafish swirl (swr) encodes Bmp2b, a member of the Bmp family required for patterning the dorsoventral axis. Zebrafish one-eyed pinhead (oep) encodes a maternally and zygotically expressed member of the EGF-CFC family essential for Nodal signaling. Both swr/bmp2b and oep mutants exhibit severe defects in myocardial development. swr/bmp2b mutants exhibit reduced or absent expression of nkx2.5, an early marker of the myocardial precursors. Embryos lacking zygotic oep (Zoep mutants) display cardia bifida and also display reduced or absent nkx2.5 expression. The zinc finger transcription factor Gata5 is an essential regulator of nkx2.5 expression. The relationships between bmp2b, oep, gata5, and nkx2.5 have been investigated. Both swr/bmp2b and Zoep mutants exhibit defects in gata5 expression in the myocardial precursors. Forced expression of gata5 in swr/bmp2b and Zoep mutants restores robust nkx2.5 expression. Moreover, overexpression of gata5 in Zoep mutants restores expression of cmlc1, a myocardial sarcomeric gene. These results indicate that both Bmp2b and Oep regulate gata5 expression in the myocardial precursors, and that Gata5 does not require Bmp2b or Oep to promote early myocardial differentiation. It is concluded that Bmp2b and Oep function at least partly through Gata5 to regulate nkx2.5 expression and promote myocardial differentiation. Thus Gata5 regulates nkx2.5 and cmlc1. Other work has suggested that Gata5 also regulates the expression of cmlc2, vmhc, gata4, gata6, and hand2. Although fgf8 is also expressed in the marginal zone of the gastrulating embryo, gata5 expression is normal in ace/fgf8 mutants and conversely, fgf8 is expressed normally in fau/gata5 mutants. Together, these data indicate that Gata5 and Fgf8 regulate myocardial differentiation independently of one another (Reiter, 2001).

A gene encoding embryonic chicken pepsinogen (ECPg), a zymogen of the digestive enzyme pepsin, is expressed specifically in epithelial cells of glands of embryonic stage proventriculus (glandular stomach) under the influence of mesenchyme. Four GATA motifs and one Sox binding motif, essential to the organ-specific expression of the gene, were found in 1.1 kb of the 5' flanking region of the ECPg gene. The expression of cGATA-5 and cSox2 in the proventriculus from day 6 to day 12 of incubation was therefore analyzed. cGATA-5 is more strongly expressed in glandular epithelial cells than in luminal epithelial cells, while cSox2 gene expression is weaker in glandular epithelial cells. Using heterologous recombination explants it was discovered that the expression of cGATA-5 and cSox2 in epithelial cells is affected by mesenchyme when the latter induces ECPg gene expression in epithelial cells. Introduction of expression constructs into epithelial cells by electroporation demonstrates that cGATA-5 upregulates transcription of a reporter luciferase gene via a cis element in the 5' flanking region of the ECPg gene. The cGATA-5 protein specifically binds to the GATA binding sites. cSox2 downregulates the activity of luciferase but not through the Sox binding motif. These results suggest that cGATA-5 positively regulates transcription of the ECPg gene and is involved in spatial regulation of the pepsinogen gene during development (Sakamoto, 2000).

Xenopus GATA-6 transcripts are first detected in the mesoderm, at the beginning of gastrulation; subsequent domains of expression include the field of cells shown to have heart-forming potential. In this region, GATA-6 expression continues only in those cells that go on to form the heart; however, a decrease occurs prior to terminal differentiation. Artificial elevation of GATA-6, but not GATA-1, prevents expression of cardiac actin, suggesting the GATA-6 blocks differentiation of heart precursors. Expression of the earlier marker XNkx-2.5 is unaffected and morphological development of the heart is initiated independent of the establishment of the contractile machinery. It is concluded that a reduction in the level of GATA-6 is important for the progression of the cardiomyogenic differentiation program and that GATA-6 may act to maintain heart cells in the precursor state. At later stages, when the elevated GATA-6 levels had decayed, differentiation ensues, but the number of cells contributing to the myocardium have increased, suggesting either that the blocked cells have proliferated or that additional cells have been recruited (Gove, 1997).

The GATA-6 transcription factor is expressed in cardiogenic cells and during subsequent stages of heart development in diverse vertebrate species. To gain insights into the molecular events that govern this heart-restricted expression, the chicken GATA-6 gene was isolated and several approaches were used to screen for associated control regions. Analysis of two chicken GATA-6/lacZ constructs in transgenic mouse embryos is particularly revealing. One GATA-6/lacZ construct, which has 1.5 kilobase pairs of upstream sequences along with the promoter and first intron, is expressed exclusively in the atrioventricular canal region of the heart. This expression pattern is novel and appears to mark specialized myocardial cells that induce underlying endocardial cells to initiate valve formation. The other GATA-6/lacZ construct, which has an additional 7.7 kilobase pairs of upstream sequences, is expressed in the ventricle and outflow tract in addition to the atrioventricular canal. The failure of these GATA-6 control regions to function as enhancers in transfected cardiac myocyte cultures underscores the importance of using transgenic approaches to elucidate transcriptional controls that function in the developing heart. Although the endogenous GATA-6 gene is expressed throughout the heart, these results indicate that this is effected in a heart region-specific manner (He, 1997).

The zinc finger transcription factors GATA4, -5, and -6 and the homeodomain protein Nkx2.5 are expressed in the developing heart and have been shown to activate a variety of cardiac-specific genes. To begin to define the regulatory relationships between these cardiac transcription factors and to understand the mechanisms that control their expression during cardiogenesis, the mouse GATA6 gene was analyzed for regulatory elements sufficient to direct cardiac expression during embryogenesis. Using beta-galactosidase fusion constructs in transgenic mice, a 4.3-kb 5' regulatory region was identified that directs transcription specifically in the cardiac lineage, beginning at the cardiac crescent stage. Thereafter, transgene expression becomes compartmentalized to the outflow tract, a portion of the right ventricle, and a limited region of the common atrial chamber of the embryonic heart. Further dissection of this regulatory region identified a 1.8-kb cardiac-specific enhancer that recapitulates the expression pattern of the larger region when fused to a heterologous promoter and a smaller 500-bp subregion that retains cardiac expression, but is quantitatively weaker. The GATA6 cardiac enhancer contains a binding site for Nkx2.5 that is essential for cardiac-specific expression in transgenic mice. These studies demonstrate that GATA6 is a direct target gene for Nkx2.5 in the developing heart and reveal a mutually reinforcing regulatory network of Nkx2.5 and GATA transcription factors during cardiogenesis (Molkentin, 2000).

In vertebrates, heart development is a complex process requiring proper differentiation and interaction between myocardial and endocardial cells. Significant progress has been made in elucidating the molecular events underlying myocardial cell differentiation. In contrast, little is known about the development of the endocardial lineage that gives rise to cardiac valves and septa. A novel in vitro model has been used to identify the molecular hierarchy of endocardial differentiation and the role of transcription factor GATA5 in endocardial development. The results indicate that GATA5 is induced at an early stage of endothelial-endocardial differentiation prior to expression of such early endocardial markers as Tie2 and ErbB3. Inhibition of either GATA5 expression or NF-ATc activation, blocks terminal differentiation at a pre-endocardial stage and GATA5 and NF-ATc synergistically activate endocardial transcription. The data reveal that transcription factor GATA5 is required for differentiation of cardiogenic precursors into endothelial endocardial cells. This, in turn, suggests that the GATA5 pathway may be relevant to early stages of valvuloseptal development: defects of this type account for the majority of human birth malformations (Nemer, 2002).

The evolutionarily conserved GATA-6 transcription factor is an early and persistent marker of heart development in diverse vertebrate species. A functionally conserved heart-specific enhancer is present upstream of the chicken GATA-6 (cGATA-6) gene. Transgenic mouse assays have been used to further characterize this regulatory module. This enhancer is activated in committed precursor cells within the cardiac crescent, and it remains active in essentially all cardiogenic cells through the linear heart stage. Although this enhancer can account for cGATA-6 gene expression early in the cardiogenic program, later in development it is not able to maintain expression throughout the heart. In particular, the enhancer is sequentially downregulated along the posterior to anterior axis, with activity becoming confined to outflow tract myocardium. Enhancers with similar properties have been shown to regulate the early heart-restricted expression of the mouse Nkx2.5 transcription factor gene. Whereas these Nkx2.5 enhancers are GATA-dependent, the cGATA-6 enhancer is Nkx-dependent. It is speculated that these enhancers are silenced to allow GATA-6 and Nkx2.5 gene expression to be governed by region-specific enhancers in the multichambered heart (Davis, 2000).

The transcriptional programs that specify the distinct components of the cardiac conduction system are poorly understood, in part due to a paucity of definitive molecular markers. A cGATA-6 gene enhancer can be used to selectively express transgenes in the atrioventricular (AV) conduction system as it becomes manifest in the developing multichambered mouse heart. Furthermore, analysis of staged cGATA-6/lacZ embryos reveals that the activity of this heart-region-specific enhancer can be traced back essentially to the outset of the cardiogenic program. Evidence suggests that this enhancer reads medial/lateral and anterior/posterior positional information before the heart tube forms and the activity of this enhancer becomes restricted at the heart looping stage to AV myocardial cells that induce endocardial cushion formation. A deeply-rooted heart-region-specific transcriptional program serves to coordinate AV valve placement and AV conduction system formation. Lastly, cGATA-6/Cre mice can be used to delete floxed genes in the respective subsets of specialized heart cells (Davis, 2001).

In the early embryonic heart the AV canal functions to delay the cardiac impulse from the atria to the ventricles. During development most of the atrial and ventricular myocardium becomes physically separated by the fibrous annulus, which is formed by the fusion of endocardial cushion derived mesenchyme and epicardially derived sulcus tissue. The only myocardial connection that remains is through the proximal part of the AVCS, the properties of which delay the (sinoatrial node generated) cardiac impulse. Without a proper delay at the AV canal, the ventricles contract too early in the cardiac cycle, a phenomenon known as ventricular pre-excitation. An increasing body of evidence indicates that the AVCS develops, at least in part, from the myocardium of the AV canal. Interestingly, the AV canal in the embryonic heart tube functions in a manner similar to the AV node in the multichambered heart. This is indicated by the fact that the tubular heart already shows an electrocardiogram with a characteristic delay of the cardiac impulse in the AV canal myocardium. Similarly, the molecular phenotype of AV canal myocardium remains relatively stable as development proceeds and resembles that seen in the mature AV node. It has been suggested that the primary heart tube consists of so-called primary myocardium, which is characterized by a gene expression profile that persists in the developing AV conduction system but is gradually lost in the flanking atrial and ventricular segments. It is not known how these expression patterns are governed. It is thus significant that an enhancer has been identified that is differentially regulated in this precise developmental context. This cGATA-6 enhancer is unique in that it drives AV canal specific expression at very early stages of heart tube formation. These results suggest that AVCS tissue may not differentiate from a common myocardial cell at a relatively late stage of development but rather may be predisposed to this specialized cell fate at the outset of the cardiogenic program (Davis, 2001).

The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner cell mass and in the trophectoderm. It is not clear whether GATA6-expression marks inner cell mass cells that will become the epiblast (giving rise to the embryo proper) or cells that will differentiate into primitive endoderm, or a mixture of both. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue. Injecting GATA6-/- embryonic stem cells into wild-type blastocysts generate a number of normal highly chimeric embryos, indicating that GATA6 is not required in the epiblast. This also endorses the notion that GATA6 is normally required in extraembryonic lineages. This conclusion is further supported by the opposite experiment, which demonstrates the inability of the wild-type embryonic stem cells to rescue the GATA6-/- phenotype. Moreover, death of the embryo (in vivo) or inner cell mass (in vitro) is not due to a cell autonomous requirement for GATA6 in the epiblast but rather to a defect in neighboring supportive tissues. Given the normality of the trophectoderm outgrowth in GATA6-/- blastocysts in vitro and the presence of parietal endoderm cells, it is likely that the primary defect lies in the visceral endoderm (Koutsourakis, 1999).

The transcription factor GATA6 is expressed in the fetal pulmonary epithelium of the developing mouse lung and loss of function studies strongly suggest that it is required for proper branching morphogenesis and epithelial differentiation. The role of GATA6 in this process was investigated by using a pulmonary epithelium specific promoter to maintain high levels of GATA6 protein during fetal lung development. Transgenic mice expressing Gata6 cDNA under the control of the human Surfactant Protein-C (SP-C) promoter were generated and their lungs were analyzed during fetal stages. Transgenic lungs exhibit branching defects as early as embryonic day (E) 14.5 and molecular analysis just before birth (E18.5) shows a lack of distal epithelium differentiation, whereas proximal epithelium is unaffected. Electron microscopic analysis and glycogen staining confirm the lack of differentiation to mature Type II cells. Thus, elevated levels of GATA6 protein affect early lung development and in analogy to other GATA factors in other tissues, GATA6 also plays a crucial role in the terminal differentiation, in this case, of the distal pulmonary epithelium (Koutsourakis, 2001).

Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. T thyroid transcription factor 1 expression is unaltered in chimeric lungs whereas SPC, a marker for type II cells that indicates distal epithelial cell differentiation, and CC10, a marker for Clara cells that indicates proximal epithelial cell differentiation, expressions are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells; this implies that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation (Keijzer, 2001).

GATA6 is the only known GATA factor expressed in the distal epithelium of the lung during development. To define the role that GATA6 plays during lung epithelial cell development, a GATA6-Engrailed dominant-negative fusion protein was expressed in the distal lung epithelium of transgenic mice. Transgenic embryos lacked detectable alveolar epithelial type 1 cells in the distal airway epithelium. These embryos also exhibited increased Foxp2 gene expression, suggesting a disruption in late alveolar epithelial differentiation. Alveolar epithelial type 2 cells, which are progenitors of alveolar epithelial type 1 cells, were correctly specified as shown by normal thyroid transcription factor 1 and surfactant protein A gene expression. However, attenuated endogenous surfactant protein C expression indicated that alveolar epithelial type 2 cell differentiation was perturbed in transgenic embryos. The number of proximal airway tubules is also reduced in these embryos, suggesting a role for GATA6 in regulating distal-proximal airway development. Finally, a functional role for GATA factor function in alveolar epithelial type 1 cell gene regulation is supported by the ability of GATA6 to trans-activate the mouse aquaporin-5 promoter. Together, these data implicate GATA6 as an important regulator of distal epithelial cell differentiation and proximal airway development in the mouse (Yang, 2002).

In vitro studies have suggested that members of the GATA and Nkx transcription factor families physically interact, and synergistically activate pulmonary epithelial- and cardiac-gene promoters. However, the relevance of this synergy has not been demonstrated in vivo. This study shows that Gata6-Titf1 (Gata6-Nkx2.1) double heterozygous (G6-Nkx DH) embryos and mice have severe defects in pulmonary epithelial differentiation and distal airway development, as well as reduced phospholipid production. The defects in G6-Nkx DH embryos and mice are similar to those observed in human neonates with respiratory distress syndromes, including bronchopulmonary dysplasia, and differential gene expression analysis reveals essential developmental pathways requiring synergistic regulation by both Gata6 and Titf1 (Nkx2.1). These studies indicate that Gata6 and Nkx2.1 act in a synergistic manner to direct pulmonary epithelial differentiation and development in vivo, providing direct evidence that interactions between these two transcription factor families are crucial for the development of the tissues in which they are co-expressed (Zhang, 2007).

Mechanisms underlying regional specification of distinct organ precursors within the endoderm, including the liver and pancreas, are still poorly understood. This is particularly true for stages between endoderm formation and the initiation of organogenesis. This report has investigated these intermediate steps downstream of the early endodermal factor Gata5, which progressively lead to the induction of pancreatic fate. TGFβ-induced factor 2 (TGIF2), encoding a homeodomain protein that belongs to the TALE superfamily of homeodomain proteins, was identifid as a novel Gata5 target; its function was identified in the establishment of the pancreatic region within dorsal endoderm in Xenopus. TGIF2 acts primarily by restricting BMP signaling in the endoderm to allow pancreatic formation. Consistently, it was found that blocking BMP signaling by independent means also perturbs the establishment of pancreatic identity in the endoderm. Previous findings demonstrated a crucial role for BMP signaling in determining dorsal/ventral fates in ectoderm and mesoderm. These results now extend this trend to the endoderm and identify TGIF2 as the molecular link between dorsoventral patterning of the endoderm and pancreatic specification (Spagnoli, 2008).

Little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart. This study shows that Wnt2 is expressed specifically in the developing inflow tract mesoderm, which generates portions of the atria and atrio-ventricular canal. Loss of Wnt2 results in defective development of the posterior pole of the heart, resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The number and proliferation of posterior second heart field progenitors is reduced in Wnt2-/- mutants. Moreover, these defects can be rescued in a temporally restricted manner through pharmacological inhibition of Gsk-3β. Wnt2 works in a feedforward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal a molecular pathway regulating the posterior cardiac mesoderm and demonstrate that cardiovascular defects caused by loss of Wnt signaling can be rescued pharmacologically in vivo (Tian, 2010).

Table of contents

serpent: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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