Table of contents

GATA-2 and GATA-3

The transcription factor GATA-2 is expressed at high levels in the nonneural ectoderm of the Xenopus embryo at neurula stages, with lower amounts of RNA present in the ventral mesoderm and endoderm. The promoter of the GATA-2 gene contains an inverted CCAAT box conserved among Xenopus laevis, humans, chickens, and mice. This sequence is essential for GATA-2 transcription during early development and the factor binding it is maternal. The DNA-binding activity of this factor is detectable in nuclei and chromatin bound only when zygotic GATA-2 transcription starts. This factor, called CBTF (CCAAT box transcription factor), has now been characterized. CBTF activity mainly appears late in oogenesis, when it is nuclear, and the complex has multiple subunits. One subunit of the factor has been identified as p122, a Xenopus double-stranded-RNA-binding protein. The p122 protein is perinuclear during early embryonic development but moves from the cytoplasm into the nuclei of embryonic cells at stage 9, prior to the detection of CBTF activity in the nucleus. Thus, the accumulation of CBTF activity in the nucleus is a multistep process. The p122 protein is expressed mainly in the ectoderm. Expression of p122 mRNA is more restricted, mainly to the anterior ectoderm and mesoderm and to the neural tube. Two properties of CBTF, its dual role and its cytoplasm-to-nucleus translocation, are shared with other vertebrate maternal transcription factors and may be general properties of these proteins (Orford, 1998).

In Xenopus, the dorsoventral axis is patterned by the interplay between active signaling in ventral territories, and secreted antagonists from Spemann's organizer. Two signals are important in ventral cells: bone morphogenetic protein-4 (BMP-4) and Wnt-8. BMP-4 plays a conserved role in patterning the vertebrate dorsoventral axis, whilst the precise role of Wnt-8 and its relationship with BMP-4 remains unclear. The role played by the GATA family of transcription factors, which are expressed in ventral mesendoderm during gastrulation and are required for the differentiation of blood and endodermal tissues, has been investigated. Ventral injection of a dominant-interfering GATA factor (called G2en), prepared from GATA-2, induces the formation of secondary axes that phenocopy those induced by the dominant-negative BMP receptor. G2en targets may include GATA factors other than GATA-2. Unlike inhibiting BMP signaling, inhibiting GATA activity in the ectoderm does not lead to neuralization. In addition, analysis of gene expression in G2en injected embryos reveals that at least one known target gene for BMP-4, the homeobox gene Vent-2, is unaffected. In contrast, the expression of Wnt-8 and the homeobox gene Vent-1 is suppressed by G2en, whilst the organizer-secreted BMP antagonist chordin becomes ectopically expressed. These data therefore suggest that GATA activity is essential for ventral cell fate and that subsets of ventralizing and dorsalizing genes require GATA activity for their expression and suppression, respectively. Finally, using G2en, it is shown that suppression of Wnt-8 expression, in conjunction with blocked BMP signalling, does not lead to head formation, suggesting that the head-suppressing Wnt signal may not be Wnt-8 (Sykes, 1998).

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. The role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo is addressed in this paper. Ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, an examination was carried out of how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which inhibits blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of one another. Thus, BMP-4 induces two transcription factors that have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage (Xu, 1999).

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, two distinct populations of progenitors for blood and endothelial cells have been located in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for hematopoietic differentiation. Both require BMP for their formation, even the Spemann organizer-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes SCL and GATA2 depends on BMP signaling, while expression of the endothelial gene Xfli1 does not. By contrast, Xfli1 expression in the adult DLP population does require BMP. These results indicate that both adult and the anterior components of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP (Walmsley, 2002).

The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. During normal development, SCL is expressed in eary sites of embryonic and fetal hematopoiesis, vascular endothelium, and specific regions of the CNS, including the midbrain, hindbrain and spinal cord. Its function in neural development is unknown. Three spatially distinct regulatory modules have been identified, each of which is both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain: the developing endothelium, the midbrain, and the hindbrain/spinal cord. In addition, GATA factor binding sites are essential for neural expression of the SCL constructs. Whereas GATA-1 is not expressed in the developing CNS, GATA-2 and GATA-2 are expressed in a spatial and temporal pattern that overlaps with SCL in the CNS and both GATA-2 and GATA-3 can bind to the SCL promoter. The midbrain element is particularly powerful, and when it was used to drive lacZ expression, details of axonal projections are revealed, implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene is highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibits a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions (Sinclair, 1999).

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. It is inferred that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation (Ko, 1991).

Acetylation of a transcription factor plays a significant role in gene regulation. GATA-3 is acetylated in T cells and a mutation introduced into amino acids 305-307 (KRR-GATA3) creates local hypoacetylation in GATA-3. Remarkably, KRR-GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T-cell homing to systemic lymphnodes, and prolonged T-cell survival after activation. These findings have significant implications in that the acetylation state of GATA-3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA-3 in T-cell survival and homing to secondary lymphoid organs (Yamagata, 2000).

PU.1 (an ETS transcription factor) and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Ralpha expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects (Anderson, 2002).

Mutations resulting in embryonic or early postnatal lethality could mask the activities of any gene in unrelated and temporally distinct developmental pathways. Targeted inactivation of the transcription factor GATA-2 gene leads to mid-gestational death as a consequence of hematopoietic failure. A 250 kbp GATA-2 yeast artificial chromosome (YAC) is expressed strongly in both the primitive and definitive hematopoietic compartments, while two smaller YACs are not. This largest YAC also rescues hematopoiesis in vitro and in vivo, thereby localizing the hematopoietic regulatory cis element(s) to between 100 and 150 kbp 5' to the GATA-2 structural gene. Introducing the YAC transgene into the GATA-2(-/-) genetic background allows the embryos to complete gestation; however, newborn rescued pups quickly succumb to lethal hydroureternephrosis, and display a complex array of genitourinary abnormalities. These findings reveal that GATA-2 plays equally vital roles in urogenital and hematopoietic development (Zhou, 1998).

What is the nature of the defect in GATA-2 expression that gives rise to such a broad spectrum of urogenital phenotypes? The most likely possibilities are either that cell-autonomous functions for GATA-2 are required in each of the affected tissues in the developing urogenital system of normal mice, or that GATA-2 directs the expression of cell signaling ligands and/or receptors which then induce the appropriate tissue remodeling and differentiation responses in those tissues. The first possibility predicts that GATA-2 would be expressed in all of the affected developing ducts and organs (which it is not), while the second possibility suggests that GATA-2 activity need only be expressed in a subset of the affected tissues, and only at specific times during embryogenesis. The latter alternative also predicts that the inducing molecules required for appropriate tissue remodeling would be under the direct or indirect regulatory influence of GATA-2. Immunohistochemical analysis of the initial stages in genitourinary development shows that GATA-2 is widely, but not ubiquitously, expressed there. Thus it is concluded that GATA-2 probably regulates both cell-autonomous and non-autonomous functions during genitourinary morphogenesis, and the identity of the ligands and/or receptors controlled by GATA-2 await discovery. Interestingly, similar phenotypes have been observed in distal hox gene mutant animals, suggesting that GATA-2 may perhaps lie in the same regulatory pathway as those genes (hox10-13) during urogenital morphogenesis (Zhou, 1998 and references).

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 a functional analysis of the FOG-2 protein was performed. The results demonstrate that (1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins; (2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and (3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, a novel N-terminal domain of FOG-2 (amino acids 1-247) has been identifed that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development (Svensson, 2000).

The transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons (Pata, 1999).

The expressions patterns of transcription factors GATA-2 and GATA-3 during early stages of embryonic development in the central nervous system (CNS) of the mouse are described. GATA-2 is expressed as early as 9 dpc in the hindbrain, in ventral rhombomere 4, and transiently in ventral rhombomere 2 (r2). From 9.5 to 11.5 dpc, activation of the gene spreads to many sites of early neuronal differentiation, such as the olfactory bulbs, the pretectum, and the oculomotor nucleus in the midbrain, a thin stripe of cells lining the floor plate from the mesencephalon to the cervical spinal cord and a ventral column of cells spanning the neural tube from rostral hindbrain and including motor neuron as well as ventral interneuron precursors. GATA-3 is expressed in a pattern very similar to that of GATA-2. Distinguishing features are the lack of expression in r2 at 9 dpc and a slight delay in its activation. In addition, GATA-2 is activated in both the ventricular and the subventricular zones of the neural tube, whereas GATA-3 is restricted mainly to the subventricular zone. Expression analyses performed on GATA-2 -/- mouse embryos between E9.5 and 10.5 dpc establish that: (1) the expression of GATA-3 in the developing CNS of the mouse embryo is dependent on the presence of GATA-2, and (2) that loss of GATA-2 leads to severe defects in neurogenesis, which strongly suggests that GATA-2 is involved, as in hematopoiesis, in the maintenance of the pool of ventral neuronal progenitors (Nardelli, 1999).

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHbeta promoter (-133 to -88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. GATA-2 transcripts and protein are present in TtT-97 thyrotropic tumors. A comigrating complex is observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrates binding specificity to the P1 region DNA probe and can be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 are combined, a slower migrating complex, indicative of a ternary protein-DNA interaction is observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulates mouse TSHbeta promoter activity 8.5-fold, while each factor alone has a minimal effect. Mutations that abrogate this functional stimulatory effect map to the P1 region. GATA-2 is shown to directly interact with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression (Gordon, 1997).

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. The appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as the molecular memory of the transient signaling events. To investigate the hypothesis that morphogen-induced transcription factors mediate the determination of pituitary cell types, the transcription factor-encoding genes initially expressed at the ventral boundary of the developing Rathke's pouch were explored. In vivo data have suggested that pituitary cell type positional determination can occur between e10.5-e12.5, long before terminally differentiated cell types appear between e15.5-e16.5. The gene encoding GATA2 exhibits ventral induction in the pituitary coincident with the closure of Rathke's pouch at e10.5 and is maintained with highest expression levels ventrally throughout early pituitary development, later becoming expressed diffusely as the adult pituitary cell populations lose spatial restriction. Based on the ventral induction of a series of transcription factors, including GATA2, an in vivo examination was carried out to see whether dorsally expanding the expression of the ventral signaling molecules BMP2 or Shh would dorsally expand specific ventrally induced genes. BMP2, normally expressed at the ventral boundary of Shh restriction out of the nascent Rathke's pouch, is required for the appearance of four pituitary cell types. Ectopic expression of BMP2/4 results in a dramatic transcriptional induction as well as dorsal expansion of GATA2 gene expression, whereas other ventrally expressed genes are not directly induced. In contrast, overexpression of Shh does not lead directly to transcriptional induction of GATA2. These data are consistent with the hypothesis that expression of GATA2 in the pituitary is selectively induced in response to the ventral BMP2 signal, with highest levels of GATA2 present in the most ventral cell type, the presumptive gonadotrope precursors (Dasen, 1999).

Unexpectedly, the program that results in the emergence of specific cell types includes a DNA binding-independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding-dependent and -independent actions of abundant determining factors contribute to generate distinct cell phenotypes. The interaction interface maps to the homeodomain of Pit1 and to a region of GATA2 containing the C-terminal DNA-binding zinc finger and an adjacent cluster of basic residues. Point mutations on the Pit1 interaction interface reveal a requirement for residues located in the N-terminal basic region (R2, K3) and the non-DNA-binding surface of the second helix of the homeodomain (P26, Q29). It is therefore suggested that a critical component of the cell type determination program is achieved through the inhibition by Pit1 of GATA2-dependent gonadotrope-specific genes while simultaneously permitting GATA2-dependent gene activation critical for establishing the thyrotrope phenotype. Two types of in vivo data support a DNA binding-independent role for Pit1: (1) targeted expression of a non-DNA-binding form of Pit1, still capable of interaction with GATA2, inhibits the gonadotrope-specific terminal differentiation program, while point mutations that abolish the Pit1-GATA2 interaction revert this inhibitory effect. (2) This hypothesis receives genetic confirmation based on developmental events in the Snell dw dwarf mouse, in which the W48C mutation in Pit1 disrupts the homeodomain structure and impairs interaction with GATA2 and in which the presumptive thyrotropes now express the GATA2-dependent gonadotrope gene activation program. This provides direct evidence that Pit1-dependent inhibition of GATA2-dependent activation of gonadotrope-specific genes is a critical component by which Pit1 controls the thyrotrope-specific program. Thus, DNA binding-independent inhibitory protein-protein interactions by the highly abundant Pit1 transcription factor, in addition to its DNA-dependent transcriptional activation roles, is a critical component of the cell type specification and suggests similar functions for other POU domain or other classes of homeodomain factors (Dasen, 1999).

The data suggest that the ventraldorsal BMP2 gradient induces GATA2 in a corresponding gradient in presumptive gonadotropes and thyrotropes and that the high levels of GATA2 in the most ventral aspect of the gland directly or indirectly restricts Pit1 gene expression out of the presumptive gonadotropes, creating the critical delineation of the gonadotrope and Pit1 cell lineages. In the absence of Pit1, GATA2 expression appears sufficient to induce the entire set of transcription factors that are typical of the gonadotrope cell type, including the transcription factors SF1, P-Frk, and Isl1. Conversely, the absence of GATA2 dorsally is critical for differentiation of Pit1+ cells to somatotrope/lactotrope fates, because the targeting of GATA2 more dorsally inhibits initial Pit1 expression and converts these cells to gonadotropes. Similarly, targeting overexpression of BMP2/4 also inhibits Pit1 expression, although the cell types fail to terminally differentiate (Dasen, 1999).

In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 (see Drosophila Serpent) during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. This study identified tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. This study shows that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 (see Drosophila Mad) phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification (Green, 2016).

ZEB (Drosophila homolog: Zn finger homeodomain 1) is a zinc finger-homeodomain protein that represses transcription by binding to a subset of E-box sequences. ZEB inhibits muscle differentiation in mammalian systems, and its Drosophila orthologue, zfh-1, inhibits somatic and cardiac muscle differentiation during Drosophila embryogenesis. ZEB also binds to the promoter of pivotal hematopoietic genes (including those encoding interleukin-2, CD4, GATA-3, and alpha(4)-integrin), and mice in which ZEB has been genetically targeted show thymic atrophy, severe defects in lymphocyte differentiation, and increased expression of the alpha(4)-integrin and CD4. ZEB contains separate repressor domains that function in T lymphocytes and muscle, respectively. The most C-terminal domain inhibits muscle differentiation in mammalian cells by specifically blocking the transcriptional activity of the myogenic factor MEF2C. The more N-terminal domain blocks activity of hematopoietic transcription factors such as c-myb, members of the ets family, and TFE-III. These results demonstrate that ZEB has evolved with two independent repressor domains that target distinct sets of transcription factors and function in different tissues (Postigo, 1999).

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc) was analysed, with a particular emphasis on intraembryonic hemogenic sites. Both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. Lmo2 mRNA is observed in all the sites endowed with a hemopoietic potential, where its expression is tightly regulated spatiotemporally. The rapid modifications of Lmo2 expression patterns suggest that it allocates specific combinations of transcription factors during key points of development. The overlapping expressions of Lmo2 and GATA-3 suggests combined functions during specific steps of definitive hemopoietic development, namely: (1) endodermal induction leading to the emergence of hemopoietic precursors in the mesoderm; (2) determination of these cells from the mesoderm, and (3) their production in the aortic region from 9 to 12 dpc as well as their release into the blood stream. Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase that determines intraembryonic precursors. A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonization. Furthermore, a strong GATA-3 signal allowed the uncovering of previously unreported mesodermal aggregates beneath the aorta. Combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation (Manaia, 2000).

CD4 T cells potentiate the inflammatory or humoral immune response through the action of Th1 and Th2 cells, respectively. The molecular basis of the differentiation of these cells from naive T cell precursors is, however, unclear. GATA-3 is selectively expressed in Th2 cells. GATA-3 is expressed at a high level in naive, freshly activated T cells and Th2 lineage cells, but subsides to a minimal level in Th1 lineage cells as naive cells commit to their Th subset. Antisense GATA-3 inhibits the expression of all Th2 cytokine genes in a Th2 clone. GATA-3 directly activated an IL-4 promoter in M12 cells. In transgenic mice, elevated GATA-3 in CD4 T cells causes Th2 cytokine gene expression in developing Th1 cells. Thus, GATA-3 is necessary and sufficient for Th2 cytokine gene expression (Zheng, 1997).

GATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of mGATA-3's contribution to the development of specific cell fates in the many tissues where it is expressed during embryogenesis. The elements controlling GATA-3 regulation can be precisely refined, using transgenic mice, to discrete cis-acting domains: within 6 kb surrounding the transcriptional initiation site, separate sequences are found to control the expression of mGATA-3 in early muscle masses, in a subset of PNS neurons, in the genital tubercle, and in the branchial arches. The branchial arch regulatory element is particularly robust; it has been located in a discrete enhancer sequence lying between nt -2832 and -2462 from the transcription initiation site. The enhancer contains potential binding sites for many well-characterized transcription factors, incluting GATA, AP2, AP1, CREG/ATF, ETS and SP1, suggesting that mGATA-3 transcriptional activity may be regulated by these proteins (or related family members) in the mesenchyme of the arches that contribute to formation of the jaw. These studies show that discrete regulatory elements required for the elaboration of complex developmental programs can be individually localized, suggesting that the developmentally transient expression of individual transcription factors collaboratively contributes to the temporal and spatial pattern of cellular differentiation leading to the formation of adult anatomy (Lieuw, 1997).

Interleukin-5 (IL-5), which is produced by CD4(+) T helper 2 (Th2) cells (but not by Th1 cells) plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4(+) T helper cells (Th1 and Th2), the molecular basis for Th1- and Th2-specific gene expression remains to be elucidated. The transcription factor GATA-3 is crucial to IL-5 promoter activation in EL-4 cells, which express both Th1- and Th2-type cytokines. GATA-3 is also critical for expression of the IL-5 gene in bona fide Th2 cells. Whereas mutations in the GATA-3 site abolish antigen- or cAMP-stimulated IL-5 promoter activation in Th2 cells, ectopic expression of GATA-3 in Th1 cells or in a non-lymphoid, non-IL-5-producing cell line activates the IL-5 promoter. During the differentiation of naive CD4(+) T cells isolated from T cell receptor transgenic mice, GATA-3 gene expression is up-regulated in developing Th2 cells, but is down-regulated in Th1 cells; antigen- or cAMP-activated Th2 cells (but not Th1 cells) express the GATA-3 protein. Thus, GATA-3 may play an important role in the balance between Th1 and Th2 subsets in immune responses. Inhibition of GATA-3 activity has therapeutic potential in the treatment of asthma and other hypereosinophilic diseases (Zhang, 1997).

Terminal deletions of chromosome 10p result in a DiGeorge-like phenotype that includes hypoparathyroidism, heart defects, immune deficiency, deafness and renal malformations. Studies in patients with 10p deletions have defined two non-overlapping regions that contribute to this complex phenotype. These are the DiGeorge critical region II, which is located on 10p13-14, and the region for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome (Mendelian Inheritance in Man number 131320), which is located more telomeric (10p14-10pter). Deletion-mapping studies have been performed in two HDR patients, and a critical 200-kilobase region has been defined that contains the GATA3 gene. This gene belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development. Investigation for GATA3 mutations in three other HDR probands has identified one nonsense mutation and two intragenic deletions that predict a loss of function, as confirmed by the absence of DNA binding by the mutant GATA3 protein. These results show that GATA3 is essential in the embryonic development of the parathyroids, auditory system and kidneys, and indicate that other GATA family members may be involved in the etiology of human malformations (Van Esch, 2000).

Members of the GATA family of zinc finger transcription factors are genetically controlled 'master' regulators of development in the hematopoietic and nervous systems. Whether GATA factors also serve to integrate epigenetic signals on target promoters is, however, unknown. The TGF-ß superfamily is a large group of phylogenetically conserved secreted factors controlling cell proliferation, differentiation, migration, and survival in multiple tissues. GATA-3, a key regulator of T helper cell development, was found to directly interact with Smad3, an intracellular signal transducer of TGF-ß. Complex formation requires a central region in GATA-3 and the N-terminal domain of Smad3. GATA-3 mediates recruitment of Smad3 to GATA binding sites independently of Smad3 binding to DNA, and the two factors cooperate synergistically to regulate transcription from the IL-5 promoter in a TGF-ß-dependent manner. Treatment of T helper cells with TGF-ß promotes the formation of an endogenous Smad3/GATA-3 nuclear complex and stimulates production of the Th2 cytokine IL-10 in a Smad3- and GATA-3-dependent manner. Through its interaction with Smad3, GATA-3 is able to integrate a genetic program of cell differentiation with an extracellular signal, providing a molecular framework for the effects of TGF-ß on the development and function of specific subsets of immune cells and possibly other cell types (Blokzijl, 2002).

GATA-2 is a zinc finger transcription factor essential for the development of hematopoiesis. While GATA-2 is generally considered to play an important role in the biology of hematopoietic stem and progenitor cells, its function within these compartments is not well understood. Both conditional expression of GATA-2 and conditional activation of a GATA-2/estrogen receptor (ER) chimera has been used to examine the effect of enforced GATA-2 expression in the development and differentiation of hematopoietic progenitors from murine embryonic stem cells. Consistent with the phenotype of GATA-2 null animals, conditional expression of GATA-2 from a tetracycline-inducible promoter enhances the production of hematopoietic progenitors. Conditional activation of a GATA-2/ER chimera produces essentially opposite effects to those observed with conditional GATA-2 expression. GATA-2 and GATA-2/ER differ in their binding activities and transcriptional interactions from other hematopoietic-associated transcription factors such as c-Myb and PU.1. While these differences in activity have been exploited to explore the transcriptional networks underlying hematopoietic cell fate determination, the results suggest that care should be taken in interpreting results obtained using only chimeric proteins (Kitajima, 2002).

Stem cells are a central feature of metazoan biology. Haematopoietic stem cells (HSCs) represent the best-characterized example of this phenomenon, but the molecular mechanisms responsible for their formation remain obscure. The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix (bHLH) transcription factor with an essential role in specifying HSCs. This study addresses the transcriptional hierarchy responsible for HSC formation by characterizing an SCL 3' enhancer that targets expression to HSCs and endothelium and their bipotential precursors, the hemangioblast. Three critical motifs have been identified that are essential for enhancer function and bind GATA-2, Fli-1 and Elf-1 in vivo. These results suggest that these transcription factors are key components of an enhanceosome responsible for activating SCL transcription and establishing the transcriptional program required for HSC formation (Gottgens, 2002).

Multipotent skin stem cells give rise to epidermis and its appendages, including hair follicles. The Lef-1/Tcf family of Wnt-regulated transcription factors plays a major role in specification of the hair shaft, but little is known about how the equally important hair channel, the inner root sheath (IRS), develops in concert to shape and guide the hair. In a microarray screen to search for transcriptional regulators of hair follicle morphogenesis, GATA-3, a key regulator of T-cell lineage determination, was identified. Surprisingly, this transcription factor is essential for stem cell lineage determination in skin, where it is expressed at the onset of epidermal stratification and IRS specification in follicles. GATA-3-null/lacZ knock-in embryos can survive up to embryonic day 18.5 (E18.5), when they fail to form the IRS. Skin grafting unveiled additional defects in GATA-3-null hairs and follicles. IRS progenitors fail to differentiate, whereas cortical progenitors differentiate, but produce an aberrant hair structure. Curiously, some GATA-3-null progenitor cells express mixed IRS and hair shaft markers. Taken together, these findings place GATA-3 with Lef-1/Wnts at the crossroads of the IRS versus hair shaft cell fate decision in hair follicle morphogenesis. This newfound function for GATA-3 in skin development strengthens the parallels between the differentiation programs governing hair follicle and lymphocyte differentiation (Kaufman, 2003).

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expresses GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD; these neuroblasts migrate from the antero-ventral otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3 (Lawoko-Kerali, 2004).

Distinct classes of serotonergic (5-HT) neurons develop along the ventral midline of the vertebrate hindbrain. A Sonic hedgehog (Shh)-regulated cascade of transcription factors has been identified that acts to generate a specific subset of 5-HT neurons. This transcriptional cascade is sufficient for the induction of rostral 5-HT neurons within rhombomere 1 (r1) that project to the forebrain, but not for the induction of caudal 5-HT neurons, which largely terminate in the spinal cord. Within the rostral hindbrain, the Shh-activated homeodomain proteins Nkx2.2 and Nkx6.1 cooperate to induce the closely related zinc-finger transcription factors Gata2 and Gata3. Gata2 in turn is necessary and sufficient to activate the transcription factors Lmx1b and Pet1, and to induce 5-HT neurons within r1. In contrast to Gata2, Gata3 is not required for the specification of rostral 5-HT neurons and appears unable to substitute for the loss of Gata2. These findings reveal that the identity of closely related 5-HT subclasses occurs through distinct responses of adjacent rostrocaudal progenitor domains to broad ventral inducers (Craven, 2004).

Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 (see Drosophila Hand) and Phox2a. The function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development was studied. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and Phox2a expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, Phox2a, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development (Tsarovina, 2004).

Gata2 is an essential hematopoietic transcriptional factor that is also expressed prominently in the nervous system. The early lethality of knockout mice due to severe anemia has largely precluded studies of gata2 neural regulation and function. The identification of zebrafish Pur alpha (Drosophila homolog: Purine-rich binding protein-alpha) and Sp8 orthologs are two factors that function to regulate neuronal expression of gata2. These factors were identified by expression cloning based on the binding activity of recombinant proteins to the previously defined gata2 cis-acting neural element. During embryogenesis, Pur alpha is expressed widely, whereas Sp8 has an overlapping pattern of expression with gata2 in the nervous system. Knockdown and ectopic expressions of Pur alpha and Sp8 indicate that these factors function, respectively, as a repressor and an activator of gata2 gene expression in the nervous system. With consideration given to the previously established roles for these factors, a model is proposed for how the transcriptional regulation of neural gata2 expression may be involved in controlling cellular proliferation in the nervous system (Penberthy, 2004).

Morpholino analysis indicates that Pur alpha plays an important role in early development in zebrafish. As far as the effects of Pur alpha morpholinos on the nervous system, such a strong arrest is generally seen during gastrulation after knockdown that the nervous system does not have time to form. However, overexpression of Pur alpha mRNA in zebrafish embryos blocks gata2 gene expression early in the ventral ectoderm and later in the nervous system. It has now been determined that Pur alpha plays an essential role in vertebrate neural development that is coupled to cellular proliferation based on gene-targeted knockout of Pur alpha in mice. Functional data is available as to the apparent trans- activity for these transcription factors regulating gata2. Together, the data support the idea that Pur alpha can function as a repressor at later stages of development (Penberthy, 2004).

Neurons in general do not proliferate. Given that it is generally accepted that the Pur factors are involved in negatively controlling DNA replication, perhaps it is one of the primary roles of Pur alpha to maintain neural cells in a differentiated state. Pur alpha has been detected in neurons and glial cells in mice with detection specifically in nonproliferating neurons. It has been shown that overexpression of Pur alpha in glioblastoma cells can inhibit the proliferation of this neural cell type. JC virus late transcription is inhibited by Pur alpha in glial cells. It has been shown that Pur alpha associates with E2F-1 to prevent E2F-1 from activating a set of genes involved in S phase (Penberthy, 2004).

By contrast, Gata2 is known to have a positive role in controlling the proliferation of a population of ventral neural progenitors, while Pur alpha is established as having a negative effect on cellular proliferation . It has been established that Sp8 is required for the maintenance of progenitor cells in the apical ectodermal ridge during limb development. This is similar to the role of Gata2 in the maintenance of progenitor cells of the nervous system and hematopoietic system. Combined with the analysis of Sp8 related to gata2 expression, a model is favored that Sp8 may be maintaining neuronal progenitor cells via the activation gata2, while Pur alpha functions as a negative regulator in nonproliferating or nonneuronal cells (Penberthy, 2004).

Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been established, although its mechanism of action is poorly understood. Detailed analysis was performed of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjkappa transcription factor to activate transcription, the aorta of the para-aortic splanchnopleura/AGM in RBPjkappa mutant embryos was examined. Specific patterns of expression of Notch receptors, ligands and Hes genes were found that were lost in RBPjkappa mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. In wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and it was demonstrate by chromatin immunoprecipitation that Notch1 specifically associates with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjkappa mutants. Consistent with a role for Notch1 in regulating Gata2, increased expression of this gene was observed in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjkappa is a crucial event for the onset of definitive hematopoiesis in the embryo (Robert-Moreno, 2005).

The ecotropic viral integration site-1 (Evi1), a common site of retroviral integration in murine myeloid tumors, is an oncogenic transcription factor in murine and human myeloid leukemia. Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, suggesting a physiological role of Evi1 in HSCs. The role and authentic target genes of Evi1 in hematopoiesis was investigated using Evi1-/- mice, which die at embryonic day 10.5. HSCs in Evi1-/- embryos are markedly decreased in numbers in vivo with defective self-renewing proliferation and repopulating capacity. Notably, expression rate of GATA-2 mRNA, which is essential for proliferation of definitive HSCs, is profoundly reduced in HSCs of Evi1-/- embryos. Restoration of the Evi1 or GATA-2 expression in Evi1-/- HSCs could prevent the failure of in vitro maintenance and proliferation of HSC through upregulation of GATA-2 expression. An analysis of the GATA-2 promoter region revealed that Evi1 directly binds to GATA-2 promoter as an enhancer. These results reveal that GATA-2 is presumably one of critical targets for Evi1 and that transcription factors regulate the HSC pool hierarchically (Yuasa, 2005).

Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5' to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP+ fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34+/c-Kit+ cells than the GFP fraction did. When cultured in vitro, the P-Sp GFP+ cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp (Kobayashi-Osaki, 2005).

The transcription factor GATA2 plays an essential role in the establishment and maintenance of adult hematopoiesis. It is expressed in hematopoietic stem cells, as well as the cells that make up the aortic vasculature, namely aortic endothelial cells and smooth muscle cells. GATA2 expression is predictive of location within the thoracic aorta; location is suggested to be a surrogate for disease susceptibility. The GATA2 gene maps beneath the Chromosome 3q linkage peak from a family-based sample set (GENECARD) study of early-onset coronary artery disease. Given these observations, the relationship of several known and novel polymorphisms within GATA2 to coronary artery disease was investigated. Five single nucleotide polymorphisms were identified that were significantly associated with early-onset coronary artery disease in GENECARD. These results were validated by identifying significant association of two of these single nucleotide polymorphisms in an independent case-control sample set that was phenotypically similar to the GENECARD families. These observations identify GATA2 as a novel susceptibility gene for coronary artery disease and suggest that the study of this transcription factor and its downstream targets may uncover a regulatory network important for coronary artery disease inheritance (Connelly, 2006; full text of article).

The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However, the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. This study shows how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis, as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver, Eklf expression is shown to be initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies, it was found that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population, followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis (Lohmann, 2008).

A Gata2 intronic enhancer confers its pan-endothelia-specific regulation

GATA-2, a transcription factor that has been shown to play important roles in multiple organ systems during embryogenesis, has been ascribed the property of regulating the expression of numerous endothelium-specific genes. However, the transcriptional regulatory hierarchy governing Gata2 activation in endothelial cells has not been fully explored. This study documents GATA-2 endothelial expression during embryogenesis by following GFP expression in Gata2-GFP knock-in embryos. Using founder transgenic analyses, a Gata2 endothelium enhancer was identified in the fourth intron and it was found that Gata2 regulation by this enhancer is restricted to the endocardial, lymphatic and vascular endothelium. Whereas disruption of three ETS-binding motifs within the enhancer diminished its activity, the ablation of its single E box extinguished endothelial enhancer-directed expression in transgenic mice. Development of the endothelium is known to require SCL (TAL1), and an SCL-E12 (SCL-Tcfe2a) heterodimer can bind the crucial E box in the enhancer in vitro. Thus, GATA-2 is expressed early in lymphatic, cardiac and blood vascular endothelial cells, and the pan-endothelium-specific expression of Gata2 is controlled by a discrete intronic enhancer (Khandekark, 2007).

In the yolk sac, the blood islands consist of a thin layer of angioblasts surrounding primitive erythrocytes. Similarly, in the aorta-gonads-mesonephros region (the initial embryonic site of definitive hematopoiesis), hematopoietic stem cells can be detected budding from the endothelium of the dorsal aorta. Given the close physical proximity of the very earliest hematopoietic and endothelial cells, it has been speculated that they originate from a common progenitor cell, which has been termed the hemangioblast. A number of transcription factors have been shown to play a role in the development of both cell lineages: for example, cloche is required for the formation of endothelial and hematopoietic progenitors in zebrafish and Scl (also known as Tal1 -- Mouse Genome Informatics), which encodes a basic helix-loop-helix transcription factor, was initially shown to be required for hematopoietic development in mice. Subsequent transgenic rescue of the hematopoietic defect in Scl-null embryos revealed a requirement for SCL in the remodeling of the yolk sac vasculature, and it has since been shown to play a role in vasculogenesis, as well as in the migration and morphogenesis of endothelial cells. Transgenic expression of SCL is able to rescue the phenotypic consequences of cloche mutation in the zebrafish, suggesting that Scl functions downstream of cloche. LMO2, a member of the LIM domain family, is required for primitive erythropoiesis in the embryo; Lmo2 ablation results in death at embryonic day (E) 9.75 secondary to hematopoietic failure. Analysis of chimeric mice bearing contributions from Lmo2-/- embryonic stem (ES) cells revealed that angiogenic remodeling of blood vessels requires Lmo2. Similarly, targeted disruption of the transcription factor Runx1 eliminates definitive hematopoiesis and results in defective angiogenesis and hemorrhaging throughout the CNS (Khandekark, 2007).

The most-widely accepted and experimentally supported model for lymphatic development has proposed that the lymphatic vasculature arises from the blood vasculature. Expression of the lymphatic endothelial hyaluronan receptor gene (Lyve1; also known as Xlkd1 -- Mouse Genome Informatics) at E9-9.5 in endothelial cells lining the anterior cardinal vein is the first sign that these cells are competent to become lymphatic endothelial cells (LECs). The lymphatic regulatory gene Prox1, encoding a homeobox transcription factor, is expressed several hours later in a subset of LYVE1+ cells in the anterior cardinal vein. Expression of the murine vascular endothelial growth factor receptor 3 gene (Vegfr3, also known as Flt4 - Mouse Genome Informatics), which binds VEGFC, is detected in blood and lymphatic vessels during early embryogenesis, but becomes largely restricted to lymphatic vessels after E14.5 (Khandekark, 2007).

Beginning at E10.5, LECs bud and migrate away from the anterior cardinal vein in a polarized non-random manner, and eventually fuse to form primitive lymph sacs from which new LECs sprout and spread into the surrounding tissues and organs. Finally, the lymphatic plexus undergoes remodeling and maturation in the terminal stages of lymphatic development. Little is known about the molecular events leading to lymphatic development, but gene-ablation studies in mice and the identification of human hereditary-lymphedema causative genes indicate that Prox1, Vegfc, Vegfr3, Foxc2 and Sox18 are requisites to the process (Khandekark, 2007).

GATA factors belong to an evolutionarily conserved family of C4 zinc-finger transcription factors that play demonstrably crucial roles in development. There are six GATA family members in vertebrates, which have historically been subdivided into two subfamilies. GATA-1, GATA-2 and GATA-3 are all important in the development of different hematopoietic lineages (erythroid, hematopoietic progenitor and T-lymphoid, respectively), among many other activities. Similarly, GATA-4, GATA-5 and GATA-6 have been shown to be involved in cardiac, genitourinary and multiple endodermal developmental events (Khandekark, 2007).

GATA-2 was originally cloned from a chicken reticulocyte cDNA library, and was shown to be expressed in a wide variety of tissues, including hematopoietic, neuronal and endothelial cells. Gata2-null mutant embryos die at mid-gestation due to a block in primitive hematopoiesis. Further examination of Gata2 gain-of-function and in vitro differentiation of Gata2-/- ES cells showed that GATA-2 plays a pivotal role in the proliferation of very early hematopoietic progenitors, underscoring the conclusions from the initial loss-of-function experiments (Khandekark, 2007).

Given that many genes involved in hematopoiesis also participate in vascular development and that GATA-2 is strongly expressed in endothelial cell lines, it was originally believed that loss of GATA-2 function would result in vascular defects. Adding further to this expectation was early evidence that many genes that appeared to be crucial for endothelial development and function are regulated via GATA-binding sites. For example, GATA sites have been implicated in the regulation of the endothelium-specific genes preproendothelin (immature form of EDN1), Pecam1, Vegfr2, eNOS (also known as Nos3 -- Mouse Genome Informatics). Mutation of a GATA-binding site in the Vegfr2 endothelium-specific enhancer completely abolished its activity in transgenic reporter assays, indicating that Vegfr2 expression is dependent on GATA activity in vivo. Surprisingly, however, the analysis of Gata2-null embryos failed to reveal any obvious defects in the vasculature at the time of their early embryonic demise, leaving the role for GATA-2 in endothelial function undefined (Khandekark, 2007).

To begin to investigate the role of GATA-2 in endothelial function, GFP expression was examined in the developing vasculature of Gata2-GFP knock-in embryos during embryogenesis. GFP was found to be expressed in cells lining arterial and venous vessels formed during vasculogenesis and angiogenesis, and that its expression continued postnatally. GFP expression was observed in budding LECs during early lymphatic development, as well as in postnatal lymphatic vessels. An endothelium-specific enhancer was identified in Gata2 intron 4 that could regulate the expression of a cis-linked reporter transgene in cardiovascular and lymphatic endothelial cells. Additionally, site-specific mutagenesis revealed that the potency of the minimal endothelium-specific enhancer is crucially dependent on an E box (CANNTG) motif. By contrast, disruption of three ETS-binding sites quantitatively reduced, but did not abolish, enhancer activity. Prior experiments showed that SCL activation is required for elaboration of the vasculature, and SCL-E12 (E12 is also known as TCFE2A -- Mouse Genome Informatics) heterodimers were shown to bind with high affinity to this crucial enhancer E box in vitro. Altogether, these data implicate ETS family members and SCL as in vivo activators of endothelium-specific Gata2 transcription (Khandekark, 2007).

GATA2 functions at multiple steps in hemangioblast development and differentiation

Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, gene expression profiles were compared between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. These studies revealed that Gata2 is a direct target of BMP4 and that enforced GATA2 expression upregulates Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, compelling evidence is provided that GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and endothelial cells (Lugus, 2007).

A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, this study demonstrates that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. These data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome (Groschel, 2014).

Gata2 and interneurons

During embryogenesis, transcription factor GATA2 is expressed in a variety of distinct cell types, and earlier experiments have shown that GATA2 is a vital regulator of both hematopoiesis and urogenital development. Despite the fact that GATA2 is expressed early and abundantly in the nervous system, there has been no demonstration of its direct participation in neurogenesis. GATA2 is expressed in the ventral spinal cord exclusively in newly generated V2 interneurons, suggesting that GATA2 might be required for the generation of this discrete neuronal population. Proof for this hypothesis was provided when the number of cells expressing V2 neuronal markers were seen to be drastically diminished in gata2 null mutant embryos. The tissue-specific enhancer that directs gata2 transcription specifically in V2 neurons is localized to a 190 bp intragenic element lying within gata2 intron 5, and this element is both necessary and sufficient to confer GATA2 spinal cord expression. The identification of a V2-specific enhancer should allow fundamental new insight into the genetic hierarchy of regulatory events that govern neurogenesis in a well-defined cell lineage (Zhou, 2000).

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including those involved in vertebrate central nervous system development. However, the role of GATA proteins in spinal cord development remains unresolved. The expression and function of two GATA proteins, GATA2 and GATA3, were examined in the developing chick spinal cord. Both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, it has been demonstrated that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, it has been demonstrated that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, these findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord (Karunaratne, 2002).

LMO4 controls the balance between excitatory and inhibitory spinal V2 interneurons: LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and LIM domain-binding protein NLI

Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. This study provides a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with bHLH factor SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and LIM domain-binding protein NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord (Joshi, 2009).

LIM-HD codes are crucial in implementing cell-type-specific transcription by directing different types of LIM-complexes in a cell context-dependent manner. These studies expand the LIM codes to include bHLH and Gata proteins as these two factors form an atypical LIM-complex via a non-DNA binding LIM factor LMO4. Unlike typical LIM-complexes such as the V2-tetramer complex, which utilize LIM-HD proteins for recognition of specific DNA response elements (Lee, 2008), SCL and Gata2 serve as the major DNA-binding components in the V2b complex. A couple of unique advantages of assembling the V2b complex can be proposed in cell fate specification (Joshi, 2009).

First, these results suggest that the V2b complex allows integration of SCL and Gata2 functions by selecting a group of target genes that bear both SCL- and GATA-recognition sites. This should ensure the expression of V2b-target genes specifically in cells coexpressing SCL and Gata2. It was found that the enhancers of Gata2 and Gata3 genes display striking similarity in that they contain reiterated bipartite elements composed of E-box (CAnnTG) and/or atypical E-box (CAnnnTG) for SCL-binding and GATA sites for recruiting Gata proteins. E-boxes and GATA sites occur relatively often in the genome due to their short sequences and serve as binding motifs for multiple bHLH and Gata factors. Thus, simultaneous recognition of paired E-box-GATA composite elements by the V2b complex is expected to provide the required stringency in choosing the target genes coregulated by SCL and Gata2 (Joshi, 2009).

Second, this study found that formation of the V2b complex facilitates the transcriptional synergy among its components by enabling the recruitment of coactivators including SSDP1. Coexpression of SSDP1 allowed a potent transcriptional activation by the V2b complex on its physiological targets, Gata2/3-enhancers. Given that SCL and Gata2 are relatively weak transcriptional activators in Gal4-DBD fusion transcription assays, the transcriptional synergy between SCL and Gata2 resulting from forming a complex may be due, at least in part, to the recruitment of SSDP1. The facilitated recruitment of SSDP1 and possibly other coactivators may account for the necessity of the V2b complex formation for inducing the V2b-IN genes (Joshi, 2009).

Regulatory interactions specifying Kolmer-Agduhr interneurons

In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr' (KA') and V3 interneurons. The differentiation of the correct number of KA' cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA' cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA' cells of the lateral floor plate and the KA' cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA' cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA' cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA' and KA' cells (Yang, 2010).

Transcriptome and phenotypic analysis reveals Gata3-dependent signalling pathways in murine hair follicles

The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. This study describes a conditional Gata3-/- mouse (K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates (Kurek, 2007).

A modular enhancer is differentially regulated by GATA and NFAT elements that direct different tissue-specific patterns of nucleosome positioning and inducible chromatin remodeling

This study investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruites distinct inducible tissue-specific enhanceosome-like complexes and directs nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer is activated by inducible binding of two NFAT/AP-1 complexes which disrupt two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer is remodeled by GATA factors which constitutively displace an upstream nucleosome (N0) and cooperate with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combine to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus is organized as an array of nucleosomes with fixed positions, but the enhancer adopts different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter are mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer (Bert, 2007; full text of article).

Gata2 is a tissue-specific post-mitotic selector gene for midbrain GABAergic neurons

Midbrain GABAergic neurons control several aspects of behavior, but regulation of their development and diversity is poorly understood. This study further refines the midbrain regions active in GABAergic neurogenesis and shows their correlation with the expression of the transcription factor Gata2. Using tissue-specific inactivation and ectopic expression, it was shown that Gata2 regulates GABAergic neuron development in the mouse midbrain, but not in rhombomere 1, where it is needed in the serotonergic lineage. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. These studies identify Gata2 as an essential post-mitotic selector gene of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of GABAergic neurons in the midbrain (Kala, 2009).

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. This study shows that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3 (see Drosophila Serpent), activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3 expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. This study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation (Haugas, 2016).

Table of contents

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

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