Transcription factor interaction in heart formation

In Drosophila, dorsal mesodermal specification is regulated by the homeobox genes tinman and bagpipe. Vertebrate homologs of tinman and bagpipe have been isolated in various species. Moreover, in vertebrates, there are at least four different genes related to tinman, which indicates that this gene has been duplicated during evolution. One of the murine homologs of tinman is the cardiac homeobox gene Csx or Nkx2.5. Gene targeting of Csx/Nkx2.5 shows that this gene is required for completion of the looping morphogenesis of the heart. However, it is not essential for the specification of the heart cell lineage. Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2.5. Possible candidates might be other members of the NK2 class of homeobox proteins, like Tix/Nkx2.6, Nkx2.3, nkx2.7, or cNkx2.8. This study reports that murine Tix/Nkx2.6 mRNA has been detected in the heart and pharyngeal endoderm. Xenopus XNkx2.3 and chicken cNkx2.3 are expressed in the heart as well as in pharyngeal and gut endoderm. In contrast, murine Nkx2.3 is expressed in the gut and pharyngeal arches but not the heart. In zebrafish and chicken, two new NK-2 class homeoproteins, nkx2.7 and cNkx2.8, have been identified. Zebrafish nkx2.7 is expressed in both the heart and pharyngeal endoderm. In chicken, cNkx2.8 is expressed in the heart primordia and the primitive heart tube and becomes undetectable after looping. No murine homologs of nkx2.7 or cNkx2.8 have been found so far. The overlapping expression pattern of NK2 class homeobox genes in the heart and the pharynx may suggest a common origin for these two organs. In the Drosophila genome, the tinman gene is linked to bagpipe, another NK family gene. A murine homolog of bagpipe, Bax/Nkx3.1, is expressed in somites, blood vessels, and the male reproductive system during embryogenesis (this study), suggesting that this gene's function may be relevant for the development of these organs. A bagpipe homolog in Xenopus, Xbap, is expressed in the gut musculature and a region of the facial cartilage during development (Tanaka, 1998).

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

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Evidence is presented from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. Zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors, as well as the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5 (Reiter, 1999).

Gata5 is capable of activating transcription from a wide range of myocardial promoters; gata5 mutants display marked defects in the expression of many genes encoding components of the myocardial sarcomere (e.g., cmlc1, cmlc2, vmhc, cardiac troponin T, tropomyosin). However, it is not clear at this point which of these genes are direct targets of Gata5 and which require intermediate Gata5-dependent regulators. In fact, there may be no clear division between these cases as Gata5 may act both directly and indirectly on a single promoter. For example, Gata5 may participate in the induction of nkx2.5 and also bind cooperatively with Nkx2.5 to regulatory elements of a wide range of myocardial genes. It is also interesting to note the different requirements various sarcomeric protein genes have for Gata5. For example, although the expression patterns of cmlc1 and cmlc2 are indistinguishable in wild-type embryos, cmlc1 expression is more severely reduced than cmlc2 expression in fau mutants. Perhaps these differences reflect different affinities of Gata5 for the respective cis regulatory elements (Reiter, 1999).

In vertebrates, heart development is a multistep process that starts with formation and patterning of the primitive heart tube and is followed by complex morphological events to give rise to the mature four-chambered heart. These various stages are characterized by distinct patterns of gene expression. Although chamber specificity and developmental regulation can be demonstrated in transgenic mice using short promoter fragments, the mechanism underlying spatial and temporal specificity within the heart remains largely unclear. Combinatorial interaction between a limited number of cardiac-specific and ubiquitous transcription factors may account for the diverse genetic inputs required to generate the complex transcriptional patterns that characterize the developing myocardium. The cardiac atrial natriuretic peptide (ANP) promoter was used to test this hypothesis. The ANP gene is transcribed in a spatial- and temporal-specific manner in the heart, and a 500 bp promoter fragment is sufficient to recapitulate both chamber and developmental specificity. This promoter is composed of three modules: a "basal" cardiac promoter that is essential for transcription in embryonic and postnatal atrial and ventricular myocytes, and two other independent modules that behave as chamber-specific enhancers. The basal cardiac promoter is the target of two cardiac-specific transcription factors, the zinc finger GATA-4 protein and the Nkx2-5 homeodomain, which bind to contiguous elements within this region. At low concentrations--a situation that likely occurs during the very first stages of cardiac cell fate determination--the two proteins synergistically activate transcription from the ANP promoter. This functional synergy requires physical interaction between the GATA-4 protein and an extended C-terminal homeodomain on Nkx2-5. This interaction, which unmasks an activation domain present just N-terminal of the homeodomain, is specific for GATA-4 and-5, but is not observed with the other cardiac GATA factor, GATA-6. Optimal synergy requires binding of both proteins to their cognate sites, although modest synergy also could be observed on heterologous promoters containing only multimerized Nkx binding sites, suggesting that Nkx2-5 is able to recruit GATA-4 into a transcriptionally active complex. The GATA/Nkx interaction, which appears to have been evolutionarily conserved in nematode, fly, and mammals, provides a paradigm for analyzing transcription factor interaction during organogenesis (Durocher, 1998).

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, a test was performed to see whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways (Lee, 1998).

The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac alpha-actin (alphaCA) promoter in fibroblasts. Nkx-2.5 is shown to also cooperate with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the alphaCA promoter. A similar cooperation also takes place on a minimal promoter, containing only multimerized Nkx-2.5 DNA binding sites (NKEs), in heterologous CV-1 fibroblasts. Transcriptional activity requires the N-terminal activation domain of Nkx-2.5 and Nkx-2.5 binding activity through its homeodomain but does not require GATA-4's activation domain. The minimal interactive regions were mapped to the homeodomain of Nkx-2.5 and the second zinc finger of GATA-4. Removal of Nkx-2.5's C-terminal inhibitory domain stimulates robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitates Nkx-2.5 DNA binding activity. The following simple model is proposed: GATA-4 induces a conformational change in Nkx-2.5 that displaces the C-terminal inhibitory domain, thus eliciting transcriptional activation of promoters containing Nkx-2.5 DNA binding targets. Therefore, alphaCa promoter activity appears to be regulated through the combinatorial interactions of at least three cardiac tissue-enriched transcription factors, Nkx-2.5, GATA-4, and SRF (Sepulveda, 1998).

The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos. Nkx2-5 expression is first detected in mesodermal cells specified to form heart at embryonic day 7.5 in the mouse and expression is maintained throughout the developing and adult heart. In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach. To investigate the mechanisms that initiate cardiac transcription during embryogenesis, the Nkx2-5 upstream region was analyzed for regulatory elements sufficient to direct expression of a lacZ transgene in the developing heart of transgenic mice. A cardiac enhancer is described. Located about 9 kilobases upstream of the Nkx2-5 gene, the enhancer fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube. Thereafter, as the atrial and ventricular chambers become demarcated, enhancer activity becomes restricted to the developing right ventricle. Transcription of Nkx2-5 in pharynx, thyroid and stomach is controlled by regulatory elements separable from the cardiac enhancer. This distal cardiac enhancer contains a high-affinity binding site for the cardiac-restricted zinc finger transcription factor GATA4. GATA4 is essential for transcriptional activity. These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart. At least two other regulatory regions that direct Nkx2-5 expression in the developing heart have also been identified and negative regulatory elements have been identified as well (Lien, 1999).

Vertebrate sequences related to tinman, such as mouse Nkx-2.5, chicken cNkx-2.5, Xenopus XNkx-2.5 and XNkx-2.3 are expressed in cardiac precursors and in tissues involved in induction of cardiac mesoderm. Mice which lack a functional Nkx-2.5 gene die due to cardiac defects. To determine the role of tinman-related sequences in heart development, both XNkx-2.3 and XNkx-2.5 were overexpressed in Xenopus embryos. The resulting embryos are morphologically normal except that they have enlarged hearts. The enlarged heart phenotype is due to a thickening of the myocardium caused by an increase in the overall number of myocardial cells (hyperplasia). Neither ectopic nor precocious expression of cardiac differentiation markers is detectable in overexpressing embryos. These results suggest that both XNkx-2.3 and XNkx-2.5 are functional homologues of tinman, responsible for maintenance of the heart field (Cleaver, 1996).

nkx-2.5 is one of the first genes expressed in the developing heart of early stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained throughout development and the gene is also expressed in the developing pharyngeal arches, spleen, thyroid and tongue. Genomic sequences flanking the mouse nkx-2.5 gene were analyzed for early developmental regulatory activity in transgenic mice. Approximately 3 kb of 5' flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and in limited regions of the developing heart at later stages. Expression also is detected in the developing spleen anlage at least 24 hours before the earliest reported spleen marker and in the pharyngeal pouches and their derivatives including the thyroid. The observed expression pattern from the -3 kb construct represents a subset of the endogenous nkx-2.5 expression pattern, which is evidence for compartment-specific nkx-2.5 regulatory modules. A 505 bp regulatory element has been identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pharynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element eliminates gene activation in the heart, pharynx and spleen primordia of transgenic embryos. The dependence of this nkx-2.5 regulatory element on GATA sites for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression. The presence of consensus binding sites for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple regulatory factors are responsible for the initial activation of nkx-2.5 in the cardiac, thyroid and spleen primordia (Searcy, 1998).

Reported here is the isolation and characterization of murine and human cDNAs encoded for by Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomains are more than 93% identical to those of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0 -8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5-11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression has also been assessed in embryos with aberrant cardiac development: mice lacking RXRalpha or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos (see Drosophila Hand) initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development downstream of Nkx2-5 and dHand (Bruneau, 2000).

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

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', the T-box-containing transcription factor Tbx5 was isolated; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells shows that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicate that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 are necessary for their interaction. Tbx5 and Nkx2-5 directly binds to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors show synergistic activation. Deletion analysis shows that both the N-terminal domain and T-box of Tbx5 are important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, does not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activates the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 start to beat earlier and express cardiac-specific genes more abundantly than do parental P19CL6 cells, whereas cell lines expressing the G80R mutant do not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development (Hiroi, 2001).

Nkx2.5/Csx and dHAND/Hand2 are conserved transcription factors that are coexpressed in the precardiac mesoderm and early heart tube and control distinct developmental events during cardiogenesis. To understand whether Nkx2.5 and dHAND may function in overlapping genetic pathways, mouse embryos lacking both Nkx2.5 and dHAND were generated. Mice heterozygous for mutant alleles of Nkx2.5 and dHAND are viable. Although single Nkx2.5 or dHAND mutants have a morphological atrial and single ventricular chamber, Nkx2.5;dHAND double mutants have only a single cardiac chamber which was molecularly defined as the atrium. Complete ventricular dysgenesis was observed in Nkx2.5;dHAND double mutants; however, a precursor pool of ventricular cardiomyocytes was identified on the ventral surface of the heart tube. Because Nkx2.5 mutants failed to activate eHAND expression even in the early precardiac mesoderm, the double mutant phenotype appears to reflect an effectively null state of dHAND and eHAND. Cell fate analysis in dHAND mutants suggests a role of HAND genes in survival and expansion of the ventricular segment, but not in specification of ventricular cardiomyocytes. These molecular analyses also reveal the cooperative regulation of the homeodomain protein, Irx4, by Nkx2.5 and dHAND. These studies provide the first demonstration of gene mutations that result in ablation of the entire ventricular segment of the mammalian heart, and reveal essential transcriptional pathways for ventricular formation (Yamagishi, 2001).

During heart development, chamber myocardium forms locally from the embryonic myocardium of the tubular heart. The atrial natriuretic factor (ANF) gene is specifically expressed in this developing chamber myocardium and is one of the first hallmarks of chamber formation. The regulatory mechanism underlying this selective expression has been investigated. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of endogenous ANF gene expression. Furthermore, this fragment is able to repress cardiac troponin I (cTnI) promoter activity selectively in the embryonic myocardium of the atrioventricular canal (AVC). In vivo inactivation of a T-box factor (TBE) or NK2-homeobox factor binding element (NKE) within the ANF fragment removes the repression in the AVC without affecting its chamber activity. The T-box family member Tbx2, encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 forms a complex with Nkx2.5 on the ANF TBE-NKE, and is able to repress ANF promoter activity. These data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC (Habets, 2002).

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. Tbx20 expression has been analyzed during murine cardiac development, and DNA-binding and transcriptional properties of Tbx20 isoforms have been assessed. Tbx20 is expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carries strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bind specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacts with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, leads to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behavior in an in vivo context. It is proposed that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development (Stennard, 2003).

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFß-Smad pathway. Foxh1−/− mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c−/− mutant hearts. Further, Mef2c is shown to be a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFß response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFß-like signals (von Both, 2004).

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. Feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state are upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT are increased, while Smad1 deletion in Nkx2-5 mutants rescue SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies are also rescued by Smad1 deletion. These findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD (Prall, 2007).

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. This study shows that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), referred to as 'cardiac TFs,' have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, chromatin occupancy by these TFs and p300 were defined genome wide and unbiased support for this hypothesis is provided. This principle was used to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. These data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers (He, 2011).

Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. This study shows that the homeoprotein Nkx2-5 and the 5'-3' exonuclease Xrn2 (see Drosophila Rat1) are involved in the regulation of alternative polyadenylation (APA) during mouse heart development (see Drosophila dorsal vessel). Nkx2-5 occupies not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affects Xrn2 binding to target loci and results in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 leads to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5+/-Xrn2+/-, but neither Nkx2-5+/- nor Xrn2+/-, newborns exhibit a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. These results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development and suggest that tissue-specific transcription factors are involved in the regulation of APA (Nimura, 2016).

The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. A systems biology study is presented integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, conclusions primarily obtained in cardiomyocyte cell culture were confirmed in a time-course of cardiac maturation in mouse around birth. Insights are provided into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, it was found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, it was found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, an Srf-centered transcription network is presented providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, this study shows the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders (Schlesinger, 2011; full text of article).

Tinman homologs: transcriptional regulation

Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. The secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4 expression in cardiac precursors, but not for gata6. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 mRNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signaling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independent of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors (Reifers, 2000).

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype the expression of the receptors transducing BMP signaling during gut development was examined. The BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, it has been found that when ectopically expressed in the gizzard, the BMP receptors act in a manner similar to BMP4. The mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation (Smith, 2000).

These data provide insight into the molecular controls of patterning events at the small intestinal-gizzard border. In the early stages of gut development, Shh expressed throughout the endoderm is secreted to signal to the overlying mesoderm, which expresses the SHH-receptor Ptc. This Shh signal causes an increase in proliferation to occur in the mesoderm throughout the gut tube, and it also causes the activation of BMP4 expression throughout the mesoderm of the intestine, but no BMP4 is activated in the gizzard. BMP4 then causes the mesoderm of the small intestine to decrease proliferation and increase apoptosis to antagonize the proliferation effects of SHH signaling, which results in the thin small intestinal mesoderm. The gizzard develops a thick mesodermal layer due both to the SHH signal and a lack of BMP signals. BMP4 expressed in the small intestinal mesoderm also diffuses across to the gizzard mesoderm to bind to BMPR1B to cause upregulation of Nkx2.5 within the posterior gizzard mesoderm and to specify the phenotype of the pyloric sphincter. Nkx2.5, a marker for the pyloric sphincter mesoderm, plays a direct role in this process, patterning the endoderm of the pyloric sphincter via some unknown signal(s). Meanwhile, BMP4 expressed in the small intestinal mesoderm, and later in the gizzard submucosa, delays smooth muscle differentiation in these tissues. Hence, BMP signaling has many important developmental roles in gut patterning. The universality of this anatomic boundary in vertebrates, expression of the molecules delineating this region, and conservation of the signaling pathways involved underscores the importance of gut development as a model system for understanding development (Smith, 2000).

In the forming vertebrate heart, bone morphogenetic protein signaling induces expression of the early cardiac regulatory gene nkx-2.5. A similar regulatory interaction has been defined in Drosophila embryos, where Dpp signaling mediated by the Smad homologs Mad and Medea directly regulates early cardiac expression of tinman. A conserved cluster of Smad consensus binding sequences has been identified in early cardiac regulatory sequences of the mouse nkx-2.5 gene. The importance of the nkx-2.5 Smad consensus region in early cardiac gene expression was examined in transgenic mice and in cultured mouse embryos. In transgenic mice, deletion of the Smad consensus region delays induction of embryonic DeltaSmadnkx-2.5/lacZ gene expression during early heart formation. Induction of DeltaSmadnkx-2.5/lacZ expression is also delayed in the outflow tract myocardium and visceral mesoderm. Targeted mutation of the three Smad consensus sequences inhibits nkx-2.5/lacZ expression in the cardiac crescent, demonstrating a specific requirement for the Smad consensus sites in early cardiac gene induction. Cultured DeltaSmadnkx-2.5/lacZ transgenic mouse embryos also exhibit delayed induction of transgene expression. In the four-chambered heart, deletion of the Smad consensus region results in expanded DeltaSmadnkx-2.5/lacZ transgene expression. Thus, the nkx-2.5 Smad consensus region can have positive or negative regulatory function, depending on the developmental context and cellular environment (Liberatore, 2002).

A target consensus binding sequence (GCCGnCGc) for Drosophila MAD and Medea has been reported based on Dpp-responsive elements in tinman, dmef2, and vestigial genes. In mice, a GCCGnCGC-like motif present in the smad6 promoter is responsive to BMP signals mediated by Smad1/5 and binds Smad5 and Smad4. This 7-bp Smad1/5-induced sequence is present with no mismatches in the mouse nkx-2.5 early cardiac regulatory element. Additional Smad-responsive regulatory elements containing the consensus CAGA are present in human plasminogen activator inhibitor type 1, c-jun, PDGF-B, CARP, and alpha2procollagen genes. Two CAGA consensus sequences are present in addition to the distal GC-rich site between alpha3059 and alpha3012 of the mouse early cardiac regulatory element. The presence of three potential Smad-responsive sequences within a short stretch of DNA is characteristic of genetic elements regulated by Smad-dependent signaling mechanisms. The mouse nkx-2.5 Smad consensus region sequence is highly conserved in human nkx-2.5 genomic DNA with 48/50 identical nucleotides. Therefore, the Smad consensus region represents a potential direct target for BMP-mediated induction of mouse nkx-2.5 gene expression. (Liberatore, 2002).

Heart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2-5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. An evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2-5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2-5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2-5 site is not required for enhancer activity. These findings demonstrate that like tinman, Nkx2-5 is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2-5 enhancer of Nkx2-5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2-5 and tinman expression during cardiogenesis in vertebrates and fruit flies (Lien, 2002).

The organization of the tinman tin-D and vertebrate Nkx2.5 enhancers was compared. There are four putative Smad4 binding sites, GTCT/AGAC, that are conserved in the AR2 enhancer. It has been shown that the tinman tin-D enhancer contains eight Mad binding sites, three of which can also be bound by Medea. The consensus sequence of the Mad and Medea binding sites is the GC-rich sequence, CGCCGC. However, for the sites that can also be bound by Medea, such as the M2 and M4 sites in the tin-D enhancer, there is an AGAC/GTCT sequence adjacent to the GC-rich sequence. This AGAC/GTCT sequence is identical to the vertebrate Smad4 binding site. Thus, it is likely that Medea actually binds to the AGAC/GTCT sequences instead of the GC-rich sequence (Lien, 2002).

Multiple Mad/Medea binding sites in the tin-D enhancer are required for dorsal mesoderm-specific activity of the enhancer. In the AR2 enhancer, the Smad4 site at -2774 is required for enhancer activity in the cardiac crescent and later in heart development. These findings reveal an evolutionarily conserved role for Smad factors in the activation of cardiac NK-type homeobox genes, and support the notion that Nkx2-5, like tinman, is a direct target of Smad proteins. Interestingly, when the mouse AR1 and AR2 enhancers with the Dpp-responsive tin-D3 enhancer of Drosophila tinman are compared, striking similarities are found among these enhancers. The essential Smad site at -2774 adjacent to the two essential GATA sites and the adjacent 3'-flanking sequences in the AR2 enhancer show high homology to the minimal Dpp response element in the tin-D enhancer. In addition, the core of the mouse AR1 enhancer contains a region with high homology to the region surrounding the essential Smad site at -2774 in the AR2 enhancer. This putative Smad site is also close to the essential GATA site in the AR1 enhancer. However, when this putative Smad site in the AR1 enhancer is mutated, enhancer activity is not abolished, suggesting there might be other redundant Smad sites present in the AR1 enhancer (Lien, 2002).

The Smad sites at the 5' end of the AR2 enhancer are not required for cardiac expression later in development and the mutant enhancer actually shows enhanced activity in the right ventricle, suggesting a negative role for Smad binding to these sites. Thus, it appears that the AR2 enhancer is a target for positive and negative regulation by Smad proteins at different stages of cardiac development. These divergent modes of regulation are likely to reflect differential associations of Smads with positive and negative cofactors that bind nearby sites in the enhancer (Lien, 2002).

Smads typically activate transcription in combination with other cofactors. Since BMPs are expressed in other regions of the embryo in addition to the cardiogenic region, the mechanism for BMP-dependent activation of Nkx2-5 must be coupled to other cell-autonomous regulators expressed prior to Nkx2-5. Understanding how BMP signaling is interpreted in mesodermal cells by cardiogenic cofactors is likely to provide insights into the molecular basis for cardiac specification. In this regard, Smad4 interacts directly with GATA-4, providing a possible molecular basis for transcriptional synergy between these factors and for directly linking cardiac gene regulation with the BMP signaling pathway (Lien, 2002).

While the transcriptional regulation of Nkx2-5 and tinman appear to be similar with respect to the dependence of the AR2 and tin-D enhancers on BMP signaling through Smad proteins, there are also fundamental differences in the regulation of these enhancers. In particular, the tinman tin-D enhancer is controlled through the combined actions of Medea and Tinman, whereas Nkx2-5 does not seem to autoregulate its own expression through the Nkx2-5 binding site in the AR2 enhancer. On the contrary, it has been suggested that Nkx2-5 negatively regulates its own expression, although no evidence was found for enhanced expression of the enhancer with the Nkx2-5 binding site mutation, as might be predicted by such a model (Lien, 2002).

The differences in regulation of tinman and Nkx2-5 transcription reflect the differences in mesoderm specification and patterning of the vertebrate and arthropod body plans. tinman is expressed throughout the nascent mesoderm of Drosophila prior to its subdivision into different sublineages. Expression of tinman in the early mesoderm is mediated by binding of Twist to a separate enhancer. Specification of the dorsal mesoderm occurs in response to Dpp signaling from the dorsal ectoderm. In contrast, Nkx2-5 expression is initiated concomitant with cardiogenic specification in response to BMP signaling from the anterior endoderm. Thus, the mechanism for BMP-dependent activation must be coupled to other cell-autonomous regulators expressed prior to Nkx2-5 itself. Understanding how BMP signaling is interpreted in mesodermal cells by cardiogenic cofactors is likely to provide insights into the molecular basis for cardiac specification (Lien, 2002).

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, a graded loss-of-function series was generated by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown results in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decrease in the expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects; this demonstrates a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 can activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. A similar Isl1 binding site has been identified in the Nkx2-5 AHF enhancer, which in transgenic mouse embryos is essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergizes with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. It is thus concluded that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression (Takeuchi, 2005).

Tinman homologs: transcriptional targets

One of the first morphological manifestations of left/right (L/R) asymmetry in mammalian embryos is a pronounced rightward looping of the linear heart tube. The direction of looping is thought to be controlled by signals from an embryonic L/R axial system. Morphological L/R asymmetry in the murine heart first becomes apparent at the linear tube stage as a leftward displacement of its caudal aspect. Beginning at the same stage, the basic helix-loop-helix (bHLH) factor gene eHand is expressed in a strikingly left-dominant pattern in myocardium, reflecting an intrinsic molecular asymmetry. In embryo hearts lacking the homeobox gene Nkx2-5, which does not loop, left-sided eHand expression is abolished. The data predict that eHand expression is enhanced in descendants of the left heart progenitor pool as one response to inductive signaling from the L/R axial system, and that eHand controls intrinsic morphogenetic pathways essential for looping (Biben, 1997).

To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, factors have been characterized that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during mammalian cardiogenesis. A 28 bp HF-1a/MEF-2 element in the MLC-2v promoter region, confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis. The ubiquitous transcription factor YB-1 binds to the HF-1a site in conjunction with a co-factor. CARP, a nuclear ankyrin-like repeat protein, forms a physical complex with YB-1 in cardiac myocytes. CARP is localized in the cardiac myocyte nucleus. CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes. carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression is first clearly detected as early as 8.5 days of development specifically in the heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homolog of Drosophila tinman, has been shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5-/- embryos, carp expression is found to be significantly and selectively reduced, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. A carp promoter-lacZ transgene is significantly reduced in Nkx2-5-/- embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway. It may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression. CARP is an early marker of cardiac differentiation that could play a role as a cofactor in the heirarchy of gene activation during that process (Zou, 1997).

Transcription of sarcomeric alpha-actin genes is developmentally regulated during skeletal and cardiac muscle development through fine-tuned control mechanisms involving multiple cooperative and antagonistic transcription factors. Among the cis-acting DNA elements recognized by these factors is the sequence CC(A/T)6GG of the serum response element (SRE), which is present in a number of growth factor-inducible and myogenic specified genes. The cardiogenic homeodomain factor, Nkx-2.5, has been shown to serve as a positive acting accessory factor for serum response factor (SRF); together, they provide strong transcriptional activation of the cardiac alpha-actin promoter. In addition, Nkx-2.5 and SRF collaborate to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts. This is accomplished by a mechanism that involves coassociation of SRF and Nkx-2.5 on intact SREs of the alpha-actin promoter. The second SRE of the avian cardiac alpha-actin promoter serves as a binding site for Nkx-2.5, SRF, and zinc finger containing GLI-Kruppel-like factor, YY1. Expression of YY1 inhibits cardiac alpha-actin promoter activity, whereas coexpression of Nkx-2.5 and SRF is able to partially reverse YY1 repression. Displacement of YY1 binding by Nkx-2.5/SRF complex occurs through mutually exclusive binding across the CaSRE2. The interplay and functional antagonism between YY1 and Nkx-2.5/SRF might constitute a developmental as well as a physiologically regulated mechanism for the modulation of modulates cardiac alpha-actin gene expression during cardiogenesis (Chen, 1997).

Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, mutant mice completely null for Csx/Nkx2.5 were generated and analyzed. Homozygous null embryos show arrest of cardiac development after looping and poor development of blood vessels. Moreover, there are severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining shows neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrate that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 is disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there are almost no ES cell-derived cardiac myocytes, while there are substantial contributions of Csx/Nk2.5-deficient cells in other organs. Whole-mount beta-gal staining of chimeric embryos shows that more than 20% contribution of Csx/Nkx2.5-deficient cells in the heart arrests cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 does not abolish initial heart looping; (2) there is no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes; (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart; (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes; (5) Csx/Nkx2.5 null cells exert dominant interfering effects on cardiac development, and (6) there are severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos (Tanaka, 1999a).

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).

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).

Regulated emigration of blood-borne leukocytes plays a defining role in lymphoid organ development, immune surveillance, and inflammatory responses. Homeobox gene Nkx2-3 is a vertebrate member of the NK-2 class of homeobox genes and a close relative of Drosophila tinman, essential for specification of cardiac and gut muscle lineages within dorsal mesoderm of the fly. Mice deficient in Nkx2-3, expressed in developing visceral mesoderm, show a complex intestinal malabsorption phenotype and striking abnormalities of gut-associated lymphoid tissue and spleen suggestive of deranged leukocyte homing. Mutant Peyer's patches are reduced in number and size; intestinal villi contain few IgA+ plasma cells, and mutant spleens are small and often atrophic, showing fused periarterial lymphoid sheaths, partially merged T and B cell zones, an absent marginal zone, and a dearth of macrophages in red pulp. Semiquantitative RT-PCR analysis and immunohistochemistry reveal down-regulation of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in endothelial cells in which Nkx2-3 is normally expressed. MAdCAM-1 is a member of the immunoglobulin superfamily, acting as an endothelial cell ligand for leukocyte homing receptors L-selectin and a4b7 integrin. These data suggest a role for a homeodomain factor in establishing the developmental and positional cues in endothelia that regulate leukocyte homing through local control of cellular adhesion and identify MAdCAM-1 as a candidate target gene of Nkx2-3 (Wang, 2000).

Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development (Chen, 2002).

An unusual homeodomain protein, HOP, is comprised simply of a homeodomain. HOP is highly expressed in the developing heart where its expression is dependent on the cardiac-restricted homeodomain protein Nkx2.5. HOP does not bind DNA and acts as an antagonist of serum response factor (SRF), which regulates the opposing processes of proliferation and myogenesis. Mice homozygous for a HOP null allele segregate into two phenotypic classes characterized by an excess or deficiency of cardiac myocytes. It is proposed that HOP modulates SRF activity during heart development; its absence results in an imbalance between cardiomyocyte proliferation and differentiation with consequent abnormalities in cardiac morphogenesis (Skin, 2002).

Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming

Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve the ability to recapitulate cell differentiation events in vitro. This study demonstrates that Nkx2.2 is part of a large repression complex in pancreatic β cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts β-cell specification. Furthermore, Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in β cells. The Nkx2.2 TN mutation results in ectopic expression of Arx inβ cells, causing β-to-α-cell transdifferentiation. A corresponding β-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent β-to-α-cell reprogramming. Notably, subsequent removal of Arx in the β cells of Nkx2.2TNmut/TNmut mutant mice reverts the β-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in β cells are the primary regulatory events required for maintaining β-cell identity (Papizan, 2011).

Additional Tinman homologs and their developmental functions

The Nkx2.6 gene belongs to the NK superfamily of homeobox genes, most closely related to the homeobox genes Nkx2.3 and Nkx2.5. The murine Nkx2.6 gene is unique among the NK family of homeobox genes because its expression is restricted to the very narrow developmental period between stages E8.5 and E10.5 of embryogenesis. The distribution of Nkx2.6 transcripts is also quite restricted spatially, with expression detected uniquely within the caudal branchial arches. Nkx2.6 is expressed in all three layers comprising the caudal branchial arches (ectoderm, mesectoderm and endoderm) with the strongest expression being detected in the surface ectoderm (Nikolova, 1997).

The homeodomain transcription factor Nkx2-3 is expressed in gut mesenchyme and spleen of embryonic and adult mice. Targeted inactivation of the Nkx2-3 gene results in severe morphological alterations of both organs and early postnatal lethality in the majority of homozygous mutants. Villus formation in the small intestine appears considerably delayed in Nkx2-3 minus fetuses due to reduced proliferation of the epithelium, while the massively increased growth of crypt cells ensues in surviving adult mutants. Interestingly, differentiated cell types of the intestinal epithelium are present in homozygous mutants, suggesting that Nkx2-3 is not required for their cell lineage allocation or migration-dependent differentiation. Hyperproliferation of the gut epithelium in adult mutants is associated with markedly reduced expression of BMP-2 and BMP-4, suggesting that these signaling molecules may be involved in mediating non-cell-autonomous control of intestinal cell growth. Spleens of Nkx2-3 mutants are generally smaller and contain drastically reduced numbers of lymphatic cells. The white pulp appears anatomically disorganized, possibly owing to a homing defect in the spleen parenchyme. Moreover, some of the Nkx2-3 mutants exhibit asplenia. Taken together these observations indicate that Nkx2-3 is essential for normal development and functions of the small intestine and spleen (Pabst, 1999).

Nkx2.5 and Nkx2.6 are murine homologs of Drosophila tinman. Their genes are expressed in the ventral region of the pharynx at early stages of embryogenesis. However, no abnormalities in the pharynges of embryos with mutations in either Nkx2.5 or Nkx2.6 have been reported. To examine the function of Nkx2.5 and Nkx2.6 in the formation of the pharynx, Nkx2.5 and Nkx2.6 double-mutant mice were generated and analyzed. Interestingly, in the double-mutant embryos, the pharynx does not form properly. Pharyngeal endodermal cells are largely missing, and the mutant pharynx is markedly dilated. Moreover, enhanced apoptosis and reduced proliferation in pharyngeal endodermal cells of the double-mutant embryos was observed. These results demonstrate a critical role of the NK-2 homeobox genes in the differentiation, proliferation, and survival of pharyngeal endodermal cells. Furthermore, the development of the atrium is less advanced in the double-mutant embryos, indicating that these two genes are essential for both pharyngeal and cardiac development (Tanaka, 2001).

Tinman homologs and congenital heart disease

Human mutations in Nkx2-5 led to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, mice were generated with a ventricular-restricted knockout of Nkx2-5. They displayed no structural defects but had progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells. Transcriptional profiling uncovered the aberrant expression of a unique panel of atrial and conduction system-restricted target genes, as well as the ectopic, high level BMP-10 expression in the adult ventricular myocardium. Further, BMP-10 is shown to be necessary and sufficient for a major component of the ventricular muscle defects. Accordingly, loss of ventricular muscle cell lineage specification into trabecular and conduction system myocytes is a new mechanistic pathway for progressive cardiomyopathy and conduction defects in congenital heart disease (Pashmforoush, 2004).

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tinman: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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