IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

ATF4 Protects the Heart From Failure by Antagonizing Oxidative Stress.

BACKGROUND: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. METHODS: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. RESULTS: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes' whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. CONCLUSIONS: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Fli1 Promotes Vascular Morphogenesis by Regulating Endothelial Potential of Multipotent Myogenic Progenitors.

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Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury.

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Cooperative Binding of ETS2 and NFAT Links Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. METHODS: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. RESULTS: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. CONCLUSIONS: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Xbp1s-FoxO1 axis governs lipid accumulation and contractile performance in heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

FoxO1-Dio2 signaling axis governs cardiomyocyte thyroid hormone metabolism and hypertrophic growth.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1-Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1-Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1-Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR.

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Fibroblast Primary Cilia Are Required for Cardiac Fibrosis.

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Glucose-regulated protein 78 is essential for cardiac myocyte survival.

Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.

The transcription factor Mesp1 interacts with cAMP-responsive element binding protein 1 (Creb1) and coactivates Ets variant 2 (Etv2) gene expression.

Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP(+) cells are derived from Mesp1-Cre(+) cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.

Fli1 acts downstream of Etv2 to govern cell survival and vascular homeostasis via positive autoregulation.

RATIONALE: Cardiovascular health depends on proper development and integrity of blood vessels. Ets variant 2 (Etv2), a member of the E26 transforming-specific family of transcription factors, is essential to initiate a transcriptional program leading to vascular morphogenesis in early mouse embryos. However, endothelial expression of the Etv2 gene ceases at midgestation; therefore, vascular development past this stage must continue independent of Etv2. OBJECTIVE: To identify molecular mechanisms underlying transcriptional regulation of vascular morphogenesis and homeostasis in the absence of Etv2. METHODS AND RESULTS: Using loss- and gain-of-function strategies and a series of molecular techniques, we identify Friend leukemia integration 1 (Fli1), another E26 transforming-specific family transcription factor, as a downstream target of Etv2. We demonstrate that Etv2 binds to conserved Ets-binding sites within the promoter region of the Fli1 gene and governs Fli1 expression. Importantly, in the absence of Etv2 at midgestation, binding of Etv2 at Ets-binding sites in the Fli1 promoter is replaced by Fli1 protein itself, sustaining expression of Fli1 as well as selective Etv2-regulated endothelial genes to promote endothelial cell survival and vascular integrity. Consistent with this, we report that Fli1 binds to the conserved Ets-binding sites within promoter and enhancer regions of other Etv2-regulated endothelial genes, including Tie2, to control their expression at and beyond midgestation. CONCLUSIONS: We have identified a novel positive feed-forward regulatory loop in which Etv2 activates expression of genes involved in vasculogenesis, including Fli1. Once the program is activated in early embryos, Fli1 then takes over to sustain the process in the absence of Etv2.

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Cardiomyocyte autophagy: metabolic profit and loss.

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide, even despite recent scientific and technological advances and comprehensive preventive strategies. The cardiac myocyte is a voracious consumer of energy, and alterations in metabolic substrate availability and consumption are hallmark features of these disorders. Autophagy, an evolutionarily ancient response to metabolic insufficiency, has been implicated in the pathogenesis of a wide range of heart pathologies. However, the precise role of autophagy in these contexts remains obscure owing to its multifarious actions. Here, we review recently derived insights regarding the role of autophagy in cardiac hypertrophy and heart failure, highlighting its effects on metabolism.

The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism.

Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.

FoxO1 in embryonic development.

Establishment of the maternal-fetal circulation during embryonic development is a fundamental process required for effective exchange of nutrients, waste products and signaling factors critical to all subsequent stages of fetal growth and development. Recent work has uncovered a previously unrecognized role of the transcription factor FoxO1 in the orchestration of molecular events underlying establishment of maternal-fetal circulatory interaction. These new data contribute to a larger body of literature implicating this protein in the governance of a wide array of processes during development and beyond.

Nkx2-5 Regulates Tdgf1 (Cripto) Early During Cardiac Development.

Congenital Heart Disease (CHD) is the most frequent and deadly birth defect. Patients with CHD that survive the neonatal period often progress to develop advanced heart failure requiring specialized treatment including cardiac transplantation. A full understanding of the transcriptional networks that direct cardiac progenitors during heart development will enhance our understanding of both normal cardiac function and pathological states. These findings will also have important applications for emerging therapies and the treatment of congenital heart disease. Furthermore, a number of shared transcriptional pathways or networks have been proposed to regulate the development and regeneration of tissues such as the heart. We have utilized transgenic technology to isolate and characterize cardiac progenitor cells from the developing mouse heart and have begun to define specific transcriptional networks of cardiovascular development. Initial studies identified Tdgf1 as a potential target of Nkx2-5. To mechanistically dissect the regulation of this molecular program, we utilized an array of molecular biological techniques to confirm that Nkx2-5 is an upstream regulator of the Tdgf1 gene in early cardiac development. These studies further define Nkx2-5 mediated transcriptional networks and enhance our understanding of cardiac morphogenesis.

ER71 directs mesodermal fate decisions during embryogenesis.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

Forkhead factor FoxO1 is essential for placental morphogenesis in the developing embryo.

Forkhead box O1 (FoxO1), a member of the Forkhead box-containing O family of transcription factors, is a key regulator of numerous genes that govern a wide array of cellular functions, including differentiation, homeostasis, and survival. However, the role of FoxO1 in development remains elusive. Here, we describe an essential and previously undefined role for FoxO1 in placental development. We demonstrate that FoxO1-null embryos up to embryonic day 9.0 (E9.0) are indistinguishable, including their morphology, cardiovascular structure, and vascular gene expression, from wild-type (WT) littermates. However, FoxO1-nulls manifested a profoundly swollen/hydropic allantois, which failed to fuse with the chorion, a phenotype that leads to subsequent cardiovascular malformation, progressive apoptotic cell death, and embryonic lethality at E10.5. Quantitative RT-PCR analysis of genes involved in placental development revealed significant attenuation of VCAM1 expression in FoxO1-null embryos. Using immunohistochemical, transcriptional, and chromatin immunoprecipitation assays, we further discovered that FoxO1 is an essential upstream regulator of the VCAM1 gene. Collectively, our findings provide critical molecular insight into a unique FoxO1-VCAM1 axis that governs placental morphogenesis, a process that is essential for subsequent normal cardiovascular development and fetal life.

Nkx2-5 represses Gata1 gene expression and modulates the cellular fate of cardiac progenitors during embryogenesis.

BACKGROUND: Recent studies suggest that the hematopoietic and cardiac lineages have close ontogenic origins, and that an early mesodermal cell population has the potential to differentiate into both lineages. Studies also suggest that specification of these lineages is inversely regulated. However, the transcriptional networks that govern the cell fate specification of these progenitors are incompletely defined. METHODS AND RESULTS: Here, we show that Nkx2-5 regulates the hematopoietic/erythroid fate of the mesoderm precursors early during cardiac morphogenesis. Using transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1, in the Nkx2-5-null embryos. We further observed that overexpression of Nkx2-5 with an Nkx2-5-inducible embryonic stem cell system significantly repressed Gata1 gene expression and suppressed the hematopoietic/erythroid potential, but not the endothelial potential, of the embryonic stem cells. This suppression was cell-autonomous, and was partially rescued by overexpressing Gata1. In addition, we demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene. CONCLUSIONS: Our results demonstrate that the hematopoietic/erythroid cell fate is suppressed via Nkx2-5 during mesodermal fate determination, and that the Gata1 gene is one of the targets that are suppressed by Nkx2-5.