A right-handed signalling pathway drives heart looping in vertebrates.

Most animals show external bilateral symmetry, which hinders the observation of multiple internal left-right (L/R) asymmetries that are fundamental to organ packaging and function. In vertebrates, left identity is mediated by the left-specific Nodal-Pitx2 axis that is repressed on the right-hand side by the epithelial-mesenchymal transition (EMT) inducer Snail1 (refs 3, 4). Despite some existing evidence, it remains unclear whether an equivalent instructive pathway provides right-hand-specific information to the embryo. Here we show that, in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline, leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right lateral plate mesoderm, converge on the asymmetric activation of the transcription factors Pitx2 and Prrx1, which integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo, and in the mouse SNAIL1 acts in a similar manner to Prrx1a in zebrafish and PRRX1 in the chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates.

The Wilms' tumor suppressor gene regulates pancreas homeostasis and repair.

The Wilms' tumor suppressor gene (Wt1) encodes a zinc finger transcription factor that plays an essential role in the development of kidneys, gonads, spleen, adrenals and heart. Recent findings suggest that WT1 could also be playing physiological roles in adults. Systemic deletion of WT1 in mice provokes a severe deterioration of the exocrine pancreas, with mesothelial disruption, E-cadherin downregulation, disorganization of acinar architecture and accumulation of ascitic transudate. Despite this extensive damage, pancreatic stellate cells do not become activated and lose their canonical markers. We observed that pharmacological induction of pancreatitis in normal mice provokes de novo expression of WT1 in pancreatic stellate cells, concomitant with their activation. When pancreatitis was induced in mice after WT1 ablation, pancreatic stellate cells expressed WT1 and became activated, leading to a partial rescue of the acinar structure and the quiescent pancreatic stellate cell population after recovery from pancreatitis. We propose that WT1 modulates through the RALDH2/retinoic acid axis the restabilization of a part of the pancreatic stellate cell population and, indirectly, the repair of the pancreatic architecture, since quiescent pancreatic stellate cells are required for pancreas stability and repair. Thus, we suggest that WT1 plays novel and essential roles for the homeostasis of the adult pancreas and, through its upregulation in pancreatic stellate cells after a damage, for pancreatic regeneration. Due to the growing importance of the pancreatic stellate cells in physiological and pathophysiological conditions, these novel roles can be of translational relevance.

Mesothelial-mesenchymal transitions in embryogenesis.

Most animals develop coelomic cavities lined by an epithelial cell layer called the mesothelium. Embryonic mesothelial cells have the ability to transform into mesenchymal cells which populate many developing organs contributing to their connective and vascular tissues, and also to organ-specific cell types. Furthermore, embryonic mesothelium and mesothelial-derived cells produce essential signals for visceral morphogenesis. We review the most relevant literature about the mechanisms regulating the embryonic mesothelial-mesenchymal transition, the developmental fate of the mesothelial-derived cells and other functions of the embryonic mesothelium, such as its contribution to the establishment of left-right visceral asymmetries or its role in limb morphogenesis.

Epicardial cell lineages and the origin of the coronary endothelium.

The embryonic epicardium generates a population of epicardial-derived mesenchymal cells (EPDC) whose contribution to the coronary endothelium is minor or, according to some reports, negligible. We have compared four murine cell-tracing models related to the EPDC in order to elucidate this contribution. Cre recombinase was expressed under control of the promoters of the Wilms' tumor suppressor (Wt1), the cardiac troponin (cTnT), and the GATA5 genes, activating expression of the R26REYFP reporter. We have also used the G2 enhancer of the GATA4 gene as a driver due to its activation in the proepicardium. Recombination was found in most of the epicardium/EPDC in all cases. The contribution of these lineages to the cardiac endothelium was analyzed using confocal microscopy and flow cytometry. G2-GATA4 lineage cells are the most frequent in the endothelium, probably due to the recruitment of circulating endothelial progenitors. The contribution of the WT1 cell lineage increases along gestation due to further endothelial expression of WT1. GATA5 and cTnT lineages represent 4% of the cardiac endothelial cells throughout the gestation, probably standing for the actual EPDC contribution to the coronary endothelium. These results suggest caution when using a sole cell-tracing model to study the fate of the EPDC.

The Insulin-like Growth Factor Signalling Pathway in Cardiac Development and Regeneration.

Insulin and Insulin-like growth factors (IGFs) perform key roles during embryonic development, regulating processes of cell proliferation and survival. The IGF signalling pathway comprises two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and six IGF binding proteins (IGFBPs) that regulate IGF transport and availability. The IGF signalling pathway is essential for cardiac development. IGF2 is the primary mitogen inducing ventricular cardiomyocyte proliferation and morphogenesis of the compact myocardial wall. Conditional deletion of the Igf1r and the insulin receptor (Insr) genes in the myocardium results in decreased cardiomyocyte proliferation and ventricular wall hypoplasia. The significance of the IGF signalling pathway during embryonic development has led to consider it as a candidate for adult cardiac repair and regeneration. In fact, paracrine IGF2 plays a key role in the transient regenerative ability of the newborn mouse heart. We aimed to review the current knowledge about the role played by the IGF signalling pathway during cardiac development and also the clinical potential of recapitulating this developmental axis in regeneration of the adult heart.

Embryonic circulating endothelial progenitor cells.

The development of vascular system in vertebrates has been traditionally explained by early vasculogenic assembly of angioblasts followed by angiogenic outgrowth of pre-existing vessels. The discovery of adult endothelial progenitor cells (Asahara et al. in Science 275(5302):964-967, 1997) challenged this view, since postnatal vascular growth could be accomplished by recruitment of circulating cells with the ability to differentiate into endothelial cells. However, the existence of embryonic circulating endothelial progenitor cells and their actual contribution to vascular development is far less known. We review in this paper the literature concerning the features, origin and physiological functions of embryonic and foetal circulating endothelial progenitors. Our review includes the early (E7.5) progenitors isolated from yolk sac, the hematovascular progenitors identified in the foetal liver, the yolk sac-derived erythro-myeloid progenitors, circulating hematopoietic cells from the G2-GATA4 lineage and the endothelial colony-forming cells isolated from the placenta and umbilical cord blood. We highlight the need of further characterization of these populations and the relationships between them.

Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections.

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

Comparative developmental biology of the cardiac inflow tract.

The vertebrate heart receives the blood through the cardiac inflow tract. This area has experienced profound changes along the evolution of vertebrates; changes that have a reflection in the cardiac ontogeny. The development of the inflow tract involves dynamic changes due to the progressive addition of tissue derived from the secondary heart field. The inflow tract is the site where oxygenated blood coming from lungs is received separately from the systemic return, where the cardiac pacemaker is established and where the proepicardium develops. Differential cell migration towards the inflow tract breaks the symmetry of the primary heart tube and determines the direction of the cardiac looping. In air-breathing vertebrates, an inflow tract reorganization is essential to keep separate blood flows from systemic and pulmonary returns. Finally, the sinus venosus endocardium has recently been recognized as playing a role in the constitution of the coronary vasculature. Due to this developmental complexity, congenital anomalies of the inflow tract can cause severe cardiac diseases. We aimed to review the recent literature on the cellular and molecular mechanisms that regulate the morphogenesis of the cardiac inflow tract, together with comparative and evolutionary details, thus providing a basis for a better understanding of these mechanisms.

A population of hematopoietic stem cells derives from GATA4-expressing progenitors located in the placenta and lateral mesoderm of mice.

GATA transcription factors are expressed in the mesoderm and endoderm during development. GATA1-3, but not GATA4, are critically involved in hematopoiesis. An enhancer (G2) of the mouse Gata4 gene directs its expression throughout the lateral mesoderm and the allantois, beginning at embryonic day 7.5, becoming restricted to the septum transversum by embryonic day 10.5, and disappearing by midgestation. We have studied the developmental fate of the G2-Gata4 cell lineage using a G2-Gata4Cre;R26REYFP mouse line. We found a substantial number of YFP+ hematopoietic cells of lymphoid, myeloid and erythroid lineages in embryos. Fetal CD41+/cKit+/CD34+ and Lin-/cKit+/CD31+ YFP+ hematopoietic progenitors were much more abundant in the placenta than in the aorta-gonad-mesonephros area. They were clonogenic in the MethoCult assay and fully reconstituted hematopoiesis in myeloablated mice. YFP+ cells represented about 20% of the hematopoietic system of adult mice. Adult YFP+ hematopoietic stem cells constituted a long-term repopulating, transplantable population. Thus, a lineage of adult hematopoietic stem cells is characterized by the expression of GATA4 in their embryonic progenitors and probably by its extraembryonic (placental) origin, although GATA4 appeared not to be required for hematopoietic stem cell differentiation. Both lineages basically showed similar physiological behavior in normal mice, but clinically relevant properties of this particular hematopoietic stem cell population should be checked in physiopathological conditions.

Coelomic epithelium-derived cells in visceral morphogenesis.

Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans.

The proepicardium keeps a potential for glomerular marker expression which supports its evolutionary origin from the pronephros.

The proepicardium is the embryonic primordium of the epicardium. This transient structure is essential for cardiac development giving rise to the epicardium and supplying the heart with vascular and cardiac connective tissue progenitors. However, their nature and evolutionary origin are poorly-known. We have suggested elsewhere (Pombal et al. Evol. Dev. 10: 210-216, 2008; Cano et al., J. Dev. Biol. 1: 3-19, 2013) that the proepicardium is an evolutionary derivative of the primordium of an ancient external pronephric glomerulus, devoid of its original excretory function. In this study, we describe for the first time expression of two podocyte markers in the chick proepicardium (glepp1 and synaptopodin) and we have shown how these podocyte markers as well as the intermediate mesoderm marker Pax2 are strongly upregulated when the proepicardium is cultured with nephrogenic inducers. Retinoic acid treatment also induced in the proepicardium expression of Hoxb4, a gene which confers to intermediate mesoderm competence to respond to nephrogenic signals. Thus, a latent nephrogenic potential persists in the proepicardium and also that its original glomerular fate can be partially rescued. The transcription factor Wt1, essential for kidney and epicardial development, plays opposite roles in both tissues, inducing epithelial-mesenchymal transition in the proepicardium and promoting epithelialization in the kidneys (Essafi et al., Dev. Cell 21: 559-574, 2011). Consistently with this antithetical function of Wt1, we have observed an upregulation of podocalyxin in the epicardium of mouse embryos with conditional deletion of the Wt1 gene, while this protein is transcriptionally activated by Wt1 in podocytes.

GATA4 loss in the septum transversum mesenchyme promotes liver fibrosis in mice.

UNLABELLED: The zinc finger transcription factor GATA4 controls specification and differentiation of multiple cell types during embryonic development. In mouse embryonic liver, Gata4 is expressed in the endodermal hepatic bud and in the adjacent mesenchyme of the septum transversum. Previous studies have shown that Gata4 inactivation impairs liver formation. However, whether these defects are caused by loss of Gata4 in the hepatic endoderm or in the septum transversum mesenchyme remains to be determined. In this study, we have investigated the role of mesenchymal GATA4 activity in liver formation. We have conditionally inactivated Gata4 in the septum transversum mesenchyme and its derivatives by using Cre/loxP technology. We have generated a mouse transgenic Cre line, in which expression of Cre recombinase is controlled by a previously identified distal Gata4 enhancer. Conditional inactivation of Gata4 in hepatic mesenchymal cells led to embryonic lethality around mouse embryonic stage 13.5, likely as a consequence of fetal anemia. Gata4 knockout fetal livers exhibited reduced size, advanced fibrosis, accumulation of extracellular matrix components and hepatic stellate cell (HSC) activation. Haploinsufficiency of Gata4 accelerated CCl4 -induced liver fibrosis in adult mice. Moreover, Gata4 expression was dramatically reduced in advanced hepatic fibrosis and cirrhosis in humans. CONCLUSIONS: Our data demonstrate that mesenchymal GATA4 activity regulates HSC activation and inhibits the liver fibrogenic process.

The evolutionary origins of chordate hematopoiesis and vertebrate endothelia.

The vertebrate circulatory system is the most complex vascular system among those of metazoans, with key innovations including a multi-chambered heart and highly specialized blood cells. Invertebrate vessels, on the other hand, consist of hemal spaces between the basal laminae of epithelia. How the evolutionary transition from an invertebrate-type system to the complex vertebrate one occurred is, however, poorly understood. We investigate here the development of the cardiovascular system of the cephalochordate amphioxus Branchiostoma lanceolatum in order to gain insight into the origin of the vertebrate cardiovascular system. The cardiac markers Hand, Csx (Nkx2-5) and Tbx4/5 reveal a broad cardiac-like domain in amphioxus; such a decentralized organization during development parallels that seen in the adult anatomy. Our data therefore support the hypothesis that amphioxus never possessed a proper heart, even transiently during development. We also define a putative hematopoietic domain, supported by the expression of the hematopoietic markers Scl and Pdvegfr. We show that this area is closed to the dorsal aorta anlages, partially linked to excretory tissues, and that its development is regulated by retinoic acid, thus recalling the aorta-gonads-mesonephros (AGM) area of vertebrates. This region probably produces Pdvegfr+ hemal cells, with an important role in amphioxus vessel formation, since treatments with an inhibitor of PDGFR/VEGFR lead to a decrease of Laminin in the basal laminae of developing vessels. Our results point to a chordate origin of hematopoiesis in an AGM-like area from where hemal Pdvegfr+ cells are produced. These Pdvegfr+ cells probably resemble the ancestral chordate blood cells from which the vertebrate endothelium later originated.

Met signaling in cardiomyocytes is required for normal cardiac function in adult mice.

Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as ß-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-ß production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.

Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2.

Epicardial-derived signals are key regulators of cardiac embryonic development. An important part of these signals is known to relate to a retinoic acid (RA) receptor-dependent mechanism. RA is a potent morphogen synthesised by Raldh enzymes, Raldh2 being the predominant one in mesodermal tissues. Despite the importance of epicardial retinoid signalling in the heart, the molecular mechanisms controlling cardiac Raldh2 transcription remain unknown. In the current study, we show that Wt1-null epicardial cells display decreased expression of Raldh2 both in vivo and in vitro. Using a RA-responsive reporter, we have confirmed that Wt1-null epicardial cells actually show reduced synthesis of RA. We also demonstrate that Raldh2 is a direct transcriptional target of Wt1 in epicardial cells. A secondary objective of this study was to identify the status of RA-related receptors previously reported to be critical to epicardial biology (PDGFRα,ß; RXRα). PDGFRα and PDGFRß mRNA and protein levels are downregulated in the absence of Wt1, but only Pdgfra expression is rescued by the addition of RA to Wt1-null epicardial cells. RXRα mRNA levels are not affected in Wt1-null epicardial cells. Taken together, our results indicate that Wt1 critically regulates epicardial RA signalling via direct activation of the Raldh2 gene, and identify a role for Wt1 in the regulation of morphogen receptors involved in the proliferation, migration, and differentiation of epicardial and epicardially-derived cells (EPDC).

Wt1-expressing progenitors contribute to multiple tissues in the developing lung.

Lungs develop from paired endodermal outgrowths surrounded by a mesodermal mesenchyme. Part of this mesenchyme arises from epithelial-mesenchymal transition of the mesothelium that lines the pulmonary buds. Previous studies have shown that this mesothelium-derived mesenchyme contributes to the smooth muscle of the pulmonary vessels, but its significance for lung morphogenesis and its developmental fate are still little known. We have studied this issue using the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1cre) crossed with the Rosa26R-EYFP reporter mouse. In the developing lungs, Wt1, the Wilms' tumor suppressor gene, is specifically expressed in the embryonic mesothelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP), allowing for colocalization with differentiation markers. Wt1cre-YFP cells were very abundant from the origin of the lung buds to postnatal stages, contributing significantly to pulmonary endothelial and smooth muscle cells, bronchial musculature, tracheal and bronchial cartilage, as well as CD34⁺ fibroblast-like interstitial cells. Thus Wt1cre-YFP mesenchymal cells show the very same differentiation potential as the splanchnopleural mesenchyme surrounding the lung buds. FSP1⁺ fibroblast-like cells were always YFP⁻; they expressed the common leukocyte antigen CD45 and were apparently recruited from circulating progenitors. We have also found defects in pulmonary development in Wt1-/- embryos, which showed abnormally fused lung lobes, round-shaped and reduced pleural cavities, and diaphragmatic hernia. Our results suggest a novel role for the embryonic mesothelium-derived cells in lung morphogenesis and involve the Wilms' tumor suppressor gene in the development of this organ.

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.

Novel Insights into the Molecular Mechanisms Governing Embryonic Epicardium Formation.

The embryonic epicardium originates from the proepicardium, an extracardiac primordium constituted by a cluster of mesothelial cells. In early embryos, the embryonic epicardium is characterized by a squamous cell epithelium resting on the myocardium surface. Subsequently, it invades the subepicardial space and thereafter the embryonic myocardium by means of an epithelial-mesenchymal transition. Within the myocardium, epicardial-derived cells present multilineage potential, later differentiating into smooth muscle cells and contributing both to coronary vasculature and cardiac fibroblasts in the mature heart. Over the last decades, we have progressively increased our understanding of those cellular and molecular mechanisms driving proepicardial/embryonic epicardium formation. This study provides a state-of-the-art review of the transcriptional and emerging post-transcriptional mechanisms involved in the formation and differentiation of the embryonic epicardium.

GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells.

In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion toward a quiescent-like state, a process called deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the deactivation and/or activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cell quiescence. In this work, we show that lack of GATA4 in adult mice caused hepatic stellate cell activation and, consequently, liver fibrosis. During regression of liver fibrosis, Gata4 was reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promoted liver fibrosis regression in CCl4-treated mice. GATA4 induced changes in the expression of fibrogenic and antifibrogenic genes, promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly repressed EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.