Slit-roundabout signaling regulates the development of the cardiac systemic venous return and pericardium.

RATIONALE: The Slit-Roundabout (Robo) signaling pathway has pleiotropic functions during Drosophila heart development. However, its role in mammalian heart development is largely unknown. OBJECTIVE: To analyze the role of Slit-Robo signaling in the formation of the pericardium and the systemic venous return in the murine heart. METHODS AND RESULTS: Expression of genes encoding Robo1 and Robo2 receptors and their ligands Slit2 and Slit3 was found in or around the systemic venous return and pericardium during development. Analysis of embryos lacking Robo1 revealed partial absence of the pericardium, whereas Robo1/2 double mutants additionally showed severely reduced sinus horn myocardium, hypoplastic caval veins, and a persistent left inferior caval vein. Mice lacking Slit3 recapitulated the defects in the myocardialization, alignment, and morphology of the caval veins. Ligand binding assays confirmed Slit3 as the preferred ligand for the Robo1 receptor, whereas Slit2 showed preference for Robo2. Sinus node development was mostly unaffected in all mutants. In addition, we show absence of cross-regulation with previously identified regulators Tbx18 and Wt1. We provide evidence that pericardial defects are created by abnormal localization of the caval veins combined with ectopic pericardial cavity formation. Local increase in neural crest cell death and impaired neural crest adhesive and migratory properties underlie the ectopic pericardium formation. CONCLUSIONS: A novel Slit-Robo signaling pathway is involved in the development of the pericardium, the sinus horn myocardium, and the alignment of the caval veins. Reduced Slit3 binding in the absence of Robo1, causing impaired cardiac neural crest survival, adhesion, and migration, underlies the pericardial defects.

Tbx18 and the fate of epicardial progenitors.

Uncovering the origins of myocardial cells is important for understanding and treating heart diseases. Cai et al. suggest that Tbx18-expressing epicardium provides a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Here we show that the T-box transcription factor gene 18 (Tbx18) itself is expressed in the myocardium, showing that their genetic lineage tracing system does not allow conclusions of an epicardial origin of cardiomyocytes in vivo to be drawn.

Notch signaling regulates smooth muscle differentiation of epicardium-derived cells.

RATIONALE: The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. OBJECTIVE: To define the role of Notch signaling in murine epicardial development. METHODS AND RESULTS: Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor ß signaling in SM differentiation of EPDCs. CONCLUSIONS: Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.

Wnt/ß-catenin signaling maintains the mesenchymal precursor pool for murine sinus horn formation.

RATIONALE: Canonical (ß-catenin [Ctnnb1]-dependent) wingless-related MMTV integration site (Wnt) signaling plays an important role in the development of second heart field-derived structures of the heart by regulating precursor cell proliferation. The signaling pathways that regulate the most posterior elongation of the heart, that is, the addition of the systemic venous return from a Tbx18(+) precursor population, have remained elusive. OBJECTIVE: To define the role of Ctnnb1-dependent Wnt signaling in the development of the cardiac venous pole. METHODS AND RESULTS: We show by in situ hybridization analysis that Wnt pathway components are expressed and canonical Wnt signaling is active in the developing sinus horns. We analyzed sinus horn (Tbx18(cre))-specific Ctnnb1 loss- and gain-of-function mutant embryos. In Ctnnb1-deficient embryos, the dorsal part of the sinus horns is not myocardialized but consists of cells with at least partial fibroblast identity; the sinoatrial node is unaffected. Stabilization of Ctnnb1 in this domain results in the formation of undifferentiated cell aggregates. Analysis of cellular changes revealed a role of canonical Wnt signaling in proliferation of the Tbx18(+) mesenchymal progenitor cell population. CONCLUSIONS: Wnt/ß-catenin signaling maintains the Tbx18(+)Nkx2-5(-) mesenchymal precursor pool for murine sinus horn formation.

Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart.

RATIONALE: The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. OBJECTIVE: To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. METHODS AND RESULTS: We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+/Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. CONCLUSIONS: The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium.

Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns.

RATIONALE: The cardiac venous pole is a common focus of congenital malformations and atrial arrhythmias, yet little is known about the cellular and molecular mechanisms that regulate its development. The systemic venous return myocardium (sinus node and sinus horns) forms only late in cardiogenesis from a pool of pericardial mesenchymal precursor cells. OBJECTIVE: To analyze the cellular and molecular mechanisms directing the formation of the fetal sinus horns. METHODS AND RESULTS: We analyzed embryos deficient for the Wt1 (Wilms tumor 1) gene and observed a failure to form myocardialized sinus horns. Instead, the cardinal veins become embedded laterally in the pleuropericardial membranes that remain tethered to the lateral body wall by the persisting subcoelomic mesenchyme, a finding that correlates with decreased apoptosis in this region. We show by expression analysis and lineage tracing studies that Wt1 is expressed in the subcoelomic mesenchyme surrounding the cardinal veins, but that this Wt1-positive mesenchyme does not contribute cells to the sinus horn myocardium. Expression of the Raldh2 (aldehyde dehydrogenase family 1, subfamily A2) gene was lost from this mesenchyme in Wt1(-/-) embryos. Phenotypic analysis of Raldh2 mutant mice rescued from early cardiac defects by retinoic acid food supply revealed defects of the venous pole and pericardium highly similar to those of Wt1(-/-) mice. CONCLUSIONS: Pericardium and sinus horn formation are coupled and depend on the expansion and correct temporal release of pleuropericardial membranes from the underlying subcoelomic mesenchyme. Wt1 and downstream Raldh2/retinoic acid signaling are crucial regulators of this process. Thus, our results provide novel insight into the genetic and cellular pathways regulating the posterior extension of the mammalian heart and the formation of its coelomic lining.

Tbx20 interacts with smads to confine tbx2 expression to the atrioventricular canal.

RATIONALE: T-box transcription factors play critical roles in the coordinated formation of the working chambers and the atrioventricular canal (AVC). Tbx2 patterns embryonic myocardial cells to form the AVC and suppresses their differentiation into chamber myocardium. Tbx20-deficient embryos, which fail to form chambers, ectopically express Tbx2 throughout the entire heart tube, providing a potential mechanism for the function of Tbx20 in chamber differentiation. OBJECTIVE: To identify the mechanism of Tbx2 suppression by Tbx20 and to investigate the involvement of Tbx2 in Tbx20-mediated chamber formation. METHODS AND RESULTS: We generated Tbx20 and Tbx2 single and double knockout embryos and observed that loss of Tbx2 did not rescue the Tbx20-deficient heart from failure to form chambers. However, Tbx20 is required to suppress Tbx2 in the developing chambers, a prerequisite to localize its strong differentiation-inhibiting activity to the AVC. We identified a bone morphogenetic protein (Bmp)/Smad-dependent Tbx2 enhancer conferring AVC-restricted expression and Tbx20-dependent chamber suppression of Tbx2 in vivo. Unexpectedly, we found in transfection and localization studies in vitro that both Tbx20 and mutant isoforms of Tbx20 unable to bind DNA attenuate Bmp/Smad-dependent activation of Tbx2 by binding Smad1 and Smad5 and sequestering them from Smad4. CONCLUSIONS: Our data suggest that Tbx20 directly interferes with Bmp/Smad signaling to suppress Tbx2 expression in the chambers, thereby confining Tbx2 expression to the prospective AVC region.

The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle.

The primary myocardium of the embryonic heart, including the atrioventricular canal and outflow tract, is essential for septation and valve formation. In the chamber-forming heart, the expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium. To gain insight into the cellular contributions of the Tbx2+ primary myocardium to the components of the definitive heart, genetic lineage tracing was performed using a novel Tbx2Cre allele. These analyses revealed that progeny of Tbx2+ cells provide an unexpectedly large contribution to the Tbx2-negative ventricles. Contrary to common assumption, we found that the embryonic left ventricle only forms the left part of the definitive ventricular septum and the apex. The atrioventricular node, but not the atrioventricular bundle, was found to derive from Tbx2+ cells. The Tbx2+ outflow tract formed the right ventricle and right part of the ventricular septum. In Tbx2-deficient embryos, the left-sided atrioventricular canal was found to prematurely differentiate to chamber myocardium and to proliferate at increased rates similar to those of chamber myocardium. As a result, the atrioventricular junction and base of the left ventricle were malformed. Together, these observations indicate that Tbx2 temporally suppresses differentiation and proliferation of primary myocardial cells. A subset of these Tbx2Cre-marked cells switch off expression of Tbx2, which allows them to differentiate into chamber myocardium, to initiate proliferation, and to provide a large contribution to the ventricles. These findings imply that errors in the development of the early atrioventricular canal may affect a much larger region than previously anticipated, including the ventricular base.

Epicardial function of canonical Wnt-, Hedgehog-, Fgfr1/2-, and Pdgfra-signalling.

AIMS: The embryonic epicardium is a source of smooth muscle cells and fibroblasts of the coronary vasculature and of the myocardium, but the signalling pathways that control mobilization and differentiation of epicardial cells are only partly known. We aimed to (re-)evaluate the relevance of canonical Wnt-, Hedgehog (Hh)-, Fibroblast growth factor receptor (Fgfr)1/2-, and platelet-derived growth factor receptor alpha (Pdgfra)-signalling in murine epicardial development. METHODS AND RESULTS: We used a T-box 18 (Tbx18)(cre)-mediated conditional approach to delete and to stabilize, respectively, the downstream mediator of canonical Wnt-signalling, beta-catenin (Ctnnb1), to delete and activate the mediator of Hh-signalling, smoothened (Smo), and to delete Fgfr1/Fgfr2 and Pdgfra in murine epicardial development. We show that epicardial loss of Ctnnb1, Smo, or Fgfr1/Fgfr2 does not affect cardiac development, whereas the loss of Pdgfra prevents the differentiation of epicardium-derived cells into mature fibroblasts. Epicardial expression of a stabilized version of Ctnnb1 results in the formation of hyperproliferative epicardial cell clusters; epicardial expression of a constitutively active version of Smo leads to epicardial thickening and loss of epicardial mobilization. CONCLUSION: Canonical Wnt-, Hh-, and Fgfr1/Fgfr2-signalling are dispensable for epicardial development, but Pdgfra-signalling is crucial for the differentiation of cardiac fibroblasts from epicardium-derived cells.

Wnt/Ctnnb1 signaling and the mesenchymal precursor pools of the heart.

Lineage tracing has shown that the different regions of the four-chambered heart of mammalian embryos derive from molecularly distinct precursor pools in a spatially and temporally tightly controlled manner. Cells of the first heart field differentiate early and form the linear heart tube of headfold-stage embryos, the future left ventricle. The right ventricle, atria, and outflow tract derive from the second heart field by recruitment and delayed local myocardial differentiation. Finally, Tbx18(+) precursors are added at the posterior cardiac pole after the chambers have been formed to generate the myocardialized aspects of the mature venous return system, including the intrapericardial parts of the caval veins and the sinoatrial node. The elongation of the linear heart tube by second heart field-derived cells requires the maintenance of highly proliferative precursor pools by a number of signaling pathways, including sonic hedgehog, fibroblast growth factor, and canonical Wnt. The molecular circuits that operate during the addition of the most posterior components from Tbx18(+) progenitors have remained elusive. It has emerged that at least one of the pathways required for proliferation of second heart field progenitors, canonical Wnt signaling, also operates in a subset of Tbx18(+) cells for formation of myocardialized caval veins. This argues for both conserved and specific regulatory modules mediating the polar extension of the cardiac tube during embryogenesis.

Partial absence of pleuropericardial membranes in Tbx18- and Wt1-deficient mice.

The pleuropericardial membranes are fibro-serous walls that separate the pericardial and pleural cavities and anchor the heart inside the mediastinum. Partial or complete absence of pleuropericardial membranes is a rare human disease, the etiology of which is poorly understood. As an attempt to better understand these defects, we wished to analyze the cellular and molecular mechanisms directing the separation of pericardial and pleural cavities by pleuropericardial membranes in the mouse. We found by histological analyses that both in Tbx18- and Wt1-deficient mice the pleural and pericardial cavities communicate due to a partial absence of the pleuropericardial membranes in the hilus region. We trace these defects to a persisting embryonic connection between these cavities, the pericardioperitoneal canals. Furthermore, we identify mesenchymal ridges in the sinus venosus region that tether the growing pleuropericardial membranes to the hilus of the lung, and thus, close the pericardioperitoneal canals. In Tbx18-deficient embryos these mesenchymal ridges are not established, whereas in Wt1-deficient embryos the final fusion process between these tissues and the body wall does not occur. We suggest that this fusion is an active rather than a passive process, and discuss the interrelation between closure of the pericardioperitoneal canals, lateral release of the pleuropericardial membranes from the lateral body wall, and sinus horn development.

The sinus venosus progenitors separate and diversify from the first and second heart fields early in development.

AIMS: During development, the heart tube grows by differentiation of Isl1(+)/Nkx2-5(+) progenitors to the arterial and venous pole and dorsal mesocardium. However, after the establishment of the heart tube, Tbx18(+) progenitors were proposed to form the Tbx18(+)/Nkx2-5(-) sinus venosus and proepicardium. To elucidate the relationship between these contributions, we investigated the origin of the Tbx18(+) sinus venosus progenitor population in the cardiogenic mesoderm and its spatial and temporal relation to the second heart field during murine heart development. METHODS AND RESULTS: Explant culture revealed that the Tbx18(+) cell population has the potential to form Nkx2-5(-) sinus venosus myocardium. Three-dimensional reconstruction of expression patterns showed that during heart tube elongation, the Tbx18(+) progenitors remained spatially and temporally separate from the Isl1(+) second heart field, only overlapping with the Isl1(+) domain at the right lateral side of the inflow tract, where the sinus node developed. Consistently, genetic lineage analysis revealed that the Tbx18(+) descendants formed the sinus venosus myocardium, but did not contribute to the pulmonary vein myocardium that developed in the Isl1(+) second heart field. By means of DiI labelling and expression analysis, the origin of the sinus venosus progenitor population was traced to the lateral rim of splanchnic mesoderm that down-regulated Nkx2-5 expression approximately 2 days before its differentiation into sinus venosus myocardium. CONCLUSION: Our data indicate that the cardiogenic mesoderm contains an additional progenitor subpopulation that contributes to the sinus venosus myocardium. After patterning of the cardiogenic mesoderm, this progenitor population remains spatially separated and genetically distinctive from the second heart field subpopulation.