Fate Mapping Identifies the Origin of SHF/AHF Progenitors in the Chick Primitive Streak

Heart development depends on the spatio-temporally regulated contribution of progenitor cells from the primary, secondary and anterior heart fields. Primary heart field (PHF) cells are first recruited to form a linear heart tube; later, they contribute to the inflow myocardium of the four-chambered heart. Subsequently cells from the secondary (SHF) and anterior heart fields (AHF) are added to the heart tube and contribute to both the inflow and outflow myocardium. In amniotes, progenitors of the linear heart tube have been mapped to the anterior-middle region of the early primitive streak. After ingression, these cells are located within bilateral heart fields in the lateral plate mesoderm. On the other hand SHF/AHF field progenitors are situated anterior to the linear heart tube, however, the origin and location of these progenitors prior to the development of the heart tube remains elusive. Thus, an unresolved question in the process of cardiac development is where SHF/AHF progenitors originate from during gastrulation and whether they come from a region in the primitive streak distinct from that which generates the PHF. To determine the origin and location of SHF/AHF progenitors we used vital dye injection and tissue grafting experiments to map the location and ingression site of outflow myocardium progenitors in early primitive streak stage chicken embryos. Cells giving rise to the AHF ingressed from a rostral region of the primitive streak, termed region ‘A’. During development these cells were located in the cranial paraxial mesoderm and in the pharyngeal mesoderm. Furthermore we identified region ‘B’, located posterior to ‘A’, which gave rise to progenitors that contributed to the primary heart tube and the outflow tract. Our studies identify two regions in the early primitive streak, one which generates cells of the AHF and a second from which cardiac progenitors of the PHF and SHF emerge.     

Patterning of the heart field in the chick

In human development, it is postulated based on histological sections, that the cardiogenic mesoderm rotates 180° with the pericardial cavity. This is also thought to be the case in mouse development where gene expression data suggests that the progenitors of the right ventricle and outflow tract invert their position with respect to the progenitors of the atria and left ventricle. However, the inversion in both cases is inferred and has never been shown directly. We have used 3D reconstructions and cell tracing in chick embryos to show that the cardiogenic mesoderm is organized such that the lateralmost cells are incorporated into the cardiac inflow (atria and left ventricle) while medially placed cells are incorporated into the cardiac outflow (right ventricle and outflow tract). This happens because the cardiogenic mesoderm is inverted. The inversion is concomitant with movement of the anterior intestinal portal which rolls caudally to form the foregut pocket. The bilateral cranial cardiogenic fields fold medially and ventrally and fuse. After heart looping the seam made by ventral fusion will become the greater curvature of the heart loop. The caudal border of the cardiogenic mesoderm which ends up dorsally coincides with the inner curvature. Physical ablation of selected areas of the cardiogenic mesoderm based on this new fate map confirmed these results and, in addition, showed that the right and left atria arise from the right and left heart fields. The inversion and the new fate map account for several unexplained observations and provide a unified concept of heart fields and heart tube formation for avians and mammals.     

The anterior heart-forming field: voyage to the arterial pole of the heart

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.     

Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field

In cardiac neural-crest-ablated embryos, the secondary heart field fails to add myocardial cells to the outflow tract and elongation of the tube is deficient. Since that study, we have shown that the secondary heart field provides both myocardium and smooth muscle to the arterial pole. The present study was undertaken to determine whether addition of both cell types is disrupted after neural crest ablation. Marking experiments confirm that the myocardial component fails to be added to the outflow tract after neural crest ablation. The cells destined to go into the outflow myocardium fail to migrate and are left at the junction of the outflow myocardium with the nascent smooth muscle at the base of the arterial pole. In contrast, the vascular smooth muscle component is added to the arterial pole normally after neural crest ablation. When the myocardium is not added to the outflow tract, the point where the outflow joins the pharynx does not move caudally as it normally should, the aortic sac is smaller and fails to elongate resulting in abnormal connections of the outflow tract with the caudal aortic arch arteries.     

The outflow tract of the heart is recruited from a novel heart-forming field.

As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease.     

The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field

The vertebrate heart arises from the fusion of bilateral regions of anterior mesoderm to form a linear heart tube. Recent studies in mouse and chick have demonstrated that a second cardiac progenitor population, known as the anterior or secondary heart field, is progressively added to the heart at the time of cardiac looping. While it is clear that this second field contributes to the myocardium, its precise boundaries, other lineages derived from this population, and its contributions to the postnatal heart remain unclear. In this study, we used regulatory elements from the mouse mef2c gene to direct the expression of Cre recombinase exclusively in the anterior heart field and its derivatives in transgenic mice. By crossing these mice, termed mef2c-AHF-Cre, to Cre-dependent lacZ reporter mice, we generated a fate map of the embryonic, fetal, and postnatal heart. These studies show that the endothelial and myocardial components of the outflow tract, right ventricle, and ventricular septum are derivatives of mef2c-AHF-Cre expressing cells within the anterior heart field and its derivatives. These studies also show that the atria, epicardium, coronary vessels, and the majority of outflow tract smooth muscle are not derived from this anterior heart field population. Furthermore, a transgene marker specific for the anterior heart field is expressed in the common ventricular chamber in mef2c mutant mice, suggesting that the cardiac looping defect in these mice is not due to a failure in anterior heart field addition to the heart. Finally, the Cre transgenic mice described here will be a crucial tool for conditional gene inactivation exclusively in the anterior heart field and its derivatives.     

Fgf8 is required for anterior heart field development

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8neo) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.     

The role of secondary heart field in cardiac development

Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart fields/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.     

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5

Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.     

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.     

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm.

Development of the arterial pole of the heart is a critical step in cardiogenesis, yet its embryological origin remains obscure. We have analyzed a transgenic mouse line in which beta-galactosidase activity is observed in the embryonic right ventricle and outflow tract of the heart and in contiguous splanchnic and pharyngeal mesoderm. The nlacZ transgene has integrated upstream of the fibroblast growth factor 10 (Fgf10) gene and comparison with the expression pattern of Fgf10 in pharyngeal mesoderm indicates transgene control by Fgf10 regulatory sequences. Dil labeling shows a progressive movement of cells from the pharyngeal arch region into the growing heart tube between embryonic days 8.25 and 10.5. These data suggest that arterial pole myocardium originates outside the classical heart field.     

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.     

Semaphorin3D regulates invasion of cardiac neural crest cells into the primary heart field

The primary heart field in all vertebrates is thought to be derived exclusively from lateral plate mesoderm (LPM), which gives rise to a cardiac tube shortly after gastrulation. The heart tube then begins looping and additional cells are added from other embryonic regions, including the secondary heart field, cardiac neural crest and the proepicardial organ. Here we show in zebrafish that neural crest cells invade and contribute cardiac myosin light chain2 (cmlc2)-positive cardiomyocytes to the primary heart field. Knockdown of semaphorin3D, which is expressed in the neural crest but apparently not in LPM, reduces the size of the primary heart field and the number of cardiomyocytes in the primary heart field by 20% before formation of the primary heart tube. Sema3D morphants have subsequent complex congenital heart defects, including hypertrophic cardiomyocytes, decreased ventricular size and defects in trabeculation and in atrioventricular (AV) valve development. Neuropilin1A, a semaphorin receptor, is expressed in LPM but apparently not in the neural crest, and nrp1A morphants have cardiac development defects. We propose that a population of sema3D-dependent neural crest cells follow a novel migratory pathway, perhaps toward nrp1A-expressing LPM, and serve as an important early source of cardiomyocytes in the primary heart field.     

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.