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.     

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.     

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.     

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.     

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.