Splanchnic mesoderm in the region described as the second heart field (SHF)is marked by Islet1 expression in the mouse embryo. The anterior part of this region expresses a number of markers, including Fgf10, and the contribution of these cells to outflow tract and right ventricular myocardium has been established. We now show that the posterior region also has myocardial potential, giving rise specifically to differentiated cells of the atria. This conclusion is based on explant experiments using endogenous and transgenic markers and on DiI labelling, followed by embryo culture. Progenitor cells in the right or left posterior SHF contribute to the right or left common atrium, respectively. Explant experiments with transgenic embryos,in which the transgene marks the right atrium, show that atrial progenitor cells acquire right-left identity between the 4- and 6-somite stages, at the time when Pitx2c is first expressed. Manipulation of Pitx2c, by gain-and loss-of-function, shows that it represses the transgenic marker of right atrial identity. A repressive effect is also seen on the proliferation of cells in the left sinus venosus and in cultured explants from the left side of the posterior SHF. This report provides new insights into the contribution of the SHF to atrial myocardium and the effect of Pitx2c on the formation of the left atrium.

Background—: Pitx2 is a homeobox transcription factor that plays a pivotal role in early left/right determination during embryonic development. Pitx2 loss-of-function mouse mutants display early embryonic lethality with severe cardiac malformations, demonstrating the importance of Pitx2 during cardiogenesis. Recently, independent genome-wide association studies have provided new evidence for a putative role of PITX2 in the adult heart. These studies have independently reported several risk variants close to the PITX2 locus on chromosome 4q25 that are strongly associated with atrial fibrillation in humans. Methods and Results—: We show for the first time that PITX2C expression is significantly decreased in human patients with sustained atrial fibrillation, thus providing a molecular link between PITX2 loss of function and atrial fibrillation. In addition, morphological, molecular, and electrophysiological characterization of chamber-specific Pitx2 conditional mouse mutants reveals that atrial but not ventricular chamber-specific deletion of Pitx2 results in differences in the action potential amplitude and resting membrane potential in the adult heart as well as ECG characteristics of atrioventricular block. Lack of Pitx2 in atrial myocardium impairs sodium channel and potassium channel expression, mediated in part by miRNA misexpression. Conclusions—: This study thus identifies Pitx2 as an upstream transcriptional regulator of atrial electric function, the insufficiency of which results in cellular and molecular changes leading to atrial electric and structural remodeling linked to arrhythmogenesis.

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. 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 ∼2 days before its differentiation into sinus venosus myocardium. 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.

Rationale: Cardiac progenitor cells from the second heart field (SHF) contribute to rapid growth of the embryonic heart, giving rise to right ventricular and outflow tract (OFT) myocardium at the arterial pole of the heart, and atrial myocardium at the venous pole. Recent clonal analysis and cell-tracing experiments indicate that a common progenitor pool in the posterior region of the SHF gives rise to both OFT and atrial myocytes. The mechanisms regulating deployment of this progenitor pool remain unknown. Objective: To evaluate the role of TBX1 , the major gene implicated in congenital heart defects in 22q11.2 deletion syndrome patients, in posterior SHF development. Methods and Results: Using transcriptome analysis, genetic tracing, and fluorescent dye-labeling experiments, we show that Tbx1 -dependent OFT myocardium originates in Hox -expressing cells in the posterior SHF. In Tbx1 null embryos, OFT progenitor cells fail to segregate from this progenitor cell pool, leading to failure to expand the dorsal pericardial wall and altered positioning of the cardiac poles. Unexpectedly, addition of SHF cells to the venous pole of the heart is also impaired, resulting in abnormal development of the dorsal mesenchymal protrusion, and partially penetrant atrioventricular septal defects, including ostium primum defects. Conclusions: Tbx1 is required for inflow as well as OFT morphogenesis by regulating the segregation and deployment of progenitor cells in the posterior SHF. Our results provide new insights into the pathogenesis of congenital heart defects and 22q11.2 deletion syndrome phenotypes.

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 Tbx2 ^Cre 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 Tbx2 ^Cre -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.

How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh^-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects.