By midgestation (E10.5), looping and heart tube extension are complete (Fig. 1.1d). Subsequent growth of the heart occurs by proliferation of atrial and ventricular cardiomyocytes in the outer curvature of the heart tube, a process known as the ballooning model of chamber morphogenesis (Fig. 1.1e). Patterned proliferation in the embryonic heart is regulated by transcription factors, in particular members of the T-box family, that are expressed in overlapping patterns in the early heart and define the atrioventricular canal and outflow tract as regions of low proliferation where endocardial cushions develop [13]. Cushions contain mesenchymal cells that are derived from the endocardium by a process of epithelial to mesenchymal transformation in response to overlying myocardial signals. The atrioventricular canal and outflow tract regulate directional blood flow through the early heart by valve-like cushion function and reduced myocardial electrical coupling. At this stage, cardiac septation initiates, dividing the heart into separate left and right chambers. The primary atrial septum converges with the developing endocardial cushions in the atrioventricular region. The addition of second heart field progenitor cells at the venous pole (Chap. 5) of the heart is essential for the formation of the muscular base of the primary atrial septum and plays a critical role in atrioventricular septation [14]. Atrioventricular cushions also connect with the interventricular septum, developing at the interface between linear heart tube and second heart field-derived myocardium, i.e., future left (systemic) and right (pulmonary) ventricles. At the arterial pole, the influx of cardiac neural crest cells (Chap. 4) is essential for the division of the outflow tract into the ascending aorta and pulmonary trunk (outlets of the left and right ventricles) through the formation of the aorticopulmonary septum and outflow tract cushion fusion (Fig. 1.1f). Defects in neural crest cell development result in a spectrum of conotruncal defects including common arterial trunk [15].

The endocardial cushions are remodeled during septation to form the definitive tricuspid, mitral, aortic, and pulmonary valves that regulate blood to and from the ventricular chambers (Fig. 1.1g). The conotruncal region rotates in a counterclockwise direction under the influence of the embryonic laterality cascade, and the aorta becomes aligned with the left ventricle through a process termed wedging. Perturbation of cardiac septation results in failure to make the exclusive connection between the ascending aorta and pulmonary trunk that is essential to separate the systemic and pulmonary circulatory systems at birth. Defects in either heart tube elongation that generates the template for septation or in the septation process itself thus result in a range of CHD. Systemic and pulmonary circulatory systems become fully isolated at birth by the closure of the oval foramen and ductus arteriosus.

Chamber development is accompanied by establishment of the definitive cardiac conduction system (Chap. 8) that regulates cardiac rhythm (Fig. 1.1h). Electrical activity is triggered by the pacemaker, or sinoatrial node, in the sinus venous region. Conduction between the atria and ventricles is delayed by the atrioventricular node, ensuring sequential atrial and ventricular contraction. The electrical signal initiates apical ventricular contraction through the atrioventricular bundle, bundle branches, and Purkinje fiber network. Conductive cells are specialized myocytes and share common progenitor cells with nonconducting myocytes [16]. Defects in the establishment of the electrical wiring of the heart result in arrhythmias and conduction blocks. The Purkinje fiber system develops adjacent to the ventricular lumen in trabecular myocytes, transient projections of myocardium that form by signal exchange between the myocardial wall and endocardium. Trabeculae are present in the mouse heart from midgestation to fetal stages when a process of compaction generates the definitive ventricular wall. Failure of this step results in noncompaction cardiomyopathy.

From E10.5, a third cell layer, the epicardium (Chap. 6), covers the external surface of the heart (Fig. 1.1i). The epicardium originates in the pro-epicardial organ and forms an epithelium from which cells undergo a mesenchymal transition and invade the myocardium, giving rise to smooth muscle cells associated with coronary arteries and cardiac fibroblasts. Signal exchange between the epicardium, epicardially derived fibroblasts, and myocardium plays an important role in regulating myocardial proliferation and differentiation. Coronary vasculature, essential for the delivery of oxygen and nutrients to the myocardium, develops during fetal stages [17]. The origin of coronary endothelial cells is controversial; however, sinus venosus endothelium and endocardial trapping during compaction are thought to be two major sources. Coronary smooth muscle cells originate from the epicardium as well as neural crest-derived cells. Coronary arteries connect with the base of the ascending aorta to form right and left coronary ostia. Abnormal proximal coronary artery patterning, often associated with conotruncal CHD, is a cause of sudden cardiac death.