In the first phase, the parietal wall of the right atrium will come into continuity with the parietal wall of the right ventricle through remodelling and posteroinferior expansion of the myocardium at the right junction. Concomitant with the development of the right atrioventricular connection, a myocardial ridge forms at the boundary between the AV canal and the embryonic right ventricle. This myocardial funnel develops to become the “tricuspid gully” [12], which at this stage does not have any leaflets of the tricuspid valve yet. It grows to become a myocardial gully that funnels atrial blood beneath the lesser curvature of the initial heart tube towards the middle of the right ventricle. Fenestrations in the floor of the gully create an additional inferior opening in the funnel, with the primary orifice of the developing tricuspid valve running parallel to the developing ventricular septum [13].

In the second phase, epicardially derived cells, myocardium and endocardial cushions all make contributions to the leaflets of the tricuspid valve [12, 13, 16]. With the appearance of the endocardial cushion, the lumen between the trabecular and compact layers of myocardium expand, leading to the formation of the anterosuperior and inferior leaflets during the seventh week of gestation. Delamination of the septal leaflet from the developing ventricular septum occurs much later during the twelfth week. The myocardial contributions to the septal leaflet come from both the myocardial gully and the ventricular septum.

In parallel to this process, the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This fusion leads to the formation of fibrous insulating tissues at approximately 7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. In the normal adult heart, the only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction [15].

Molecular pathways involved in the pathogenesis of Ebstein anomaly are likely to impact the cellular and morphological events of tricuspid valve formation at very early stages. Several signalling and transcription cascades are therefore prime candidates based on their spatiotemporal expression patterns or known functions in heart morphogenesis.

Gain- and loss-of-function studies, tissue explant studies and expression patterns point towards an important role of BMP (bone morphogenetic protein) signalling in endothelial-mesenchymal transformation of endocardial cushions. BMP2 is strongly expressed in AV cushions, whereas the major BMP receptors, including BmprII, Alk2 (Acvr1, activin A receptor, type 1) and Alk3 (Acvr3) are widely expressed in the entire heart, with no clear spatial restriction [17, 18]. Using a GATA (GATA binding protein) 6-Cre driver mouse, Gaussin et al. conditionally deleted Alk3 in the developing AV canal [19]. Interestingly, lineage analysis showed a clear contribution of AV canal cardiac myocytes to the future tricuspid mural and posterior leaflets, but not to the tricuspid septal leaflet. The mitral septal leaflet and the atrial border of the annulus fibrosus were also stained. When Alk3 was deleted in these cells, defects were seen in the same leaflets, i.e., the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall and the annulus fibrosus was disrupted resulting in ventricular preexcitation. These defects provide in fact some overlap with Ebstein anomaly and demonstrate that signalling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal is required for the development of both the AV valves and annulus fibrosus, but they do not replicate the hallmark of the condition, namely, the apical displacement and non-delamination of the septal leaflet of the tricuspid valve. Conceivably, expression of Cre in the GATA6-Cre driver mouse may become progressively restricted in primary myocardium and thus insufficient to label all AV canal myocardial cells.

However, in extrapolation of these results, it can be presumed that key processes possibly involved in Ebstein anomaly are at least in part mediated through BMP signalling in the developing atrioventricular myocardium, including the processes leading to proper electric insulation of ventricles and atria [19]. Within the same morphological processes and molecular pathway, it was observed that positioning of the endocardial cushions in the ventricular lumen and formation of early trabeculae are part of one remodelling process which is under control of TGF-β2 (transforming growth factor, beta 2) [20]. In the Tgf-beta2-KO mouse, remodelling of the early myocardium is disturbed as indicated by defective trabeculae formation, persistence of valvular myocardium, disturbed myocardial phenotypes and differential defects at left and right side of the AV canal [20]. Bmp2 and Tgf-beta2 themselves are under genetic control of Tbx (T-box) 20, disruption of which leads to defective AV canal myocardial and valve development [21].

Epicardially derived cells (EPDC) are known to contribute to the developing fibroblast population in the heart [22, 23]. EPDCs contribute to the development of the AV canal and the annulus fibrosus and populate the ventricular myocardial wall and interventricular septum [24, 25]. While early development of the myocardial compact layer is independent of EPDCs, the subsequent increase in wall thickness is impaired when epicardial development is disrupted [26, 27]. Using the mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED) 10 until birth, Wessels et al. found that there is a dramatic disparity of the contribution of epicardial- versus endocardial-derived cells to the different leaflets of the right-sided AV valve: the parietal AV leaflets have a very strong epicardial contribution, whereas the septal leaflet of the tricuspid valve (and all leaflets derived from the major cushions) hardly have any epicardial contribution [16]. The differential contribution of EPDCs and other cell sources to the various leaflets of the atrioventricular valves, as well as their roles in myocardial growth and maturation, thus helps to understand how individual components of this region of the heart may be differentially affected in Ebstein anomaly. A corresponding cell population that heavily populates the septal leaflet of the tricuspid valve and also participates in the remodelling of the “tricuspid gully” [12], however, remains elusive to date. Equally unknown are the precise molecular factors that drive the apoptosis of the myocardial layer in the developing valves and their subsequent excavation and delamination [28, 29].