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Cellular and molecular left-right differences that are present in the mesodermal heart fields suggest that the heart is lateralized from its inception. Left-right asymmetry persists as the heart fields coalesce to form the primary heart tube, and overt, morphological asymmetry first becomes evident when the heart tube undergoes looping morphogenesis. Thereafter, chamber formation, differentiation of the inflow and outflow tracts, and position of the heart relative to the midline are additional features of heart development that exhibit left-right differences. Observations made in human clinical studies and in animal models of laterality disease suggest that all of these features of cardiac development are influenced by the embryonic left-right body axis. When errors in left-right axis determination happen, they almost always are associated with complex congenital heart malformations. The purpose of this review is to highlight what is presently known about cardiac development and upstream processes of left-right axis determination, and to consider how perturbation of the left-right body plan might ultimately result in particular types of congenital heart defects.

In Xenopus, several TGF betas, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left-right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGF betas. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGF beta receptor(s) are involved in mediating left-side TGF beta signaling. We report here that similar to effects of ectopic TGF betas, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGF beta(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGF betas, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGF betas is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGF betas to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.

Valvuloseptal morphogenesis of the primitive heart tube into a four-chambered organ requires the formation of endocardial cushion tissue. The latter is the outcome of an inductive interaction in which endocardial (endothelial) cells are induced to transform into mesenchyme by paracrine signals secreted by the adjacent myocardium. In this study, we propose that transforming endothelial/mesenchymal cells themselves secrete a factor-TGFbeta-3-that functions in an autocrine mode to promote/sustain mesenchyme formation and possibly in a paracrine manner to amplify the original (myocardial) inductive event. Cushion mesenchyme-conditioned medium, previously demonstrated to be an endogenous source of autocrine, migration-promoting factors, was found in the present study to contain TGFbeta-3, as detected by immunoblot analysis. Immunoneutralization of TGFbeta-3 in preparations of cushion mesenchyme-conditioned medium resulted in a failure of treated target endocardial cells to migrate as mesenchyme, whereas inclusion of a control antibody did not inhibit the migration-promoting activity of the conditioned medium. Similar to treatment with the conditioned medium, direct addition of TGFbeta-3 to target endocardial cells also elicited invasive migration but only in cultures which had been activated in vivo by inductive interaction with the myocardium prior to treatment. Selective inhibition of TGFbeta-3-mediated autocrine signaling in continuous cocultures of endocardium plus myocardium resulted in endocardial cells which did not migrate, even though they had expressed early markers associated with endocardial cell activation (e.g., alpha-smooth muscle actin, ES/130, and TGFbeta-3). Collectively, these results suggest that (i) two signaling pathways, myocardial and endocardial, are required to start and complete epithelial-mesenchymal transformation in cushion-forming regions of the heart and (ii) the endocardial pathway signals through iteration of TGFbeta-3 and is not functionally redundant to the myocardial pathway.