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Circulation Research. 2005;96:274-276
doi: 10.1161/01.RES.0000157574.65952.b8
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(Circulation Research. 2005;96:274.)
© 2005 American Heart Association, Inc.

Editorials

Wnt, a Driver of Myocardialization?

Maurice J.B. van den Hoff, Antoon F.M. Moorman

From the Molecular and Experimental Cardiology Group, Department of Anatomy and Embryology, Academic Medical Center, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands

Correspondence to Dr Maurice J.B. van den Hoff, Department of Anatomy and Embryology, Academic Medical Center, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. E-mail m.j.vandenhoff{at}amc.uva.nl

See related article, pages 292–299


Key Words: Lp mouse • Wnt growth factors • signal transduction

In this current issue of Circulation Research, Phillips and coworkers1 carefully analyze the cardiovascular defect observed in the naturally occurring Loop-tail (Lp) mouse strain, which is best known for its severe neural tube defects.2 The genetic alteration in the Lp mouse was identified several years ago by two independent groups and appears to be a missense mutation in the Van Gogh–like 2 (VANGL2) gene (S464N).3,4 Moreover, a second mouse strain was identified with another mutation in the VANGL2 gene (D255E) which has a similar phenotype.5 Vangl2 encodes a 521-aa protein which is predicted to contain four transmembrane regions, both the N and C termini protruding into the cytoplasm, and a C-terminal PDZ domain. The ancestral gene, Van Gogh (VANG), was initially identified in Drosophila as a regulator of cell/tissue polarity during development. Cell polarization is coordinated not only by Vang/Vangl but also by other components of the Wnt-Frizzled signaling pathway (see reviews6,7). Which Wnt growth factors are involved in triggering the signal transduction is not yet known, though Wnt11 is a potential candidate based on zebrafish studies.8 In vertebrates a second homologue of Vang was cloned, which is referred to as Van Gogh–like 1. Vangl1 and 2 are structurally similar. In vitro assays showed that both Vangl1 and 2 are able to directly interact through their PDZ domain with all three known members of the Wnt intracellular signal transducer Deshevelled (Dvl). Moreover, both known Vangl2 mutations were found to disrupt binding to Dvl, with Dvl1 being most sensitive.9 Dvl is not specifically involved in the Wnt-planar cell polarity (PCP) pathway, but was also found to transduce signals in the canonical Wnt-ß-catenin and the non-canonical Wnt-Ca2+ pathway.

The group of Dr Henderson had previously shown that in all Vangl2 homozygous mutant mice a double-outlet right ventricle with an obligatory ventricular septal defect is present as well.2 In this respect it is of relevance that the majority of all congenital malformations of the cardiovascular system in humans (~1% of newborn babies, 20% of spontaneous abortions, and 10% of stillbirths) involve abnormal development of the septal structures.10,11 Analyzing the cardiac phenotype of Lp-mouse, the group of Dr Henderson now provides data that suggest that the Wnt-PCP pathway is operational during outflow tract septation.1 As yet, the Wnt-PCP pathway was not found to be of relevance in cardiovascular development. Before discussing the potential involvement of the Wnt-PCP pathway in outflow tract septation, we first provide shortly a morphological context because the views on heart development have considerably deepened with the introduction of molecular, cell biological, and computer-aided visualization techniques in the last decades.

The heart-forming regions are located in the anteriolateral mesoderm, but were found to extend more medially and posteriorly than the initially described "classic" heart-forming regions. Folding of the embryo has as consequence that the heart forming regions meet at the anterior side of the embryo, forming the cardiac crescent. Owing to this folding process, cells that initially were located at the medial side of the heart-forming region are now located at the lateral side of the cardiac crescent, and visa-versa the initial lateral cells are now positioned medially. At the anterior side of the cardiac crescent, a trough and subsequently a linear heart tube is formed. This linear heart tube comprises an outer myocardial layer and an inner endocardial layer, both derived from the cardiogenic mesoderm. The primary heart tube does not yet encompass all cardiac compartments, in mouse it only contains the left ventricle (see review12). During subsequent development the heart tube elongates due to recruitment of cardiogenic mesoderm into the cardiac lineage. The mesodermal cells that are recruited to the heart were recently visualized in an elegant study using the Islet1 gene by the group of Dr Evans.13 At the posterior side of the heart tube the recruited mesoderm is located posteriorly to the cardiac crescent, whereas at the anterior side it appears medially to the cardiac crescent. With the elongation of the heart tube, the tube bends to the right resulting in an S-shaped tube. At the outer curvatures of this S-shaped heart the cardiac atrial and ventricular chambers form by local differentiation and proliferation, whereas the inflow, atrioventricular canal, and outflow tract, as well as the inner curvatures of the S-shaped tube, retain the initial phenotype (see review14).

During septation the bent heart tube is separated into a right or pulmonary and a left or systemic halve. The atrial chamber is divided into a left and right component by the formation of the atrial septum, which grows as a crescent shape from the roof of the atrial chamber myocardium. The ventricle is divided by the formation of the interventricular septum, which elongates due to apposition of cardiomyocytes at the apical side of the septum. What remains to be septated are the remnants of the initial primary heart tube. Within these parts of the heart extensive extracellular matrix deposits are laid down in between the myocardial and endocardial layers, which are molded into two cushions in the atrioventricular canal and two cushions in the outflow tract (see review15). The cushions fuse and separate the atrioventricular canal, the interventricular foramen, and the outflow tract into a left and a right component. Although these cushions are initially acellular, they become filled with mesenchyme which is derived from the endocardium, neural crest, and epicardium. In the formed heart the valves are largely composed of endocardial derived mesenchyme.16 Considering the extent of the cushions, one would anticipate that large mesenchymal septa would be present in the formed adult heart, which is, however, not the case (Figure). During development the mesenchyme is replaced by myocardium. This process is referred to as myocardialization (see review17), and pertinent to the study of Phillips and colleagues.1



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  		Relation of the muscular vs mesenchymal outlet septum. A, An adult human heart in which both ventricular cavities are opened to expose the interventricular septum and the outlet septum. The muscular outlet septum is indicated encircled by a dotted line. B, A schematic representation of the forming heart at 4 to 5 weeks of human development. In blue the ballooning chambers are indicated. In gray the remnants of the initial linear heart tube are shown which are covered by cushions (yellow). The curved dark gray arrows illustrate the left and right bloodstreams. The cushions running through the proximal portion of the outflow tract and connecting to the interventricular septum will form during subsequent development the mesenchymal outlet septum. The arrow points to the region in the embryonic heart which is mesenchymal but appears to be muscular in the formed adult heart. The process underlying this mesenchymal to myocardial conversion is referred to as myocardialization. Ao indicates Aorta; avc, atrioventricular canal; ias, interatrial septum; ivs, interventricular septum; LA, left atrium; LV, left ventricle; oft, outflow tract; pt, pulmonary trunk; RA, right atrium; RV, right ventricle.

Myocardialization was retrieved from oblivion in the late 1990s.18,19 Myocardialization becomes apparent when cardiomyocytes that flank cushion mesenchyme loose their epithelial context and protrude into the flanking mesenchyme. Subsequently, networks of cardiomyocytes that are intermingled with cushion mesenchyme are formed. The cardiomyocytes in these networks are slender and spindle-shaped and are contiguous with the flanking myocardium. Within the heart myocardialization contributes to the formation of the muscular atrioventricular canal septum and the outlet septum, which is initiated at E12 in mice in the atrioventricular canal and slightly later in the outflow tract. In human and chicken essentially the same is observed, though in chicken also the tricuspid valve becomes myocardial. When explants of mouse or chicken outflow tracts or atrioventricular canals are cultured on thick collagen matrices, they form spontaneously myocardial networks into the collagen, provided the proper stages are cultured. Early stages do not form myocardial networks spontaneously but can do so when induced with factors present in medium that is conditioned by late explants that do form networks spontaneously.19–21 Bone morphogenetic proteins and/or fibroblast growth factors were found to be necessary but not sufficient to induce myocardialization in vitro.22,23 Based on these observations it was concluded that myocardium formation in the cushions is caused by a redistribution of existing cardiomyocytes and regulated by secreted factors. Nevertheless, the inducing substance remains elusive.

However, when chicken cushions were cocultured in the presence of a late explant, the cushion mesenchyme was found to differentiate into heart muscle cells, suggesting that recruitment or differentiation is also involved.20 Morphological observations, showing that smooth muscle markers precede the expression of myocardial markers during heart muscle formation in the cushions support this finding.24,25 In several labs the origin and fate of the mesenchymal and myocardial cells in the outflow tract are currently being investigated using a mouse genetic system based on Cre/lox recombination.

Myocardialization is only observed to occur in cushion mesenchyme which is found inside the heart tube and never in the epicardial mesenchyme which covers the heart at the outside. Thus, the polarity of the cardiomyocytes of the outflow tract during myocardialization needs to be regulated precisely; the cardiomyocytes need to go to the inside and not to the outside. The Wnt-PCP pathway might be an excellent candidate signaling pathway to regulate this directional movement. The finding that the outlet septum does not become myocardial in Vangl2 mutant mice strengthens this suggestion. The fact that Lp-mice have severe neural tube abnormalities led to the obvious suggestion that the cardiovascular defects were caused by aberrant invasion of the outflow tract by cardiac neural crest cells (see review26). However, extensive analysis of various neural crest markers showed that this is not likely to be the case.2 The study of Phillips and coworkers1 shows the presence of various Wnt signaling components in the developing OFT, suggesting an involvement of the Wnt-PCP pathway in myocardialization. This analysis showed that in both controls and mutant mice1 the growth factor Wnt5a is expressed in the cushion mesenchyme just before the initiation of myocardialization, and2 the growth factor Wnt11, the transmembrane protein Vangl2, and the intracellular signal transducers Dvl2 and Rock1 are expressed in a comparable expression pattern in the outflow tract myocardium and myocardializing cardiomyocytes. The fact that in the mutant mice a loss-of-function mutation is present in the VANGL2 gene and the cardiomyocytes initiate myocardialization suggests that either Vangl2-mediated signaling is not involved in the initial coordination of the polarity of the myocardializing cardiomyocytes or that the onset is rescued because of redundancy. A potential factor which might be able to rescue nonfunctioning Vangl2 is Vangl1, which has been shown to be structurally and functionally similar to Vangl2. Whether the latter possibility is an option can be evaluated when the expression pattern of Vangl1 is determined in the outflow tract.

However, not all expression patterns determined in control and mutant mice are similar. The expression pattern of the downstream intracellular signaling molecule RhoA differs between wild-type and mutant mice. RhoA was found to be most abundantly expressed at the interface of the cushion mesenchyme with the myocardium in wild-type mice, whereas in mutant mice this abundant expression was absent. Comparing adjacent sections, it is suggested that Rock1 and RhoA are coexpressed in the cardiomyocytes protruding into the mesenchyme in control mice but not in mutant mice. This finding would suggest that Wnt-PCP signaling is disrupted at a more downstream level as well in mutant mice, because in the absence of RhoA Rock1 is not activated. This secondary effect of loss-of-function of Vangl2 might underlie the absence of myocardium formation in the mesenchymal outlet septum of Lp mice.

The outflow tract is not the only place in the developing heart where myocardialization is observed. Myocardialization is also observed during formation of the atrioventricular canal. It would be of interest to evaluate whether in the atrioventricular canal of Lp-mouse a normal muscular septum is formed, and, moreover, to evaluate whether a similar signaling mechanism is operational in this region as well. Nonetheless, these observations provide not only an excellent starting point for further morphological analysis but also to evaluate whether the Wnt-PCP is operational in vivo. A first biological experiment might be to re-express RhoA in the Rock1 expressing cardiomyocytes of the Lp-mouse, to restore myocardium formation in the outlet septum. When myocardialization of the outlet septum can be rescued in the Lp-mouse, the signaling pathway should be further elucidated with respect to receptors and ligands.

Knowledge of the molecular mechanisms that regulate cardiomyocyte migration and differentiation during development might provide exciting therapeutic inroads toward repopulation of fibrotic areas in infarcted or diseased hearts with flanking healthy cardiomyocytes.


*    Acknowledgments
 
M.J.B.v.d.H. and A.F.M.M. are financially supported by the Netherlands Heart Foundation M96.002.


*    Footnotes
 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.


*    References
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 *References
 

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Related Article:

Vangl2 Acts via RhoA Signaling to Regulate Polarized Cell Movements During Development of the Proximal Outflow Tract
Helen M. Phillips, Jennifer N. Murdoch, Bill Chaudhry, Andrew J. Copp, and Deborah J. Henderson
Circ. Res. 2005 96: 292-299. [Abstract] [Full Text] [PDF]




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