N-cadherin relocalization during cardiac trabeculation
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Author contributions: A.V.C. and D.Y.R.S. designed research; A.V.C., S.M.A., and H.-M.M. performed research; R.F. contributed new reagents/analytic tools; A.V.C., S.M.A., and D.Y.R.S. analyzed data; and A.V.C. and D.Y.R.S. wrote the paper.
Significance
The process of trabeculation is central to heart development and maturation, as it allows the increase in muscle mass before the formation of coronaries. This complex process involves a number of morphological changes in a subset of cardiomyocytes, resulting in their delamination from the compact layer. As cardiomyocytes delaminate, they must also remain attached to the compact layer. We identified Erb-b2 receptor tyrosine kinase 2-mediated relocalization of N-cadherin (Cdh2) as a mechanism underlying the formation of cell–cell junctions between trabecular and compact layer cardiomyocytes. Interestingly, we found that blood flow and heart contraction are also essential for the localization of Cdh2 molecules. These studies further our understanding of the complex cell biological processes underlying the maturation of the vertebrate heart.
Abstract
During cardiac trabeculation, cardiomyocytes delaminate from the outermost (compact) layer to form complex muscular structures known as trabeculae. As these cardiomyocytes delaminate, the remodeling of adhesion junctions must be tightly coordinated so cells can extrude from the compact layer while remaining in tight contact with their neighbors. In this study, we examined the distribution of N-cadherin (Cdh2) during cardiac trabeculation in zebrafish. By analyzing the localization of a Cdh2-EGFP fusion protein expressed under the control of the zebrafish cdh2 promoter, we initially observed Cdh2-EGFP expression along the lateral sides of embryonic cardiomyocytes, in an evenly distributed pattern, and with the occasional appearance of punctae. Within a few hours, Cdh2-EGFP distribution on the lateral sides of cardiomyocytes evolves into a clear punctate pattern as Cdh2-EGFP molecules outside the punctae cluster to increase the size of these aggregates. In addition, Cdh2-EGFP molecules also appear on the basal side of cardiomyocytes that remain in the compact layer. Delaminating cardiomyocytes accumulate Cdh2-EGFP on the surface facing the basal side of compact layer cardiomyocytes, thereby allowing tight adhesion between these layers. Importantly, we find that blood flow/cardiac contractility is required for the transition from an even distribution of Cdh2-EGFP to the formation of punctae. Furthermore, using time-lapse imaging of beating hearts in conjunction with a Cdh2 tandem fluorescent protein timer transgenic line, we observed that Cdh2-EGFP molecules appear to move from the lateral to the basal side of cardiomyocytes along the cell membrane, and that Erb-b2 receptor tyrosine kinase 2 (Erbb2) function is required for this relocalization.
To maximize its function, the heart undergoes a series of morphological changes during development, with trabeculation being one of the main processes (1–5). Trabeculae initially appear as myocardial ridges in the outer curvature of the ventricle, and they are important for cardiac function, as evidenced by studies of hypo- and hypertrabeculation models (3, 6–11). Previous studies have shown that Erbb2 signaling is essential for trabeculation (3, 6, 7, 10, 11). Moreover, disturbing blood flow or cardiac contractility in the ventricle perturbs trabeculation (4, 12, 13), suggesting an important role for mechanical forces in this process. Our understanding of the cellular mechanisms regulating trabeculation remains fairly limited. Cardiomyocytes in the early heart tube show an epithelium-like morphology (3, 14). During trabeculation, some cardiomyocytes delaminate and enter the trabecular layer, where they join other trabecular cardiomyocytes to form ridge-like structures (3, 15). Previous studies have shown that the most proximal cardiomyocytes in the trabecular layer remain tightly attached to the basal side of compact layer cardiomyocytes (3, 15). Some of the key molecules involved in cell–cell adhesion belong to the cadherin family, and they also play crucial roles in epithelial cell morphology and behavior, including cell migration (16–18). Studies of epithelial cells in culture have shown that changes in cell morphology are accompanied by the extensive remodeling of cell–cell junctions; for example, cell–cell adhesion can be remodeled by regulating the expression and/or endocytic recycling of cadherin (19–23). Mechanical tension plays a crucial role in regulating the size of cell–cell junctions, and local tension generated by the actomyosin network may also modulate cell–cell junction remodeling (24–26). These forces can activate vinculin and stabilize E-cadherin/VE-cadherin-mediated cell–cell adhesion (27–29). Additional studies have shown that cadherin punctae formed by the clustering of E-cadherin along cell–cell boundaries can increase in size during the maturation of cell–cell junctions, and that they are important for cell–cell adhesion and force transmission in vivo (26, 30). How E-cadherin molecules come together and form these punctae has been under intense investigation (31): Some studies have suggested that E-cadherin molecules are able to move along lateral membranes (32, 33), and one of the common themes emerging is the importance of the cortical actin cytoskeleton in their formation (26, 31).
Cdh2 (N-cadherin) adhesive junctions play an important role in mechanical coupling between cardiomyocytes (34, 35). Despite its potential importance, no detailed analysis has been carried out on the organization of Cdh2 during heart development. As it is amenable to high-resolution imaging during its formation and maturation, the zebrafish heart is a good model to study the reorganization of Cdh2 under the influence of mechanical forces and during cardiomyocyte delamination (15, 36). Here we show that Cdh2 molecules first appear on the lateral sides of cardiomyocytes, where they cluster to form punctae. As trabeculation is initiated, Cdh2 molecules appear on the basal side of compact layer cardiomyocytes, possibly by moving from the lateral sides along the cell membrane. We also show that blood flow and/or cardiac contractility play an essential role in the distribution of Cdh2, although one distinct from the role played by Erbb2 signaling, a critical regulator of cardiac trabeculation.
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Acknowledgments
We thank Darren Gilmour for providing the cadherin transgenic lines; Arica Beisaw, Yu Hsuan Carol Yang, Michelle Collins, and Rashmi Priya for discussions and/or critical reading of the manuscript; Radhan Ramadass for help with confocal microscopy, as well as feedback on the manuscript; and Sharon Meaney-Gardian, Sabine Fischer, Carmen Büttner, Nana Fukuda, Sophie Mucenieks, and Marianne Ploch for excellent assistance.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606385113/-/DCSupplemental.
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