N-cadherin relocalization during cardiac trabeculation

Anoop Cherian

Ryuichi Fukuda

Sruthy Augustine

Hans Maischein

Didier Stainier

Supplementary Material

^{Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany}

^{To whom correspondence should be addressed. Email:}ed.gpm.nb-ipm@reiniats.reidid.

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 16, 2016 (received for review April 29, 2016)

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.

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 16, 2016 (received for review April 29, 2016)

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.

Keywords: N-cadherin, trabeculation, heart development, Erbb2 signaling, Cdh2-EGFP

Significance

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.

Abstract

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.

Supplementary File

Click here to view.^{(354K, avi)}

Supplementary File

Click here to view.^{(342K, avi)}

Supplementary File

Click here to view.^{(1.0M, avi)}

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.

Acknowledgments

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.

Footnotes

References

1. Sedmera D, et al Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts. Am J Physiol Heart Circ Physiol. 2003;284(4):H1152–H1160.[PubMed][Google Scholar]
2. Stankunas K, et al Endocardial Brg1 represses ADAMTS1 to maintain the microenvironment for myocardial morphogenesis. Dev Cell. 2008;14(2):298–311.[Google Scholar]
3. Liu J, et al A dual role for ErbB2 signaling in cardiac trabeculation. Development. 2010;137(22):3867–3875.[Google Scholar]
4. Peshkovsky C, Totong R, Yelon DDependence of cardiac trabeculation on neuregulin signaling and blood flow in zebrafish. Dev Dyn. 2011;240(2):446–456.[PubMed][Google Scholar]
5. Staudt D, Stainier DUncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu Rev Genet. 2012;46:397–418.[PubMed][Google Scholar]
6. Gassmann M, et al Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature. 1995;378(6555):390–394.[PubMed][Google Scholar]
7. Lee KF, et al Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature. 1995;378(6555):394–398.[PubMed][Google Scholar]
8. Meyer D, Birchmeier CMultiple essential functions of neuregulin in development. Nature. 1995;378(6555):386–390.[PubMed][Google Scholar]
9. Suri C, et al Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996;87(7):1171–1180.[PubMed][Google Scholar]
10. Morris JK, et al Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development. Neuron. 1999;23(2):273–283.[PubMed][Google Scholar]
11. Tidcombe H, et al Neural and mammary gland defects in ErbB4 knockout mice genetically rescued from embryonic lethality. Proc Natl Acad Sci USA. 2003;100(14):8281–8286.[Google Scholar]
12. Chi NC, et al Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol. 2008;6(5):e109.[Google Scholar]
13. Samsa LA, et al Cardiac contraction activates endocardial Notch signaling to modulate chamber maturation in zebrafish. Development. 2015;142(23):4080–4091.[Google Scholar]
14. Trinh LA, Stainier DYFibronectin regulates epithelial organization during myocardial migration in zebrafish. Dev Cell. 2004;6(3):371–382.[PubMed][Google Scholar]
15. Staudt DW, et al High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation. Development. 2014;141(3):585–593.[Google Scholar]
16. Harris TJ, Tepass UAdherens junctions: From molecules to morphogenesis. Nat Rev Mol Cell Biol. 2010;11(7):502–514.[PubMed][Google Scholar]
17. Takeichi MDynamic contacts: Rearranging adherens junctions to drive epithelial remodelling. Nat Rev Mol Cell Biol. 2014;15(6):397–410.[PubMed][Google Scholar]
18. Niessen CM, Leckband D, Yap ASTissue organization by cadherin adhesion molecules: Dynamic molecular and cellular mechanisms of morphogenetic regulation. Physiol Rev. 2011;91(2):691–731.[Google Scholar]
19. Lochter A, et al Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells. J Cell Biol. 1997;139(7):1861–1872.[Google Scholar]
20. Marambaud P, et al A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions. EMBO J. 2002;21(8):1948–1956.[Google Scholar]
21. Maretzky T, et al ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation. Proc Natl Acad Sci USA. 2005;102(26):9182–9187.[Google Scholar]
22. Steinhusen U, et al Cleavage and shedding of E-cadherin after induction of apoptosis. J Biol Chem. 2001;276(7):4972–4980.[PubMed][Google Scholar]
23. Yilmaz M, Christofori GEMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 2009;28(1-2):15–33.[PubMed][Google Scholar]
24. Lecuit T, Yap ASE-cadherin junctions as active mechanical integrators in tissue dynamics. Nat Cell Biol. 2015;17(5):533–539.[PubMed][Google Scholar]
25. Lecuit T, Lenne PF, Munro EForce generation, transmission, and integration during cell and tissue morphogenesis. Annu Rev Cell Dev Biol. 2011;27:157–184.[PubMed][Google Scholar]
26. Maître JL, et al Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science. 2012;338(6104):253–256.[PubMed][Google Scholar]
27. Yonemura S, Wada Y, Watanabe T, Nagafuchi A, Shibata Malpha-Catenin as a tension transducer that induces adherens junction development. Nat Cell Biol. 2010;12(6):533–542.[PubMed][Google Scholar]
28. Huveneers S, et al Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J Cell Biol. 2012;196(5):641–652.[Google Scholar]
29. le Duc Q, et al Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner. J Cell Biol. 2010;189(7):1107–1115.[Google Scholar]
30. Truong Quang BA, Mani M, Markova O, Lecuit T, Lenne PFPrinciples of E-cadherin supramolecular organization in vivo. Curr Biol. 2013;23(22):2197–2207.[PubMed][Google Scholar]
31. Yap AS, Gomez GA, Parton RGAdherens Junctions Revisualized: Organizing Cadherins as Nanoassemblies. Dev Cell. 2015;35(1):12–20.[PubMed][Google Scholar]
32. Kametani Y, Takeichi MBasal-to-apical cadherin flow at cell junctions. Nat Cell Biol. 2007;9(1):92–98.[PubMed][Google Scholar]
33. Wang YC, Khan Z, Kaschube M, Wieschaus EFDifferential positioning of adherens junctions is associated with initiation of epithelial folding. Nature. 2012;484(7394):390–393.[Google Scholar]
34. Ferreira-Cornwell MC, et al Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart. J Cell Sci. 2002;115(Pt 8):1623–1634.[PubMed][Google Scholar]
35. Bagatto B, Francl J, Liu B, Liu QCadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development. BMC Dev Biol. 2006;6:23.[Google Scholar]
36. Beis D, Stainier DYIn vivo cell biology: Following the zebrafish trend. Trends Cell Biol. 2006;16(2):105–112.[PubMed][Google Scholar]
37. Revenu C, et al Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation. Development. 2014;141(6):1282–1291.[PubMed][Google Scholar]
38. Merzlyak EM, et al Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods. 2007;4(7):555–557.[PubMed][Google Scholar]
39. Donà E, et al Directional tissue migration through a self-generated chemokine gradient. Nature. 2013;503(7475):285–289.[PubMed][Google Scholar]
40. Khmelinskii A, et al Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat Biotechnol. 2012;30(7):708–714.[PubMed][Google Scholar]
41. Liu Z, et al Mechanical tugging force regulates the size of cell-cell junctions. Proc Natl Acad Sci USA. 2010;107(22):9944–9949.[Google Scholar]
42. Sehnert AJ, et al Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat Genet. 2002;31(1):106–110.[PubMed][Google Scholar]
43. Wheelock MJ, Shintani Y, Maeda M, Fukumoto Y, Johnson KRCadherin switching. J Cell Sci. 2008;121(Pt 6):727–735.[PubMed][Google Scholar]
44. El-Amraoui A, Petit CCadherins as targets for genetic diseases. Cold Spring Harb Perspect Biol. 2010;2(1):a003095.[Google Scholar]
45. Martin ACPulsation and stabilization: Contractile forces that underlie morphogenesis. Dev Biol. 2010;341(1):114–125.[PubMed][Google Scholar]
46. Cavey M, Rauzi M, Lenne PF, Lecuit TA two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature. 2008;453(7196):751–756.[PubMed][Google Scholar]
47. Wu SK, et al Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia. Nat Cell Biol. 2014;16(2):167–178.[PubMed][Google Scholar]
48. Reischauer S, Arnaout R, Ramadass R, Stainier DYActin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture. Circ Res. 2014;115(10):845–856.[Google Scholar]
49. Lin YF, Swinburne I, Yelon DMultiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart. Dev Biol. 2012;362(2):242–253.[Google Scholar]
50. Takeuchi JK, et al Chromatin remodelling complex dosage modulates transcription factor function in heart development. Nat Commun. 2011;2:187.[Google Scholar]
51. Trinh A, et al A versatile gene trap to visualize and interrogate the function of the vertebrate proteome. Genes Dev. 2011;25(21):2306–2320.[Google Scholar]