Requisite Role for Nck Adaptors in Cardiovascular Development, Endothelial-to-Mesenchymal Transition, and Directed Cell Migration

Abstract

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development.

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ACKNOWLEDGMENTS

We thank Rong Wang (University of California at San Francisco, San Francisco, CA) and Tony Pawson and Julie Ruston (Samuel Lunenfeld Research Institute, Toronto, ON, Canada) for sharing mouse lines and expertise and Louise Larose (McGill University, Montreal, QC, Canada) for kindly providing Nck antibodies. We also acknowledge Ken Harpal (Mount Sinai Hospital, Toronto, ON, Canada) for assistance with embryo sectioning and Dan Dumont (Sunnybrook Research Institute, Toronto, ON, Canada) as well as members of the N. Jones laboratory for helpful comments on the manuscript. We are also grateful to Martha Smith and the University of Guelph Central Animal Facility staff for their assistance with animal maintenance.

This work was supported by an operating grant from the Canadian Institutes of Health Research (MOP number 93526) and an Early Researcher Award (to N.J.). N.J. is the recipient of a Natural Sciences and Engineering Research Council (NSERC) University Faculty Award and a New Investigator Award from the Kidney Research Scientist Core Education and National Training (KRESCENT) Program and is a Tier 2 Canada Research Chair.