Cell Adhesion Mediated by VCAM–ITGa9 Interactions Enables Lymphatic Development

Objective—Adhesive ligand–receptor interactions play key roles in blood vessel angiogenesis but remain poorly characterized during lymphatic vessel growth. In this study, we use genetic approaches in both fish and mice to address the roles of cell surface integrin ligand vascular cell adhesion molecule (VCAM) and its 2 receptors, integrins a9 and a4, during lymphatic vascular development. Approach and Results—Conditional deletion of the Vcam gene was used to test VCAM function in lymphatic growth in midgestation mice. Morpholino knockdown and cRNA rescue of the 2 zebrafish vcam alleles, as well as integrins a9 and 4, were used to test the role of these ligands and receptors during lymphatic growth in the developing fish. We show that VCAM is essential for lymphatic development in the zebrafish embryo and that integrin α9 (Itga9) rather than Itga4 is the required VCAM receptor in the developing fish. VCAM is expressed along lines of lymphatic migration in the mouse intestine, but its loss only retards lymphatic growth. Conclusions—These studies reveal an unexpected role for cell–cell adhesion mediated by Itga9–VCAM interactions during lymphatic development in the fish but not in the mouse. We propose that the relative importance of cellular adhesive ligands is magnified under conditions of rapid tissue growth when the cell number increases faster than cell matrix, such as in the early zebrafish embryo. Lymphatic vessels develop in all vertebrates where they function to drain fluid and cells that leak from the closed blood vasculature into the interstitium.1 Lymphatic development has been studied in detail using genetic models in the mouse and fish.2 These studies reveal that lymphatic endothelial cells (LECs) arise from venous blood endothelial cells and then migrate away from blood vessels to form primary lymphatic structures that sprout to create a network of lymphatic vessels. In the mouse, this primary structure is the lymph sac,3 whereas in the fish, it is a line of parachordal lymphangioblasts.4 In both fish and mice, lymphatic vessels arise in response to conserved molecular cues, such as the secreted factors VEGF-C and CCBE1.5–10 However, the molecular mechanisms by which LECs rapidly migrate to generate this network remain incompletely understood.