We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

Lymphangiogenesis is a dynamic process that involves the sprouting of lymphatic endothelial cells (LECs) from veins to form lymphatic vessels. Vegfr3 signalling, through its ligand Vegfc and the extracellular protein Ccbe1, is essential for the sprouting of LECs to form the trunk lymphatic network. In this study we determined whether Vegfr3, Vegfc and Ccbe1 are also required for development of the facial and intestinal lymphatic networks in the zebrafish embryo. Whereas Vegfr3 and Ccbe1 are required for the development of all lymphatic vessels, Vegfc is dispensable for facial lymphatic sprouting but not for the complete development of the facial lymphatic network. We show that zebrafish vegfd is expressed in the head, genetically interacts with ccbe1 and can rescue the lymphatic defects observed following the loss of vegfc. Finally, whereas knockdown of vegfd has no phenotype, double knockdown of both vegfc and vegfd is required to prevent facial lymphatic sprouting, suggesting that Vegfc is not essential for all lymphatic sprouting and that Vegfd can compensate for loss of Vegfc during lymphatic development in the zebrafish head.