The activity of the Vegfc/Vegfr3 signaling pathway is critical for the formation of lymphatic vessels from pre-existing vasculature (lymphangiogenesis). The first lymphatic endothelial precursor cells in the embryo bud from the dorsal walls of the cardinal veins under the control of Vegfc/Vegfr3 signaling. We previously performed a forward genetic screen for lymphatic mutants in zebrafish and identified collagen and calcium-binding EGF domains-1 (ccbe1) as indispensable for lymphangiogenesis. Ccbe1 acts at the same developmental stage as Vegfc/Vegfr3 signaling and the role of CCBE1 is highly conserved in mice and humans, with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam Syndrome). Despite the importance of CCBE1 in lymphatic development and disease, the mechanism by which it acts has remained elusive.

Here we show that ccbe1 genetically interacts with vegfc and vegfr3 in double and triple heterozygous mutant zebrafish. Furthermore, phenotypes driven by increased Vegfc/Vegfr3 signaling in the absence of Dll4 function are suppressed in ccbe1 mutant embryos further validating the capability of Ccbe1 to modulate Vegfc signaling outcomes. Finally, we optimized cross-reacting antibodies that detect the phosphorylated kinase domain of zebrafish Vegfr3. We demonstrate, using immunofluorescence of embryonic tissues and immunoprecipitation of whole embryo lysates, that normal Vegfr3 signaling during embryonic lymphangiogenesis requires ccbe1.

Taken together, these data identify Ccbe1 as a modulator of the Vegfc/Vegfr3 pathway in the developing embryo. Given theconservation of ccbe1 phenotypes from fish to man, we predict that this role is likely to be conserved.