Netrin-4 Acts as a Pro-angiogenic Factor during Zebrafish Development

Netrins form a heterogeneous family of laminin-related molecules with multifunctional activities. Netrin-4, the most distant member of this family, is related to the laminin beta chain and has recently been proposed to play an important role in embryonic and pathological angiogenesis. However, the data reported so far lead to the apparently contradictory conclusions supporting Netrin-4 as either a pro- or an anti-angiogenic factor. To elucidate this controversy, Netrin-4 was analyzed for a vascular activity in both cell-based models (human umbilical vein endothelial cells and human umbilical artery endothelial cells) and two zebrafish models: the wild-type AB/Tu strain and the transgenic Tg(fli1a:EGFP)(y1) strain. We show that Netrin-4 is expressed in endothelial cells and in the zebrafish vascular system. We also show evidence that Netrin-4 activates various kinases and induces various biological effects directly linked to angiogenesis in vitro. Using a morpholinos strategy, we demonstrate that Netrin-4 expression is crucial for zebrafish vessel formation and that a blood vessel formation defect induced by netrin-4 morpholinos can be partially rescued through drug delivery leading to protein kinase activation. Together these data underscore the crucial role of Netrin-4 in blood vessel formation and the involvement of protein kinases activation in Netrin-4-induced biological effects related to vascular development.