Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.

Summary Currently, it remains controversial how vascular endothelial progenitor cells (angioblasts) establish their arterial or venous fates. We show using zebrafish that the arterial progenitors of the major axial vessels originate earlier and closer to the midline than the venous progenitors. Both medial and lateral progenitor populations migrate to distinct arterial and venous positions and not into a common precursor vessel as previously suggested. Overexpression of VEGF or Hedgehog (Hh) homologs results in the partially randomized distribution of arterial and venous progenitors within the axial vessels. We further demonstrate that the function of the Etv2 transcription factor is required at earlier stages for arterial development than for venous. Our results argue that the medial angioblasts undergo arterial differentiation because they receive higher concentration of Vegf and Hh morphogens than the lateral angioblasts. We propose a revised model of arterial-venous differentiation that explains how angioblasts choose between an arterial and venous fate.

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein.

ETS transcription factor ETV2 / Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.