Vascular endothelial growth factor a (Vegfa) is essential for blood vessel formation and can induce activation of numerous signaling effectors in endothelial cells. However, it is unclear how and where these function in developmental contexts during vascular morphogenesis. To address this issue, we have visualized activation of presumptive Vegfa effectors at single-cell resolution in zebrafish blood vessels. From these studies, we find that phosphorylation of the serine/threonine kinase ERK (pERK) preferentially occurs in endothelial cells undergoing angiogenesis, but not in committed arterial endothelial cells. pERK in endothelial cells was ectopically induced by Vegfa and lost in Vegfa signaling mutants. Both chemical and endothelial autonomous inhibition of ERK prevented endothelial sprouting, but did not prevent initial artery differentiation. Timed chemical inhibition during angiogenesis caused a loss of genes implicated in coordinating tip/stalk cell behaviors, including flt4 and, at later stages, dll4. ERK inhibition also blocked excessive angiogenesis and ectopic flt4 expression in Notch-deficient blood vessels. Together, these studies implicate ERK as a specific effector of Vegfa signaling in the induction of angiogenic genes during sprouting.
The authors declare no competing or financial interests.
M.S. designed and performed experiments for all aspects of this work and contributed to the editing of the manuscript. T.B. performed in situ hybridization experiments and contributed to maintenance of zebrafish lines described in this study. A.Q. designed and generated the insUAS constructs. I.M. contributed to identification and maintenance of zebrafish lines described in this study. L.J.Z. performed statistical analyses. N.D.L. designed experiments and wrote the manuscript.
This work was supported by grants from the National Heart, Lung, and Blood Institute (NHLBI) [R01HL093467 and R01HL122599 to N.D.L.]; by the Uehara Memorial Foundation (M.S.); and by the Japan Society for the Promotion of Science (JSPS). Deposited in PMC for release after 12 months.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.137919.supplemental
- Received March 21, 2016.
- Accepted August 23, 2016.