Spry2 represses vascular branching. (A) Aortic rings from 3- to 5-week-old mice (Spry+/− and Spry−/−, respectively) and from age-matched wild-type control littermates (Spry+/+) were embedded in Matrigel and treated with control medium (Control) and pro-angiogenic BBE (200 μg/mL) with or without miR-27b targeting or control MOs (MO-27b and MO-C). Vascular sprouting was documented after 4 days. (B) The average sprout length was measured in 6-8 samples with ImageJ software. *P < .0035 for the difference from noninduced wild-type controls and **P < .0002 for the difference from BBE-stimulated wild-type controls. (C) Fertilized zebrafish eggs were injected with mRNA encoding Spry2 (200 ng/injection) or control mRNA and the embryos were photographed at 36 hpf. Note the lack of T-shaped cells at the tips of the ISVs. (D) Confocal images of the embryos overexpressing Spry2 and control (C). Note the lack of T-shaped tip cells and decreased filopodia formation. (E) Quantification of the experiment in panel D. The percentage of vessels with T-shaped tip cells at the end was determined in the trunk area of 5 representative embryos. *P < 5 × 10−7, P < .0007, P < .009, and P < .0002, respectively, for the differences from the controls. (F) Zebrafish embryos were injected with MO-27b, MO-Spry2, or both. Confocal images of the vasculature are shown. Note premature branching and increased filopodia formation in the Spry2 morphants and normalized phenotype of the double morphants. (G) Quantitative analysis of the experiment in panel F. *P < .00086 for the difference from controls; **P < .002 for the difference from MO-27b alone.
