miR-27b regulates arterial-venous segregation. (A-F) miR-27b, Dll4, and Spry2 were silenced and arterial-venous segregation observed at 32 hours. (A) Segregation of the DA and PCV in control morphants. (B-D) Defective PCV morphogenesis in the embryos injected with MO-27b (B), MO-Dll (C), and MO-Spry2 (D). (E-F) Rescue of PCV morphogenesis in double morphants. (G-H) miR-27b inhibitors increase arterial phenotype. miR-27b was blocked with MO-27b (G) or miR-Zip lentiviral vectors (H) and EphB2 and EphB4 were measured by Western blot. (I) miR-27b regulates Flt4. In situ hybridization for the whole-mount zebrafish embryo. (J-M) Dll4 and Spry2 maintain arterial phenotype. Dll4 and Spry2 were silenced in cultured microvascular endothelial cells using lentivirally expressed shRNA (J-K). Distinct shRNA sequences are indicated as c1 and c2. (L-M) Endothelial cells infected with control vector, Dll4, or Spry shRNA were left untreated or treated with 1 ng/mL of VEGF, and the expression of EphB2, EphB4, and Flt4 was measured by Western blot.