Fig. 3. Yolk sac vessel configuration and arterial marker expression following perfusion. (A) 12 ss and (B) 19 ss embryos. Whole-mount in situ hybridization with the indicated markers. (A,B) Prior to (A) and just after (B) perfusion, ephrinB2 (A) and NRP1 (B) label the posterior (arterial) pole (arrowheads; A,B). (C-E) 30 ss embryos. (C) Intracardiac FITC-Dextran injection. The left side of the yolk sac is shown. Blood from the vitelline artery (red arrow) flows directly into veins (blue arrow), without passing through a true capillary bed. (D) NRP1 labels the vitelline artery (red arrow), but not the vein (blue arrow). (E) The right side of the yolk sac is shown. Ephrin-B2 expression closely resembles NRP1 expression. The future territory of the posterior vitelline vein has downregulated NRP1 and Ephrin-B2 expression (arrowheads, D and E). (F) 40 ss embryo, the left side of the yolk sac is shown. In vivo protein binding of EphB4-Fc to arteries (A) expressing ephrinB2. Veins (V) are negative and are already interlaced with arteries at this stage. Scale bars: 420 µm in A,B; 400 µm in C-E; 190 µm in F.