TEM antigen expression by proangiogenic hindbrain macrophages. (A-B) Wild-type hindbrains (11.5 dpc) were double-labeled for IB4 and NRP1 to visualize the subventricular zone, including the SVP; the boxed areas in panels A, A′, and A′′ are shown in higher magnifications in panels B, B′, and B′′, respectively; yellow indicates colocalization in panels A′′ and B′′. Many NRP1-positive macrophages (arrows in B,B′′) interacted with endothelial tip cells (arrowheads in panel B′′); a NRP1-positive macrophage bridging neighboring tip cells is indicated with a wavy arrow in panels B and B′′. (C-G) Double labeling for NRP1 and the macrophage markers CD11b (CD18/MAC-1), F4/80, or IBA1 (C-E), the vascular endothelial marker PECAM (F), or the neural progenitor marker SOX2 (G) established that NRP1 was expressed in all 3 cell types. Yellow indicates coexpression of NRP1 with macrophage and endothelial markers on the cell surface; in contrast, NRP1 surrounds the SOX2-positive neural progenitor nuclei. The images shown in panels F and G are stacks through the SVP or neural progenitor layer only. (H-I) The specificity of the NRP1 antibody was established by immunolabeling a Nrp1-deficient hindbrain, obtained from a littermate embryo of the wild-type shown in panels A and B. (J-K) Like NRP1, TIE2 (red) colocalized with IB4 (green) on a filopodia-bearing endothelial tip cell (arrowhead) and interacting tissue macrophage (arrow). Scale bars represent 25 μm (except A-A′′, 100 μm).