NRP1 expression by tissue macrophages is not essential for brain vascularization. (A) A E10.5 hindbrain with a constitutively active Csf1r-iCre transgene and the Rosa26^Yfp reporter was triple labeled for YFP (green), NRP1 (red), and IB4 (blue), shown together (A) and as single channels (A′-A′′′). The solid arrow indicates a tissue macrophage, the clear arrow a NRP1-positive macrophage process; the solid arrowhead indicates a tip cell. Scale bar represents 50 μm. (B,C) Double labeling of E11.5 control Nrp1^fl/– and mutant Csf1r-iCre;Nrp1^fl/– hindbrains for NRP1 (red) and IB4 (blue) shown together (B,C) and as single NRP1 channels (B′,C′). A NRP1-positive tissue macrophage is indicated with an arrow in (B,B′); NRP1-negative tissue macrophages are indicated with asterisks in (C,C′). Scale bar represents 25 μm. (D,E) PECAM immunohistochemistry of E12.5 littermate hindbrains of the indicated genotypes; scale bar represents 100 μm. (F) Quantitation of SVP branchpoints at E12.5; error bars represent SD; n.s., not significant.