NRP1 and ABL1 promote PXN phosphorylation during retinal angiogenesis in the mouse. (A) A schematic representation of retinal angiogenesis illustrates how vessels (red) expand from the retinal center toward the periphery (indicated by arrows). Vessels are guided by an astrocyte-derived FN network (green) ahead of the vascular front, but FN subsequently overlaps partially with the vessel pattern (red with green outline). (B) Immunolabeling for FN together with the vascular marker IB4 demonstrates angiogenesis in the P6 retina. Bar, 400 µm. The area shown in higher magnification is indicated with a dotted square in the first panel. (C) Reduced NRP1 levels in Nrp1^+/− compared with Nrp1^+/+ littermates, shown by immunoblot quantification of NRP1 protein levels in P6 lungs. NRP1 levels were normalized to VE-cadherin levels and expressed as percentage relative to wild type (mean ± SEM; n ≥ 3). *, P < 0.05, Student’s t test. (D–H) Single confocal slices through the vascular front in retinas of Nrp1^+/+, Nrp1^+/− littermate mice and tamoxifen-inducible EC-specific Nrp1-null mice injected with tamoxifen from P2 to P5 (D) or treated with vehicle or 100 mg/kg/d Imatinib on days P4 and P5 (G). Retinas were immunostained for pPXN Y118 and IB4. Bar, 15 µm. In D and G (right), Imaris software was used to mask areas not labeled for IB4 to reveal pPXN staining in ECs. Quantification of vascular pPXN in optical z-stacks after applying a mask to isolate IB4-positive areas in Nrp1^+/− (E), tamoxifen-inducible EC-specific Nrp1-null mutants (F), and Imatinib-treated mice (H) relative to controls. Mean pixel values of pPXN relative to IB4 are expressed as percentage of control ± SEM (n = 3). *, P < 0.05, Student’s t test.