Defective retinal vascularization in Sema6A^−/− mice. (A) Retinal whole mounts from paraformaldehyde-fixed eyes of P4 Sema6A-null and wild-type littermates; representative images. (B) Average radial length of retinal vessels from the optic nerve to the periphery was measured in P4 paired littermates (n = 7; mean ± SEM). The results are expressed as percentage retina occupied by vessels. (C) Number of branching points/mm² retinal vessels was measured in P4 paired littermates (n = 7; mean ± SEM). (D) Sema6A immunostaining of P4 wild-type and Sema6A-null retinas. Isolectin B4 (IB4) was used for visualization of retinal vessels; nuclei are visualized with DAPI. (E) Magnified section of retina from panel D showing Sema6A expression in wild-type retinal vessels (IB4-positive) and in ganglion cells. The arrows point to endothelial filopodia expressing Sema6A. Comparative images from wild-type and Sema6A-null tissues were obtained and processed by using the same settings.