Finally, to exclude functional selleck compound compensation for SEMA signaling through NRP1 by NRP2, we examined mice deficient in NRP2 (Nrp2−/−) or in SEMA signaling through both neuropilins (Nrp1Sema−/− Nrp2−/− mutants; Gu et al., 2003). The size and organization of both optic tracts was normal in seven out of seven Nrp2 null and two out of two compound neuropilin mutants ( Figures 4C and 4D). We conclude that SEMA signaling through neuropilins is not essential for RGC pathfinding at the mouse optic chiasm. Because loss of SEMA signaling cannot explain the optic chiasm defects of Nrp1 null mice, we asked if the alternative NRP1 ligand

VEGF164 regulates RGC pathfinding. To address this possibility, we analyzed Vegfa120/120 mice, which cannot make NRP1-binding VEGF164 or VEGF188, but

express VEGF120 to support blood vessel formation ( Ruhrberg et al., 2002). Anterograde DiI labeling revealed that 13/14 Vegfa120/120 mutants displayed a range of RGC axon pathfinding errors that were strikingly similar to those caused by loss of NRP1, but were never seen in any of 13 wild-type littermates ( Figure 4E). Thus, wholemount preparations showed that both the ipsilateral and contralateral optic tracts were defasciculated in the mutants, with the majority of axons organized into two discrete bundles; consequently, the characteristic asymmetry in the width of the optic tracts was lost ( Figure 4E). Moreover, the ipsilateral index was increased significantly CB-839 mw in

the mutants, suggesting an increase unless in the proportion of axons that projected ipsilaterally, similar to Nrp1 null mutants (Vegfa+/+, 0.09 ± 0.01; versus Vegfa120/120, 0.29 ± 0.07; p < 0.01; Figure 4F). Coronal sections through DiI-labeled brains ( Figure 4G) and neurofilament immunofluorescence staining ( Figure 4H) did not reveal additional guidance errors. Based on the striking phenotypic similarities between Nrp1 and Vegfa120/120 mutants (compare Figures 2A–2D with Figures 4E–4G), we conclude that VEGF164 is the principal NRP1 ligand that promotes RGC axon crossing at the optic chiasm and optic tract organization. Because VEGF-A signaling through FLK1 (KDR/VEGFR2) has been proposed to regulate retinal progenitor cell proliferation and differentiation in the chick (Hashimoto et al., 2006), we examined the expression pattern of VEGF-A and its receptors in the developing eye. Vegfa was expressed in the neural retina during the period of RGC development ( Figure S3A). Its main vascular VEGF-A receptors, FLT1 (VEGFR1) and FLK1, were expressed by choroidal and hyaloid blood vessels, as expected ( Figure S3B, arrowheads). In addition, Flk1, but not Flt1, was expressed in the neuroblastic layer of the retina ( Figure S3B). We therefore examined if a defective retinal architecture contributes to the RGC pathfinding errors in Vegfa120/120 mutants. However, labeling of retinas from E15.