There are two types of secreted neuropilin ligands, class 3 SEMAs and VEGF164 (reviewed by Schwarz and Ruhrberg, 2010). Class 3 SEMAs bind the neuropilin a1 domain through their conserved SEMA domain, while VEGF164 binds the b1 domain (Figure 3A). VEGF164 is one of three major VEGF isoforms, named according to the number of amino acids in the

mature protein, and binds to NRP1 via an exon 7-encoded domain that is not present in VEGF120 (Figure 3B; Gitay-Goren learn more et al., 1996, Soker et al., 1996 and Soker et al., 1998). It is not known if the larger VEGF188 also binds NRP1, because VEGF188 cannot be produced for biochemical studies. To determine the expression pattern of class 3 SEMAs versus VEGF-A at the optic chiasm, we performed in situ hybridization on sections through the optic chiasm at E12.5 and E14.5 (Figure 3C). We found that none of the five SEMA genes examined were expressed anywhere near the chiasm at

E12.5 (Figure 3D). At E14.5, Sema3b or Sema3f expression was still not detectable anywhere near the chiasm, Pfizer Licensed Compound Library order and the expression domains of Sema3a, Sema3c, and Sema3e in the diencephalon were positioned far posterior to the RGC axon path ( Figure 3D). By contrast, in situ hybridization demonstrated expression of Vegfa at the chiasmatic midline ( Figure 3E). At E12.5, when the first RGC axons begin to grow into the diencephalon, Vegfa was expressed already at the ventral midline, where the chiasm is destined to form (asterisks in Figure 3E). Moreover, expression was strong near the area where RGC axons were extending through the chiasm at E14.5 and was maintained in this area until at least E17.5 ( Figure 3E). Vegfa is therefore expressed in a pattern that is consistent with a role in RGC axon guidance at the optic chiasm. Our in situ hybridization studies suggested that the main NRP1-binding SEMA, Sema3a, was not expressed at the site where the optic chiasm forms. Because Histone demethylase we could not exclude

the possibility that SEMA3A diffuses from distant sites of expression into the chiasmatic region, we examined RGC axon guidance in Sema3a null mutants ( Taniguchi et al., 1997). Anterograde DiI labeling demonstrated that the size and organization of both optic tracts was normal in all four Sema3a null mutants examined ( Figures 4A and 4B). Together with the expression study, these results establish that NRP1 does not function as a SEMA3A receptor during RGC axon guidance in the mouse. We next asked whether functional redundancy of SEMA3A with other NRP1-binding class 3 SEMAs, such as those whose expression pattern we had not examined, was responsible for the lack of phenotype in Sema3a null mutants. To address this possibility, we took advantage of a mouse mutant that carries point mutations in the a1 domain of NRP1 that abolish the binding of all class 3 SEMAs, but not VEGF164, to NRP1 (Nrp1Sema−/− mice; Gu et al., 2003; Figure 3A).