, 1996, Tran et al., 2007 and Yazdani and Terman, 2006). Previous work has shown that Sema5A and Sema5B can act as guidance cues to either attract or repel processes belonging to different neuronal populations (Goldberg et al., 2004, Hilario et al., 2009, Kantor et al., 2004, Lett et al., 2009 and Oster et al., 2003). We generated mice harboring knockout alleles

of Sema5A and Sema5B by targeting exon 6 of Sema5A and exon 2 of Sema5B, each of which encode the first 41 or 51 amino acids, respectively, of these proteins (see Figure S1 available BTK inhibitor online). Our Sema5A and Sema5B mutant mice lack full-length Sema5A and Sema5B proteins ( Figures S1G and S1H). Unlike the early embryonic lethality observed in previously generated Sema5A null mice (in a mixed 129/NMRI genetic JAK2 inhibitor drug background) ( Fiore et al., 2005), we found that in a 129/C57BL/6 mixed genetic background, our Sema5A−/−, Sema5B−/−, and Sema5A−/−; Sema5B−/− mice are viable and fertile. This difference could be due to either the utilization of different targeting strategies and/or mouse genetic backgrounds. These results strongly suggest that our Sema5A and Sema5B mutant mice are null

mutants. Sema5A−/−; Sema5B−/− mice exhibit severe defects in the stereotypic neurite arborization of multiple amacrine cell types. In Sema5A−/−; Sema5B−/− mice, tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, which predominantly stratify within the S1 sublamina of the IPL in wild-type (WT) retinas ( Figure 1I), exhibit dramatic mistargeting within both the INL and OPL ( Figure 1L). Similarly, vGlut3-expressing amacrine cells, which mostly stratify within the S2/S3 sublaminae medroxyprogesterone of the IPL in WT retinas ( Figure 1M), show severe neurite mistargeting within both the IPL and INL in Sema5A−/−; Sema5B−/− mice ( Figure 1P). In addition, AII amacrine cells (labeled with Disabled-1 [Dab-1]), cholinergic amacrine cells (labeled with choline acetyltransferase [ChAT]), calretinin-positive cells, and calbindin-positive cells all exhibit

pronounced ectopic neurite extension toward the outer retina in these mutant mice ( Figures 2A–2H). Importantly, these defects are observed with full penetrance and expressivity in Sema5A−/−; Sema5B−/− animals (n = 12 Sema5A−/−; Sema5B−/− mice; n = 12 WT mice). Sema5B−/− mice also exhibit neurite arborization defects involving these same neuronal subtypes ( Figures 1K and 1O; data not shown), although these phenotypes are less severe than those seen in Sema5A−/−; Sema5B−/− mice. Sema5A−/− mice, and also Sema5A+/−; Sema5B+/− mice, did not show defects in these same classes of retinal neurons ( Figures 1J and 1N and Figure S2; data not shown). These results suggest that Sema5A and Sema5B play redundant roles in regulating multiple amacrine cell neurite arborization events in vivo.