We also analyzed Sema-2bC4;PlexB double null mutant embryos ( Figure 2G) and Sema-2abA15;PlexB double null mutant embryos (that are null for Sema2a, Sema2b, and PlexB) ( Figure 2H); both genotypes exhibit 1D4-i

defects identical to those observed in PlexB−/− single mutants and Sema-2abA15 homozygous mutants with equal penetrance ( Figure 2I), indicating that both Sema-2a and Sema-2b function in the same genetic pathway as PlexB. Interestingly, Sema-2aB65/+,Sema-2bC4/+ trans-heterozygous mutant embryos exhibit a much lower penetrance of CNS longitudinal connective defects than embryos of either single mutant ( Figures 2E and 2I), suggesting that Sema-2a and Sema-2b functions are distinct and contribute to different aspects of intermediate

longitudinal connection formation. To complement our genetic analyses we next performed alkaline phosphatase (AP)-tagged ligand binding assays on live dissected embryos (Fox and Zinn, 2005). Kinase Inhibitor Library datasheet We first confirmed that AP alone does not bind to the CNS of dissected Drosophila embryos in our assay (data not shown). We then observed that Sema-2a-AP and Sema-2b-AP both bound to endogenous CNS receptors in dissected wild-type embryos ( Figures 2J and 2L), but not to endogenous CNS receptors in PlexB−/− mutants DAPT cost ( Figures 2K and 2M). Compared to Sema-2a-AP, Sema-2b-AP bound more robustly to endogenous CNS receptors ( Figure 2N). We also expressed PlexB in a Drosophila S2R+ cell line and observed that Sema-2b-AP bound strongly to these cells

but not to PlexA-expressing S2R+ cells ( Figures S2G and S2D), as observed previously Dipeptidyl peptidase for Sema-2a ( Ayoob et al., 2006) ( Figures S2B–S2F). These ligand-receptor binding specificities correlate well with the functions of these proteins in CNS longitudinal track formation. PlexB−/− and PlexA−/− mutant embryos exhibit distinct CNS longitudinal tract defects ( Ayoob et al., 2006 and Winberg et al., 1998b), and Sema-1a−/− mutants have defects similar to those observed in PlexA−/−, but not PlexB−/−, mutants ( Yu et al., 1998) ( Figures S2H and S2I). In addition, we observed that Sema-1a, Sema-2b double null mutants and Sema-1a;PlexB double null mutants both show disorganization of the 1D4-l and 1D4-m tracts ( Figures S2J and S2K), further supporting the idea that Sema-1a-PlexA and Sema-2b-PlexB signaling direct distinct aspects of embryonic longitudinal tract formation. Taken together, these results show that Sema-2a and Sema-2b signaling through the PlexB receptor accounts for most, if not all, PlexB functions in embryonic CNS intermediate longitudinal tract formation. We next assessed Sema-2b protein distribution in Drosophila embryos using a polyclonal antibody specific for Sema-2b (L.B.S., Y. Chou, Z.W., T. Komiyama, C.J. Potter, A.L.K., K.C. Garcia, and L.L., unpublished data). Sema-2b is weakly expressed on CNS commissures and more robustly on two longitudinal pathways ( Figure 3B).