At 16 hr APF, just prior to the arrival of adult ORN axons, Sema-2a and Sema-2b were highly enriched medially and ventromedially within the antennal lobe (Figures 2A1 and 2A2). Sema-2a and Sema-2b showed similar distribution patterns, although there was a subtle difference upon quantification: RGFP966 cell line the Sema-2a gradient
was steeper in the ventromedial antennal lobe whereas that of Sema-2b was more gradual and extended further into the dorsolateral antennal lobe (Figure 2C). By comparison, the pan-neuropil marker N-cadherin was broadly distributed across the entire antennal lobe (Figure 2A3), as were the distributions of all three proteins when quantified along the orthogonal dorsomedial-ventrolateral axis (Figure 2C). In sema-2a sema-2b homozygous double mutant flies (see below) at 16 hr APF, Sema-2a and Sema-2b staining in the antennal lobe was undetectable ( Figure 2B), confirming both the specificity of the Sema-2 antibodies and the absence of protein in these mutants. In addition, these antibodies did not cross react, as sema-2a or sema-2b single mutants only lacked Sema-2a or
-2b antibody staining, respectively (data not shown). We also examined expression of Sema-2a and Sema-2b at 0 hr, 6 hr, and 12 hr APF and found that their distribution patterns during these earlier time learn more points were similar to those described above for 16 hr APF ( Figure S2). These expression studies suggest that Sema-2a and Sema-2b can be used as cues for PN dendrite targeting along the dorsolateral-ventromedial axis. At 16 hr APF, the ventromedial enrichment of Sema-2a/2b is in opposition to the previously shown dorsolateral-high Sema-1a gradient (Komiyama et al., 2007). Where Sema-2a was high, Sema-1a Thymidine kinase was low; where Sema-1a was high, Sema-2a was low (Figure 2D). These opposing expression
patterns, in addition to our binding data, suggested that Sema-1a and Sema-2a/2b may function together during PN dendrite targeting to segregate PN dendrites along this axis. The onset of localized Sema-2a and Sema-2b expression (Figure S2) preceded that of Sema-1a (∼6–12 hr APF) (Komiyama et al., 2007), consistent with a hypothesis that Sema-2a and Sema-2b instruct Sema-1a mediated PN dendrite targeting to the dorsolateral antennal lobe. To test the requirement for secreted Sema-2a and Sema-2b in PN dendrite targeting, we utilized two P-element insertions at the sema-2a locus ( Kolodkin et al., 1993) and a piggyBac insertion into sema-2b ( Thibault et al., 2004). All mutations resulted in a complete loss of corresponding proteins during PN dendrite targeting as assessed by antibody staining ( Figures 2B and S2).