Both full-length and reconstituted fragments of PlexB transduce signal in trajectory choice. (A) In wild-type pupal brains at 24hAPF, ORN axons form the dorsolateral (DL) and ventromedial (VM) trajectories circumnavigating the antennal lobe (left panels). Overexpression of PlexB in ORNs shifts ORN axons to the DL trajectory (right panels). ORN axons were labeled by pan-ORN Peb-GAL4 (Sweeney et al., 2007) driven mtdTomato expression. Antennal lobes were co-stained with a neuropil marker N-cadherin (NCad). (B) Fluorescence intensity ratios of ORN axon trajectories (DL/VM) in wild-type and PlexB overexpression brains at 24hAPF. Geometric means: control, 0.68; WT OE, 0.94. ‘OE’ hereafter denotes ORN-specific overexpression of PlexB variants. (C–K) Fluorescence intensity ratios of ORN axon trajectories (DL/VM) for respective PlexB variants. Geometric means: ∆Sema, 0.68; GAPmut, 1.01; ∆Rac1, 0.73; Rac1mut, 0.84; ∆Rho1, 0.81; Uncleav, 1.04; CleavSec, 0.69; CleavTMCyto, 0.65; CleavSec + CleavTMCyto, 0.82. Sample sizes are noted in parentheses. Significance among multiple groups in Figure 3B–K was determined by one-way ANOVA with Tukey’s test for multiple comparisons. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Images are shown as maximum z-projections of confocal stacks. Scale bars, 10 µm. Axes, D (dorsal), L (lateral).