(A) In wild-type pupal brains at 24 hr after puparium formation (24hAPF), ORN axons bifurcate and form the dorsolateral (DL) and ventromedial (VM) trajectories (arrowheads) circumscribing the antennal lobe (AL). ORN axons were labeled by the pan-ORN Peb-GAL4 (Sweeney et al., 2007) driven mtdTomato expression. Antennal lobes were co-stained with a neuropil marker N-cadherin (NCad). (B) In plexB homozygous mutant, ORN axons preferentially choose the DL trajectory. (C) Fluorescence intensity ratios of ORN axon trajectories (DL/VM) in wild-type and plexB–/– brains at 24hAPF. Geometric means: control, 0.73; plexB–/–, 1.53. (D) Design of the PlexB-GAL4: a T2A-GAL4 cassette (Diao et al., 2015) was inserted into a MiMIC locus in the first coding intron of PlexB. SA, splicing acceptor. Hsp70, terminator sequence of Hsp70. SD, splicing donor (not functional because mRNAs terminate at the Hsp70 terminator). (E) PlexB-GAL4 labels the antennal lobe and nearby brain structures at 24hAPF. The signal from ORNs is not detectable. Dotted circle, antennal lobe (AL). (F) Intersection of PlexB-GAL4 and eyFLP (FLP in ORNs) labels both DL and VM trajectories. (G) DL/VM fluorescence intensity ratios of the pan-ORN GAL4 (Peb-GAL4) and the PlexB-GAL4 intersected with eyFLP. Geometric means: Peb-GAL4, 0.73; PlexB-GAL4∩eyFLP, 0.61. (H) Design of the PlexB conditional tag: a cassette including a splicing acceptor (SA), the PlexB coding sequence after the first exon, FRT-FLAG-Stop-FRT-V5-Stop, and the PlexB 3’-UTR sequence, was inserted into a MiMIC locus in the first coding intron of PlexB. (I, J) FLAG staining of 24hAPF brains of PlexB-Tag alone (I) or PlexB-Tag with eyFLP (J). (K, L) V5 staining of 24hAPF brains of PlexB-Tag alone (K) or PlexB-Tag with eyFLP (L). (M) DL/VM fluorescence intensity ratios: ORN axon distributions were calculated based on the Peb-GAL4 > mtdTomato signal (as in Figure 1A); ORN-specific PlexB protein distributions were calculated based on the V5 staining of PlexB-Tag with eyFLP (as in Figure 1L). Geometric means: ORN axon distribution, 0.73; ORN-specific PlexB protein distribution, 1.41. Sample sizes are noted in parentheses. Significance between two groups was determined by an unpaired, two-tailed t-test. ****p<0.0001. Images are shown as maximum z-projections of confocal stacks. Scale bars, 10 μm. Axes, D (dorsal), L (lateral).