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Fly stocks and genetics. Fly stocks and crosses were maintained in standard medium at 25°C, whereas RNAi experiments were performed at 29°C. To discover molecules involved in bilateral sensory map formation, an RNAi screen was conducted against several cell adhesion molecules using pebbeled-Gal4 [a gift from L. Luo (28)]. To perform Nrg RNAi knockdown in ORNs, glia, and AL-associated neurons, the following stocks were obtained from Bloomington Drosophila Stock Center (BDSC) or received as gift: ORN drivers, Sg18.1-Gal4 (43) (BDSC 6405), atonal-Gal4 (a gift from B.A. Hassan), and amos-Gal4 [a gift from T. Chihara (29)]; ORN-specific drivers and markers were provided by B. Dickson and L. Vosshall (44, 45); LN drivers, OK107-Gal4 [BDSC 854 (18)] and OK371-Gal4 [a gift from H. Aberle (17)]; Gal4 and LexA driver lines having expression in subset of LNs were selected on the basis of expression pattern (19) and were obtained from BDSC; glia-specific drivers, repo-Gal4 (BDSC 7415), 442-Gal4 [a gift from T. Préat (16)], PN-specific GH146-Gal4 (46), UAS-NrgRNAi (BDSC 38215), GH146-QF, and QUAS-mtdTomato-3×HA (BDSC 30037). Stocks used for Nrg mutant and intracellular domain deletion constructs (P[acman] constructs) were the following: nrg849 (BDSC 35827), nrg14, and P[acman] constructs were provided by J. Pielage (22). For visualizing axons and synaptic terminals and co-labeling of two distinct cell types, reporters of different binary systems were used: UAS-mCD8::GFP (42), UAS-Brp::GFP (BDSC 36291), UAS-mCherry (BDSC 27392), 10XUAS-mCD8::RFP,13X LexAop2-mCD8::GFP (BDSC 32229), GH146-QF, QUAS-mtdTomato-3xHA (BDSC 30037). For generating single-cell clones, hs-FLPm5 (41) (BDSC 56799 and BDSC 35534) and UAS-Flybow1.1B (41) (BDSC 56803) constructs were used. For cell ablation, the following stocks were used: UAS-DTI (BDSC 25039) and UAS-hid (a gift from J. R. Nambu).

Immunohistology. Dissection of the larval, pupal, or adult brains was carried out in 1× phosphate-buffered saline (PBS) and fixed in 2% freshly prepared paraformaldehyde (PFA) (prepared in 1× PBS) for 60 min for larval and pupal brains and 90 min for adult brains at room temperature. The fixative was removed, and the brains were washed four times with 0.3% PBTx (Triton X-100 in 1× PBS) for 15 min each at room temperature. Blocking of the samples was performed for 60 min at room temperature in 10% goat serum, prepared in 0.3% PBTx, and incubated with primary antibody overnight at 4°C. Following four times washing, 15 min each, samples were incubated with secondary antibody overnight at 4°C. After four times washing, 15 min each, samples were mounted in VECTASHIELD (Vector Laboratories), an antifade mounting medium for confocal microscopy. Both primary and secondary antibodies were diluted in 10% goat serum. Fluorescent samples were analyzed using a Leica TCS SP5II confocal microscope. To process and analyze images and quantify phenotypes, the open source tool ImageJ, Adobe Photoshop, and Imaris (Bitplane) were used.

Primary antibodies used for this study were rat anti–N-cadherin extracellular domain [DN-Ex no.8, 1:10; Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Flamingo (1:5; DSHB), mouse anti–Neuroglian-180 (BP 104, 1:10; DSHB), rabbit anti-GFP (green fluorescent protein) (1:1000; Invitrogen), and mouse anti-Fasciclin2 (1:5; DSHB). Secondary antibodies used were as follows: goat anti-rabbit Alexa 488 (1:500), goat anti-rat Alexa 568 (1:300), goat anti-rat Alexa 647 (1:500), goat anti-mouse Alexa 568 (1:300), and avidin Alexa 488 (1:40). For general nuclear staining, TOTO-3 (1:5000) was used. All secondary antibodies were obtained from Invitrogen.

Unilateral antennal backfills. All experiments were performed on adult flies. Mosquitoes (Anopheles arabiensis) were provided by International Atomic Energy Agency Laboratories, Seibersdorf Laboratories, Austria. Almost all diptera species were collected within the state of Vienna area, with the exception of Hermetia illucens, which was caught in South Tyrol. Species identity of fly samples was performed by a diptera determination key to reach family level and further specific literature to refine the taxon. In cases where a genus level could not be reached or there were remaining uncertainties, the help of experts on the diptera forum (www.diptera.info) was claimed. Living flies were first anesthetized via CO2 and then inserted into 200- or 1000-μl plastic pipette tips, where parts of the tips have been cut off according to the body size so that the head could stick out of the tip. The flies were immobilized with plasticine. In a petri dish, the loaded pipette tip was placed on a plasticine cube with a small indentation for the fly head. To apply the tracer, a wall was built around the antenna with vaseline, creating a small cavity and leaving only one antenna exposed. In all flies, the right antenna was cut, at the base of the third segment, and completely submerged with a drop of neuronal backfill tracer—2% neurobiotin (Vector Laboratories) diluted in Millipore water. The cavity was then completely covered with vaseline to prevent desiccation, and the petri dish was kept in 4°C for 90 min. Afterward, fly heads were removed and processed with the abovementioned immunohistology process for visualization on the same day (47). DN-cadherin was used as a general neuropil marker, and anti-Nrg was used to label the olfactory commissure.

Flyclear. Three- to 5-day-old adult flies were fixed in 4% PFA at 4°C for 90 min, followed by three times washing with 1× PBS at 4°C for 20 min. Furthermore, the flies were dipped in Solution-1 (48) for 5 days at 37°C. Flies with the complete depigmentation of the compound eyes were further processed and washed with 1× PBS for three times at 25°C. Last, the samples were immersed in Solution-2 (48) for a minimum of 1 day at 25°C and mounted in VECTASHIELD (Vector Laboratories) for imaging using a Leica TCS SP5II confocal microscope.

Cell ablation. For the genetic ablation of cPINs, two different reagents were used. The expression of temperature-sensitive diphtheria toxin (UAS-DTI) was induced directly from Gal4 driver line, and its expression was developmentally controlled by temperature shifts. In the case of UAS-hid, Gal4 expression was controlled via Gal80ts. In both cases, the crosses were raised at 18°C. Late third instar larvae were picked and kept at 29°C to allow transgene expression. The adult flies were dissected 3 days after eclosion.

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