Fly husbandry and stocks
Drosophila melanogaster were reared on a standard cornmeal medium at 25 °C under a 12 h:12 h light:dark cycle. To enhance transgene expression levels, flies from all genetic perturbation experiments, including control groups, were shifted to 29 °C shortly before puparium formation. Both male and female flies were used. The w[1118] strain was used as wild type, with ages ranging from larva stage to seven days old adults. Detailed genotypes for each experiment are listed in Supplementary Table 1.
Generation of cDNA constructs and transgenic flies
Complementary DNA (cDNA) encoding proteins used in this study were obtained from different resources. Toll2 cDNA was amplified from the cDNA library of w1118 pupal brain extracts using Q5 hot-start high-fidelity DNA polymerase (New England Biolabs) as previously described32; Ptp10D cDNA was amplified from clone RE52018 (DGRC 9073; https://dgrc.bio.indiana.edu//stock/9073; RRID:DGRC_9073); Fili cDNA was amplified from pUAST-attB-SP-V5-Fili-Flag plasmid27; kek1 cDNA was amplified from clone GH23277 (DGRC 1263019; https://dgrc.bio.indiana.edu//stock/1263019; RRID:DGRC_1263019); kirre cDNA was amplified from genomic DNA extraction of UAS-kirre.C-HA fly59 (RRID:BDSC_92196) using DNeasy blood and tissue kit (QIAGEN). Sequence-verified coding regions were assembled into pUAST-attB-mtdT-3xHA60, pUAST-attB-SP-V5-Fili-Flag32 or pJFRC19-13XLexAop2-IVS-myr::GFP (Addgene plasmid #26224) backbones using the NEBuilder HiFi DNA assembly master mix (New England Biolabs) to generate pUAST-attB-Toll2-Flag, pUAST-attB-Ptp10D-3xHA, pUAST-attB-kek1-3xHA, pUAST-attB-kirre-3xHA, and pJFRC19-13XLexAop2-IVS-Toll2 plasmids. Transgenic flies for overexpression experiments were generated by BestGene with microinjection of plasmids into the VK5 site.
Generation of conditional tags
Endogenous conditional tag flies were generated using CRISPR knock-in with modifications from a previous strategy38. To increase the efficiency of knock-in, we incorporated the short repair templates flanked by guide RNA (gRNA) target sites61 and the gRNA into a single plasmid TOPO-HR1-FRT-3xHA-Stop-FRT-3xMyc-Stop-loxP-mCherry-loxP-HR2-pU6-gRNA or TOPO-HR1-FRT-V5-Stop-FRT-Flag-Stop-loxP-mCherry-loxP-HR2-pU6-gRNA. In the plasmid, HR1 and HR2 are the 150-bp genomic sequences of upstream and downstream of the target genes stop codon, respectively; gRNA sequences were designed by the flyCRISPR Target Finder tool that targeting stop codons of the genes, and were cloned into the backbone of pU6-BbsI-chiRNA vector62,63 to make the pU6-gRNA. The plasmids were synthesized by Synbio Technologies and were microinjected in house into nos-Cas9 flies64. All mCherry+ progenies were individually balanced and the loxP-flanked mCherry cassettes were then removed by crossing each line to balancer expressing Cre (Bloomington Drosophila Stock Center, RRID:BDSC 1092). To detect cell-type-specific expression level, we used ey-Flp65 for ORNs and VT033006-GAL4;UAS-FLP66 for PNs.
Generation of the sparse driver and single-neuron genetic manipulations
The FRT100-Stop-FRT100 element was cloned into the 78H05-p65AD plasmid (in backbone pBPp65ADZpUw) to generate plasmid 78H05-FRT100-Stop-FRT100-p65AD as previously reported67. The plasmid was integrated into the VK27 site. To perform the sparse genetic manipulations, flies including VA1d-ORN sparse driver (31F09-GAL4DBD, 78H05-FRT100-Stop-FRT100-p65AD), hsFLP (heat shock protein promoter-driven FLP), reporter (UAS-mCD8-GFP), and knockdown or overexpression transgenes were raised at 29 °C. To induce sparse manipulation, the flies were collected at 0–6 h APF and heat shocked for 1–2 h in a water bath67 at 37 °C.
Immunostaining
Fly brain dissection, fixation and immunostaining were performed according to the published protocol68. For primary antibodies, we used rat anti-N-cadherin (1:40; DN-Ex#8, Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1000; GFP-1020, Aves Labs), rabbit anti-DsRed (1:500; 632496, Clontech), mouse anti-rat CD2 (1:200; OX-34, Bio-Rad), rabbit anti-HA (1:100, 3724S, Cell Signaling), mouse anti-HA (1:100, 2367S, Cell Signaling), rabbit anti-MYC (1:250, 2278S, Cell Signaling) and mouse anti-V5 (1:250, R960-25, Thermo Fisher Scientific). Donkey secondary antibodies conjugated to Alexa Fluor 405/488/568/647 or Cy3 (Jackson ImmunoResearch or Thermo Fisher) were used at 1:250. For the staining of conditional tag for Hbs and Fili in PNs, the routine protocol described above failed to detect MYC signal from the background, probably owing to low expression of endogenous proteins in vivo. Alexa 488 Tyramide SuperBoost kit (Thermo Fisher) was used to amplify the immunostaining signal by following the manufacture’s protocol.
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