Fig. 5: Rdl expressed in insulin-producing cells is involved in WAKE deficiency-dependent male-male courtship behaviour.

a1 The blue and yellow circles represent the patterns of ilp2-LexA and rdl^2-1-Gal4 expression, respectively. The overlapping cells that express 4 × SNAPs are limited to the regions where rdl^2-1-Gal4 drives UAS-Bxb1 expression, as the transcriptional stop cassette is removed to allow LexA-induced expression, which represents the SNAP proteins that can be labelled specifically with molecular probes. a2 Green indicates a pattern of expression for chemical labelling with 4 × SNAPs, zoomed in on the cell bodies of IPCs, resulting from the intersection of ilp2-LexA and the rdl^2-1-Gal4 driver (n = 6 for each). b Self-interaction of WAKE-RG::HA and Rdl was visualized in IPCs using an in situ proximity ligation assay (PLA). Comparison of representative images showing staining with anti-HA (b1) or anti-Rdl (b2) antibody as negative controls, respectively (n = 6 for each). Strong PLA signals for anti-HA and anti-Rdl (green in b3). Scale bars, 20 µm. c There was a significant difference between the Courtship index of untreated controls and ilp2-GeneSwitch tester males exhibiting rdl dsRNA expression due to treatment with RU486. n = 18 for test group no. 1–6, p < 0.0001, Kruskal–Wallis test. ^****p < 0.0001, post hoc Dunn’s multiple comparisons test. Downregulation of WAKE in IPCs and simultaneous overexpression of Rdl significantly suppressed male–male courtship behavioural activity. n = 24, 24, 25, 22 (from left to right) for test group no. 7–10. ^*p < 0.05, and ^**p < 0.01, two-tailed Mann–Whitney U-test. The expression of Rdl alone in IPCs did not affect on male-female courtship behaviour. n = 18, 18, 25 (from left to right) for test group no. 11–13. p > 0.05 (n.s.), one-way ANOVA using F-test, F(2, 58) = 0.2656, p = 0.7677. Scatterplots include ± SEM for all data points. Source data are provided as a Source Data file.