Fig. 1.

Ubx is necessary for normal flight maintenance in adult Drosophila. (A) Confocal images of the whole VNC of adult Drosophila showing expression of Ubx protein in specific regions of the adult VNC (T1-T3, indicate the respective thoracic segments). (B) Strategy for conditional neuronal downregulation of Ubx in the adult. Flies expressing Ubx^RNAi under temperature-sensitive Gal80ts repression, were exposed to elevated temperatures (30°C) upon hatching to allow active downregulation of Ubx. At permissive temperature (18°C) Gal80ts protein is functional and represses expression of Ubx^RNAi (‘Ubx ON’); conversely, expression of Ubx^RNAi is activated in cells at temperatures above 30°C (‘Ubx OFF’). Behavioral experiments were performed on adult males and females in Ubx ON/OFF modes. (C–E) Evaluation of flight performance on tethered Drosophila. (C) Representative flight duration is indicated by video snapshots of tethered WT, pan-neuronal (Tub-Gal80ts;Elav-Gal4, Ubx^RNAi), and dopaminergic (Tub-Gal80ts;TH-Gal4, Ubx^RNAi) Ubx^RNAi expressing flies, at 30°C. The orange arrow indicates the induction stimulus (air-puff), and WT flies are able to fly above 10 min compared to Ubx^RNAi expressing flies. (D and E) Flight duration represented as a heat map, and as averages of flight duration at week 2, for flies expressing Ubx^RNAi in different nerve cells (Ubx/elav/Vglut/TH/Cha>Ubx^RNAi) compared to controls (UAS-Ubx^RNAi) under Gal80ts repression (week 1 = 6–7 d; week 2 = 8–10 d; week 3 = 14–15 d; n > 20). (F) Cartoon illustrating the forced flight test procedure (for details, see Materials and Methods). Flies are shown as brown dots on the cylinder and membrane. (G) Average forced flight index at week 2 of flies expressing Tub-Gal80ts; Ubx^RNAi in different neuronal populations and reared at 30°C compared to controls (Tub-Gal80ts; Ubx^RNAi) (Each data point represents a group of n > 12 flies). (H) Representative raw images of GCaMP fluorescence (warmer color) in flight muscles. (I) Representative calcium signal traces in dorsal longitudinal flight muscle (Dlm) from control (Mef2^LexA,GCaMP6m^LexAOP/Tub-Gal80^ts;TH-Gal4/FLP) and experimental (Mef2^LexA,GCaMP6m^LexAOP/Tub-Gal80^ts; TH-Gal4/Ubx^RNAi) flies. (J) Normalized mean of GCaMP signals maximum during sustained flight (n = 5–10). All error bars represent SEM. Significant values in all figures based on Mann–Whitney U test or one-way ANOVA with the post hoc Tukey–Kramer test: ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001.