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. 2019 Feb 6;8:e43482. doi: 10.7554/eLife.43482

Figure 7. Transient loss of Eyeless during development impairs adult fly navigation.

(A) Timing of Eyeless reduction in INP lineages. Transient inactivation of ts.Gal80 (29°C heat pulse; gray bar) results in transient EyelessRNAi during the time in which E-PG neurons are normally generated. (B,C) The manipulation in A does not alter CX neuropil morphology as seen by nc82 (neuropil) staining (EB, shown; other neuropils, data not shown). (D) Schematic of experimental apparatus for sun navigation experiments. The wing stroke amplitudes of a tethered, flying fly were monitored with an IR camera; the stroke difference determined the angular velocity of a 2.4° sun stimulus. Modified from (Giraldo et al., 2018). (E) Example flight (top panel) and summary data (bottom panel) from ts.EyelessRNAi control with no heat pulse. Top panel: left plot shows headings over 5 min flight; 0° is sun position in front of fly. Right histogram is distribution of headings in this example flight; sideways green triangle is the mean. Bottom panel: summary data, with each 5 min flight represented by radial lines. The angle of each line is the mean flight heading. The length of each line is vector strength of flight, varying from 0 (center of circle; no stimulus stabilization) to 1 (edge of circle; perfect stabilization). Each fly flew for two 5 min flights separated by a 5 min rest period. Cyan line shows mean heading, across flights with vector strength >0.2, as well as 95% confidence interval, calculated via resampling across flies. 44 flights, 22 flies total. (F) Example and summary data from ts.mcherryRNAi control. Same plotting convention as (E). 48 flights, 24 flies. (G) Example and summary data from ts.EyelessRNAi flies with heat pulse. Same plotting convention as (E,F). 50 flights, 25 flies. (H) Cumulative probability distribution of vector strengths from both control groups (black and gray) and from experimental group (red). There was no significant difference between means (ts.EyelessRNAi heat pulse, 0.34, red; ts.EyelessRNAi no heat pulse, 0.31,black;ts.mcherryRNAi heat pulse 0.30,gray; p>0.1, permutation test). (I) Cumulative probability distribution of mean absolute headings. The heading distribution for the experimental group, ts.EyelessRNAi heat pulse, was skewed significantly to frontal headings (mean 63.7°, 37 flights in 23 flies with vector strength >0.2) compared to control distributions (p<0.01, permutation test; ts.EyelessRNAi no heat pulse, mean 107.9°, 35 flights in 19 flies; ts.mcherryRNAi, mean 106.9°, 31 flights in 21 flies). Scale bars, 10µm.

Figure 7.

Figure 7—figure supplement 1. Sun navigation is impaired when E-PG neurons are silenced; stripe fixation behavior is not altered by the loss of Eyeless.

Figure 7—figure supplement 1.

(A–D) Silencing E-PG neurons impairs the flies’ capacity to maintain an arbitrary heading to a bright spot resembling the sun. (A) Flight headings to sun stimulus in empty-Split/UAS Kir 2.1 control flies. Plotting convention same as Figure 7E–G. N = 72 flights, 36 flies. (B) Flight headings to sun stimulus in ss00096 Split-Gal4 (E-PG neurons)/UAS-Kir2.1 flies. N = 50 flights, 25 flies. (C) Cumulative probability distribution of vector strength values for flights to sun stimulus. empty Split-Gal4 UAS-Kir2.1 (black line), mean = 0.43, N = 72 flights, 36 flies; ss00096 Split-Gal4/UAS-Kir2.1 (purple line), mean = 0.32, N = 50 flights, 25 flies. p=0.001, permutation test for significant difference in mean. (D) Cumulative probability distribution of mean absolute heading for flights to sun stimulus. empty Split-Gal4 UAS-Kir2.1 (black line), mean = 52.7°, N = 61 flights, 36 flies; ss00096 Split-Gal4/UAS-Kir2.1 (purple line), mean = 32.2°, N = 37 flights, 23 flies. p=0.011, permutation test for significant difference in mean. (E–H) Transient removal of Eyeless in larvae at the time of E-PG development (as in Figure 7) has no effect on stripe fixation behavior in adult flies. Plotting conventions as in Figure 7E–G. (E) Schematic of behavioral apparatus; flies controlled rotation of a dark vertical stripe with their wing strokes. (F) Control ts.EyelessRNAi no heat pulse. N = 19 flies. Flight headings are tightly clustered around 0°, where stripe is in front of fly. (G) Control ts.mcherryRNAi flies with heat pulse. N = 18 flies. (H) Experimental ts.EyelessRNAi flies with heat pulse. Heading distribution remains centered around 0°. N = 21 flies. (I) Cumulative probability distribution of vector strength values for stripe stimuli with control groups (black and gray) and experimental group (red). We observed no significant difference between means for all genotypes.