Figure 10. Reaching behavior resulting from activation of LC10 subtypes.

(A) Representative video images of a fly exhibiting reaching behavior in the single-fly assay. Time stamps indicate milliseconds (ms) after the start of reaching. (B) Comparison of lobula layer patterns and penetrance of reaching upon optogenetic activation for 15 LC10 split-GAL4 lines. The images show reconstructed views of CsChrimson expression in the lobula (generated using Vaa3D and manually aligned using the anti-Brp reference marker). Approximate layer positions are indicated on the left. Scale bar represents 10 µm. CsChrimson expression using two additional LC10 driver lines (SS00941 and SS00942) resulted in unexpected uncoordinated behaviors in response to optogenetic activation that precluded analyses of reaching behavior. These lines, which are related as they only differ from each other in the insertion site of the AD hemidriver (see Supplementary file 1B), were therefore excluded from further analyses. (C) Single cell labeling reveals subtype expression patterns of LC10 driver lines. Overall lobula expression of an LC10 split-GAL4 line (SS02664) (top panel; displayed as in B) and examples of MCFO labeled single cells from this line (bottom panels) are shown. LC10 subtypes (indicated below the images) of single cells were assigned based on layer pattern, arbor size and shape as follows: LC10c cells mainly arborize in layer Lo5B with some processes in the adjacent layers. LC10a cells also have arbors in Lo5B, but differ from LC10c by having additional processes in the more distal layers Lo2, Lo3 and Lo4. LC10b and LC10d differ from LC10a and LC10c by having major arbors in Lo6 and only few processes in Lo5B. Their distal arbors reach Lo4. LC10b cells are wider than LC10d cells and show many small varicosities (presumably presynaptic sites; compare Figure 5—figure supplement 2E) in Lo6. LC10 neurons also differ in their axonal paths in the AOTu: LC10a and LC10d axons run both dorsally and ventrally, and LC10b and LC10c only ventrally (Figure 3G; also see Figure 10—figure supplement 1). Scale bars represent 10 µm. (D) Reaching penetrance observed upon activation of the LC10 split-GAL4 drivers and various control lines in the single-fly assay. Split-GAL4 drivers are grouped by expression patterns in the LC10 subtypes: either subtype a and/or d, or neither a nor d. Driver lines were included in the ‘a and/or d’ category if two or more cells of these types were identified in MCFO experiments (see Figure 10—figure supplement 2). Colors representing various controls and split-GAL4 driver lines are the same as those in Figure 9.

DOI: http://dx.doi.org/10.7554/eLife.21022.026

Figure 10—figure supplement 1. Expression patterns of LC10 split-GAL4 driver lines.

Brain (A) and VNC (B) expression patterns using 20xUAS-CsChrimson-mVenus in attP18 visualized with an anti-GFP antibody (green) and a neuropil marker (anti-Brp, magenta) are shown. While some driver lines also had expression in a few non-LC10 cells, none of these additional neurons was consistently present in lines with the reaching behavior. Imaging parameters and brightness or contrast adjustments were identical for each brain/VNC pair but not across all images. OL0020B and OL0023B images are the same as those shown in Figure 2 and Figure 2—figure supplement 1. Scale bar represents 50 µm. Original confocal stacks are available from www.janelia.org/split-GAL4.

DOI: http://dx.doi.org/10.7554/eLife.21022.027

Figure 10—figure supplement 2. Stochastic single cell labeling reveals LC10 subtype expression patterns of the LC10 split-GAL4 driver lines.

(A) Examples (10 per driver line) of MCFO-labeled LC10 cells from five different lines. Subtype classification is indicated for each cell (white lowercase letters). Anti-Brp pattern (used to facilitate comparison of layer positions across specimens) is shown in grey; views were generated using Vaa3D. Layer patterns of these projection images were manually aligned using the anti-Brp marker. To do so, the scale of some images was slightly adjusted to compensate for the non-uniform depth of the lobula. Approximate layer positions are indicated in the bottom right panel. Scale bar represents 10 µm. (B) Summary of LC10 MCFO results. For each driver line, the number of MCFO-labeled LC10 cells of each subtype was determined for the indicated number of fly brain samples. Some LC10 cells that were located at an edge of the lobula neuropil or overlapped with other labeled cells and thus could not be identified were excluded from the counts. Total cells refer to the number of all LC10 cells that were assigned a subtype. Subtypes that were only seen as single labeled cell are in parentheses in the summary.

DOI: http://dx.doi.org/10.7554/eLife.21022.028