A split-GAL4 driver line resource for Drosophila neuron types

Abstract

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system (CNS) have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila CNS and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell-type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

Keywords: D. melanogaster; GAL4; central nervous system; confocal microscopy; driver line; neuroscience; split-GAL4; targeting.

Figure 1.. Example cell-type-specific lines.

(A) Split-GAL4 line SS52577 is expressed in P-FNv neurons arborizing in the protocerebral bridge, fan-shaped body, and nodulus (Wolff and Rubin, 2018). (B) Split-GAL4 line MB043C is expressed primarily in PAM-α1 dopaminergic neurons that mediate reinforcement signals of nutritional value to induce stable olfactory memory for driving wind-directed locomotion and higher-order learning (Aso et al., 2014; Ichinose et al., 2015; Aso and Rubin, 2016; Aso et al., 2023; Yamada et al., 2023). (C) Split-GAL4 line SS40265 is expressed in members of the 8B(t1) cluster of cholinergic neurons that connect the lower tectulum neuropil of the prothorax with the gnathal neuropil and the ventral most border of the vest neuropil of the brain ventral complex. (D) Split-GAL4 line SS60203 is expressed in ascending neurons likely innervating the wing neuropil. (E) Split-GAL4 line SS47938 is expressed in LBL40, mediating backwards walking (Feng et al., 2020; same sample used in Figure 5b, CC-BY license). (F) Split-GAL4 line SS36564 is expressed in female-specific aIPg neurons (F1) and not observed in males (F2; Schretter et al., 2020). (G) MCFO of split-GAL4 line SS72207 with specific expression in DNg34, a cell type described in Namiki et al., 2018. Scale bars, 50 µm. See Figure 1—source data 1 for more line information and Supplementary file 1 for images of all cell-type-specific lines.

Figure 2.. Examples of line quality levels.

The 3060 cell-type lines were scored for expression. (A) Quality level 1 (1767 lines): Split-GAL4 line OL0015B (Wu et al., 2016) is specifically and strongly expressed in a single cell type. Occasional weak expression may be seen in other cells. (B) Quality level 2 (1232 lines): Split-GAL4 line SS59643 (Wolff et al., 2024) has expression in two cell types. Occasional weak expression may be seen in other cells. (C) Quality level 3 (26 lines): Split-GAL4 line SS59643 (Wolff et al., 2024) has expression in three or more cell types. (D) Quality level 4 (34 lines): Split-GAL4 line SS61022 has specific expression but weak or variable labeling efficiency. See Figure 2—figure supplement 1 for examples of variable expression. (E) Quality level 5: IS36417 is an Initial Split combination not selected for stabilization. Groups of neurons are visible, but the cell type of interest was not labeled with sufficient specificity for further work. Such lines were only included in the raw image collection. Scale bars, 50 µm.

Figure 2—figure supplement 1.. Example expression variability in Quality level 4 lines.

Five samples from split-GAL4 line SS61022 show variable expression. (A–C) Male flies. (D, E) Female flies. Scale bars, 50 µm.

Figure 3.. Spatial distribution of cell-type lines.

(A–D) Images of one male and one female sample from 3029 cell-type rescreening lines were aligned to JRC2018 Unisex (Bogovic et al., 2020), segmented from background (see Methods), binarized, overlaid, and maximum intensity projected, such that brightness indicates the number of lines with expression. All images were scaled uniformly to a maximum brightness equal to 206 lines on Fiji’s ‘royal’ LUT (scale inset in D). This saturated a small portion of the male antennal mechanosensory and motor center (AMMC) that reached a peak value of 260 lines per voxel, for the purpose of better visualizing the rest of the central nervous system (CNS). (A) Female CNS. (B) Male CNS. (C) Female image stack minus male, then maximum intensity projected. (D) Male image stack minus female, then maximum intensity projected. (E, F) Split-GAL4 line SS56987 (Schretter et al., 2020) is sex-specifically expressed in pC1d neurons that largely lie within the region of the female central brain highlighted in (C). Male and female images have different brightness scales. (G, H) Split-GAL4 line SS35230 (Shuai et al., 2024) is sex-specifically expressed in abdominal ganglion neurons that largely lie within the region of the female ventral nerve cord (VNC) highlighted in (C). Male and female images have different brightness scales. All scale bars, 50 µm.

Figure 4.. Split-GAL4 workflow and FlyLight data release statistics.

(A) Typical workflow of predicting and characterizing split-GAL4 combinations. (B) Example with split-GAL4 line SS23880 (Garner et al., 2023).