Publications reporting split-GAL4 lines from the adult cell-type-specific collection. Publications are listed by year. The number of lines from the collection in each publication is listed, along with the CNS regions and/or cell types most commonly labeled. Many of these publications describe additional lines that were not included in the collection described here.

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 & 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 & 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 Fig 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 Supplemental file 1 for more line information and Figure 1 Supplemental file 2 for a movie of all cell-type-specific lines.

Examples of line quality levels.

The 3060 cell type lines were scored for expression.

(A) Quality level 1 (1725 lines): Split-GAL4 line OL0015B (Wu, Nern, et al., 2016) is specifically and strongly expressed in a single cell type. Occasional weak expression may be seen in other lines.

(B) Quality level 2 (1292 lines): Split-GAL4 line SS59643 (Wolff, et al., in prep) has expression in two unrelated cell types that are easily distinguished anatomically. Other lines may have more than two types.

(C) Quality level 3 (45 lines): Split-GAL4 line SS45635 (Minegishi, et al., 2023) has specific expression but weak or variable labeling efficiency.

(D) Quality level 4: 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.

Spatial distribution of cell type lines.

(A and B) 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 in the (A) female and (B) male CNS.

(C) Female image stack minus male, maximum intensity projected.

(D) Male image stack minus female, maximum intensity projected.

All images were scaled uniformly to a maximum brightness equal to 300 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 491 lines per pixel, for the purpose of better visualizing the rest of the CNS. Scale bars, 50 µm.

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).

(C) Summary of FlyLight adult fly line and image releases. Includes descriptions of Jenett et al., 2012; Dionne et al., 2018; Tirian & Dickson, 2017; publications in Table 1; and stock distribution by Bloomington Drosophila Stock Center and Janelia Fly Facility. Section in green is specific to publications from Table 1 and this publication. Image counts are unique between categories, whereas line counts overlap extensively between categories. “Image tiles” are considered as unique 3D regions, with each MCFO tile captured using two LSM image stacks. Stock shipments count each shipment of each stock separately. Raw and processed images are available at https://www.janelia.org/gal4-gen1, https://gen1mcfo.janelia.org, https://splitgal4.janelia.org, and many can be searched based on anatomy at https://neuronbridge.janelia.org.