1. Neuroscience

A split-GAL4 driver line resource for Drosophila neuron types

  1. Geoffrey W Meissner  Is a corresponding author
  2. Allison Vannan
  3. Jennifer Jeter
  4. Kari Close
  5. Gina M DePasquale
  6. Zachary Dorman
  7. Kaitlyn Forster
  8. Jaye Anne Beringer
  9. Theresa Gibney
  10. Joanna H Hausenfluck
  11. Yisheng He
  12. Kristin Henderson
  13. Lauren Johnson
  14. Rebecca M Johnston
  15. Gudrun Ihrke
  16. Nirmala A Iyer
  17. Rachel Lazarus
  18. Kelley Lee
  19. Hsing-Hsi Li
  20. Hua-Peng Liaw
  21. Brian Melton
  22. Scott Miller
  23. Reeham Motaher
  24. Alexandra Novak
  25. Omotara Ogundeyi
  26. Alyson Petruncio
  27. Jacquelyn Price
  28. Sophia Protopapas
  29. Susana Tae
  30. Jennifer Taylor
  31. Rebecca Vorimo
  32. Brianna Yarbrough
  33. Kevin Xiankun Zeng
  34. Christopher T Zugates
  35. Heather Dionne
  36. Claire Angstadt
  37. Kelly Ashley
  38. Amanda Cavallaro
  39. Tam Dang
  40. Guillermo A Gonzalez III
  41. Karen L Hibbard
  42. Cuizhen Huang
  43. Jui-Chun Kao
  44. Todd Laverty
  45. Monti Mercer
  46. Brenda Perez
  47. Scarlett Rose Pitts
  48. Danielle Ruiz
  49. Viruthika Vallanadu
  50. Grace Zhiyu Zheng
  51. Cristian Goina
  52. Hideo Otsuna
  53. Konrad Rokicki
  54. Robert R Svirskas
  55. Han SJ Cheong
  56. Michael-John Dolan
  57. Erica Ehrhardt
  58. Kai Feng
  59. Basel EI Galfi
  60. Jens Goldammer
  61. Stephen J Huston
  62. Nan Hu
  63. Masayoshi Ito
  64. Claire McKellar
  65. Ryo Minegishi
  66. Shigehiro Namiki
  67. Aljoscha Nern
  68. Catherine E Schretter
  69. Gabriella R Sterne
  70. Lalanti Venkatasubramanian
  71. Kaiyu Wang
  72. Tanya Wolff
  73. Ming Wu
  74. Reed George
  75. Oz Malkesman
  76. Yoshinori Aso  Is a corresponding author
  77. Gwyneth M Card  Is a corresponding author
  78. Barry J Dickson  Is a corresponding author
  79. Wyatt Korff  Is a corresponding author
  80. Kei Ito  Is a corresponding author
  81. James W Truman  Is a corresponding author
  82. Marta Zlatic  Is a corresponding author
  83. Gerald M Rubin  Is a corresponding author
  84. FlyLight Project Team
  1. Janelia Research Campus, Howard Hughes Medical Institute, United States
  2. Institute of Zoology, University of Cologne, Germany
  3. Queensland Brain Institute, University of Queensland, Australia
  4. Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, United States
  5. Department of Cell & Molecular Biology, University of California, Berkeley, United States
https://doi.org/10.7554/eLife.98405.3
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Cite this article
  1. Geoffrey W Meissner
  2. Allison Vannan
  3. Jennifer Jeter
  4. Kari Close
  5. Gina M DePasquale
  6. Zachary Dorman
  7. Kaitlyn Forster
  8. Jaye Anne Beringer
  9. Theresa Gibney
  10. Joanna H Hausenfluck
  11. Yisheng He
  12. Kristin Henderson
  13. Lauren Johnson
  14. Rebecca M Johnston
  15. Gudrun Ihrke
  16. Nirmala A Iyer
  17. Rachel Lazarus
  18. Kelley Lee
  19. Hsing-Hsi Li
  20. Hua-Peng Liaw
  21. Brian Melton
  22. Scott Miller
  23. Reeham Motaher
  24. Alexandra Novak
  25. Omotara Ogundeyi
  26. Alyson Petruncio
  27. Jacquelyn Price
  28. Sophia Protopapas
  29. Susana Tae
  30. Jennifer Taylor
  31. Rebecca Vorimo
  32. Brianna Yarbrough
  33. Kevin Xiankun Zeng
  34. Christopher T Zugates
  35. Heather Dionne
  36. Claire Angstadt
  37. Kelly Ashley
  38. Amanda Cavallaro
  39. Tam Dang
  40. Guillermo A Gonzalez III
  41. Karen L Hibbard
  42. Cuizhen Huang
  43. Jui-Chun Kao
  44. Todd Laverty
  45. Monti Mercer
  46. Brenda Perez
  47. Scarlett Rose Pitts
  48. Danielle Ruiz
  49. Viruthika Vallanadu
  50. Grace Zhiyu Zheng
  51. Cristian Goina
  52. Hideo Otsuna
  53. Konrad Rokicki
  54. Robert R Svirskas
  55. Han SJ Cheong
  56. Michael-John Dolan
  57. Erica Ehrhardt
  58. Kai Feng
  59. Basel EI Galfi
  60. Jens Goldammer
  61. Stephen J Huston
  62. Nan Hu
  63. Masayoshi Ito
  64. Claire McKellar
  65. Ryo Minegishi
  66. Shigehiro Namiki
  67. Aljoscha Nern
  68. Catherine E Schretter
  69. Gabriella R Sterne
  70. Lalanti Venkatasubramanian
  71. Kaiyu Wang
  72. Tanya Wolff
  73. Ming Wu
  74. Reed George
  75. Oz Malkesman
  76. Yoshinori Aso
  77. Gwyneth M Card
  78. Barry J Dickson
  79. Wyatt Korff
  80. Kei Ito
  81. James W Truman
  82. Marta Zlatic
  83. Gerald M Rubin
  84. FlyLight Project Team
(2025)
A split-GAL4 driver line resource for Drosophila neuron types
eLife 13:RP98405.
https://doi.org/10.7554/eLife.98405.3
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4 figures, 3 tables and 3 additional files

Figures

Figure 1
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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 1—source data 1

Spreadsheet of cell-type-specific adult and larval split-GAL4 lines.

Lines are listed with available cell-type annotations. For details of confidence levels and expression in multiple cell types, refer to the original publication listed for the line. Where applicable, entries specify the appropriate DOI or in preparation manuscript to cite.

https://cdn.elifesciences.org/articles/98405/elife-98405-fig1-data1-v2.xlsx
Figure 1—source data 2

Spreadsheet of metadata for rescreened cell-type-specific adult split-GAL4 lines.

Images are listed by fly sample. Related brain and ventral nerve cord (VNC) images from the same sample have the same slide code. Each sample lists its source line, full genotype, sex, etc.

https://cdn.elifesciences.org/articles/98405/elife-98405-fig1-data2-v2.xlsx
Figure 1—source data 3

Spreadsheet of image metadata for raw data release.

Images are listed by sample as in Figure 1—source data 2. Includes a tab detailing genotypes and other information for UAS and LexAop effectors.

https://cdn.elifesciences.org/articles/98405/elife-98405-fig1-data3-v2.xlsx
Figure 2 with 1 supplement
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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
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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
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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 3—source data 1

Spreadsheet for coverage analysis.

3D histograms were calculated for the brain and ventral nerve cord (VNC) heat maps in males and females. Percent coverage over a specified threshold was calculated. The brain and VNC were further subdivided into regions with individual histograms and weighted average coverage levels.

https://cdn.elifesciences.org/articles/98405/elife-98405-fig3-data1-v2.xlsx
Figure 4
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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).

Tables

Table 1
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 central nervous system (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.

Publication DOIFirst author(s)YearCitationSplit-GAL4 linesAnatomical region/cell types for lines
10.1016/j.neuron.2013.05.024Tuthill, Nern2013Tuthill et al., 201322Lamina (optic lobe)
10.1016/j.neuron.2014.05.017Feng, Palfreyman2014Feng et al., 20141SAG ascending neurons
10.7554/eLife.04577Aso2014Aso et al., 201463Mushroom body
10.7554/eLife.16135Aso2016Aso and Rubin, 20162Mushroom body
10.7554/eLife.21022Wu, Nern2016Wu et al., 201656Lobula columnar neurons (optic lobe)
10.1016/j.neuron.2017.03.010Strother2017Strother et al., 20179T4 neurons and inputs (optic lobe)
10.1016/j.neuron.2017.05.036von Reyn2017von Reyn et al., 20171Lobula columnar neuron LC4 (optic lobe)
10.1038/nature24626Klapoetke2017Klapoetke et al., 20173LPLC2 neurons and inputs (optic lobe)
10.7554/eLife.24394Takemura2017Takemura et al., 20171CT1 neurons (optic lobe)
10.1002/cne.24512Wolff2018Wolff and Rubin, 201846Central complex
10.7554/eLife.34272Namiki2018Namiki et al., 2018137Descending neurons
10.1016/j.cub.2019.01.009Jovanic2019Jovanic et al., 20192Larval anemotaxis and adult descending neuron
10.7554/eLife.43079Dolan2019Dolan et al., 20192Lateral horn
10.1016/j.cub.2020.07.083Wang,Wang2020Wang et al., 2020a5Descending neurons DNp13
10.1016/j.neuron.2020.08.006Turner-Evans2020Turner-Evans et al., 20202Central complex
10.1038/s41467-020-19936-xFeng2020Feng et al., 202080Leg motor MDN targets
10.1038/s41586-020-2055-9Wang, Wang2020Wang et al., 2020b9Descending neurons oviDN
10.1371/journal.pone.0236495Bogovic2020Bogovic et al., 20201Brain
10.7554/eLife.50901Davis, Nern2020Davis et al., 202040Optic lobe
10.7554/eLife.57685Morimoto2020Morimoto et al., 202010Central brain targets of lobula LC6 neurons
10.7554/eLife.58942Schretter2020Schretter et al., 202012Central brain pC1d aIPg
10.1038/s41586-020-2972-7Wang1, Wang2021Wang et al., 20217Descending neurons vpoDN vpoEN
10.1101/2021.07.23.453511Mais2021Mais et al., 20211Central brain pC1e
10.7554/eLife.66039Hulse, Haberkern, Franconville, Turner-Evans2021Hulse et al., 20211Central complex
10.7554/eLife.71679Sterne2021Sterne et al., 202167Subesophageal zone
10.7554/eLife.71858Kind, Longden, Nern, Zhao2021Kind et al., 20213R7 and R8 photoreceptor targets (optic lobe)
10.1016/j.cub.2022.01.008Namiki, Ros2022Namiki et al., 20229Descending neurons flight
10.1016/j.cub.2022.06.019Baker2022Baker et al., 202228Central brain AMMC, WED, AVLP, and PVLP
10.1016/j.neuron.2022.02.013Klapoetke2022Klapoetke et al., 20222Lobula LC18 and LC25 neurons (optic lobe)
10.1101/2022.12.14.520178Zhao2022Zhao et al., 20221H2 neurons (optic lobe)
10.1038/s41586-023-06271-6Vijayan2023Vijayan et al., 20232Descending neurons oviDN
10.1101/2023.05.31.542897Ehrhardt, Whitehead2023Ehrhardt et al., 2023164Dorsal VNC
10.1101/2023.06.07.543976Cheong, Eichler, Stuerner2023Cheong et al., 202311VNC premotor
10.1101/2023.06.21.546024Isaacson2023Isaacson et al., 20236Lobula plate LPC and LLPC (optic lobe)
10.1101/2023.10.16.562634Zhao2023Zhao et al., 20232MeLp2 and LPi4b neurons (optic lobe)
10.1101/2023.11.29.569241Garner, Kind2023Garner et al., 20235Medulla to AOTU (MeTu) neurons (optic lobe)
10.25378/janelia.23726103Minegishi2023Minegishi et al., 202352Ascending neurons (see also Chen et al., 2023a)
10.1038/s41467-023-43566-8Longden2023Longden et al., 20231L1 neurons (optic lobe)
10.1016/j.cub.2024.01.015Lillvis2024Lillvis et al., 202422VNC song generation
10.1016/j.cub.2024.01.071Cheong, Boone, Bennett2024Cheong et al., 20241Ascending neurons flight
10.1038/s41586-024-07222-5Gorko2024Gorko et al., 20243Neck motor neurons
10.1101/2024.03.15.585289Schretter2024Schretter et al., 20243Aggression circuit
10.1101/2024.04.16.589741Nern2024Nern et al., 2024320Optic lobe
10.1101/2024.10.21.619448Wolff2024Wolff et al., 2024240Central complex
10.7554/eLife.90523Rubin2024Rubin and Aso, 202421Mushroom body output neurons
10.7554/eLife.94168.3Shuai2024Shuai et al., 2024168Mushroom body
Dionne et al., in prepDionne 81Accessory medulla (optic lobe) and clock
Rubin et al., in prepRubin 50Anterior optic tubercle
This paper 1285
Total 3060
Table 2
Summary of FlyLight adult fly line and image releases.

Includes descriptions of Jenett et al., 2012; Dionne et al., 2018; Tirian and Dickson, 2017; publications in Table 1; and stock distribution by Bloomington Drosophila Stock Center and Janelia Fly Facility. Section with italic text 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.

Gen1 GAL4Gen1 LexAGen1 MCFOAD/DBDIS screenSS screenSS MCFOSS polarityTotal
Lines938915005155888235,70889867823705067,562
Samples imaged9389150074,363NA36,18216,97455,70237,526231,636
20× image tiles18,891296629,780NA72,34633,92466,48461,787286,178
40× image tiles00111,563NA0000111,563
63× image tiles0022,372NA0077,03949,117148,528
Samples with 63× images008489NA0031,84917,61957,957
Stocks currently available28711781NA8358NA3013NANA16,023
Janelia & Bloomington stock shipments as of September 2024167,08229,227NA69,546NA40,067NANA305,922
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)MCFO-1; hsPESTOPT_attP3_ 3stop1_X_0036; (w, pBPhsFlp2::PEST in attP3;; pJFRC201- 10XUASFRT >STOP > FRT-myr::smGFP-HA in VK00005,pJFRC240- 10XUAS-FRT>STOP > FRT- myr::smGFP-V5-THS-10XUASFRT >STOP > FRT- myr::smGFPFLAG in su(Hw)attP1/TM3,Sb)Nern et al., 2015RRID:BDSC_64085 (Janelia stock 1117734) 
Genetic reagent (Drosophila melanogaster)MCFO-2; pBPhsFLP_PEST_ HAV5_FLAG_OLLAS_ X3_0095; (w, pBPhsFlp2::PEST in attP3;; pJFRC210- 10XUASFRT >STOP > FRT-myr::smGFP-OLLAS in attP2, pJFRC201- 10XUAS-FRT>STOP > FRT- myr::smGFP-HA in VK0005, pJFRC240-10XUAS- FRT>STOP > FRT-myr::smGFP-V5-THS10XUAS- FRT>STOP > FRT-myr::smGFPFLAG in su(Hw)attP1/TM2)Nern et al., 2015RRID:BDSC_64086 (Janelia stock 3022015) 
Genetic reagent (Drosophila melanogaster)MCFO-4; 57C10wt_attp8_ 3stop1; (w, R57C10-Flp2 in su(Hw)attP8;; pJFRC201-10XUASFRT >STOP > FRT-myr::smGFP-HA in VK00005,pJFRC240- 10XUAS- FRT>STOP > FRT-myr::smGFP-V5-THS-10XUASFRT >STOP > FRT-myr::smGFP-FLAG in su(Hw)attP1)Nern et al., 2015RRID:BDSC_64088 (Janelia stock 1116898) 
Genetic reagent (Drosophila melanogaster)MCFO-5; 57C10PEST_attp8_ 3stop1; (w, R57C10- Flp2::PEST in su(Hw)attP8;; pJFRC201- 10XUAS- FRT>STOP > FRT-myr::smGFPHA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRTmyr::smGFP- V5-THS-10XUAS-FRT>STOP > FRTmyr::smGFP- FLAG in su(Hw)attP1/TM2)Nern et al., 2015RRID:BDSC_64089 (Janelia stock 1116876) 
Genetic reagent (Drosophila melanogaster)MCFO-6; 57C10L_attp8_ 4stop1; (w, R57C10-FlpL in su(Hw)attp8;; pJFRC210-10XUASFRT >STOP > FRT- myr::smGFP-OLLAS in attP2, pJFRC201- 10XUAS- FRT>STOP > FRT-myr::smGFP-HA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRT-myr::smGFP- V5-THS10XUAS-FRT>STOP > FRT-myr::smGFPFLAG in su(Hw)attP1/TM2)Nern et al., 2015RRID:BDSC_64090 (Janelia stock 1116894) 
Genetic reagent (Drosophila melanogaster)MCFO-7; 57C10PEST_attp18_ 4stop1; (w, R57C10- Flp2::PEST in attp18;; pJFRC210-10XUASFRT >STOP > FRT-myr::smGFP-OLLAS in attP2, pJFRC201- 10XUAS-FRT>STOP > FRT-myr::smGFPHA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRTmyr::smGFP-V5-THS-10XUAS-FRT>STOP > FRTmyr::smGFP-FLAG in su(Hw)attP1/TM2)Nern et al., 2015RRID:BDSC_64091 (Janelia stock 1116875) 
Genetic reagent (Drosophila melanogaster)MCFO-3 derivative; 57C10L_brp_SNAP_ MCFO_X23_0117; (w, R57C10-FlpL in su(Hw)attP8; brp::Snap / CyO; pJFRC201-10XUAS-FRT>STOP > FRT-myr::smGFPHA in VK00005,pJFRC240-10XUAS- FRT>STOP > FRTmyr::smGFP-V5-THS-10XUAS- FRT>STOP > FRTmyr::smGFP-FLAG in su(Hw)attP1/TM6B)Nern et al., 2015; Kohl et al., 2014RRID:BDSC_64087 (Janelia stock 3023700) 
Genetic reagent (Drosophila melanogaster)57C10PEST_brp_SNAP_ MCFO_X23_0099; (w, R57C10- Flp2::PEST in attP18; brp::Snap / CyO; pJFRC201-10XUASFRT >STOP > FRT-myr::smGFP- HA in VK00005,pJFRC240- 10XUAS-FRT>STOP > FRT-myr::smGFP-V5-THS-10XUASFRT >STOP > FRT- myr::smGFP-FLAG in su(Hw)attP1/TM6B)Nern et al., 2015(Janelia stock 3023701) 
Genetic reagent (Drosophila melanogaster)MCFO-1 derivative; pBPhsFlp2_PEST_ brp_SNAP_ MCFO_0128; (w, pBPhsFlp2::PEST in attP3; brp::Snap / CyO; pJFRC201- 10XUAS-FRT>STOP > FRT-myr::smGFPHA in VK00005,pJFRC240-10XUAS- FRT>STOP > FRTmyr::smGFP-V5-THS-10XUAS- FRT>STOP > FRTmyr::smGFP-FLAG in su(Hw)attP1/TM6B)Nern et al., 2015; Kohl et al., 2014RRID:BDSC_64085 (Janelia stock 3023951) 
Genetic reagent (Drosophila melanogaster)pJFRC2-10XUAS-IVS-mCD8::GFPPfeiffer et al., 2010RRID:BDSC_32185 (Janelia stock 1115125) 
Genetic reagent (Drosophila melanogaster)UAS_Chrimson_Venus_X_0070; (20XUAS-CsChrimson-mVenus trafficked in attP18)Klapoetke et al., 2014RRID:BDSC_55134 (Janelia stock 1150416) 
Genetic reagent (Drosophila melanogaster)UAS_CsChrimson_Venus_X_0107; (UAS-Syt-HA, 20XUAS-CsChrimson-mVenus trafficked in attP18)Klapoetke et al., 2014; Robinson et al., 2002(Janelia stock 3028408) 
Genetic reagent (Drosophila melanogaster)hsPESTOPT_attP3_3stop1_X_0036; (MCFO-1; (w, pBPhsFlp2::PEST in attP3;; pJFRC201- 10XUASFRT >STOP > FRT-myr::smGFP-HA in VK00005,pJFRC240- 10XUAS-FRT>STOP > FRT- myr::smGFP-V5-THS-10XUASFRT >STOP > FRT- myr::smGFPFLAG in su(Hw)attP1/TM3,Sb))Nern et al., 2015RRID:BDSC_64085 (Janelia stock 1117734) 
Genetic reagent (Drosophila melanogaster)57C10L_brp_SNAP_MCFO_X23_0117; (MCFO-3 derivative; (w, R57C10-FlpL in su(Hw)attP8; brp::Snap / CyO; pJFRC201-10XUAS-FRT>STOP > FRT-myr::smGFPHA in VK00005,pJFRC240-10XUAS- FRT>STOP > FRTmyr::smGFP-V5-THS-10XUAS- FRT>STOP > FRTmyr::smGFP-FLAG in su(Hw)attP1/TM6B))Kohl et al., 2014; Nern et al., 2015(Janelia stock 3023700) 
Genetic reagent (Drosophila melanogaster)57C10PEST_attp8_3stop1; (MCFO-5; (w, R57C10- Flp2::PEST in su(Hw)attP8;; pJFRC201- 10XUAS- FRT>STOP > FRT-myr::smGFPHA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRTmyr::smGFP- V5-THS-10XUAS-FRT>STOP > FRTmyr::smGFP- FLAG in su(Hw)attP1/TM2))Nern et al., 2015RRID:BDSC_64089 (Janelia stock 1116876) 
Genetic reagent (Drosophila melanogaster)57C10L_attp8_4stop1; (MCFO-6; (w, R57C10-FlpL in su(Hw)attp8;; pJFRC210-10XUASFRT >STOP > FRT- myr::smGFP-OLLAS in attP2, pJFRC201- 10XUAS- FRT>STOP > FRT-myr::smGFP-HA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRT-myr::smGFP- V5-THS10XUAS-FRT>STOP > FRT-myr::smGFPFLAG in su(Hw)attP1/TM2))Nern et al., 2015RRID:BDSC_64090 (Janelia stock 1116894) 
Genetic reagent (Drosophila melanogaster)57C10PEST_attp18_4stop1; (MCFO-7; (w, R57C10- Flp2::PEST in attp18;; pJFRC210-10XUASFRT >STOP > FRT-myr::smGFP-OLLAS in attP2, pJFRC201- 10XUAS-FRT>STOP > FRT-myr::smGFPHA in VK00005, pJFRC240-10XUAS-FRT>STOP > FRTmyr::smGFP-V5-THS-10XUAS-FRT>STOP > FRTmyr::smGFP-FLAG in su(Hw)attP1/TM2))Nern et al., 2015RRID:BDSC_64091 (Janelia stock 1116875) 
Genetic reagent (Drosophila melanogaster)LexAop_IVS-myr_3_0008; (pJFRC19-13XLexAop2-IVS-myr::GFP in attP2)Pfeiffer et al., 2012RRID:BDSC_32209 (Janelia stock 1116736) 
Genetic reagent (Drosophila melanogaster)UAS_IVS-mCD8_3_0007; (pJFRC2-10XUAS-IVS-mCD8::GFP)Pfeiffer et al., 2010RRID:BDSC_32185 (Janelia stock 1115125) 
Genetic reagent (Drosophila melanogaster)HAV5_X_0083; pJFRC200-10XUAS-IVS-myr::smGFP-HA (attP18), pJFRC216-13XLexAop2-IVS-myr::smGFP-V5 (su(Hw)attP8; Dr e /TM6B)Nern et al., 2015(Janelia stock 3020172) 
Genetic reagent (Drosophila melanogaster)IVS-myr-FLAG_Syt-HA_3_0055; (w;;5XUAS-IVS-myr::smFLAG in VK00005, pJFRC51-3XUAS-IVS-Syt::smHA in su(Hw)attP1)Aso et al., 2014(Janelia stock 3001064) 
Genetic reagent (Drosophila melanogaster)UAS_IVS_myr_3_0009; (pJFRC12-10XUAS-IVS-myr::GFP in attP2)Pfeiffer et al., 2010(Janelia stock 1115116) 
Genetic reagent (Drosophila melanogaster)TLN-V5_myr-FLAG_Syt-HA_23_0037; (w; 3XpJFRC-TLN-smV5 in su(Hw)attP5; 5XUAS-IVS-myr::smFLAG in VK00005_pJFRC51-3XUAS-IVS-Syt::smHA in su(Hw)attP1/CyO::TM6b)Nern et al., 2015(Janelia stock 2600002) 
Genetic reagent (Drosophila melanogaster)TLN-V5_myr-FLAG_Syt-HA_23_0038; (w; 3XpJFRC-TLN-smV5 in su(Hw)attP5; 5XUAS-IVS-myr::smFLAG in VK00005_pJFRC51-3XUAS-IVS-Syt::smHA in su(Hw)attP2/CyO::TM6b)Nern et al., 2015(Janelia stock 2600003) 
Genetic reagent (Drosophila melanogaster)UAS_IVS_mCD8_3_0045; (pJFRC2-10XUAS-IVS-mCD8::GFP in VK00005)Pfeiffer et al., 2010(Janelia stock 1115339) 
Genetic reagent (Drosophila melanogaster)IVS_myr_GFP_X_0072; (pJFRC200-10XUAS-IVS-myr::smGFP-HA in attP18)Nern et al., 2015RRID:BDSC_62145 (Janelia stock 1116624) 
Genetic reagent (Drosophila melanogaster)UAS_IVS_mCD8_3_0073; (pJFRC7-20XUAS-IVS-mCD8::GFP in attP2)Pfeiffer et al., 2010RRID:BDSC_32194 (Janelia stock 1115387) 
Genetic reagent (Drosophila melanogaster)LexAop2_Chrimson_Venus_X_0082; (13XLexAop2-CsChrimson-mVenus trafficked in attP18)Klapoetke et al., 2014(Janelia stock 1150410) 
Genetic reagent (Drosophila melanogaster)57C10L_suHwattP8_HAV5_FLAG_0098; (R57C10-FlpL in su(Hw)attP8;; pJFRC201-10XUAS-FRT>STOP > FRT-myr::smGFP-HA in VK0005, pJFRC240-10XUAS-FRT>STOP > FRT-myr::smGFP-V5-THS-10XUAS-FRT>STOP > FRT-myr::smGFP-FLAG in su(Hw)attP1/TM2)Nern et al., 2015RRID:BDSC_64087 (Janelia stock 3022016) 
AntibodyAnti-Brp mouse monoclonal nc82Developmental Studies Hybridoma Bank (DSHB)RRID:AB_23148661:30
AntibodyAnti-HA rabbit monoclonal C29F4Cell Signaling Technologies: 3724SRRID:AB_15495851:300
AntibodyAnti-FLAG rat monoclonal DYKDDDDK Epitope Tag AntibodyNovus Biologicals: NBP1-06712RRID:AB_16259811:200
AntibodyDyLight 550 conjugated anti-V5 mouse monoclonalAbD Serotec: MCA1360D550GARRID:AB_26875761:500
AntibodyAnti-rat IgG (H&L) Goat Polyclonal Antibody ATTO 647N ConjugatedRockland: 612-156-120RRID:AB_108933861:300
AntibodyAlexa Fluor 594 AffiniPure Donkey Polyclonal Anti-Rabbit IgG (H+L)Jackson ImmunoResearch Labs: 711-585-152RRID:AB_23406211:500
AntibodyAnti-Green Fluorescent Protein (GFP) Rabbit Polyclonal Antibody, UnconjugatedThermo Fisher Scientific: A-11122RRID:AB_2215691:1000
AntibodyGoat Polyclonal anti-Rabbit IgG (H+L) Highly Cross- Adsorbed Antibody, Alexa Fluor 488Thermo Fisher Scientific: A-11034RRID:AB_25762171:800
AntibodyGoat Polyclonal anti-Mouse IgG (H+L) Highly Cross- Adsorbed Antibody, Alexa Fluor 568Thermo Fisher Scientific: A-11031RRID:AB_1446961:800
AntibodyAnti-HA High Affinity; Rat monoclonal antibody (clone 3F10)Roche: 11867423001RRID:AB_3909181:100
AntibodyCy2-AffiniPure Goat Anti-Mouse IgG (H+L) (min X Hu,Bov,Hrs,Rb,Rat Sr Prot)Jackson ImmunoResearch Labs: 115-225-166RRID:AB_23387461:600
AntibodyCy3-AffiniPure Goat Anti-Rabbit IgG (H+L) (min X Hu,Ms,Rat Sr Prot)Jackson ImmunoResearch Labs: 111-165-144RRID:AB_23380061:1000
Software, algorithmJanelia WorkstationRokicki et al., 2025 
Software, algorithmNeuronBridge codebaseClements et al., 2024; Clements et al., 2021 
Software, algorithmFijiSchindelin et al., 2012; https://fiji.scRRID:SCR_0022852 
Software, algorithmAffinity Designerhttps://affinity.serif.com/designer/RRID:SCR_016952 

Additional files

Supplementary file 1

Images of cell-type-specific adult split-GAL4 lines.

Images are averaged color depth MIPs from rescreened and raw collection SS screen and polarity neuron channel images (Otsuna et al., 2018). Images were inverted, overlayed on a 2D outline of JRC2018, and composited. Depth color scale for inverted color depth MIPs is on first page, running from yellow on the anterior brain (or ventral VNC) to blue on the posterior brain (or dorsal VNC).

https://cdn.elifesciences.org/articles/98405/elife-98405-supp1-v2.pdf
Supplementary file 2

Guide to FlyLight data.

A guide to interpreting the images at https://gen1mcfo.janelia.org and https://splitgal4.janelia.org further describes data organization, labeling, and imaging methods. Images at https://www.janelia.org/gal4-gen1 are processed as described in Jenett et al., 2012.

https://cdn.elifesciences.org/articles/98405/elife-98405-supp2-v2.pdf
MDAR checklist
https://cdn.elifesciences.org/articles/98405/elife-98405-mdarchecklist1-v2.docx

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  1. Geoffrey W Meissner
  2. Allison Vannan
  3. Jennifer Jeter
  4. Kari Close
  5. Gina M DePasquale
  6. Zachary Dorman
  7. Kaitlyn Forster
  8. Jaye Anne Beringer
  9. Theresa Gibney
  10. Joanna H Hausenfluck
  11. Yisheng He
  12. Kristin Henderson
  13. Lauren Johnson
  14. Rebecca M Johnston
  15. Gudrun Ihrke
  16. Nirmala A Iyer
  17. Rachel Lazarus
  18. Kelley Lee
  19. Hsing-Hsi Li
  20. Hua-Peng Liaw
  21. Brian Melton
  22. Scott Miller
  23. Reeham Motaher
  24. Alexandra Novak
  25. Omotara Ogundeyi
  26. Alyson Petruncio
  27. Jacquelyn Price
  28. Sophia Protopapas
  29. Susana Tae
  30. Jennifer Taylor
  31. Rebecca Vorimo
  32. Brianna Yarbrough
  33. Kevin Xiankun Zeng
  34. Christopher T Zugates
  35. Heather Dionne
  36. Claire Angstadt
  37. Kelly Ashley
  38. Amanda Cavallaro
  39. Tam Dang
  40. Guillermo A Gonzalez III
  41. Karen L Hibbard
  42. Cuizhen Huang
  43. Jui-Chun Kao
  44. Todd Laverty
  45. Monti Mercer
  46. Brenda Perez
  47. Scarlett Rose Pitts
  48. Danielle Ruiz
  49. Viruthika Vallanadu
  50. Grace Zhiyu Zheng
  51. Cristian Goina
  52. Hideo Otsuna
  53. Konrad Rokicki
  54. Robert R Svirskas
  55. Han SJ Cheong
  56. Michael-John Dolan
  57. Erica Ehrhardt
  58. Kai Feng
  59. Basel EI Galfi
  60. Jens Goldammer
  61. Stephen J Huston
  62. Nan Hu
  63. Masayoshi Ito
  64. Claire McKellar
  65. Ryo Minegishi
  66. Shigehiro Namiki
  67. Aljoscha Nern
  68. Catherine E Schretter
  69. Gabriella R Sterne
  70. Lalanti Venkatasubramanian
  71. Kaiyu Wang
  72. Tanya Wolff
  73. Ming Wu
  74. Reed George
  75. Oz Malkesman
  76. Yoshinori Aso
  77. Gwyneth M Card
  78. Barry J Dickson
  79. Wyatt Korff
  80. Kei Ito
  81. James W Truman
  82. Marta Zlatic
  83. Gerald M Rubin
  84. FlyLight Project Team
(2025)
A split-GAL4 driver line resource for Drosophila neuron types
eLife 13:RP98405.
https://doi.org/10.7554/eLife.98405.3