Data from: Identification of markers facilitating the study of support cells in adult olfactory sensilla in Drosophila
Data files
Sep 16, 2026 version files 47.51 GB
Abstract
A major barrier to studying mature olfactory support cells in Drosophila has been the lack of validated markers to distinguish and manipulate these cells in adult flies. We systematically identify and characterize tools to differentially label specific support cells: thecogen, trichogen, and tormogen cells in the antenna. We include validation of svT2A-GAL4 as a broad marker which labels all olfactory support cells. We confirm that a commonly utilized marker, ASE5-GAL4, labels olfactory tormogen cells, which are also strongly labeled by an anti-Su(H) antibody. We demonstrate that a prospero antibody broadly and exclusively marks olfactory thecogen cells, unlike the previously reported and use marker nompA-GAL4. The pros-positive cells are also labeled by a recently developed prosT2A-GAL4 line, but surprisingly, they are distinct from those labeled by a widely used pros-GAL4 line, which instead primarily labels olfactory neurons. We also generate and validate the first reporter line for olfactory trichogen cells, atkGAL4. Additionally, we identify a reporter line to label olfactory epithelial cells, Obp19d-GAL4. Finally, we provide evidence that each tool may function similarly in the maxillary palp, a secondary olfactory organ. Our findings establish a framework for distinguishing and manipulating olfactory support cell classes in the adult fly antenna, a crucial step towards understanding their individual contributions to sensillar function.
Access this dataset on Dryad, Dataset DOI: 10.5061/dryad.msbcc2gdh
This dataset contains raw confocal microscopy image files of Drosophila antenna which are stored in Nikon's NIS-Elements (.nd2) format. A data organization spreadsheet and quantification spreadsheet are stored in Excel (.xlsx) format. These were used to support the following findings.
- We confirmed a previously used ASE5-GAL4 reporter broadly labels olfactory tormogen cells.
- Determined that nompA is restricted to a subset of thecogen cells.
- Identified a better marker, prospero, that is expressed broadly in olfactory thecogen cells.
- Developed new artichoke reporter line as the first tool to label olfactory trichogen cells.
- We report that a shaven reporter line broadly labels all olfactory support cells and an Obp19d reporter line labels olfactory epithelial cells.
- Additionally, we demonstrate that these individual tools to mark olfactory support cells may function similarly in the maxillary palp, a secondary olfactory organ.
Description of the data and file structure
File: Support_Cell_Paper_Data_Organization_for_Repository-updated.xlsx
Description: A detailed 'Support_Cell_Paper_Data_Organization_for_Repository-updated.xlsx' spreadsheet is included to identify individual .nd2 data files. Each worksheet corresponds to one set of findings from the associated article, which are summarized below.
- Description of Figure 1: ASE5-GAL4 is a broad marker of olfactory tormogen cells
- Description of Figure 2: nompA is restricted to a subset of olfactory thecogen cells
- Description of Figure 3: Prospero antibody is a marker of olfactory thecogen cells in adult flies
- Description of Figures 4: Prospero-CRIMIC-GAL4 as a tool to label olfactory thecogen cells in adult flies
- Description of Figure 5: Artichoke is a marker for olfactory trichogen cells and validation of new atkGAL4 reporter
- Description of Figure 6: Obp19d-GAL4 reporter as an epithelial cell marker
- Description of Figure 7: Expression of new olfactory support cell markers in the maxillary palp
- Description of Figure S1: Shaven broadly labels antennal support cells.
- Note: Data for Figure S1A are found within the data folder for Figure 1D-1F. Data for Figures S1B-S1C are found within the data folder for Figure 1E.
Each worksheet describes individual experiments supporting a particular finding (ex. Figure 1). Each worksheet contains individual experiments listing the antibodies and genotypes used for the antennal microscopy. It also lists the raw .nd2 confocal image files that were collected with those experimental parameters on each experimental date, using the precise name found on the .nd2 confocal image dataset.
File: Counting_spreadsheet_for_support_cells_respository_updated.xlsx
Description: Manual quantification of fluorophore co-localization from the associated publication is available in the 'Counting_spreadsheet_for_support_cells_respository_updated.xlsx' spreadsheet. Quantified .nd2 files are listed by their precise name. The channels that were counted are described. The number of overlapping versus non-overlapping cells in each section is included.
File: Repository_Submission.zip
Description: This dataset the raw confocal microscopy image files are stored in Nikon's NIS-Elements (.nd2) format. Image files show Drosophila antennal sections from flies of various genotypes immunostained with various antibodies. Each raw .nd2 file contains z-stack images for one antenna and one condition. Data is organized by main finding folders, individual experiment subfolders, and folders by imaging date. The file names and dates correspond to those in the "Support_Cell_Paper_Data_Organization_for_Repository-updated.xlsx". As described below in "File naming", names of each raw .nd2 file contain partial information on genotypes, antibodies, and their association with different fluorescent channels in the imaging file. A full description is found in the "Support_Cell_Paper_Data_Organization_for_Repository-updated.xlsx" spreadsheet.
File naming
Raw .nd2 files contain multiple fluorescent channels. More details for individual folders follows below. This section describes common names and abbreviations used for naming files.
The fluorescent signal channels imaged are as follows:
- Green: also labeled as 488, GFP, G, mcd8-GFP, mcd8GFP
- Red: also labeled as 568, RFP, anti-RFP, R, RedStinger (RS), mCherry, mCherrynls, mCherry.nls, mcd8RFP, mcd8-RFP, mCD8;;RFP, mCD8-RFP, 10xRFP
- Magenta: also labeled as far red, FR
- DAPI: also labeled as Blue
- Each image file also contains the Differential Interference Contrast (DIC) channel of the antennal tissue.
Some marker genes and antibodies used in this study may be abbreviated in file names.
Abbreviations for marker genes are as follows:
- ASE5: Autoregulatory Socket Enhancer 5 region of the Suppressor of Hairless gene
- NompA, nompA: nompA gene
- prospero, pros, pros-CRIMIC, CRIMIC-pros, CRIMIC-prospero: prospero gene
- atk, ATK: artichoke gene
- Obp19d, obp19d, 0bp19d: Odorant binding protein 19d gene
- sv, SV, sv-CRIMIC, svGal4: shaven gene
- Osi23: Osiris 23 gene
Abbreviations for antibodies are as follows:
- anti-SuH, anti-Su(H), anti-su(H), antiSu(H), m a-Su(H), Su(h): Suppressor of Hairless antibody
- anti-elav, antielav, rat anti-elav, rat a-elav, elav: elav antibody
- anti-pros, anti-prosp, antiprospero, anti-prospero, m a-pros, m anti-pros, prosp: prospero antibody
- anti-Ir8a, Ir8a: Ir8a antibody
Seven main folders contained within the 'Repository_Submission.zip' file are named corresponding to the cells they focus on and main markers.
- Figure 1. Tormogen (ASE5)
- Note: Data for Figure S1A are found within the data folder for Figure 1D-1F. Data for Figures S1B-S1C are found within the data folder for Figure 1E.
- Figure 2. Thecogen (nompA)
- Figure 3. Thecogen (anti-Pros)
- Figure 4. Thecogen (pros-GAL4)
- Figure 5. Trichogen (atk)
- Figure 6. Epithelial (Obp19d)
- Figure 7. Palps
Each main folder contained within the 'Repository_Submission.zip' contains subfolders to organize the data by its respective subfigure in the associated publication. Each subfigure subfolder contains folders of raw data named by the date in which the biological repeat was carried out.
Folders (main findings), Subfolders (individual experiments), and replicate dates are listed below.
- Figure 1. Tormogen (ASE5)
- 1D-1F and S1A
- 1-28-25
- 3-2-25
- 11-22-24
- File naming convention:
- 1333 is an internal stock number and therefore not relevant for analysis
- ASE5-mCherrynls = ASE5 marker in the Red channel
- anti-SuH-GFP, anti-Su(H)-GFP = Su(H) antibody in the Green channel
- anti-elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 1E and S1B and S1C
- 2-11-26
- 2-18-26
- 2-22-26
- File naming convention:
- 1531x1266 is an internal stock number and therefore not relevant for analysis
- sv-Red = sv marker in the Red channel
- m a-su(H)-G, m a-su(H)-488 = Su(H) antibody in the Green channel
- rat a-elav-FR = elav antibody in the Magenta channel
- n=# = independent antennal sample captured during the imaging session
- 1G-1H-1I-1J-1K
- 7-14-26
- 8-28-25
- 10-15-25
- 10-30-25
- File naming convention:
- 1498 is an internal stock number and therefore not relevant for analysis
- ASE5-mCD8;;RFP, ASE5-mCD8-RFP, ASE5-10xRFP = ASE5 marker in the Red channel
- anti-Ir8a-GFP, Ir8a-GFP = Ir8a antibody in the Green channel
- DAPI, DAPI-Blue = DAPI counterstain in the Blue channel
- a# = independent antennal sample captured during the imaging session
- 1D-1F and S1A
- Figure 2. Thecogen (nompA)
- 2C-2D
- 1-27-25
- 2-17-25
- 12-13-24
- File naming convention:
- 1213 is an internal stock number and therefore not relevant for analysis
- NompA-RS, nompA-RS, nompA-red = nompA marker in the Red channel
- anti-Ir8a green, anti-Ir8a-GFP = Ir8a antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 2C-2D
- Figure 3. Thecogen (anti-Pros)
- 3C
- 5-23-25
- 5-30-25
- 6-6-25
- File naming convention:
- 1213 is an internal stock number and therefore not relevant for analysis
- nompA-RS, nompA-redstinger = nompA marker in the Red channel
- prosp-GFP, antiprospero-GFP = prospero antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 3D
- 5-23-25
- 6-6-25
- 11-22-24
- File naming convention:
- 1335 is an internal stock number and therefore not relevant for analysis
- prospero-mcherry, pros-mCherrynls = prospero marker in the Red channel
- prosp-GFP, antiprosp-GFP, anti-pros-GFP = prospero antibody in the Green channel
- anti-elav-FR, Elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 3E
- 2-18-26
- 2-22-26
- 3-5-26
- File naming convention:
- 1531x1266 is an internal stock number and therefore not relevant for analysis
- sv-Red = sv marker in the Red channel
- m a-pros-488 = prospero antibody in the Green channel
- n=# = independent antennal sample captured during the imaging session
- 3F
- 1-22-25
- 2-20-25
- 12-12-24
- File naming convention:
- 1333 is an internal stock number and therefore not relevant for analysis
- ASE5-mCherrynls, ASE5-mCherry.nls = ASE5 marker in the Red channel
- anti-pros-GFP, anti-prospero-GFP = prospero antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 3C
- Figure 4. Thecogen (pros-GAL4)
- 4A
- 5-23-25
- 6-6-25
- 11-22-24
- File naming convention:
- 1335 is an internal stock number and therefore not relevant for analysis
- prospero-mcherry, pros-mCherrynls = prospero marker in the Red channel
- prosp-GFP, antiprosp-GFP, anti-pros-GFP = prospero antibody in the Green channel
- anti-elav-FR, Elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 4B
- 3-17-26
- 3-24-26
- 4-3-26
- File naming convention:
- 1647x1266 is an internal stock number and therefore not relevant for analysis
- sv-R = sv marker in the Red channel
- m anti-pros-G = prospero antibody in the Green channel
- rat anti-elav-FR = elav antibody in the Magenta channel
- n=# = independent antennal sample captured during the imaging session
- 4D-4F
- 1-5-25
- 1-15-26
- 7-10-26
- 10-12-25
- File naming convention:
- 78970x887, 1647x887, 1505x887 are internal stock numbers and therefore not relevant for analysis
- CRIMIC-pros-mcd8RFP, CRIMIC-prospero-mcd8RFP, pros-CRIMIC-mcD8;;RFP = prospero marker in the Red channel
- DAPI-blue = DAPI counterstain in the Blue channel
- a# = independent antennal sample captured during the imaging session
- 4A
- Figure 5. Trichogen (atk)
- 5C
- 9-6-25
- 10-17-25
- 10-30-25
- File naming convention:
- 393x1487, 1522 are internal stock numbers and therefore not relevant for analysis
- Osi23-GFP, Osi23-mcd8-GFP = Osiris 23 gene in the Green channel
- a# = independent antennal sample captured during the imaging session
- 5D
- 9-6-25
- 10-17-25
- 10-30-25
- File naming convention:
- 1237 is an internal stock number and therefore not relevant for analysis
- ATK-GFP, ATK-mcd8-GFP = artichoke marker in the Green channel
- a# = independent antennal sample captured during the imaging session
- 5E
- 5-23-25
- 6-6-25
- 6-13-25
- File naming convention:
- 1334 is an internal stock number and therefore not relevant for analysis
- atk-mcherry, ATK-mcherry = artichoke marker in the Red channel
- antielav-GFP, elav-GFP = elav antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 5F
- 1-22-25
- 3-2-25
- 12-13-24
- File naming convention:
- 1334 is an internal stock number and therefore not relevant for analysis
- atk-mCherrynls, atk-mCherry.nls = artichoke marker in the Red channel
- anti-SuH-GFP= Su(H) antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 5G
- 1-22-25
- 2-20-25
- 12-12-24
- File naming convention:
- 1334 is an internal stock number and therefore not relevant for analysis
- atk-mCherrynls, atk-mCherry.nls = artichoke marker in the Red channel
- anti-prospero, anti-pros = prospero antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 5H-5I-5J-5K-5L-5M
- 8-28-25
- 10-15-25
- 10-30-25
- File naming convention:
- 1499 is an internal stock number and therefore not relevant for analysis
- ATK-10xRFP, atk-mCD8-RFP = artichoke marker in the Red channel
- anti-Ir8a-GFP, Ir8a-GFP = Ir8a antibody in the Green channel
- DAPI/DAPI-Blue = DAPI counterstain in the Blue channel
- a# = independent antennal sample captured during the imaging session
- 5C
- Figure 6. Epithelial (Obp19d)
- 6C and F
- 1-22-25
- 3-2-25
- 7-2-25
- 12-13-24
- File naming convention:
- 1300 is an internal stock number and therefore not relevant for analysis
- Obp19d-mCherrynls, Obp19d-mCherry.nls, obp19d-mcherry = Obp19d marker in the Red channel
- anti-SuH-GFP, antiSu(H)-GFP = Su(H) antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 6D
- 3-16-26
- 4-1-26
- 4-2-26
- 7-10-26
- File naming convention:
- 1112x889 is an internal stock number and therefore not relevant for analysis
- Obp19d-mCherry, 0bp19d-mcherry, Obp19d-mCD8;;RFP = Obp19d marker in the Red channel
- a# = independent antennal sample captured during the imaging session
- 6E
- 5-23-25
- 6-6-25
- 6-13-25
- File naming convention:
- 1300 is an internal stock number and therefore not relevant for analysis
- obp19d-mcherry = Obp19d marker in the Red channel
- elav-GFP = elav antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 6G
- 1-22-25
- 2-20-25
- 7-2-25
- 12-12-24
- File naming convention:
- 1300 is an internal stock number and therefore not relevant for analysis
- Obp19d-mCherrynls, Obp19d-mCherry.nls, obp19d-mcherry = Obp19d marker in the Red channel
- anti-pros-GFP, anti-prospero-GFP, antiprospero-GFP = prospero antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 6C and F
- Figure 7. Palps
- 7A
- 1-30-26
- 2-11-26
- 12-5-25
- File naming convention:
- 1531x1266 is an internal stock number and therefore not relevant for analysis
- SV-CRIMIC-mcherry, sv-Red, RFP-svGal4 = sv marker in the Red channel
- elav-GFP, rat a-elav-G rat a-elav-488 = elav antibody in the Green channel
- a#, n=# = independent antennal sample captured during the imaging session
- 7B
- 6-6-25
- 6-26-25
- 7-11-25
- File naming convention:
- 1335 is an internal stock number and therefore not relevant for analysis
- prospero-mcherry = prospero marker in the Red channel
- antiprosp-GFP = prospero antibody in the Green channel
- elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 7C
- 3-17-26
- 3-24-26
- 4-3-26
- File naming convention:
- 1647x1266 is an internal stock number and therefore not relevant for analysis
- sv-R = sv marker in the Red channel
- m anti-pros-G = prospero antibody in the Green channel
- Rat anti-elav-FR = elav antibody in the Magenta channel
- n=# = independent antennal sample captured during the imaging session
- 7D
- 6-26-25
- 6-26-25 (2)
- 6-26-25 (3)
- File naming convention:
- 1334 is an internal stock number and therefore not relevant for analysis
- ATK-mcherry = artichoke marker in the Red channel
- Elav-GFP = elav antibody in the Green channel
- SV-GFP = shaven antibody in the Green channel
- Elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 7E
- 6-26-25
- 6-26-25 (2)
- 6-26-25 (3)
- File naming convention:
- 1333 is an internal stock number and therefore not relevant for analysis
- ASE5-mcherry = ASE5 marker in the Red channel
- Su(h)-gfp = Su(H) antibody in the Green channel
- Elav-FR = elav antibody in the Magenta channel
- a# = independent antennal sample captured during the imaging session
- 7F
- 6-6-25
- 6-26-25
- File naming convention:
- 1300 is an internal stock number and therefore not relevant for analysis
- Obp19d-mcherry, obp19d-mcherry = Obp19d marker in the Red channel
- elav-gfp, elav-GFP = elav antibody in the Green channel
- a# = independent antennal sample captured during the imaging session
- 7A
Software
Files were visualized with ImageJ/FIJI 2.18.0/1.54p software and analyzed with the included Cell Counter plugin. The software can be downloaded for free at the following link. https://imagej.net/downloads
How to open raw .nd2 files:
- Open the ImageJ/FIJI application.
- Drag and drop a raw .nd2 file to the application control panel.
- A Bio-Formats Import Options panel will open, select OK to open the image.
How to visualize multiple fluorescent channels at once:
- Navigate to 'Image --> Color --> Channels Tool'
- When the Channels panel opens, select 'Composite' from the drop down menu.
- Fluorescent channels can be selected or deselected for visualization with each check box.
How to access the Cell Counter plugin and initialize images:
- Navigate to 'Plugins --> Analyze --> Cell Counter --> Cell Counter'
- When the Cell Counter panel opens, along with the image for analysis open, select 'Initialize' to load the image into the Cell Counter.
- A separate window named 'Counter Window -' followed by the image file name will open.
Antennal immunohistochemistry
Equal numbers of male and female flies of a single genotype were aged to ~7 days post-eclosion. Their heads were encased in OCT (Tissue-Tek) in a silicone embedding mold, frozen over dry ice, and separated from their bodies. Blocks of tissue were stored at -80˚C until sectioning. 20 μm thick tissue sections were collected at -20˚C. The following steps were performed at room temperature unless otherwise specified. Tissue sections were fixed for 10 minutes in 1X PBS containing 4% paraformaldehyde. Residual fixative was removed with three 5-minute washes in 1X PBS. Tissue was permeabilized with 1X PBS containing 0.2% Triton X-100 (PBST), followed by blocking in PBST with 1% BSA. Primary antibodies were diluted into PBST with 1% BSA, and primary antibody solution was pipetted under bridged coverslips. Slides were incubated overnight at 4˚C
The following day, primary antibody solution was tapped away onto a Kimwipe, and residual was removed with three-10 minute PBST washes. Secondary antibodies were diluted 1:500 in PBST with 1% BSA and pipetted under bridged coverslips, followed by a two-hour incubation in the dark. Following incubation, excess antibody solution was removed, and slides were washed in PBST three times for 5 minutes. Slides were dried for 30 minutes in the dark prior to mounting with VectaShield Mounting Medium with DAPI (Vector Labs, #1200-10) or ProLong Diamond Mountant (Invitrogen, #P3965). Stacks of images with a 0.5 μm z-step size were collected for stained antennal sections using a Nikon AXR confocal microscope in the UConn Advanced Light Microscopy Facility. Images were analyzed using ImageJ/FIJI software. Quantitative analysis was done on ten consecutive sections from each antennal stack. All quantification is reported as the mean ± SEM. The ImageJ/FIJI cell counter tool was used to manually label cells during quantification. All manual quantification was recorded in the 'Counting_spreadsheet_for_support_cells_respository.xlsx' spreadsheet. Each experiment was repeated at least three independent days.
Imaging endogenous expression of fluorophores
A shortened protocol was used to visualize atkGAL4>UAS-mCD8::GFP, Osi23GAL4>UAS-mCD8::GFP, prosT2A-GAL4>10XUAS-mCD8::RFP, and Obp19d-GAL4>10XUAS-mCD8::RFP antennae because an antibody was not applied in these experiments. Blocks of tissue were prepared and cryosectioned in the same way as described above. Antennal sections were fixed for 10 minutes in 4% paraformaldehyde in 1X PBS and washed three times for 5 minutes with 1X PBS. Slides were then dried for 30 minutes in the dark prior to mounting. The sections were imaged with a confocal microscope using the same protocol as described above. Each experiment was repeated at least three independent days.