DOI: 10.5061/dryad.k0p2ngfg0
title: Confocal image stacks of GFP expression in Drosophila forelegs driven by Gal4 driver expression in foreleg motor neurons

description: Like the vertebrate spinal cord, the insect ventral nerve cord (VNC)
 mediates limb sensation and motor control. We applied automated tools for
 electron microscopy volume alignment, neuron segmentation, and synapse
 prediction toward creating a connectome of an adult female Drosophila VNC.
 To interpret a connectome, it is crucial to know its relationship with the
 rest of the body. We therefore mapped the muscle targets of leg and wing
 motor neurons in the connectome by comparing their morphology to genetic
 driver lines, dye fills, and X-ray nano-tomography of the fly leg and
 wing. The dataset made available here contains the confocal image stacks
 of the legs of each driver line. We imaged only driver lines for which we
 found an image of a single motor neuron labeled by MCFO, from the
 collection on the https://neuronbridge.janelia.org website. Thus, the
 dendritic morphology of the MNs that are visible in these images stacks
 can be found by cross referencing the alphanumeric code for the Gal4
 driver line.
description: Confocal imaging Fly prothoracic (front)
 legs were immersed in a 4% formaldehyde (PFA) PBS solution for 20 minutes,
 followed by three rinses in PBS with 0.2% Triton X-100 (PBT). The legs
 were then incubated in a PBS solution containing 1:50 phalloidin
 (Alexa-phalloidin-647, Fisher A222287) and the following reagents that
 improve tissue penetrance: 1% Triton X-100, 0.5% DMSO, 0.05 mg/ml Escin
 (Sigma-Aldrich, E1378), and 3% normal goat serum. Legs were incubated for
 one week at 4 °C with occasional rocking. After staining, legs were rinsed
 3x with PBS-Tx, 1 rinse with PBS, and were mounted onto slides in
 Vectashield. To image MNs innervating the coxal muscles in the thorax, the
 fly was fixed as above, then hemisected along the parasagital plane with a
 fine razor blade in Tissue-Tek O.C.T. compound (Sakura 4583) frozen for 10
 seconds on dry ice. Hemisected thoraces were rinsed 3x in PBT and stained
 as above. Mounted legs or thoraxes were imaged on a
 Confocal Olympus FV1000. At least one image stack of each segment of the
 leg was acquired. If GFP was expressed in a motor neuron in a particular
 segment, two image stacks of the segment were acquired. Images are
 available upon request. Image stacks were processed in FIJI (Rueden et
 al., 2017). Fly strains for genetically labeling motor
 neurons We screened a large collection of VNC neurons
 sparsely labeled with the multi-color Flp-out (MCFO) technique to identify
 GAL4 driver or split-GAL4 hemidriver lines labeling leg MNs (Meissner et
 al., 2020). The confocal images in Figures A2-A17 come from a resulting
 collection of 75 lines driving expression of GFP expression in the leg
 (Table A2). An additional 31 lines were imaged that showed no MN
 expression in the leg. For “Gen1” Janelia GAL4 lines, the genotype was
 P{&lt;GMR&gt;-GAL4}attP2/P{20XUAS-06XGFP}attP2. For VDRC Vienna
 Tile DBD hemidrivers, the genotype was
 TI{2A-p65(AD)::Zip+}VGlut[2A-p65AD]/+;
 P{y[+t7.7]w[+mC]=&lt;VT&gt;-GAL4.DBD}attP2/P{20XUAS-06XGFP}attP2. Data format Image stacks are stored as .tif files. The files are named according to &lt;driver line code&gt;_&lt;leg segment&gt;_&lt;sample number&gt;_&lt;date&gt;.tif. Stacks are typically two-color, with magenta indicating phalloidin staining of muscles, and green indicating GFP expression in the leg.
description: # Confocal image stacks of GFP expression in Drosophila forelegs driven by
 Gal4 lines labelling foreleg motor neurons
 [https://doi.org/10.5061/dryad.k0p2ngfg0](https://doi.org/10.5061/dryad.k0p2ngfg0) Like the vertebrate spinal cord, the insect ventral nerve cord (VNC) mediates limb sensation and motor control. We applied automated tools for electron microscopy volume alignment, neuron segmentation, and synapse prediction toward creating a connectome of an adult female *Drosophila* VNC. To interpret a connectome, it is crucial to know its relationship with the rest of the body. We therefore mapped the muscle targets of leg and wing motor neurons in the connectome by comparing their morphology to genetic driver lines, dye fills, and X-ray nano-tomography of the fly leg and wing. The dataset made available here contains the confocal image stacks of the legs of each driver line.  ## Description of the data and file structure Meissner et al. ([https://doi.org/10.7554/eLife.80660](https://doi.org/10.7554/eLife.80660)) described a searchable database of sparse labeling of neurons in the central nervous system of the fly. We screened VNC neurons sparsely labeled with the multi-color Flp-out (MCFO) technique to identify GAL4 driver or split-GAL4 hemidriver lines labeling leg MNs (Meissner et al., 2020). The confocal images in Figures A2-A17 come from a resulting collection of 75 lines driving expression of GFP expression in the leg (Table A2). An additional 31 lines were imaged that showed no MN expression in the leg. For “Gen1” Janelia GAL4 lines, the genotype was P{-GAL4}attP2/P{20XUAS-06XGFP}attP2. For VDRC Vienna Tile DBD hemidrivers, the genotype was TI{2A-p65(AD)::Zip+}VGlut[2A-p65AD]/+; P{y[+t7.7]w[+mC]=-GAL4.DBD}attP2/P{20XUAS-06XGFP}attP2. This repository consists of a folder for each gal4 line. The dendritic morphology of the MNs that are visible in these images stacks can be found by cross referencing the alphanumeric code for the Gal4 driver line against the MCFO collection at neuronbridge.janelia.org. Image stacks are stored as .tif files, which can be opened with ImageJ (FIJI). The files are named according to _.tif. Stacks are typically two-color, with magenta indicating phalloidin staining of muscles, and green indicating GFP expression in the leg. ## **Sharing/Access information** The analysis of the image stacks is shown in the preprint, [Tools for comprehensive reconstruction and analysis of Drosophila motor circuits](https://www.biorxiv.org/content/10.1101/2022.12.15.520299v1.full), specifically the Supplemental Appendix, the atlas of MNs in the Female Adult Nerve Cord dataset. [https://doi.org/10.1101/2022.12.15.520299](https://doi.org/10.1101/2022.12.15.520299)