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.

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{<GMR>-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]=<VT>-GAL4.DBD}attP2/P{20XUAS-06XGFP}attP2.

Data format

Image stacks are stored as .tif files. The files are named according to <driver line code>_<leg segment>_<sample number>_<date>.tif. Stacks are typically two-color, with magenta indicating phalloidin staining of muscles, and green indicating GFP expression in the leg.