Isolating neurons in the ventral nerve cord (VNC).

(A) The fly central nervous system (gray) with the ventral nerve cord (VNC) highlighted in black. Illustrations of wing motoneuron and haltere sensory afferent shown in blue and purple, respectively. (B) Top-down (dorsal) view of VNC showing example neuron types: wing motoneuron (blue), descending neuron (black), and haltere-to-wing neuropil interneuron (orange). Boundaries between the pro-, meso-, and metathoracic neuromeres—i.e. T1, T2, and T3—are also shown (gray dotted lines and labels). (C) Schematic of VNC neuropils. Abbreviations used: T1 (prothoracic segment), T2 (mesothoracic segment), T3 (metathoracic segment), VAC (ventral association center), mVAC (medial ventral association center), and AMNp (accessory mesothoracic neuropil). (D) Example usage of the split-GAL4 technique for narrowing driver line expression profile (white) in the VNC (orange). R59G07 (D1) and R50G08 (D2) are used to drive half of the GAL4 transcription factor in SS54506 (D3), resulting in a sparse expression pattern. (E) Multiple interneurons segmented from the expression pattern of SS54506 using multicolor flip-out (MCFO).

Morphology of DLM power muscle motoneurons.

(A1) Color MIP of full expression pattern of a split line targeting DLMns, SS44039, crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A2) Segmented images of DLM wing motoneurons in upright VNCs. VNCs were aligned to the JRC 2018 Unisex template. (A3) Transverse views of the segmented neurons shown in A2. (A4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A5) Segmented muscle images. (B-F) Multicolor flipout (MCFO) was used to separate the power motoneurons, isolating individual cells where possible. Males were used for all motoneuron images.

Morphology of DVM power muscle motoneurons.

(A1) Color MIP of full expression pattern of a split line targeting DVMns, SS31950, crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A2) Segmented images of DVM wing motoneurons in upright VNCs. VNCs were aligned to the JFC 2018 Unisex template. (A3) Transverse views of the segmented neurons shown in A2. (A4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A5) Segmented muscle images. (B-H) Multicolor flipout (MCFO) was used to separate the power motoneurons, isolating individual cells where possible. Males were used for all motoneuron images.

Morphology of tergopleural wing steering muscle motoneurons targeted by our sparse split lines.

(A-C1) Color MIPs of full expression patterns of the split lines (respectively SS51528, SS41052, SS47120), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-C2) Segmented images of tergopleural wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-C3) Transverse views of the segmented neurons shown in A-C2. (A-C4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-C5) Segmented muscle images. (D-F) segmented images of steering motoneurons including side views. Males were used for all motoneuron images except tpN, as our tpN line lacked expression in males.

Morphology of other indirect wing steering muscle motoneurons targeted by our sparse split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS47204, SS47125), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Segmented images of other indirect wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-B3) Transverse views of the segmented neurons shown in A-B2. (A-B4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B5) Segmented muscle images. (C-D) segmented images of steering motoneurons including side views. Males were used for all motoneuron images.

Morphology of basalar wing steering muscle motoneurons targeted by our sparse split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS40980, SS45772, SS45779), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Segmented images of basalar wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-B3) Transverse views of the segmented neurons shown in A-B2. (A-B4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B5) Segmented muscle images. (C-D) segmented images of steering motoneurons including side views. Males were used for all motoneuron images.

Morphology of first axillary wing steering muscle motoneurons targeted by our sparse split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS41039, SS45782), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Segmented images of first axillary wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-B3) Transverse views of the segmented neurons shown in A-B2. (A-B4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B5) Segmented muscle images. (C-D) segmented images of steering motoneurons including side views. Males were used for all motoneuron images.

Morphology of third axillary wing steering muscle motoneurons targeted by our sparse split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS41027, SS45779, SS41027), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Segmented images of third axillary wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-B3) Transverse views of the segmented neurons shown in A-B2. (A-B4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B5) Segmented muscle images. (C-D) segmented images of steering motoneurons including side views. Males were used for all motoneuron images.

Morphology of fourth axillary wing steering muscle motoneurons targeted by our sparse split lines.

(A-C1) Color MIPs of full expression patterns of the split lines (SS32023,SS37253, SS49039), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-C2) Segmented images of fourth axillary wing motoneurons in upright VNCs. Multicolor flipout (MCFO) was used to separate left and right neurons. VNCs were aligned to the JFC 2018 Unisex template. (A-C3) Transverse views of the segmented neurons shown in A-C2. (A-C4) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-C5) Segmented muscle images. (D-F) segmented images of steering motoneurons including side views. Males were used for all motoneuron image.

Optogenetic stimulation of wing motor neurons evokes changes to flight kinematics.

(A) Schematic of tethered flight measurement apparatus. Tethered flies are positioned in front of a display screen that presents both open- and closed-loop visual stimuli. The fly is illuminated near-infrared LEDs and filmed by three cameras recording at 100 fps. An additional red LED (617 nm) provides optogenetic stimulation in 100 ms pulses. Stills from two of the three cameras recording the tethered fly in flight. Top panel (side view) illustrates the forward (fwd) and backward (back) deviation angles; bottom panel (bottom view) illustrates the stroke amplitude. (B) Averaged wing kinematics for two flies undergoing 50 repetitions of a closed loop trial with 100 ms optogenetic pulse. Dark lines and envelopes show the mean and 95% confidence interval, respectively. Traces in blue correspond to a i2-GAL4>UAS-CsChrimson fly; traces in gray show an example genetic control, SS01062>UAS-CsChrimson, where SS01062 is an empty split Gal4 line. (D-G) Statistics across flies for the four kinematic variables shown in (C): stroke amplitude (D), wingbeat frequency (E), forward deviation angle (F), and backward deviation angle (G). Open circles show per-fly measurements; bars and horizontal lines show interquartile range and population median, respectively. The number of flies per genotype is shown between E and G. Significance is determined via Wilcoxon rank sum test with Bonferroni correction (***, p<0.001; **, p<0.01; *, p<0.05).

Chronic silencing of wing motoneurons results in courtship song deficits.

(A) Image from a typical courtship assay, showing a male (above) extending its left wing to sing to a female (below). (B) Example trace of song recording from a control group fly (SS01055>UAS-Kir2.1). Bouts of sine and pulse song are labelled on the left and right of the trace, respectively. (C) Example slow (top) and fast (bottom) pulse modes for a single fly. Thick black lines show the mean pulse shape; thin gray lines show individual pulses. (D) Courtship song statistics for motoneuron driver lines crossed to UAS-Kir2.1. Rows show different song parameters: total fraction of the trial spent singing (top), fraction of song spent singing pulse mode song (middle), and fraction of song spent singing sine mode song (bottom). Open circles show per-fly measurements; bars and horizontal lines show interquartile range and population median, respectively. The number of flies per genotype is given in D, with the sample sizes for control and experimental groups on the top and bottom, respectively. (E)€ Analysis of pulse type. The top and middle and rows show waveforms for slow and fast pulse modes, respectively, for each genotype in C (blue), overlaid onto control (dark gray). Thick line shows the grand mean across flies; envelope gives 95% confidence interval for mean from bootstrap. Bottom row shows the fraction of pulses that are classified as slow for each genotype. Significance assigned using the Wilcoxon rank sum test and Fisher’s exact test (***, p<0.001; **, p<0.01; *, p<0.05).

Morphology of haltere motoneurons targeted by our sparse split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS51523, SS41075), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B3) Segmented muscle images. (C-D) segmented images of haltere motoneurons.

Morphology of haltere motoneurons targeted by our broad split lines.

(A-B1) Color MIPs of full expression patterns of the split lines (SS37231, SS47195), crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. (A-B2) Images of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-B3) Segmented muscle images. (C-E) segmented images of haltere motoneurons.

Morphology of VUMs in our split lines.

(A1-E1) Color MIPs of the full expression pattern of each split (SS40867, SS40868, SS42385, SS45766, SS51508) in the VNC of males crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, aligned to the JRC 2018 VNC Unisex template. Scale bar in A1 represents 50 μm. Wing and haltere neuropils are indicated by dashed white outlines. (A-E2) Medial views of the muscles and their motoneuron innervation in thoraxes stained with phalloidin conjugated with Alexa 633. Phalloidin staining is shown in blue, GFP in green, and nc82 (Bruchpilot, to label the nervous system) in gray. (A-E3) Segmented medial muscle images. (A-E4) Lateral views of the muscles and their motoneuron innervation. (A-E5) Segmented lateral muscle images. Males were used for all images.

Morphology of individual VUMs and a VPM in our splits, and a table of expression in our T2VUM splits.

Segmented multicolor flipout images aligned to the JRC 2018 VNC Unisex template. The driver lines were SS40867, SS46645, SS42385, SS40867, SS51508, SS48268 respectively.

Morphology of identified intrasegmental interneurons.

MCFO was used to separate interneurons. Each neuron is shown from three views—horizontal (top left), sagittal (bottom left), and transverse (bottom right)— along with neuron name and hemilineage identity (top right). Images taken from preparations of male flies, segmented to isolate individual neurons, and aligned to the JRC 2018 Unisex VNC template. The driver lines used are: SS44314, SS43546, SS36094, SS36094, SS44314, SS31472, SS49042, SS33409, SS49042, SS25511.

Morphology of identified intersegmental interneurons.

MCFO was used to separate interneurons. Each neuron is shown from three views—horizontal (top left), sagittal (bottom left), and transverse (bottom right)— along with neuron name and hemilineage identity (top right). Images taken from preparations of male flies, segmented to isolate individual neurons, and aligned to the JRC 2018 Unisex VNC template. The driver lines used are: SS54495, SS48619, SS60603, SS31899, SS32400, SS54480, SS49807, SS54495, VT014604 (no single cell images were produced from the split GAL4 driver lines targeting this cell, SS31309, SS30330, SS54480, SS54474 or SS54495, so a generation 1 GAL4 image was used), SS25553.

Comparison of interneuron single-cell light microscopy images and MANC electron microscopy matches.

Panels (A-I) each correspond to a single cell type, showing the skeletonized light microscopy (LM) image (black) and the skeleton of the best match obtained from electron microscopy data (red; MANC connectome). The labels in each panel give the neuron name according to our nomenclature (black) and the body ID for the cell in MANC (red).

Sexually dimorphic anatomy in split lines targeting two hemilineages.

A shows the male expression pattern of a split targeting 3B t1, crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, not aligned B-E show the male expression patterns of 17A t2 in four different split lines, crossed with pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005. F-J show the female expression patterns of these same lines, crossed with UAS-CsChrimson. All images are aligned to the JRC 2018 VNC template.