Proprioceptive neurons on the Drosophila wing.

(A) The cell bodies and dendrites of sensory neurons are in the periphery, on the wing and wing hinge, and their axons project to the ventral nerve cord (VNC). Before entering the VNC, the sensory axons fasciculate together and enter through the Anterior Dorsomedial Nerve (ADMN). (B) Proprioceptors on the wing include campaniform sensilla (CS), chordotonal organs (CO), and a hair plate (HP). Each campaniform sensillum dome is innervated by a single sensory neuron, as is each hair in a hair plate. A chordotonal organ is made up of a group of sensory neurons with supporting cells that fix them to the underside of the cuticle (shown in blue). Blue asterisks (bottom right) indicates a single external hair in the hair plate. Images show the membrane-bound fluorescent label mCD8::GFP to highlight each proprioceptor type. See Table 3 for details on which proprioceptors are labeled by which driver lines. Scale bars are 10 μm. (C) Location of sensory neurons on the wing and wing hinge. The location of sensory neurons and the number of campaniform sensilla in each field are based on confocal images and a prior study (Dinges et al., 2021). A subset of sclerites and other structures that make up the wing hinge are included as landmarks: pterale C (ptC), the anterior nodal wing process (ANWP, which also features three CS), the parascutal shelf (ps), and the second axillary (ax ii). (D) We reconstructed each sensory axon in the ADMN wing nerve to visualize its full morphology and analyze downstream connectivity in the VNC. More information on each of these steps in Methods and Azevedo et al., 2024. In the nerve cross-section, the motor domain and margin bristle domains are highlighted by outlined gray masks.

ADMN Sensory axon identification and nomenclature

Literature characterizing peripheral anatomy of wing mechanosensory neurons

Driver lines labeling wing sensory neurons

Numbers in the table indicate how many neurons are labeled, e.g. 4 of 24 radius CO neurons for the first driver line, 10A07-GAL4.

Postsynaptic connectivity and morphology of wing sensory axons.

(A) Connectivity matrix based on the left wing proprioceptors and postsynaptic neurons in the VNC. Only partners with at least five synapses from a single proprioceptor are shown. For simplicity, we do not show: (1) a descending neuron that is postsynaptic to sensory neurons (0.1% of the proprioceptive outputs), (2) a single non-motor efferent neuron (0.1%); and unproofread or fragment neurons, (9.7%). Postsynaptic neurons are classified as either motor neurons, sensory neurons, VNC intrinsic neurons, or ascending neurons (axons project to the brain). Within each class, postsynaptic neurons were then sorted according to which wing proprioceptor they receive the most synapses from. The number of synapses is displayed on a log scale. (B) Cosine similarity matrix of the 126 left wing axons not from margin bristles. Axons are ordered by agglomerative clustering. Boxes indicate clusters with similar morphology, with the number next to each cluster indicating the morphology clusters in (C-E). Filled green boxes indicate morphologies identified in the project. See Table 1 and Methods for details on matching axon morphologies to prior literature.

Campaniform sensilla on the tegula target the tonic wing steering motor neuron b1.

(A) Connectivity between previously uncharacterized wing sensory axons and wing steering motor neurons. Wing steering motor neurons (columns) are grouped by motor modules, which are groups of motor neurons that receive a high degree of synaptic input from shared presynaptic partners and are therefore likely to be co-activated (Lesser et al., 2024). (B) The left b1 motor neuron with circles showing predicted synapse locations from the FANC EM volume. (C) 3D reconstructions of the left b1 motor neuron (black) and all the sensory axons from which it receives direct synaptic input. Inset: three example individual axons from the left wing to demonstrate the variation in axon branching. (D) Ultrastructure of putative electronic synapses: these sensory axons feature densely packed mitochondria at terminals near the b1 motor neuron. (E) A similarly high density of mitochondria is also seen at axon terminals of a wing contralateral haltere interneuron (w-ChiN), which likely have electrical synapses onto b1 based on dye-fill experiments (Trimarchi and Murphey, 1997). (F) Axon branching pattern in VNC. Axons are from two morphological clusters (#6 and #7 from Figure 2). Below: rotated view of the VNC. (G) Maximum projection from FlyLight Z-Stack of images of the driver line 13B12-GAL4. Projection that is crossing the midline is from a different sensory neuron that enters through the posterior dorsal medial nerve and innervates a thorax bristle. (H) Expression in the periphery. Maximum projection from confocal z-stack showing sensory neurons that innervate the campaniform sensilla field on the tegula. The driver line also labels two tegula hair plate hairs, but their axon morphology is distinct (see Figure 4). Wing hinge abbreviations: anterior nodal wing process (ANWP), first axillary (ax i).

Tegula hair plate.

(A) 3D reconstructed axons. Above: population of axons with similar morphology (black) and VNC volume (gray). Below: rotated view to show how the axons split to scoop around the dorsal and ventral edges of the wing neuropil. (B) Axon branching pattern in VNC. Axons are from morphological cluster #10 in Figure 2. Maximum projection from the FlyLight MCFO collection of the driver line 16C09-GAL4. (C) Expression in the periphery. Maximum projection from confocal Z-Stack showing sensory neurons that innervate the hairs of the tegula hair plate. Red arrow indicates an external hair plate hair. Wing hinge abbreviations: anterior nodal wing process (ANWP), first axillary (ax i).

Tegula chordotonal organ.

(A) 3D reconstructed axons. Axons are from two morphological clusters (#17 and #18 in Figure 2). (B) Axon branching pattern in VNC. Maximum projection from FlyLight Z-Stack of images of the driver line 60D12-GAL4. (C) Expression in the periphery. Maximum projection from confocal Z-Stack showing sensory neurons that innervate the chordotonal organ in the tegula. There are two clusters of neurons, which are differentiated by their separate attachment points within the tegula. 60D12-GAL4 labels neurons from both clusters. (D) Maximum z-projection of the proximal wing co-labeling iav-GAL4 with ChAT-LexA. ChAT-LexA labels all sensory neurons (green, nuclear stain) and iav-GAL4 labels the radius CO but not the tegula CO (red, nuclear stain). (E) Phalloidin labels the actin-rich cap cells that are part of chordotonal organs. Asterisk indicates muscle that is also labeled by phalloidin.

Radius chordotonal organ.

(A) 3D reconstructed axons. Axons are from the morphological clusters #3, #4, and #5 in Figure 2. Green arrow indicates the characteristic lateral projection found in each neuron. (B) A sparse driver line, 10A07-GAL4, labels a subset of neurons that make up the radius chordotonal organ. For other driver lines that label radius chordotonal neurons, see Table 3. (C) Peripheral expression of 10A07-GAL4 > UAS-mCD8::GFP. (D) Peripheral anatomy of the radius chordotonal organ, which is better shown by a broad driver line, 15F10-GAL4 > UAS-mCD8::GFP. The radius chordotonal organ attaches to the ventral inner wall of the radius by cap cells (blue). A blue arrow is shown across the confocal images and cartoons to orient to the “pocket” in the radius near the CO cell bodies.

Sensory axons near the wing hinge.

(A) 3D reconstructed axons. Axons belong to the morphological cluster #10 from Figure 2. (B) Axon branching pattern in VNC. Maximum projection from the FlyLight MCFO collection of the driver line 37D11-GAL4. (C) Expression in the periphery. Top: maximum projection from confocal Z-Stack of a broader driver line, 10G03-GAL4, to show the morphology of the sensory neurons at the base of the parascutal shelf. Below: maximum projection from confocal Z-Stack of the sparse driver line 37D11-GAL4 > UAS-mCD8::GFP showing neurons labeled at the base of the parascutal shelf. The asterisk marks an innervated bristle on the thorax. (D) Pterale C is not an innervated sclerite. Pterale C was previously predicted to be innervated based on experiments in which an electrode placed at the base of pterale C recorded signals in response to wing vibration (Miyan and Ewing, 1984). We found no neurons innervating pterale C, but we did observe that the axon bundle from the radius passes directly under pterale C, which could explain previously published results.

Campaniform sensilla in the same field have unique axons.

(A) The ventral radius C (v.Rad.C) field of campaniform sensilla is on the ventral side of the more distal part of the radius. The field has four to five domes, the fifth dome is proposed to be its own individual dome as it is farther apart from the other four and its orientation is slightly different (Dinges et al., 2021). (B) Summary of the CS within v.Rad.C that are labeled by sparse GAL4 lines shown in C. (C) Peripheral expression in specific campaniform sensilla from sparse driver lines (each row). Maximum projection from confocal z-stack showing expression in the periphery from each sparse driver line. CS in v.Rad.C are labeled one through four, as in (B) to show which CS is innervated in each image. (D) Pairs of images showing (left) A depth-colored single channel MCFO Z-Stack from the FlyLight collection (Meissner et al., 2023), with the wing axon highlighted in the image. Contrast of z-sections was optimized to emphasize visual clarity of wing axons, see Methods for details. (right) The reconstructed axon from EM that best matches the morphology, depth-colored and aligned to the same template as the FlyLight images. (E) Postsynaptic connectivity of axons with morphologies that match those found for v.Rad.C. Postsynaptic connectivity is more similar for axons with similar morphologies than from the same CS field.

Pipeline for matching 3D reconstructed axons to sensory neurons on the wing and wing hinge.

Driver lines were identified by their sparse expression in the wing nerve, then crossed to a membrane-bound GFP (UAS-mCD8::GFP), imaged with a confocal microscope, and then compared with literature for identification. For details on matching peripheral wing anatomy to literature, see Table 2