Peripheral anatomy and central connectivity of proprioceptive sensory neurons in the Drosophila wing

Reviewer #1 (Public review):

Summary:

Lesser et al provide a comprehensive description of Drosophila wing proprioceptive sensory neurons at the electron microscopy resolution. This "tour-de-force" provides a strong foundation for future structural and functional research aimed at understanding wing motor control in Drosophila with implications for understanding wing control across other insects.

Strengths:

(1) The authors leverage previous research that described many of the fly wing proprioceptors, and combine this knowledge with EM connectome data such that they now provide a near-complete morphological description of all wing proprioceptors.

(2) The authors cleverly leverage genetic tools and EM connectome data to tie the location of proprioceptors on the wings with axonal projections in the connectome. This enables them to both align with previous literature as well as make some novel claims.

1. In addition to providing a full description of wing proprioceptors, the authors also identified a novel population of sensors on the wing tegula that make direct connections with the B1 wing motor neurons, implicating the role of the tegula in wing movements that was previously underappreciated.

(4) Despite being the most comprehensive description so far, it is reassuring that the authors clearly state the missing elements in the discussion.

Weaknesses:

(1) The authors do their main analysis on data from the FANC connectome but provide corresponding IDs for sensory neurons in the MANC connectome. I wonder how the connectivity matrix compares across FANC and MANC if the authors perform a similar analysis to the one they have done in Figure 2. This could be a valuable addition and potentially also pick up any sexual dimorphism.

(2) The authors speculate about the presence of gap junctions based on the density of mitochondria. I'm not convinced about this, given that mitochondrial densities could reflect other things that correlate with energy demands in sub-compartments.

(3) I'm intrigued by how the tegula CO is negative for iav. I wonder if authors tried other CO labeling genes like nompc. And what does this mean for the nature of this CO. Some more discussion on this anomaly would be helpful.

(4) The authors conclude there are no proprioceptive neurons in sclerite pterale C based on Chat-Gal4 expression analysis. It would be much more rigorous if authors also tried a pan-neuronal driver like nsyb/elav or other neurotransmitter drivers (Vglut, GAD, etc) to really rule this out. (I hope I didn't miss this somewhere.)

Overall, I consider this an exceptional analysis that will be extremely valuable to the community.

Reviewer #2 (Public review):

Summary:

Lesser et al. present an atlas of Drosophila wing sensory neurons. They proofread the axons of all sensory neurons in the wing nerve of an existing electron microscopy dataset, the female adult fly nerve cord (FANC) connectome. These reconstructed sensory axons were linked with light microscopy images of full-scale morphology to identify their origin in the periphery of the wing and encoded sensory modalities. The authors described the morphology and postsynaptic targets of proprioceptive neurons as well as previously unknown sensory neurons.

Strengths:

The authors present a valuable catalogue of wing sensory neurons, including previously undescribed sensory axons in the Drosophila wing. By providing both connectivity information with linked genetic drive lines, this research facilitates future work on the wing motor-sensory network and applications relating to Drosophila flight. The findings were linked to previous research as well as their putative role in the proprioceptive and nerve cord circuitry, providing testable hypotheses for future studies.

Weaknesses:

(1) With future use as an atlas, it should be noted that the evidence is based on sensory neurons on only one side of the nerve cord. Fruit flies have stereotyped left/right hemispheres in the brain and left/right hemisegments in the nerve cord. The comparison of left and right neurons of the nervous system can give a sense of how robust the morphological and connectivity findings are. Here, the authors have not compared the left and right side sensory axons from the wing nerve, leaving potential for developmental variability across samples and left/right hemisegments.

(2) Not all links between the EM reconstructions and driver lines are convincing. To strengthen these, for all EM-LM matches in Figures 3-7, rotated views of the driver line (matching the rotated EM views) should be shown to provide a clearer comparison of the data. In particular, Figure 3G and Figure 7B are not very convincing based on the images shown. MCFO imaging of the driver lines in Figure 3G and 7B would make this position stronger if a clone that matches the EM reconstruction could be identified.

(3) Figure 7B looks like the driver line might have stochastic expression in the sensory neuron, which further reduces confidence in the result shown in Figure 7C. Is this expression pattern in the wing consistently seen? Many split-GAL4s have stochastic expressions. The evidence would be strengthened if the authors presented multiple examples (~4-5) of each driver line's expression pattern in the supplement.

(4) Certain claims in this work lack quantitative evidence. On line 128, for instance, "Overall, our comprehensive reconstruction revealed many morphological subgroups with overlapping postsynaptic partners, suggesting a high degree of integration within wing sensorimotor circuits." If a claim of subgroups having shared postsynaptic partners is being made, there should have been quantitative evidence. For example, cosine similar amongst members of each group compared to the cosine similarity of shuffled/randomised sets of axons from different groups. The heat map of cosine similarity in Figure 2B alone is not sufficient.

(5) Similarly, claims about putative electrical connections to b1 motor neurons are very speculative. The authors state that "their terminals contain very densely packed mitochondria compared to other cells", without providing a quantitative comparison to other sensory axons. There is also no quantitative comparison to the one example of another putative electrical connection from the literature. Further, it should be noted that this connection from Trimarchi and Murphey, 1997, is also stated as putative on line 167, which further weakens this evidence. Quantification would strongly strengthen this position. Identification of an example of high mitochondrial density at a confirmed electrical connection would be even better. In the related discussion section "A potential metabolic specialization for flight circuitry", it should be more clearly noted that the dense mitochondria could be unrelated to a putative electrical connection. If the authors have an alternative hypothesis about the mitochondria density, this should be stated as well.

(6) It would be appropriate to cite previous work using a similar strategy to match sensory axons to their cell bodies/dendrites at the periphery using driver lines and connectomics (see Figure 5 for example in the following paper: https://doi.org/10.7554/eLife.40247 ).

The methods section is very sparse. For the sake of replicability, all sections should be expanded upon.

Reviewer #3 (Public review):

Summary:

The authors aim to identify the peripheral end-organ origin in the fly's wing of all sensory neurons in the anterior dorsomedial nerve. They reconstruct the neurons and their downstream partners in an electron microscopy volume of a female ventral nerve cord, analyse the resulting connectome, and identify their origin with a review of the literature and imaging of genetic driver lines. While some of the neurons were already known through previous work, the authors expand on the identification and create a near-complete map of the wing mechanosensory neurons at synapse resolution.

Strengths:

The authors elegantly combine electron microscopy, neuron morphology, connectomics, and light microscopy methods to bridge the gap between fly wing sensory neuron anatomy and ventral nerve cord morphology. Further, they use EM ultrastructural observations to make predictions on the signaling modality of some of the sensory neurons and thus their function in flight.

The work is as comprehensive as state-of-the-art methods allow to create a near-complete map of the wing mechanosensory neurons. This work will be of importance to the field of fly connectomics and modelling of fly behavior, as well as a useful resource to the Drosophila research community.

Through this comprehensive mapping of neurons to the connectome, the authors create a lot of hypotheses on neuronal function, partially already confirmed with the literature and partially to be tested in the future. The authors achieved their aim of mapping the periphery of the fly's wing to axonal projections in the ventral nerve cord, beautifully laying out their results to support their mapping.

The authors identify the neurons in a previously published connectome of a male fly ventral nerve cord to enable cross-individual analysis of connections. Further, together with their companion paper, Dhawan et al. 2025, describing the haltere sensory neurons in the same EM dataset, they cover the entire mechanosensory space involved in Drosophila flight.

Weaknesses:

The connectomic data are only available upon request; the inclusion of a connectivity table of the reconstructed neurons would aid analysis reproducibility and cross-dataset comparisons.