Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster

Reviewer #1 (Public review):

This paper presents a set of tools that will pave the way for a comprehensive understanding of the circuits that control wing motion in flies during flight or courtship. These tools are mainly focused on wing motor neurons and interneurons, as well as a few motor neurons of the haltere. This paper and the library of driver lines described within it will serve as a crucial resource in the pursuit of understanding how neural circuits give rise to behavior. Overall, I found the paper well-written, the figures are quite nice, and the data from the functional experiments convincing. I do not have many major concerns, but a few suggestions that I think will make the paper easier to understand.

I think the introduction could use some reorganization, as right now I found it quite difficult to follow. For example, lines 85-88 seem to fit more naturally at the end of the next paragraph, compared to the current location of those sentences, which feels rather disjointed. I would suggest introducing the organization of the wing motor system (paragraphs 3 and 4) and then discussing the VNC (paragraph 2) before moving on to describe the neurons within the VNC that may control wing motion. Additionally, lines 141-144, which describe the broad subdivisions of the VNC, can be moved up to where the VNC is first introduced.

One of my major takeaways from the paper is the call to examine the premotor circuits that govern wing motion. For that reason, I was surprised that there was little mention of the role of sensory input to these circuits. As the authors point out in the discussion, the haltere, for example, provides important input to the wing steering system. I recognize that creating driver lines for the sensory neurons that innervate the VNC is well beyond the scope of this project. I would just like some clarification in the text of the role these inputs play in structuring wing motion, especially as some act at rapid timescales that possibly forgo processing by the very circuits detailed here. This brings up a related issue: if the roles of the interneurons that are presynaptic to the wing motor neurons are "largely unexplored," with how much confidence can we say that they are the key for controlling behavior? To be sure, this has been demonstrated quite nicely in the case of courtship, but in flight, I think the evidence supporting this argument is less clear. I suggest the authors rephrase their language here.

Reviewer #2 (Public review):

Summary:

In this study, the researchers generated an impressive collection of sparse split GAL4 driver lines that target wing-relevant cell types. They then characterized the cell types according to function, development, and morphology. This resource is a necessary companion to the fly ventral nerve cord connectomes. The fly connectomes enable biologically-constrained hypothesis generation, but we need genetic reagents like the ones generated here in order to test those hypotheses and understand the biological limits of what we can learn from connectomes. This project identifies wing-relevant cell types and generates a library of driver lines to provide genetic access to small populations of these cell types. The study also characterizes these cell types according to developmental lineage and morphology, and performs functional analyses on some of the cell types, including single pairs of motor neurons.

Strengths:

The genetic toolkit that the authors produce is rigorous and well-documented, and will be broadly useful. Further, they bolster the utility of the resource by characterizing cell types according to developmental lineage, morphology, and connectome nomenclature. The authors successfully produce a foundation for future studies of the functional organization of neural circuits. In particular, the driver lines created in this study match the specificity of the connectome, providing a necessary resource to functionally test predictions from the connectomes.

Weaknesses:

The manuscript includes several broad statements about certain questions being "unexplored" (e.g., lines 71, 129). However, the authors cite papers (e.g., Harris 2015 and Lillvis 2024) that directly address these topics. To better support the narrative, it would be helpful to more accurately summarize the key findings from these prior studies. For example, the Harris paper found behavioral correlates of hemilineage activation. By using the sparse toolkit you have created, it may be possible to dissect behavior into finer-grained modules or specific movements, providing deeper insight into how complex behaviors are produced by the nervous system.

There are a few places where the current manuscript does not acknowledge the post-connectome universe it now exists in. For example, line 600: the morphology of DVMns in Drosophila had never been described, and line 762: revealed diversity within hemilineages which had not previously been reported. Although this manuscript was in progress before the VNC connectomes were released, they are now published, and the current manuscript should reflect this development.

The authors focus on some well-characterized "Named Neurons" e.g., ChINs and the PSI. This focused approach makes sense, but the authors miss the opportunity to point out a major strength of the toolkit they have produced: we are now less constrained by studying only these Named Neurons. With this new resource, we have genetic access to sparse sets of neurons that are likely just as important but were previously inaccessible.

Reviewer #3 (Public review):

Summary:

This paper provides a catalogue of 195 well-documented Drosophila strains with sparse and cell-type-specific GAL-4 expression in the adult ventral nerve cord (VNC). The focus is on motor neurons, interneurons, and modulatory unpaired neurons in the dorsal VNC neuropils that drive motor control of the wings. Intersegmental sensory and interneurons are not included. The expression patterns of all 195 fly strains are exceptionally well-characterized and catalogized. Compelling links to hemi-lineages and connectomics data are well documented, and some solid functional data demonstrate the applicability of the GAL4 strains in genetic silencing and optogenetic activation experiments. In sum, this catalogue provides a fantastic toolkit for future functional analyses of motor control centers in the dorsal VNC.

Strengths:

Particularly noteworthy is that the authors did a tremendous job in identifying and catalogizing the correspondences between the neurons in their catalogue and the MANC connectomics dataset, as well as with the respective hemi-lineages. The catalogue has been generated with exquisite care and is impressive.

Weaknesses:

There are no significant weaknesses. I have only minor recommendations on definitions and naming of neuron types, the text on the optogenetic experiments, and the comparison to other insects.