Classification and genetic targeting of cell types in the primary taste and premotor center of the adult Drosophila brain
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Evaluation Summary:
This manuscript will be of interest to scientists working on adult Drosophila behavior, especially as it relates to a region called subesophageal zone. This area is an important integration center for different nervous system functions, including taste information processing and motor control of mouth parts and body movements. Specifically, it provides genetic tools (sparse gal-4 lines) that target different cell types in the subesophageal zone for future functional analysis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Neural circuits carry out complex computations that allow animals to evaluate food, select mates, move toward attractive stimuli, and move away from threats. In insects, the subesophageal zone (SEZ) is a brain region that receives gustatory, pheromonal, and mechanosensory inputs and contributes to the control of diverse behaviors, including feeding, grooming, and locomotion. Despite its importance in sensorimotor transformations, the study of SEZ circuits has been hindered by limited knowledge of the underlying diversity of SEZ neurons. Here, we generate a collection of split-GAL4 lines that provides precise genetic targeting of 138 different SEZ cell types in adult Drosophila melanogaster , comprising approximately one third of all SEZ neurons. We characterize the single-cell anatomy of these neurons and find that they cluster by morphology into six supergroups that organize the SEZ into discrete anatomical domains. We find that the majority of local SEZ interneurons are not classically polarized, suggesting rich local processing, whereas SEZ projection neurons tend to be classically polarized, conveying information to a limited number of higher brain regions. This study provides insight into the anatomical organization of the SEZ and generates resources that will facilitate further study of SEZ neurons and their contributions to sensory processing and behavior.
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Author Response:
Reviewer #1:
The manuscript is well-written and easy to follow. The authors are thorough in their characterization, shown both through the text itself and the figures. Most of the comments relate to the narrative structure itself and are merely suggestions. Overall, this work represents an important resource for the community and especially to people working on the role of the SEZ in feeding and motor behaviors.
Specific comments and suggestions:
• The authors give a very nice overview of the SEZ and the split-Gal4 technique. However, they spend much less time discussing the rationale behind using the cell body numbers within subesophageal neuromeres. This to me assumes two extremely different kinds of readers, one relatively new to Drosophila research and the other relatively well-versed. Since this technique is …
Author Response:
Reviewer #1:
The manuscript is well-written and easy to follow. The authors are thorough in their characterization, shown both through the text itself and the figures. Most of the comments relate to the narrative structure itself and are merely suggestions. Overall, this work represents an important resource for the community and especially to people working on the role of the SEZ in feeding and motor behaviors.
Specific comments and suggestions:
• The authors give a very nice overview of the SEZ and the split-Gal4 technique. However, they spend much less time discussing the rationale behind using the cell body numbers within subesophageal neuromeres. This to me assumes two extremely different kinds of readers, one relatively new to Drosophila research and the other relatively well-versed. Since this technique is crucial to the approach used throughout the manuscript and significant in the authors labeling about 1/3 of the region, I would suggest the authors to give a brief summary and justification as to why they decided to use this neuromere labeling technique, and spend more time in the discussion (perhaps in the paragraphs between lines 352-386) talking about the pros and cons of this technique (is it expected to label fewer than 50% of the neurons? How may this complement the EM and FAFB dataset, and what are the advantages and disadvantages using the technique employed here?).
We now provide a brief introduction to the approach in the results section (lines 82-96) and include additional pros and cons of the approach in the discussion (lines 369-383). We expect that this approach labels the vast majority of SEZ neurons.
Related suggestions:
o Line 81: elaborate on deutocerebral contributions
We have moved this to discussion (lines 374-377). We clarify that not much is known about deutocerebral contributions.
o Lines 84-85: along similar lines, Hox gene drivers
We altered this sentence to be clear to a general audience (lines 86-90).
• Figure 9: having a color legend in the figure itself will facilitate understanding of this figure. I think it would be nice to have visual examples of interneurons, projection neurons, and so forth. Perhaps when the authors first describe neurons in Group 1, instead of marking "first half of the group" (line 210) the authors can explicitly name the neuron types (peep, doublescoop, etc.)
We now include a color legend as well as a new figure with visual examples of polarity (Figure 10 – figure supplement 1). As suggested, we changed the text to explicitly name the neuron types in Group 1 that are interneurons versus projection neurons (lines 241-243).
• In the polarity section of the discussion, it would be interesting to have additional remarks relating to how to determine whether these identified neurons are thought to be ascending and why. Since one of the authors has previously characterized some ANs, perhaps comparisons to this work would be helpful to readers new to this region of the brain.
We now include a brief definition of ascending neurons in the results section (lines 149-150) and note that ascending neurons were not included in the collection.
• The parallel structures used in characterizing Groups 1 through 6 are very useful. However, I think that when the authors relate each group to previous works, this might fit better in the Discussion section.
We altered this section of the results to move speculation about group function to the Discussion (lines 421-445), as recommended.
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Evaluation Summary:
This manuscript will be of interest to scientists working on adult Drosophila behavior, especially as it relates to a region called subesophageal zone. This area is an important integration center for different nervous system functions, including taste information processing and motor control of mouth parts and body movements. Specifically, it provides genetic tools (sparse gal-4 lines) that target different cell types in the subesophageal zone for future functional analysis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript is well-written and easy to follow. The authors are thorough in their characterization, shown both through the text itself and the figures. Most of the comments relate to the narrative structure itself and are merely suggestions. Overall, this work represents an important resource for the community and especially to people working on the role of the SEZ in feeding and motor behaviors.
Specific comments and suggestions:
• The authors give a very nice overview of the SEZ and the split-Gal4 technique. However, they spend much less time discussing the rationale behind using the cell body numbers within subesophageal neuromeres. This to me assumes two extremely different kinds of readers, one relatively new to Drosophila research and the other relatively well-versed. Since this technique is crucial …
Reviewer #1 (Public Review):
The manuscript is well-written and easy to follow. The authors are thorough in their characterization, shown both through the text itself and the figures. Most of the comments relate to the narrative structure itself and are merely suggestions. Overall, this work represents an important resource for the community and especially to people working on the role of the SEZ in feeding and motor behaviors.
Specific comments and suggestions:
• The authors give a very nice overview of the SEZ and the split-Gal4 technique. However, they spend much less time discussing the rationale behind using the cell body numbers within subesophageal neuromeres. This to me assumes two extremely different kinds of readers, one relatively new to Drosophila research and the other relatively well-versed. Since this technique is crucial to the approach used throughout the manuscript and significant in the authors labeling about 1/3 of the region, I would suggest the authors to give a brief summary and justification as to why they decided to use this neuromere labeling technique, and spend more time in the discussion (perhaps in the paragraphs between lines 352-386) talking about the pros and cons of this technique (is it expected to label fewer than 50% of the neurons? How may this complement the EM and FAFB dataset, and what are the advantages and disadvantages using the technique employed here?).
Related suggestions:
o Line 81: elaborate on deutocerebral contributions
o Lines 84-85: along similar lines, Hox gene drivers• Figure 9: having a color legend in the figure itself will facilitate understanding of this figure. I think it would be nice to have visual examples of interneurons, projection neurons, and so forth. Perhaps when the authors first describe neurons in Group 1, instead of marking "first half of the group" (line 210) the authors can explicitly name the neuron types (peep, doublescoop, etc.)
• In the polarity section of the discussion, it would be interesting to have additional remarks relating to how to determine whether these identified neurons are thought to be ascending and why. Since one of the authors has previously characterized some ANs, perhaps comparisons to this work would be helpful to readers new to this region of the brain.
• The parallel structures used in characterizing Groups 1 through 6 are very useful. However, I think that when the authors relate each group to previous works, this might fit better in the Discussion section.
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Reviewer #2 (Public Review):
The authors aimed to generate a novel set of split-Gal4 drivers to access neurons located within the SEZ region of the adult fruit fly brain. This paper achieved its goals - (i) it generated a novel collection of SEZ specific Gal4 drivers and (ii) provided the framework for scientists to generate additional SEZ specific split Gal4 drivers. Figures 2-8 were beautiful and very compelling, a strength of the manuscript. I especially liked the regional specificity of the different SEZ cell types. A weakness was the heavy jargon used, what felt like an incomplete explanation of how the individual cell types were determined, and no "raw" data to back up the polarity quantifications. Regardless, the authors achieved their aims and their results have many exciting future implications for Drosophila behavioralists.
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Reviewer #3 (Public Review):
Sterne et al generates 138 sparse gal-4 lines that target different types of cells in the subesophageal zone (SEZ) of Drosophila adults. Using bioinformatic tools, the different cells types are clustered into six domains based on neuron morphology. These include presumptive sensory, motor and interneurons, and in selected cases provide tools to target neurons that were identified previously by stochastic methods. The main strength of the paper is providing tools for the Drosophila community interested in SEZ and behavioral processes related to this region of the fly nervous system. It is doing a great service. What this paper does not provide are functional and synaptic information of these lines, and the coverage is estimated to be about 30%. The tools provided here will nicely complement EM connectivity …
Reviewer #3 (Public Review):
Sterne et al generates 138 sparse gal-4 lines that target different types of cells in the subesophageal zone (SEZ) of Drosophila adults. Using bioinformatic tools, the different cells types are clustered into six domains based on neuron morphology. These include presumptive sensory, motor and interneurons, and in selected cases provide tools to target neurons that were identified previously by stochastic methods. The main strength of the paper is providing tools for the Drosophila community interested in SEZ and behavioral processes related to this region of the fly nervous system. It is doing a great service. What this paper does not provide are functional and synaptic information of these lines, and the coverage is estimated to be about 30%. The tools provided here will nicely complement EM connectivity analysis of the adult SEZ.
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