A library of lineage-specific driver lines connects developing neuronal circuits to behavior in the Drosophila Ventral Nerve Cord
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eLife Assessment
This work presents an important genetic toolkit for Drosophila neurobiologists to access and manipulate neuronal lineages during development and adulthood. The evidence supporting the fidelity of this toolkit is convincing. This work will interest Drosophila neurobiologists in general, and some of the genetic tools may be used outside the nervous system. The conceptual approaches used in this paper are likely transferable to other fields as comparable data and genomic methods are obtained.
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Abstract
Understanding the developmental changes in neuronal lineages is crucial to elucidate how they assemble into functional neural networks. Studies investigating nervous system development in model systems have focused on only on a few regions of the central nervous system due to the limited availability of genetic drivers that target specific neuronal lineages throughout development and adult life. This has hindered our understanding of how distinct neuronal lineages interconnect to form neuronal circuits during development. Here, we present a split-GAL4 library composed of genetic driver lines, which we generated via editing the genomic locus of lineage-specific transcription factors and demonstrate that we can use this library to specifically target most individual neuronal hemilineages in the Drosophila ventral nerve cord (VNC) throughout development and into adulthood. Using these genetic driver lines, we found striking morphological changes in neuronal processes within a lineage during metamorphosis. We also demonstrated how neurochemical features of neuronal classes can be quickly assessed. Lastly, we documented behaviors elicited in response to optogenetic activation of individual neuronal lineages and generated a comprehensive lineage-behavior map of the entire fly VNC. Looking forward, this lineage-specific split-GAL4 driver library will provide the genetic tools needed to address the questions emerging from the analysis of the recent VNC connectome and transcriptome datasets.
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eLife Assessment
This work presents an important genetic toolkit for Drosophila neurobiologists to access and manipulate neuronal lineages during development and adulthood. The evidence supporting the fidelity of this toolkit is convincing. This work will interest Drosophila neurobiologists in general, and some of the genetic tools may be used outside the nervous system. The conceptual approaches used in this paper are likely transferable to other fields as comparable data and genomic methods are obtained.
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Reviewer #1 (Public review):
The ventral nerve cord (VNC) of organisms like Drosophila is an invaluable model for studying neural development and organisation in more complex organisms. Its well-defined structure allows researchers to investigate how neurons develop, differentiate, and organise into functional circuits. As a critical central nervous system component, the VNC plays a key role in controlling motor functions, reflexes, and sensory integration.
Particularly relevant to this work, the VNC provides a unique opportunity to explore neuronal hemilineages - groups of neurons that share molecular, genetic, and functional identities. Understanding these hemilineages is crucial for elucidating how neurons cooperate to form specialized circuits, essential for comprehending normal brain function and dysfunction.
A significant …
Reviewer #1 (Public review):
The ventral nerve cord (VNC) of organisms like Drosophila is an invaluable model for studying neural development and organisation in more complex organisms. Its well-defined structure allows researchers to investigate how neurons develop, differentiate, and organise into functional circuits. As a critical central nervous system component, the VNC plays a key role in controlling motor functions, reflexes, and sensory integration.
Particularly relevant to this work, the VNC provides a unique opportunity to explore neuronal hemilineages - groups of neurons that share molecular, genetic, and functional identities. Understanding these hemilineages is crucial for elucidating how neurons cooperate to form specialized circuits, essential for comprehending normal brain function and dysfunction.
A significant challenge in the field has been the lack of developmentally stable, hemilineage-specific driver lines that enable precise tracking and measurement of individual VNC hemilineages. The authors address this need by generating and validating a comprehensive, lineage-specific split-GAL4 driver library.
Strengths and weaknesses
The authors select new marker genes for hemilineages from previously published single-cell data of the VNC. They generate and validate specific and temporally stable lines for almost all the hemilineages in the VNC. They successfully achieved their aims, and their results support their conclusions. This will be a valuable resource for investigating neural circuit formation and function.
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Reviewer #2 (Public review):
It is my pleasure to review this manuscript from Stoffers, Lacin, and colleagues, in which they identify pairs of transcription factors unique to (almost) every ventral nerve cord hemilineage in Drosophila and use these pairs to create reagents to label and manipulate these cells. The advance is sold as largely technical-as a pipeline for identifying durably expressed transcription factor codes in postmitotic neurons from single cell RNAseq data, generating knock-in alleles in the relevant genes, using these to match transcriptional cell types to anatomic cell types, and then using the alleles as a genetic handle on the cells for downstream explication of their function. Yet I think the work is gorgeous in linking the expression of genes that are causal for neuron-type-specific characteristics to the …
Reviewer #2 (Public review):
It is my pleasure to review this manuscript from Stoffers, Lacin, and colleagues, in which they identify pairs of transcription factors unique to (almost) every ventral nerve cord hemilineage in Drosophila and use these pairs to create reagents to label and manipulate these cells. The advance is sold as largely technical-as a pipeline for identifying durably expressed transcription factor codes in postmitotic neurons from single cell RNAseq data, generating knock-in alleles in the relevant genes, using these to match transcriptional cell types to anatomic cell types, and then using the alleles as a genetic handle on the cells for downstream explication of their function. Yet I think the work is gorgeous in linking the expression of genes that are causal for neuron-type-specific characteristics to the anatomic instantiations of those neurons. It is astounding that the authors are able to use their deep collective knowledge of hemilineage anatomy and gene expression to match 33 of 34 transcriptional profiles. Together with other recent studies, this work drives a major course correction in developmental biology, away from empirically identified cell type "markers" (in Drosophila neuroscience, often genomic DNA fragments that contain enhancers found to be expressed in specific neurons at specific times), and towards methods in which the genes that generate neuronal type identity are actually used to study those neurons. Because the relationship between fate and form/function is built into the tools, I believe that this approach will be a trojan horse to integrate the fields of neural development and systems neuroscience.
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Reviewer #3 (Public review):
Summary:
Soffers et al. developed a comprehensive genetic toolkit that enables researchers to access neuronal hemilineages during developmental and adult time points using scRNA-seq analysis to guide gene cassette exchange-based or CRISPR-based tool building. Currently, research groups studying neural circuit development are challenged with tying together findings in the development and mature circuit function of hemilineage-related neurons. Here, authors leverage publicly available scRNA-seq datasets to inform the development of a split-Gal4 library that targets 32 of 34 hemilineages in development and adult stages. The authors demonstrated that the split-Gal4 library, or genetic toolkit, can be used to assess the functional roles, neurotransmitter identity, and morphological changes in targeted cells. The …
Reviewer #3 (Public review):
Summary:
Soffers et al. developed a comprehensive genetic toolkit that enables researchers to access neuronal hemilineages during developmental and adult time points using scRNA-seq analysis to guide gene cassette exchange-based or CRISPR-based tool building. Currently, research groups studying neural circuit development are challenged with tying together findings in the development and mature circuit function of hemilineage-related neurons. Here, authors leverage publicly available scRNA-seq datasets to inform the development of a split-Gal4 library that targets 32 of 34 hemilineages in development and adult stages. The authors demonstrated that the split-Gal4 library, or genetic toolkit, can be used to assess the functional roles, neurotransmitter identity, and morphological changes in targeted cells. The tools presented in this study should prove to be incredibly useful to Drosophila neurobiologists seeking to link neural developmental changes to circuit assembly and mature circuit function. Additionally, some hemilineages have more than one split-Gal4 combination that will be advantageous for studies seeking to disrupt associated upstream genes.
Strengths:
Informing genetic tool development with publicly available scRNA-seq datasets is a powerful approach to creating specific driver lines. Additionally, this approach can be easily replicated by other researchers looking to generate similar driver lines for more specific subpopulations of cells, as mentioned in the Discussion.
The unification of optogenetic stimulation data of 8B neurons and connectomic analysis of the Giant-Fiber-induced take-off circuit was an excellent example of the utility of this study. The link between hemilineage-specific functional assays and circuit assembly has been limited by insufficient genetic tools. The tools and data present in this study will help better understand how collections of hemilineages develop in a genetically constrained manner to form circuits amongst each other selectively.
Weaknesses:
Although cell position, morphology (to some extent), and gene expression are good markers to track cell identity across developmental time, there are genetic tools available that could have been used to permanently label cells that expressed genes of interest from birth, ensuring that the same cells are being tracked in fixed tissue images.
Although gene activation is a good proxy for assaying neurochemical features, relying on whether neurochemical pathway genes are activated in a cell to determine its phenotype can be misleading given that the Trojan-Gal4 system commandeers the endogenous transcriptional regulation of a gene but not its post-transcriptional regulation. Therefore, neurochemical identity is best identified via protein detection. (strong language used in this section of the paper).
The authors mainly rely on the intersectional expression of transcription factors to generate split-Gal4 lines and target hemilineages specifically. However, the Introduction (Lines 97-99) makes a notable point about how driver lines in the past, which have also predominantly relied on the regulatory sequences of transcription factors, lack the temporal stability to investigate hemilineages across time. This point seems to directly conflict with the argument made in the Results (Lines 126-127) that states that most transcription factors are stably expressed in hemilineage neurons that express them. It is generally known that transcription factors can be expressed stably or transiently depending on the context. It is unclear how using the genes of transcription factors in this study circumvents the issue of creating temporally stable driver lines.
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