An updated catalogue of split-GAL4 driver lines for descending neurons in Drosophila melanogaster

Reviewer #1 (Public review):

Summary:

This manuscript by Zung et al. describes a curated library of genetic lines labeling a class of important neurons called Descending Neurons in the fruit fly, Drosophila melanogaster. These neurons are especially important in their critical role in relaying information from the brain to motor circuits within the ventral nerve cord - the insect analogy of the vertebrate spinal cord. The authors screened through a vast resource of Gal4 lines to generate 500 new genetic lines that allow for the precise labeling of 190 (40%) of all Descending Neurons. The tools introduced here will allow researchers to perform precise circuit dissection of the exact roles these neurons play in linking the brain to the ventral nerve cord.

Strengths:

This manuscript represents an important follow-up to the author's 2018 paper in the extension of the genetic toolkit from 178 genetic lines that target 65 Descending Neuron (DN) classes to 806 lines that target 190 DN classes. The presentation of this toolkit is comprehensive with confocal images, informative classifications of lines based on specificity/consistency, and identification of the neuron types - when possible - in the EM dataset.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

Summary:

Descending neurons (DNs) are critical nodes in the neural computation underlying sensorimotor transformation. Building on their earlier work, the authors have substantially expanded the genetic resources for labeling these cell types in D. melanogaster, offering a valuable public resource.

Strengths:

The authors identified 146 additional DN types and generated 500 new DN driver lines, expanding the genetic reagents from labeling 98 cell types to 244, representing approximately 50% of all DN types estimated by EM connectomes. While the EM connectomes offer unprecedented resolution of neuronal cell types and their connectivity, genetic access to these cell types remains essential for studying their functions and testing hypotheses. Given the broad interest in DNs, the reagents generated in this study will be of important value for addressing a wide range of questions in sensorimotor transformation.

The organization of the dataset is overall intuitive and comprehensive. The authors also provided clear information and guidance on accessing the relevant resources, such as stack images and fly lines. In addition, the authors have thoughtfully handled the information updated from the earlier collection they generated (Namiki et al. 2018) and incorporated previously published DN lines, providing a consolidated and up-to-date resource for the DN community.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #3 (Public review):

Summary:

This study provides the Drosophila community with a large collection of new split-Gal4 descending neuron genetic lines. They extend previous efforts to characterize and identify genetic lines for this important class of neurons by providing images of descending neurons and a metric for genetic lines based on specificity and consistency. Their discussion highlights several applications of this collection, for example, to understand the function of new descending neurons through optogenetic and/or physiological characterization. They also helpfully discuss caveats, encouraging users of this collection to validate expression patterns and to be careful when interpreting optogenetic experimental results, considering potential off-target labeling in the lines. Overall, members of the Drosophila community interested in understanding the function of descending neurons and their role in behavior will find this a helpful resource.

Strengths:

(1) The authors extend the previous genetic access of descending neurons in Drosophila to over 800 split-Gal4 lines and 190 cell types (nearly half of the known population of descending neurons). The authors update and at times correct the previous identification of descending neurons from a previous, large-scale analysis. The authors extend and, at times, correct previous efforts at characterizing these neurons.

(2) Clear images of descending neurons labeled by new genetic lines are presented in the main figure papers for reference.

(3) This study classifies lines labeling descending neurons using a quality score to indicate specificity and consistency. They provide this for the entire set of genetic lines, a valuable assessment for researchers interested in targeting these neurons for optogenetic or physiological characterization.

Weaknesses:

Although this paper represents a substantial effort and useful contribution to the Drosophila community, a few weaknesses, primarily regarding the specificity and reliability of genetic lines, remain:

(1) The authors state that optogenetic activation of DN types using the new split-GAL4 lines is expected to reliably activate the target neurons with virtually no off-target effects in the rest of the central nervous system. More data supporting this conclusion, including both qualitative and quantitative anatomical evidence, would strengthen this claim.

(2) The authors do recommend that researchers using these lines examine expression patterns themselves to evaluate line cleanliness and consistency, but some analysis by the authors would be useful, for example, providing guidelines for best practices to perform this evaluation.

(3) Changes in expression patterns after several generations are noted by the authors, weakening confidence somewhat in the long-term usefulness of this collection of genetic lines.