multiplex-detection-gene-expression-intact-drosophila-brain-using

Multiplex Detection of Gene Expression in the Intact <em>Drosophila</em> Brain Using Expansion-Assisted Iterative Fluorescence <em>In Situ</em> Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Howard Hughes Medical Institute

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 inÂ each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy ofÂ dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function.

A GAL4-driver line resource for Drosophila neurobiology.

We report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.

A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individual neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time.

An unbiased template of the Drosophila brain and ventral nerve cord.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of  neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

A searchable image resource of  GAL4 driver expression patterns with single neuron resolution.

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system (CNS) have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in  and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult  CNS and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell-type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

Article	Year
A GAL4-driver line resource for Drosophila neurobiology. Cell reports\| PubMed ID: 23063364	2012
A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila. Cell reports\| PubMed ID: 25088417	2014
An unbiased template of the Drosophila brain and ventral nerve cord. PloS one\| PubMed ID: 33382698	2020
A searchable image resource of GAL4 driver expression patterns with single neuron resolution. eLife\| PubMed ID: 36820523	2023
A split-GAL4 driver line resource for neuron types. eLife\| PubMed ID: 39854223	2025

Jennifer Jeter

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