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

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila 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.

Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.

Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons.

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.

Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis.

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.

Genetic mosaic analysis in the nervous system.

Genetic mosaic techniques provide a powerful tool for dissecting gene function in the intricate genetic networks that underlie the formation and function of nervous systems. For instance, it is possible to make individual cells or groups of cells homozygous for mutations of interest at specific points during an organism's development. It is also possible to resolve lineage relationships and to characterize cellular morphology and connectivity. Current techniques for creating genetically mosaic organisms incorporate improved controls over clone induction, identification, and/or mosaic tissue characterization.

Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts.

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

A GAL4-driver line resource for Drosophila neurobiology.

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of 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.

Baboon/dSmad2 TGF-beta signaling is required during late larval stage for development of adult-specific neurons.

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2-mediated TGF-beta signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult-specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis.