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Flybow – multicolour cell-labelling in Drosophila

07 February 2011

NIMR scientists have devised a genetic multi-colour cell labelling approach for Drosophila, called Flybow, to facilitate the visualization of cells in neural circuits with single cell resolution. The research is published in Nature Methods.

The shapes of individual neurons provide important insights into their identity, specific connectivity and functions within developing and adult neural circuits. However, anatomical and functional analyses of neuronal and glial subtypes in Drosophila are often hampered by the lack of enhancer elements that are specific for one single cell type. Moreover, because of the stochastic nature of available single cell-labelling techniques, visualization of individual cells in complex neural networks often relies on the analysis of a large number of samples.

To overcome these limitations Dafni Hadjieconomou and Iris Salecker (pictured) in NIMR’s Division of Molecular Neurobiology have collaborated with Shay Rotkopf and Barry J. Dickson in the Research Institute of Molecular Pathology (Vienna), Cyrille Alexandre in NIMR’s Division of Developmental Neurobiology and Donald M. Bell in NIMR’s Confocal Image Analysis Laboratory. They have generated three Drosophila variants of the mouse Brainbow system. This technique, developed in 2007 by Jean Livet et al. (Nature 450, 56), uses spectral fluorescent protein variants for multicolour labelling of individual cells within the same sample.

The approach for Drosophila, called Flybow, is based on the Brainbow-2 strategy but it has its own distinct features. These take advantage of available genetic techniques in Drosophila and support both anatomical and functional studies of genetically accessible cell populations in the nervous system and beyond.

Sequences encoding different fluorescent proteins were arranged in pairs within cassettes each flanked by recombination sites. Flybow uses the Gal4/UAS binary system to regulate transgene expression and an inducible modified Flp/FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters in different cell populations. Furthermore, all fluorescent proteins were membrane-tethered to promote labelling of fine processes. This study uses the fly visual system, the embryonic nervous system and the wing imaginal disc to validate the approach. It also shows that Flybow is compatible with available genetic tools to facilitate functional studies.

The idea for Flybow emerged after hearing the inspiring talk of Josh Sanes at a scientific meeting in Ascona a few years ago, in which he presented the mouse Brainbow system developed in his and Jeff Lichtman’s laboratories. We felt that there was a need for such an approach also in the Drosophilist’s toolbox. Flybow could find a variety of applications in the future and, for instance, facilitate the tracing and identification of neuron subtypes within neural circuits that are defined by the expression of neurotransmitters or transcription factors linked to specific behaviors. Because Flybow can be combined with genetic studies of mutant phenotypes, we hope that it will also help to identify the molecular mechanisms that underly the assembly of neural circuits as one path towards understanding their function.

Iris Salecker

Activity of Flybow 1.1 (FB1.1) in the 3rd instar larval (A) and adult visual system (B, C).

Click image to view at full-size

A pan-neuronal driver was used to drive expression of the FB1.1 transgene in the optic lobe. Heat-induced expression of Flp recombinase triggers excision and inversion events within the transgene leading to the expression of four fluorescent proteins (enhanced Green fluorescent protein (EGFP), mCitrine, mCherry and Cerulean tagged with V5) in different neurons in the same sample. FB1.1 provides sufficient resolution to trace and identify individual neuron subtypes based on their characteristic projection patterns.