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Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster

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To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence 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 within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.

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Figure 1: Schematic of Flybow variants.
Figure 2: Activity of FB1.0 and FB1.1 transgenes.
Figure 3: Expression of FB1.1 transgenes in distinct cell populations.
Figure 4: FB2.0 facilitates sparse labeling of cells within a Gal4-expressing cell population.
Figure 5: Combining Flybow and MARCM for functional mosaic analysis.

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  • 16 February 2011

    In the version of this article initially published online, accession codes were not included. The error has been corrected for the print, PDF and HTML versions of this article.


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We thank W. Joly and E. Ober (MRC National Institute for Medical Research) for sharing fluorescent protein cDNAs; Y. Voziyanov (University of Texas) for the altered-specificity Flp and FRT reagents; T. Hummel (University of Muenster), K. Keleman (Research Institute of Molecular Pathology), I. Miguel-Aliaga (University of Cambridge) and the members of the Bloomington Drosophila Stock Center for fly stocks; W. Joly for his technical advice for cloning; H. Apitz for help with dissections and mounting of some samples; and E. Ober, J.P. Vincent, S. Tozer, H. Apitz, E. Richardson, B. Richier, K. Timofeev and N. Shimosako for critical comments on the manuscript. Basic research at the Research Institute of Molecular Pathology is funded by Boehringer Ingelheim (S.R. and B.J.D.). The work at the National Institute for Medical Research is supported by the MRC (COIM1175, D.M.B.; U117581332, D.H. and I.S.).

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Authors and Affiliations



I.S., B.J.D., D.H. and C.A. designed the Flybow strategy. D.H. cloned the Flybow constructs, D.H. and I.S. generated the transgenic fly stocks, and D.H. conducted the experimental analysis. S.R. and B.J.D. developed the modified Flp-FRT system, and provided the original pKC26 UAS vector and the wild-type Flp-out cassette. C.A. provided expert advice for cloning, and D.M.B. provided expert advice for image acquisition and analysis. I.S. and D.H. wrote the manuscript in interaction with all contributing authors.

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Correspondence to Iris Salecker.

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The authors declare no competing financial interests.

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Hadjieconomou, D., Rotkopf, S., Alexandre, C. et al. Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat Methods 8, 260–266 (2011).

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