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

Journal name:
Nature Methods
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Published online
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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.

At a glance


  1. Schematic of Flybow variants.
    Figure 1: Schematic of Flybow variants.

    (ac) Pairs of fluorescent protein–encoding cDNAs are arranged in opposing orientations and flanked by mFRT71 sites (black arrowheads). Fluorescent proteins are membrane-tethered either by a Cd8a (cd8) or the palm-myr (pm) sequence of Lyn kinase. Fluorescent protein sequences are followed by SV40 and hsp70Ab polyadenylation (pA) signals. Constructs were subcloned into a modified pKC26 UAS vector, which contains ten UAS (10xUAS) sites and a short attB recognition sequence. mFlp5 under the control of the heat-shock promoter (hs-mFlp5) induces inversions of DNA cassettes by recombining mFRT71 sites in opposing orientations, or excisions (Flp-out) by recombining mFRT71 sites placed in the same orientation. FB1.0 (a) consists of one invertible cassette encoding two fluorescent proteins (mCherry and Cerulean-V5). FB1.1 (b) contains two invertible cassettes, each encoding two fluorescent proteins (EGFP and mCitrine; mCherry and Cerulean-V5). FB2.0 (c) contains an additional stop cassette, flanked by canonical FRT sites (white arrowheads) facing in the same orientation, which can be excised by wild-type Flp. The stop cassette consists of lamin cDNA, followed by two HA tag sequences and hsp70Aa and hsp27 polyadenylation signals.

  2. Activity of FB1.0 and FB1.1 transgenes.
    Figure 2: Activity of FB1.0 and FB1.1 transgenes.

    (a,b) Schematic of the third instar larval (3L) (a) and adult visual system (b). Shown are R1–R8 photoreceptor axons, lamina neurons (ln) L1–L5, and the medulla neuron (mn) and lobula neuron and lobula plate neuron subtypes Tm, TmY, Dm and T, which are main target neuron subtype classes. Glial subtypes include epithelial glia (eg), marginal glia (mg), medulla glia (meg) and medulla neuropil glia (mng)15. GMC, ganglion mother cell; LPC, lamina precursor cells; MF, morphogenetic furrow; Nb, neuroblasts; and OPC, outer proliferation center. (c) Schematic showing that FB1.0 enables expression of Cerulean-V5 instead of mCherry in a Gal4-expressing cell population upon heat-shock induction of mFlp5. (d,e) Confocal images of a larval eye disc (d) and optic lobe (e) with some R cells and lamina neurons expressing Cerulean-V5. la, lamina; me, medulla. (f) Schematic showing that FB1.1 leads to expression of EGFP, mCitrine, mCherry and Cerulean-V5. (g,h) Confocal images of larval R cells (g) and of lineages of younger (y, asterisks) and individual older (o, arrowheads) medulla neurons (h) expressing different fluorescent proteins. (il) Differentially labeled adult neuron subtypes14: lamina neurons L5 (i,j), lineage-related ascending T2–T5 neurons (k), an amacrine Dm neuron and the transmedullary neurons Tm18 (arrows) and TmY5a (arrowheads) (l). lo, lobula; lop, lobula plate. (m,n) Sagittal view of a live stage 16 embryo with clusters and single neurons expressing mCitrine or mCherry (arrowheads). Arrow, cluster expressing EGFP and mCherry owing to perdurance; asterisk, unlabeled cluster of Cerulean-V5–expressing neurons. Insets, live growth cone extending from the ventral nerve cord (VNC) into the peripheral nervous system (PNS). (o,p) Flat preparation of a fixed VNC with large (arrows) or smaller (arrowheads) growth cones exploring lateral (l) tracts and anterior (ac) or posterior (pc) commissures. (q) PNS neurons, including the lateral chordotonal organ (lch). elav-Gal4c155 was used as pan-neuronal driver. EGFP, mCitrine and mCherry were detected using endogenous fluorescence signals and Cerulean using immunolabeling with antibody to V5. Scale bars, 50 μm (d,e,gq) and 20 μm (insets, n).

  3. Expression of FB1.1 transgenes in distinct cell populations.
    Figure 3: Expression of FB1.1 transgenes in distinct cell populations.

    (ae) Confocal images showing third instar larval (3L) and adult R1–R8 photoreceptor axons labeled using GMR-Gal4. R1–R6 growth cones (arrowheads) in the lamina (la), and young (double arrowheads) and mature (arrows). R8 growth cones in the medulla (me) are highlighted in a,b. Adult R8 and R7 terminals in a column express the same fluorescent protein (arrowhead) or combinations of two fluorescent proteins (asterisks) (ce). (fi) Single optical sections (f,g) and a 10 μm z-stack projection of the mCitrine channel (h) of adult optic lobes in which MzVum-Gal4 drives FB1.1 expression in medulla neurons. One neuron (arrow; g,i) traced through a series of consecutive sections had TmY5a neuron-like features; asterisks indicate additional or absent branches compared to reported morphology14. (j,k) Epithelial (eg) and marginal (mg) glia in the lamina, and medulla neuropil glia (mng) at the distal medulla neuropil border were labeled with different fluorescent proteins in third instar larval (j) and adult (k) optic lobes using repo-Gal4 and FB1.1. In k, fluorescence signals in the lamina above the white line were reduced relative to those in the medulla. (lo) Higher magnification of the image in k, showing elaborate shapes of epithelial and medulla neuropil glia. (p) Area with overlapping medulla neuropil glial cell branches (arrowhead) in a medulla cross-section. (q,r) en-Gal4–driven expression of different fluorescent proteins in epithelial cell clones in the posterior (p) compartment of wing discs. d, dorsal. Scale bars, 50 μm (a,c,fk,pr) and 10 μm (b,d,e,lo).

  4. FB2.0 facilitates sparse labeling of cells within a Gal4-expressing cell population.
    Figure 4: FB2.0 facilitates sparse labeling of cells within a Gal4-expressing cell population.

    (a) Schematic showing that upon heat induction, Flp excises the upstream FRT stop cassette to enable reporter expression in a subset of Gal4-expressing cells. Induction of hs-mFlp5 randomizes the fluorescent protein selection as in FB1.1. Expression of four fluorescent proteins is restricted to cells with overlapping Gal4, Flp and mFlp5 activities. (be) Confocal images showing that elav-Gal4c155 in conjunction with FB2.0 led to fluorescent protein expression in only a small subset of R cells in eye discs posterior to the morphogenic furrow (b), and of lamina and medulla neurons (mn) in larval (c) and adult (d,e) optic lobes. Labeling of fewer cells facilitated the identification of neuron subtypes in the dense neuropils of the lamina (la), medulla (me), lobula (lo) and lobula plate (lop). Surrounded by mCherry-expressing medulla neurons, lamina neurons subtype L3 can be identified by mCitrine expression in the adult medulla. Scale bars, 50 μm.

  5. Combining Flybow and MARCM for functional mosaic analysis.
    Figure 5: Combining Flybow and MARCM for functional mosaic analysis.

    (a) Schematic of Flp-FRT–mediated mitotic recombination in trans during the G2-M phase of the cell cycle and subsequent chromosome segregation, which leads to the loss of the Gal80 repressor in one of the daughter cells, enabling reporter gene expression. This Gal80-free cell is homozygous for any mutation located on the homologous chromosome arm (vertical bar on black chromosome). mFlp5-mFRT71–mediated recombination of the FB1.1 transgene in cis leads to the stochastic expression of one of four fluorescent proteins in progeny not expressing Gal80. (bf) Confocal images of adult optic lobes (b,c) and higher magnifications of medulla neuropil (df) showing that elav-Gal4c155 in conjunction with FB1.1 drives expression of EGFP, mCitrine and mCherry in wild-type control and CadNM19 homozygous mutant neurons in the adult lamina (la), medulla (me), lobula (lo) and lobula plate (lop). ln, lamina neurons. R-cell axons were labeled with the photoreceptor-specific antibody mAb24B10 (blue). Shown are mCitrine-expressing lamina neuron L1 innervating layers M1 and M5 (d) and mCherry-expressing lamina neurons L5 terminating in the M1, M2 and M5 layers (e) in controls. In the latter, axonal arbors can be distinguished from overlapping branches of neighboring neurons expressing EGFP or mCitrine (arrowhead). Also shown are mCherry-expressing L1 and L5 neurons with aberrant projections in the absence of CadNM19 (f). (g) Schematic illustrating the axonal arborizations of control and CadNM19 homozygous mutant L1 and L5 neurons. Scale bars, 50 μm (b,c) and 10 μm (df).

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Corrected online 16 February 2011
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Author information


  1. Medical Research Council (MRC) National Institute for Medical Research, Division of Molecular Neurobiology, London, UK.

    • Dafni Hadjieconomou &
    • Iris Salecker
  2. Research Institute of Molecular Pathology, Vienna, Austria.

    • Shay Rotkopf &
    • Barry J Dickson
  3. MRC National Institute for Medical Research, Division of Developmental Neurobiology, London, UK.

    • Cyrille Alexandre
  4. MRC National Institute for Medical Research, Confocal Image Analysis Laboratory, London, UK.

    • Donald M Bell
  5. Present address: Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel.

    • Shay Rotkopf


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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