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Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns

Nature Methods volume 8, pages 253–259 (2011)Cite this article

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A Corrigendum to this article was published on 28 August 2015

A Corrigendum to this article was published on 30 August 2012

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Abstract

We developed a multicolor neuron labeling technique in Drosophila melanogaster that combines the power to specifically target different neural populations with the label diversity provided by stochastic color choice. This adaptation of vertebrate Brainbow uses recombination to select one of three epitope-tagged proteins detectable by immunofluorescence. Two copies of this construct yield six bright, separable colors. We used Drosophila Brainbow to study the innervation patterns of multiple antennal lobe projection neuron lineages in the same preparation and to observe the relative trajectories of individual aminergic neurons. Nerve bundles, and even individual neurites hundreds of micrometers long, can be followed with definitive color labeling. We traced motor neurons in the subesophageal ganglion and correlated them to neuromuscular junctions to identify their specific proboscis muscle targets. The ability to independently visualize multiple lineage or neuron projections in the same preparation greatly advances the goal of mapping how neurons connect into circuits.

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Figure 1: Schematic of the dBrainbow construct.
Figure 2: Comparison of endogenous and antibody-based fluorescence of UAS-dBrainbow flies.
Figure 3: Expression of UAS-dBrainbow in three projection neuron lineages.
Figure 4: dBrainbow labeling of lineages or individual neurons in different colors.
Figure 5: Expression of UAS-dBrainbow in motor neurons that connect the subesophageal ganglion to the proboscis muscles in sections of a single example of an hs-cre; UAS-dBrainbow; R12D05-GAL4 fly.

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NCBI Reference Sequence

Change history

  • 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.

  • 30 August 2012

    The schematic of the dBrainbow vector construct presented in the original Figure 1 of the paper and its description had incorrect epitope tags. This error has not been corrected in the HTML or PDF versions of the article, but a correct version of the figure as well as full details of the error can be found in the corrigendum.

  • 03 August 2015

    In the version of this article initially published, the sequence reported for the dBrainbow construct was incorrect. The blue fluorescent protein was reported as EBFP2; it is mTFP1. The mKO2 protein is tagged with a V5 epitope and not a Myc epitope, and the EGFP protein is tagged with HSV, not with V5, as detailed in a previous correction to this paper. The red channel reflects endogenous mKO2 fluorescence, not anti-Myc staining. The mKO2 protein is produced by a codon-optimized sequence. These errors have been corrected in the HTML and PDF versions of the article, and the correct sequence is provided.

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Acknowledgements

We thank A. Arnold, E. Shumsky and A. Soell for help with imaging and spectral separation; B. Pfeiffer, A. Nern and G. Rubin (Janelia Farm Research Campus) for sharing vectors and recombinase lines before publication and for the gift of R12D05-GAL4; V. Hartenstein, B. Gerber, T. Lee, B. Baker and P. Keller for helpful discussions about biological applications of dBrainbow; S. Albin, A. Seeds and E. Hoopfer for scientific discussion of the project; D. Grover for statistical advice; and K. Basler, R. Yagi and C. Lehner (University of Zurich) and M. Siegal (New York University) for additional Cre lines.

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Author notes

  1. Stefanie Hampel and Phuong Chung: These authors contributed equally to this work.

Authors and Affiliations

  1. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Stefanie Hampel, Phuong Chung, Claire E McKellar, Donald Hall, Loren L Looger & Julie H Simpson

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  1. Stefanie Hampel
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Contributions

S.H. designed and performed cloning, tested constructs in S2 cells and made the figures. P.C. performed the fly genetics, immunohistochemistry and confocal imaging. C.E.M. generated and analyzed the subesophageal ganglion and proboscis data. D.H. generated the recombinant fly stocks. L.L.L. advised on selection of fluorescent proteins, construct design and the conversion from endogenous fluorescence to antibody. J.H.S. conceived the project, cloned initial test constructs and wrote the paper with help from S.H., P.C., C.E.M. and L.L.L.

Corresponding author

Correspondence to Julie H Simpson.

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The authors declare no competing financial interests.

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Hampel, S., Chung, P., McKellar, C. et al. Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nat Methods 8, 253–259 (2011). https://doi.org/10.1038/nmeth.1566

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