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Brain Development pp 137–152Cite as

Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

Abstract

Visualization of single neurons and glia, as well as neural lineages within their complex environment is a pivotal step towards uncovering the mechanisms that control neural circuit development and function. This chapter provides detailed technical information on how to use Drosophila variants of the mouse Brainbow-2 system, called Flybow, for stochastic labeling of individual cells or lineages with different fluorescent proteins in one sample. We describe the genetic strategies and the heat shock regime required for induction of recombination events. Furthermore, we explain how Flybow and the mosaic analysis with a repressible cell marker (MARCM) approach can be combined to generate wild-type or homozygous mutant clones that are positively labeled in multiple colors. This is followed by a detailed protocol as to how to prepare samples for imaging. Finally, we provide specifications to facilitate multichannel image acquisition using confocal microscopy.

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References

  1. Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA et al (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450(7166):56–62

    Article  CAS  Google Scholar 

  2. Hampel S, Chung P, McKellar CE, Hall D, Looger LL, Simpson JH (2011) Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nat Methods 8(3):253–259

    Article  CAS  Google Scholar 

  3. Hadjieconomou D, Rotkopf S, Alexandre C, Bell DM, Dickson BJ, Salecker I (2011) Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat Methods 8(3):260–266

    Article  CAS  Google Scholar 

  4. Richier B, Salecker I (2015) Versatile genetic paintbrushes: Brainbow technologies. Wiley Interdiscip Rev Dev Biol 4(2):161–180

    Article  CAS  Google Scholar 

  5. Nern A, Pfeiffer BD, Rubin GM (2015) Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system. Proc Natl Acad Sci U S A 112(22):E2967–E2976

    Article  CAS  Google Scholar 

  6. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415

    CAS  PubMed  Google Scholar 

  7. Voziyanov Y, Konieczka JH, Stewart AF, Jayaram M (2003) Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site. J Mol Biol 326(1):65–76

    Article  CAS  Google Scholar 

  8. Liaw CW, Zamoyska R, Parnes JR (1986) Structure, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene. J Immunol 137(3):1037–1043

    CAS  PubMed  Google Scholar 

  9. Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569):913–916

    Article  CAS  Google Scholar 

  10. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448(7150):151–156

    Article  CAS  Google Scholar 

  11. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572

    Article  CAS  Google Scholar 

  12. Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22(4):445–449

    Article  CAS  Google Scholar 

  13. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909

    Article  CAS  Google Scholar 

  14. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276(31):29188–29194

    Article  CAS  Google Scholar 

  15. Shimosako N, Hadjieconomou D, Salecker I (2014) Flybow to dissect circuit assembly in the Drosophila brain. Methods Mol Biol 1082:57–69

    Article  CAS  Google Scholar 

  16. Goedhart J, van Weeren L, Hink MA, Vischer NO, Jalink K, Gadella TW Jr (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7(2):137–139

    Article  CAS  Google Scholar 

  17. Hadjieconomou D, Timofeev K, Salecker I (2011) A step-by-step guide to visual circuit assembly in Drosophila. Curr Opin Neurobiol 21(1):76–84

    Article  CAS  Google Scholar 

  18. Hayashi S, Ito K, Sado Y, Taniguchi M, Akimoto A, Takeuchi H et al (2002) GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4 enhancer traps. Genesis 34(1-2):58–61

    Article  CAS  Google Scholar 

  19. Timofeev K, Joly W, Hadjieconomou D, Salecker I (2012) Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila. Neuron 75(1):80–93

    Article  CAS  Google Scholar 

  20. Apitz H, Salecker I (2018) Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9(1):2295

    Article  Google Scholar 

  21. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW et al (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5(6):545–551

    Article  CAS  Google Scholar 

  22. Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA et al (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun 3:751

    Article  Google Scholar 

  23. Green EW, Fedele G, Giorgini F, Kyriacou CP (2014) A Drosophila RNAi collection is subject to dominant phenotypic effects. Nat Methods 11(3):222–223

    Article  CAS  Google Scholar 

  24. Vissers JH, Manning SA, Kulkarni A, Harvey KF (2016) A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth. Nat Commun 7:10368

    Article  CAS  Google Scholar 

  25. Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22(3):451–461

    Article  CAS  Google Scholar 

  26. Enriquez J, Venkatasubramanian L, Baek M, Peterson M, Aghayeva U, Mann RS (2015) Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes. Neuron 86(4):955–970

    Article  CAS  Google Scholar 

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Acknowledgments

We thank J. Goedhart for sharing the mTurquoise and mTurquoise2 cDNA. D. Hadjieconomou developed the original and C sets of FB transgenes. N. Shimosako generated the B set of transgenes and validated the hs-mFLP5MH insertions. The original FB approach was developed in collaboration with B.J. Dickson, S. Rotkopf, C. Alexandre, and D.M. Bell. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001151), the UK Medical Research Council (FC001151), and the Wellcome Trust (FC001151), and by the UK Medical Research Council (U117581332).

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Authors and Affiliations

  1. Visual Circuit Assembly Laboratory, The Francis Crick Institute, London, UK

    Emma L. Powell & Iris Salecker

Authors
  1. Emma L. Powell
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  2. Iris Salecker
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Corresponding author

Correspondence to Iris Salecker .

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Editors and Affiliations

  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G. Sprecher

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Powell, E.L., Salecker, I. (2020). Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

  • eBook Packages: Springer Protocols

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