RNA Detection pp 163-175 | Cite as

Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction

  • Joshua S. Titlow
  • Lu Yang
  • Richard M. Parton
  • Ana Palanca
  • Ilan Davis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1649)

Abstract

The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.

Key words

smFISH Single molecule fluorescence in situ hybridization Structured Illumination Super-resolution imaging 3D-SIM Drosophila melanogaster Larval neuromuscular junction mRNA localization Synapse 

Notes

Acknowledgments

We thank Talila Volk (Weizmann Institute of Science, Rehovot, Israel) for the Msp300 antibody; Flybase and the Bloomington Drosophila Stock Center for resources. We also thank David Ish-Horowicz and members of the Davis lab for discussions and comments on the manuscript. This work was supported by a Wellcome Trust Senior Basic Biomedical Research Fellowship (096144) to I.D., Wellcome Trust Strategic Awards (091911 and 107457/Z/15/Z) supporting advanced microscopy at Micron Oxford (http://micronoxford.com), and a Clarendon scholarship to LY.

References

  1. 1.
    Femino AM et al (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590CrossRefPubMedGoogle Scholar
  2. 2.
    Raj A et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol 714:3–13CrossRefPubMedGoogle Scholar
  4. 4.
    Dubnau J, Tully T (1998) Gene discovery in Drosophila: new insights for learning and memory. Annu Rev Neurosci 21:407–444CrossRefPubMedGoogle Scholar
  5. 5.
    Skoulakis EM, Grammenoudi S (2006) Dunces and da Vincis: the genetics of learning and memory in Drosophila. Cell Mol Life Sci 63(9):975–988CrossRefPubMedGoogle Scholar
  6. 6.
    Walkinshaw E et al (2015) Identification of genes that promote or inhibit olfactory memory formation in Drosophila. Genetics 199(4):1173–1182CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pradhan SJ et al (2012) The conserved P body component HPat/Pat1 negatively regulates synaptic terminal growth at the larval Drosophila neuromuscular junction. J Cell Sci 125(Pt 24):6105–6116CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nesler KR et al (2013) The miRNA pathway controls rapid changes in activity-dependent synaptic structure at the Drosophila melanogaster neuromuscular junction. PLoS One 8(7):e68385CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Abbaszadeh EK, Gavis ER (2016) Fixed and live visualization of RNAs in Drosophila oocytes and embryos. Methods 98:34–41CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Trcek T et al (2015) Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat Commun 6:7962CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Karr J et al (2009) Regulation of glutamate receptor subunit availability by microRNAs. J Cell Biol 185(4):685–697CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gardiol A, St Johnston D (2014) Staufen targets coracle mRNA to Drosophila neuromuscular junctions and regulates GluRIIA synaptic accumulation and bouton number. Dev Biol 392(2):153–167CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Packard M et al (2015) Nucleus to synapse Nesprin1 railroad tracks direct synapse maturation through RNA localization. Neuron 86(4):1015–1028CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gustafsson MG et al (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94(12):4957–4970CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190(2):165–175CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Herbert AD, Carr AM, Hoffmann E (2014) FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis. PLoS One 9(12):e114749CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Mueller F et al (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4):277–278CrossRefPubMedGoogle Scholar
  18. 18.
    Ball G et al (2015) SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci rep 5:15915CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Verstreken P, Ohyama T, Bellen HJ (2008) FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction. Methods Mol Biol 440:349–369CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Brent JR, Werner KM, McCabe BD (2009) Drosophila larval NMJ dissection. J Vis Exp (24):–1107Google Scholar
  21. 21.
    Smith, R. and J.P. Taylor, (2011). Dissection and imaging of active zones in the Drosophila neuromuscular junction. J Vis Exp, (50): 2676Google Scholar
  22. 22.
    Dobbie IM et al (2011) OMX: a new platform for multimodal, multichannel wide-field imaging. Cold Spring Harb Protoc 2011(8):899–909CrossRefPubMedGoogle Scholar
  23. 23.
    Demmerle J et al (2015) Assessing resolution in super-resolution imaging. Methods 88:3–10CrossRefPubMedGoogle Scholar
  24. 24.
    Muller M et al (2016) Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nat Commun 7:10980CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Allan C et al (2012) OMERO: flexible, model-driven data management for experimental biology. Nat Methods 9(3):245–253CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Burel JM et al (2015) Publishing and sharing multi-dimensional image data with OMERO. Mamm Genome 26(9–10):441–447CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Li S et al (2016) Metadata management for high content screening in OMERO. Methods 96:27–32CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gopal A et al (2012) Visualizing large RNA molecules in solution. RNA 18(2):284–299CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kner P et al (2010) High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing. J Microsc 237(2):136–147CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Authors and Affiliations

  • Joshua S. Titlow
    • 1
  • Lu Yang
    • 1
  • Richard M. Parton
    • 1
  • Ana Palanca
    • 1
  • Ilan Davis
    • 1
  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK

Personalised recommendations