Advertisement

Lighting Up Gene Activation in Living Drosophila Embryos

  • Carola Fernandez
  • Mounia LaghaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

Abstract

With its rapid development, ease of collection, and the presence of a unique layer of nuclei located close to the surface, the Drosophila syncytial embryo is ideally suited to study the establishment of gene expression patterns during development. Recent improvements in RNA labeling technologies and confocal microscopy allow for visualizing gene activation and quantifying transcriptional dynamics in living Drosophila embryos. Here we review the available tools for mRNA fluorescent labeling and detection in live embryos and precisely describe the overall procedure, from design to mounting and confocal imaging.

Key words

Live imaging Transcription Embryo Drosophila mRNA MS2/MCP system 

Notes

Acknowledgments

We are very grateful to J. Dufourt for critical reading and insightful suggestions on the manuscript. We thank M. Bellec, M. Dejean, and A. Trullo for helpful discussions. We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, “Investments for the future”). The ERC SyncDev and HFSP-CDA grants supported this work.

References

  1. 1.
    Garcia HG, Tikhonov M, Lin A et al (2013) Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr Biol 23:2140CrossRefGoogle Scholar
  2. 2.
    Lucas T, Ferraro T, Roelens B et al (2013) Live imaging of Bicoid-dependent transcription in Drosophila embryos. Curr Biol 23:2135CrossRefGoogle Scholar
  3. 3.
    Lucas T, Tran H, Perez Romero CA et al (2018) 3 minutes to precisely measure morphogen concentration. PLoS Genet 14:e1007676CrossRefGoogle Scholar
  4. 4.
    Chen H, Levo M, Barinov L et al (2018) Dynamic interplay between enhancer–promoter topology and gene activity. Nat Genet 50:1296–1303CrossRefGoogle Scholar
  5. 5.
    Bothma JP, Garcia HG, Esposito E et al (2014) Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc Natl Acad Sci U S A 111:10598CrossRefGoogle Scholar
  6. 6.
    Esposito E, Lim B, Guessous G et al (2016) Mitosis-associated repression in development. Genes Dev 30(13):1503–1508CrossRefGoogle Scholar
  7. 7.
    Ferraro T, Esposito E, Mancini L et al (2016) Transcriptional memory in the Drosophila embryo. Curr Biol 26:212–218CrossRefGoogle Scholar
  8. 8.
    Dufourt J, Trullo A, Hunter J et al (2018) Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs. Nat Commun 9:5194CrossRefGoogle Scholar
  9. 9.
    Fukaya T, Lim B, Levine M (2017) Rapid rates of pol II elongation in the Drosophila embryo. Curr Biol 27:1387–1391CrossRefGoogle Scholar
  10. 10.
    Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell 166:358CrossRefGoogle Scholar
  11. 11.
    Lim B, Heist T, Levine M et al (2018) Visualization of transvection in living Drosophila embryos. Mol Cell 70:287–296.e6CrossRefGoogle Scholar
  12. 12.
    Lim B, Fukaya T, Heist T et al (2018) Temporal dynamics of pair-rule stripes in living Drosophila embryos. Proc Natl Acad Sci 115(33):10430CrossRefGoogle Scholar
  13. 13.
    Bertrand E, Chartrand P, Schaefer M et al (2017) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437–445CrossRefGoogle Scholar
  14. 14.
    Larson DR, Zenklusen D, Wu B et al (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475–478CrossRefGoogle Scholar
  15. 15.
    Urbanek MO, Galka-marciniak P, Olejniczak M et al (2014) RNA imaging in living cells methods and applications. RNA Biol 11(8):1083–1095CrossRefGoogle Scholar
  16. 16.
    Pichon X, Lagha M, Mueller F et al (2018) A growing toolbox to image gene expression in single cells: sensitive approaches for demanding challenges. Mol Cell 71(3):468–480CrossRefGoogle Scholar
  17. 17.
    Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics 166:1775–1782CrossRefGoogle Scholar
  18. 18.
    Ren X, Sun J, Housden BE et al (2013) Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A 110:19012CrossRefGoogle Scholar
  19. 19.
    Ferraro T, Lucas T, Clémot M et al (2016) New methods to image transcription in living fly embryos: the insights so far, and the prospects. Wiley Interdiscip Rev Dev Biol 5(3):296–310CrossRefGoogle Scholar
  20. 20.
    Halstead JM, Lionnet T, Wilbertz JH et al (2015) Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science 347:1367–1671CrossRefGoogle Scholar
  21. 21.
    Forrest K, Gavis E (2003) Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr Biol 13:1159–1168CrossRefGoogle Scholar
  22. 22.
    Huang A, Amourda C, Zhang S et al (2017) Decoding temporal interpretation of the morphogen bicoid in the early drosophila embryo. elife 6:1–21Google Scholar
  23. 23.
    Weil TT, Parton RM, Davis I (2010) Making the message clear: visualizing mRNA localization. Trends Cell Biol 20:380–390CrossRefGoogle Scholar
  24. 24.
    Fusco D, Accornero N, Lavoie B et al (2003) Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr Biol 13:161CrossRefGoogle Scholar
  25. 25.
    Katsani KR, Karess RE, Nathalie Dostatni VD (2008) In vivo dynamics of Drosophila nuclear envelope components. Mol Biol Cell 19:3652–3666CrossRefGoogle Scholar
  26. 26.
    Trullo et al., in preparationGoogle Scholar
  27. 27.
    Tran H, Perez-romero CA, Ferraro T et al (2018) LiveFly: a toolbox for the analysis of transcription dynamics in live Drosophila embryos. Methods Mol Biol 1863:183–195CrossRefGoogle Scholar
  28. 28.
    Tantale K, Mueller F, Kozulic-Pirher A et al (2016) A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat Commun 7:12248CrossRefGoogle Scholar
  29. 29.
    Femino AM, Fay FS, Fogarty K et al (1998) Visualization of single RNA transcripts in situ. Science 280:585–590CrossRefGoogle Scholar
  30. 30.
    Keller PJ, Schmidt AD, Santella A et al (2010) Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods 7:637–642CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institut de Génétique Moléculaire de MontpellierUniversity of Montpellier, CNRSMontpellier, Cedex 5France

Personalised recommendations