Skip to main content
SpringerLink
Log in

Imaging Cytonemes in Drosophila Embryos

  • Protocol
  • First Online:
Morphogen Gradients

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1863))

Abstract

Conserved morphogenetic signaling proteins disperse across tissues to generate signal and signaling gradients, which in turn are considered to assign positional coordinates to the recipient cells. Recent imaging studies in Drosophila model have provided evidence for a “direct-delivery” mechanism of signal dispersion that is mediated by specialized actin-rich signaling filopodia, named cytonemes. Cytonemes establish contact between the signal-producing and target cells to directly exchange and transport the morphogenetic proteins. Although an increasing amount of evidence supports the critical role of these specialized signaling structures, imaging these highly dynamic 200 nm-thin structures in the complex three-dimensional contour of living tissues is challenging. Here, we describe the imaging methods that we optimized for studying cytonemes in Drosophila embryos.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Change history

  • 07 December 2018

    The book was published with the following errors: In Chapter 1, figure 1 was stretched. This misconfiguration has now been modified.

References

  1. Entchev EV, Schwabedissen A, González-Gaitán M (2000) Gradient formation of the TGF-β Homolog Dpp. Cell 103:981–992. https://doi.org/10.1016/S0092-8674(00)00200-2

    Article  CAS  PubMed  Google Scholar 

  2. Teleman AA, Cohen SM (2000) Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103:971–980

    Article  CAS  PubMed  Google Scholar 

  3. Goentoro LA, Reeves GT, Kowal CP et al (2006) Quantifying the Gurken morphogen gradient in Drosophila oogenesis. Dev Cell 11:263–272. https://doi.org/10.1016/j.devcel.2006.07.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Strigini M, Cohen SM (2000) Wingless gradient formation in the Drosophila wing. Curr Biol 10:293–300

    Article  CAS  PubMed  Google Scholar 

  5. Wolpert L (1969) Positional information and the spatial pattern of cellular differentiation. J Theor Biol 25:1–47

    Article  CAS  PubMed  Google Scholar 

  6. Rogers KW, Schier AF (2011) Morphogen gradients: from generation to interpretation. Annu Rev Cell Dev Biol 27:377–407. https://doi.org/10.1146/annurev-cellbio-092910-154148

    Article  CAS  PubMed  Google Scholar 

  7. Wolpert L (2016) Positional information and pattern formation. Curr Top Dev Biol 117:597–608. https://doi.org/10.1016/bs.ctdb.2015.11.008

    Article  PubMed  Google Scholar 

  8. Müller P, Rogers KW, Yu SR et al (2013) Morphogen transport. Development 140:1621–1638. https://doi.org/10.1242/dev.083519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Christian JL (2012) Morphogen gradients in development: from form to function. Wiley Interdiscip Rev Dev Biol 1:3–15. https://doi.org/10.1002/wdev.2

    Article  CAS  PubMed  Google Scholar 

  10. Schwank G, Dalessi S, Yang S-F et al (2011) Formation of the long range Dpp morphogen gradient. PLoS Biol 9:e1001111. https://doi.org/10.1371/journal.pbio.1001111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhou S, Lo W-C, Suhalim JL et al (2012) Free extracellular diffusion creates the Dpp morphogen gradient of the Drosophila wing disc. Curr Biol 22:668–675. https://doi.org/10.1016/j.cub.2012.02.065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yu SR, Burkhardt M, Nowak M et al (2009) Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461:533–536. https://doi.org/10.1038/nature08391

    Article  CAS  PubMed  Google Scholar 

  13. Dubrulle J, Pourquié O (2004) fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427:419–422. https://doi.org/10.1038/nature02216

    Article  CAS  PubMed  Google Scholar 

  14. Shilo B-Z, Haskel-Ittah M, Ben-Zvi D et al (2013) Creating gradients by morphogen shuttling. Trends Genet 29:339–347. https://doi.org/10.1016/j.tig.2013.01.001

    Article  CAS  PubMed  Google Scholar 

  15. Entchev EV, Schwabedissen A, Gonzalez-Gaitan M (2000) Gradient formation of the TGF-beta homolog Dpp. Cell 103:981–991

    Article  CAS  PubMed  Google Scholar 

  16. Belenkaya TY, Han C, Yan D et al (2004) Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans. Cell 119:231–244. https://doi.org/10.1016/j.cell.2004.09.031

    Article  CAS  PubMed  Google Scholar 

  17. Kornberg TB, Roy S (2014) Cytonemes as specialized signaling filopodia. Development 141:729–736. https://doi.org/10.1242/dev.086223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kornberg TB (2017) Distributing signaling proteins in space and time: the province of cytonemes. Curr Opin Genet Dev 45:22–27. https://doi.org/10.1016/j.gde.2017.02.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ramírez-Weber FA, Kornberg TB (1999) Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97:599–607

    Article  PubMed  Google Scholar 

  20. Hsiung F, Ramirez-Weber F-A, Iwaki DD, Kornberg TB (2005) Dependence of Drosophila wing imaginal disc cytonemes on decapentaplegic. Nature 437:560–563. https://doi.org/10.1038/nature03951

    Article  CAS  PubMed  Google Scholar 

  21. Roy S, Hsiung F, Kornberg TB (2011) Specificity of Drosophila cytonemes for distinct signaling pathways. Science 332:354–358. https://doi.org/10.1126/science.1198949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sato M, Kornberg TB (2002) FGF is an essential mitogen and chemoattractant for the air sacs of the drosophila tracheal system. Dev Cell 3:195–207

    Article  CAS  PubMed  Google Scholar 

  23. Roy S, Huang H, Liu S, Kornberg TB (2014) Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein. Science 343:1244624–1244624. https://doi.org/10.1126/science.1244624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Huang H, Kornberg TB (2016) Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes. elife 5:197. https://doi.org/10.7554/eLife.18979

    Article  CAS  Google Scholar 

  25. Roy S, Kornberg TB (2011) Direct delivery mechanisms of morphogen dispersion. Sci Signal 4:pt8. https://doi.org/10.1126/scisignal.2002434

    Article  PubMed  Google Scholar 

  26. Rojas-Rios P, Guerrero I, Gonzalez-Reyes A (2012) Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain Germline stem cells in Drosophila. PLoS Biol 10:e1001298–e1001213. https://doi.org/10.1371/journal.pbio.1001298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Roy S, Kornberg TB (2015) Paracrine signaling mediated at cell-cell contacts. BioEssays 37:25–33. https://doi.org/10.1002/bies.201400122

    Article  CAS  PubMed  Google Scholar 

  28. Kornberg TB (2014) Cytonemes and the dispersion of morphogens. Wiley Interdiscip Rev Dev Biol 3:445–463. https://doi.org/10.1002/wdev.151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Du L, Sohr A, Ge Y, et al (2018) Feedback regulation of cytoneme-mediated transport shapes a tissue-specific FGF morphogen gradient. eLife. https://doi.org/10.7554/eLife.38137

  30. Sanders TA, Llagostera E, Barna M (2013) Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 497:628–632. https://doi.org/10.1038/nature12157

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Huang H, Kornberg TB (2015) Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-notch signaling to the air sac primordium. elife 4:e06114. https://doi.org/10.7554/eLife.06114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Holzer T, Liffers K, Rahm K et al (2012) Live imaging of active fluorophore labelled Wnt proteins. FEBS Lett 586(11):1638–1644. https://doi.org/10.1016/j.febslet.2012.04.035

    Article  CAS  PubMed  Google Scholar 

  33. Stanganello E, Scholpp S (2016) Role of cytonemes in Wnt transport. J Cell Sci 129:665–672. https://doi.org/10.1242/jcs.182469

    Article  CAS  PubMed  Google Scholar 

  34. Buszczak M, Inaba M, Yamashita YM (2016) Signaling by cellular protrusions: keeping the conversation private. Trends Cell Biol 26:526–534. https://doi.org/10.1016/j.tcb.2016.03.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Du L, Roy S (2017) Cytonemes mediate formation of a morphogen gradient of FGF during branching morphogenesis of Drosophila trachea. Mol Biol Cell 28:3727 (abstract #M37)

    Google Scholar 

  36. Seijo-Barandiarán I, Guerrero I, Bischoff M (2015) In vivo imaging of hedgehog transport in Drosophila epithelia. Methods Mol Biol 1322:9–18. https://doi.org/10.1007/978-1-4939-2772-2_2

    Article  PubMed  Google Scholar 

  37. Stocker H, Gallant P (2008) Getting started: an overview on raising and handling Drosophila. Methods Mol Biol 420:27–44. https://doi.org/10.1007/978-1-59745-583-1_2

    Article  PubMed  Google Scholar 

  38. Du L, Zhou A, Patel A Rao M, Anderson K, Roy S (2017) Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis. Dev Biol 427:35-48. https://doi.org/10.1016/j.ydbio.2017.05.009

    Article  CAS  PubMed  Google Scholar 

  39. Yu D, Gustafson WC, Han C et al (2014) An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging. Nat Commun 5:3626. https://doi.org/10.1038/ncomms4626

    Article  CAS  PubMed  Google Scholar 

  40. Ghabrial A, Luschnig S, Metzstein MM, Krasnow MA (2003) Branching morphogenesis of the Drosophila tracheal system. Annu Rev Cell Dev Biol 19:623–647. https://doi.org/10.1146/annurev.cellbio.19.031403.160043

    Article  CAS  PubMed  Google Scholar 

  41. Chen W, Huang H, Hatori R, Kornberg TB (2017) Essential basal cytonemes take up hedgehog in the Drosophila wing imaginal disc. Development 144:3134–3144. https://doi.org/10.1242/dev.149856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sutherland D, Samakovlis C, Krasnow MA (1996) Branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87:1091–1101

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. T.B. Kornberg and the Bloomington Stock Center for reagents, colleagues and lab members, especially Alex Sohr for reading the manuscript and valuable suggestions, UMD Imaging core facility, and Dr. A.E. Beaven for assistance in the imaging core. Funding from NIH: R00HL114867 and R35GM124878 to S.R.

Author information

Authors and Affiliations

  1. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA

    Lijuan Du & Sougata Roy

Authors
  1. Lijuan Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sougata Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sougata Roy .

Editor information

Editors and Affiliations

  1. Integrated Microscopy Core, Baylor College of Medicine, Houston, TX, USA

    Julien Dubrulle

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Du, L., Roy, S. (2018). Imaging Cytonemes in Drosophila Embryos. In: Dubrulle, J. (eds) Morphogen Gradients. Methods in Molecular Biology, vol 1863. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8772-6_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8772-6_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8771-9

  • Online ISBN: 978-1-4939-8772-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation