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Long-term in vivo imaging of Drosophila larvae

Abstract

The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology and responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, longer-term live imaging has been technically challenging because of a lack of efficient methods for immobilizing larvae for extended periods. We describe here a simple procedure for anesthetization and uninterrupted long-term in vivo imaging of the epidermis and other larval organs, including gut, imaginal discs, neurons, fat body, tracheae, muscles and hemocytes, for up to 8 h. We also include a procedure for probing cell properties by laser ablation. We provide a survey of the effects of different anesthetics, demonstrating that short exposure to diethyl ether is the most effective for long-term immobilization of larvae. This protocol does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and subcellular processes in larvae.

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Fig. 1: Effect of different anesthetics on larval survival.
Fig. 2: Outline of the experimental procedure.
Fig. 3: Overview of equipment required to anesthetize larvae.
Fig. 4: Construction of the larval cage.
Fig. 5: Construction of anesthetization chamber.
Fig. 6: Anesthetization and mounting the larvae for live imaging.
Fig. 7: Live imaging of inner organs and subcellular organelles.
Fig. 8: Epidermis of Drosophila larva.
Fig. 9: Signs of reduced larval vitality.
Fig. 10: Single- and multi-cell laser wounding in the larval epidermis.
Fig. 11: Live imaging of insulin/PIP3/FOXO signaling and the effect of reduced insulin receptor signaling on PIP3.

Data availability

All data generated and analyzed during the current study are available from the corresponding authors upon request.

References

  1. 1.

    Dobie, K. W. et al. Identification of chromosome inheritance modifiers in Drosophila melanogaster. Genetics 157, 1623–1637 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Hales, K. G., Korey, C. A., Larracuente, A. M. & Roberts, D. M. Genetics on the fly: a primer on the Drosophila model system. Genetics 201, 815–842 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Wieschaus, E. & Nusslein-Volhard, C. The Heidelberg Screen for pattern mutants of Drosophila: a personal account. Annu. Rev. Cell Dev. Biol. 32, 1–46 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Abreu-Blanco, M. T., Verboon, J. M. & Parkhurst, S. M. Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling. J. Cell Biol. 193, 455–464 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Tsai, C. R., Wang, Y. & Galko, M. J. Crawling wounded: molecular genetic insights into wound healing from Drosophila larvae. Int. J. Dev. Biol. 62, 479–489 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Razzell, W., Wood, W. & Martin, P. Swatting flies: modelling wound healing and inflammation in Drosophila. Dis. Model. Mech. 4, 569–574 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Losick, V. P., Fox, D. T. & Spradling, A. C. Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium. Curr. Biol. 23, 2224–2232 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Belacortu, Y. & Paricio, N. Drosophila as a model of wound healing and tissue regeneration in vertebrates. Dev. Dyn. 240, 2379–2404 (2011).

    Article  CAS  Google Scholar 

  9. 9.

    Dar, A. C., Das, T. K., Shokat, K. M. & Cagan, R. L. Chemical genetic discovery of targets and anti-targets for cancer polypharmacology. Nature 486, 80–84 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Enomoto, M., Siow, C. & Igaki, T. Drosophila as a cancer model. Adv. Exp. Med. Biol. 1076, 173–194 (2018).

    Article  CAS  Google Scholar 

  11. 11.

    Das, T. K. & Cagan, R. L. A Drosophila approach to thyroid cancer therapeutics. Drug Discov. Today Technol. 10, e65–e71 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Sonoshita, M. et al. A whole-animal platform to advance a clinical kinase inhibitor into new disease space. Nat. Chem. Biol. 14, 291–298 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    McGurk, L., Berson, A. & Bonini, N. M. Drosophila as an in vivo model for human neurodegenerative disease. Genetics 201, 377–402 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Moloney, A., Sattelle, D. B., Lomas, D. A. & Crowther, D. C. Alzheimer’s disease: insights from Drosophila melanogaster models. Trends Biochem. Sci. 35, 228–235 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Piper, M. D. W. & Partridge, L. Drosophila as a model for ageing. Biochim. Biophys. Acta Mol. Basis Dis. 1864, 2707–2717 (2018).

    Article  CAS  Google Scholar 

  16. 16.

    Ugur, B., Chen, K. & Bellen, H. J. Drosophila tools and assays for the study of human diseases. Dis. Model. Mech. 9, 235–244 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kam, Z., Minden, J. S., Agard, D. A., Sedat, J. W. & Leptin, M. Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy. Development 112, 365–370 (1991).

    CAS  PubMed  Google Scholar 

  18. 18.

    Wang, S. & Hazelrigg, T. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400–403 (1994).

    Article  CAS  Google Scholar 

  19. 19.

    Ninov, N. & Martin-Blanco, E. Live imaging of epidermal morphogenesis during the development of the adult abdominal epidermis of Drosophila. Nat. Protoc. 2, 3074–3080 (2007).

    Article  CAS  Google Scholar 

  20. 20.

    Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J. & Rasse, T. M. Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses. Nat. Protoc. 2, 3285–3298 (2007).

    Article  CAS  Google Scholar 

  21. 21.

    Martin, J. L. et al. Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss. Elife 7, e36248 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Weavers, H., Franz, A., Wood, W. & Martin, P. Long-term in vivo tracking of inflammatory cell dynamics within Drosophila pupae. J. Vis. Exp. 2018, e57871 (2018).

    Google Scholar 

  23. 23.

    Antunes, M., Pereira, T., Cordeiro, J. V., Almeida, L. & Jacinto, A. Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding. J. Cell Biol. 202, 365–379 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Weavers, H., Evans, I. R., Martin, P. & Wood, W. Corpse engulfment generates a molecular memory that primes the macrophage inflammatory response. Cell 165, 1658–1671 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Larson, D. E., Liberman, Z. & Cagan, R. L. Cellular behavior in the developing Drosophila pupal retina. Mech. Dev. 125, 223–232 (2008).

    Article  CAS  Google Scholar 

  26. 26.

    Weitkunat, M., Brasse, M., Bausch, A. R. & Schnorrer, F. Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo. Development 144, 1261–1272 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Bosveld, F. et al. Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis. Nature 530, 495–498 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Sauerwald, J., Backer, W., Matzat, T., Schnorrer, F. & Luschnig, S. Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles. Elife 8, e48857 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Nienhaus, U., Aegerter-Wilmsen, T. & Aegerter, C. M. In-vivo imaging of the Drosophila wing imaginal disc over time: novel insights on growth and boundary formation. PLoS One 7, e47594 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Ghannad-Rezaie, M., Wang, X., Mishra, B., Collins, C. & Chronis, N. Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae. PLoS One 7, e29869 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Heemskerk, I., Lecuit, T. & LeGoff, L. Dynamic clonal analysis based on chronic in vivo imaging allows multiscale quantification of growth in the Drosophila wing disc. Development 141, 2339–2348 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Andlauer, T. F. & Sigrist, S. J. In vivo imaging of the Drosophila larval neuromuscular junction. Cold Spring Harb. Protoc. 2012, 481–489 (2012).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Cevik, D. et al. Chloroform and desflurane immobilization with recovery of viable Drosophila larvae for confocal imaging. J. Insect Physiol. 117, 103900 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Kakanj, P. et al. Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing. Nat. Commun. 7, 12972 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Beati, H. et al. The adherens junction-associated LIM domain protein Smallish regulates epithelial morphogenesis. J. Cell Biol. 217, 1079–1095 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Griffin, R., Binari, R. & Perrimon, N. Genetic odyssey to generate marked clones in Drosophila mosaics. Proc. Natl Acad. Sci. USA 111, 4756–4763 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Germani, F., Bergantinos, C. & Johnston, L. A. Mosaic analysis in Drosophila. Genetics 208, 473–490 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Makhijani, K. et al. Precision optogenetic tool for selective single- and multiple-cell ablation in a live animal model system. Cell Chem. Biol. 24, 110–119 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Stein, D. S. & Stevens, L. M. Maternal control of the Drosophila dorsal-ventral body axis. Wiley Interdiscip. Rev. Dev. Biol. 3, 301–330 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Tepass, U. et al. Shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia. Genes Dev. 10, 672–685 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Cox, R. T., Kirkpatrick, C. & Peifer, M. Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J. Cell Biol. 134, 133–148 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Uemura, T. et al. Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev. 10, 659–671 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Kuhn, H., Sopko, R., Coughlin, M., Perrimon, N. & Mitchison, T. The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos. Development 142, 3869–3878 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Mirth, C. K. & Shingleton, A. W. Integrating body and organ size in Drosophila: recent advances and outstanding problems. Front. Endocrinol. (Lausanne) 3, 49 (2012).

    Article  Google Scholar 

  45. 45.

    Vollmer, J., Casares, F. & Iber, D. Growth and size control during development. Open Biol. 7, 170190 (2017).

  46. 46.

    Johnston, L. A. & Gallant, P. Control of growth and organ size in Drosophila. Bioessays 24, 54–64 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Nijhout, H. F. The control of growth. Development 130, 5863–5867 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Badouel, C. & McNeill, H. Apical junctions and growth control in Drosophila. Biochim. Biophys. Acta 1788, 755–760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Diaz-de-la-Loza, M. D. et al. Apical and basal matrix remodeling control epithelial morphogenesis. Dev. Cell 46, 23–39 e25 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Tumaneng, K., Russell, R. C. & Guan, K. L. Organ size control by Hippo and TOR pathways. Curr. Biol. 22, R368–379 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Beira, J. V. & Paro, R. The legacy of Drosophila imaginal discs. Chromosoma 125, 573–592 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Baena-Lopez, L. A., Nojima, H. & Vincent, J. P. Integration of morphogen signalling within the growth regulatory network. Curr. Opin. Cell Biol. 24, 166–172 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Miles, W. O., Dyson, N. J. & Walker, J. A. Modeling tumor invasion and metastasis in Drosophila. Dis. Model. Mech. 4, 753–761 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Stefanatos, R. K. & Vidal, M. Tumor invasion and metastasis in Drosophila: a bold past, a bright future. J. Genet. Genomics 38, 431–438 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Das, T. K. & Cagan, R. L. Non-mammalian models of multiple endocrine neoplasia type 2. Endocr. Relat. Cancer 25, T91–T104 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Rudrapatna, V. A., Cagan, R. L. & Das, T. K. Drosophila cancer models. Dev. Dyn. 241, 107–118 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Bangi, E., Garza, D. & Hild, M. In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila. J. Chem. Biol. 4, 55–68 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Willoughby, L. F. et al. An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery. Dis. Model. Mech. 6, 521–529 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Gasque, G., Conway, S., Huang, J., Rao, Y. & Vosshall, L. B. Small molecule drug screening in Drosophila identifies the 5HT2A receptor as a feeding modulation target. Sci. Rep. 3, srep02120 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Burra, S., Wang, Y., Brock, A. R. & Galko, M. J. Using Drosophila larvae to study epidermal wound closure and inflammation. Methods Mol. Biol. 1037, 449–461 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Galko, M. J. & Krasnow, M. A. Cellular and genetic analysis of wound healing in Drosophila larvae. PLoS Biol. 2, E239 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Mishra, B. et al. Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae. J. Vis. Exp. 2014, e50998 (2014).

  63. 63.

    Zhang, Y. et al. In vivo imaging of intact Drosophila larvae at sub-cellular resolution. J. Vis. Exp. 2010, e2249 (2010).

    Google Scholar 

  64. 64.

    Rasse, T. M. et al. Glutamate receptor dynamics organizing synapse formation in vivo. Nat. Neurosci. 8, 898–905 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Zirin, J. et al. Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles. Dev. Biol. 383, 275–284 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Takats, S., Varga, A., Pircs, K. & Juhasz, G. Loss of Drosophila Vps16A enhances autophagosome formation through reduced Tor activity. Autophagy 11, 1209–1215 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Wodarz, A., Hinz, U., Engelbert, M. & Knust, E. Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67–76 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Lawrence, P. A., Bodmer, R. & Vincent, J. P. Segmental patterning of heart precursors in Drosophila. Development 121, 4303–4308 (1995).

    CAS  PubMed  Google Scholar 

  71. 71.

    Wang, Y. et al. Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis. Curr. Biol. 25, 2215–2227 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Huang, J., Zhou, W., Dong, W., Watson, A. M. & Hong, Y. From the cover: directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering. Proc. Natl Acad. Sci. USA 106, 8284–8289 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Cohen, E. Chitin synthesis and inhibition: a revisit. Pest Manag. Sci. 57, 946–950 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Maimon, I. & Gilboa, L. Dissection and staining of Drosophila larval ovaries. J. Vis. Exp. 2011, e2537 (2011).

    Google Scholar 

  75. 75.

    Eijkelenboom, A. & Burgering, B. M. FOXOs: signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 14, 83–97 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. & Edgar, B. A. Drosophila’s insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2, 239–249 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Forster, D. & Luschnig, S. Src42A-dependent polarized cell shape changes mediate epithelial tube elongation in Drosophila. Nat. Cell Biol. 14, 526–534 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Nehme, N. T. et al. A model of bacterial intestinal infections in Drosophila melanogaster. PLoS Pathog. 3, e173 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Jiang, H. et al. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell 137, 1343–1355 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Zeng, X., Chauhan, C. & Hou, S. X. Characterization of midgut stem cell- and enteroblast-specific Gal4 lines in Drosophila. Genesis 48, 607–611 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Micchelli, C. A. & Perrimon, N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475–479 (2006).

    Article  CAS  Google Scholar 

  82. 82.

    Tanimoto, H., Itoh, S., ten Dijke, P. & Tabata, T. Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Mol. Cell 5, 59–71 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. 83.

    St Pierre, S. E., Galindo, M. I., Couso, J. P. & Thor, S. Control of Drosophila imaginal disc development by rotund and roughened eye: differentially expressed transcripts of the same gene encoding functionally distinct zinc finger proteins. Development 129, 1273–1281 (2002).

    Google Scholar 

  84. 84.

    Jenett, A. et al. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep. 2, 991–1001 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. 85.

    Kambadur, R. et al. Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 12, 246–260 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Lin, D. M. & Goodman, C. S. Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13, 507–523 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Torroja, L., Packard, M., Gorczyca, M., White, K. & Budnik, V. The Drosophila beta-amyloid precursor protein homolog promotes synapse differentiation at the neuromuscular junction. J. Neurosci. 19, 7793–7803 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Gronke, S. et al. Control of fat storage by a Drosophila PAT domain protein. Curr. Biol. 13, 603–606 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Goto, A., Kadowaki, T. & Kitagawa, Y. Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects. Dev. Biol. 264, 582–591 (2003).

    Article  CAS  Google Scholar 

  90. 90.

    Stramer, B. et al. Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration. J. Cell Biol. 168, 567–573 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Lee, S. & Kolodziej, P. A. The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion. Development 129, 1509–1520 (2002).

    Article  CAS  Google Scholar 

  92. 92.

    Ranganayakulu, G., Schulz, R. A. & Olson, E. N. Wingless signaling induces nautilus expression in the ventral mesoderm of the Drosophila embryo. Dev. Biol. 176, 143–148 (1996).

    Article  CAS  Google Scholar 

  93. 93.

    Klein, P. et al. Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J. 33, 341–355 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. 94.

    Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. & Brand, A. H. Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2, 7–12 (2000).

    Article  CAS  Google Scholar 

  95. 95.

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

    Article  CAS  Google Scholar 

  96. 96.

    Pfeiffer, B. D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. 97.

    Morin, X., Daneman, R., Zavortink, M. & Chia, W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl Acad. Sci. USA 98, 15050–15055 (2001).

    Article  CAS  Google Scholar 

  98. 98.

    Oda, H. & Tsukita, S. Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells. J. Cell Sci. 114, 493–501 (2001).

    CAS  PubMed  Google Scholar 

  99. 99.

    Cai, D. et al. Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157, 1146–1159 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. 100.

    Royou, A., Sullivan, W. & Karess, R. Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity. J. Cell Biol. 158, 127–137 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. 101.

    Martin, A. C., Kaschube, M. & Wieschaus, E. F. Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495–499 (2009).

    Article  CAS  Google Scholar 

  102. 102.

    Buszczak, M. et al. The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175, 1505–1531 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. 103.

    Harris, T. J. & Peifer, M. Adherens junction-dependent and -independent steps in the establishment of epithelial cell polarity in Drosophila. J. Cell Biol. 167, 135–147 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. 104.

    Barolo, S., Castro, B. & Posakony, J. W. New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques 36, 436–441 (2004).

    Article  CAS  Google Scholar 

  105. 105.

    Polychronidou, M., Hellwig, A. & Grosshans, J. Farnesylated nuclear proteins Kugelkern and lamin Dm0 affect nuclear morphology by directly interacting with the nuclear membrane. Mol. Biol. Cell 21, 3409–3420 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Katsani, K. R., Karess, R. E., Dostatni, N. & Doye, V. In vivo dynamics of Drosophila nuclear envelope components. Mol. Biol. Cell 19, 3652–3666 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Feng, K., Palfreyman, M. T., Hasemeyer, M., Talsma, A. & Dickson, B. J. Ascending SAG neurons control sexual receptivity of Drosophila females. Neuron 83, 135–148 (2014).

    Article  CAS  Google Scholar 

  108. 108.

    Okajima, T., Xu, A., Lei, L. & Irvine, K. D. Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding. Science 307, 1599–1603 (2005).

    Article  CAS  Google Scholar 

  109. 109.

    Okajima, T., Reddy, B., Matsuda, T. & Irvine, K. D. Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling. BMC Biol. 6, 1 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. 110.

    Pilling, A. D., Horiuchi, D., Lively, C. M. & Saxton, W. M. Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol. Biol. Cell 17, 2057–2068 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. 111.

    Bardet, P. L. et al. A fluorescent reporter of caspase activity for live imaging. Proc. Natl Acad. Sci. USA 105, 13901–13905 (2008).

    Article  Google Scholar 

  112. 112.

    Nezis, I. P. et al. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J. Cell Biol. 190, 523–531 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. 113.

    Arsham, A. M. & Neufeld, T. P. A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway. PLoS One 4, e6068 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. 114.

    Rusten, T. E. et al. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev. Cell 7, 179–192 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. 115.

    Chang, Y. Y. & Neufeld, T. P. An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation. Mol. Biol. Cell 20, 2004–2014 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. 116.

    Hatan, M., Shinder, V., Israeli, D., Schnorrer, F. & Volk, T. The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures. J. Cell Biol. 192, 307–319 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Grieder, N. C., de Cuevas, M. & Spradling, A. C. The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127, 4253–4264 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. 118.

    Megraw, T. L., Kilaru, S., Turner, F. R. & Kaufman, T. C. The centrosome is a dynamic structure that ejects PCM flares. J. Cell Sci. 115, 4707–4718 (2002).

    Article  CAS  Google Scholar 

  119. 119.

    Zirin, J., Nieuwenhuis, J. & Perrimon, N. Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. PLoS Biol. 11, e1001708 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. 120.

    Klapholz, B. et al. Alternative mechanisms for talin to mediate integrin function. Curr. Biol. 25, 847–857 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. 121.

    Benton, R. & St Johnston, D. A conserved oligomerization domain in Drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity. Curr. Biol. 13, 1330–1334 (2003).

    Article  CAS  Google Scholar 

  122. 122.

    Khuong, T. M., Habets, R. L., Slabbaert, J. R. & Verstreken, P. WASP is activated by phosphatidylinositol-4,5-bisphosphate to restrict synapse growth in a pathway parallel to bone morphogenetic protein signaling. Proc. Natl Acad. Sci. USA 107, 17379–17384 (2010).

    Article  Google Scholar 

  123. 123.

    Karpova, N., Bobinnec, Y., Fouix, S., Huitorel, P. & Debec, A. Jupiter, a new Drosophila protein associated with microtubules. Cell Motil. Cytoskeleton 63, 301–312 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. 124.

    Venken, K. J. et al. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nat. Methods 8, 737–743 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. 125.

    Garelli, A. et al. Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat. Commun. 6, 8732 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to M. Kakanj for her photographic and graphical support. We thank S. Roth, N. Riddiford, A. Schauss, F. Papagiannouli, V. Böhm and C. Lesch for critical reading of manuscript, comments and helpful discussions. We thank A. Schauss, P. Zentis and C. Jüngst from the CECAD imaging facility in Cologne (University of Cologne, Cluster of Excellence in Ageing Research) for support and the Bloomington, VDRC and DGGR stock centers for fly strains. This work was supported by grants from the European Regional Development Fund and the German state North Rhine-Westphalia (NRW im Ziel 2) to S.A.E. and L.P., a CMMC grant to M.L. and S.A.E., a CECAD grand by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy, grant EXC-2030/1-390661388 to M.L. and an EMBL research fellowship to P.K.

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Authors

Contributions

P.K. conceived, designed and developed the long-term live imaging and laser ablation protocol for Drosophila larvae; performed all experiments; and prepared the figures and tables. P.K., M.L. and L.P. analyzed and discussed the data and drafted the manuscript. S.A.E. provided input on wound healing analysis and discussed the data.

Corresponding authors

Correspondence to Parisa Kakanj, Linda Partridge or Maria Leptin.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Greg Macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Kakanj, P. et al. Nat. Commun. 7, 12972 (2016): https://doi.org/10.1038/ncomms12972

Beati, H. et al. J. Cell Biol. 217, 1079–1095 (2018): https://doi.org/10.1083/jcb.201610098

Supplementary information

Supplementary Video 1 | Live imaging of mitochondria during epidermal wound healing.

Live imaging of mitochondria (green) dynamics during epidermal wound healing. Single-cell wound in the dorsal epidermis of Drosophila L3 larva expressing mito-GFP (green) and DsRed2-Nuc (magenta) to mark mitochondria and nuclei in the epidermis: A58>mito-GFP; DsRed2-Nuc (w1118; UAS-mito-HA-GFP/+; A58-Gal4, DsRed2-Nuc/+). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 7a-b.

Reporting Summary

Supplementary Video 2 | Live imaging of ER vesicle trafficking.

Live imaging of ER vesicle (green) trafficking between ER and Golgi in the epidermis of Drosophila L3 larva expressing e22c>; RFP-KDEL (w1118; e22c-Gal4/+; UASp-RFP.KDEL/+). KDEL is an ER marker, encodes a sequence to prevent secretion of the protein form ER or retrieval of ER proteins from the Golgi. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7c-d.

Supplementary Video 3 | Live imaging of fat body.

Live imaging of fat body (green) in the L3 larvae expressing c7>mCD8-GFP (w1118; c7-Gal4/+; UAS-mCD8-GFP/+). The mCD8-GFP marks the cell membranes. Each frame is a single plane. Scale bars: first movie 80 µm and second movie 20 μm. Corresponds to Fig. 7e-f.

Supplementary Video 4 | Live imaging of trachea.

Live imaging of trachea (green) in the early L3 larvae in two individuals both expressing btl>GFP (w1118; btl-Gal4, UAS-GFP; +). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bar: (a,b) 17 μm. Corresponds to Fig. 7g-h.

Supplementary Video 5 | Live imaging of wing imaginal disc.

First movie shows a live imaging through the z-stacks of dorsal compartment of wing imaginal disk (grey) in late L3 larva expressing hh>mCD8-GFP (w1118; +; hh-Gal4, UAS-mCD8-GFP/+). The mCD8-GFP marks the cell membranes. Each frame is a single plane of z-stacks (54 stacks with 1µm space). Scale bar: 50 μm. Second movie shows a short live imaging of entire imaginal disk (green) in early L3 larvae expressing esg>GFP (w1118; esg-Gal4, UAS-GFP/+; +). Each frame is a single plane. Scale bar: 100 μm. Corresponds to Fig. 7i-k.

Supplementary Video 6 | Live imaging of peripheral neurons.

Live imaging of peripheral neuron (black) in the L3 larva expressing mCD8-GFP (black) to mark cell membranes of the neurons: elav>mCD8-GFP (w1118, elave-Gal4/UAS-mCD8-GFP; UAS-mCD8-GFP/+; UAS-mCD8-GFP/+). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bar: 15 μm. Corresponds to Fig. 7m.

Supplementary Video 7 | Live imaging of a chordotonal organ.

Live imaging of lateral pentascolopidial (Lch5) organ (green) in L3 larva expressing mCD8-GFP (green) to mark cell membranes: elav>mCD8-GFP (w1118, elave-Gal4/UAS-mCD8-GFP; UAS-mCD8-GFP/+; UAS-mCD8-GFP/+). The Lch5 organ is a particular chordotonal organ, which plays a role by proprioceptive locomotion control. Each frame is a single plane. Scale bar: 8 μm.

Supplementary Video 8 | Live imaging of midgut enterocytes.

Live imaging of midgut enterocytes cells (green) in the L3 larva expressing NP1>GFP-Atg8a; foxo (w1118; NP1-Gal4/UAS-GFP-Atg8a; UAS-foxo/+). Overexpression of FOXO in the midgut enterocytes induces autophagy (green dots are autophagosomes). The fine-focus was changed during the live imaging to image through different planes. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7n.

Supplementary Video 9 | Live imaging of midgut interstitial cells.

Live imaging of midgut interstitial cells (green) in the L3 larva expressing NP1>GFP-Atg8a; foxo (w1118; NP1-Gal4/UAS-GFP-Atg8a; UAS-foxo/+). Overexpression of FOXO induces autophagy in the midgut interstitial cells (green dots are autophagosomes). The fine-focus was changed during the live imaging to image through different planes. Each frame is a single plane. Scale bar: 20 μm. Corresponds to Fig. 7o.

Supplementary Video 10 | Live imaging of hemocytes.

Live imaging of haemocytes in the L3 larva expressing Hml>dsRed (magenta, in the first movie) to mark the entire cytoplasm of the haemocytes and Jupiter (green, in the second movie) to mark the microtubules of the haemocytes: Hml>dsRed (w1118; Hml-Gal4/+; UAS-dsRed/+) and Jupiter-GFP trap (w1118; +; Jupiter-GFP). Each frame is a merge of 21 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 7p-q.

Supplementary Video 11 | Live imaging of muscle.

First movie shows live imaging of dorsal muscles (green) in L3 larva expressing MHC>GFP-Atg8a; foxo (w1118; MHC-Gal4/UAS-GFP-Atg8a; UAS-foxo/+), in which high level of FOXO induces autophagy (green dots are autophagosomes). Second movie shoes live imaging of dorsal muscles (green) in L3 larva expressing Nrg-GFP t

Supplementary Video 12 | Single- and multicell laser ablation and wound healing process.

Live imaging of single-cell (first movie) and multi-cell (second movie) laser wounding in the dorsal epidermis of L3 larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) to mark cell membrane and nuclei in the epidermis: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 10.

Supplementary Video 13 | Dynamic insulin/PIP3/FOXO signaling during wound healing.

Redistribution of PIP3 reporter, tGPH (green) and FOXO (magenta) shuttling after wounding in the cells directly surrounding the wound. Wound healing was performed in the dorsal epidermis of L3 larva expressing tGPH (tubulin:GFP-PH) and foxo-mCherry (w-; tGPH; endo-dfoxo-v3-mCherry). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 11a.

Supplementary Video 14 | Effect of reduced insulin receptor signaling.

Lowering of insulin receptor signalling in the larvae by expressing a dominant negative version of the insulin receptor (InRDN), reduced accumulation of the PIP3-reporter tGPH (back) at the wound edges and slows down the wound healing processes. Transgene genotypes: control (w1118; tGPH/+; A58-Gal4/+) and A58>InRDN (w1118; tGPH/+; A58-Gal4/UAS- InRDN). Each frame is a merge of 57 planes spaced 0.28 μm apart. Scale bar: 20 μm. Corresponds to Fig. 11b.

Supplementary Video 15 | Long exposure to anesthetic increases larval lethality during live imaging.

The early L3 instar larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) was exposed for 6 min instead of 3.5 min to diethyl ether. A single-cell laser wound was made in the dorsal epidermis. The larva died during the experiment. Transgene genotypes of larvae: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Projections of a time-lapse series. Each frame is merged from 57 planes spaced 0.28 μm apart. Scale bars: 20 μm. Corresponds to Fig. 9g.

Supplementary Video 16 | Insufficient immobilization for long-term live imaging.

Example of a larva that did not remain immobile for the entire experiment. The reason for the larva waking up could be that (i) the exposure to diethyl ether was to short or (ii) the larvae in larval cage were too crowed or (iii) they were not completely dried (Table 5, Step 17). A single-cell laser wound was produced in the dorsal epidermis of a larva expressing Src-GFP (green) and DsRed2-Nuc (magenta) and live imaged. Transgene genotypes of larvae: A58>Src- GFP,DsRed2-Nuc (w1118; +; A58-Gal4, UAS-Src-GFP, UAS-DsRed2-Nuc/+). Projections of a time-lapse series in early L3 larvae. Each frame is merged from 57 planes spaced 0.28 μm apart. Scale bars: 20 μm.

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Kakanj, P., Eming, S.A., Partridge, L. et al. Long-term in vivo imaging of Drosophila larvae. Nat Protoc 15, 1158–1187 (2020). https://doi.org/10.1038/s41596-019-0282-z

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