Culturing Drosophila Egg Chambers and Investigating Developmental Processes Through Live Imaging

  • Lathiena Manning
  • Michelle Starz-GaianoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1328)


Drosophila oogenesis provides many examples of essential processes in development. A myriad of genetic tools combined with recent advances in culturing egg chambers ex vivo has revealed several surprising mechanisms that govern how this tissue develops, and which could not have been determined in fixed tissues. Here we describe a straightforward protocol for dissecting ovaries, culturing egg chambers, and observing egg development in real time by fluorescent microscopy. This technique is suitable for observation of early- or late-stage egg development, and can be adapted to study a variety of cellular, molecular, or developmental processes. Ongoing analysis of oogenesis in living egg chambers has tremendous potential for discovery of new developmental mechanisms.


Drosophila melanogaster oogenesis Live imaging Ex vivo egg culturing Fluorescent microscopy Developmental genetics Cell migration Morphogenesis 



This work was funded in part by a Department of Education Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017), and by an NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036), IMSD Meyerhoff Graduate Fellowship, to L.M. and a National Science Foundation CAREER Award (IOS-1054422) to M.S.G. We appreciate helpful comments on the manuscript from Dr. N. Sanchez-Alberola, G. Wunderlin, E. Desai, and D. DiMercurio, and we thank Dr. J. McDonald and Dr. A.C.C. Jang for sharing culturing information.


  1. 1.
    He L, Wang X, Montell DJ (2011) Shining light on Drosophila oogenesis: live imaging of egg development. Curr Opin Genet Dev 21:612–619CrossRefPubMedGoogle Scholar
  2. 2.
    Mavrakis M, Pourquie O, Lecuit T (2010) Lighting up developmental mechanisms: how fluorescence imaging heralded a new era. Development 137:373–387CrossRefPubMedGoogle Scholar
  3. 3.
    Parton RM, Valles AM, Dobbie IM et al (2010) Live cell imaging in Drosophila melanogaster. Cold Spring Harb Protoc 2010:pdb.top75CrossRefPubMedGoogle Scholar
  4. 4.
    Hudson AM, Cooley L (2014) Methods for studying oogenesis. Methods 68:207–217PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Bastock R, St. Johnston D (2008) Drosophila oogenesis. Curr Biol 18:R1082–R1087CrossRefPubMedGoogle Scholar
  6. 6.
    Horne-Badovinac S, Bilder D (2005) Mass transit: epithelial morphogenesis in the Drosophila egg chamber. Dev Dyn 232:559–574CrossRefPubMedGoogle Scholar
  7. 7.
    Zhao T, Graham OS, Raposo A et al (2012) Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis. Science 336:999–1003PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Forrest KM, Gavis ER (2003) Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr Biol 13:1159–1168CrossRefPubMedGoogle Scholar
  9. 9.
    Weil TT, Forrest KM, Gavis ER (2006) Localization of bicoid mRNA in late oocytes is maintained by continual active transport. Dev Cell 11:251–262CrossRefPubMedGoogle Scholar
  10. 10.
    Bianco A, Poukkula M, Cliffe A et al (2007) Two distinct modes of guidance signalling during collective migration of border cells. Nature 448:362–365CrossRefPubMedGoogle Scholar
  11. 11.
    Prasad M, Montell DJ (2007) Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging. Dev Cell 12:997–1005CrossRefPubMedGoogle Scholar
  12. 12.
    Tekotte H, Tollervey D, Davis I (2007) Imaging the migrating border cell cluster in living Drosophila egg chambers. Dev Dyn 236:2818–2824CrossRefPubMedGoogle Scholar
  13. 13.
    Cai D, Chen SC, Prasad M et al (2014) Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157:1146–1159PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Majumder P, Aranjuez G, Amick J et al (2012) Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration. Curr Biol 22:363–372PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Starz-Gaiano M, Melani M, Wang X et al (2008) Feedback inhibition of Jak/STAT signaling by apontic is required to limit an invasive cell population. Dev Cell 14:726–738CrossRefPubMedGoogle Scholar
  16. 16.
    Ramel D, Wang X, Laflamme C et al (2013) Rab11 regulates cell-cell communication during collective cell movements. Nat Cell Biol 15:317–324PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Wang X, He L, Wu YI et al (2010) Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat Cell Biol 12:591–597PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    He L, Wang X, Tang HL et al (2010) Tissue elongation requires oscillating contractions of a basal actomyosin network. Nat Cell Biol 12:1133–1142PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Spracklen AJ, Fagan TN, Lovander KE et al (2014) The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis. Dev Biol 393:209–226CrossRefPubMedGoogle Scholar
  20. 20.
    Ferreira T, Prudencio P, Martinho RG (2014) Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis. Dev Biol 394:277–291CrossRefPubMedGoogle Scholar
  21. 21.
    Cox RT, Spradling AC (2003) A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130:1579–1590CrossRefPubMedGoogle Scholar
  22. 22.
    McLean PF, Cooley L (2013) Protein equilibration through somatic ring canals in Drosophila. Science 340:1445–1447CrossRefPubMedGoogle Scholar
  23. 23.
    Haigo SL, Bilder D (2011) Global tissue revolutions in a morphogenetic movement controlling elongation. Science 331:1071–1074PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Dorman JB, James KE, Fraser SE et al (2004) bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis. Dev Biol 267:320–341CrossRefPubMedGoogle Scholar
  25. 25.
    Osterfield M, Du X, Schüpbach T et al (2013) Three-dimensional epithelial morphogenesis in the developing Drosophila egg. Dev Cell 24:400–410PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Lewellyn L, Cetera M, Horne-Badovinac S (2013) Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila. J Cell Biol 200:721–729PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Weil TT, Parton RM, Davis I (2012) Preparing individual Drosophila egg chambers for live imaging. J Vis Exp (60): e3679Google Scholar
  28. 28.
    Prasad M, Jang AC, Starz-Gaiano M et al (2007) A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging. Nat Protoc 2:2467–2473CrossRefPubMedGoogle Scholar
  29. 29.
    Pokrywka NJ (2013) Live imaging of GFP-labeled proteins in Drosophila oocytes. J Vis Exp (73): 50044Google Scholar
  30. 30.
    Morris LX, Spradling AC (2011) Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary. Development 138:2207–2215PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Snapp EL, Iida T, Frescas D et al (2004) The fusome mediates intercellular endoplasmic reticulum connectivity in Drosophila ovarian cysts. Mol Biol Cell 15:4512–4521PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Airoldi SJ, McLean PF, Shimada Y et al (2011) Intercellular protein movement in syncytial Drosophila follicle cells. J Cell Sci 124:4077–4086PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Buszczak M, Paterno S, Lighthouse D et al (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175:1505–1531PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Karpova N, Bobinnec Y, Fouix S et al (2006) Jupiter, a new Drosophila protein associated with microtubules. Cell Motil Cytoskeleton 63:301–312CrossRefPubMedGoogle Scholar
  35. 35.
    Fichelson P, Moch C, Ivanovitch K et al (2009) Live-imaging of single stem cells within their niche reveals that a U3snoRNP component segregates asymmetrically and is required for self-renewal in Drosophila. Nat Cell Biol 11:685–693CrossRefPubMedGoogle Scholar
  36. 36.
    Martinez-Campos M, Basto R, Baker J et al (2004) The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J Cell Biol 165:673–683PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Peel N, Stevens NR, Basto R et al (2007) Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation. Curr Biol 17:834–843PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Morin X, Daneman R, Zavortink M et al (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15050–15055PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Conduit PT, Brunk K, Dobbelaere J et al (2010) Centrioles regulate centrosome size by controlling the rate of Cnn incorporation into the PCM. Curr Biol 20:2178–2186CrossRefPubMedGoogle Scholar
  40. 40.
    Minestrini G, Mathe E, Glover DM (2002) Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis. J Cell Sci 115(4):725–736PubMedGoogle Scholar
  41. 41.
    Royou A, Field C, Sisson JC et al (2004) Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol Biol Cell 15(2):838–850PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Shimada Y, Yonemura S, Ohkura H et al (2006) Polarized transport of Frizzled along the planar microtubule arrays in Drosophila wing epithelium. Dev Cell 10:209–222CrossRefPubMedGoogle Scholar
  43. 43.
    Cliffe A, Poukkula M, Rørth P (2009) Culturing Drosophila egg chambers and imaging border cell migration. Nat Protoc 10:289Google Scholar
  44. 44.
    Datta SR, Vasconcelos ML, Ruta V et al (2008) The Drosophila pheromone cVA activates a sexually dimorphic neural circuit. Nature 452:473–477CrossRefPubMedGoogle Scholar
  45. 45.
    Huang J, Zhou W, Dong W et al (2009) Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering. Proc Natl Acad Sci 106(20):8284–8289PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Davis I, Parton RM (2006) Selection of appropriate imaging equipment and methodology for live cell imaging in Drosophila. CSH Protoc doi: 10.1101/pdb.ip21
  47. 47.
    Stonko D, Manning L, Starz-Gaiano M, Peercy B (2015) A force-based biophysical model of collective cell migration in a three-dimensional, heterogeneous environment. PLoS One. 10(4):e0122799Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of Maryland Baltimore CountyBaltimoreUSA

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