Dissection, Fixation, and Standard Staining of Adult Drosophila Ovaries

Studies of the Drosophila ovary have provided significant insight into the molecular and cellular processes that control cell function, tissue organization, and animal development. To characterize mutants with defects in oogenesis or to observe the distribution of gene products involved in egg production, the ovaries must be carefully extracted and prepared for analysis. This chapter describes the manual dissection of ovaries from adult Drosophila females, followed by standard fixation and staining of the isolated tissue. Specifically, this chapter provides procedures for simple DNA and F-actin staining to assess cell and tissue morphology, as well as immunostaining to localize proteins of interest.

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References

  1. Hinnant TD, Merkle JA, Ables ET (2020) Coordinating proliferation, polarity, and cell fate in the Drosophila female germline. Front Cell Dev Biol 8:19. https://doi.org/10.3389/fcell.2020.00019ArticleGoogle Scholar
  2. Mahowald A, Kambysellis M (1980) Oogenesis. In: Ashburner M, Wright T (eds) The genetics and biology of Drosophila, 2nd edn. Academic Press, pp 141–225 Google Scholar
  3. McLaughlin JM, Bratu DP (2015) Drosophila melanogaster oogenesis: an overview. In: Bratu DP, McNeil GP (eds) Drosophila oogenesis. Springer New York, New York, pp 1–20 Google Scholar
  4. Merkle JA, Wittes J, Schüpbach T (2020) Signaling between somatic follicle cells and the germline patterns the egg and embryo of Drosophila. In: Current topics in developmental biology. Elsevier, pp 55–86 Google Scholar
  5. Spradling A (1993) Developmental genetics of oogenesis. In: Bate M, Martinez-Arias A (eds) The development of Drosophila melanogaster. Cold Spring Harbor Press, Cold Spring Harbor, pp 1–70 Google Scholar
  6. Blatt P, Martin ET, Breznak SM, Rangan P (2020) Post-transcriptional gene regulation regulates germline stem cell to oocyte transition during Drosophila oogenesis. In: Current topics in developmental biology. Elsevier, pp 3–34. https://doi.org/10.1016/bs.ctdb.2019.10.003ChapterGoogle Scholar
  7. Drummond-Barbosa D (2019) Local and physiological control of germline stem cell lineages in Drosophila melanogaster. Genetics 213:9–26. https://doi.org/10.1534/genetics.119.300234ArticleCASGoogle Scholar
  8. Eliazer S, Buszczak M (2011) Finding a niche: studies from the Drosophila ovary. Stem Cell Res Ther 2:45. https://doi.org/10.1186/scrt86ArticleGoogle Scholar
  9. Flora P, McCarthy A, Upadhyay M, Rangan P (2017) Role of chromatin modifications in Drosophila germline stem cell differentiation. In: Arur S (ed) Signaling-mediated control of cell division. Springer International Publishing, pp 1–30 Google Scholar
  10. Riechmann V (2017) In vivo RNAi in the Drosophila follicular epithelium: analysis of stem cell maintenance, proliferation, and differentiation. In: Zhang B (ed) RNAi and small regulatory RNAs in stem cells. Springer New York, New York, pp 185–206 ChapterGoogle Scholar
  11. Slaidina M, Gupta S, Banisch TU, Lehmann R (2021) A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries. Genome Res 31:1938–1951. https://doi.org/10.1101/gr.274340.120ArticleGoogle Scholar
  12. Wu X, Tanwar PS, Raftery LA (2008) Drosophila follicle cells: morphogenesis in an eggshell. Semin Cell Dev Biol 19:271–282. https://doi.org/10.1016/j.semcdb.2008.01.004ArticleCASGoogle Scholar
  13. Ables ET, Hwang GH, Finger DS et al (2016) A genetic mosaic screen reveals ecdysone-responsive genes regulating Drosophila oogenesis. G3 Bethesda 6:2629–2642. https://doi.org/10.1534/g3.116.028951ArticleCASGoogle Scholar
  14. Czech B, Preall JB, McGinn J, Hannon GJ (2013) A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway. Mol Cell 50:749–761. https://doi.org/10.1016/j.molcel.2013.04.007ArticleCASGoogle Scholar
  15. Gans M, Audit C, Masson M (1975) Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster. Genetics 81:683–704. https://doi.org/10.1093/genetics/81.4.683ArticleCASGoogle Scholar
  16. Hayashi R, Wainwright SM, Liddell SJ et al (2014) A genetic screen based on in vivo RNA imaging reveals centrosome-independent mechanisms for localizing gurken transcripts in Drosophila. G3 Bethesda 4:749–760. https://doi.org/10.1534/g3.114.010462ArticleCASGoogle Scholar
  17. Jagut M, Mihaila-Bodart L, Molla-Herman A et al (2013) A mosaic genetic screen for genes involved in the early steps of Drosophila oogenesis. G3 Bethesda. https://doi.org/10.1534/g3.112.004747
  18. Jambor H, Surendranath V, Kalinka AT et al (2015) Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. eLife 4:e05003. https://doi.org/10.7554/eLife.05003ArticleGoogle Scholar
  19. Jia D, Soylemez M, Calvin G et al (2015) A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. Sci Rep 5:12328. https://doi.org/10.1038/srep12328ArticleCASGoogle Scholar
  20. Lee M-C, Skora AD, Spradling AC (2017) Identification of genes mediating Drosophila follicle cell progenitor differentiation by screening for modifiers of GAL4::UAS variegation. G3 Bethesda 7:309–318. https://doi.org/10.1534/g3.116.036038ArticleCASGoogle Scholar
  21. Sanchez CG, Teixeira FK, Czech B et al (2016) Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation. Cell Stem Cell 18:276–290. https://doi.org/10.1016/j.stem.2015.11.004ArticleCASGoogle Scholar
  22. Schüpbach T, Wieschaus E (1989) Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics 121:101–117. https://doi.org/10.1093/genetics/121.1.101ArticleGoogle Scholar
  23. Schüpbach T, Wieschaus E (1991) Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129:1119–1136 ArticleGoogle Scholar
  24. Tootle TL, Williams D, Hubb A et al (2011) Drosophila eggshell production: identification of new genes and coordination by Pxt. PLoS One 6:e19943. https://doi.org/10.1371/journal.pone.0019943ArticleCASGoogle Scholar
  25. Wittes J, Schüpbach T (2019) A gene expression screen in Drosophila melanogaster identifies novel JAK/STAT and EGFR targets during oogenesis. G3 Bethesda 9:47–60. https://doi.org/10.1534/g3.118.200786ArticleCASGoogle Scholar
  26. Abbaszadeh EK, Gavis ER (2016) Fixed and live visualization of RNAs in Drosophila oocytes and embryos. Methods 98:34–41. https://doi.org/10.1016/j.ymeth.2016.01.018ArticleCASGoogle Scholar
  27. Cetera M, Lewellyn L, Horne-Badovinac S (2016) Cultivation and live imaging of Drosophila ovaries. In: Dahmann C (ed) Drosophila. Springer New York, New York, pp 215–226 ChapterGoogle Scholar
  28. Dai W, Montell DJ (2016) Live imaging of border cell migration in Drosophila. In: Jin T, Hereld D (eds) Chemotaxis. Springer New York, New York, pp 153–168 ChapterGoogle Scholar
  29. Haack T, Bergstralh DT, St Johnston D (2013) Damage to the Drosophila follicle cell epithelium produces “false clones” with apparent polarity phenotypes. Biol Open 2:1313–1320. https://doi.org/10.1242/bio.20134671ArticleGoogle Scholar
  30. Hudson AM, Cooley L (2014) Methods for studying oogenesis. Methods 68:207–217. https://doi.org/10.1016/j.ymeth.2014.01.005ArticleCASGoogle Scholar
  31. Peters NC, Berg CA (2016) In vitro culturing and live imaging of Drosophila egg chambers: a history and adaptable method. In: Nezis IP (ed) Oogenesis. Springer New York, New York, pp 35–68 ChapterGoogle Scholar
  32. Shalaby NA, Buszczak M (2017) Live-cell imaging of the adult Drosophila ovary using confocal microscopy. In: Buszczak M (ed) Germline stem cells. Springer New York, New York, pp 85–91 ChapterGoogle Scholar
  33. Thompson L, Randolph K, Norvell A (2015) Basic techniques in Drosophila ovary preparation. In: Bratu DP, McNeil GP (eds) Drosophila oogenesis. Springer New York, New York, pp 21–28 ChapterGoogle Scholar
  34. Wong LC, Schedl P (2006) Dissection of Drosophila ovaries. J Vis Exp 52. https://doi.org/10.3791/52-v
  35. Zimmerman SG, Peters NC, Altaras AE, Berg CA (2013) Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat Protoc 8:2158–2179. https://doi.org/10.1038/nprot.2013.136ArticleCASGoogle Scholar
  36. Hsu H-J, Drummond-Barbosa D (2017) A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines. Gene Expr Patterns 23–24:13–21. https://doi.org/10.1016/j.gep.2017.01.001ArticleCASGoogle Scholar
  37. Buszczak M, Paterno S, Lighthouse D et al (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175:1505–1531. https://doi.org/10.1534/genetics.106.065961ArticleCASGoogle Scholar
  38. Morin X, Daneman R, Zavortink M, Chia W (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci 98:15050–15055. https://doi.org/10.1073/pnas.261408198ArticleCASGoogle Scholar
  39. Quiñones-Coello AT, Petrella LN, Ayers K et al (2007) Exploring strategies for protein trapping in Drosophila. Genetics 175:1089–1104. https://doi.org/10.1534/genetics.106.065995ArticleCASGoogle Scholar
  40. Rørth P (1998) Gal4 in the Drosophila female germline. Mech Dev 78:113–118. https://doi.org/10.1016/S0925-4773(98)00157-9ArticleGoogle Scholar
  41. He L, Wang X, Montell DJ (2011) Shining light on Drosophila oogenesis: live imaging of egg development. Curr Opin Genet Dev 21:612–619. https://doi.org/10.1016/j.gde.2011.08.011ArticleCASGoogle Scholar
  42. Pokrywka NJ (2013) Live imaging of GFP-labeled proteins in Drosophila oocytes. J Vis Exp:50044. https://doi.org/10.3791/50044

Acknowledgments

The author thanks Olivier Devergne, Makayla Gomperts, Julia Wittes, and McKenzie Young for their contributions to this protocol.