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A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging

Nature Protocols volume 2, pages 2467–2473 (2007)Cite this article

Abstract

This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes ∼24 h while the preparation of egg chambers for live imaging requires only 15–20 min.

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Figure 1: Schematic illustration of egg chamber dissection.
Figure 2: Stages of egg chamber development.
Figure 3: Schematic drawing of the culture chamber as viewed from the top and as viewed from the side (lateral view).
Figure 4: Time lapse series of micrographs of stage 9 egg chambers of genotype slbo-GAL4,UAS mCD8::GFP.

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Acknowledgements

This work was supported by National Institutes of Health grants GM46425 and GM73164 to D.J.M.

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Authors and Affiliations

  1. Department of Biological Chemistry, Center for Cell Dynamics, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, 21205-2185, Maryland, USA

    Mohit Prasad, Anna C-C Jang, Michelle Starz-Gaiano, Mariana Melani & Denise J Montell

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  1. Mohit Prasad
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  2. Anna C-C Jang
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  3. Michelle Starz-Gaiano
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  4. Mariana Melani
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  5. Denise J Montell
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Corresponding author

Correspondence to Denise J Montell.

Supplementary information

Supplementary Video 1

Normal Border Cell Migration Dynamics (MOV 10331 kb)

Supplementary Video 2

Another example of wild type border cell migration (MOV 3616 kb)

Supplementary Video 3

Abnormal egg chamber development and arrest of border cell migration (MOV 37606 kb)

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Prasad, M., Jang, AC., Starz-Gaiano, M. et al. A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging. Nat Protoc 2, 2467–2473 (2007). https://doi.org/10.1038/nprot.2007.363

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