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

Prasad, Mohit; Jang, Anna C.C.; Starz‐Gaiano, Michelle; Melani, Mariana; Montell, Denise J.

doi:10.1038/nprot.2007.363

Cited by 166 publications

(145 citation statements)

References 22 publications

Supporting

Mentioning

145

Contrasting

Order By: Relevance

“…Living egg chambers were prepared for real-time imaging of border cell migration as described (56). Image acquisition is described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Assaker

Ramel²,

Wculek³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

107

View full text Add to dashboard Cite

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. In this study, we show that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, we demonstrate that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, our data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration.

show abstract

“…Living egg chambers were prepared for real-time imaging of border cell migration as described (56). Image acquisition is described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Assaker

Ramel²,

Wculek³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

107

View full text Add to dashboard Cite

show abstract

“…Egg chambers for live imaging of follicle cell mitoses were processed as previously described (Prasad et al, 2007). Ovaries were dissected in Schneider's Drosophila medium (Invitrogen).…”

Section: Live Imagingmentioning

confidence: 99%

Intercellular protein movement in syncytialDrosophilafollicle cells

Airoldi

McLean

Shimada

et al. 2011

Journal of Cell Science

View full text Add to dashboard Cite

SummaryRing canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. Here, we show by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, we show that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. We show for the first time that somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline.

show abstract

“…Ovaries from various fly strains and experiments were dissected in PBS, and optionally cultured per Prasad et al (2007). Briefly, ovarioles were gently removed from the muscle sheath and placed into a culture chamber containing Schneider's media supplemented with 200 lg ml 21 insulin, 15% FBS (v/v), and 0.63 penicillin/streptomycin, pH 7.0 for 4 h. Fixation was performed using devitellinizing buffer with 5% formaldehyde under heptane for 5 min.…”

Section: Discussionmentioning

confidence: 99%

“…Culturing fly ovaries in vitro for 2 h in media containing the TOR inhibitor RAP resulted in a dose-dependent increase in mid-stage egg chambers undergoing these degenerative steps . Hypothesizing that viral ssRNA contributes to the ovarian response, ovaries were cultured after Prasad et al (2007) in Poly:U, a synthetic RNA molecule known to activate innate immune response(s) in other systems (Diebold et al, 2004). After 2 h of culture, ovaries were fixed and stained with DAPI (DNA) and phalloidin (actin), and signs of egg chamber destruction were assessed as performed previously Fig.…”

Section: Ssrna Induces Egg Chamber Destructionmentioning

confidence: 99%

Oocyte destruction is activated during viral infection

Thomson

Schneemann

Johnson

2012

Genesis

View full text Add to dashboard Cite

Summary: Viral infection has been associated with a starvation-like state in Drosophila melanogaster. Because starvation and inhibiting TOR kinase activity in vivo result in blocked oocyte production, we hypothesized that viral infection would also result in compromised oogenesis. Wild-type flies were injected with flock house virus (FHV) and survival and embryo production were monitored. Infected flies had a doseresponsive loss of fecundity that corresponded to a global reduction in Akt/TOR signaling. Highly penetrant egg chamber destruction mid-way through oogenesis was noted and FHV coat protein was detected within developing egg chambers. As seen with in vivo TOR inhibition, oogenesis was partially rescued in loss of function discs large and merlin mutants. As expected, mutants in genes known to be involved in virus internalization and trafficking [Clathrin heavy chain (chc) and synaptotagmin] survive longer during infection. However, oogenesis was rescued only in chc mutants. This suggests that viral response mechanisms that control fly survival and egg chamber survival are separable. The genetic and signaling requirements for oocyte destruction delineated here represent a novel host-virus interaction with implications for the control of both fly and virus populations. genesis 50:453-465, 2012. V V C 2011 Wiley Periodicals, Inc.

show abstract

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

Cited by 166 publications

References 22 publications

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Intercellular protein movement in syncytialDrosophilafollicle cells

Oocyte destruction is activated during viral infection

Contact Info

Product

Resources

About