Nuclear Migration in the Drosophila Oocyte

Summary

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Abstract

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Introduction

For several years, the Drosophila oocyte has emerged as a model system to study nuclear migration. The Drosophila oocyte develops in a multicellular structure called the egg chamber. Egg chambers encompass 16 germ cells (15 nurse cells and the oocyte) surrounded by an epithelial layer of follicular somatic cells. Egg chamber development has been subdivided into 14 stages (Figure 1A), during which the oocyte will grow and accumulate reserves necessary for the early development of the embryo. During the development, upon microtubule reorganization and asymmetric transport of maternal determinants, the oocyte polarizes alon....

Protocol

1. Imaging medium preparation

1. Prepare fresh media on the day of use. Pipette 200 µL of Schneider medium (containing L-Glutamine and 0.40 g/L of NaHCO₃ complemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin).
2. Supplement with 30 µL of insulin 10 mg/mL.
3. Add 4 µL of heat-inactivated fetal calf serum.

2. Observation-chamber preparation

1. With a pipette tip, apply a smal.......

Discussion

Other protocols describe how to prepare and culture Drosophila egg chambers ex vivo for live-imaging assay^12,13. The novelty of this protocol is the use of an imaging chamber constructed using a hollowed aluminum slide, a coverslip, and an O₂/CO₂ permeable membrane. The main advantage of this set-up is to limit the movement in Z without exerting pressure on the sample. Thus, the oocyte can still move freely, and this is why first, t.......

Materials

Name	Company	Catalog Number	Comments
Anesthetize CO2 pad	Dutscher	789060	Anesthetize flies
Coverslip (24x50 mm)	Knittel Glass	VD12450Y100A	Observation-chamber preparation
Forceps Dumont #5	Carl Roth	K342.1	Dissection
Stainless steel needles	Entosphinx	20	Dissection
Heat-inactivated fetal calf serum	SIGMA-ALDRICH	F7524	Imaging medium
Insulin solution bovine pancreas	SIGMA-ALDRICH	10516 - 5ml	Imaging medium
Penicilin/Streptomycin solution	SIGMA-ALDRICH	P0781	Imaging medium
Permeable membrane	Leica	11521746	Observation-chamber preparation
Schneider Medium	Pan Biotech	P04-91500	Imaging medium
Silicon grease	BECKMAN COULTER	335148	Observation-chamber preparation
Spinning disk confocal	Zeiss	CSU-X1	Nuclear migration observation
Voltalef oil 10S	VWR	24627 - 188	Observation-chamber preparation