Nuclear Migration in the Drosophila Oocyte

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.

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.

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....

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.......

Before migration, the nucleus is dynamic and oscillates around a central position during a period defined as pre-migration. These small movements reflect a balance of pushing and pulling forces that maintain equilibrium in the middle of the oocyte. By quantifying the trajectories of the nuclei, we have shown that the APM and LPM trajectories had similar proportions. We define the nature of the trajectory by the first contact between the nucleus and the plasma membrane⁶. Thus, the nucleus reaches e.......

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.......

We are extremely grateful to Jean-Antoine Lepesant and Nicolas Tissot who originally developed the protocol and shared some graphical elements of Figure 3 with us. We thank Fanny Roland-Gosselin who took the photos of Figure 4. We also thank other lab members for helpful discussions that contributed to the amelioration of this technique and Nathaniel Henneman for his comments that helped to improve this manuscript. We acknowledge the ImagoSeine core facility of the Institute Jacques Monod, member of France-BioImaging (ANR-10-INBS-04). Maëlys Loh is supported by a PhD fellowship from the French Ministry of Research (MESRI). Antoine Guichet and Fred Bernard were su....