The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Summary

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Abstract

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

Introduction

Each Drosophila ovary is composed of ~16 ovarioles, or chains of sequentially maturing egg chambers or follicles. Each follicle is composed of a single oocyte, 15 germ line derived nurse or support cells, and ~650 somatic cells termed follicle cells (Figure 1A). Drosophila oogenesis is divided into 14 morphologically defined stages of development¹. Each stage of follicle development is observed many times within a single fly, making it relatively easy to isolate a substantial number of stage-specific follicles.

The mid-to-late stages of oogenesis (Stages 10B-14) are particularly well suited....

Protocol

1. Preparing Drosophila Prior to Stage Isolation

1. Make wet yeast paste by combining 50 g of active dry yeast with 90 ml of distilled water. Mix with a spatula to combine. Let the mixture stand for ~30 min before assessing consistency. The consistency should be just thick enough to adhere to the side of a fly vial but not run down the side. To achieve the desired consistency it may be necessary to add up to 10 ml of additional water or a small amount of dry yeast. Store in a covered container at 4 °C. The stored yeast can be used for ~1-2 weeks.
2. Collect <24 hr old adult flies (both males and females) of the desired ....

Results

When isolating specific stages of Drosophila follicle development it is essential to be able to accurately distinguish the different morphological stages. This is somewhat challenging for S10A and S10B, as the nurse cells and the oocyte each take up half the length of the follicle at these stages (Figure 1B compared to 1C). However, S10A follicles are shorter in length than S10B follicles, as the S10B follicles are fully elongated and thus equal in length to a S14 follicle (

Discussion

The Drosophila follicle is composed of only a small number of cell types, making it ideal for both morphologic and molecular analyses. Furthermore, due to the structure of the ovary, it is relatively easy to obtain large numbers of specific stages of follicle development with a common dissecting scope and minimal training. As each stage represents a short temporal window, stage isolation can provide significant molecular insights into the developmental processes occurring during that stage. For example, we have .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Active Dry Yeast	Genesee Scientific	62-103	Any source of Active Dry Yeast is fine
Grace’s Insect Media	Lonza	04-457F
Heat Inactivated Fetal Bovine Serum	Atlanta Biologicals	S11050H	Any Heat Inactivated FBS should work
10x Pen/Strep	Gibco/Invitrogen	15140-122
Pin Vises and Needles	Ted Pella, Inc.	13561-10
Spot Plate, Nine Well	Corning	7220-85
#5 Dumont forceps	Fine Science Tools	11252-20
24 multi-well plates	Becton Dickinson	35 3226	Any 24-well tissue culture dish should work
Coverslip Bottom Dishes (35mm)	MatTek Corporation	P35G-1.0-14-C	Coverslip thickness will depend on the microscope/objective being used
Glass pipettes	Corning	7095B-5x (for transferring follicles)
Glass pipettes - long	Corning	7095B-9 (for producing pulled pipettes)
Sample pestle (1.5 μl; RNase/DNase free)	Research Products International	199228	Any plastic pestle that fits 1.5 μl microfuge tubes can be used
Trizol	Invitrogen	15596-018