Dissection and Staining of Drosophila Larval Ovaries

Summary

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Abstract

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) ^{1, 2}. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches ^3-12.

Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar ^13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs ^{7, 16, 18, 19}. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.

Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100μm across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Protocol

1. Egg laying

1. Five days before dissection: allow mated females to lay eggs for 2-4 hours on fresh food supplemented with yeast. To get synchronized and well-developed larvae, it is important not to have overcrowded cultured (about 30 eggs/ 25mm vial). Typically, 7-16 females are used per vial, depending on how well they lay.

2. Selecting larvae

1. Prepare a 9-well glass dissecting dish filled with Ringer's medium (128mM NaCl, 2mM Kcl, 1.8mM CaCl₂, 4mM MgCl₂, 35.5mM Sucrose, 5mM Hepes pH 6.9).
2. Prepare cell strainers in a six-well plate containing Ringer's medium and place it on ....

Discussion

This video demonstrates an isolation and staining protocol of late third instar larval ovaries. To perform this protocol routinely and reliably, attention should be paid to the following points:

1. For synchronized and well-developed larvae, over crowding must be avoided.
2. To avoid loss of the small translucent ovaries, make sure to dissect the fat body intact. This will also help in locating the ovaries at the mounting stage, particularly when mounting younger (second or beginning of third instar).......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	JT Baker
Kcl	Merck & Co., Inc.
CaCl2	Sigma-Aldrich
MgCl2	Merck & Co., Inc.
Sucrose	JT Baker
Hepes	Sigma-Aldrich
PBS	Sigma-Aldrich
Triton X-100	Sigma-Aldrich
Albumin Bovine Fraction V	MP Biomedicals	160069
Dumont biology tweezers 5 dumstar polished	Fine Science Tools	11295-10
Nickel plated pin holder	Fine Science Tools	26018-17
s.s minutien pins 0.1mm diam, 10mm long	Fine Science Tools	26002-10
9 well plates 85X100 mm, 22mm o.d.x7mm deep	Corning	7220-85
Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST	Leica Microsystems
6 well plates	Costar	3516
Slides	Menzel-Glaser	798
Cover slips	Corning	2940-223
Mounting media	Vectashield	H-1200
Cell strainer	Falcon BD	FAL352350
1B1 antibody	Developmental Studies Hybridoma Bank
Anti-Traffic Jam	Laboratory of Dr. Dorothea Godt
Anti-Vasa	Laboratory of Dr. Ruth Lehmann
Anti β-Galactosidase	Cappel
Secondary Antibodies	Jackson ImmunoResearch