Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Summary

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Abstract

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs' asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Introduction

Live imaging of biological processes is instrumental in obtaining direct experimental evidence. The combination of advanced confocal microscopy and the optimization of methodologies permits the exploration of multiple biological events with high precision. Optimizing steps in protocols such as tissue manipulation and dissection, sample preparation and preservation, and microscopy acquisition settings is critical to maximize the reliability and robustness of the results obtained. Here, we present a protocol to monitor samples for extended imaging, which is specially focused on Drosophila melanogaster ovaries. The Drosophila melanogaster ovary is an ex....

Protocol

NOTE: Step 1.4 must be done at least 2 days in advance. Steps 2.1 and 2.2 can be done 1 day in advance.

1. Pre-experimental setup I

1. Prepare 100 µL aliquots of streptomycin/penicillin antibiotic mix (10,000 U/mL penicilin and 10 mg/mL streptomycin) and store at -20 °C until use.
2. Prepare 15 mL of the following solutions using autoclaved pure water, store them at 4 °C, and use them within a month: Sodium bicarbonate (NaHCO₃) 0.1 M p.......

Discussion

Here we present a protocol to monitor the Drosophila melanogaster GSC niche for a prolonged period of time, up to 16 h. Most of the protocols to film Drosophila biological processes ex vivo focus on shorter time windows and multiple examples can be found for imaginal wing discs^15,16,17, embryo gonads^18,19 or ovaries²⁰

Materials

Name	Company	Catalog Number	Comments
9-well glass plate	Corning	7220-85	n/a
Calcium chloride dihydrate	Sigma Aldrich	C3881	CaCl2·2H2O; To prepare Ringer's solution
Cell-Tak	Corning	354240	Cell and Tissue Adhesive used to attach cells or tissue sections to many types of surfaces, including plastic, glass, metal, FEP Polymer, and biological materials.
Dumont #5 Forceps	Finescience	11252-20	n/a
Dumont #55 Forceps	Finescience	11255-20	Dimensions 0.05 mm x 0.02 mm and length 11 cm
Fetal Bovine Serum	Gibco	10500-064	Qualified, heat inactivated, E.U.-approved, South America Origin
Glass bottom dishes	MatTek	P35GC-1.5-10-C	35 mm Dish, No. 1.5 Coverslip, 10 mm Glass Diameter, Poly-D-Lysine Coated
HEPES	Sigma Aldrich	H4034	To prepare Ringer's solution
Magnesium chloride	Sigma Aldrich	M1028	MgCl2; To prepare Ringer's solution
Needle Holder	Roboz	RS-6061	Light weight, hollow stainless steel handle; 4 3/4" Long.
Nikon SMZ18 binocular	Nikon		https://www.microscope. healthcare.nikon.com/products/stereomicroscopes-macroscopes/smz25-smz18
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122	10,000 U/mL
Potassium chloride	Sigma Aldrich	P3911	KCl; To prepare Ringer's solution
Schneider's Drosophila Medium	Biowest	L0207	Cell culture medium for insect cells
Sodium bicarbonate	Sigma Aldrich	S8875	NaHCO3; ≥99.5%, powder
Sodium chloride	Sigma Aldrich	S9888	NaCl; To prepare Ringer's solution
Sucrose	Sigma Aldrich	S9378	≥99.5% (GC); To prepare Ringer's solution
Tungsten Dissection Needle	Roboz	RS-6064	0.25 mm, Ultra Fine, 1 Micron Tip (Pk 10)
Tween 20	Sigma Aldrich	P9416	for molecular biology, viscous liquid
Yeast powder	Sigma Aldrich	51475	n/a