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 excellent model system for the analyses of a wide range of developmental processes. Among many others, this reproductive organ of the fruit fly Drosophila melanogaster contains a very well-defined adult stem cell niche. This GSC niche sustains the development of the female gametes during adulthood. The Drosophila ovaries are composed of approximately 18 ovarioles, where egg chambers are developed in the germarium. In the tip of each germarium, 2-4 GSCs are maintained in a somatic cellular niche mainly formed by a terminal filament of 8-10 cells, a rosette of 5-8 cap cells, and 2-3 anterior escort cells (Figure 1A). This somatic niche provides the GSCs with essential signals and physical support to maintain their stemness, control proliferation, and prevent differentiation^1,2,3,4,5.

The GSCs normally divide asymmetrically to generate a new stem cell that keeps contact with the somatic niche and a daughter cell, the cystoblast (CB), which loses direct contact with the somatic cap cells and differentiates. GSCs and CBs contain a highly dynamic and cytoplasmic organelle, the spectrosome, whose main function is the correct orientation of the mitotic spindle during mitosis⁶. The CB divides 4 times with incomplete cytokinesis to develop 16-cell cysts, where one of the germline cells specifies into the oocyte and the other 15 cells become nurse cells. The CB spectrosome grows into a branched structure called the fusome which connects the 16-cell interconnected germline cells. The spectrosome is enriched in small vesicles and skeletal proteins such as the serine-threonine kinase Par-1 and the membrane component Hu-li tai shao (Hts)⁷. During the GSC cell cycle, the spectrosome grows by the addition of new material and changes its shape, allowing the identification of the G1, S, G2, and M phases (Figure 1B)^8,9.

We have recently implemented an ex vivo culture method of the Drosophila germarium that allows imaging of live GSCs for up to 16 h. Since these cells divide once every 15.5 h on average⁸, this method permits the filming of large portions of the GSC cell cycle. Thus, in combination with other tools, our culturing method has allowed the description of spectrosome morphology during the GSC cell cycle and the analysis of the duration of the different cell cycle phases in vivo⁸ (Figure 1C). Here, we provide a detailed protocol of this extended live imaging method supported by a step-by-step guided video that describes the methodology (Figure 2).

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 pH 8.0 and 0.1% Tween 20.
3. Prepare the following culture medium and solution in a laminar flow hood to avoid contamination. Use sterilized filter tips and tubes.
   1. Prepare 2 mL aliquots of undiluted fetal bovine serum (FBS) and store at -20 °C until use in aseptic conditions.
   2. Prepare Schneider's Drosophila medium supplemented with 15% FBS and 0.6% streptomycin/penicillin antibiotic mix. Prepare 7 mL aliquots and store at 4 °C. One aliquot is enough for two MatTek plates (see section 2).
      1. Prepare Ringer's solution (128 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 4 mM MgCl₂, 35.5 mM Sucrose, 5 mM Hepes in autoclaved pure water; pH 6.9). Prepare 1 mL aliquots and store at -20 °C until use.
4. Collect 1-2 days old flies expressing constitutively and ubiquitously the Par-1 protein fused to GFP (poly Ubiquitin-mGFP6::par-1 line) into a new tube with fly food supplemented with yeast powder. Culture them for 2 days at 25 °C in an incubator before dissection.
   NOTE: This step can be done with any fluorescently tagged reporter of interest.

2. Glass bottom plate coating and preparation

1. Place a 3 µL-drop of a specific Cell-Tak adhesive (henceforth referred to as cell and tissue adhesive) in the center of the coverslip of a 35-mm poly-D-lysine coated plate (glass bottom dish, see Table of Materials) without manual spreading. Use sterile tips.
   NOTE: The cell and tissue adhesive should be stored at 4 °C. In the case of experiments with two genetic conditions (typically, control and mutant conditions), place two individual, well-separated drops of the adhesive in the same glass bottom plate. This will ensure that both genetic backgrounds are imaged in the same conditions.
2. Immediately after, add an equal volume (3 µL in this protocol setup) of 0.1 M NaHCO₃ pH 8.0 carefully into the 3 µL adhesive drop as recommended by the manufacturer and mix by pipetting.
3. For complete evaporation, incubate the plate at room temperature (RT) for 20 min and then keep at 4 °C overnight to be used the next day. Alternatively, incubate the prepared plate at 37 °C for 25 min until complete evaporation and proceed directly to the next step. In both cases, maintain the plate with its cover. Use sterile tips.
4. Allow the prepared glass bottom dish to reach RT by placing it on the bench for 15-20 min before use.
5. Wash 3 times the prepared plate carefully. Avoid touching and disturbing the adhesive layer(s). In each wash, add 6 µL of autoclaved pure water and then remove the water using a clean tip for each of the three washes to eliminate residues of the NaHCO₃ solution.
6. Allow the complete evaporation of the water by placing the clean and prepared plate at RT while dissecting for 5-10 min with its lid on to avoid dust particles.

3. Drosophila ovary dissection and ovariole isolation and mounting

NOTE: The ovary dissection is performed in Ringer's solution (instead of Schneider's medium) without FBS to prevent the presence of proteins that could interfere with the sticking of the sample tissue to the adhesive.

1. Clean a three-well dissection dish, forceps, and dissection needles with 70% ethanol before starting.
2. Add a sufficient volume (~200 µL) of Ringer's solution in each of the three wells.
3. Dissect the ovaries of 5 yeast-fed females in 200 µL of Ringer's solution in the first well with the help of two pairs of forceps following standard procedures^10,11. Grab the Drosophila female at the intersection of the thorax and abdomen with one of the forceps. Then, gently tear the cuticle at the posterior abdomen and pull the abdomen cuticle until the two ovaries are exposed.
4. Transfer the dissected ovaries to the second well to reduce contamination with remnants of other tissues or debris resulting from dissection.
5. Remove the muscle sheath of 15-20 ovarioles with the help of a stereomicroscope (zoom 8X-13.5X) with light-emitting diode (LED) illumination and fine-point forceps. Use one pair of forceps to anchor the entire body of the ovary, and use the second one to smoothly grab a late-stage egg chamber and gently stretch until the muscle breaks and lags behind.
   NOTE: It is essential to avoid damage to the germaria just by reducing the contact with this part of the ovaries. Remove egg chambers older than stages 8-9.
6. Transfer one by one the 15-20 muscle sheath-free ovarioles using the forceps to the third well containing 200 µL of Ringer's medium.
7. Pre-wet a 20-200 µL tip with 0.1% Tween 20 by pipetting. This averts the ovarioles' adhesion to the tip's plastic walls.
8. Transfer 6 µL of of Ringer's solution containing the muscle sheath-free ovarioles using a pre-wetted tip to the prepared adhesive plate (kept at RT).
   NOTE: Avoid all contact between the tip and the adhesive layer to prevent germaria detachment after ovariole deposition (next step). Approximately 30% of germaria do not adhere as expected.
9. To ensure that the transferred ovarioles adhere to the adhesive surface, carefully press them down to the bottom of the Ringer's drop until they contact the adhesive surface using a dissection needle or forceps.
   NOTE: Do not try to rearrange the ovarioles once they settle.
10. Fill the MatTek plate carefully with 3 mL of Schneider's medium supplemented with the 15% FBS and 0.6% streptomycin/penicillin antibiotic mix (step 1.3). To prevent the detachment of the ovarioles, add slowly 1 mL at a time around the outer edge of the plate.
11. Transport the plate containing the muscle sheath-free ovarioles on a flat surface to the microscope station.

4. Live imaging using a confocal microscope

1. Place the plate on the 63x objective of a spinning-disk microscope or a confocal microscope (similar equipment can be used) and focus the sample. Select the middle z plane for each germarium.
2. Configure the following experimental parameters: capture type: 3D time-lapse, adjust the laser power of 70/100, excitation wavelength: 488 nm, range of the z-stack: 30 µm and distance between Z-stacks: 1.2 µm, duration: 17 h, interval between time-points: every 10 min.
3. If available, using a multi-position option, redefine the XY coordinates of the samples and select a Z-plane in the middle of each germarium, as the z-stacks will be centered in this position. Start the acquisition.
   NOTE: As the normal width of a germarium is 30 µm, each Z-stack will be ~25-27 sections. Depending on the confocal, at least 25 germaria can be imaged within the 10 min intervals. Normally, 40x/1.30 NA or 63x/1.40 NA oil-immersion, planapo-corrected objectives are used.
4. Analyze the images in time and Z-axis to visualize specific changes in the spectrosome morphology using Imaris or ImageJ (Fiji) software^12,13. Specific times on their observation are displayed (Figure 1B'). To mount the movies, manually select the best Z plane at each time point.

Results

With this extended live imaging protocol, we can record the asymmetric mitosis of female GSCs inside their niche without apparent biological disturbances. To do so, we use germaria expressing ubiquitously the GFP::Par-1 protein, which discriminates between five distinct spectrosome morphologies through the GSC cell cycle: Round, Plug, Bar, Fusing, and Exclamation point (Figure 1B,C). In a more detailed description, we observed that the strong GFP:Par1 signal of Round-G2 spec...

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