We are interested in discovering new cellular and molecular mechanisms controlling the stemness of cells. Our main aim is to decipher how stem cells avoid differentiation in the niches. Since we are working under experimental conditions, it is essential to preserve tissue integrity to ensure that GSCs receive a physiological behavior.
Our protocol maintains a healthy and functional GSC niche enabling the monitoring of dividing GSCs as they would be in vivo. The use of extended life imaging allow us to study the entire cell cycle of GSCs and spectrosome dynamics. Specifically, we successfully characterize the spectrosome changes across the cell cycle, a task that was previously difficult to achieve with fixed images or shorter imaging periods.
Knowing how to perform mainly experiments without affecting it's dynamics allows us to explore how the niche cells communicate. We want to study regulatory mechanisms of gene expression and cellular metabolism mediated by direct interaction between mRNAs, metabolic enzymes in the biology of the stem cells. To begin, prepare 100 microliter aliquots of the streptomycin penicillin antibiotic mix at a concentration of 10, 000 units per milliliter.
Prepare all other required reagents for the procedure and store them appropriately. Collect one to two day old flies expressing the ubiquitously and constitutively expressed par one fused to GFP into a new tube. Culture the flies for two days at 25 degrees Celsius in an incubator before dissection.
Place a three microliter drop of the specific Cell-Tak adhesive in the center of the cover slip of a 35 millimeter poly-D-lycine-coated plate. Immediately add an equal volume of 0.1 molar sodium bicarbonate with a pH of eight to the three microliter adhesive drop. Mix the solution with a pipette.
For complete evaporation, incubate the plate with it's cover at room temperature for 20 minutes. Then store the plate at four degrees Celsius overnight. After thawing the plate the next day, wash it three times with six microliters of autoclave pure water carefully without touching and disturbing the adhesive layer.
Let the water evaporate completely at room temperature for five to 10 minutes. To begin, culture the flies and prepare the glass bottom plate with the coating. Then clean a three well dissection dish, forceps and dissection needles with 70%ethanol.
Add approximately 200 microliters of ringer solution to each of the three wells of the dissection dish. Using forceps, grab the Drosophila female at the intersection of the thorax and abdomen. Gently tear the cuticle at the posterior abdomen and pull it back to expose the ovaries.
After dissecting the ovaries, transfer them to the next well to reduce contamination with tissue remnants, or debris from the dissection. Under a stereo microscope with LED illumination, use one pair of fine forceps to anchor the entire ovary and the second pair to grab a late stage egg chamber. Gently stretch until the muscle sheath breaks and lags behind.
Using forceps, transfer the 15 to 20 muscle sheath-free ovarials one by one to the third well containing 200 microliters of ringers medium. Pre-wet a 200 microliter tip with 0.1%Tween 20 to prevent the ovarials from sticking to the tip wall. Transfer six microliters of ringer solution containing the muscle sheath-free ovarials using the pre-wetted tip to the prepared adhesive plate at room temperature.
Using a dissection needle or forceps, carefully press the ovarials to the bottom of the ringer's drop to ensure contact with the adhesive surface. Next, add three milliliters of Schneider's medium, supplemented with 15%FBS and 0.6%Streptomycin penicillin antibiotic mix to the MatTek plate. Transport the plate containing the muscle sheath-free ovarials on a flat surface to the microscope station.
Place the plate on the 63x objective of a spinning disc microscope or a confocal microscope. Focus the sample and select the middle Z plane for each germanium. To configure the experimental parameters, set the capture type to 3D time lapse.
Adjust the laser power to 70%of the maximum. Set the excitation wavelength to 488 nanometers. The Z stack range to 30 micrometers and the distance between Z stacks to 1.2 micrometers.
Set the interval between time points to every 10 minutes and the duration to 17 hours. If available, use the multi-position option to redefine the XY of the samples. Select the Z plane in the middle of each germarium so that the Z stacks will be centered at this position and start the acquisition.
Analyze the images in time and along the Z axis to visualize changes in the spectrosome morphology using Imaris or Image J software. To create the movies, manually select the best Z plane at each time point. The strong GFP par one signal of the round G2 spectro zone was significantly reduced during the G2 MG1 phases.
During early prophase GFP par one left the spectrosome and filled the cytoplasm indicating the germline stem cell entry into mitosis. After mitosis and nuclear envelope reformation, the GFP par one signal gradually recovered in the round G1 Spectro cycle. Denovo edition of GFP par one to the round G1 spectrosome led to the formation of plug, bar and fusing spectrosome morphologies.
