Maintenance of epithelial tissues requires continuous removal of damaged and dying cells without disrupting barrier function. To accomplish this epithelial extrude dying cells by forming and contracting a ring of actin and myosin in the cell surrounding the dying cell to eject it while closing any gaps that may have resulted from its exit. In this video article, A method to induce and image apoptotic cell extrusion from the epidermis of larval zebra fish in real time.
In shown this is achieved by injection of a red fluorescent protein labeled F actin probe into one cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis to visualize acting filaments in epithelial tissues. Day four, post personalization larvae that express both GFP and RFP are then treated with G four 18 to induce apoptosis in the epidermis. Actin dynamics and epithelial cell shape changes that occur during extrusion are visualized by time-lapse imaging on a spinning disc confocal microscope.
The main advantage of this technique over existing methods like immuno force and analyses, is that we can observe rapid changes and actin dynamics that occur during the extrusion process if a in a living epithelial tissue. In this video, we will show you a procedure for imaging extrusion of apoptotic cells from the epidermis of developing zebra fish in real time. We use this procedure in our laboratory to understand how cells coordinately rearrange their acton cytoskeleton to remove apoptotic cells while maintaining barrier function and how disrupting the in extrusion process may lead to certain disease states.
So let's get started To visualize acting dynamics during cell extrusion in the epidermis of developing zebrafish begin by thawing previously transcribed RNA on ice. We transcribe Mr.mRNA encoding red fluorescent protein fused to the calp in homology domain of utrophin and act in binding protein. Add phenol red to the RNA at a one-to-one ratio for a final RNA concentration of around 30 nanograms per microliter and load one microliter of solution into a pulled capillary needle by back filling.
Collect around 75 1 cell stage C-K-G-F-P embryos in E three buffer. Pipette the collected embryos into the microinjection mold with a five and three quarter pasta pipette and gently orient the embryos in the trough with FST Dumont. Number five, forceps work quickly to avoid having to inject the RNA into multi-stage embryos under a standard laboratory dissecting stereo microscope.
Use a pressure controlled micro injector to inject the RNA into the yoke of the embryos as fossil of phenol. Red should be visible in the embryo after injection. Be sure to inject at least 50 embryos for each experiment.
Sort the embryos to remove any unfertilized or damaged eggs and incubate all remaining embryos at 28 degrees Celsius. After 24 hours, use a fluorescent dissecting microscope to select zebrafish embryos that have a high level of GFP fluorescence in the epidermis and RFP labeled acting fluorescence. Incubate the selected embryos at 28 degrees Celsius until ready to proceed with the drug treatment to induce apoptosis exposure to the amino glycoside.
Antibiotic G four 18 or medicine causes extrusion of apoptotic cells from the epidermis of the developing zebrafish larvae. Importantly, this treatment only works on larvae that are four days post fertilization and older to induce cell extrusion. Collect up to 25 4 day post fertilization zebrafish larvae expressing both GFP and RFP into a 35 by 10 millimeter culture dish containing three milliliters of E three medium.
Carefully replace the medium with three milliliters of E three containing one milligram per milliliter of G four 18 and incubate larva in the drug for four hours in the dark at 28 degrees Celsius. Then remove the media and replace with prewarm E three media containing 0.02%trica and proceed to mounting the larvae to mount the larvae for imaging. Transfer up to three larvae to a 1.5 milliliter fendor tube and remove most of the E three medium.
Using a cut pipette tip pipette 120 microliters of 1%low melt aros into the tube mix. And then gently pipette the larvae and aro mixture onto the cover glass in the bottom of a matech culture dish. Using an FST pin holder with an insert pin or tungsten needle attached, quickly orient the larvae so they are straight and flat against the cover glass.
Allow the aose to solidify. Then gently fill the dish with E three medium containing 0.02%trica. We have found that the entire process of apoptotic cell extrusion from the epidermis of the developing zebrafish larvae takes approximately 20 minutes.
Here we demonstrate how to set up a time-lapse imaging experiment using a spinning disc confocal microscope and the and or IQ software to collect a series Z planes through a single layer of the zebrafish epidermis. First, power up the argon and helium neon lasers microscope camera and epi fluorescent lamp within the and or IQ software. Create a time series protocol containing the wavelengths to be imaged.
The frequency of the intervals between image captures and the number of times the capture should be repeated Gently place the mattec dish containing the specimen on the microscope stage and then use a 20 times objective with transmitted light to focus on the larvae. Move the 40 times water immersion objective into the correct position. Using this objective, we can film approximately 20 to 25 cells per field, which allows us to follow cellular behaviors during extrusion, while also resolving subcellular acting dynamics.
Carefully place a drop of water on the top of the 40 times objective and quickly place the mattec dish on top. Identify the specimen using brightfield illumination and then use 488 nanometer epi fluorescence to focus specifically on the GFP positive epidermis. Switch to confocal scanning mode.
Adjust the laser intensity and exposure time for each of the channels accordingly. Take care to balance the laser power and time of exposure for optimal detection of your signal and to prevent any phototoxicity. To identify extruding cells, use epi fluorescence to visualize the GFP labeled epidermis and identify a group of cells in a classic rosette pattern consisting of a collection of cells surrounding a small round cell in the middle.
Additionally, there should be an accumulation of the RFP labeled actin visible as a ring around the small round cell in the middle, a hallmark of cell extrusion. Once an extruding cell has been identified, quickly set the upper and lower limits for the Z Series and the step size. Then begin acquiring four D confocal data sets to view the data and to visualize contraction of the actin ring and epithelial behaviors that occur around the dying cell.
Over time, make 3D projections of the Z series and save the data as a movie file. This step can be performed using a variety of software packages. This figure shows the expression of RFP UTR CH in the epidermis of a four day post fertilization CK GFP zebrafish larvae.
Here still frame Z projections from a time lapse movie that follow live acting dynamics. During the process of epithelial cell extrusion are shown the arrows denote the ring of actin formed by neighboring cells, which contracts and closes over the course of two minutes. Following this procedure, we can use other methods in combination with this technique, such as the use of chemical inhibitors or genetic mutations to better understand how altering extrusion may lead to certain disease states.
Stemming from the failure to clear apoptotic cells, we've just shown you how to set up a time-lapse imaging experiment to visualize the extrusion process from the epidermis of the developing zebrafish. When performing this procedure, it's important to remember to limit the amount of light exposed to your specimen to prevent any photo bleaching or toxicity. So that's it.
Thank you for watching and good luck with your experiments.
