The overall goal of this procedure is to image cell shape change in living drosophila embryos. This is accomplished by first generating and expanding transgenic fly stocks that express GFP tagged protein probes. The second step of the procedure is to transfer these flies to embryo collection cups.
The third step of the procedure is to collect embryos. The fourth step is to coate and mount the embryo. Ultimately, results can be obtained that show cell shape changes such as cellular by time-lapse confocal imaging of the mounted embryos.
The main advantage of this technique over existing methods, such as fixed tissue imaging, is that the dynamic aspects of cell shape changes can be captured in the context of living embryos. Although the method described here is tailored to esophagus ization, it can be adapted and applied to other cell shape changes that accompany early fly development, such as ventral furrow formation, germ band extension, trache, agenesis, and dorsal closure. Individuals new to this technique often find it a challenge to manipulate and mount the embryos under the dissecting microscope.
My graduate advisor, Anna Marie Soki, and I will be demonstrating this procedure. This procedure starts by making a cup for collecting embryos using a razor cut the bottom off of a 100 milliliter tricorn beaker, making the edge as smooth as possible. Trim the three corners off of the top to make the cup easier to handle.
Next, cut a square of wire mesh six centimeters by six centimeters working in a fume hood. Place a piece of heavy duty aluminum foil on a prewarm hot plate and place a square of wire mesh on top of the aluminum foil. Push the cut bottom edge of the cup firmly onto the hot mesh.
Wait for a few seconds and lift the cup with the mesh now attached. If the foil also sticks, peel it off. Call the cup overnight the next day.
Remove excess mesh with scissors and sand off any sharp edges with fine grain sandpaper. The next step is to make a yeast paste, which will promote embryo production by the female.Flies. Fill a small cup with roughly equal parts red star active dry yeast and distilled water, and stir until the yeast is dissolved.
The paste should approximate the consistency of wet peanut butter. When the yeast paste is ready, remove the apple juice agar plates from the four degrees Celsius storage. A description of how to make the apple juice agar plates is in the accompanying manuscript streak.
The apple juice plates with the yeast paste and wait at least an hour for the plates to warm up to room temperature. Fold circular filter paper in half. Then in half again.
Trim the paper to eight to nine centimeters in diameter. Make accordion folds in the quarter circle. Unfold the circle, invert it, and insert it in a collection cup until it touches the mesh.
Make sure that the paper is secure in the cup so that it does not fall and crush the flies. Now it's time to transfer the flies. Take a stock bottle of flies that has had two weeks to reproduce This bottle should have more than enough flies to fill one embryo collection cup with a minimum of 50 females and 30 males.
For best laying use flies that are less than five days post hatching. Label the collection cup with the stock name and date. Invert the bottle of flies and gently shake it over the collection cup to transfer the flies into the cup, immediately cover the cup with a prepared apple juice plate.
Secure the plate to the cup with a rubber band set The cup mesh shied up at room temperature in an area with direct light. Make sure that no shadows fall on the cup as the flies will not lay well in the dark. After two hours, collect the embryos by replacing the apple juice plate with a new plate to our collections at room temperature.
Work well for imaging cellular. Next, prepare a mounting chamber for the embryos. To do this cuss a piece of double-sided tape two to three centimeters long and place it on a slide aligning in the long axes of the tape and slide.
Cut a second, two to three centimeter long piece of double-sided tape and layer it on top of the other piece of tape, making sure their edges are aligned. Using a razor blade, make two cuts, approximately three millimeters apart in the center of the tape and perpendicular to the long axis of the slide. Remove the tape between the cuts to make a channel pipette drop of halo carbon 27 oil into the channel.
This channel is where the embryos will be mounted. Coate the embryos by covering the apple juice plate with 50%bleach for 30 to 60 seconds. Collect embryos in a cell, strainer and wash vigorously with distilled water from a squirt bottle.
Dab the strainer on paper towels. Use a moist paint brush to transfer 10 to 50 coated embryos to a clean apple juice plate that has no yeast paste. Wick away water with the torn edge of a paper towel and immediately cover the embryos with a small amount of halo carbon 27 oil.
Move the apple juice plate to a dissecting microscope using transmitted light. Stage the embryos according to the morphological guidelines of bounds. Use forceps to transfer five mitotic cycle 11 or 12 embryos corresponding to bound stage four to the channel of the mounting chamber.
Arrange the embryos in a line with the dorsal and ventral sides visible and their lateral side down. Embryos are easily arranged in this orientation in oil alone. To avoid crowding the embryos together, leave at least half an embryo length between embryos.
Lay one edge of a 25 by 25 millimeter cover slip on the double sided tape at one side of the channel aligning one edge of the cover slip with one edge of the tape. Drop the cover slip to cover the channel. If air is caught underneath the cover slip, apply a small amount of oil at the edge of the cover.
Slip capillary action will pull the oil into the channel and push out the air. Place the mounting chamber on the confocal microscope and find the embryos by transmitted light. Then switch to confocal imaging to focus on a region of interest for cellular image in a single plane near the middle of the embryo.
To follow Invagination dynamics, use 30 to 62nd intervals for time-lapse imaging. After acquiring the images, analyze them using an image analysis package such as ni h's freeware package called image J.The data can then be presented as movies, sequences, or CH graphs. To image a cell shape change other than cellular, it may be necessary to mount embryos in a specific orientation that is not easily maintained in oil alone.
To do so, use an alternate mounting method to the one described previously. Begin by making embryo glue by combining 20 centimeters of double-sided tape with 250 microliters of heptane in a ation vial. Place this insulation vial on a mutator or rotating platform and mix overnight.
The next day, dip a yellow pipette tip into the embryo glue and then trace the pipette tip along a slide leaving a trail of glue while the heptane from the embryo glue is evaporating. Align the staged embryos on a block of agar such that the embryo's surface to be imaged is facing the agar. For example, to image cell shape change.
During farrow formation, mount the embryos with their ventral side facing the agar. Invert the slide with the embryo glue and gently press the trail of glue against the embryos on the agar block. Now invert the slide again.
Most of the embryos should be stuck to the slide in the appropriate orientation. Next, add two layers of double-sided tape on either side of the embryos. Cover them with halo carbon 27 oil and apply a cover slip.
The embryos are now ready for imaging. These photos show time-lapse imaging of cellular embryos are mounted with dorsal and ventral sides, clearly visible and are image near their middle To follow the plasma membrane in imaginations in cross-section, the embryo shown here expresses a GFP myosin two probe, which concentrates at the tips of the plasma membrane.Invaginations. Tracking the ingression of this front over time gives the rate at which the plasma membrane invaginates the zero minute time point corresponds to cellular onset shortly after the 56 minute time point gastro starts on the ventral side of the embryo.
This time lapse movie of cellular started in the prior mitotic cycle, 13, capturing pseudo cleavage, furrow regression, and continued until gastro movements were seen on the ventral side of the embryo. Once mastered, the mounting and imaging can be done in 60 to 90 minutes if performed properly. Of course, embryo collection requires additional time and must be coordinated with the mounting and imaging.
While attempting this procedure, it's important to do everything possible to maintain good embryo health. For example, don't over bleach the embryos and limit their laser exposure while imaging following this procedure. More sophisticated imaging techniques such as photo bleaching and photo switching can be used to answer additional questions like, what are the cytoskeletal and membrane dynamics that accompany a given cell shape change?
