The overall goal of this procedure is to visualize in vivo reporter for epidermal wound response in drosophila embryo. This is accomplished by first collecting the developmental stage where the reporter activity can be consistently detected. The second step of the procedure is to puncture the embryo using a glass needle and microinjection apparatus.
The third step is to anesthetize the embryo for visualization of the reporter. The final step is to validate the reporter localization by detecting the expression pattern of RNA probes. Ultimately, the results can show the specific activation of the epidermal wound response reporter in only the cells surrounding the site of puncture injury through confocal microscopy.
The main advantage of this technique over existing methods that involve looking at wound closure is that we can see in living embryos gene expression that's triggered by puncture wounding. This method can help answer key questions in the tissue repair field, such as how cells surrounding an injury regulate an immediate wound response to promote Repair. The implications of this technique extend towards therapy or diagnosis of tissue regeneration because the wound response regulation is the initial step.
Wound healing can be assayed in any stage of development. However, by stage 17, the cuticle is too mature. Known wound reporters will not be activated.
So to demonstrate, stage 15 to 16 will be wounded to harvest embryos. Collect two to three day old adult flies, 50 females and 25 males. Transfer them to an embryo collection cage with a fresh plate of apple juice, agar and a dollop of yeast paste.
House the cage at 25 degrees Celsius each morning for the next three days. Replace the plate with a fresh one. On the afternoon of the third day, allow eggs to accumulate on a fresh plate for two hours.
Store this plate for 14 hours at 25 degrees Celsius. A two hour lay should yield about 200 embryos the next day. Add a few milliliters of water to the plate and swirl it gently.
Then using a paintbrush, transfer the water and embryos from the plate to a nylon mesh basket in a collection tube. Now make a shallow bleach bath and soak the embryos in it for two minutes. This step removes the CORs, the eggs.
Shell swirl the bath a few times to prevent the embryos from clumping. After two minutes, rinse off embryos with flowing water and petri dish rinsing for five seconds each. Then transfer the embryo basket to a paper towel to dry off the embryos.
Next, using a dissecting needle, move about 50 embryos from the mesh to an agar plate made with green food dye. The dye is for increasing knee eggs visibility. Under a dissecting scope, make two parallel rows of embryos about five millimeters apart.
Position the rows apart by the width of an embryo to allow for gas exchange. Next, carefully press a slide with double stick tape onto the embryos so that the rows are perpendicular to the length of the slide. To reduce their internal pressure, allow the embryos to air dry.
This way they won't burst when they're wounded. After 25 minutes, the first prepared slide should be dry. Now, cover the embryos with two to three drops of halo carbon oil to prevent further dehydration and immediately proceed with wounding the dry embryos.
Place the embryo slide in the injection microscope stage. Find the embryos in the field of view and practice moving the stage of the inverted microscope. Now focus on middle plane of embryo along the dorsal ventral axis and bring the top embryo of the first aligned row into the field of view.
Next, carefully place and secure a pulled needle in the needle holder. Use the micro manipulator to move the needle down to the slide. Align the needle with the embryo in the same focal plane.
Now, do not adjust the microm manipulator any further. Now move the stage to puncture the embryo through and through. Then move to the next embryo in the row.
After wounding the first row, move the needle completely up and away from the stage. Then rotate the slide and continue puncturing embryos in the second row. Once all wounded, the embryos can be immediately fixed for inside you hybridization or antibody staining.
To examine fluorescent reporters wait four to six hours and transfer the embryo to a new on the new slide, place glass beads around the embryos. Then add a few drops of 50%One phenoxy, two propanol, which is an anesthetic, and place a cover slip on the embryos for micro injections. Load the injection needle from the rear with one microliter of chemical solution plus dye, such as 0.6 molar hydrogen peroxide.
Allow capillary action to draw the chemical solution to the tip of the needle. Next, break the tip of the needle against the edge of a cover slip in halo carbon oil mix. First, bring the mounted needle into focus with the edge of the angled cover slip at not quite 90 degrees.
Next, gently move the cover slip edge across the tip of the needle until it breaks. Then apply pressure on the microinjection apparatus and check that the solution flows out of the needle. If it doesn't flow, break the needle open a little more.
Now, perform the same procedure used to puncture embryos only. This time, micro inject the solution simultaneously with the puncture. Ideally, inject 10 nanoliters.
If too much solution is injected into the embryo, the Vitale membrane will burst while following the fixation procedure in the text protocol, pay careful attention to the following steps. Use a glass pipette, not plastic to rinse the heptane over the slide and use glass to swirl the embryos into the center of the glass Petri dish. Later, shake the embryos in fixative for 25 minutes at 220 to 230 RPM.
After shaking, let the bubbles at the interface pop before completely removing the bottom aqueous phase. Also, be careful not to pull up the embryos. After adding the methanol, the wounded embryos should cluster at the interface later, spread the embryos out along the surface of the plate like so, and transfer them to a double stick tape glass slide like so.
Then carefully push the embryos with the dissecting needle to pop the Vitale membrane and proceed with the dehydration. Wild type flies were transformed with fluorescent reporters fused to DOPA decarboxylase or tyrosine hydroxylase enhancers and subjected to the described assay. Under brightfield, it was possible to see the wound site while under fluorescence illumination, it was possible to see DCD wound reporter activity.
The tyrosine hydroxylase reporter showed a similar result. Both images were collected on a Leica SP two confocal microscope with a 20 x objective. The procedure was performed in a loss of function flow two mutant.
Background, DOPA decarboxylase reporter activity was expanded throughout epidermal cells in gain of function flow two mutants generated with ubiquitous expression in all cells. Inhibition of the reporter was noted throughout the epidermal cells. Next embryos were simultaneously wounded and injected with a solubilized compound and udine blue dye hydrogen peroxide and water expanded DOPA decarboxylase reporter activity in the epidermal cells, as did an injection of methyl B cyclodextrin in sodium hydroxide using inside U hybridization.
Transcriptional activation of wound response genes was assay endogenous DOPA decarboxylase. RNA expression was seen to be localized around the wound site and of course, tyrosine hydroxylase, RNA transcripts were also found to accumulate at the wound site. Once mastered, this technique can be done in 60 minutes if performed properly.
While attempting this procedure, it's important not to spend too much time aligning embryos and move forward with the next steps in the procedure.
