The overall goal of this procedure is to induce benign and invasive tumors in drosophila, and then visualize them by dissecting the larval cephalic complex. This begins with setting up across between the desired strains and isolating the larval progeny, bearing either the invasive or benign tumors. The next step is to dissect the selected larvae.
To expose the cephalic complex by first cutting them two thirds of the way down, securing them, and then turning them inside out. Next fat body and gut tissues are cleaned off, and then any nerves still attached to the ventral nerve cord and the larval epidermis are also broken. The final step is to image the cephalic complex or fix it for downstream applications.
Ultimately, results can show GFP tagged invasive or benign tumors through the use of fluorescence microscopy. This method can help answer some of the key questions in cancer biology. For example, you may want to understand which genes could inhibit progression of tumors in an intact organism.
Once the required genetic stocks have been obtained, prepare a starter culture of the tester strain. Also prepare a starter culture for the tested stock. Cross 10 female virgins from the tester stock with 10 males from the tested stock.
Allow the flies to mate and lay eggs for 24 to 48 hours at 25 degrees Celsius, and then transfer them to a fresh vial to produce more progeny. Monitor the cross vials for eggs and first instar larvae. Set up a new cross.
If the collected vials show poor egg deposition. If the collected vials start drying out, add just three to four drops of autoclave distilled water to keep the culture moist. Continue monitoring the vials until wandering.
Third instar larvae are present. Then proceed with monitoring them under a fluorescence stereo microscope. Using the same procedure, collect flies bearing invasive tumors using the listed stocks by culture day five or six, wandering third instar larvae will be available from the benign tumor cultures.
However, they will not be available until day 10 from the invasive tumor cultures. To collect the third instar larvae, wet a fine paintbrush with distilled water and scrape them off the wall of the vial. Place the larvae into a depression slide with cold one XPBS, and then using a wet paint brush.
Scrape off any food material from their epidermis. For a few minutes of immobilization, place the larva on the plate at minus 20 degrees Celsius for 30 minutes. For a longer immobilization time, place the larvae in a culture vial with only fly nap on a wand.
Plug the culture vial and let it stand for 40 minutes. Next, transfer the immobilized larvae to a glass slide. Add a drop of light halo carb oil and cover the larvae with a cover glass.
Now, observe the larvae under a fluorescent stereo microscope. For GFP expression, select the green fluorescent larvae and proceed with the dissection. Benign tumor bearing larvae will fluoresce green in the anterior region where the cephalic complex is present.
Invasive tumors will also fluoresce green in the anterior region of the larvae at the cephalic complex. If possible, be sure to photo document the fluorescing tumors. So the translucent epidermis of the larva makes it difficult to observe the extent of migration of the tumors, and that is why we must dissect the tumors.
Begin with using forceps to transfer a fresh larva with either tumor. Type to a well of a dissection dish containing one milliliter of cold one XPBS. Hold the larvae down using a pair of forceps at about two thirds the distance from the anterior end.
Using another pair of forceps smartly separate the posterior third of the larvae and discard it. It then let go of the anterior two thirds. As the pressure inside the larvae is released, its contents will ooze out of the body cavity.
Using forceps, remove the tissues that have oozed out of the body cavity. Next, hold the mouth hook of the larva and push it into the larva epidermis. Using the other pair of forceps completely invert the larvae.
Now with the forceps, gently and carefully, remove the fat body salivary glands, gut, the wing disc complex and any tracheal tubes. The cephalic complex may now be visible attached to the mouth hook. The brain hemispheres and the VNC are still connected to the larval epidermis via nerves emanating from the VNC.
With forceps, gently break these connections. Then remove any excess fat body or other tissues so that the cephalic complex becomes clearly visible. Attached to the larval epidermis at the mouth hooks.
Leave the cephalic complex as it is for further processing prior to visualization. Otherwise, to directly visualize the cephalic complex, detach it from the epidermis and place it in a drop of glycerol based mounting media such as vector shield. Using the described protocol, tumors were induced in the eye.
Anin disc, benign tumors remained localized to the eye anin disc where they are induced. Notice that the GFP labeled tissue has not migrated to contiguous organs like the ventral nerve cord in the cephalic complex bearing invasive tumors from a day 10 larva overgrowth of tumor tissue was noted. Also, as pointed out by the red arrow, the tumors invaded contiguous organs like the VNC and mouth hooks.
After watching this video, you should have a good understanding of how to genetically induce benign and invasive tumors, and then visualize them by dissecting the cephalic complex intra ano gastro.
