多色 FlpOut 技術の高分解能単一細胞の形態とショウジョウバエにおけるグリア細胞の細胞間相互作用の研究への応用

The overall goal of this technique is to visualize single cell morphologies in complex anatomical tissues, thus simplifying the study of cell cell interaction in Drosophila. This method can help to answer key questions regarding complex morphologies and cell cell interactions using the fruit fly. The main advantage of this technique is that it can be adapted to the study of single cell morphologies in any tissue and at any developmental stage.

Demonstrating the procedure will be Anja Kieser, a technician in our laboratory. The MCFO technique modifies the standard FlpOut technique as follows. A heat shock FLP recombinase and different reporters remain under UAS control, however, each reporter has a common backbone of a myristoylated superfolder GFP, in which copies of an epitope tag have been inserted.

The resulting non-fluorescent proteins are named spaghetti monster GFPs and are identified with antibodies. Depending on the number of excisions, a different combination of epitopes gets expressed. Because the HSP promoter is slightly active at 25 degrees Celsius, set up crosses at 18 degrees Celsius where it is truly inactive.

Then, wait around 20 days to obtain the F1 generation. Sort the F1 females and males into separate vials. To heat shock them, first transfer them into fresh food vials when they are three to four days old.

Younger flies can be too delicate to heat shock. Next, transfer the vials into a 37 degree Celsius water bath. Submerge them deeply to ensure a homogenous heat shock.

For sparse labeling of glial cells, start with five to eight minutes of heat shock and then optimize. The optimal heat shock time will sparsely label the target cells and its relation depends on the specific GAL4 driver in use. After the heat shock, lay the vials horizontally on the bench for a few minutes to cool them down.

Now, start maintaining the flies at 25 degrees Celsius in the same vials. After two days, reporter expression will be optimal and the flies can be dissected. In preparation, put three deep depression wells on ice.

Fill one well with 70%ethanol, one with PBS and one with S2 cell culture medium. Then, load a three centimeter dissecting dish lined with black silicone with cold S2.Conduct the entire dissections in S2 to keep the tissues healthy. Now, anesthetize the flies with carbon dioxide.

Then, using forceps or a feather, put the flies in cold 70%ethanol for about 30 seconds followed by cold PBS for about 30 seconds and then transfer them into cold S2.Thus, wash all the flies to be dissected. Up to 10 flies of one genotype is usually enough. Now, within 30 minutes, dissect out all the brains.

First, detach the head from the rest of the body and discard the body. Keep the head held with the off hand through the entire dissection or it will be very difficult to regain a grip on it. Next, pull out one eye by gripping the tissue only just below the retina.

Then pull off the other eye and thus expose the brain and surrounding tissues. Next, remove all the tracheal tissue and remaining cuticle around the brain, until the brain tissue appears clean. All the tracheal tissue must be removed for optimal staining results.

This is all that is needed to prepare the brain for fixation. Proceed with dissecting all the brains, while keeping the dissected brains in cold S2.Try to have up to 10 brains prepared per microcentrifuge tube for optimal staining. Transfer the isolated brains using a P10 pipette tip into a 200 microliter microcentrifuge tube with fixative solution.

Never handle the brains using forceps. Incubate the tissues protected from light. Avoid aspirating the brains during solution transfers.

To remove the fixative, use three or more 15 minute washes with washing solution. Then block the tissues with blocking solution for 30 minutes or longer. Next, replace the blocking solution with primary antibodies diluted in wash solution and incubate the brains overnight at four degrees Celsius.

To remove the primary antibodies, use three one hour washes in wash solution. Next, add secondary fluorophore conjugated antibodies in wash solution and incubate the brains overnight at four degrees Celsius or for four hours at room temperature. To completely wash off the unbound antibodies, use three one hour washes in wash solution followed by a longer wash with PBS.

Then mount the brains. Prepare two cover slips with an imagining spacer. In the spacer and on the extra cover slip, apply 10 microliters of mounting medium with an anti-fade agent.

Next, using a P10 pipette, transfer the brains to the cover slip, depositing them next to the medium along with some PBS. Do not let the tissue dry out. Now, carefully move the brains into the mounting medium using the pipette and finally move them to the medium in the spacer and arrange them with forceps.

Then, remove the adhesive liner on the spacers and attach a cover slip. Secure the cover slip with a little pressure using forceps and proceed immediately with imaging or store the slides at minus 20 degrees Celsius for later analysis. Using the described methods, three different membrane tagged reporters are kept silent by transcriptional terminators flanked by FRT sites.

When the animals were heat shocked, the FLP recombinase was expressed and randomly removed the terminators, leading to the expression of different combinations of reporters. Overall, the different reporter combinations provide seven different colors, thus multiple single cell morphologies can be visualized and studied individually. Various heat shock times were tested with EG-GAL4 and ALG-GAL4.

The single cell labeling was analyzed in the antennal lobe region of the brain. The EG-GAL4 driver expressed in ensheathing glial cells that all ensheathed on the surface of the antennal lobe. By comparison, astrocyte-like glia targeted by the ALG-GAL4, covered mostly non-overlapping areas of the antennal lobe.

After watching this video, you should have a good understanding of how to label single cells in solution. In order to understand their complex and anatomical relationships using Drosophila and in principle and in model organism in which the binary system GAL4 UIS may be applied. While attempting this procedure for the first time, it's important to remember first to optimize the heat shock time and to follow as much as possible the single steps of the staining protocol in order to obtain the best results.