The protocol facilitate the dissection of GFP-labeled indirect flight muscle from Drosophila at various time point during pupae development to assess changes in gene and protein expression. Using these techniques, a high enriched flight muscle sample with minimal RNA protein degradation can be achieved that is suitable for RT-PCR, western blot, or omic approaches. These specialized dissections require practice and skill, but the number of pupae that can be dissected and the quality of the samples improve with experience.
Visual demonstration of this dissection is critical for determining how to distinguish flight muscles from non-muscle tissues and facilitating the rapid isolation of flight muscles from pupal specimens. To stage the pupae, use a wetted paintbrush to collect and transfer pre-pupae into a 60 millimeter Petri dish, and sex the pupae under a binocular microscope. Males are identified by the presence of testes, which appear as translucent globes on the otherwise opaque pupae.
After labeling the dish with the time, date, and genotype of the flies, age the pupae in a 25 to 27 degree Celsius incubator until the appropriate stage. For IFM dissection before 48 H APF, use a wetted paintbrush to transfer the appropriately staged pupae to a black dissecting dish about 2/3 filled with chilled PBS. Under the fluorescent dissecting microscope, use a 5 forceps to push one of the pupae to the bottom of the black dissecting dish.
Adjust the microscope zoom and focus until the pupae can be clearly visualized. Using one pair of forceps to grasp the anterior of the pupae, poke the pupae with a single tip of a second pair of forceps, slightly off-center in the abdomen, just behind the thorax. Use the first pair of forceps to remove the anterior half of the pupal case, and to pinch the exposed pupae just behind the thorax to separate the abdomen from the thorax.
Next, gently squeeze the anterior part of the thorax to expose the fluorescently labeled IFMs. Then, use the forceps to discard the remaining carcass to the opposite side of the dish, and dissect the subsequent pupae in the same manner. When all of the pupae have been dissected, use the forceps to collect the IFM fibers, and organize the fibers into a pile at the bottom of the black dissecting dish.
Use the forceps to push any debris out of the field of view, and remove any non-IFM muscles, fat, cuticles, or other unwanted tissue from the samples. Then, use a clipped pipette tip to transfer the pile of IFMs into a 1.5 milliliter microcentrifuge tube containing 250 microliters of chilled PBS. For IFM dissection after 48 H APF, use a lightly wetted paintbrush to transfer the staged pupae onto a strip of double-sided sticky tape mounted on a microscope slide.
Orient the pupae in a line, ventral side down, and anterior toward the bottom of the slide. When all of the pupae have been placed, use forceps to tease apart and open the first pupal case above the anterior spiracles, and gently slide a pair of forceps dorsally, toward the posterior, cutting the pupal case as the forceps move. Free the pupae from the opened case, and immediately transfer the pupae to a drop of PBS on a second microscope slide.
When all of the pupae are freed, use fine scissors to cut the abdomen of the pupae away from the thorax, and push the abdomens into a separate pile. When all of the thoraxes have been removed, use a piece of tissue paper to remove the majority of the PBS and the pile of abdomens. Add a drop of fresh, chilled PBS to the remaining thoraxes, before using the scissors to cut from the head along the longitudinal body axis in a single motion to divide the thorax in half.
When all of the pupae have been dissected, use the 5 forceps to select one of the hemisections, and gently insert the tips of one forceps above and below the middle of the IFMs. Holding the first pair of forceps still, use fine scissors to cut one end of the IFM away from the cuticle and tendons, before rotating the pupae 180 degrees, to allow the other end of the IFM to be cut free from the cuticle and tendons. Use forceps to transfer the IFM bundle from the thorax to the edge of PBS bubble, using water tension to hold the bundle in place.
Then, push the carcass to the opposite side of the slide, and dissect the rest of the IFMs in the same manner. When all of the IFMs have been collected, use the 5 forceps to remove any jump muscle or cuticle fragments that may have found their way into the samples. Then use water tension to gently capture the dissected IFMs between a pair of forceps, and place the IFMs in a 1.5 milliliter microcentrifuge tube containing 250 microliters of chilled PBS.
mRNA sequencing data from BRUNO1 IR IFMs shows changes in expression on the gene-unit level compared to wildtype sequencing data. Using whole proteome mass spectrometry on dissected IFMs, a similar regulation is observed at the protein and protein isoform levels. After the insertion of a protein trap line into the final intron of Mhc, the incorporation of the GFP-labeled protein can be observed in IFM as two dots on either side of the M-line at 90 H APF, while in leg muscle tissue the GFP signal can be observed uniformly across the sarcomere M-line.
When using RT-PCR on dissected IFM, an Mhc isoform switch from the GFP-labeled exon 34 to 37 event to the unlabeled exon 34 to 35 event, can be detected between 30 and 48 hours after puparium formation at 27 degrees Celsius. From mRNA sequencing data, leg and jump muscle tissues express all three Mhc isoforms, maintaining expression of the GFP-labeled isoform in mature muscle. Both Spalt major mutant and BRUNO1 IR-IFM, failed to turn off expression of the GFP-labeled Mhc isoform as they develop, resulting in an isoform expression profile resembling that of leg and jump muscle tissue.
Remember that the dissections should be performed in under 30 minutes to prevent RNA degradation. And, to take care to limit contamination by other cell types. This method generates IFM-enriched samples suitable for biochemical approaches like RT-PCR, or western blotting, as well as omic style approaches, like mass spectrometry, high throughput sequencing, chromatin conformation capture, and metabolomics.
These dissections can complement the powerful genetic approaches in Drosophila with systems-level data for investigating the fundamental mechanisms of development and gene regulation.
