Hello, my name is Joshua Krupp. I'm a postdoctoral fellow in the laboratory of Joel Levine at the University of Toronto at Mississauga. This video article will be demonstrating a dissection technique that I developed for the isolation of the adult cytes from the fruit lid esophagal mal gaster In fruit flies As another insects.
A waxy layer composed of hydrocarbon compounds, coats outer surface of the cuticle. This waxy layer provides resistance against desiccation and protects the animal against other environmental challenges. Several of these particular hydrocarbon compounds also serve as semiochemical signals.
As such, they mediate hormonal communications and influence behavior. The cytes, the cells of gel be demonstrating how to dissect are considered to be a primary site for the production of these particular hydrocarbon compounds. The dissected preparation demonstrated here will allow for the detailed molecular and genetic analysis of the cytes from an adult fruit fly.
For this dissection procedure, some basic dissection tools are required. First, a Pasteur pipette is required for dispensing chilled insect media. Second tungsten filament is needed for making a fine dissection needle to produce this needle that the tungsten filament is first inserted through a fine bore hypodermic needle.
This provides a tungsten filament with needed support. The tsin is then passed slowly through a flame. This acts to burn off some of the tsin at the tip, creating a very fine and very sharp needle.
Next, a so guard dissection plate is needed, so guard comes in several shades of gray, or it can be transparent. The so guard provides a soft dissection surface into which dissection pins can be inserted. Lastly, a set of fine number five, dissection forceps as needed.
I keep on hand an old blunted pair of forceps for handling dissection pins. This keeps my newer set of forceps from being damaged. The following procedure prepares the abdomen for the subsequent removal of the cytes.
The same procedure can be used for preparations of the other tissues attached to the inner surface of the cuticle. These include the heart and fat body. We'll be dissecting a 60 day old wild type male.
The flies first anesthetized on CO2 quickly. The fly is removed to the dissection plate. The fly is then secured to the dissection plate.
By placing a fine dissection pin through the thorax, the legs and wings of the fly are removed while removing the third set of legs and opening an cuticle is created at the point just anterior to where the thorax meets the abdomen. The abdomen of the fly is then secured in place by placing a second dissection pin through the genital segment. Next, the fly is covered with shield, shield and san insect cell.
Medium trapped air is removed by drawing it off with a PEs tour pipette. Next, using forceps, an incision is made along the ventral midline of the abdomen. The incision runs from the opening and the cuticle created by removing the legs to the genital segment.
The guts and the gonads can now be removed from the abdomen. The cuticle and trachea connecting the abdomen to the thorax are now severed. The only tissue connecting the abdomen to the thorax is the heart.
The anterior most corners of the abdominal cuticle are now pinned flat. Once the first pin is inserted, the thorax can be removed. Pulling the cuticle top pin flat, the posterior corners of the Cuticle, The pin in the genital segment may need to be repositioned to make the cuticle lay flat.
That completes the first part of the dissection. Next, I will demonstrate how to remove the cytes. The abdomen filet preparation exposes the internal surface of the cuticle and attached tissues.
These tissues include the heart, fat body, the tracheal system, body wall, muscles, epidermis, and the cytes. The heart lies along the dorsal midline fat body. The opaque tissue covers most of the internal cuticular surface trache.
The white tubular structures extend from the lateral spherical openings and make extensive branches, which infiltrate these other tissues lying beneath the fat body. The pigmented cytes, amber and color radiate from the midline to the lateral edge of the cuticle in the posterior region of each segment, the cytes lie directly beneath the darkly tanned regions of the cuticle and can be difficult to identify without prior knowledge of their location. In each abdominal segment, a prominent leaf of fat body lies anterior to the cytes.
This fat body tissue often covers the cytes of the segment preceding it. The dorsal vessel and the fat body must first be removed in order to expose the cytes. The first step to dissect adult cytes is to sever the tracheal trunks emanating from the sphericals.
Using the tungsten dissection needle, remove the fat body and heart by severing the connections to the inner surface of the cuticle. Again, using the dissection needle to do so, position the point of the dissection needle between the fat body and cytes and drag it from the lateral edge of the cuticle through to the midline, moving underneath the heart and continuing to the opposite side, progressing from the posterior to the anterior. Remove the fat body tissue and the heart from the cuticle of each successive segment.
If performed carefully, the cytes remain attached to the cuticle and the fat body and heart can be removed. Whole I body wall muscles strap the cytes to the inner surface of the cuticle. Using the dissection needle, sever the longitudinal body wall muscles of each segment.
To do so, drag the dissection needle along the posterior side of the endocyte strand, gently lifting the strand from the surface of the cuticle. The ribbon like cyte strands should come away from the cuticle and can be laid to the side while the remaining cytes are removed. It is important to note that the amber pigmentation of the cytes clearly distinguishes these cells from the surrounding tissue.
The intensity of this pigmentation increases with the age of the fly. It's also worth noting that a basal lamina and sheath cytes this extracellular material provides support to the cell clusters and prevents the cells from becoming dissociated during the dissection. The basal lamina also aids in the removal of extraneously adhered tissues by creating a weak point where these adhesions can be easily disrupted.
Once removed from the cuticle and lying on the dissection plate, it may be necessary to clean off any adhered to fat body tissue from the cytes. This is done by simply placing a dissection needle between the fat body tissue and the cytes and severing the connection by pushing the needle down against the dissection plate. Gather the cytes strands by simply poking them with the end of the dissection needle.
Once stuck to the end of the dissection needle, cytes can be pulled across the surface of the culture medium. The dissected cytes can be placed directly into cell lysis buffer appropriate for either RNA or protein extraction. That completes the cyte dissection.
Thank you for watching this video article. I hope you'll find the dissection techniques demonstrated here helpful to your studies. Good luck with Your experiments.
