Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Organisms show a tremendous variety in their shapes. We are highly interested in the molecular and the cellullar mechanisms underlying the formation, diversification, and evolution of these shapes. To understand this, we have been investigating how fate-determined the cells form the final shape of the adult leg in Drosophila.

Using live imaging, we have found an unexpectedly surprising behavior of cells. During the final shape formation of the leg, the epithelial cells transiently formed the special structure as we named the Parthenon-like structure. This structure seems to be a general feature of epithelial cells as they establish their final shape For long term live imaging during the pupa stage, it is important to remove the puparium while maintaining moisture.

Our method, which uses a glass bottom dish and a drop of water is stable, reliable, and easy to handle, even for beginners. Our results demonstrate that cells undergo unexpected dynamic shape changes during the final shape formation process. To accurately understand the final shape formation mechanisms, it is essential to thoroughly grasp the continuous changes in the cell shape and investigate the mechanisms behind these changes and their importance to morphogenesis.

The next questions we want to address are how the Parthenon-like structure is transiently formed, and how it contributes to the finer shape formation. To begin, collect white pupae of the desired strain of Drosophila melanogaster from vials and place them into dishes or empty vials using a paintbrush. Incubate the collected white pupae at 25 degrees Celsius for at least 14 hours until they reach the desired stage for observation.

Using a paintbrush soaked in distilled water, carefully remove glue and fly food from the surface of the puparium. Then place the cleaned pupae on cleaning wipes and leave them for several minutes to dry. Now attach a piece of double-sided tape to a glass slide and place the dried pupae on the double-sided tape with the ventral side facing down.

Using a dried paintbrush, gently push the dorsal side of the pupae to improve adhesion to the double-sided tape. Under a stereo microscope, use a pair of forceps to carefully open the operculum of the puparium. Insert one tip of the forceps into the space between the pupa and the puparium from the edge of the opened operculum.

Then using the forceps, grasp the puparium and pull it outward until it breaks open. Lift the torn fragments of the puparium and adhere them to the double-sided tape. To begin, prepare Drosophila melanogaster pupal leg for imaging.

Depending on the lens used, apply one microliter of distilled water or silicone oil to the bottom of a glass bottom dish. Then soak a paintbrush in distilled water and gently scoop up the pupa. Place the pupa with the ventral side facing down onto the distilled water or silicone oil in the dish.

Using a micropipette, add 10 microliters of distilled water near the edge of the glass portion of the dish. Using a syringe, apply a circle of silicone grease around the edge of the glass portion of the dish. Then place a cover slip onto the silicone grease to seal the dish.

After turning on the confocal microscope and software, place the glass bottom dish with the pupa onto the microscope stage and image the desired area of interest. The epithelial cells in the tarsus formed apicobasal basal projections and basal connections, which are reminiscent of the Parthenon between 16 hours and 27.5 hours after puparium formation. Progressive reduction in epithelial thickness was observed from 41 hours to 60 hours after puparium formation, even after the leg tissue outline became more defined.