The overall goal of this procedure is to prepare Driss Pupi for live cell imaging of dividing thoracic sensory organ progenitor or SOP cells during metamorphosis. This is accomplished by first setting up the appropriate genetic cross to obtain progeny puy for live cell imaging. The second step of the procedure is to collect oph puy at the appropriate stage to visualize asymmetrically dividing neural progenitor cells.
The third step of the procedure is to remove the pupil case in order to mount the pupa for imaging. The final step of the procedure is to mount the pupa between slide and cover slip and proceed to live cell imaging. Ultimately, results can be obtained that show the dynamics of fluorescently tagged proteins during asymmetric cell division and differentiation of wild type or mutant sensory organ cells through either epi fluorescence or confocal microscopy.
The main advantage of this technique over existing methods like immunohistochemistry is that protein dynamics can be followed in real time and over the course of several hours in an intact pipa From across of the desired gal four line and GFP tagged fusion protein. Under UAS control identify puy that have undergone ation within the hour. These puy will possess characteristic pupil morphology but lack pigmentation.
Selected puy can either be transferred to a fresh file or can be marked on the cross file to remember which ones were white. 18 hours later when the puy reaches the appropriate stage, place a piece of double-sided tape onto a microscope slide and adhere the pupil case to the slide ventral side down under the dissecting microscope. Identify and grasp the edge of the URM using a pair of forceps and gently lifted off, revealing the head of the immature fly.
Being careful not to puncture the body of the fly. Gently tear the side of the pupil case and to remove it completely to reveal the thorax and anterior portion of the abdomen. Continue to carefully tear the same side towards the posterior and pull the pupil case to the opposite side and stick it to the tape.
Now that the abdomen is fully exposed, gently push a soft bristled brush under the head of the pupa and lift it from the rest of the pupil case. Place the pupa onto a microscope slide dorsal side up Position the pupa at a 45 degree angle so that the cover slip will touch the thoracic SOP cells region. First, make an 18 by 18 millimeter square frame out of watman filter paper by cutting a 10 by 10 millimeter window in the center.
Immerse the frame in water until saturated and then place around the pupa. Use a five cc syringe to apply a uniform layer of silicone grease around the frame such that the layer's thickness is only slightly greater than the pupil diameter. Pipette approximately one microliter of water on the center of a 22 by 22 millimeter cover slip and position the cover slip such that the droplet touches the pupil nodom.
Press gently to form a complete seal and flat surface against the pupa. Now that the live pupa has been mounted, it can be imaged using confocal microscopy by combining the dissection and mounting techniques. With live imaging, one can visualize the accumulation of actin GFP fusion protein at the cleavage furrow of a mitotic SOP cell.
Over time here, the distribution of partner of Num RFP and RAB 5G FP can be compared across two SOP daughter cells. Note that while partner of NUM RFP is asymmetrically distributed, RAB 5G FP is distributed equally in both cells at late pupil stages. Differentiated external sensory organs can be visualized menos sensory sockets and hairs exhibit autofluorescence and are shown here overlapping with L-G-L-G-F-P expressed in a subset of SOP cells.
Using the Markem system Once mastered, this technique can be done in less than 10 minutes if it is performed properly. Following this procedure, other methods like immunohistochemistry can be performed in order to answer additional questions like identifying sulfates of sensory organ cells with specific antibody markers or colocalization study of GFP fusion proteins with endogenous proteins.
