The overall goal of the following experiment is to analyze the development and survival of mosaic photoreceptor neurons in the retina of living drosophila. This is achieved by crossing a fly stock, carrying a mutation recombined on an FRT chromosome with a tomato G-F-P-F-L-P-F-R-T stock. In the progeny, mosaic clones are generated by mitotic recombination during development.
As a second step living flies are embedded into aeros, which maintains the fly immobile. Next, the retina of the immobilized living fly is imaged with a confocal microscope in order to view mosaic photoreceptor neurons, all photoreceptors express the green fluorescent protein, GFP, the wild type and heterozygous photoreceptors express the red fluorescent protein tomato on the merge image. Homozygous mutant receptors are green, whereas wild type and heterozygous photoreceptors are yellow.
Results are obtained that show developmental defects and degeneration of photoreceptor neurons based on the visualization of the same fly over days to weeks. The tomato, G-F-P-F-L-P-F-A method can help answer quick question in the neuronal degeneration field and demonstrate the requirement of specific genes for photoreceptor survival and function in the adult fly. This method is also useful for addressing S issues in mutants affecting development, such as those in which the establishments of PNA cell polarity is affected In comparison with classical histological sectioning of the retina.
The first advantage of this method is that it is quick and therefore can be used for ice throughput screening. The second advantage of this method is that it is done in living drosophila, which enables time course analysis of photoreceptor degeneration at a single cell level and over several weeks. For photoreceptor visualization by the cornea neutralization technique, flies must be immobilized onto aros plates.
Begin by filling a squirt bottle with water and place it on ice. Next, prepare 200 milliliters of 1.5%aros and keep it at 55 degrees Celsius until it is used. Now, anesthetize the flies with CO2 for at least a minute.
Pour the warm aros solution into a 35 millimeter or 65 millimeter Petri dish, and immediately place the anesthetized drosophila into the aros. Start with 10 flies per dish. When learning the technique with practice, as many as 20 flies can be screened simultaneously.
Under a dissecting microscope, orient a drosophila on its side with forceps. Push one wing into the aros such that the wing and half of the body are embedded in the aros. Stick the other wing onto the surface of the aros.
The head is not embedded. If the flies are not embedded deeply enough, the fly will be free to move its head causing problems. High temperatures of the aros may damage the corneas, but if the aros is too cool, it will be difficult to embed the fly.
Once all the flies are embedded, transfer the dish to ice and allow the agros to solidify. Once the aros is solidified, return the dish to the scope and orient each head such that one eye is exposed to the immersion objective. When the pseudo pupil, the dark spot is at the center of the eye, the head is well oriented.
Also, remove any legs or risi obscuring the eye. The orientation step is a critical step that aims to find the region of the eye with the widest field of focus. Photoreceptors, usually it's the center of the eye.
After orienting all the eyes, cover the flies with ice, cold water and put the dish on ice to maintain anesthesia until the flies are visualized. To visualize the photo receptors begin by placing the Petri dish on a glass slide attached to the microscope stage so that it is possible to smoothly manipulate the dish position on the stage. Plunge the immersion objective into the water of the Petri dish and position the head of the fly under the excitation beam.
Start with the GFP filter. If it is difficult to distinguish the body parts of the fly, particularly the distal part of the abdomen and the head, then look directly at the stage and reposition the drosophila. Move the stage up and down until the beam converges on the eye.
When the eye is at the right level, it reflects the excitation light. Looking through the eyepiece or at the screen center the field of view on the eye and focus below the cornea. Thus visualizing the fluorescent photo receptors.
To improve the confocal image, open the pinhole wider than is usually recommended for the objective. For example, use a value of 2 0 4 rather than the default 98 for the aperture of the pinhole. Confocal microscopy provides a good alternative technique to standard fluorescence microscopy because the images have less background and a wider field of focused photoreceptors to follow the fate of an individual photoreceptor within the same eye over a period of time.
First, reduce the temperature of the aros to 45 degrees Celsius to maximize the animal's survival. Second, only place one drosophila per dish so that the flies are not mixed up During observations. Also, be sure to orient the fly on the same side to view the same eye.
Then under the confocal microscope, recognize the same photoreceptor clone by its shape and by the polarity of the eye. If the clone of interest is misplaced in the field of view, reorient the eye underwater to store the fly. To visualize again later, drain the water from the dish.
Then gently pull the fly out of the aros with forceps, then dry the drosophila on a tissue and transfer it to a vial. Return the vial to 25 degrees Celsius, making sure the fly does not get stuck in the food. The described tomato, G-F-P-F-L-P-F-R-T method was implemented as a rapid screen for clonal mutations in photo receptors.
The screen uncovered developmental defects affecting various processes and photoreceptor neuron cell death. Results are given in detail online. Seven in absentia is required for photoreceptor recruitment, particularly for R seven, one of the inner photoreceptors.
The loss of seven in absentia resulted in the loss of the inner photoreceptors and some outer photoreceptors. Grainy head is involved in planar cell polarity establishment. It was found that some Oma TIA are inverted in grainy head mutant clones, and that grainy head is required in R three photoreceptors for correct planar cell polarity establishment crumbs and apical membrane protein required for rhabdo.
Morphogenesis was found to lead to irregular, larger, or smaller photoreceptors when mutated. Using the described protocol, longitudinal tracking of individual photoreceptors was used to follow their fate during adulthood. This uncovered a mutation of fat P that induces progressive photoreceptor degeneration in adulthood.
In mosaics wild type photoreceptors were unaffected. This method was possible because the individual photoreceptors could be re-identified by the morphology and polarity of the clone they belong to. To confirm the role of fat P in the fat P mutant, the degeneration of photoreceptor neurons was prevented by expressing wild type fat P in the outer photoreceptors.
In the mutant background, After watching this video, you should have a good understanding of how to visualize mosaic photoreceptor, neuro neuros in living a do to study in neuro degeneration and development.
