Somatic Mosaic Analysis In The Eye

Somatic clones in the eye can be generated by X ray-induced mitotic recombination; and can be genetically marked by the cell-autonomous white gene. For any gene you want to analyze by generating clones, you want to use the marker white gene at a cytological map position which is close to the gene of interest. This is conveniently done by choosing an appropriate P-element transformant; stock in which the P vector contains the white gene and has integated in the genome cytologically close to the gene of interest, which will be called gene X. Three criteria should be considered before choosing the transformant stock. One, the white gene should be located cyologically close to gene X. Two, it is preferable if the white gene is more proximal to the centromere than gene X. Third, one copy of the white gene in this transformant stock should produce enough pigment in the photoreceptor cells that it can be easily seen in semi-thin or thick sections. This last consideration can be rapidly surmised by looking at flies hemizygous for the white gene under a dissecting microscope. Their eye color should be indistinguishable from wild type. Alternatively, you could fix and section eyes from hemizygous flies and score pigment in photoreceptor cells. However this last criterion is most important when analyzing mosaic ommatidia and is not important if you just want to look at the phenotype of the entire clone.

Make a fly stock which is homozygous for a mutant allele of gene X and which is homozygous white minus. We use the null allele of white, w-1118. Obviously if the mutant allele of gene X is lethal, it has to be balanced over a chromosome with a dominant marker.

The strategy is to put the chromosome with mutant gene X in trans to the chromosome containing the white gene. Recombination events between sister chromosomes during mitosis are induced by X irradiation. If recombination occurs between the centromere and the white gene it will produce a daughter cell that is homozygous mutant for white and gene X. Replication will expand cell numbers to the point that in the eye a white patch is seen in a red eye.

Make a mass fly cross between w-; X- and w-; P[w+]. I prefer to mate 400 of each genotype putting 100 pairs in one bottle. Incubate 24 hours at 25íC. Transfer flies to fresh bottles. These can either be egg laying bottles or regular bottles. I prefer egg laying bottles because the parameters for irradiation are more consistent. If you use egg laying bottles, use plates containing yeast-glucose medium (see end of section). Collect embryos for 12 hours at 25íC. Repeat collection. Incubate plates or bottles for a further 42 hours at 25íC. The flies will now be 48 +/-6 hours old and will be late first instar larvae. Inducing recombination at this time gives a balance between reasonable clone size and frequency of clones (about 1 in 20 flies). If you want to generate larger clones, irradiate at an earlier time, but be prepared to generate clones at a lower frequency.

Irradiate larvae in plates or unstoppered bottles with 1000 rads (115 kV, 5 mA). If using egg-laying plates, cut the plates in halves or quarters and place each piece in a bottle. When the flies have eclosed, they can be screened for clones. Most white clones appear black under a dissecting microscope. Fix and section the eyes as described in Section 19. However, before the flies are sacrificed, shine a bright light on them for a few minutes. This causes the pigment granules in the photoreceptor cells to migrate apically and makes it easier to score for white pigment in cells R7 and R8. Cut 1 to 4 micron thick sections and collect all of the sections serially. This is important as pigment may only be visible in R7 and R8 cells for one or a few sections. Do not stain the eye sections but mount in DPX and view under phase contrast. Pigment granules appear as black specks adjecant to each cell's rhabdomere.

Recipe for Yeast-Glucose Plates: