Genetic mosaic screens in Drosophila mushroom bodies

Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. In neurons whose progenitors can be efficiently targeted for mitotic recombination, genetic mosaic screens can be used to systematically uncover cell-autonomous genes that are required for development or function. This technique involves the generation of numerous FRT lines carrying various independent mutations, followed by derivation and phenotypic analysis of MARCM clones using these mutant FRT lines in combination with an MARCM-enabling stock that carries all the other genetic elements required for MARCM. Mutants of interest are recovered based on the MARCM phenotypes, which are imaged live using diverse fluorescent markers. Mutant genes that underlie the phenotypes of interest can then be identified by conventional genetics including derivation and analysis of series of recombinant chromosomes. Besides chemical mutagenesis, genes on a particular FRT chromosome may be randomly disrupted by P element insertion. This protocol describes procedures specifically used for genetic mosaic screens in the mushroom bodies (MBs).