Reverse genetics by loss-of-function mosaic analysis in Drosophila

Abstract

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. One major application of MARCM, as described here, is to study cell-autonomous function(s) of a gene within single cells or a group of cells in otherwise unperturbed organisms. A mutation of interest distal to one FRT site is put in trans to a tubP-GAL80-containing chromosome arm that carries the same FRT. The resulting MARCM clones, which are negative for tubP-GAL80 and thus specifically marked, will become homozygous for the mutation in otherwise heterozygous organisms. By including a UAS-transgene, one can perform rescue experiments in the mutant MARCM clones. Conversely, if the mutation is placed on the same chromosome arm as tubP-GAL80, MARCM-labeled cells will be homozygous wild-type and may lie adjacent to sister cells that are homozygous mutant. This variant, called reverse MARCM, allows one to determine non-cell-autonomous effects of a mutation.