Generation of standard wild-type MARCM clones for analysis of drosophila brain development

Abstract

The generation and analysis of clones of cells with a genotype different from the rest of an organism has been used for studying molecular mechanisms underlying development. 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. This permits unique labeling of specific homozygous daughter cells, and thus makes mosaic analysis possible in the complex nervous system. The principle of MARCM involves the generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues, therefore allowing GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, it is necessary to generate organisms carrying at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. Induction of FLP in neural precursors can be temporally controlled using a heat-shock promoter or spatially regulated using a tissue-specific promoter. Mitotic recombination in a neuroblast (NB) may yield a multicellular NB clone (progeny of the renewed NB) or a two-cell clone (progeny of the derived ganglion mother cell [GMC]). Single-cell clones can be obtained following mitotic recombination in GMCs. Phenotypic analysis of mosaic brains can be greatly simplified using a GAL4 driver that is only expressed in neurons of interest. One can also mark different subcellular structures of the clones using distinct UAS reporters. This protocol outlines the steps involved in standard MARCM analysis.