This application of mosaics is especially useful for the study of genes that are essential for the viability of the whole organism because specifically removing their function in isolated clones does not necessarily impair overall survival. This chapter discusses the use of clones of cells to study the phenotype of mutations in isolated patches of tissue, within an otherwise wild-type. The present protocol provides a detailed description of a standard MARCM analysis of brain development that includes generation of MARCM stocks and crosses, induction of clones, brain dissection at various stages of development, immunohistochemistry, and confocal microscopy, and can be modified for similar experiments involving mitotic clones. A related technique, the twin spot generator, generates positively labeled clones without the use of a repressor, thus minimizing the lag time between clone induction and appearance of label. MARCM can be combined with any UAS-based construct, such as localized GFP fusions to visualize subcellular compartments, genes for rescue and ectopic expression, and modifiers of neural activity. Variations of the technique include labeling of both sister clones (twin spot MARCM), the simultaneous use of two different drivers within the same clone (dual MARCM), as well as the use of different repressible transcription systems (Q-MARCM). Mosaic analysis with a repressible cell marker (MARCM) generates positively labeled, wild-type or mutant mitotic clones by unequally distributing a repressor of a cell lineage marker, originally tubP-driven GAL80 repressing the GAL4/UAS system. More importantly, since EMS mutations have been notoriously difficult to map to specific genes in the past, I briefly discuss mapping methods that allow rapid identification of the causative mutations. These F1 clonal screens allow rapid phenotypic screening within clones of mutant cells induced at specific developmental stages and in tissues of interest, bypassing the pleiotropic effects of isolated mutations. These screens combine a classical EMS (ethyl methanesulfonate) chemical mutagenesis protocol along with clonal analysis via FRT-mediated mitotic recombination. Here, I describe how to perform F1 clonal forward genetic screens in Drosophila to identify novel components of the Notch signaling pathway. Given that the developmental context of signaling is important, these screens should be customized to specific cell populations or tissues. A powerful strategy to accomplish this task is based on genetic screens. ![]() To unravel the complexity of Notch signaling, it is important to identify the different components of the Notch signaling pathway. The functional versatility of the Notch signaling pathway is tightly linked with the complexity of its regulation in different cellular contexts. Notch signaling controls a wide range of developmental processes, including proliferation, apoptosis, and cell fate specification during both development and adult tissue homeostasis.
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