Regulated growth during normal development is contingent on generating the appropriate number of cells. Mechanisms must exist that restrict cell proliferation in vivo. A genetic screen has been devised in Drosophila that allows the identification of loss-of-function mutations that give cells even a subtle growth advantage over their neighbors. Mutations are induced on a paternally derived chromosome arm that includes a proximally located FRT element. The FLP recombinase is expressed from the eyeless promoter, which induces mitotic recombination at high frequency in the eye primordium. The wild type chromosome (inherited from the mother) also contains an FRT element and a mini-white gene. Thus, following mitotic recombination, the mutant clone is white and the twin-spot generated by the same recombination event is red. Mutations that result in increased proliferation give rise to eyes, which contain more white tissue than red tissue. A pilot screen has been conducted of more than 20,000 chromosomes; mutations were identified that cause a relative overproliferation of mutant cells. Mutations recovered from the screen include alleles of net, N-cadherin and nerp1, a novel complementation group.
This proposal outlines strategies for conducting a comprehensive screen of the second chromosome for mutations of this nature. Mutations will be classified by complementation analysis and by testing for interactions with mutations in genes encoding known cell cycle regulators. Those mutations that are lethal or have a visible phenotype will be localized by a combination of deletion mapping and meiotic mapping strategies. A detailed molecular and phenotypic characterization of three of the loci identified in the screen will be conducted. net and N-cadherin have already been cloned by others. The nerp1 locus will be cloned. In each case, it will be determined whether the overproliferation phenotype is due to a loss-of-function or whether it requires specific alterations in those genes. For each of these three loci, the patterns of cell divisions will be examined in mutant embryos and in mutant tissue in the imaginal disc. The effects of these mutations on the expression patterns of known cell cycle regulators will be investigated and alterations in the kinetics of cell cycle progression in mutant tissue will be studied. Finally, the effect of overexpressing each of these genes on progression through specific phases of the cell cycle will be analyzed. These studies will potentially identify a number of negative regulators of the cell cycle and will provide insights into the mechanisms by which three of these loci regulate cell proliferation.
