A mosaic screen for growth-promoting genes identifies the small GTPase Rheb. H. Stocker 1, B. Schindelholz 2, P. Daram 2, S. Breuer 2, E. Hafen 1. 1) Zoological Institute, University of Zurich, Zurich, Switzerland; 2) The Genetics Company Inc., Wagistrasse 27, CH-8952 Schlieren, Switzerland.
Mutations in components of the insulin receptor signaling cascade impair cellular growth in a cell-autonomous manner. Clones of mutant cells bear a severe growth disadvantage and remain small compared to their wild-type sister clones. If mitotic recombination is forced to occur by the constant supply of Flp recombinase and the sister clone is eliminated by means of a cell-lethal mutation, these clones can, however, cover substantial fractions of whole organs. Mosaic flies with impaired insulin receptor signaling specifically in the head show a very characteristic phenotype. While their bodies are of normal size, their eyes and heads are dramatically reduced (the so-called pinhead phenotype). We wished to identify mutations in growth-promoting genes based on similar phenotypes.
To this end, 50000 mosaic flies were scored for size defects and 321 mutations on the right arm of the third chromosome giving rise to smaller eyes were recovered. The isolation of 48, 18, and 13 alleles of the genes encoding the insulin receptor, the catalytic subunit of PI3K, and dAkt/PKB, respectively, proved the feasibility of our screening strategy.
In addition, we identified ten alleles of the gene encoding the small GTPase Rheb. Whereas loss of Rheb function results in a severe growth deficit, overexpression of Rheb stimulates cellular growth. We show that the overexpression phenotype is independent of insulin receptor signaling, but that it depends on the function of S6K. Furthermore, it is counteracted by the co-expression of TSC1 and TSC2, indicating that Rheb might be a component of the TSC-TOR-S6K signaling branch.