sensitized genetic background has led to the identification of additional components in those pathways. These new components have been difficult to discover, because they are either stored in the egg and thus show a maternal perdurance or they are used in multiple pathways, the consequence being that their mutant phenotype diverges from that seen in any one pathway or is generally lethal. In this section, the committee will describe sensitized mutants in some detail, because it believes that they will be useful in the future for toxicant assays.
The screen used by Simon et al. (1991) has set the pattern for many subsequent screens (and the committee will later draw attention to the potential use of this strategy for assaying toxicants). The Sevenless gene encodes a tyrosine kinase receptor that is required specifically for the formation of rhabdomere 7, one of eight light-receptor cells in each ommatidium of the Drosophila compound eye. The authors conducted a genetic screen on flies bearing weak, temperature-sensitive mutations in the Sevenless gene. They screened for genes for which inactivation of one copy caused the Sevenless phenotype. Hence, even if such genes are needed in multiple tissues, the activity of the remaining intact gene would suffice in all but the eye. Increases or decreases in gene activity in the tyrosine kinase pathway would affect the intermediate eye phenotype of the fly strain used. The screen detected a series of genes functioning downstream of the receptor, including homologs of Ras, Raf1, and a guanine nucleotide exchange factor (Figure 7-3). The dose-dependence of the Ras mutation suggested that a small (two-fold) reduction downstream should modify the rough-eye phenotype imparted by the mutant Ras and do so only in the eye. The suppressors so discovered are the Drosophila homologs of components of the MAP kinase pathway. These are used by many tissues, so their mutations would be recessive lethal. This study provided the basis for integrating the downstream effector pathway for many tyrosine kinase receptor functions.
Since this initial screen, screens using sensitized genetic backgrounds have become commonplace in developmental genetics of flies. For example, the initial set of downstream functions of the decapentaplegic pathway (a TGFβ signaling pathway), now called SMADs, was discovered in a screen for mutations that enhanced the phenotype of a weak Dpp allele (Raftery et al. 1995). Mutations in the Notch pathway have identified additional components functioning downstream of the receptor (Xu and Artavanis-Tsakonas 1990).
A major difficulty in identifying components of these critical signaling pathways is that they play essential roles in many developmental processes; hence, mutations in the genes involved are lethal. Genetic approaches have been developed in Drosophila to circumvent that phenomenon. The site-specific recombinase system from yeast (FRT-FRP system, Golic and Lindquist 1989) has been particularly useful (Figure 7-4). In the case of essential genes that are expressed during oogenesis, germ-line clones that are homozygous for a lethal mutation are made in the background of an ovary expressing a dominant female sterile mutation. These clones, lacking the dominant female sterile mutation, are