The Notch pathway is one of a small group of signaling systems which underlie metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest due to its fundamental roles in a range of developmental and disease-related processes. Despite intensive analysis, Notch signaling is not well understood, and it appears that additional pathway components need to be identified and characterized. This proposal is designed to address this issue through the characterization of newly-isolated loci that may be implicated in Notch signaling.
After ligand activation of the Notch receptor, an intracellular segment (N IC ) translocates to the nucleus. N IC associates with the Suppressor of Hairless [Su(H)] and Mastermind (Mam) proteins during target gene activation. Mam contains an N-terminal basic domain that interacts with N IC and more carboxy terminal acidic regions that likely function in transcriptional activation. Truncated versions of Mam that lose these carboxy regions, but retain the basic domain, elicit dominant negative phenotypes when they are overexpressed under GAL4-UAS regulation in the wing, eye, notum and embryo. These phenotypes derive from a depression of Notch pathway function that is associated with diminished activation of target loci. Loss or gain of function for Notch pathway genes can enhance or suppress truncation-associated phenotypes (respectively), suggesting that truncation expression sensitizes the system. Such conditions are appropriate for genetic modifier screens, and two different screens have been performed using the Mam truncations: Enhancer-Promoter (EP) overexpression, and EMS mutagenesis. Both screens rovided a number of modifier loci that need to be further characterized.
Three major objectives are proposed here: 1. The initial overexpression screen was limited to a few thousand inserts, therefore EP -based screening will continue. 2. Several loci identified as modifiers in the EP screen are promising candidates for Notch pathway loci, and more detailed studies of these loci will be initiated. 3. In collaboration with the Artavanis lab, ~ 66 modifier complementation groups (enhancers and suppressors) were found in the EMS screen. Various strategies will be used to sort out the most promising complementation groups, and a small number will be selected for subsequent SNP mapping and molecular cloning.
Intellectual Merit: The Notch pathway is a critical component of metazoan development, and Mam plays an essential role in the function of this pathway. A more complete characterization of loci described in this proposal will ultimately provide significant information on signal ransduction mechanisms.
Broader Impacts: This project typically involves a number of students who learn a range of molecular genetic methods and principles during their training. The lab has integrated a very diverse group of students and technicians, including women, African Americans, and Asians. Five undergraduate research students are coauthors on papers from our progress in the last funding period: this includes two women and two Asians.
