Signaling mechanisms that regulate attractive axon guidance at the CNS midline

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

The establishment of proper neuron connections during development is essential for coordinated behavior. Axons, the wires of the nervous system, are guided to their targets by attractive and repulsive cues. This project will investigate the mechanisms that promote axon attraction to the midline of the fly central nervous system. Similar to the vertebrate floor plate, the fly midline is an intermediate target for navigating axons, which must decide whether or not to cross the midline. Netrin attracts axons to the midline through receptors of the Deleted in Colorectal Carcinoma (DCC) family (frazzled in Drosophila). However, it is unclear how Frazzled/DCC signaling mediates axon attraction and what additional mechanisms contribute to midline circuit assembly. Defining these mechanisms will enrich understanding of how guidance molecules work in the intact animal and have the potential to suggest therapeutic targets for disorders of the nervous system. The project includes outreach activities that are aimed at increasing high school scientific literacy and aptitude. Each summer, a high school teacher will spend six weeks in the laboratory, where he/she will collaborate with lab personnel to conduct original research and design a teaching module to bring back to the classroom. To define mechanisms mediating axon attraction, this proposal seeks 1) to identify genes that act with Fra to control midline guidance, 2) to determine how the novel role of Fra in activating gene transcription is regulated by receptor proteolysis, and 3) to test if nuclear localization and transcriptional activation function of the Fra intracellular domain (ICD) are required for Fra-dependent gene expression. A sensitized genetic screen will identify new components that act with Fra to promote midline crossing, and molecular genetic approaches will be used to investigate a novel role for Semaphorin1a (identified in our screen) during midline guidance. Molecular and genetic approaches will be used to test the importance of proteolysis of the Fra receptor. Candidate genes from the screen will be tested for roles in Fra-dependent transcription. In vitro assays in insect and yeast cells will be used to delimit sequences that are necessary and sufficient for nuclear localization, nuclear export, and transactivation function of the Fra ICD. Finally, in vivo requirements for these elements will be tested by generating mutated forms of the Fra receptor, expressing them as transgenes, and assaying their ability to promote midline axon attraction and target gene expression.