Role of proteoglycan-mediated trans-axonal signaling in pre-target topographic sorting - PROJECT SUMMARY Precise wiring of axonal projections into topographic maps facilitates the transfer of information between brain regions and is thus critical for brain function. An important mechanism contributing to topographic map for- mation is pre-target axon sorting, whereby axons become topographically ordered along tracts before they reach their target. In the visual system, for instance, dorsal and ventral retinal axons respectively segregate into the ventral and dorsal branches of the optic tract before reaching the tectum. While pre-target axon sorting has an instructive role in topographic mapping, how it is established during development remains poorly under- stood. Using the unique transparency and accessibility of the zebrafish embryo, we previously showed that axon sorting in the visual system is achieved by a pruning mechanism that eliminates axons that missort along the optic tract. Whereas ventral axons never misroute, some dorsal axons wrongfully navigate along the dorsal branch of the tract but then stop and rapidly degenerate. Our new results further demonstrate that pioneer ven- tral axons instruct the degeneration of the dorsal axons that missort, demonstrating that axonal pruning is initi- ated by signaling among growing axons themselves. The heparan sulfate proteoglycan Glypican-3 (Gpc3) and the adhesion molecule Teneurin-3 (Tenm3) act cooperatively along pioneer ventral axons to induce the degen- eration of missorted dorsal axons. On the other hand, the adhesion G protein-coupled receptor (GPCR) Latro- philin 3.1 (Lphn3.1) signals cell-autonomously along missorted dorsal axons to initiate their pruning. While our studies uncovered a unique trans-axonal signaling for selective axon pruning, several important questions re- main unsolved. First, the mechanism by which Tenm3 and Gpc3 instruct axonal pruning remains unknown. Missorted dorsal axons elongate in the wrong branch for several hours before stopping and degenerating, indi- cating that they become responsive to Tenm3 signaling. Here, we will test the hypothesis that Gpc3 initiates this temporal response by controlling the onset of Tenm3 signaling from ventral axons. We will use a combina- tion of biochemical, genetic and high resolution imaging approaches to test whether Gpc3 interacts with Tenm3 in a heparan sulfate-dependent manner and regulates its trafficking to, or processing at, the plasma membrane of ventral axons. A second question is how Lphn3.1 instructs selective axon pruning. It remains unknown how Lphn3.1 signals and whether its function is specific or shared by other latrophilins. Here, we will test whether other latrophilins can compensate for the loss of Lphn3.1, and identify which features of Lphn3.1 are necessary for its activity. Finally, we will explore which signaling pathways act downstream of Lphn3.1 by testing whether Cyfip2, a cytoplasmic protein required for pruning missorted dorsal axons, mediates Lphn3.1 signaling. Alto- gether, our proposed studies will be the first to determine how trans-axonal signaling between pioneer and fol- lower axons establishes pre-target axon sorting in the visual system. They will fill a major gap in our knowledge of developmental axon pruning and tract formation, two processes essential for precisely wiring neural circuits.
