The association of axons into bundles, each of which terminates in a well defined and distinct target area, is a general organizational principal of all developing nervous systems. Although ubiquitous, there is little understanding of the molecular mechanisms through which this organization occurs, nor is its role in axonal targeting well understood. In this study we will take advantage of several advantageous properties of the developing olfactory system in embryonic zebrafish to study the mechanism and function of axonal bundling. These advantages include highly accessible and rapidly developing embryos, easy transgenesis that makes it possible to fluorescently label subsets of neurons with a particular target, easy mutagenesis which makes it straightforward to study the function of specific targeted genes, clear embryos that allow fluorescent axon trajectories to be visualized and reconstructed with light microscopy, and finally, a relatively simplified olfactory circuitry that preserves all of the key features of more complex vertebrate systems that are more difficult to study. In this project we will use two lines of fish, one in which a class of olfactory sensory axons is labeled with a green fluorescent protein, and another in which a different class is labeled with a red fluorescent protein. The axons from each class bundle together with their own kind as they extend between the epithelium in which they originate to their target, the olfactory bulb. Once they reach the bulb, they terminate in mutually exclusive and reproducible target regions. Using the red and green fluorescent markers, we have purified sensory neurons from each of these two classes and identified all of the mRNAs and their relative expression levels in each class. From this information we identified specific genes that are differentially expressed between the two classes. We are now in the process of making fish lines in which each of these differentially expressed genes is inactivated. Our goal is to introduce these mutations into the fluorescently labeled fish lines. We will measure both the degree of selective axonal bundling of the two classes of axons and the ultimate fidelity of their targeting in the bub in mutant as compared to control embryos. These studies will identify molecular determinants of selective bundling and target acquisition that are of general applicability to understanding how circuits form in the developing vertebrate nervous system.