Project Summary The nervous system regulates many aspects of animal behavior and physiology, and its function relies on the generation of intricate networks of cellular connections. Failure to establish or maintain the proper connections can have profound health consequences, including intellectual and developmental disabilities, requiring that robust developmental mechanisms exist to ensure their precise organization. A common organizational motif in nervous systems is the topographic map, in which the spatial relationships of neurons in a projecting field matches the spatial organization of the target field. By leveraging the optical, embryological and genetic accessibility of the zebrafish, we have discovered a novel temporal mechanism of topographic map formation that is different from existing “chemoaffinity” models that involve the molecular matching of spatial coordinates. Vagus motor axons emerge sequentially, with anterior axons emerging several hours earlier than posterior axons. In a parallel temporally regulated process in the head periphery, pharyngeal arches appear in an anterior-to-posterior appearance sequence. The outgrowing vagus axons then innervate the pharyngeal arches sequentially from anterior to posterior, resulting in a topographic map in which anterior neurons innervate more anterior targets and posterior neurons innervate more posterior targets. In this application, I propose to investigate the molecular mechanisms underlying these two parallel temporal processes. In Aim 1 I will determine how axon initiation is regulated at the cellular level in vivo, and will identify the differentially expressed genes that promote axonogenesis in anterior vagus neurons and/or delay it in posterior neurons. In Aim 2 I will investigate the role of the motor neuron chemoattractant HGF as the sequentially expressed pharyngeal arch attractant that is required for vagus topographic mapping. This work will establish new mechanisms that regulate axon specification and targeting, and will elucidate how the coordinated regulation of temporal developmental events can guide the development of complex topographic maps in vivo.