Cellular signals within discrete compartments generate, shape, and reshape development, and are required for proper physiological function. Unfortunately it is difficult to study complex spatio-temporal signals in vivo. Although powerful, traditional electrophysiological approaches lack the necessary spatial resolution to delineate these processes. To circumvent this limitation, we utilize genetically-encoded indicators that can be localized to different subcellular compartments. Specfically we study how discrete subcellular signals, such as Ca2+ influx and vesicle release in sensory hair cells affect auditory and vestibular development and function. In hair cells, mechanosensitive responses are shaped by distinct sources of Ca2+: mechanosensitive Ca2+ -permeable channels in the hair bundle, voltage-gated Ca2+ channels at the synapse, and Ca2+ storage and release from mitochondria and ER. All of these Ca2+ signals shape vesicle release and ultimately hair-cell function. To examine these complex signals in vivo, we use the zebrafish model system. The zebrafish is an ideal system for optical imaging due to the ease of generating transgenics, and the fact that larvae are born and develop transparently. Furthermore, the imaging of hair cells is further simplified in zebrafish due to the presence of a lateral line system. The lateral line system is composed of clusters of superficial hair cells called neuromasts that are readily visualized and physically stimulated. Using this system we can precisely monitor Ca2+ signals in the hair-cell cytoplasm, hair bundle, presynaptic density, and monitor synaptic vesicle release. We plan to combine in vivo imaging of Ca2+and vesicle fusion, confocal and electron microscopy, genetics, and pharmacology to characterize how discrete signals shape sensory function and development in an intact system.