PROJECT SUMMARY/ABSTRACT Our long-term goal is to understand the cellular and genetic rules governing how endothelial cells (EC), vascular smooth muscle cells (VSMC) and sympathetic neurons (SN) interact within the artery. SNs innervate vascular SMCs and control normal vascular functions such as circulation, respiration, body temperature, and metabolism. Thus, aberrant regulation of the vascular-mural-sympathetic (EC-VSMC-SN) interaction underlies numerous cardiovascular pathologies which affect millions of people worldwide. Although much progress has been made in identifying the molecular signaling controlling the individual EC, VSMC, and SN vascular functions, less is known about the players that allow the three cell types to work as a unit. This lack of knowledge is underscored by the limited strategies available to improve vascular function by manipulating the sympathetic vascular unit. Here we will use a basic science approach to address this problem through experiments designed to unravel the cellular and genetic mechanisms that govern normal EC-VSMC-SN unit formation. We will first understand, via live-cell imaging microscopy, how blood flow coordinate EC-VSMC-SN unit assembly at the dorsal aorta in zebrafish (Aim 1) and then dissect the function of the identified flow-depended genetic programs in each cells to allow the neurovascular coupling (Aim 2). Finally, in Aim 3, thanks to our successful collaboration with Drs. Anne Eichmann, Martin Schwartz and Martina Brueckner, the signaling and cellular study of the EC- VSMC-SN unit will be translated in the mammalian cardiovascular system. Given the therapeutic potential of manipulating sympathetic innervation, our studies will have a broad, positive impact on identifying novel targetable signaling pathways to control sympathetic function.