PROJECT SUMMARY / ABSTRACT Cilia, microtubule-based organelles found on nearly all eukaryotic cells, coordinate numerous signaling cascade that are essential for vertebrate development and disease. Mutations in primary cilia are associated with numerous cardiovascular defects, most notably major congenital heart diseases (CHD). Further, ciliary defects are known to cause heterotaxy, a human disorder of abnormal left-right (LR) asymmetric body patterning that commonly affects the heart and is tightly correlated with CHD. During embryogenesis, proper LR asymmetric development requires motile cilia that move in a coordinated fashion to generate the initial signal to break LR symmetry: leftward flow of extra-embryonic fluid in a structure called the “left-right organizer” (LRO). Although leftward flow in the LRO is necessary and sufficient for LR development, how this flow is sensed and transduced into phenotypic LR asymmetry remains unclear. Strikingly, we have generated preliminary data in the zebrafish LRO that suggests immotile cilia may function as calcium-signaling compartments that coordinate downstream Nodal signaling via gap junctions during LR development. To elucidate this potential mechanism, we propose three scientific aims that combine state-of-the-art in vivo microscopy, optogenetic tools, machine learning data analysis approaches, zebrafish genetics and molecular embryology. In Aim 1, we will combine light-sheet microscopy and optogenetic actuators to regulate calcium dynamics in the LRO of zebrafish. Combined with new quantitative machine learning analysis tools, this approach will enable us to definitively address whether intra- ciliary calcium signaling is sufficient and instructive for LR development. In Aim 2, we will investigate the molecular machinery that underlies cilia-mediated calcium signaling in LR development. Specifically, we will examine how the Pkd1l1-Pkd2 polycystin complex interact with one another to mediate calcium signaling in the zebrafish LRO. In Aim 3, we will investigate how cilia-mediated calcium signaling is transduced from the cilium to the cytoplasm by Invs, a calcium binding protein which localizes to the base of LRO cilia and is required for LR development. Completion of these studies will resolve the role of the cilium as mechanosensitive antennae that sense and translate extra-embryonic fluid flow into calcium signals that build the LR axis and lead to a greater understanding of the mechanisms that drive situs of the heart.