The development of non-random asymmetry along the left-right axis is a unique feature of vertebrate development. Defects in this process in mouse and man commonly affect the development of the heart and result in severe congenital cardiac anomalies. The goal of this proposal is to understand the mechanism by which embryonic cilia create and signal left-right positional information. Motility of embryonic cilia is driven by the axonemal dynein, left right dynein (lrd). Lrd is essential and specific to the development of left-right asymmetry. When it is defective, the normally motile monocilia found on the node (organizer) of the embryo at the time of gastrulation are paralyzed. Motile node cilia generate directional flow of the extraembryonic fluid surrounding the node (nodal flow), and there is no nodal flow in mice with defective lrd. These observations suggest a direct role for motile 9+0 node cilia in the initiation of LR asymmetry. The development of LR asymmetry is also abnormal in mice with defects in the polycystin gene Pkd2, which functions in kidney monocilia as a mechanotransducer by mediating an intracellular calcium signal in response to fluid flow in the renal tubule. We have shown that Polycystin-2 protein localizes to a subset of node monocilia that are non-motile. Finally, we observe an asymmetric perinodal calcium signal in e7.75 mouse embryos. These data suggest that embryonic cilia may be required to both create and sense nodal flow. In this proposal, we will first test whether there are two classes of monocilia at the node. First, we will study the structure of node monocilia by immunofluorescence and transmission electron microscopy. Using Cre-lox technology, we will generate mice that have deleted all node monocilia, and compare their LR development to that observed in mice lacking only motile node monocilia. We will determine the role of asymmetric calcium signaling in the development of LR asymmetry by observing the effect of mutations affecting nodal flow, and of artificial nodal flow, on the asymmetric perinodal calcium signal. The position of asymmetric perinodal calcium signaling in the pathway of LR development will be tested by examining the effect of artificially introduced calcium signals on LR phenotype in cultured embryos. Finally, we will evaluate the role of the calcium-permeable cation channel polycystin-2 in LR development. Pkd2-/- mice will be examined for morphology of node cilia, and for asymmetric perinodal calcium signals in order to determine whether nodal flow produces asymmetric perinodal calcium signals via the polycystin channel. ? ? ?
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