Intraciliary calcium directs cardiac left-right asymmetry

Yale University, New Haven CT

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Abstract

? DESCRIPTION (provided by applicant): Cilia are central to the development of vertebrate left-right (LR) asymmetry, and failure to establish normal LR asymmetry results in the human heterotaxy syndrome (Htx) and severe congenital heart disease (CHD). Htx is amongst the most lethal forms of CHD in humans, and mutations affecting at least 12 genes affecting cilia structure and function have been identified in patients with Htx and other CHD. This proposal investigates the cilium as a distinct calcium signaling compartment, and investigates the role intraciliary calcium plays in heart development. We will define the mechanism by which intraciliary calcium establishes cardiac LR asymmetry. In vertebrate LR development, cilia at the left-right organizer (LRO) beat to generate directional flow of extraembryonic fluid, which is sensed by cilia and directs asymmetrical gene expression. Several lines of evidence link calcium, cilia and LR development: the ciliary calcium channel polycystin-2 (Pkd2) at the LRO is essential for LR development, and in mouse and zebrafish, a left-biased increase of cytoplasmic calcium correlates with LR development and is dependent on both flow and Pkd2. Despite the established importance of cilia, flow, polycystins and cytoplasmic calcium signaling in LR development, the mechanisms linking these components, and further to the downstream signaling cascade driving asymmetric cardiac morphogenesis, are not understood. We have developed a novel method to visualize and manipulate intraciliary calcium in cultured cells and zebrafish embryos. In this proposal, we will use this to address the physiology and function of intraciliary calcium in LR and heart development. We hypothesize that the cilium is a distinct cellular compartment with respect to calcium signaling, and that intraciliary calcium is the link between ciliary motility at the LRO and asymmetric cardiac morphogenesis. In Aim 1, we will characterize intraciliary calcium in-vivo in cultured cells and zebrafish embryos and examine the response of intraciliary calcium to ciliary motility and whether intraciliary calcium requires the polycystin channel in LR development. In Aim 2, we will dissect how intraciliary calcium regulates LR development by analyzing how intraciliary calcium regulates asymmetric gene expression. In Aim 3, we will test whether the ankyrin-repeat protein Inversin transduces intraciliary calcium in cultured renal epithelial cells, zebrafish and mice with mutations affectin inversin. In summary, these experiments provide a new paradigm for ciliary signaling in LR and heart development, and develop techniques that will be broadly applicable to the study of cilia biology.