Optogenetic regulation of intra-ciliary calcium signaling in cardiac situs development

项目摘要/摘要 纤毛是几乎在所有真核细胞中发现的以微管为基础的细胞器，它协调许多信号级联。 对于脊椎动物的发育和疾病来说是必不可少的。初级纤毛的突变与 许多心血管缺陷，最突出的是重大先天性心脏病(CHD)。此外，睫状体缺陷症 已知会导致异位畸形，这是一种人类异常的左右(LR)不对称体型障碍， 通常影响心脏，并与冠心病密切相关。在胚胎发育过程中，适当的LR不对称 发育需要活动的纤毛以协调的方式移动，以产生打破LR的初始信号 对称性：胚胎外液体向左流动的结构称为“左-右组织者”(LRO)。虽然 LRO中向左的流动对于LR的发展是必要的和充分的，这种流动是如何被感知和传递的 转化为表型的LR不对称性仍不清楚。引人注目的是，我们已经在斑马鱼中产生了初步数据 LRO表明，静止的纤毛可能作为钙信号隔间来协调下游 在LR发育过程中通过缝隙连接传递节点信号。为了阐明这一潜在的机制，我们建议 结合最先进的活体显微镜、光遗传学工具、机器学习数据的三个科学目标 分析方法、斑马鱼遗传学和分子胚胎学。在目标1中，我们将结合光片 显微镜和光遗传致动器来调节斑马鱼LRO中的钙动力学。与新的 量化机器学习分析工具，这种方法将使我们能够明确地解决内部 纤毛钙信号对LR的发育是充分的和有指导意义的。在目标2中，我们将调查 纤毛介导的钙信号在LR发育中的分子机制。具体来说，我们将 研究Pkd1l1-PKD2多囊蛋白复合体如何相互作用以介导细胞内钙信号转导 斑马鱼LRO。在目标3中，我们将研究纤毛介导的钙信号是如何从纤毛转导的。 InVS是一种钙结合蛋白，定位于LRO纤毛的底部，是 LR开发。这些研究的完成将解决纤毛作为机械敏感触角的作用。 这种感觉并将胚胎外液体流动转化为钙信号，从而构建LR轴并导致 对驱动心脏部位的机制有更深入的了解。

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.