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

心脏位置发育中纤毛内钙信号传导的光遗传学调控

基本信息

批准号：
10504671
负责人：
Shiaulou Yuan
金额：
$ 55.51万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10504671
关键词：
Abdomen Address Affect Binding Binding Sites Biological Assay Biology Body Patterning Calcium Calcium Channel Calcium Oscillations Calcium Signaling Calcium-Binding Proteins Calmodulin Calmodulin-Binding Proteins Cardiac Cardiac development Cardiovascular system Cations Cell physiology Cells Chest Cilia Cilium Microtubule Complex Congenital Abnormality Congenital Heart Defects Cues Cytoplasm Data Data Analyses Defect Development Diagnostic Disease Embryo Embryology Embryonic Development Eukaryotic Cell Extracellular Fluid Gap Junctions Gene Expression Genetic Handedness Heart Human Image Instruction Ions Knowledge Lead Left Light Liquid substance Live Birth Liver Lung Machine Learning Mediating Microscopy Modeling Molecular Morphogenesis Mutation Myocardial Natural regeneration Nodal Organ Organelles PKD2 protein Patient-Focused Outcomes Pattern Perception Phenotype Play Positioning Attribute Process Proteins Regulation Reporter Research Role Signal Transduction Situs Inversus Structure Syndrome Techniques Testing Therapeutic Transducers Translating Vascular Diseases WNT Signaling Pathway Work Zebrafish base calcium indicator cardiogenesis ciliopathy cilium motility congenital heart disorder fluid flow improved outcome in vivo in vivo calcium imaging innovation mutant novel optogenetics receptor spatiotemporal tool

项目摘要

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.

项目概要/摘要纤毛是几乎所有真核细胞中发现的基于微管的细胞器，协调众多信号级联对于脊椎动物的发育和疾病至关重要。许多心血管缺陷，尤其是主要的先天性心脏病（CHD）此外，还有纤毛缺陷。已知会导致异位症，这是一种人类左右（LR）不对称身体模式失调的疾病在胚胎发生过程中，适当的 LR 不对称通常会影响心脏并与 CHD 密切相关。发育需要活动纤毛以协调的方式移动以产生破坏 LR 的初始信号对称性：胚胎外液体在称为“左右组织体”（LRO）的结构中向左流动。 LRO 中的向左流动对于 LR 发展是必要且充分的，如何感知和转换这种流动令人惊讶的是，我们已经在斑马鱼中获得了初步数据。 LRO表明不动的纤毛可能充当协调下游的钙信号室为了阐明这种潜在机制，我们提出了 LR 发育过程中通过间隙连接的节点信号传导。结合最先进的体内显微镜、光遗传学工具、机器学习数据的三个科学目标在目标 1 中，我们将结合光片分析方法、斑马鱼遗传学和分子胚胎学。显微镜和光遗传学致动器与新的相结合来调节斑马鱼 LRO 中的钙动态。定量机器学习分析工具，这种方法将使我们能够明确地解决内部是否睫状体钙信号传导对于 LR 发育来说是充分且具有指导意义的。在目标 2 中，我们将研究具体而言，我们将研究 LR 发育中纤毛介导的钙信号传导的分子机制。检查 Pkd1l1-Pkd2 多囊蛋白复合物如何相互作用以介导钙信号传导在目标 3 中，我们将研究斑马鱼 LRO 是如何从纤毛转导纤毛介导的钙信号传导的。通过 Invs 进入细胞质，Invs 是一种钙结合蛋白，定位于 LRO 纤毛的基部，是 LR 开发的完成将解决纤毛作为机械敏感触角的作用。感知胚胎外液流并将其转化为钙信号，从而构建 LR 轴并导致更好地了解驱动心脏位置的机制。