The Role of Cardiomyocyte Polarity in Cardiac Morphogenesis

 描述（由申请人提供）：心脏是第一个发育的器官，这一过程涉及多个细胞命运决定和形态学过程。其中的关键是特定形态学事件的方向，如小梁形成和分隔朝向心脏内腔。这些过程的失败可能导致先天性心脏病，这是死亡和发病的主要原因。本提案的目的是确定非典型蛋白激酶C Iota（Prkci）和下游PAR极性机制在单细胞和整个器官水平指导心肌极化的机制。中心假设是来自内皮细胞和心胶质的方向线索引导Prkci和Par复合体将梭形器和腔心肌的细胞分裂平面朝向心脏腔定向并推动小梁形成。该假设是基于申请人实验室中产生的强有力的初步数据而制定的，并以三个特定目的进行测试：1）确定Prkci如何在心室小梁形成期间指导心肌细胞增殖和分化; 2）定义Prkci控制Par复合物和纺锤体机制以调节腔心肌细胞中极化细胞分裂的分子机制;和3）定义引导心肌极化的非细胞自主诱导信号传导线索。在第一个目标下，进行单细胞克隆分析以确定Prkci如何指导定向细胞分裂。高度创新的细胞标记和基因组编辑技术与尖端的光谱共聚焦显微镜相结合，以确定Prkci依赖的极化细胞分裂在小梁形成起始中的作用。在第二个目的中，申请人确定Prkci如何控制下游Par机制，并探索该途径在人干细胞衍生的肌细胞中指导极化细胞分裂的潜力。在第三个目标中，申请人确定了进入Prkci及其相互作用伙伴的上游诱导线索，并将从发育生物学获得的知识应用于干细胞生物学。这些研究的基本原理是，他们是第一个解决如何高度保守的PAR复合物定向有丝分裂纺锤体和轴的细胞分裂，以控制心肌对齐在体内心脏发生和体外细胞分化。基于干细胞的再生心脏病学方法的出现以及确保移植细胞或构建体的适当细胞排列的必要性使得这项研究非常重要。因此，该项目的总体影响是提供一个机制的理解，如何高度保守的Par机制指导极化细胞分裂，以控制正常的心脏器官发生。这项研究提供了心脏发育中关键形态学事件的基本理解，可应用于再生心血管医学，其中有必要扩大功能性CM的数量，并促进其与天然心肌的对齐和细胞整合。

 DESCRIPTION (provided by applicant): The heart is the first organ to develop, a process that involves multiple cell fate decisions and morphological processes. Key among these is the orientation of specific morphological events such as trabeculation and septation toward the heart lumen. Failure of these processes can result in congenital heart disease, a major cause of mortality and morbidity. The objective of this proposal is to identify the mechanism whereby atypical Protein Kinase C Iota (Prkci) and the downstream PAR polarity machinery direct myocardial polarization at the single cell and whole organ levels. The central hypothesis is that directional cues from the endocardium and cardiac jelly direct Prkci and the Par complex to orient the spindle apparatus and the cell division plane of luminal myocardial toward the heart lumen and propel trabecular formation. This hypothesis has been formulated on the basis of strong preliminary data produced in the applicant's laboratory and is tested with three specific aims: 1) determine how Prkci directs cardiomyocyte proliferation and differentiation during ventricular trabeculation; 2) define the molecular mechanism through which Prkci controls the Par complex and the spindle machinery to regulate polarized cell division in luminal myocardial cells; and 3) define the non-cell autonomous inductive signaling cues that direct myocardial polarization. Under the first aim, single cell clonal analysis is performed to determine how Prkci directs oriented cell division. Highly innovative cell labeling and genome editing techniques are coupled with cutting-edge spectral confocal microscopy to define the role of Prkci-dependent polarized cell division in the initiation of trabeculation. In the second aim, the applicant determines how Prkci controls the downstream Par machinery and explores the potential of this pathway to direct polarized cell division in human stem cell derived myocytes. In the third aim, the applicant identifies the upstream inductive cues that feed into Prkci and its interacting partners and adapts the knowledge gained from developmental biology to stem cell biology. The rationale for these studies is that they are the first to address how the highly conserved PAR complex orients the mitotic spindle and axis of cell division to control myocardial alignment during in vivo cardiogenesis and in vitro cellular differentiation. The advent of stem cell based approaches to regenerative cardiology and the necessity of ensuring the proper cellular alignment of transplanted cells or constructs make this research highly significant. Thus, the overall impact of this project is to provide a mechanistic understanding of how the highly conserved Par machinery directs polarized cell division to control normal cardiac organogenesis. This study provides a fundamental understanding of a key morphological event in cardiac development that can be applied to regenerative cardiovascular medicine where it is necessary to expand the number of functional CMs and promote their alignment and cellular integration with the native myocardium.