Molecular and Cellular Mechanisms of Trabeculation

描述(申请人提供)：在发育过程中，心脏从心肌细胞的上皮层转变为对其功能至关重要的复杂的三维结构。许多形态发生过程促进了这种转变，其中一个过程导致了心脏小梁的形成，即心肌壁中的片状肌肉结构。在冠状动脉循环系统形成之前，小梁在增加心肌质量方面起着关键作用，并且是心室传导系统的前体。此外，小梁形成失败或室壁紧实化失败可导致先天性心肌病。尽管这些结构很重要，但它们是如何形成的仍有许多悬而未决的问题。在这个提案中，我们将使用斑马鱼系统可用的强大的实时成像和转基因方法来研究小梁形成的细胞和分子决定因素。目前尚不清楚单个肌细胞是如何进入小梁层的。我们的初步数据表明，小梁是通过分层过程形成的，但确切的细胞机制仍不清楚。我们假设，心肌细胞利用心尖收缩这一保守的发育过程退出致密层，形成小梁片。为了检验这一假设，我们将 利用荧光蛋白的镶嵌表达结合活体成像来监测斑马鱼脑室内单个心肌细胞的细胞形状变化。此外，我们将使用实时成像和免疫荧光相结合的方法来检测肌动蛋白和肌球蛋白在这一过程中的行为。只有心室肌内的一些细胞对小梁层有贡献，这些细胞是如何选择的目前尚不清楚。Notch通路在发育过程中扮演着许多角色，并且已知在各种环境中调节不对称的细胞命运决定。我们的初步数据表明，心肌细胞内的Notch信号仅限于 紧凑层。我们假设Notch信号的激活阻止了细胞对小梁的贡献。我们建议通过监测Notch随时间的激活来验证这一假设，使用在Notch响应启动子下表达GFP的转基因斑马鱼品系。此外，我们将使用Notch通路主要激活物和抑制物的嵌合表达来确定Notch激活是否对于防止小梁合并是必要的和充分的。通过使用斑马鱼系统中可用的强大成像和遗传工具，这一提议将阐明在细胞水平上控制小梁形成的机制。

DESCRIPTION (provided by applicant): Over the course of development, the heart transforms from an epithelial layer of myocytes to a complex, three-dimensional structure critical for its function. A number of morphogenetic processes contribute to this transformation, and one of these processes leads to the formation of the cardiac trabeculae, sheet-like muscular structures in the ventricular myocardial wall. Trabeculae play a critical role in increasing myocardial mass before the development of a coronary circulatory system, and serve as precursors for the ventricular conduction system. Additionally, failure to form trabeculae or failure of ventricular compaction can cause congenital cardiomyopathies. Despite the importance of these structures, there remain many open questions about how they are formed. In this proposal, we will use the powerful live imaging and transgenic approaches available with the zebrafish system to examine the cellular and molecular determinants of trabecular formation. How individual myocytes enter the trabecular layer is currently unknown. Our initial data suggest that trabeculae form via a process of delamination, but the precise cellular mechanisms remain unclear. We hypothesize that myocytes use the conserved developmental process of apical constriction to exit the compact layer and form trabecular sheets. To test this hypothesis, we will use mosaic expression of fluorescent proteins combined with live imaging to monitor the cell shape changes of individual cardiomyocytes within the zebrafish ventricle. Additionally, we will use a combination of live imaging and immunofluorescence to examine the behavior of actin and myosin during this process. Only some cells within the ventricular myocardium contribute to the trabecular layer, and how these cells are chosen is currently unknown. The Notch pathway plays many roles in development, and is known to regulate asymmetric cell fate decisions in a variety of contexts. Our initial data suggest that Notch signaling within myocytes is restricted to the compact layer. We hypothesize that activation of Notch signaling prevents cells from contributing to trabeculae. We propose to test this hypothesis by monitoring Notch activation over time using a transgenic zebrafish line expressing GFP under a notch-responsive promoter. Further, we will use mosaic expression of dominant activators and repressors of the Notch pathway to determine whether Notch activation is necessary and sufficient for preventing trabecular incorporation. By using the powerful imaging and genetic tools available in the zebrafish system, this proposal will shed light on the mechanisms controlling trabecular formation at a cellular level.