Molecular and Cellular Mechanisms of Trabeculation

描述（由申请人提供）：在发育过程中，心脏从肌细胞的上皮层转变为对其功能至关重要的复杂三维结构。许多形态发生过程有助于这种转变，其中一个过程导致心脏小梁的形成，心室心肌壁中的片状肌肉结构。小梁在冠状动脉循环系统发育前增加心肌质量方面起关键作用，并作为心室传导系统的前体。此外，无法形成小梁或心室致密化失败可导致先天性心肌病。尽管这些结构很重要，但关于它们是如何形成的，仍然存在许多悬而未决的问题。在这个建议中，我们将使用强大的活体成像和转基因方法与斑马鱼系统，以检查小梁形成的细胞和分子决定因素。单个肌细胞如何进入小梁层目前尚不清楚。我们最初的数据表明，小梁形成通过一个过程中的分层，但精确的细胞机制仍不清楚。我们推测，肌细胞利用保守的顶端收缩的发育过程退出致密层，形成小梁片。为了验证这个假设，我们将 利用荧光蛋白的镶嵌表达结合实时成像来监测斑马鱼心室内单个心肌细胞的细胞形状变化。此外，我们将使用活成像和免疫荧光的组合来检查肌动蛋白和肌球蛋白在这个过程中的行为。只有心室肌内的一些细胞对小梁层有贡献，目前还不知道这些细胞是如何被选择的。Notch通路在发育中起着许多作用，并且已知在各种情况下调节不对称的细胞命运决定。我们最初的数据表明，肌细胞内的Notch信号传导仅限于 致密层。我们假设Notch信号的激活阻止了细胞对小梁的贡献。我们建议测试这一假设，通过监测Notch激活随时间的推移，使用转基因斑马鱼线表达GFP下的notch响应启动子。此外，我们将使用镶嵌表达的显性激活剂和抑制剂的Notch途径，以确定是否Notch激活是必要的和足够的，以防止小梁掺入。通过使用斑马鱼系统中可用的强大的成像和遗传工具，该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.