Molecular and Cellular Mechanisms of Trabeculation

描述（由申请人提供）：在发育过程中，心脏从肌细胞上皮层转变为复杂的三维结构，对其功能至关重要。许多形态发生过程有助于这种转变，其中一个过程导致心小梁的形成，心小梁是心室心肌壁的片状肌肉结构。小梁在冠脉循环系统形成前心肌质量增加中起关键作用，是心室传导系统的前体。此外，小梁形成失败或心室压实失败可引起先天性心肌病。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。在这个建议中，我们将使用强大的实时成像和转基因方法与斑马鱼系统来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。我们的初步数据表明小梁是通过分层形成的，但确切的细胞机制尚不清楚。我们假设肌细胞使用保守的顶端收缩发育过程退出致密层并形成小梁片。为了验证这个假设，我们将

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.