Numb Family Proteins Regulate Trabecular Development

描述(申请人提供)：心血管疾病是导致发病率和死亡率的主要原因。其中一种疾病是左心室致密化不全(LVNC)，即小梁不能进行紧实化。小梁是从心肌延伸到心腔的片状结构，其功能是有效地泵送。缺乏小梁会导致小鼠胚胎死亡，而过度小梁会导致人类心肌病和心力衰竭。它的临床相关性以小梁性非致密性心肌病为例，在这种疾病中，小梁不能进行致密化，大约50万美国人患有这种疾病。尽管这种形态形成过程的基本性质及其关键的临床意义，但调节小梁形成的细胞和分子机制尚不清楚。在小梁形成过程中，心肌细胞伸入心脏凝胶中，并垂直于心壁。我们删除了小鼠心脏细胞内适配器蛋白Numblike及其同源基因Numblike，称为Numb Family Proteins(NFP)，以产生心肌NFP双基因敲除(MDKO)，发现MDKO心脏表现出各种心脏缺陷，包括明显的小梁起始和心肌细胞分化中断。三维(3D)成像显示MDKO中的心肌细胞不能突入心腔。使用谱系追踪/镶嵌小鼠模型，我们发现大多数NFP零克隆未能定位到小梁。我们的中心假设是，NFP通过N-钙粘素(N-CAD)依赖的机制控制定向迁移和定向细胞分裂(OCD)来调节小梁的启动和分化，这一假设将通过两个目标进行验证。目的I：确定NFP是否通过控制定向迁移和强迫症来调节骨小梁的起始。我们将使用Brain弓小鼠进行多色谱系追踪，在小梁培养之前，心脏管中的单个细胞将被基因标记，这使我们能够分析每个标记的心肌细胞后代的定位，并跟踪它们在小梁培养过程中的动态。时间推移4D成像将用于观察心肌细胞如何启动小梁细胞。我们将确定强迫症是否是一种不对称的细胞分裂，并有助于小梁状心肌细胞的分化。这一目的将揭示骨小梁起始和形态发生的细胞基础。目的II：确定NFP是否通过稳定N-CAD来调节骨小梁形成。我们将确定迁移和N-CAD表达所需的Numb的功能残基/结构域。上位性分析和转基因品系挽救将用于确定N-CAD和Numb之间的遗传互作。这一目标将开始揭示NFP如何调控小梁形成的分子机制。这些目标的完成有望对体内骨小梁起始和心肌细胞分化的细胞和分子机制以及NFP在这些过程中的功能提供新的见解，最终将为治疗非致密性心肌病和形态发生相关的先天性心脏病(CHD)提供机制基础。

DESCRIPTION (provided by applicant): Cardiovascular diseases are leading causes of morbidity and mortality. One such disease is left ventricular noncompaction (LVNC), in which trabeculae fail to undergo compaction. Trabeculae are sheet-like structures extending from the myocardium to the heart lumen and function to pump efficiently. A lack of trabeculation causes embryonic lethality in mice and excess trabeculation causes cardiomyopathy and heart failure in human. Its clinical relevance is exemplified by trabecular non-compaction cardiomyopathy, in which trabeculae fail to undergo compaction and about half a million Americans suffer from this disease. Despite the fundamental nature of this morphogenic process and its critical clinical implication, the cellular and molecular mechanisms that regulate trabeculation are not clear. During trabecular initiation, myocardial cells protrude into the cardiac jelly and are oriented perpendicular to the heart wall. We deleted the intracellular adaptor protein Numb and its homolog Numblike, known as Numb Family Proteins (NFP) in mouse heart to generate myocardium NFP double knockout (MDKO) and found that MDKO hearts displayed a variety of cardiac defects, including a marked disruption in the trabecular initiation and cardiomyocyte differentiation. 3-dimentional (3D) imaging indicates that cardiomyocytes in MDKO fail to protrude into the heart lumen. Using lineage tracing/mosaic mouse model, we found that the majority of the NFP null clones failed to localize to the trabeculae. Our central hypothesis is tha NFP regulate trabecular initiation and differentiation by controlling directional migration and oriented cell division (OCD) via N-cadherin (N-CAD) dependent mechanisms, which will be tested by two aims. Aim I: Determine whether NFP regulate trabecular initiation by controlling directional migration and OCD. We will perform multicolor lineage tracing using the Brainbow mouse in which single cells in the heart tube will be genetically labeled before trabeculation, which allows us to analyze the localization of each labeled cardiomyocyte descendants and track their dynamics during trabeculation. Time-lapse 4D imaging will be applied to observe how cardiomyocytes initiate trabecula. We will identify if OCD is an asymmetric cell division and contributes to trabecular cardiomyocyte differentiation. This aim will reveal the cellular basis fo trabecular initiation and morphogenesis. Aim II: Determine whether NFP regulate trabeculation by stabilizing N-CAD. We will determine the functional residues/domains of Numb that are required for migration and N-CAD expression. Epistasis analysis and transgenic line rescuing will be applied to determine the genetic interaction between N-CAD and Numb. This aim will begin to reveal molecular mechanisms of how NFP regulate trabeculation. Completion of these aims is expected to yield novel insights into the cellular and molecular mechanisms of trabecular initiation and cardiomyocyte differentiation in vivo, and the functions of NFP in these processes, which will ultimately provide a mechanistic basis for treating non-compaction cardiomyopathy and morphogenesis related congenital heart defects (CHD).