Early Cardiac Progenitors

描述(申请人提供)：哺乳动物的心脏在胚胎发育早期形成，其形成缺陷是先天性心脏缺陷(CHDS)的根源，影响1-2%的活产。在成年人中，心脏病是西方世界的头号杀手，导致 相当大的健康负担。了解心脏的组成成分对于确定先天性心脏病的病因和创造新的基于细胞的再生策略来治疗心脏病至关重要。从多能细胞中识别和分离心脏前体细胞将有助于鉴定和扩增心肌细胞以用于再生策略。我们确定Smarcd3(也称为BAF60c)的表达是预先指定的中胚层群体的候选早期标记，该群体主要产生发育中的心脏细胞。利用转基因报告基因分析和诱导型Cre介导的谱系示踪，我们发现Smarcd3在小鼠前中胚层的早期表达是早期特定心脏前体的特异性标记，先于任何其他心脏前体标记的表达。我们假设Smarcd3标志着小鼠胚胎中最早的特定心脏祖细胞，并且这种特殊的中胚层群体具有导致优先分化为心肌细胞的独特属性。我们将在三个具体目标上检验这一假设。目标1：实现 定义以Smarcd3表达为标志的早期心脏谱系。使用转基因报告系，我们将描绘Smarcd3谱系和先前特征的心脏谱系之间的关系。我们将使用可诱导的Cre标记，结合多色的“彩虹”报告鼠来定义早期的Smarcd3血统及其后代之间的克隆关系。利用荧光报告转基因小鼠株系，我们将纯化Smarcd3表达的前体细胞，目的2。确定激活早期Smarcd3表达的转录和信号通路。在这个目标中，我们将定义在早期心脏祖细胞中指导Smarcd3表达的基因和信号输入。我们将使用小鼠的瞬时转基因和分化的ES细胞来定义激活Smarcd3表达的转录输入。使用在Smarcd3调节区控制下表达EGFP的转基因ES细胞，我们将识别指导激活的信号输入集 在这个心脏前体的早期人群中，Smarcd3的基因表达水平很高。目的3：明确导致早期心脏谱系分化的信号通路。我们将确定其重要性 BMP和Wnt信号在心脏组织和胚胎中胚层分化中的作用，以及这些途径的时间需求。这一结果将对理解心脏分化所需的诱导线索至关重要。这项研究所产生的知识将对我们理解导致心脏形成的最早的命运决定至关重要。

DESCRIPTION (provided by applicant): The mammalian heart forms early in embryogenesis, and defects in its formation are at the root of congenital heart defects (CHDs), affecting 1-2% of live births. In adults, heart disease is the number one killer in the Western world, resulting in a considerable health burden. Understanding the building blocks of the heart is critical to define the etiology of CHDs and to create novel cell-based regeneration strategies to treat heart disease. Identifying and isolating cardiac precursors from pluripotent cells would allow characterization and expansion of cardiac cells for regenerative strategies. We identified expression of Smarcd3 (also known as Baf60c) as a candidate early marker of a prespecified mesoderm population that gives rise predominantly to cells of the developing heart. Using transgenic reporter assays and inducible Cre-mediated lineage tracing, we showed early expression of Smarcd3 in anterior mesoderm of the mouse is a specific marker of the earliest specified cardiac precursors, preceding expression of any other cardiac precursor marker. We hypothesize that Smarcd3 marks the earliest specific cardiac progenitors in the mouse embryo, and that this specialized mesodermal population has distinct properties that lead to preferential differentiation into cardiomyocytes. We will test this hypothesis in three Specific Aims. Aim 1: To define the early cardiac lineage marked by Smarcd3 expression. Using transgenic reporter lines, we will delineate the relationship between the Smarcd3 lineage and previously characterized cardiac lineages. We will use inducible Cre labeling, combined with multicolor "rainbow" reporter mice to define the early Smarcd3 lineage and the clonal relationships between its descendents. Using fluorescent reporter transgenic mouse lines, we will purify Smarcd3-expressing progenitors Aim 2. To determine the transcriptional and signaling pathways that activate early Smarcd3 expression. In this aim, we will define the genetic and signaling inputs that direct Smarcd3 expression in early cardiac progenitors. We will use transient transgenesis in mouse and differentiating ES cells to define the transcriptional inputs that activate Smarcd3 expression. Using transgenic ES cells expressing EGFP under control of the Smarcd3 regulatory region, we will identify the sets of signaling inputs that direct activation of Smarcd3 in this early population of cardiac precursors. Aim 3: To define signaling pathways that lead to cardiac differentiation of the early cardiac lineage. We will determine the importance of BMP and Wnt signaling in the differentiation of cardiac tissue from embryonic mesoderm, as well as the temporal requirement for these pathways. The results will be critical to understand the inductive cues required for cardiac differentiation. The knowledge generated from this study will be crucial to our understanding of the earliest fate decisions that lead to the formation of te heart.