Early Cardiac Progenitors

摘要 哺乳动物的心脏在胚胎发育的早期就形成了，而心脏形成的缺陷是先天性心脏病的根源 心脏缺陷(CHD)，影响1-2%的活产儿。冠心病中受干扰的基因调控网络 已经开始从大规模的人类基因研究中得到阐明。许多导致冠心病的基因是 转录调节因子，其中几个也与成人心脏病的遗传学有关，例如 心肌病和心律失常。全面了解心脏祖细胞的诞生和第一步 这导致胚胎心脏的形成对冠心病和成人心脏病都有影响。 心脏在胚胎发生中的起源被定义为始于中胚层，在 原肠形成。我们之前定义了Smarcd3的一个特定增强子，该增强子标记了一个非常受限的亚群 Mesp1谱系标记的中胚层，几乎只对心脏有贡献。胚胎发育是一种高度 动态过程，通过复杂的协调细胞运动定位器官前体 形态发生，并控制对强大的形态发生信号梯度的反应。虽然动态的方面 心脏的形态发生已经在鸟类胚胎中可视化，这是我们对心脏祖细胞的了解 缺乏小鼠胚胎的出现和行为。我们已经能够培养和想象活的血统- 标记的小鼠胚胎，并将利用这一令人兴奋的方法来研究哺乳动物的心脏发育。 我们假设早期心脏的分化、迁移和对早期心脏形成的贡献 祖细胞受特定的基因调控网络的调节，该网络由时间调节的DNA组成 调控元件和一系列转录调控因子的协调功能。我们将对此进行测试 假设有两个具体的目的。 具体目标1：在全基因组范围内定义控制早期黄斑狼疮出现的基因调控网络 心脏祖细胞。我们将定义全基因组调控网络，控制规范、迁移、 转基因报告系结合单细胞RNA-seq对早期心脏前体细胞分化的研究 和单细胞ATACseq(染色质可及性)在正常和突变的胚胎心脏发育中的作用 具体目的2.用活体胚胎显像法观察早期心脏祖细胞的出生和迁移过程。 在这个目标中，我们将使用血统标记的小鼠胚胎的活胚胎光片显微成像来 在4D中可视化心脏祖细胞的实时动态出现、他们的迁移和他们的集体行为 在心脏形态发生的早期。我们将进一步在4D早期心脏祖细胞的活胚胎中成像 迁移受损(Mesp1基因敲除胚胎)和第一或第二心脏分化缺陷的背景 FIELS(Tbx5和MEF2C基因敲除胚胎)。 从转录因子在基因组中的足迹，到转录本，再到活体细胞，我们的项目将阐明 在单细胞水平上，最早的心脏前体的分子性质。

SUMMARY 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. The gene regulatory networks that are disturbed in CHDs have begun to be elucidated from large-scale human genetic studies. Many of the genes causing CHD are transcriptional regulators, and several of these are also associated with genetics of adult heart disease, such as cardiomyopathy and arrhythmias. A complete understanding of the birth of cardiac progenitors and the first steps that lead to the formation of the embryonic heart has implications for both CHD and adult heart disease. The origins of the heart in embryogenesis have been defined as beginning in mesoderm that arises during gastrulation. We previously defined a specific enhancer of Smarcd3 that labels a very restricted subpopulation of Mesp1 lineage-labeled mesoderm, which contribute almost exclusively to the heart. Embryogenesis is a highly dynamic process, whereby complex coordinated cell movements position organ precursors for precise morphogenesis, and control the response to potent morphogenetic signaling gradients. While aspects of dynamic cardiac morphogenesis have been visualized in the avian embryo, our knowledge of cardiac progenitor emergence and behaviour in the mouse embryo is lacking. We have been able to culture and image live lineage- labeled mouse embryos, and will leverage this exciting approach to investigate mammalian cardiac development. We hypothesize that differentiation, migration, and contribution to early heart formation of early cardiac progenitors are regulated by a specific gene regulatory network composed of temporally-modulated DNA regulatory elements and the coordinated function of a cascade of transcriptional regulators. We will test this hypothesis in two Specific Aims. Specific Aim 1: To define genome-wide the gene regulatory networks that control the emergence of early cardiac progenitors. We will define genome-wide regulatory networks that control the specification, migration, and differentiation of early cardiac precursor using transgenic reporter lines combined with single cell RNA-seq and single cell ATACseq (for chromatin accessibility) in normal and mutant embryonic heart development Specific Aim 2. To visualize the birth and migration of early cardiac progenitors with live embryo imaging. In this aim, we will use live embryo light sheet microscopic imaging of lineage-labeled mouse embryos to visualize in 4D the live dynamic emergence of cardiac progenitors, their migration, and their collective behaviours during early cardiac morphogenesis. We will further image in live embryos in 4D early cardiac progenitors in the context of impaired migration (Mesp1 knockout embryos) and defective differentiation of first or second heart fields (Tbx5 and Mef2c knockout embryos). From transcription factor footprints in the genome, to transcriptomes, to live cells in vivo, our project will elucidate at the single cell level the molecular nature of the earliest cardiac precursors.