Early Cardiac Progenitors

总结 哺乳动物的心脏在胚胎发育早期就形成了，其形成过程中的缺陷是先天性心脏病的根源。 心脏缺陷（CHD），影响1-2%的活产婴儿。CHD中基因调控网络受到干扰 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，其中一些也与成人心脏病的遗传学有关，如 心肌病和心律失常完整了解心脏祖细胞的诞生和第一步 导致胚胎心脏形成的基因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， 原肠胚形成我们先前定义了Smarcd 3的特异性增强子，其标记非常有限的亚群 的Mesp 1谱系标记的中胚层，这几乎完全有助于心脏。胚胎发生是一个高度 一个动态的过程，通过复杂的协调细胞运动定位器官前体， 形态发生，并控制对有效的形态发生信号梯度的反应。虽然动态方面 心脏的形态发生已经在鸟类胚胎中显现出来，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成像活体血统- 标记的小鼠胚胎，并将利用这种令人兴奋的方法来研究哺乳动物心脏发育。 我们假设，早期心脏细胞的分化、迁移和对早期心脏形成的贡献， 祖细胞受特定基因调控网络的调控，该网络由时间调节的DNA组成 调节元件和转录调节子级联的协调功能。我们将测试这个 两个具体目标的假设。 具体目标1：在全基因组范围内确定控制早期乳腺癌发生的基因调控网络。 心脏祖细胞我们将定义全基因组调控网络，控制规范，迁移， 使用转基因报告细胞系结合单细胞RNA-seq的早期心脏前体细胞的分化 正常和突变胚胎心脏发育中的单细胞ATACseq（染色质可及性） 具体目标2。用活胚胎显像观察早期心脏祖细胞的出生和迁移。 在这个目标中，我们将使用活胚胎光片显微成像的谱系标记的小鼠胚胎， 在4D中可视化心脏祖细胞的动态出现，它们的迁移和它们的集体行为 在早期心脏形态发生中。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（Mesp 1敲除胚胎）和第一或第二心脏分化缺陷的背景 场（Tbx 5和Mef 2c敲除胚胎）。 从基因组中的转录因子足迹到转录组，再到体内的活细胞，我们的项目将阐明 在单细胞水平上，最早的心脏前体细胞的分子性质。

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.