Molecular Circuits in the Hematopoietic Stem Cell Niche

在哺乳动物中，造血干细胞(HSCs)最初起源于一种特殊的血液内皮细胞， 发育中的胚胎主动脉，迁移到胎肝并在其中扩张，最终定植于骨骼 骨髓，在成人生活中支持造血。这些不同的解剖位置 专门的微环境，支持发育的成熟、扩展，并最终 造血干细胞自我更新与分化的平衡。促进形成和发展的转录程序 造血干细胞和祖细胞(HSPC)的分化已经被广泛询问，但有很多 关于造血微环境中的支持性壁龛细胞和 细胞-细胞相互作用的机制，规定了HSC在发育、迁移、倒伏 和胎儿骨龛的扩张，以及骨骼的最终静止、自我更新和分化 骨髓。在我们的初步数据中，我们收集了包括内皮细胞在内的多种细胞类型的证据 细胞、间充质细胞、巨噬细胞、神经脊衍生物和体节作为 造血利基。我们将收集全面的“组学”数据，对基因表达计划进行分类 在腹主动脉-性腺-中肾发育过程中的不同造血龛细胞内 (AGM)、胎肝、骨髓和胎盘(目标1)。我们的方法从tomo-seq开始，它使我们能够 发现像内皮细胞这样具有区域特异性的细胞群体特有的基因表达模式 专业化。我们将通过单细胞rna-seq和原位检测来验证facs纯化细胞中的细胞特异性表达。 杂交，并将在实验中使用吗啡和CRISPR击倒来记录功能 易驯服的斑马鱼模型。然后我们使用ATAC-seq来定义这些基因周围的功能性开放染色质， 和基序寻找软件，以确定作为候选驱动因素的DNA结合调控因素 造血细胞的命运。我们将使用计算管道并开发新的算法来分析这些 数据(目标2)。来自目标1和目标2的假说将通过构建新的报告菌株来检验 斑马鱼和小鼠，以及携带合成记者和驱动程序的工程多能干细胞(AIM 3)。我们的目标是定义在HSC的关键时期中指定利基细胞的分子电路 出现和扩展，并探索利基元素和HSPC之间的串扰。我们希望收集到 对驱动造血形成和成熟的分子机制的独特见解 胚胎发育，并提高我们对HSC维持、静止、自我更新的认识 和差异化。

In mammals, hematopoietic stem cells (HSCs) first arise from a specialized hemogenic endothelium that lines the developing embryonic aorta, migrate to and expand in the fetal liver, and ultimately colonize the bone marrow, which supports hematopoiesis throughout adult life. These distinct anatomic locations harbor specialized microenvironments that support the developmental maturation, expansion, and ultimately the balance of self-renewal and differentiation of HSCs. The transcriptional programs that promote formation and differentiation of hematopoietic stem and progenitor cells (HSPCs) have been widely interrogated, but much remains to be learned about the supportive niche cells of the hematopoietic microenvironment and the mechanisms of cell-cell interaction that specify HSC emergence during development, HSC migration, lodging, and expansion in fetal niches, and the ultimate quiescence, self-renewal, and differentiation in the bone marrow. In our preliminary data, we have gathered evidence for number of cell types, including endothelial cells, mesenchymal cells, macrophages, neural crest derivatives, and somites as components of the hematopoietic niche. We will gather comprehensive “omics” data to catalogue the gene expression programs within the distinct hematopoietic niche cells that occur during development in the aorta-gonad-mesonephros (AGM), fetal liver, bone marrow, and placenta (aim 1). Our approach begins with tomo-seq, which enables us to discover gene expression patterns unique to cell populations like endothelium that have region-specific specialization. We will validate cell-specific expression in FACS purified cells by single cell RNA-seq and in situ hybridization, and will document functionality using morpholino and CRISPR knock-down in the experimentally tractable zebrafish model. We then use ATAC-seq to define functional open chromatin around these genes, and motif-finding software to identify DNA-binding regulatory factors that are candidate drivers of hematopoietic cell fate. We will employ a computational pipeline and develop novel algorithms to analyze these data (aim 2). Hypotheses emerging from aims 1 and 2 will be tested by constructing novel reporter strains of zebrafish and mice, as well as engineered pluripotent stem cells carrying synthetic reporters and drivers (aim 3). Our goal is to define the molecular circuitry that specifies niche cells during the critical periods of HSC emergence and expansion, and to probe cross-talk between niche elements and HSPCs. We hope to glean unique insights into the molecular mechanisms that drive hematopoietic formation and maturation during embryonic development, and to enhance our understanding of HSC maintenance, quiescence, self-renewal and differentiation.