Molecular Circuits in the Hematopoietic Stem Cell Niche

在哺乳动物中，造血干细胞（HSC）首先来自于一个专门的造血内皮细胞， 发育中的胚胎主动脉，迁移到胎儿肝脏并在其中扩张，最终在骨骼中定居 骨髓，它支持整个成年期的造血。这些不同的解剖位置 专门的微环境，支持发育成熟，扩展，并最终 平衡HSC的自我更新和分化。转录程序，促进形成和 造血干细胞和祖细胞（HSPCs）的分化已经被广泛质疑，但 关于造血微环境的支持性小生境细胞和 细胞-细胞相互作用的机制，这些机制规定了HSC在发育过程中的出现，HSC迁移，倒伏， 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，我们收集了许多细胞类型的证据，包括内皮细胞， 细胞、间充质细胞、巨噬细胞、神经嵴衍生物和体节作为细胞的组分。 造血生态位我们将收集全面的“组学”数据， 在发育过程中出现在性腺-中肾的不同造血小生境细胞内 (AGM)、胎肝、骨髓和胎盘（aim 1）。我们的方法从tomo-seq开始， 为了发现细胞群体特有的基因表达模式，如内皮细胞， 专业化我们将通过单细胞RNA-seq和原位杂交验证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啉代和CRISPR敲低记录功能性。 一种易于驾驭的斑马鱼模型。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， 和基序发现软件来识别DNA结合调节因子，这些因子是 造血细胞命运我们将采用一个计算管道，并开发新的算法来分析这些 数据（目标2）。目标1和2中的假设将通过构建新的报告菌株进行测试。 斑马鱼和小鼠，以及携带合成报告基因和驱动基因的工程多能干细胞（aim 3）。我们的目标是确定在HSC的关键期内指定小生境细胞的分子电路 出现和扩张，并探讨生态位元件和HSPC之间的串扰。我们希望收集 独特的见解，推动造血形成和成熟的分子机制， 胚胎发育，并加强我们对HSC的维持，静止，自我更新的理解 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.