Molecular Circuits in the Hematopoietic Stem Cell Niche

在哺乳动物中，造血干细胞 (HSC) 首先产生于一种专门的造血内皮细胞，该内皮细胞排列在 发育中的胚胎主动脉，迁移到胎儿肝脏并在其中扩张，最终定植于骨骼 骨髓，支持整个成年时期的造血功能。这些独特的解剖位置隐藏着 支持发育成熟、扩展以及最终发育的专门微环境 HSC 自我更新和分化的平衡。促进形成和的转录程序 造血干细胞和祖细胞（HSPC）的分化已被广泛质疑，但很多 关于造血微环境的支持性生态位细胞和 细胞间相互作用的机制，指定发育过程中 HSC 的出现、HSC 迁移、倒伏、 胎儿生态位的扩张和最终的静止、自我更新和骨骼的分化 骨髓。在我们的初步数据中，我们收集了多种细胞类型的证据，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组成部分 造血生态位。我们将收集全面的“组学”数据来对基因表达程序进行分类 在主动脉-性腺-中肾发育过程中出现的独特造血生态位细胞内 (AGM)、胎儿肝脏、骨髓和胎盘（目标 1）。我们的方法始于 tomo-seq，它使我们能够 发现具有区域特异性的细胞群（如内皮细胞）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 敲除来记录功能 易于驯服的斑马鱼模型。然后我们使用 ATAC-seq 定义这些基因周围的功能性开放染色质， 和基序寻找软件来识别 DNA 结合调节因子，这些因子是候选驱动因素 造血细胞的命运。我们将采用计算管道并开发新颖的算法来分析这些 数据（目标 2）。目标 1 和 2 中出现的假设将通过构建新的报告菌株来检验 斑马鱼和小鼠，以及携带合成报告基因和驱动基因的工程多能干细胞（目标 3）。我们的目标是定义在 HSC 关键时期指定利基细胞的分子电路 的出现和扩展，并探讨利基元素和 HSPC 之间的串扰。我们希望能收集到 对驱动造血形成和成熟的分子机制有独特的见解 胚胎发育，并增强我们对 HSC 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.