Characterizing Novel Neural Crest Derived Regulators of Hematopoietic Stem Cell Specification

项目摘要 造血干细胞(HSCs)在生物体的整个生命周期中产生所有血统，并在临床上 作为用于治疗血液病的骨髓移植的重要治疗剂。然而， 由于很难找到免疫相容的捐赠者，移植治疗的可用性受到限制。这个 从诱导的多能干细胞(IPSCs)体外产生大量自体HSCs的能力是一种 主要生物医学目标，并有可能消除细胞可获得性问题。迄今为止，它还没有 有可能产生真正的造血干细胞，具有高植入潜力和多谱系重建。一 从IPSC产生HSC的可能方法将是重构全套HSC规范信号 然而，在体外，识别这些信号需要进一步询问控制信号的分子机制 体内的HSC规范。在脊椎动物胚胎中，造血干细胞来源于腹侧的血源性内皮(HE) 胚胎背主动脉(DA)的底板。我的工作是使用斑马鱼的发育造血模型 已有研究表明，主干神经脊(NC)细胞为HSC的HE规范提供了所需的诱导线索。至 确定HSC规范的新型NC衍生调节器，目标是为在 在体外，我进行了树干NC和血管内皮细胞的转录图谱筛选。使用NC特定目标 斑马鱼胚胎中基因的缺失，我将确定候选基因识别的分子机制 在屏幕上指示HSC规范。此外，我还鉴定了HE表达的G蛋白偶联 受体GPR182和可能的辅助受体Ramp2，作为HSC规范的新调节因子。重要的是 GPR182或RAMP2的敲除直接复制NC消融对HSC规范的影响， 提示GPR182可能介导了NC来源的HSC规范信号。使用有针对性的击倒和 蛋白质组学，我将在HSC规范的控制下鉴定GPR182和Ramp2。主干NC的导数， 尤其是交感神经元和间充质干细胞，控制着成年HSCs的动员。这个 HSC规范中对NC衍生品的要求表明这些细胞之间存在终身信令关系 类型。我将使用永久的血统追踪方法来确定HSC规格和对照成人 HSC动员需要相同类型的NC衍生物。本建议书的指导阶段将在 圣犹大儿童研究医院，由威尔逊·克莱门茨博士领导，并将确认HSC的候选监管机构 指定并专注于为独立阶段产生突变/转基因斑马鱼品系。这个 独立相将阐明新型调节剂指导HSC规范的分子机制 以及关于NC衍生品的永久谱系追踪。机构资源和学术环境以及 我的提案中概述的计划课程将确保我成功地过渡到独立。

Project Summary Hematopoietic stem cells (HSCs) generate all blood lineages throughout the life of an organism and are clinically important as the therapeutic agents of bone marrow transplants used to treat hematological disorders. However, the availability of transplant therapy is limited by difficulty in finding immunologically compatible donors. The ability to generate large numbers of autologous HSCs in vitro from induced pluripotent stem cells (iPSCs) is a major biomedical objective and has the potential to eliminate problems of cell availability. To date, it has not been possible to generate bona fide HSCs with high engraftment potential and multi-lineage reconstitution. One possible means of generating HSCs from iPSCs would be to reconstitute the full set of HSC specification signals in vitro, however identifying these signals requires further interrogation of the molecular mechanisms controlling HSC specification in vivo. In vertebrate embryos, HSCs arise from hemogenic endothelium (HE) lining the ventral floor of the embryonic dorsal aorta (DA). My work using the zebrafish model of developmental hematopoiesis has shown that trunk neural crest (NC) cells provide required inductive cues for HSC specification to the HE. To identify novel NC derived regulators of HSC specification with the goal of informing efforts to generate HSCs in vitro, I conducted a transcriptional profiling screen of trunk NC and endothelial cells. Using NC specific targeted deletion of genes in zebrafish embryos, I will determine the molecular mechanisms by which candidates identified in the screen instruct HSC specification. Additionally, I have identified the HE expressed G-protein coupled receptor Gpr182 and putative co-receptor Ramp2, as novel regulators of HSC specification. Importantly, knockdown of either gpr182 or ramp2 directly phenocopy the effect of NC ablation on HSC specification, suggesting that Gpr182 could mediating a NC derived HSC specification signal. Using targeted knock out and proteomics, I will characterize Gpr182 and Ramp2 in the control of HSC specification. Derivatives of trunk NC, specifically sympathetic neurons and mesenchymal stem cells, control mobilization of adult HSCs. The requirement for NC derivatives in HSC specification suggests a lifelong signaling relationship between these cell types. I will employ permanent lineage tracing approaches to determine if HSC specification and the control adult HSC mobilization require the same types of NC derivatives. The mentored phase of this proposal will occur at St. Jude Children’s Research Hospital, under Dr. Wilson Clements and will confirm candidate regulators of HSC specification and focus on generation of mutant/transgenic zebrafish lines for the independent phase. The independent phase will elucidate the molecular mechanisms by which novel regulators instruct HSC specification and on permanent lineage tracing of NC derivatives. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.