Characterizing Novel Neural Crest Derived Regulators of Hematopoietic Stem Cell Specification

项目摘要 造血干细胞（HSC）在生物体的整个生命过程中产生所有血液谱系，并且在临床上是 作为用于治疗血液疾病的骨髓移植的治疗剂是重要的。然而，在这方面， 移植治疗的可用性受到难以找到免疫相容供体的限制。的 从诱导多能干细胞（iPSC）体外产生大量自体HSC的能力是一种重要的生物学特性。 主要的生物医学目的，并有可能消除细胞可用性的问题。迄今为止， 已经可以产生具有高移植潜力和多谱系重建的真正的HSC。一 从iPSC产生HSC的可能方法是重建HSC特化信号的完整集合 然而，在体外，识别这些信号需要进一步询问控制这些信号的分子机制。 体内HSC质量标准。在脊椎动物胚胎中，HSC来自腹侧衬里的生血内皮（HE） 胚胎背主动脉（DA）的底部。我的工作是用斑马鱼的造血发育模型 已经表明，躯干神经嵴（NC）细胞提供所需的诱导线索HSC规格的HE。到 鉴定HSC特化的新NC衍生调节剂，目的是为在以下条件下产生HSC的努力提供信息： 在体外，我进行了主干NC和内皮细胞的转录谱筛选。使用NC特定靶向 删除基因在斑马鱼胚胎，我将确定的分子机制，候选人确定 在屏幕上指示HSC规格。此外，我还鉴定了HE表达的G蛋白偶联物， 受体Gpr 182和推定的辅助受体Ramp 2，作为HSC特化的新型调节剂。重要的是， gpr 182或ramp 2的敲低直接表型复制NC消融对HSC特化的影响， 这表明Gpr 182可以介导NC衍生的HSC特化信号。使用有针对性的淘汰， 蛋白质组学方面，我将描述Gpr 182和Ramp 2在HSC特化控制中的作用。主干NC的衍生物， 特别是交感神经元和间充质干细胞，控制成体HSC的动员。的 HSC规范中对NC衍生物的要求表明这些细胞之间的终身信号传导关系 类型我将采用永久性谱系追踪方法来确定HSC特异性和对照成人是否 HSC动员需要相同类型的NC衍生物。本提案的辅导阶段将在 圣威尔逊·克莱门茨（Wilson Clements）博士领导下的裘德儿童研究医院（Jude Children's Research Hospital）将确认HSC的候选监管机构 本发明的目的在于说明并集中于产生用于独立阶段的突变体/转基因斑马鱼系。的 独立的阶段将阐明新的调节剂指导HSC特化的分子机制 以及NC衍生物的永久谱系追踪。机构资源和学术环境， 我的建议中概述的计划课程将确保我成功过渡到独立。

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.