Investigating the Role for Mir-223 in Hematopoietic Stem Cell Emergence

研究 Mir-223 在造血干细胞出现中的作用

• 批准号: 9272268
• 负责人: Dionna Kasper
• 金额: $ 5.92万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-19 至 2019-04-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272268
• 关键词: 3' Untranslated Regions; Address; Adult; Animal Model; Aorta; Behavior; Binding; Binding Sites; Biological; Biological Assay; Biology; Blood Cells; Blood Vessels; Cardiovascular Physiology; Cardiovascular system; Cell Line; Cell Lineage; Cells; Data; Defect; Development; Dissection; Dorsal; Embryo; Endothelial Cells; Endothelium; Fertilization; Gene Targeting; Generations; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Hematological Disease; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Imagery; In Vitro; Knock-out; Knowledge; Maintenance; Methods; MicroRNAs; Modeling; Molecular; Mus; Optics; Pathway interactions; Pattern; Phenocopy; Population; Process; Production; Publishing; Regenerative Medicine; Regulation; Reporter; Reporting; Repression; Research; Role; Signal Pathway; Specific qualifier value; Stem cells; Testing; Therapeutic; Time; To specify; Transgenic Organisms; Transplantation; Untranslated RNA; Up-Regulation; Vertebrates; Work; Zebrafish; base; blood treatment; cell type; experimental study; granulocyte; in vivo; innovation; leukemia; leukemia treatment; leukemogenesis; monocyte; mutant; neutrophil; novel; overexpression; progenitor; promoter; public health relevance; reconstitution; regenerative therapy; reverse genetics; self-renewal; sensor; spatiotemporal; stem; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): All blood cell types that make up the adult cardiovascular system are created and maintained by self-renewing, multipotent hematopoietic stem/progenitor cells (HSPCs). Conserved across vertebrates, HSPCs are specified early in development, arising from a unique population of vascular cells called the hemogenic endothelium. Given the difficulties in the ex vivo differentiation and expansion of HSPCs from pluripotent stem cells for regenerative therapies, it is readily apparent that knowledge about HSPC specification is significantly lacking. This gap in understanding stems from the challenges in visualizing HSPC emergence in mammalian embryos that develop internally. The proposed study will address these biological problems with the zebrafish model because the transparency and external fertilization of zebrafish embryos will allow for the molecular dissection of the mechanisms that control this dynamic endothelial to hematopoietic transition (EHT) in vivo. Small, non-coding microRNAs (miRNAs) are key regulators of stem cell and cardiovascular biology because of their ability to post-transcriptionally silence a diverse array of target genes. While miRNA regulation is crucial for HSPC self-renewal, maintenance, and differentiation into multiple lineages, the role for miRNAs in hemogenic endothelial specification and HSC emergence is largely unexplored. From a reverse genetic screen to identify the cardiovascular function of endothelial-enriched miRNAs in zebrafish, mir-223 was identified as a novel candidate regulator of HSPC specification because the loss of mir-223 led to an expansion of HSPCs at the onset of HSPC induction. Thus, the goal of this proposal is to directly test the hypothesis that mir-223 functions to negatively regulate the transition from endothelial to hematopoietic cell fates. By employing an innovative strategy to create a mir-223 fluorescent reporter, mir-223 localization patterns will be assessed for the first time in vivo and correlated with the dynamic emergence of HSPCs from the hemogenic endothelium in zebrafish. The proposed research will also examine whether mir-223 functions in HSPC production by visualizing changes in hemogenic endothelial specification and cellular behaviors of emerging HSPCs when mir-223 activity is lost or elevated. Finally, the genes that are directly targeted by mir-223 during EHT will be identified by their upregulation in mir-223 mutants compared to wildtype hemogenic endothelial cells. Altogether, this proposed work will implicate miRNA regulation as a fundamental mechanism of HSPC specification, and will better define the genetic network regulating this process. Importantly, these studies will establish the zebrafish mir-223 mutant as a new leukemogenesis animal model, and will have the potential to instruct regenerative medicine approaches in the ex vivo production of HSPCs for the treatment of leukemia and other blood disorders.

 描述（由申请人提供）：构成成人心血管系统的所有血细胞类型均由自我更新的多能造血干/祖细胞（HSPC）产生和维持。在脊椎动物中保存，HSPC在发育早期被指定，由称为生血内皮的独特血管细胞群产生。鉴于HSPC从多能干细胞离体分化和扩增用于再生疗法的困难，显而易见的是，关于HSPC特化的知识显著缺乏。这种理解上的差距源于在内部发育的哺乳动物胚胎中可视化HSPC出现的挑战。拟议的研究将解决斑马鱼模型的这些生物学问题，因为斑马鱼胚胎的透明性和外部受精将允许对体内控制这种动态内皮向造血转变（EHT）的机制进行分子解剖。 小的非编码microRNA（miRNAs）是干细胞和心血管生物学的关键调节因子，因为它们能够转录后沉默多种靶基因。 虽然miRNA调控对于HSPC自我更新、维持和分化成多个谱系至关重要，但miRNA在生血内皮特化和HSC出现中的作用在很大程度上尚未探索。从反向遗传筛选以鉴定斑马鱼中内皮富集的miRNA的心血管功能，mir-223被鉴定为HSPC特化的新型候选调节剂，因为mir-223的缺失导致HSPC在HSPC诱导开始时扩增。因此，该提议的目标是直接测试mir-223的功能是负调节从内皮细胞到造血细胞命运的转变的假设。通过采用一种创新的策略来创建一个mir-223荧光报告，mir-223定位模式将首次在体内进行评估，并与斑马鱼生血内皮HSPC的动态出现相关。拟议的研究还将通过观察mir-223活性丧失或升高时新生HSPC的生血内皮特异性和细胞行为的变化来检查mir-223是否在HSPC产生中起作用。最后，在EHT期间由mir-223直接靶向的基因将通过与野生型生血内皮细胞相比其在mir-223突变体中的上调来鉴定。总而言之，这项拟议的工作将涉及miRNA调控作为HSPC特化的基本机制，并将更好地定义调控这一过程的遗传网络。重要的是，这些研究将建立斑马鱼mir-223突变体作为一种新的白血病发生动物模型，并将有可能指导再生医学方法在体外生产HSPC用于治疗白血病和其他血液疾病。