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

 描述(申请人提供)：构成成人心血管系统的所有血细胞类型都是由自我更新的多潜能造血干细胞/祖细胞(HSPC)创造和维持的。HSPC在脊椎动物中是保守的，是在发育早期特定的，起源于一种称为血源性内皮的独特的血管细胞群。鉴于从多能干细胞体外分化和扩增HSPC用于再生治疗的困难，很明显，关于HSPC规范的知识非常缺乏。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。这项拟议的研究将解决斑马鱼模型中的这些生物学问题，因为斑马鱼胚胎的透明度和体外受精将允许在体内分子解剖控制这种动态内皮到造血转变(EHT)的机制。小的、非编码的microRNAs(MiRNAs)是干细胞和心血管生物学的关键调节因子，因为它们能够在转录后沉默一系列不同的靶基因。 虽然miRNA调控对于HSPC的自我更新、维持和分化为多个谱系至关重要，但miRNAs在造血内皮细胞规范和HSC出现中的作用在很大程度上尚不清楚。通过反向遗传筛选鉴定斑马鱼富含内皮细胞的miRNAs的心血管功能，mir-223被确定为HSPC规范的一个新的候选调节子，因为mir-223的丢失导致HSPC在HSPC诱导开始时扩张。因此，这项提议的目标是直接测试这一假设，即mir-223功能是负向调节从内皮细胞到造血细胞命运的转变。通过采用一种创新的策略来创建mir-223荧光报告程序，将首次在体内评估mir-223定位模式，并将其与斑马鱼血液内皮细胞中HSPC的动态出现相关联。拟议的研究还将通过可视化mir-223活性丧失或升高时新出现的HSPC的血源性内皮规格和细胞行为的变化，来检查mir-223是否在HSPC的生产中发挥作用。最后，在EHT期间被mir-223直接靶向的基因将通过与野生型血液内皮细胞相比在mir-223突变体中上调来识别。总之，这项拟议的工作将暗示miRNA调控是HSPC规范的基本机制，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致病动物模型，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。

 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.