Molecular and functional dissection of zebrafish hematopoietic stem cell niche

斑马鱼造血干细胞生态位的分子和功能解剖

• 批准号: 8010760
• 负责人: David Traver
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-18 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010760
• 关键词: Adult; Aorta; Behavior; Binding; Biological Models; Birth; Blood; Blood Cells; Candidate Disease Gene; Cell Lineage; Cell physiology; Cells; Complement; Daughter; Development; Dissection; Embryo; Emigrations; Environment; Event; Fetus; Flow Cytometry; Fluorescent in Situ Hybridization; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genetic; Gonadal structure; Growth; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Image; Immigration; In Vitro; Individual; Kidney; Label; Lasers; Life; Maps; Marrow; Mesonephric structure; Microscopy; Molecular; Molecular Profiling; Patients; Pattern; Population; Pronephric structure; Regulation; Research Personnel; Route; Signal Transduction; Stem Cell Development; Stem cells; System; Techniques; Technology; Testing; Time; Tissues; Transgenes; Transgenic Animals; Transgenic Organisms; Translating; Translations; Transplantation; Travel; Zebrafish; gene therapy; human embryonic stem cell; in utero; in vivo; insight; migration; mutant; novel; programs; prospective; research study; self-renewal; spatiotemporal; stem cell fate specification; stem cell niche; stem cell population

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) exist as rare populations within blood-forming tissues that both self-renew and generate all blood cell lineages for life. HSCs are born in the embryo, but relatively little is known regarding their ontogeny and early function. This is due, in large part, to these events occurring in utero in the mammalian fetus. In contrast, the zebrafish, with externally fertilized and transparent embryos, presents an ideal model system to observe directly and manipulate genetically vertebrate HSC formation and function. I have recently pioneered prospective isolation approaches, transplantation technology and in vivo imaging for the study of zebrafish HSCs. With fluorescent transgenes targeted to hematopoietic precursors, these approaches will now be used in combination for unprecedented study of the genesis of HSCs. We will perform multiplex fluorescent in situ hybridization (FISH) to dissect the molecular signatures of both HSCs and their microenvironmental niche. In addition to these static analyses, we will image the behavior of HSCs by confocal time lapse microscopy in living, multicolor transgenic animals to witness the birth of HSCs for the first time. Putative HSC subsets will be prospectively isolated by flow cytometry and tested functionally by transplantation into zebrafish blood mutants. Finally, a precise characterization of the molecular determinants and in vivo behavior of embryonic HSCs will provide the framework to systematically dissect the mechanisms of HSC support by the niche. Insight into the genetic regulation of embryonic HSC function will be translated to the adult hematopoietic system. Novel findings regarding the environmental interactions of HSCs will ultimately be examined for conservation in mammalian systems. The translation of the developmental mechanisms required to maintain and instruct stem cell function has great potential in both the in vitro generation of HSCs from human ES cells and in ex vivo manipulation of patient HSCs for cell and gene therapy.

描述（由申请人提供）：造血干细胞（HSC）作为造血组织中的罕见群体存在，它们既能自我更新，又能产生生命所需的所有血细胞谱系。HSC在胚胎中出生，但对其个体发育和早期功能知之甚少。这在很大程度上是由于哺乳动物胎儿在子宫内发生的这些事件。相反，斑马鱼，外部受精和透明的胚胎，提出了一个理想的模型系统，直接观察和遗传操纵脊椎动物HSC的形成和功能。我最近开创了斑马鱼HSC研究的前瞻性分离方法，移植技术和体内成像。随着荧光转基因靶向造血前体，这些方法现在将结合使用前所未有的研究造血干细胞的发生。我们将进行多重荧光原位杂交（FISH）来剖析HSC及其微环境生态位的分子特征。除了这些静态分析外，我们还将通过共聚焦延时显微镜在活体转基因动物中对HSC的行为进行成像，以首次见证HSC的诞生。将通过流式细胞术前瞻性分离推定的HSC亚群，并通过移植到斑马鱼血液突变体中进行功能测试。最后，一个精确的表征胚胎HSC的分子决定因素和在体内的行为将提供一个框架，系统地剖析HSC的支持机制的生态位。对胚胎HSC功能的遗传调控的深入了解将被翻译到成人造血系统。关于HSC与环境相互作用的新发现最终将在哺乳动物系统中进行保护研究。维持和指导干细胞功能所需的发育机制的翻译在从人ES细胞体外生成HSC和用于细胞和基因治疗的患者HSC的离体操作中具有巨大的潜力。