Dissection of zebrafish hematopoietic stem cell niche

描述(申请人提供)：造血干细胞(HSCs)在造血组织中作为稀有群体存在，既能自我更新，又能为生命生成所有血细胞谱系。造血干细胞是在胚胎中出生的，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这些事件发生在哺乳动物胎儿的子宫内。相比之下，斑马鱼拥有体外受精和透明的胚胎，为直接观察和操纵脊椎动物HSC的形成和功能提供了一个理想的模型系统。最近，我开创了用于斑马鱼HSCs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于造血祖细胞，这些方法现在将被联合使用，对造血干细胞的起源进行前所未有的研究。我们将进行多重荧光原位杂交(FISH)来分析这两种HSC及其微环境的分子特征。除了这些静态分析外，我们还将通过共聚焦时间推移显微镜在活的、多色转基因动物中成像HSCs的行为，以首次见证HSCs的诞生。可能的HSC亚群将通过流式细胞术进行分离，并通过移植到斑马鱼血液突变体中进行功能测试。最后，对胚胎HSC的分子决定因素和体内行为的精确表征将为系统地剖析利基支持HSC的机制提供框架。对胚胎HSC功能的遗传调控的洞察将转化为成人的造血系统。有关HSCs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。维持和指导干细胞功能所需的发育机制的翻译在从人类ES细胞体外生成HSCs以及在体外操纵患者HSCs进行细胞和基因治疗方面都具有巨大的潜力。

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.