Dissection of zebrafish hematopoietic stem cell niche

描述（由申请人提供）：造血干细胞（hsc）作为造血组织中的稀有群体存在，既能自我更新，又能产生生命所需的所有血细胞谱系。造血干细胞在胚胎中出生，但对其个体发生和早期功能的了解相对较少。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里。相比之下，斑马鱼具有外部受精和透明的胚胎，是直接观察和操纵遗传脊椎动物HSC形成和功能的理想模型系统。我最近开创了前瞻性分离方法，移植技术和斑马鱼造血干细胞的体内成像研究。随着针对造血前体的荧光转基因，这些方法现在将被用于对造血干细胞发生的前所未有的研究。我们将进行多重荧光原位杂交（FISH）来解剖造血干细胞及其微环境生态位的分子特征。除了这些静态分析外，我们将通过共聚焦时移显微镜在活的多色转基因动物中对造血干细胞的行为进行成像，首次见证造血干细胞的诞生。假定的HSC亚群将通过流式细胞术进行前瞻性分离，并通过移植到斑马鱼血液突变体中进行功能测试。最后，对胚胎造血干细胞的分子决定因素和体内行为的精确描述将为系统剖析生态位支持造血干细胞的机制提供框架。对胚胎造血干细胞功能的遗传调控的深入研究将被应用于成人造血系统。关于造血干细胞的环境相互作用的新发现将最终在哺乳动物系统中进行保护。翻译维持和指导干细胞功能所需的发育机制，无论是在体外从人类胚胎干细胞生成造血干细胞，还是在体外操作患者造血干细胞进行细胞和基因治疗，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.