CHANGE OF GRANTEE INSTITUTION - Deconstructing The Hematopoietic Stem Cell Niche

项目摘要/摘要 造血干细胞(HSC)生态位是一个专门的微环境，支持终身自我 造血干细胞的更新及其产生所有血细胞谱系的能力。许多不同的细胞类型和 分子因素与哺乳动物骨髓中的HSC生态位有关。然而，到目前为止， 直接对骨髓进行可视化的挑战排除了严格、系统地研究 促进造血干细胞植入的细胞间相互作用。斑马鱼提供了无与伦比的环境 HSCs与其支持的壁龛细胞之间的动态相互作用可以在实验上得到 以任何其他生物体都不可能达到的分辨率和吞吐量进行操作和直接可视化。这个 这项提议的首要目标是在斑马鱼胚胎中使用活细胞成像，结合新的 基因表达分析和组织特异性基因破坏技术，以阐明新的细胞和 干细胞在其生态位内植入所需的分子机制。第一个的目标 目的是集成多个空间和组织特定的基因表达数据集，以生成全面的 斑马鱼尾部造血组织(CHT)的基因表达图谱，这是一个类似于 哺乳动物的胎肝。由此得到的细胞特异性基因表达图谱将用于指导功能 CHT内移植HSC所需的细胞间相互作用的研究。这个基因 表达数据将被用来产生一个报告基因，专门标记CHT中的基质细胞。 此外，这些基因表达数据将与特定于组织的CRISPR系统结合使用，以 确定介导巨噬细胞-HSC相互作用的细胞黏附分子，以及HSC所必需的 在红隧进行嫁接。第二个目标是研究氧化应激对体内HSC生态位的影响。 红隧。这将通过用诱导氧化应激的化合物联合处理胚胎来实现。 使用活细胞染料，可以可视化活性氧物种(ROS)。此外，两个组件的功能 已知在维持低ROS水平中起作用的基因(sepp1a和nrros)将用损失- 功能和过度表达实验。这些研究可以确定新的治疗靶点，以增强 血液病移植治疗中造血干细胞的植入和维持。 作为博士后研究员，Hagedorn博士将在Leonard Zon博士的实验室进行他的研究 著名的血液学家和干细胞生物学家。在遗传和活细胞方面有很强的背景 Hagedorn博士将扩展他的专业知识，包括空间基因表达和 组织特异性基因破坏。在宗博士和一个杰出的指导委员会的指导下， Hagedorn构建了一项严格的研究和培训计划，这将使他在 以及奖项的独立阶段。波士顿儿童医院和哈佛医学院的环境 学校将为哈格多恩博士成为一名成功的独立科学家提供理想的环境。

Project Summary/Abstract The hematopoietic stem cell (HSC) niche is a specialized microenvironment that supports the life-long self- renewal of HSCs and their ability to produce all blood cell lineages. A number of different cell types and molecular factors have been associated with HSC niches in the mammalian bone marrow. To date, however, the challenges of directly visualizing the bone marrow have precluded a rigorous, systematic investigation of the cell-cell interactions that promote the niche engraftment of HSCs. Zebrafish offer an unparalleled setting in which the dynamic interactions between HSCs and their supporting niche cells can be experimentally manipulated and directly visualized at a resolution and throughput not possible in any other organism. The overarching goal of this proposal is to use live cell imaging in the zebrafish embryo, in combination with new technologies in gene expression analysis and tissue-specific gene disruption, to elucidate novel cellular and molecular mechanisms that are required for the engraftment of HSCs within their niche. The goal of the first aim is to integrate multiple spatial and tissue-specific gene expression datasets to generate a comprehensive map of gene expression within the zebrafish caudal hematopoietic tissue (CHT), a transient HSC niche akin to the mammalian fetal liver. The resulting map of cell-specific gene expression will be used to guide functional investigations of the cell-cell interactions that are required for HSC engraftment within the CHT. This gene expression data will be used to generate a reporter transgene that specifically labels stromal cells in the CHT. In addition, this gene expression data will be used, in combination with a tissue-specific CRISPR system, to identify the cellular adhesion molecules that mediate macrophage-HSC interactions and are required for HSC engraftment in the CHT. The second aim will investigate the effects of oxidative stress on the HSC niche within the CHT. This will be done by treating embryos with compounds that induce oxidative stress in combination with a live cell dye that permits visualization of reactive oxygen species (ROS). Additionally, the function of two genes (sepp1a and nrros), with known roles in maintaining low ROS levels, will be investigated using loss-of- function and overexpression experiments. These studies could identify novel therapeutic targets to enhance the engraftment and maintenance of HSCs during transplantation-based treatment of blood disorders. As a postdoctoral fellow, Dr. Hagedorn will conduct his research in the laboratory of Dr. Leonard Zon, a renowned hematologist and stem cell biologist. Building on a strong background in genetic and live cell imaging, Dr. Hagedorn will expand his expertise to include new technologies in spatial gene expression and tissue-specific gene disruption. Under the guidance of Dr. Zon and an exceptional mentoring committee, Dr. Hagedorn has constructed a rigorous research and training plan that will allow him to succeed in the mentored and independent phases of the award. The environment at Boston Children's Hospital and Harvard Medical School will provide the ideal surroundings for Dr. Hagedorn to become a successful independent scientist.