Regulation of zebrafish hematopoiesis by the sinusoidal endothelial cell niche

窦状内皮细胞生态位对斑马鱼造血的调节

• 批准号: 9926235
• 负责人: Bradley Wayne Blaser
• 金额: $ 15.08万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926235
• 关键词: Address; Adult; Affect; Bar Codes; Behavior; Biology; Blood; Blood Cells; Blood Circulation; Bone Marrow; CXCL12 gene; Cell Communication; Cell Count; Cell Survival; Cell division; Cell physiology; Cells; Chronic; Clonal Hematopoietic Stem Cell; Clonality; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Confocal Microscopy; Data; Development; Drug Targeting; Embryonic Development; Endothelial Cells; Engraftment; Event; Experimental Models; Exposure to; Expression Profiling; Failure; Gene Expression; Genes; Goals; Growth; Health; Hematologic Neoplasms; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; IL8 gene; IL8RA gene; IL8RB gene; Image Analysis; KDR gene; Kidney; Knock-out; Label; Laboratories; Marrow; Mediating; Mitosis; Mitotic; Modeling; Molecular; Optics; Organism; Patient Care; Patients; Physiological; Proteins; Recovery; Regulation; Residencies; Risk Factors; Role; Safety; Shapes; Signal Pathway; Signal Transduction; Site; Specificity; Stress; Stromal Cell-Derived Factor 1; Supporting Cell; System; Techniques; Testing; Tissue-Specific Gene Expression; VEGFA gene; WNT5A gene; Work; Zebrafish; base; cell type; chemotherapy; cytokine; digital imaging; endothelial stem cell; experimental study; extracellular; gain of function; hematopoietic stem cell expansion; hematopoietic stem cell quiescence; hematopoietic stem cell self-renewal; improved; loss of function; microscopic imaging; new therapeutic target; novel; novel therapeutics; overexpression; preservation; promoter; receptor; response; self-renewal; single cell sequencing; single-cell RNA sequencing; stem cell biology; transcriptome sequencing; ultraviolet irradiation

Project Summary/Abstract The hematopoietic microenvironment is made up of a number of niches that regulate hematopoietic stem cell (HSC) function in health and under conditions of stress. Sinusoidal endothelial cells comprise a niche that is necessary for HSC recovery after myelotoxic chemotherapy and engraftment after hematopoietic stem cell transplantation (HSCT). A sinusoidal endothelial cell niche is transiently present in the caudal hematopoietic territory (CHT) of the developing zebrafish (Danio rerio), where it supports HSC survival and expansion before hematopoiesis shifts to the adult kidney marrow. The overarching goal of this proposal is to identify new factors that are important for HSC-endothelial cell interactions within the CHT and to understand how these interactions affect HSC expansion, HSC clonal diversity and the biology of the niche itself. The first aim addresses how HSCs and other blood cells use the angiogenic cytokine, CXCL8, to stimulate the growth of the CHT and induce expression of molecules that favor HSC retention, survival and proliferation. In gain-of-function experiments, CXCL8 will be expressed at high levels in HSCs and other blood lineages using specific gene promoters which have been previously identified and cloned. An endothelial cell-specific CRISPR-based system will be used to knock out the putative CXCL8 receptors, CXCR1 and CXCR2, in order to understand the receptor specificity for CXCL8 within the CHT. HSC engraftment, mitotic rate, and cell-cell interactions within the CHT as well changes in the size and function of the niche itself will be quantified in the gain- and loss-of-function experiments using confocal microscopy and digital image analysis. The goal of the second aim is to identify new extracellular signaling factors that specifically mediate HSC-endothelial cell interactions within the CHT and to determine how the niche affects HSC clonality via these factors. Interacting HSCs and endothelial cells expressing a photoconvertible protein within the CHT will be identified and permanently labeled using UV irradiation. Single photoconverted cells will be sorted and gene expression profiles specific for interacting HSCs and endothelial cells will be generated by RNA sequencing and comparing to gene expression profiles from non-interacting, control cells. The functional role of factors specific to the HSC-endothelial cell interaction will be tested using gain- and loss-of-function experiments as described in the first aim. In addition, the role of these factors in regulating the clonal diversity of the HSC pool will be assessed in gain- and loss-of-function experiments using an optical barcoding approach. These studies have the potential to identify new therapeutic targets for improving HSC numbers and clonal diversity in patients with blood disorders and those who are recovering from chemotherapy or HSCT.

项目摘要/摘要 造血微环境由许多调节造血茎的细分市场组成 细胞（HSC）在健康状况和压力条件下的功能。正弦内皮细胞包括一个小众 对于造血干细胞后骨髓毒性化疗和植入后，HSC恢复是必需的 移植（HSCT）。正弦内皮细胞生态位瞬时存在于尾造血 发展中国家（Danio Rerio）的领土（CHT），它支持HSC生存和扩张之前 造血转移到成年肾脏骨髓。该提案的总体目标是确定新因素 对于CHT内的HSC - 内层细胞相互作用和了解这些相互作用的重要性很重要 影响HSC扩展，HSC克隆多样性和利基本身的生物学。第一个目的解决了如何 HSC和其他血细胞使用血管生成细胞因子CXCL8刺激CHT的生长并诱导 有利于HSC保留，存活和增殖的分子的表达。在功能收益实验中， CXCL8将使用特定的基因启动子在HSC和其他血统中以高水平表达 以前已被识别和克隆。内皮细胞特异性CRISPR的系统将用于 删除推定的CXCL8受体CXCR1和CXCR2，以了解受体特异性 CXCL8在CHT中。 HSC植入，有丝分裂率和CHT内的细胞 - 细胞相互作用也会改变 利基本身的大小和功能将在使用功能丧失实验中进行量化 共聚焦显微镜和数字图像分析。第二个目标的目标是确定新的细胞外 信号传导因素专门介导CHT内的HSC - 内层细胞相互作用，并确定如何 利基市场通过这些因素影响HSC克隆性。相互作用的HSC和表达的内皮细胞 CHT内的光旋转蛋白将被鉴定并使用UV辐照永久标记。单身的 光转向细胞将进行分类，并针对相互作用的HSC和内皮特异 细胞将通过RNA测序和与非相互作用的基因表达谱进行比较，从而产生细胞。 控制细胞。特定于HSC - 内皮细胞相互作用的因素的功能作用将使用 第一个目标中所述的功能丧失实验。另外，这些因素在 调节HSC池的克隆多样性将在使用功能丧失实验中评估 一种光条形码方法。这些研究有可能确定改进的新治疗靶标 血液疾病患者的HSC数量和克隆多样性以及正在从 化学疗法或HSCT。