Regulation of zebrafish hematopoiesis by the sinusoidal endothelial cell niche

项目摘要/摘要 造血微环境由许多调节造血干的小生境组成。 细胞(HSC)在健康和应激条件下发挥作用。肝窦内皮细胞构成了一个利基 是骨髓毒性化疗后HSC恢复和造血干细胞植入所必需的 移植(HSCT)。尾端造血区内有一内皮细胞巢，呈正弦状。 斑马鱼(Danio Rerio)发育中的领地(CHT)，在那里它支持HSC的生存和扩张 造血转移到成人的肾脏骨髓。这项提议的首要目标是确定新的因素 对于CHT内HSC-内皮细胞的相互作用以及了解这些相互作用是如何进行的 影响HSC扩增、HSC克隆多样性和生态位本身的生物学。第一个目标是解决如何 造血干细胞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 表达有利于HSC保留、存活和增殖的分子。在功能增益实验中， CXCL8将利用特定的基因启动子在HSC和其他血统中高水平表达，这些基因启动子 已被鉴定和克隆。基于内皮细胞特异性CRISPR的系统将用于 敲除可能的CXCL8受体CXCR1和CXCR2，以了解CXCR8受体的特异性 红隧内的CXCL8。HSC在CHT内的植入、有丝分裂速率和细胞-细胞相互作用以及变化 生态位本身的大小和功能将在功能增益和功能损失实验中使用 共焦显微镜和数字图像分析。第二个目标是确定新的细胞外 特异性介导CHT内HSC-内皮细胞相互作用的信号因子及其确定方式 生态位通过这些因素影响HSC的克隆性。肝星状细胞与血管内皮细胞的相互作用 将使用紫外线照射来识别和永久标记CHT中的可光转化蛋白质。单人 将对光转化细胞进行分类，并针对HSCs和内皮细胞相互作用的基因表达谱进行分析 细胞将通过RNA测序并与非相互作用的基因表达谱进行比较而产生， 控制单元。肝星状细胞-血管内皮细胞相互作用的特定因子的功能作用将通过 如第一个目标中所述的功能增益和损耗实验。此外，这些因素在 调节HSC池的克隆多样性将在获得和失去功能的实验中使用 一种光学条形码方法。这些研究有可能确定改进的新治疗靶点。 血液病患者和恢复期患者HSC数量和克隆多样性的研究 化疗或造血干细胞移植。

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.