Developing a competitive hematopoietic repopulating assay in zebrafish

项目概述/摘要：造血干细胞（hsc）精确调节自我更新和分化之间的平衡，以产生生物体所需的适当数量的成熟血细胞。当指导这些决定的基因失调时，不受控制的自我更新会导致造血恶性肿瘤。表征调节造血干细胞功能的决策过程对于理解白血病的发生以及增强用于治疗许多人类癌症和血液疾病的造血干细胞移植策略至关重要。我的实验室正在利用斑马鱼动物模型来发现造血干细胞的新基因调控因子。我开发了一种免疫匹配的转基因斑马鱼模型，以复制克隆CG2斑马鱼竞争性再种群的小鼠模型。为了比较突变型斑马鱼hsc与野生型hsc的相对移植潜能，将具有荧光色阳性骨髓细胞的转基因CG2:ubiquitin-GFP和CG2:ubiquitin-DsRedE2供体鱼移植到荧光色阴性克隆受体中，实现长期、多系移植。对胚胎HSC发育异常的斑马鱼突变体进行初步中试筛选，发现两个单倍体不足突变体在亚致死辐射损伤后骨髓造血前体细胞恢复受损。磷脂酶C γ - 1 (plc？1)和组蛋白去乙酰化酶1 （hdac1）先前已被确定在自我更新中起作用，我们的数据表明它们可能对调节HSC稳态很重要。我将通过验证PLC？1和HDAC1单倍体功能不全会导致造血移植受损。此外，使用克隆斑马鱼供体的随机插入诱变，我将进行无偏见的正向诱变筛选，以确定改变HSC植入的基因。来自杂合和/或纯合突变成人的骨髓将用于我的竞争性造血再填充试验，以确定那些骨髓再填充偏向竞争性供体之一的突变体。我相信这项检测将识别出对造血移植多个方面重要的已知和未知基因。这项工作将产生突变斑马鱼，作为工具来解剖决定HSC功能的复杂调控网络。表征这些基因将加速我们对体内和移植后正常HSC功能的理解，并为引起白血病的机制提供新的见解。

DESCRIPTION (provided by applicant): Developing a competitive hematopoietic repopulating assay in zebrafish Project Summary/Abstract Hematopoietic stem cells (HSCs) precisely regulate the balance between self-renewal and differentiation to generate appropriate numbers of mature blood cells required by an organism. When genes that direct these decisions are dysregulated, unchecked self-renewal can result in hematopoietic malignancy. Characterizing the decision-making processes regulating HSC function is critically important to understand leukemogenesis and also to enhance HSC transplantation strategies used to treat many human cancers and blood diseases. My laboratory is capitalizing on the zebrafish animal model to uncover new genetic regulators of HSCs. I have developed an immune- matched transgenic zebrafish model to phenocopy murine models of competitive repopulation using clonal CG2 zebrafish. To compare the relative engraftment potential of mutant zebrafish HSCs with wild type HSCs, transgenic CG2:ubiquitin-GFP and CG2:ubiquitin-DsRedE2 donor fish with fluorochrome positive marrow cells are transplanted into fluorochrome negative clonal recipients, resulting in long term, multi-lineage engraftment. A preliminary pilot screen of zebrafish mutants with abnormal embryonic HSC development identified two haploinsufficient mutants with impaired recovery of hematopoietic precursor cells in the marrow after sublethal radiation injury. Both phospholipase C gamma1 (plc?1) and histone deacetylase 1 (hdac1) have previously been identified as playing a role in self-renewal, and our data suggest they may be important for regulating HSC homeostasis. I will characterize and refine our competitive hematopoietic repopulating assay by testing the hypothesis that PLC?1 and HDAC1 haploinsufficiency will result in impaired hematopoietic engraftment. In addition, using random insertional mutagenesis of clonal zebrafish donors, I will perform an unbiased forward mutagenesis screen to identify genes that alter HSC engraftment. Marrow from heterozygous and/or homozygous mutant adults will be used in my competitive hematopoietic repopulating assay to identify those mutants with skewed marrow repopulation favoring one of the competitive donors. I am confident this assay will identify known and unknown genes important for multiple aspects of hematopoietic engraftment. This work will generate mutant zebrafish to use as tools to dissect the complex regulatory network that determines HSC function. Characterizing these genes will accelerate our understanding of normal HSC function in vivo and after transplantation, as well as providing new insights into the mechanisms that cause leukemia.