Developing a competitive hematopoietic repopulating assay in zebrafish

描述(由申请人提供)：在斑马鱼项目摘要/摘要中开发一种竞争性的造血再填充试验造血干细胞(HSCs)精确地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，不加控制的自我更新可能会导致血液系统的恶性肿瘤。确定调节HSC功能的决策过程对于了解白血病的发生以及加强用于治疗许多人类癌症和血液疾病的HSC移植策略至关重要。我的实验室正在利用斑马鱼的动物模型来发现HSCs的新的遗传调控因素。我开发了一种免疫匹配的转基因斑马鱼模型，以克隆CG2斑马鱼为竞争种群的表型小鼠模型。为了比较突变斑马鱼HSCs与野生型HSCs的相对植入能力，将CG2：ubiquitin-GFP和CG2：ubiquitin-DsRedE2转基因供体鱼的荧光阳性骨髓细胞移植到荧光阴性克隆受体体内，实现了长期、多系植入。对胚胎HSC发育异常的斑马鱼突变体进行初步中试筛选，发现两个单倍体缺陷突变体在亚致死性辐射损伤后骨髓造血祖细胞恢复受损。磷脂酶C-Gamma1(plc？1)和组蛋白去乙酰基酶1(HDAC1)都被认为在自我更新中起作用，我们的数据表明它们可能对调节HSC的动态平衡起重要作用。我将通过测试PLC？1和HDAC1单倍体不足会导致造血植入受损的假设来表征和完善我们的竞争性造血再填充试验。此外，利用克隆斑马鱼供体的随机插入突变，我将进行无偏正向突变筛选，以确定改变HSC植入的基因。来自杂合子和/或纯合子突变成人的骨髓将用于我的竞争性造血再繁殖试验，以鉴定那些骨髓再繁殖倾斜的突变个体，这些突变有利于竞争性捐赠者之一。我相信，这项测试将识别对造血植入的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，作为工具来剖析决定HSC功能的复杂调控网络。确定这些基因的特征将加速我们对体内和移植后正常的HSC功能的理解，并为白血病的发病机制提供新的见解。

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.