Identification of Regulators of Hematopoietic Stem Cells Using Zebrafish Genetics

描述(申请人提供)：候选人概述：我在造血干细胞生物学方面的研究背景，加上我在斑马鱼遗传学和造血学方面的发展专长，为我提供了进行拟议研究的知识。结合研究计划，完成这项拨款中提出的职业发展计划对我实现我的最终目标至关重要，即领导一个独立的研究小组，在其内源性微环境中研究造血干细胞。 研究描述：为了应对血液系统的急性损伤，造血干细胞(HSC)被刺激增殖和分化，以再生受损的血液系统。如果不加以控制，不受控制的扩散可能会导致白血病疾病状态。更深入地了解成人HSC的体内调控将有助于改进各种血液和癌症疾病的治疗。人们对这一过程背后的遗传因素知之甚少。在亚致死剂量照射后，首先返回的血细胞群体是多系前体细胞(MLP)，因此我们利用MLP恢复的速度和程度作为造血干/祖细胞(HSPC)激活的替代表型。照射后短暂刺激Wnt信号通路可增强HSPC的激活。Wnt信号通路参与了正常和恶性造血的发生，但Wnt在HSC体内激活的条件和机制仍存在争议。为了发现Wnt介导的HSPC激活的修饰因子，我们采用了候选和筛选的方法。通过相互作用研究，我们确定了BMP和Wnt信号通路之间的协同关系，以促进HSPC的激活。同时，我们实施了一种正向遗传筛选来识别成人HSPC激活的新调节因子。我们在成年斑马鱼中进行了敏化的F1显性增强子筛选，以寻找促进Wnt介导的HSPC扩张的因素。对600多条鱼进行了筛选，初步确定了19个潜在的突变体，其中4个已经确认了遗传特征。在进一步对突变体进行表型鉴定之前，我们将通过定位克隆来鉴定受影响的基因。由于体内HSPC激活分析不能直接解决Wnt或BMP刺激后或在新发现的基因突变中HSC扩增的自主性，需要进行额外的实验。为了解决这个问题，在目标1和目标2中，我们将首先利用定量的造血细胞移植试验来描述对长期HSC和更成熟的多系祖细胞的影响。其次，我们将检测具有突变血细胞或突变壁龛细胞的嵌合动物中HSPC的激活，以确定HSC现象的内在或外在性质。最后，我们将阐明突变体中改变的成分与Wnt和BMP信号通路之间的潜在相互作用。这些研究将揭示体内微调调节HSC增殖的途径之间的联系。在临床环境中利用这一知识可以改进各种血液和癌症疾病的治疗方法。拟议研究的完成将导致开发一种系统，通过基因筛选发现成人生态位内特定的HSC调节因子，通过对受影响基因的定位克隆识别因子，并将这些因子放置在已知信号通路的背景下。在未来，这一策略可以应用于HSC生物学的其他方面。 公共卫生相关性：对造血干细胞(HSC)体内调控的更深入了解将有助于改进各种血液和癌症疾病的治疗。新型调节剂的发现将拓宽可能的治疗靶点。我将利用斑马鱼遗传学来发现影响HSC扩张的新因素。

DESCRIPTION (provided by applicant): Candidate Summary: My research background in hematopoietic stem biology plus my developing expertise in zebrafish genetics and hematopoiesis provide me with the knowledge to perform the proposed research. Combined with the research plan, completion of the career development plan put forward in this grant is essential for me to reach my ultimate goal of directing an independent research group studying hematopoietic stem cells within their endogenous microenvironments. Research Description: In response to acute injuries to the blood system, hematopoietic stem cells (HSC) are stimulated to proliferate and differentiate to regenerate the damaged blood system. If left unchecked, uncontrolled proliferation could result in a leukemic disease state. Greater insight into the in vivo regulation of adult HSC will lead to improved treatment for a variety of hematologic and cancer disorders. The genetic factors underlying this process are poorly understood. Following sublethal irradiation, the first population of blood cells to return is the multilineage precursors (MLP), thus we utilized the rate and extent of MLP recovery as a surrogate phenotype for the activation of hematopoietic stem and progenitor cell (HSPC). Transient stimulation of the Wnt signaling pathway after irradiation led to an enhanced HSPC activation. The Wnt signaling pathway has been implicated in both normal and malignant hematopoiesis, but the there is still controversy surrounding the in vivo requirement and mechanism of Wnt activation in HSC. To uncover modifiers of Wnt-mediated HSPC activation, we employed candidate and screening approaches. Through interaction studies, we defined a synergistic relationship between the BMP and Wnt signaling pathways to increase HSPC activation. In parallel, we implemented a forward genetic screen to identify novel regulators of adult HSPC activation. We performed a sensitized F1 dominant enhancer screen in adult zebrafish for factors that enhance Wnt-mediated HSPC expansion. Over 600 fish were screened, 19 potential mutants were initially identified, and 4 of these have confirmed inherited traits. Prior to further phenotypic characterization in the mutants, we will identify the affected genes by positional cloning. As In vivo HSPC activation assays do not directly address the HSC autonomy of the expansions following Wnt or BMP stimulation or in the newly identified genetic mutants, additional experiments need to be performed. To resolve this question, in both Aims 1 and 2, we will first utilize quantitative hematopoietic cell transplantation assays to delineate effects on long-term HSC versus more mature multilineage progenitors. Secondly, we will examine HSPC activation in chimeric animals that have either mutant blood cells or mutant niche cells to determine the HSC intrinsic or extrinsic nature of the phenomena. Finally, we will elucidate the potential interplay between the altered components in the mutants and the Wnt and BMP signaling pathways. These studies will reveal the connections among pathways during the fine-tuned in vivo regulation of HSC proliferation. Exploitation of this knowledge in the clinical setting can improve therapies for the treatment of a variety of hematologic and cancer disorders. Completion of the proposed research will result in the development of a system to discover regulators specific for HSC within the adult niche through genetic screens, the identification of the factors by positional cloning of the affected genes, and placement of these factors within the context of known signaling pathways. In the future, this strategy can be applied to other aspects of HSC biology. PUBLIC HEALTH RELEVANCE: Greater insight into the in vivo regulation of hematopoietic stem cells (HSC) will lead to improved treatment for a variety of hematologic and cancer disorders. Identification of novel regulators will broaden possible therapeutic targets. I will use zebrafish genetics to discover new factors modifying HSC expansion.