Identification of Regulators of Hematopoietic Stem Cells Using Zebrafish Genetics

候选人摘要 我在造血干细胞生物学方面的研究背景，以及我在斑马鱼方面的开发专长 遗传学和造血学为我提供了进行拟议研究的知识。 结合调研计划，完成本次提出的职业发展计划 格兰特对我实现领导一个独立研究小组的最终目标至关重要 在其内源性微环境中研究造血干细胞。 研究说明 为了应对血液系统的急性损伤，造血干细胞(HSC)被刺激以 增殖和分化，以再生受损的血液系统。如果不进行检查， 不受控制的增殖可能导致白血病疾病状态。对体内的更深入的了解 对成人HSC的调节将导致对各种血液病和癌症的改善治疗 精神错乱。人们对这一过程背后的遗传因素知之甚少。继亚致死之后 辐射后，第一批返回的血细胞是多系前体细胞(MLP)，因此我们 利用MLP回收率和程度作为激活的替代表型 造血干祖细胞(HSPC)。Wnt信号通路的瞬时刺激 照射后HSPC活性增强。Wnt信号通路一直是 与正常和恶性造血有关，但目前仍存在争议 围绕肝星状细胞中Wnt活化的体内要求和机制。揭开真相 对于Wnt介导的HSPC激活的修饰物，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之间的协同关系 增加HSPC激活的信号通路。同时，我们实现了一个正向遗传 筛选新的成人HSPC激活调节因子。我们做了一次敏化的F1 成年斑马鱼显性增强子筛选增强Wnt介导HSPC的因素 扩张。对600多条鱼进行了筛选，初步鉴定出19个潜在的突变体，其中4个 这些都证实了遗传特征。在突变体中进行进一步的表型鉴定之前， 我们将通过定位克隆来识别受影响的基因。在活体内HSPC激活检测不能 直接解决WNT或BMP刺激后或在 新发现的基因突变，还需要进行更多的实验。要解决这个问题 问题：在目标1和目标2中，我们将首先利用数量造血细胞移植 描述对长期HSC和更成熟的多系祖细胞的影响的试验。 其次，我们将检查具有突变血细胞的嵌合动物中HSPC的激活情况 或突变的利基细胞，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。这些研究将揭示不同途径之间的联系 体内微调期间对HSC增殖的调节。利用这一知识在 临床环境可以改进各种血液病和癌症的治疗方法 精神错乱。拟议研究的完成将导致开发一个系统，以 通过基因筛查，发现成人利基体内针对HSC的特定调节因子 通过定位克隆受影响的基因来识别因子，并放置这些因子 已知信号通路背景下的因素。未来，这一策略可以应用于 HSC生物学的其他方面。

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.