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.
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.
