Mechanisms of Flow-driven Transcriptional Control of Hematopoietic Stem Cell Development by YAP

项目摘要/摘要 造血干细胞(HSCs)能够产生所有的红系、髓系和淋巴系血液 有机体的细胞。再加上自身独特的自我更新能力，成功移植健康 造血干细胞是目前唯一可以完全替代和恢复患者血液系统的治疗方法。 患有白血病和淋巴瘤。尽管有这种需要，但目前还不能有效地在体外创造或培养造血干细胞， 这表明支持它们在体内生长和发育的外在因素从现有的 协议。我们实验室以前的工作表明，血液流动是一种必不可少的非遗传环境 脊椎动物胚胎中产生HSC所需的信号，部分通过刺激HSC的机械激活而介导 YAP转录因子(Tf)。这项建议意在解决身体上、基因上 以及机械激活、YAP驱动的HSC产生的分子机制。YAP，虽然是一个强有力的 基因表达的共激活因子，本身缺乏与DNA结合的能力。了解背后的分子逻辑 FLOW/YAP驱动的造血，第一个目标是利用化学、物理和遗传扰动 活体斑马鱼胚胎中剪切应力和循环拉伸的变化，以评估这些单独成分的影响 血液动力对血液内皮细胞(HE)产生HSC的影响。在这上面还会加一些组织- 野生型斑马鱼分选HE的特异转录和全基因组YAP/DNA相互作用图谱， 血流缺陷和YAP-/-动物(血流正常)以区分依赖血流的基因调控 依赖于YAP的模块和转录靶点。假设驱动的候选TF将在体内进行测试，并 体外评估YAP相互作用能力并发现正常YAP依赖所需的关键伙伴 造血术。在第二个目标中，斑马鱼系统将被用于研究候选膜- 局部蛋白质，Piezos和整合素，作为连接血流动力和YAP激活的组件。这些 研究将为血流如何激活YAP提供一个全面的“从膜到核”的范例 指导发育中的造血，这可能会改善目前生成或扩增造血干细胞的努力。 作为博士后研究员，萨格登博士将在特里斯塔·诺斯博士的实验室进行他的研究 波士顿儿童医院。她在发育造血的外在调节方面的专业知识，以及 由乔治·戴利博士(干细胞生物学和血液学专家)和一位强大的 顾问团队为职业发展和研究提供了一个特别好的支持环境 训练。萨格登博士将在发育遗传学和活体成像方面建立坚实的背景，通过增加新的 转录因子/DNA相互作用图谱、转录组学和蛋白质体外研究方法的技术 互动。严格的研究和培训计划为成功奠定了基础，无论是在被指导的还是在 奖项的独立阶段。波士顿儿童医院和哈佛医学院的环境 将为萨格登博士成为一名成功的独立科学家提供理想的环境。

Project Summary/Abstract Hematopoietic stem cells (HSCs) are capable of producing all erythroid, myeloid and lymphoid blood cells of an organism. Coupled with their unique capacity for self-renewal, successful transplantation of healthy HSCs is the only therapy currently available that can completely replace and restore the blood system in patients with leukemia and lymphoma. Despite this need, HSCs presently cannot be efficiently created or cultured in vitro, suggesting that extrinsic factors supporting their growth and development in vivo are lacking from existing protocols. Previous work from our lab demonstrated that blood flow is an essential non-genetic environmental cue required for HSC production in vertebrate embryos, mediated in part by stimulating mechanical activation of the Yes-associated protein (YAP) transcription factor (TF). This proposal intends to resolve the physical, genetic and molecular mechanisms underlying mechanically-activated, YAP-driven HSC production. YAP, while a potent co-activator of gene expression, lacks DNA-binding ability of its own. To understand the molecular logic behind flow/YAP-driven hematopoiesis, the goal of the first aim is to employ chemical, physical and genetic perturbation of shear stress and cyclic stretch in live zebrafish embryos to assess the impact of these individual components of hemodynamic force on HSC production from hemogenic endothelium (HE). To this will be added tissue- specific transcriptomic and genome-wide YAP/DNA interaction profiling from sorted HE from wildtype zebrafish, flow-deficient and yap-/- animals (with normal blood flow) in order to discriminate flow-dependent gene regulatory modules and transcriptional targets that rely on YAP. Hypothesis-driven candidate TFs will be tested in vivo and in vitro to evaluate YAP-interaction ability and uncover key partners required for normal YAP-dependent hematopoiesis. In the second aim, the zebrafish system will be used to investigate candidate membrane- localized proteins, Piezos and Integrins, as components linking hemodynamic forces with YAP activation. These studies stand to provide a comprehensive “membrane-to-nucleus” paradigm for how blood flow activates YAP to guide developmental hematopoiesis, which may improve current efforts to generate or expand HSCs. As a postdoctoral fellow, Dr. Sugden will conduct his research in the laboratory of Dr. Trista North at Boston Children's Hospital. Her expertise in extrinsic regulation of developmental hematopoiesis, together with dedicated co-mentorship by Dr. George Daley (an expert in stem cell biology and hematology) and a strong advisory team provide an exceptionally well-supported environment for career development and research training. Dr. Sugden will build on a solid background in developmental genetics and live-imaging, by adding new technologies in transcription factor/DNA interaction profiling, transcriptomics and in vitro methods to study protein interactions. A rigorous research and training plan lay the groundwork for success, both in the mentored and independent phases of the award. The environment at Boston Children's Hospital and Harvard Medical School will provide the ideal surroundings to support Dr. Sugden to become a successful independent scientist.