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

项目总结/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 一个有机体的细胞。再加上它们独特的自我更新能力，成功移植健康的 HSC是目前唯一可以完全替代和恢复患者血液系统的疗法 白血病和淋巴瘤尽管有这种需要，但目前不能有效地在体外产生或培养HSC， 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，血流是一个重要的非遗传环境， 在脊椎动物胚胎中HSC产生所需的提示，部分通过刺激机械激活 Yes相关蛋白（雅普）转录因子（TF）。这项建议旨在解决物理，遗传 以及机械活化的YAP驱动的HSC产生的分子机制。雅普，虽然一个强大的 基因表达的辅激活因子，缺乏自身的DNA结合能力。去理解背后的分子逻辑 流动/YAP驱动的造血，第一个目标的目标是采用化学，物理和遗传扰动 的剪切应力和周期性拉伸活斑马鱼胚胎，以评估这些单独的组件的影响 血流动力学对造血内皮细胞（HE）生成HSC的影响。再加上组织- 来自野生型斑马鱼的分选HE的特异性转录组和全基因组雅普/DNA相互作用谱， 流动缺陷和雅普-/-动物（具有正常血流），以区分流动依赖性基因调节 依赖于雅普的模块和转录靶。假设驱动的候选TF将在体内进行测试， 在体外评估YAP相互作用能力，并揭示正常YAP依赖性 造血在第二个目标中，斑马鱼系统将用于研究候选膜- 局部蛋白质，Piezos和整合素，作为连接血液动力学力与雅普激活的组分。这些 这些研究为血流如何激活雅普提供了一个全面的“膜-核”范例 指导造血发育，这可能会改善目前的努力，以产生或扩大造血干细胞。 作为博士后研究员，Sugden博士将在Trista North博士的实验室进行研究， 波士顿儿童医院她在发育性造血的外在调节方面的专长，以及 专门的共同指导博士乔治戴利（在干细胞生物学和血液学专家）和一个强大的 顾问团队为职业发展和研究提供了非常良好的支持环境 训练Sugden博士将在发育遗传学和活体成像方面的坚实背景基础上， 转录因子/DNA相互作用谱分析、转录组学和研究蛋白质的体外方法 交互.一个严格的研究和培训计划奠定了成功的基础，无论是在指导和 独立的奖项。波士顿儿童医院和哈佛医学院的环境 将提供理想的环境，以支持萨格登博士成为一个成功的独立科学家。

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.