Clonal dynamics of the blood stem cell niche

项目摘要 造血干细胞和祖细胞（HSPC）生态位受克隆性造血干细胞和祖细胞（HSPC）影响的机制 血液疾病如骨髓增生异常综合征（MDS）仍然知之甚少。而且 内皮细胞和基质细胞（HSPC生态位的主要成分）的异质性和克隆反应， 体内MDS仍未探索。为了解决这些问题，我开发了一个新的斑马鱼模型的MDS驱动 原癌基因CMYC在血细胞中特异性过表达。另外，我的基因 追踪名为GESTALT的斑马鱼品系到携带两个荧光报告基因的双转基因斑马鱼品系 允许特异性纯化小生境内皮细胞和基质细胞。这样，我创建了一个新的GESTALT系列， 在斑马鱼胚胎发育期间允许基于CRISPR-CAS9的条形码，纯化成体骨髓 小生境细胞和通过测序回收小生境DNA条形码。结合这些新的工具，我诱导MDS， 对斑马鱼进行条形码化，并读出内皮细胞和基质细胞的克隆性和转录组。我发现 骨髓增生异常综合症中，基质细胞克隆选择性扩增，内皮细胞发生转录重塑。 鉴于这些数据，我假设MDS重塑了HSPC的克隆性和转录谱， 微生态位和HSPC-微生态位相互作用中涉及的机制促进疾病进展。下 在伦纳德Zon博士的指导下，我将研究MDS使用 计算机模拟计算方法的组合，遗传（GESTALT）和基于颜色（Zebrabow）的谱系 示踪、共聚焦显微镜和体内嵌合诱变。一旦我建立了我的实验室，我将建立一个 多学科团队，以深化我的计算分析，并扩大我的体内遗传和生化 MDS中的生态位参与的克隆机制的扰动。我的首要目标是找出 HSPC生态位特异性的靶向机制，其将预防和/或阻止MDS进展。这 K99/R 00奖将使我能够发展新的技术技能，参加课程，这将提高我的能力 管理一个实验室，并参加会议，这将扩大我的网络和我的知识， 血液病模型、斑马鱼谱系追踪与共聚焦显微镜配对和斑马鱼 诱变我召集的科学咨询委员会包括以下领域的专家： 造血，谱系追踪和干细胞生物学，并与Zon博士一起沿着，将给我关于我的 研究和职业发展。这些拟议的研究和职业发展活动将为以下方面铺平道路： 我将成为一名独立调查员，发现和研究负责 造血干细胞龛介导的造血疾病进展。

PROJECT SUMMARY The mechanisms by which the hematopoietic stem and progenitor (HSPC) niche is affected by clonal hematological disorders such as myelodysplastic syndrome (MDS) remain poorly understood. Furthermore, the heterogeneity and clonal response of endothelial and stromal cells (the main components of the HSPC niche) in MDS in vivo remain unexplored. To tackle these aspects, I developed a new zebrafish model of MDS by driving the protooncogene CMYC overexpression specifically in blood cells. Additionally, I crossed a genetic lineage tracing zebrafish line called GESTALT to a double transgenic zebrafish line carrying two fluorescent reporters allowing to purify specifically niche endothelial and stromal cells. This way, I created a new GESTALT line that permits CRISPR-CAS9 based barcoding during zebrafish embryonic development, purification of adult marrow niche cells and recovery of niche DNA barcodes by sequencing. Combining these novel tools, I induced MDS in barcoded zebrafish and read out the clonality and the transcriptome of endothelial and stromal cells. I discovered that clones of stromal cells selectively expand, and endothelial cells are transcriptionally remodeled in MDS. Given these data, I hypothesize that MDS remodels the clonality and transcriptional profile of the HSPC niche and that mechanisms involved in HSPC-niche interactions promote disease progression. Under the mentorship of Dr. Leonard Zon, I will investigate the mechanisms by which MDS remodels the niche using a combination of in silico computational approaches, genetic (GESTALT) and color based (Zebrabow) lineage tracing, confocal microscopy and in vivo mosaic mutagenesis. Once I establish my laboratory, I will build a multidisciplinary team to deepen my computational analyses and broaden my in vivo genetic and biochemical perturbations of the clonal mechanisms of niche involvement in MDS. My overarching goal is to identify novel targetable mechanisms specific to the HSPC niche that would prevent and/or halt MDS progression. This K99/R00 award will enable me to develop new technical skills, participate in courses that will improve my ability to manage a laboratory, and attend conferences that will broaden my network and my knowledge of hematological disease modeling, Zebrabow lineage tracing paired with confocal microscopy and zebrafish mutagenesis. The scientific advisory committee I have put together includes experts in the fields of hematopoiesis, lineage tracing, and stem cell biology and, along with Dr. Zon, will give me feedback on my research and career progress. These proposed research and career development activities will pave the way for me to become an independent investigator discovering and studying new mechanisms responsible for hematopoietic disorders progression mediated by the blood stem cell niche.