Clonal dynamics of the blood stem cell niche

项目总结 克隆影响造血干/祖细胞生态位的机制 血液学疾病，如骨髓增生异常综合征(MDS)，仍然知之甚少。此外， HSPC生态位的主要成分内皮细胞和基质细胞的异质性和克隆反应 体内的MDS仍未被探索。为了解决这些方面的问题，我开发了一种新的斑马鱼模型，通过驱动 原癌基因cmyc在血细胞中高表达。此外，我还跨越了一个基因谱系 追踪携带两个荧光记者的双转基因斑马鱼品系格式塔 允许特定地提纯利基内皮细胞和基质细胞。这样，我创建了一条新的格式塔线条 允许在斑马鱼胚胎发育、成年骨髓纯化期间基于CRISPR-Cas9的条形码 壁龛细胞和通过测序恢复壁龛DNA条形码。结合这些新工具，我在 对斑马鱼进行条形码编码，并读出内皮细胞和基质细胞的克隆性和转录组。我发现 在MDS中，基质细胞的克隆选择性地扩张，内皮细胞在转录上重塑。 鉴于这些数据，我假设MDS重塑了HSPC的克隆性和转录特征 以及HSPC-生态位相互作用中涉及的机制促进疾病进展。在……下面 在Leonard Zon博士的指导下，我将调查MDS通过使用 结合电子计算方法，遗传(格式塔)和基于颜色(斑马)的血统 示踪、共聚焦显微镜和体内镶嵌诱变。一旦我建立了我的实验室，我将建造一个 多学科团队，深化我的计算分析，拓宽我的体内遗传和生化 MDS中涉及生态位的克隆机制的扰动。我的首要目标是识别小说 防止和/或阻止MDS进展的针对HSPC利基的有针对性的机制。这 K99/R00奖将使我能够发展新的技术技能，参加将提高我的能力的课程 管理一个实验室，参加会议，这将扩大我的人脉和我的知识 血液学疾病建模、斑马弓血统追踪与共聚焦显微镜和斑马鱼配对 诱变。我组建的科学咨询委员会包括以下领域的专家 造血学、血统追踪和干细胞生物学，并将与Zon博士一起，给我关于我的 研究和事业的进步。这些拟议的研究和职业发展活动将为 我将成为一名独立调查员，发现和研究负责 造血系统疾病的进展由血液干细胞区介导。

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.