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Zebrafish Genetics to Identify Genes Involved in Hematopoiesis

斑马鱼遗传学鉴定参与造血的基因

基本信息

批准号：
8730626
负责人：
DHVANIT INDRAVADAN SHAH
金额：
$ 15.86万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-15 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8730626
关键词：
ATP phosphohydrolase Adult Affect Alleles Aminolevulinic Acid Anemia Animal Model Award Biochemical Biogenesis Biological Assay Biology Blood Blood Vessels Candidate Disease Gene Cell Count Cell Differentiation process Cells Child Chromosome Mapping Communities Complex Congenital Abnormality Data Defect Development Developmental Biology Dianisidine Diploidy Embryo Environment Erythrocytes Erythroid Erythroid Cells Erythropoiesis Erythropoietic Protoporphyria Exhibits Face Finding of Mean Corpuscular Hemoglobin Foundations Functional disorder Future Genes Genetic Genetic Markers Genetic Screening Genetic Transcription Globin Goals Haploidy Hematocrit procedure Hematological Disease Hematopoiesis Hematopoietic Heme Hemochromatosis Hemoglobin Human Human Genetics Human Genome Hypochromic anemia Iron Iron Metabolism Disorders Knowledge Lesion Link Medical Mentored Research Scientist Development Award Mentors Mesoderm Cell Mitochondria Modeling Molecular Mutagenesis Myelogenous National Institute of Diabetes and Digestive and Kidney Diseases Nitrosourea Compounds Organ Outcome Pathway interactions Poisson Distribution Randomized Regulation Research Personnel Role Scientist Signal Transduction Staining method Stains Stem Cell Development Stem cells Synteny Time Variant World Health Organization Zebrafish base effective therapy genetic analysis hematopoietic tissue improved insight iron metabolism loss of function mRNA Expression mutant novel positional cloning progenitor public health relevance self-renewal stem success tool

项目摘要

DESCRIPTION (provided by applicant): The goal of this project is to offer novel insights into heme synthesis and hematopoietic pathways that can improve treatment of congenital and acquired anemia. Despite decades of efforts to understand genetic blood disorders, every year 400,000 newly born babies worldwide face dreadful complications in the absence of effective therapies. Thus, better understanding and knowledge of heme synthesis, iron metabolism, and hematopoietic stem/progenitor revcell (HSPC) biogenesis is required. The zebrafish, with its close synteny to the human genome and its conserved molecular pathways regulating the development of hematopoietic tissues, offers a powerful tool to understand human blood disorders. Based on our Poisson distribution analyses, we hypothesized that the genetic screens for anemic mutants are not at saturation to study hematologic diseases. We employed de novo ethyl nitrosourea (ENU) based genetic screens and identified five unique zebrafish anemic mutants, including sauvignon blanc (sav) and pinotage (pnt). pnt exhibits hypochromic anemia. pnt has a defect in hemoglobinisation while preserving red cell numbers. In contrast, sav shows anemia due to a lack of erythroid cells. sav lacks expression markers for HSPC (scl/tal1), myeloerythroid progenitor (gata-1), erythroid ((-globin) and myeloid (mpo) cells. Expression of vascular markers such as, fli-1 is preserved in sav. These data suggest that the pathophysiology of sav is confined to hematopoietic cell development and not to mesodermal cells. The genetic mapping, positional cloning, loss-of-function, mRNA expression levels, and cRNA over-expression studies have revealed a novel functional gene for the pnt locus. We have identified closely linked (~0 cM) genetic markers flanking the unique sav locus. In order to further characterize the importance of the disrupted genes in pnt and sav mutants and their involvement in genetic blood disorders, we propose: 1) To analyze the biochemical function of the pnt gene in heme synthesis. The pathophysiology of the pnt gene in hypochromic anemia will be ascertained by analyzing mechanisms regulating transport and incorporation of iron into heme or [Fe-S] cluster synthesis using mammalian and other model organisms. 2) To identify the sav gene and its function in hematopoiesis. The positional cloning, loss-of-function, cRNA over-expression studies and mutational analyses will be performed to identify the sav gene. We will perform assays to derive cell intrinsic or extrinsic role of sav in the regulation of hematopoiesis, hematopoietic stem/progenitor cell development, stem cell self-renewal, and cell differentiation. In the long term, the successful outcomes of these projects will lay strong foundations to understand human genetic blood diseases and to discover new opportunities for improved therapies. The potential of zebrafish genetics, expert guidance of mentors and scientific advisors, and the environment of Harvard Medical Community will certainly provide the candidate a unique opportunity to evolve as a successful independent investigator.

描述(由申请人提供)：这个项目的目标是提供关于血红素合成和造血途径的新见解，以改善先天性和获得性贫血的治疗。尽管几十年来人们一直在努力了解遗传性血液疾病，但在缺乏有效治疗的情况下，全世界每年仍有40万新生儿面临可怕的并发症。因此，需要更好地了解和了解血红素合成、铁代谢和造血干/祖细胞(HSPC)的生物发生。斑马鱼与人类基因组关系密切，其调控造血组织发育的保守分子途径为理解人类血液疾病提供了强有力的工具。基于我们的泊松分布分析，我们假设贫血突变的遗传筛查不是饱和的，以研究血液疾病。我们采用了基于新亚硝酸乙酯(ENU)的遗传筛选，鉴定了5个独特的斑马鱼贫血突变体，包括白苏维翁(Sav)和Pinotage(Pnt)。PNT表现为低色素性贫血。PNT在血红素化方面存在缺陷，同时保留了红细胞数量。相比之下，SAV显示由于缺乏红系细胞而引起的贫血。SAV缺乏HSPC(scl/tal1)、粒系祖细胞(GATA-1)、红系(-珠蛋白)和髓系(MPO)细胞的表达标志。血管标志物如Fli-1的表达在SAV中得到保留。这些数据表明，SAV的病理生理学仅限于造血细胞发育，而不是中胚层细胞。遗传作图、位置克隆、功能丧失、mRNA表达水平和cRNA过度表达研究揭示了PNT基因座的一个新的功能基因。我们已经确定了紧密连锁(~0 cM)的遗传标记位于独特的SAV基因座的两侧。为了进一步研究突变基因在PNT和SAV突变中的重要性及其在遗传性血液疾病中的作用，我们提出：1)分析PNT基因在血红素合成中的生化功能。低色素性贫血中PNT基因的病理生理机制将通过哺乳动物和其他模式生物的[Fe-S]簇合成或血红素转运和整合的调节机制来确定。2)鉴定SAV基因及其在造血中的作用。将进行定位克隆、功能丧失、cRNA过度表达研究和突变分析，以鉴定sav基因。我们将进行细胞分析，以确定SAV在造血调控、造血干/祖细胞发育、干细胞自我更新和细胞分化中的内在或外在作用。从长远来看，这些项目的成功成果将为了解人类遗传性血液疾病和发现改进治疗的新机会奠定坚实的基础。斑马鱼遗传学的潜力，导师和科学顾问的专家指导，以及哈佛医学界的环境，肯定会为候选人提供一个独特的机会，让他成为一名成功的独立研究员。