Zebrafish Genetics to Identify Genes Involved in Hematopoiesis

斑马鱼遗传学鉴定参与造血的基因

• 批准号: 8326104
• 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
• 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/8326104
• 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

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. PUBLIC HEALTH RELEVANCE: The success of this application will advance the field of human genetic blood disorders by identifying novel genes defective in zebrafish genetic mutants, pinotage (pnt) and sauvignon blanc (sav), and establishing their role in iron disorders and hematopoiesis. The expert guidance and environment available to Dr. Shah will help him to successfully complete his project and make him competitive to become an independent investigator. The appeal and the potential of future outcomes of this project satisfy the objectives of NIDDK sponsored Mentored Scientist Development Award (K01) mechanism.

描述（由申请人提供）：该项目的目标是为血红素合成和造血途径提供新的见解，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，但全球每年仍有40万新生儿在缺乏有效治疗的情况下面临可怕的并发症。因此，需要更好地理解和了解血红素合成、铁代谢和造血干细胞/祖细胞（HSPC）的生物发生。斑马鱼与人类基因组具有密切的同源性，其保守的调节造血组织发育的分子途径为了解人类血液疾病提供了有力的工具。基于泊松分布分析，我们假设贫血突变体的基因筛选尚未达到研究血液病的饱和状态。我们采用基于ENU的基因筛选方法鉴定了5种独特的斑马鱼贫血突变体，包括长相思（sav）和皮诺塔奇（pnt）。PNT表现为低色素贫血。PNT在保留红细胞数量的同时存在血红蛋白化缺陷。相反，sav表现为红细胞缺乏引起的贫血。sav缺乏HSPC （scl/tal1）、髓系祖细胞（gata-1）、红系（-球蛋白）和髓系（mpo）细胞的表达标记。在sav中保留了fli-1等血管标志物的表达。这些数据表明，sav的病理生理局限于造血细胞的发育，而不是中胚层细胞。遗传定位、定位克隆、功能缺失、mRNA表达水平和cRNA过表达研究揭示了pnt位点的一个新的功能基因。我们在独特的sav位点两侧鉴定出紧密相连（~0 cM）的遗传标记。为了进一步表征pnt和sav突变体中被破坏基因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。pnt基因在低色素贫血中的病理生理将通过哺乳动物和其他模式生物分析铁转运和纳入血红素或[Fe-S]簇合成的机制来确定。2)确定sav基因及其在造血中的功能。将进行定位克隆、功能缺失、cRNA过表达研究和突变分析来鉴定sav基因。我们将进行实验，以获得sav在造血、造血干细胞/祖细胞发育、干细胞自我更新和细胞分化调节中的细胞内在或外在作用。从长远来看，这些项目的成功成果将为了解人类遗传血液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，以及哈佛医学院的环境，肯定会为候选人提供一个独特的机会，使其成为一名成功的独立研究者。