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: 8627722
• 负责人: DHVANIT INDRAVADAN SHAH
• 金额: $ 0.1万
• 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/8627722
• 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.

描述（由申请人提供）：该项目的目标是提供血红素合成和造血途径的新见解，可以改善先天性和获得性贫血的治疗。尽管数十年来一直在努力了解遗传性血液疾病，但由于缺乏有效的治疗方法，全球每年仍有40万新生儿面临可怕的并发症。因此，需要更好地理解和血红素合成，铁代谢和造血干/祖细胞（HSPC）生物合成的知识。斑马鱼与人类基因组的同线性关系及其调控造血组织发育的保守分子途径，为了解人类血液疾病提供了有力的工具。基于我们的泊松分布分析，我们假设贫血突变体的遗传筛查在研究血液病方面还没有达到饱和。我们采用了基于从头乙基亚硝基脲（ENU）的遗传筛选，并鉴定了五种独特的斑马鱼贫血突变体，包括长相思（sauvignon blanc）（sav）和皮诺泰奇（pnt）。PNT表现出低色素性贫血。PNT在保持红细胞数量的同时具有血红蛋白化缺陷。相反，sav由于缺乏红系细胞而显示贫血。sav缺乏HSPC（scl/tal 1）、骨髓红系祖细胞（加塔-1）、红系（β-珠蛋白）和骨髓（mpo）细胞的表达标记。血管标记物如fli-1的表达在sav中得以保留。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、定位克隆、功能丧失、mRNA表达水平和cRNA过表达研究揭示了pnt基因座的新功能基因。我们已经确定了紧密连锁（~0 cM）的遗传标记侧翼的独特的sav基因座。为了进一步阐明pnt和sav突变体中被破坏基因的重要性及其在遗传性血液病中的作用，我们提出：1）分析pnt基因在血红素合成中的生化功能。pnt基因在低色素性贫血中的病理生理学将通过使用哺乳动物和其他模式生物分析调节铁转运和铁掺入血红素或[Fe-S]簇合成的机制来确定。2)鉴定sav基因及其在造血中的功能。将进行定位克隆、功能丧失、cRNA过表达研究和突变分析，以鉴定sav基因。我们将进行测定以推导出sav在调节造血、造血干细胞/祖细胞发育、干细胞自我更新和细胞分化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性血液病和发现改进疗法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，发展成为一个成功的独立调查员。