GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

斑马鱼胚胎发育的遗传分析

• 批准号: 7734866
• 负责人: PU PAUL LIU
• 金额: $ 67.45万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734866
• 关键词: Address; Adult; Affect; Anemia; Anti-Sense Probes; Biological Models; Blood; Blood Cells; Blood Circulation; Blood Platelets; Cell Cycle; Cell Cycle Progression; Cell Death; Cell Lineage; Cells; Child; Chromosome Mapping; Chromosomes; Code; Cytokinesis; DNA Binding; Data; Defect; Development; Disease; Down Syndrome; Embryo; Embryonic Development; Erythroid; Family; Fertilization; Fishes; GATA1 gene; GATA1 protein; Genes; Genetic; Genetic Screening; Genome; Goals; Haploidy; Hela Cells; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemoglobin; Hemorrhage; Homologous Gene; Human; In Situ Hybridization; Injection of therapeutic agent; Kidney; Knock-out; Knockout Mice; Lead; Lesion; Location; Lymphocyte; MYB gene; Maintenance; Mammals; Marrow; Messenger RNA; Microtubules; Modeling; Mummies; Mus; Mutate; Mutation; Myelogenous; Myeloid Progenitor Cells; Myelopoiesis; Numbers; Optics; Phenotype; Play; Point Mutation; Population; Process; Proteins; Proto-Oncogene Proteins c-myb; RNA; RNA Helicase; RNA Splicing; Regulation; Role; Series; Signal Pathway; Staging; Staining method; Stains; Survival Rate; Tubulin; Vertebrates; Visual; Work; Yeasts; Zebrafish; cytokine; day; dosage; erythroid differentiation; fetal; genetic analysis; interest; knock-down; knockout animal; leukemia; leukemia/lymphoma; leukemogenesis; member; mouse model; mutant; novel; plastin; positional cloning; progenitor; promoter; receptor; small hairpin RNA; tool; transcription factor

During this fiscal year, we have worked on three separate projects: 1. Characterization of a runx1 knockout zebrafish model Traditional and conditional knockout mouse models have shown that Runx1 is absolutely necessary for the emergence of hematopoietic stem cells (HSCs) during embryogenesis but not for their maintenance and differentiation during adult hematopoiesis. However, its role in the initiation of adult HSCs can not be addressed due to the embryonic lethality of the knockout and the presence of Runx1 during the critical embryonic period in the conditional knockout animals. Zebrafish provide an alternate model system to study hematopoiesis due to their optical clarity and ability to perform genetic screens. By TILLING (Targeting Induced Local Lesions IN Genomes) we identified a truncation mutation, W84X, in zebrafish runx1 gene. Homozygous null embryos develop normal primitive hematopoiesis but no definitive hematopoiesis during the larval stage. In contrast to the embryonic lethality of knockout mice, 20% of runx1 null zebrafish embryos survive to adulthood and sustain multilineage adult hematopoiesis. These data show that as suggested recently in mammals, definitive hematopoiesis in zebrafish also occurs in two stages: larval (equivalent to fetal) and adult. Using cd41-GFP, we demonstrate that HSCs initiate and migrate to the appropriate locations in the absence of runx1. This was a surprising finding and would not have been uncovered in the mouse models due to their stringent requirement for blood circulation during embryogenesis. 2. Truncation mutation of dhx8, a member of the DEAH family of RNA helicases, results in cell cycle and hematopoietic defects in zebrafish embryos Embryonic hematopoiesis in both zebrafish and mammals is a tightly regulated process that is coordinated by a series of transcription factors. In order to identify novel genes required for embryonic hematopoiesis, particularly those affecting early stages as well as myelopoiesis, we conducted a whole mount RNA in situ hybridization screen of ENU-mutagenized F2 haploid embryos with antisense probes for cbfb and l-plastin, genes expressed in the hematopoietic stem cells and myeloid lineage cells, respectively. One mutant line, mummy (mmy), was identified from this screen that showed reduced scl, cbfb expression and total lack of l-plastin expression. In addition, mummy mutants have reduced gata1 and hemoglobin expression. As both erythroid and myeloid lineages are affected, these results suggested that the defect in mmy is at or above HSC level. The mmy mutant embryos also suffer from widespread cell death and die around 30 hpf. Genetic mapping and positional cloning have identified the mutated gene as dhx8, an RNA helicase of the DEAH family. Morpholino injection has confirmed that knock-down of dhx8 can produce a similar phenotype in wild-type embryos. The yeast homolog of dhx8, prp22, functions during mRNA splicing. Our results show that mmy mutants are defective in the splicing of some hematopoietic genes. In addition, when dhx8 was knocked-down in HeLa cells using shRNA, cells were observed with a multi-nucleated phenotype with visible signs of chromosome bridges connecting them clearly demonstrating a cell cycle defect. When stained with tubulin anti-body, mmy mutant embryos showed increased cell cycle defects as characterized by disorganized micro-tubules and multiple spindle poles formations. Together, our results suggest that dhx8 is not only involved in mRNA splicing, its expression is also essential for correct cell cycle progression. Loss of dhx8 expression can lead to cytokinesis defects in HeLa cells as well as cell cycle defects in mmy mutant embryos. These observations provide a mechanism by which loss of dhx8 expression can lead to hematopoietic defects and embryonic lethality in zebrafish embryos. 3. Differential requirement for gata1 between primitive and definitive stages of hematopoiesis in the zebrafish The GATA1 transcription factor plays an essential role in hematopoiesis. Gata1 knockout in mice results in blockage of erythroid and megakaryocytic developments. In humans, mutations in gata1 are associated with anemia and leukemia development in children with Down syndrome. To further investigate GATA1 function in hematopoiesis and leukemogenesis we generated fish carrying gata1 point mutations that led to either reduced (T301K) or complete loss (R339X) of DNA binding. Fish embryos homozygous for the T301K mutation had blood circulation and normal survival rate while embryos homozygous for the R339X mutation were bloodless and died around 11-15 days post fertilization (dpf). Embryos compound-heterozygous for these two mutations (gata1-T301K/R339X) lacked blood circulation initially but recovered circulation between 7-14 dpf, suggesting that primitive hematopoiesis requires higher GATA1 dosages than definitive hematopoiesis. In situ hybridization showed a GATA1 dosage-dependent expansion of myeloid marker l-plastin and the hematopoietic stem cell marker c-myb. Visual observations of gata1-T301K/R339X, gata1-R339X/R339X and wild type embryos carrying the green fluorecent protein (GFP) driven by either the cd41 (expressed in thrombocytes) or gata1 (marking erythroid lineage) promoter showed that the number of thrombocytes is reduced in gata1-T301K/R339X embryos initially and absent in gata1-R339X/R339X embryos, and that gata1 is required for maintenance and differentiation of the erythroid/thromboid progenitors but not for their initiation. FACS analysis of adult gata1-T301K/R339X whole kidney marrow showed an increase of the populations containing precursors and lymphocytes. We are generating a mouse model carrying a Gata1 mutation equivalent to the zebrafish T301K substitution and will use this mouse model to investigate if primitive hematopoiesis in zebrafish and mouse require similar GATA1 dosage.

在本财政年度，我们开展了三个独立的项目： 1.一个Runx 1基因敲除斑马鱼模型的特性研究 传统的和条件性基因敲除小鼠模型已经表明，Runx 1是绝对必要的造血干细胞（HSC）在胚胎发生过程中的出现，但不是他们的维持和分化在成人造血。然而，由于基因敲除的胚胎致死性和Runx 1在条件性基因敲除动物的关键胚胎期的存在，其在成体HSC启动中的作用不能得到解决。斑马鱼提供了一个替代的模型系统来研究造血，由于其光学清晰度和执行遗传筛选的能力。通过TILLING（Targeting Induced Local Lesions IN Genomes）技术，我们在斑马鱼runx 1基因中发现了一个截短突变W84 X。纯合子无效胚胎发育正常的原始造血，但在幼虫阶段没有确定的造血。与基因敲除小鼠的胚胎致死率相反，20%的runx 1基因敲除斑马鱼胚胎存活至成年，并维持多系成体造血。这些数据表明，正如最近在哺乳动物中提出的那样，斑马鱼的造血也发生在两个阶段：幼虫（相当于胎儿）和成年。使用cd 41-GFP，我们证明了HSC启动和迁移到适当的位置在runx 1的情况下。这是一个令人惊讶的发现，并且在小鼠模型中不会被发现，因为它们在胚胎发育期间对血液循环有严格的要求。 2. RNA解旋酶DEAH家族成员dhx 8的截短突变导致斑马鱼胚胎细胞周期和造血缺陷 斑马鱼和哺乳动物的胚胎造血是一个由一系列转录因子协调的严格调控的过程。为了确定新的基因所需的胚胎造血，特别是那些影响早期阶段以及骨髓，我们进行了一个完整的安装RNA原位杂交筛选ENU诱变的F2单倍体胚胎与cbfb和l-plastin，基因表达的造血干细胞和髓系细胞，分别反义探针。一个突变株系mummy（mmy）从该筛选中被鉴定，其显示scl、cbfb表达降低和l-质体表达完全缺失。 此外，木乃伊突变体减少了gata 1和血红蛋白的表达。由于红系和髓系都受到影响，这些结果表明mmy的缺陷处于或高于HSC水平。mmy突变的胚胎也遭受广泛的细胞死亡，大约在30 hpf左右死亡。遗传作图和定位克隆已确定突变基因为dhx 8，DEAH家族的RNA解旋酶。 吗啉代注射已经证实dhx 8的敲低可以在野生型胚胎中产生类似的表型。dhx 8的酵母同源物prp 22在mRNA剪接期间起作用。我们的结果表明mmy突变体在某些造血基因的剪接中存在缺陷。此外，当使用shRNA在HeLa细胞中敲低dhx 8时，观察到细胞具有多核表型，并且连接它们的染色体桥的明显迹象清楚地表明细胞周期缺陷。微管蛋白抗体染色显示，mmy突变体胚胎细胞周期缺陷增加，其特征是微管排列紊乱和多个纺锤极形成。总之，我们的研究结果表明，dhx 8不仅参与mRNA剪接，其表达也是正确的细胞周期进程所必需的。dhx 8表达缺失可导致HeLa细胞中的胞质分裂缺陷以及mmy突变胚胎中的细胞周期缺陷。这些观察提供了dhx 8表达缺失可导致斑马鱼胚胎造血缺陷和胚胎致死的机制。 3.斑马鱼造血原始期和定型期对gata 1的需求差异 GATA 1转录因子在造血中起着重要作用。小鼠Gata 1基因敲除导致红细胞和巨核细胞发育受阻。在人类中，gata 1突变与唐氏综合征儿童的贫血和白血病发展相关。为了进一步研究GATA 1在造血和白血病发生中的功能，我们产生了携带gata 1点突变的鱼，这些突变导致DNA结合减少（T301 K）或完全丧失（R339 X）。 T301 K突变纯合子的鱼胚胎具有血液循环和正常的存活率，而R339 X突变纯合子的胚胎是无血的，并且在受精后（dpf）约11-15天死亡。这两种突变（gata 1-T301 K/R339 X）的复合杂合胚胎最初缺乏血液循环，但在7-14 dpf之间恢复循环，这表明原始造血需要比确定造血更高的GATA 1剂量。原位杂交显示GATA 1剂量依赖性扩增的髓系标志物l-plastin和造血干细胞标志物c-myb。目视观察携带由cd 41驱动的绿色荧光蛋白（GFP）的gata 1-T301 K/R339 X、gata 1-R339 X/R339 X和野生型胚胎。（在血小板中表达）或gata 1（标记红细胞谱系）启动子显示，血小板的数量在gata 1-T301 K/R339 X胚胎中最初减少，而在gata 1-R339 X/R339 X胚胎中不存在，并且GATA 1是维持和分化红细胞/血栓样祖细胞所需的，但不是它们的起始所需的。成人gata 1-T301 K/R339 X全肾骨髓的FACS分析显示含有前体细胞和淋巴细胞的群体增加。我们正在生成一种携带Gata 1突变的小鼠模型，该突变相当于斑马鱼T301 K置换，并将使用该小鼠模型研究斑马鱼和小鼠的原始造血是否需要相似的GATA 1剂量。