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.

在这个财政年度，我们从事三个单独的项目： 1。runx1敲除斑马鱼模型的表征 传统的和有条件的敲除小鼠模型表明，Runx1对于胚胎发生过程中造血干细胞（HSC）的出现绝对必要，而不是在成年造血期间其维持和分化。但是，由于敲除的胚胎致死性以及在条件敲除动物中关键的胚胎时期内的胚胎杀伤力以及Runx1的存在，因此无法解决其在成年HSC的启动中的作用。斑马鱼为研究造血的替代模型系统，因为它们的光学清晰度和执行遗传筛查的能力。通过耕种（基因组中靶向局部病变），我们在斑马鱼Runx1基因中鉴定出截断突变，W84X。纯合子零胚胎会形成正常的原始造血，但在幼虫阶段没有明确的造血。与基因敲除小鼠的胚胎致死性相反，Runx1 Null斑马鱼的胚胎中有20％幸存到成年并维持多琳的成年造血。这些数据表明，正如最近在哺乳动物中所建议的那样，斑马鱼中的确定造血作用也分为两个阶段：幼虫（相当于胎儿）和成人。使用CD41-GFP，我们证明HSC在没有RUNX1的情况下启动并迁移到适当的位置。这是一个令人惊讶的发现，由于小鼠模型在胚胎发生过程中对血液循环的严格要求，因此不会被发现。 2。DHX8的截断突变，DHX8是RNA解旋酶DEAH家族的成员，导致斑马鱼胚胎中的细胞周期和造血缺陷 斑马鱼和哺乳动物中的胚胎造血是一个严格调节的过程，由一系列转录因子协调。为了确定胚胎造血所需的新基因，尤其是那些影响早期阶段和骨髓骨的基因，我们对eNu-核酸含量的F2单倍体胚胎的原位RNA进行了整个RNA，用抗敏感探针对CBFB和L-倍蛋白进行了抗固定探针，并在l- plastin中表达了l-plastin，在血液中表达了l-plaste，在血液素细胞中表达了血液素细胞和肌体素。从该屏幕中鉴定出一条突变线木乃伊（MMY），该屏幕显示SCL，CBFB表达降低，总缺乏L-普拉斯汀表达。 另外，木乃伊突变体减少了GATA1和血红蛋白表达。由于红细胞和髓样谱系都受到影响，因此这些结果表明MMY的缺陷在HSC水平上或高于HSC水平。 MMY突变胚胎也患有广泛的细胞死亡，死亡约30 hpf。遗传映射和位置克隆已将突变的基因确定为DHX8，DHX8是DEAH家族的RNA解旋酶。 Morpholino注射已经证实，DHX8的敲除可以在野生型胚胎中产生类似的表型。 MRNA剪接过程中DHX8的酵母同源物在MRNA剪接过程中起作用。我们的结果表明，MMY突变体在某些造血基因的剪接中有缺陷。另外，当使用shRNA在HeLa细胞中撞倒DHX8时，用多核表型观察细胞，并具有可见的染色体桥梁符号，将它们连接起来清楚地证明了细胞周期缺陷。当用微管蛋白抗体染色时，MMY突变体胚胎显示出增加的细胞周期缺陷，其特征是小管混乱和多个纺锤形杆形成。总之，我们的结果表明DHX8不仅参与mRNA剪接，而且表达对于正确的细胞周期进程也是必不可少的。 DHX8表达的丧失会导致HeLa细胞中的细胞因子缺陷以及MMY突变胚胎中的细胞周期缺陷。这些观察结果提供了一种机制，通过该机制，DHX8表达的丧失可以导致斑马鱼胚胎中的造血缺陷和胚胎致死性。 3。在斑马鱼中造血的原始阶段和确定阶段之间对GATA1的差异要求 GATA1转录因子在造血中起着至关重要的作用。小鼠的GATA1敲除导致红细胞和巨核细胞发展的阻塞。在人类中，GATA1中的​​突变与唐氏综合症儿童的贫血和白血病发育有关。为了进一步研究造血和白血病发生中的GATA1功能，我们产生了携带GATA1点突变的鱼，导致DNA结合的降低（T301K）或完全损失（R339x）。 T301K突变纯合的鱼类胚胎具有血液循环和正常的存活率，而R339X突变纯合子的胚胎是无血的，受精后11-15天死亡（DPF）。这两个突变（GATA1-T301K/R339X）的胚胎杂合化最初缺乏血液循环，但在7-14 dPF之间恢复了循环，这表明原始造血需要比确定的造血剂更高的GATA1剂量。原位杂交显示髓样标记L-普拉斯汀和造血干细胞标记C-MYB的GATA1剂量依赖性膨胀。 GATA1-T301K/R339X，GATA1-R339X/R339X和野生型胚胎的视觉观察以及由CD41驱动的绿色荧光蛋白（GFP）的野生型胚胎（以血小板细胞表达）或GATA1（标记成等级IS促进剂）显示了该促进症的促进症，该促进症的数量是GATA1-T301K/R339X胚胎最初在GATA1-R339X/R339X胚胎中不存在，并且该GATA1是维持和分化红细胞/势力型祖先所必需的，而不是为了启动。 FACS对成年GATA1-T301K/R339X全肾骨髓的分析显示，含有前体和淋巴细胞的种群增加。我们正在生成一个携带与斑马鱼T301K取代等效的GATA1突变的小鼠模型，并将使用该小鼠模型研究斑马鱼和小鼠中的原始造血是否需要类似的GATA1剂量。