GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

斑马鱼胚胎发育的遗传分析

• 批准号: 7594303
• 负责人: PU PAUL LIU
• 金额: $ 101.03万
• 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/7594303
• 关键词: Adult; Affinity; Alleles; Anemia; Binding; Blood; Blood Cells; Blood Circulation; Blood Island; Blood Platelets; C-terminal; Cells; Code; DNA; Data; Defect; Development; Disease; Embryo; Embryonic Development; Erythroid Cells; Ethylnitrosourea; Fertilization; Fishes; Gelshift Analysis; Genes; Genetic; Genetic Screening; Goals; Hand; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Human; In Situ; In Situ Hybridization; Kidney; Lead; MYB gene; Mammals; Missense Mutation; Modeling; Mutagenesis; Mutation; Partner in relationship; Phase; Proteins; Proto-Oncogene Proteins c-myb; Recovery; Regulation; Signal Pathway; Site; Staging; Stem cells; Techniques; Thymus Gland; Transgenic Organisms; Vertebrates; Zebrafish; Zinc Fingers; cytokine; day; dosage; genetic analysis; hematopoietic tissue; interest; leukemia; leukemia/lymphoma; mutant; null mutation; positional cloning; receptor; tool; transcription factor

Embryonic or primitive hematopoiesis in the zebrafish takes place in the intermediate cell mass (ICM), while definitive or adult hematopoiesis takes place in the kidney. A new transition site of definitive hematopoiesis was recently identified and was called caudal hematopoietic tissue (CHT), or posterior blood island (PBI). Using lineage tracing techniques the hematopoietic cells originating from ICM were shown to transit through CHT and eventually populate kidney and thymus. However, the lineage relationship of the cells at these sites and the genetic control of early hematopoiesis in the zebrafish remain to be resolved. Transcription factors Gata1 and Runx1 are required for primitive and definitive hematopoiesis respectively in mammals, and are likely candidates as key hematopoietic regulators in the zebrafish. By ENU mutagenesis and reverse genetic screening, we have generated a zebrafish runx1 mutant line with a truncation mutation, W84X, in the runt homology domain and a hypomorphic gata1 mutant line with a missense mutation, T301K, in the C-terminal zinc finger domain. We used the new gata1 hypomorphic allele in combination with the previously characterized gata1 null mutation, vlad tepes (vlt) to assess the requirements for gata1 during primitive and definitive hematopoiesis. Gel-shift analysis showed that the T301K gata1 protein had reduced binding affinity for DNA as opposed to complete lack of binding by the vlt mutant protein. This reduced activity is sufficient for hematopoieisis since gata1T301K/T301K embryos had normal circulation at all stages and survived to adulthood, while gata1vlt/vlt embryos never developed circulating blood cells and died around 11-15 days post fertilization (dpf). On the other hand, compound heterozygous gata1T301K/vlt embryos lacked circulation until 7 dpf, regained circulation around 8-11dpf and survived normally to adulthood. Analysis of markers for definitive hematopoiesis by in situ hybridzationan and crossing with transgenic Tg(cd41-GFP) fish indicated that definitive hematopoiesis was normal. These data suggest dosage effect of gata1 function during primitive and definitive stages of hematopoiesis, indicating that partial gata1 activity was sufficient for definitive hematopoiesis. Furthermore, we identified two phases of definitive hematopoiesis by characterization of the runx1 truncation mutation. runx1W84X/W84X embryos had normal circulation until 7dpf, gradually lost circulation around 8-11dpf, stayed bloodless until 20-25dpf and the surviving embryos regained circulation, while majority of them died during the bloodless phase. Approximately twenty percent of runx1W84X/W84X embryos survived to adulthood. By in situ hybridization, definitive hematopoietic stem cell markers, runx1 and c-myb, were not detectable in the runx1 mutant embryos. However, crossing with transgenic Tg(cd41-GFP) fish showed that cd41+ stem cells of definitive hematopoiesis were retained in the runx1W84X/W84X embryos and migrated from ICM to CHT and then to kidney as wildtype clutch-mates. In runx1 mutant Tg(gata1-GFP) and Tg(cd41-GFP) embryos the bloodless phase was accompanied by lack of gata1-GFP+ erythroid cells and cd41-GFP+ circulating thrombocytes, which reappeared after recovery of visible circulating blood cells. These data suggest that there are two phases of definitive hematopoiesis: larval and adult, and that runx1 is absolutely required for the larval stage. In conclusion, we have identified three stages of hematopoiesis in the zebrafish and revealed the differential dosage requirement for gata1 and runx1 during these three stages.

斑马鱼的胚胎或原始造血发生在中间细胞团（ICM）中，而最终或成体造血发生在肾脏中。 最近发现了一个新的造血过渡部位，称为尾侧造血组织（CHT）或后血岛（PBI）。 使用谱系追踪技术，显示来自ICM的造血细胞通过CHT转运并最终分布在肾脏和胸腺。然而，这些位点的细胞的谱系关系和斑马鱼早期造血的遗传控制仍有待解决。 转录因子Gata 1和Runx 1分别是哺乳动物原始造血和永久造血所必需的，并且可能是斑马鱼中关键造血调节因子的候选者。通过ENU诱变和反向遗传学筛选，我们已经产生了一个斑马鱼runx 1突变系的截断突变，W84 X，在runt同源结构域和一个亚纯型gata 1突变系的错义突变，T301 K，在C-末端锌指结构域。 我们使用新的gata 1亚型等位基因结合先前表征的gata 1无效突变vlad tepes（vlt）来评估原始和确定性造血过程中对gata 1的需求。凝胶位移分析表明，T301 K gata 1蛋白与DNA的结合亲和力降低，而vlt突变体蛋白则完全不结合。这种降低的活性足以进行造血，因为gata 1 T301 K/T301 K胚胎在所有阶段都有正常的循环并存活至成年，而gata 1vlt/vlt胚胎从未发育出循环血细胞并在受精后（dpf）约11-15天死亡。另一方面，复合杂合gata 1 T301 K/vlt胚胎在7 dpf之前缺乏循环，在8- 11 dpf左右恢复循环并正常存活至成年。 通过原位杂交和与转Tg（cd 41-GFP）基因鱼杂交，对确定性造血的标记进行分析，结果表明，确定性造血是正常的。 这些数据表明，在造血的原始和定型阶段的gata 1功能的剂量效应，表明部分gata 1活性是足够的定型造血。 此外，我们确定了两个阶段的确定性造血特征的runx 1截断突变。 runx 1 W84 X/W84 X胚胎在7 dpf前循环正常，8- 11 dpf时逐渐失去循环，20- 25 dpf时无血，存活胚胎恢复循环，但大部分胚胎在无血期死亡。大约20%的runx 1 W84 X/W84 X胚胎存活至成年。 通过原位杂交，确定的造血干细胞标记，runx 1和c-myb，在runx 1突变胚胎中检测不到。然而，与转基因Tg（cd 41-GFP）鱼杂交表明，永久造血的cd 41+干细胞保留在runx 1 W84 X/W84 X胚胎中，并从ICM迁移到CHT，然后作为野生型交配动物迁移到肾脏。 在runx 1突变体Tg（gata 1-GFP）和Tg（cd 41-GFP）胚胎中，无血期伴随着缺乏gata 1-GFP+红系细胞和cd 41-GFP+循环血小板，这在恢复可见的循环血细胞后再次出现。这些数据表明，有两个阶段的决定性造血：幼虫和成人，和runx 1是绝对需要的幼虫阶段。总之，我们已经确定了斑马鱼造血的三个阶段，并揭示了在这三个阶段中gata 1和runx 1的不同剂量需求。