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.

斑马鱼中的胚胎或原始造血作用发生在中间细胞肿块（ICM）中，而确定的或成年的造血发生在肾脏中。 最近确定了一个新的确定造血的过渡点，被称为尾造血组织（CHT）或后血岛（PBI）。 使用谱系追踪技术，源自ICM的造血细胞通过CHT传播，最终填充了肾脏和胸腺。但是，这些位点细胞的谱系关系以及斑马鱼早期造血的遗传控制仍有待解决。 转录因子GATA1和RUNX1分别是哺乳动物中原始和确定的造血作用所必需的，并且可能是斑马鱼中的关键造血调节剂。通过ENU诱变和反向遗传筛选，我们在Runt同源性结构域中产生了斑马鱼Runx1突变线，其W84X和型型肌t301k在runt同源性结构域中，在c-terminal zinc Finder Zinc Finger Domain中，T301K，T301K。 我们使用了新的GATA1型肌层等位基因与先前特征的GATA1 NULL突变（VLAD TEPES（VLT））一起评估原始和确定的造血期间GATA1的需求。凝胶转移分析表明，T301K GATA1蛋白的结合亲和力降低了DNA的结合亲和力，而不是完全缺乏VLT突变蛋白的结合。由于GATA1T301K/T301K胚胎在所有阶段都正常循环并幸存到成年，而GATA1VLT/VLT胚胎从未发育过循环血细胞并在受肥后11-15天死亡（DPF）。另一方面，复合杂合的GATA1T301K/VLT胚胎缺乏循环，直到7 dpf，在8-11dpf左右恢复了循环，并正常幸存到成年。 通过原位杂交剂和与转基因TG（CD41-GFP）鱼交叉对确定造血的标记分析表明，确定性造血是正常的。 这些数据表明在造血的原始和确定阶段，GATA1功能的剂量效应，表明部分GATA1活性足以用于确定的造血。 此外，我们通过表征runx1截断突变来确定确定的造血的两个阶段。 Runx1W84X/W84X胚胎的循环正常，直到7dpf逐渐失去8-11dpf，一直保持无血流动，直到20-25DPF，而幸存的胚胎恢复了循环，而大多数人在无血阶段死亡。 Runx1w84x/W84x胚胎的大约20％存活到成年。 通过原位杂交，在Runx1突变体胚胎中无法检测到明确的造血干细胞标记，RUNX1和C-MYB。然而，与转基因TG（CD41-GFP）鱼交叉表明，确定性造血的CD41+干细胞保留在Runx1W84X/W84X胚胎中，并从ICM到CHT迁移，然后迁移到CHT，然后以Wildtype离合器伴侣的身份迁移到肾脏。 在Runx1突变体TG（GATA1-GFP）和TG（CD41-GFP）胚胎中，无血相伴随着缺乏GATA1-GFP+红细胞细胞和CD41-GFP+循环血小板细胞，在可见的循环血细胞中恢复后重新出现。这些数据表明，确定性造血的两个阶段：幼虫和成人，而幼虫阶段绝对需要Runx1。总之，我们已经确定了斑马鱼中造血的三个阶段，并在这三个阶段揭示了GATA1和RUNX1的差异剂量需求。