Identifying factors that promote clonal dominance in zebrafish hematopoiesis

 描述（由申请人提供）：造血干细胞和祖细胞（HSPC）中的特异性体细胞突变提供了增殖优势，导致克隆造血以及骨髓增殖性疾病（MPD）和急性髓性白血病（AML）的发展。对TET 2或DNMT 3A突变患者的研究表明，造血可以是克隆性的而没有病理学，这表明克隆性扩增可能是疾病发展的先决条件。目前尚不清楚这些突变的哪种组合对于促进克隆优势是必要的或足够的，或者突变事件的顺序对于疾病进展是否至关重要。斑马鱼已经成为造血系统恶性肿瘤，特别是急性淋巴细胞白血病的一个很好的模型。MPD和AML的模型显示低克隆率，没有明显的AML，尽管在这些模型中是否建立了非病理性克隆优势尚不清楚。这将是有趣的，以评估正常的造血干细胞的克隆性作为遗传扰动发生在癌前状态。病毒克隆标记将是有帮助的，但斑马鱼肾骨髓中存在的相对少量的HSC需要不太复杂的克隆标记系统。为了在克隆水平上研究HSPCs，我们优化了用于斑马鱼造血的Brainbow系统。该系统将克隆分析扩展到红细胞谱系，其在斑马鱼中有核，但在哺乳动物中没有。在不同的胚胎阶段，用他莫昔芬处理普遍表达Brainbow构建体并含有血液特异性CreERT 2的斑马鱼。使处理的鱼生长至成年，并对外周血和骨髓样品进行共聚焦和FACS分析以观察彩色条形码。使用对Brainbow荧光蛋白的标准化强度的自动聚类分析，我们在治疗后8个月的所有血液谱系中检测到多个和独特的条形码化细胞（平均4-8个）。该系统中重组的效率允许标记20-80%的成熟血池。将Brainbow标记的接近有限稀释的全肾骨髓移植到辐射受体中显示出强的单克隆植入，表明HSC被标记。使用这个系统，我们将调查突变是必要的或足够的克隆优势的产生。我们的研究将提供对调节正常和癌前造血的早期克隆事件的了解。

 DESCRIPTION (provided by applicant): Specific somatic mutations in hematopoietic stem and progenitor cells (HSPCs) provide a proliferative advantage, leading to clonal hematopoiesis and the development of myeloproliferative disorders (MPDs) and acute myeloid leukemia (AML). Studies of patients with TET2 or DNMT3A mutations show that hematopoiesis can be clonal without pathology, suggesting that clonal expansion may be a prerequisite for disease development. It is unclear which combination of these mutations are necessary or sufficient to promote clonal dominance or whether the order of the mutational events are critical for disease progression. The zebrafish has emerged as an excellent model for hematopoietic malignancies, notably acute lymphoblastic leukemias. Models of MPDs and AML show low penetrance with no overt AML, although it is not clear in these models whether a non-pathological clonal dominance is established. It would be interesting to evaluate clonality of normal HSCs as genetic perturbations occur in the premalignant state. Viral clonal marking would be helpful, but the relatively small number of HSCs present in the zebrafish kidney marrow require a less complex clonal labeling system. To investigate HSPCs at the clonal level, we have optimized the Brainbow system for use in zebrafish hematopoiesis. This system extends clonal analysis to the erythrocyte lineage, which is nucleated in zebrafish but not in mammals. Zebrafish that ubiquitously express the Brainbow construct and contain a blood specific CreERT2 were treated with tamoxifen at various embryonic stages. Treated fish were grown to adulthood, and peripheral blood and marrow samples were subjected to confocal and FACS analysis to observe color barcodes. Using automated cluster analysis on normalized intensities of the Brainbow fluorescent proteins, we detected multiple and unique barcoded cells (average of 4-8) in all lineages of the blood as far as 8 months post treatment. The efficiency of recombination in this system allows for labeling of 20-80% of the mature blood pool. Transplants of Brainbow-labeled whole kidney marrow near limiting dilution into irradiated recipients showed strong monoclonal engraftment, suggesting that HSCs are being labeled. Using this system, we will investigate mutations that are necessary or sufficient for generation of clonal dominance. Our studies will provide insight on early clonal events that regulate normal and premalignant hematopoiesis.