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)中的特定体细胞突变提供了增殖优势，导致克隆性造血和骨髓增生性疾病(MPD)和急性髓系白血病(AML)的发展。对TET2或DNMT3A突变患者的研究表明，造血可以在没有病理的情况下克隆，这表明克隆性扩张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是促进克隆显性所必需的或足够的，也不清楚突变事件的顺序是否对疾病进展至关重要。斑马鱼已经成为治疗血液系统恶性肿瘤，特别是急性淋巴细胞性白血病的极佳模型。MPDS和AML模型显示低外显性，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显性尚不清楚。当基因扰动发生在癌前状态时，评估正常造血干细胞的克隆性将是有趣的。病毒克隆标记将是有帮助的，但斑马鱼肾脏骨髓中存在的HSCs数量相对较少，需要一个不那么复杂的克隆标记系统。为了在克隆水平上研究HSPC，我们优化了用于斑马鱼造血的脑弓系统。该系统将克隆分析扩展到红细胞谱系，在斑马鱼中有核，但在哺乳动物中没有。斑马鱼普遍表达脑弓结构并含有血液特异的CreERT2，在不同的胚胎阶段用他莫昔芬治疗。将处理过的鱼培育到成年，并对外周血和骨髓样本进行共聚焦和流式细胞仪分析，以观察颜色条形码。使用脑弓荧光蛋白归一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独特的条形码细胞(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。将接近有限稀释的Brainrow标记的全肾骨髓移植到受照射的受者体内，显示出很强的单克隆性植入，这表明HSCs正在被标记。利用这个系统，我们将研究产生克隆显性所必需的或充分的突变。我们的研究将为调节正常和癌前造血的早期克隆事件提供洞察力。

 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.