GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

最近的研究表明，发育中的小鼠胚胎的主动脉-性腺-中肾(AGM)区的血源性内皮细胞产生了大多数胎肝和骨髓造血细胞。此外，造血的最早步骤，即从血源性内皮中产生造血干细胞(HSC)，是由RUNX1控制的。然而，目前还不清楚RUNX1是否是所有最终的HSCs出现所必需的，无论是在胚胎中还是在成人骨髓中。通过对ENU处理的斑马鱼进行大规模DNA测序，我们培育了一个RUNX1基因截断突变(W84X)的斑马鱼品系。对该品系的鉴定表明，胎儿(或斑马鱼的幼体)和成体造血共享一个共同的干细胞库，但RUNX1仅是启动幼体造血所必需的。Runx1W84X/W84X的胚胎完全阻断了仔鱼(相当于胎肝)的造血，80%的Runx1W84X/W84X仔鱼因无血表型而死亡。值得注意的是，20%的runx1W84X/W84X幼虫恢复了血液循环，并成长为多血统成虫造血的可育成虫。我们发现在Runx1W84X/W84X胚胎中保留了造血干细胞，并且在没有RUNX1的情况下能够启动成人造血。这些发现表明，尽管胎儿和成人的造血具有共同的前体，但它们的启动需要不同的调节程序(Sood等人，血液2010)。 与NIH化学基因组中心合作，我们已经对大约150万种化学物质进行了RUNX1-CBFbeta相互作用抑制剂的筛选，这已被证明是几种类型的人类急性髓系白血病的关键相互作用。经过初步筛选和随后的确认/验证实验，已经确定了130多个候选化合物。我们开发了一种基于斑马鱼的检测方法，以确定候选化合物是否在体内阻止RUNX1-CBFβ相互作用。本实验旨在将不同血细胞类型(如血小板的cd41-GFP和淋巴细胞的lck-GFP)中表达GFP的转基因斑马鱼胚胎与这些化合物孵育，观察GFP+细胞数量所反映的造血缺陷。如果候选化合物阻断RUNX1的功能，胚胎中的GFP+细胞预计会减少，因为RUNX1是确定的造血所必需的。通过这种方法，我们已经鉴定了三种结构上相关的化合物，它们能够减少胚胎中循环中的GFP+细胞，基本上是表型复制RUNX1突变胚胎，而其他情况下胚胎看起来正常。三种化合物中的一种已经应用于我们的小鼠白血病模型，并表明它可以减轻小鼠的白血病负担，验证了我们在斑马鱼模型中预筛选候选化合物的方法。此外，我们正在使用这些化合物来确定斑马鱼胚胎造血过程中RUNX1的时间需求。

Recent studies showed that hemogenic endothelial cells in the aortic-gonado-mesonephros (AGM) region of the developing mouse embryo give rise to most fetal liver and bone marrow hematopoietic cells. In addition, the earliest step of hematopoiesis, the emergence of hematopoietic stem cells (HSC) from the hemogenic endothelium, is controlled by Runx1. However, it is not clear whether Runx1 is required for the emergence of all definitive HSCs, both in the embryos and in the adult bone marrow. Through large scale DNA sequencing of ENU-treated zebrafish, we developed a zebrafish line with a truncation mutation (W84X) in the runx1 gene. Characterization of this line showed that fetal (or larval in zebrafish) and adult hematopoiesis share a common stem cell pool, but runx1 is only required for the initiation of larval hematopoiesis. The embryos of the runx1W84X/W84X fish had complete block of larval (equivalent to fetal liver) hematopoiesis and 80% of the runx1W84X/W84X larvae died due to their bloodless phenotype. Strikingly, 20% runx1W84X/W84X larvae resumed blood circulation and grew up to fertile adults with multi-lineage adult hematopoiesis. We found that hematopoietic stem cells were retained in the runx1W84X/W84X embryos and were able to initiate adult hematopoiesis in the absence of runx1. These findings suggest that even though fetal and adult hematopoiesis share common precursors, their initiation requires distinct regulatory programs (Sood et al., Blood 2010). In collaboration with NIH Chemical Genomics Center we have conducted a screen of roughly 1 quarter million chemicals for inhibitors of RUNX1-CBFbeta interaction, which has been demonstrated to be a critical interaction for several types of human acute myeloid leukemia. Over 130 candidate compounds have been identified after the initial screen and subsequent confirmation/validation experiments. We have developed a zebrafish-based assay to determine if the candidate compounds block RUNX1-CBFbeta interaction in vivo. The assay is to incubate transgenic zebrafish embryos with GFP expression in various blood cell types (such as cd41-GFP and lck-GFP for thrombocytes and lymphocytes, respectively) with the compounds and observe for hematopoietic defects as reflected by the number of GFP+ cells. The GFP+ cells are expected to decrease in the embryos if Runx1 function is blocked by the candidate compounds, since Runx1 is required for definitive hematopoiesis. Through this approach we have identified three structurally related compounds that were able to reduce circulating GFP+ cells in the embryos, which essentially phenocopied the runx1 mutant embryos, while otherwise the embryos looked normal. One of the three compounds has been administered to our mouse leukemia model and showed that it could reduce leukemia burden in the mice, validating our approach to pre-screen the candidate compounds in the zebrafish model. In addition, we are using these compounds to determine the temporal requirement of runx1 during zebrafish embryonic hematopoiesis.