GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

最近的研究表明，小鼠胚胎发育中的AGM区的生血内皮细胞产生大多数胎肝和骨髓造血细胞。此外，造血的最早步骤，造血干细胞（HSC）从造血内皮细胞的出现，是由Runx1控制。 然而，目前尚不清楚Runx1是否是胚胎和成人骨髓中所有定形HSC出现所必需的。 通过对ENU处理的斑马鱼进行大规模DNA测序，我们开发了一种在runx1基因中具有截短突变（W84X）的斑马鱼品系。这条线的表征表明，胎儿（或斑马鱼的幼虫）和成人造血共享一个共同的干细胞池，但runx1只需要启动幼虫造血。 runx1W84X/W84X鱼的胚胎具有完全阻断的幼虫（相当于胎肝）造血，并且80%的runx1W84X/W84X幼虫由于其无血表型而死亡。 引人注目的是，20%的runx1W84X/W84X幼虫恢复了血液循环，并生长为具有多谱系成体造血的可育成体。我们发现造血干细胞保留在runx1W84X/W84X胚胎中，并且能够在缺乏runx1的情况下启动成人造血。 这些发现表明，即使胎儿和成人造血具有共同的前体，它们的启动也需要不同的调节程序（Sood等人，Blood 2010）。 我们与NIH化学基因组学中心合作，对大约25万种化学物质进行了RUNX1-CBFbeta相互作用抑制剂的筛选，RUNX1-CBFbeta相互作用已被证明是几种类型的人类急性髓性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，已确定了130多种候选化合物。我们已经开发了一种基于斑马鱼的测定方法，以确定候选化合物是否在体内阻断RUNX1-CBFbeta相互作用。该测定是将在各种血细胞类型（例如分别用于血小板和淋巴细胞的cd41-GFP和lck-GFP）中具有GFP表达的转基因斑马鱼胚胎与化合物一起孵育，并观察由GFP+细胞的数量反映的造血缺陷。如果Runx1功能被候选化合物阻断，则预期GFP+细胞在胚胎中减少，因为Runx1是确定性造血所需的。通过这种方法，我们已经确定了三种结构相关的化合物，它们能够减少胚胎中的循环GFP+细胞，这些细胞基本上模仿了runx1突变胚胎，而其他胚胎看起来正常。这三种化合物中的一种已被用于我们的小鼠白血病模型，并表明它可以减少小鼠的白血病负担，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.