MLL in Hematopoiesis and Leukemia in the Zebrafish Model

描述（由申请人提供）：以11q23染色体带MLL基因易位为特征的婴儿白血病和化疗相关白血病是具有独特临床和生物学特征的毁灭性疾病，通常是致命的疾病。MLL编码一种具有转录抑制和激活功能的复杂癌蛋白。易位涉及多个配对基因，并产生5‘-MLL-Partner-3’重排、5‘-Partner-MLL-3’重排和由于一个等位基因参与易位而导致的MLL单倍不足。斑马鱼胚胎的独特属性使得完整动物体内正常和异常造血发育过程的可视化与其他动物模型不同。此外，斑马鱼胚胎非常适合模拟MLL，因为婴儿白血病发生中的MLL易位起源于子宫。到目前为止，还没有在斑马鱼身上进行过mll的研究。我们克隆了斑马鱼的mll同源基因，发现人类mll的所有关键功能域都高度保守。我们发现，mll转录本是母体提供给胚胎的，在斑马鱼的整个生命周期中表达，并且在小鼠造血细胞和其他表达mll的组织中可以检测到。我们证明，mll - morpholino敲低表型可以影响mll -/-小鼠的外部表型、细胞凋亡和贫血。我们发现，由此产生的mll缺失不仅导致同源盒辅助因子减少和细胞周期基因表达改变，而且导致许多造血基因在不同血细胞区室中过表达和过表达，这表明mll与以前未知的靶点有新的联系。此外，血细胞基因表达的改变引起了严重的血细胞畸形。虽然在前体细胞/髓细胞室中，lmo2、scl、cmyb、ikaros、rag2和gata1等基因过表达，但在红细胞部分检测到gata1和其他红细胞基因的表达减少，这将mll耗尽引起的贫血与整个红细胞程序的失调联系起来。这导致假设MLL在造血系统的发育控制中具有深刻的多谱系作用，MLL对包括抑制和激活在内的基因表达的时间和细胞类型特异性调节是有序规范造血祖细胞和干细胞发育所必需的。MLL缺失会导致血细胞谱系基因异位和异步过表达和过表达，导致造血系统发育不良。本项目试图通过对整个生物体的研究以及对整个生物体细胞的细胞和分子研究来调查这一假设，这些研究只可能在斑马鱼中进行，以确定MLL最重要的细胞，以及MLL易位何时开始转化。

DESCRIPTION (provided by applicant): Infant leukemias and chemotherapy related leukemias characterized by translocations of the MLL gene at chromosome band 11q23 are devastating, often fatal diseases with unique clinical and biological features. MLL encodes a complex oncoprotein with transcriptional repression and activation functions. The translocations involve many partner genes and generate 5'-MLL-Partner-3' rearrangements, 5'-Partner-MLL-3' rearrangements and MLL haploinsufficiency due to involvement of one allele in the translocation. Unique attributes of zebrafish embryos enable in vivo visualization of normal and abnormal hematopoietic developmental processes in intact animals like no other animal models. Moreover, zebrafish embryos are well suited to model MLL because MLL translocations in infant leukemogenesis originate in utero. Until now, no studies of mll had been done in zebrafish. We cloned the zebrafish mll ortholog and showed high conservation of all of the critical functional domains of human MLL. We found that mll transcripts are maternally supplied to the embryo, expressed during the entire zebrafish lifespan, and detectable in hematopoietic cells and other tissues where Mll is expresed in mice. We demonstrated that mll morpholino knockdown phenocopies the external phenotype, apoptosis and anemia of Mll-/- mice. We made the striking observations that the resulting mll depletion caused not only reduced homeobox cofactor and altered cell cycle gene expression, but also overexpression and underexpression of many hematopoietic genes in different blood cell compartments, suggesting new links of mll to previously unknown targets. Furthermore, the changes in blood cell gene expression caused profound blood cell dysmorphologies. While overexpressed genes in the precursor/myeloid compartment featured lmo2, scl, cmyb, ikaros, rag2 and gata1, reduced expression of gata1 and other red cell genes was detected in the erythroid fraction, linking the anemia from mll depletion to deregulation of an entire erythroid program. This leads to the hypothesis that MLL has profound multi-lineage roles in the developmental control of the hematopoietic system, that temporal and cell-type specific regulation of gene expression by MLL including repression and activation is required for orderly specification of hematopoietic progenitor and stem cell development, and that loss of MLL causes ectopic and asynchronous overexpression and underexpression of blood cell lineage genes and ineffective development of the hematopoietic system. This project endeavors to investigate this hypothesis by exploiting whole organism studies and cellular and molecular studies on cells from whole organisms that are only possible in zebrafish to pinpoint the cells where MLL is most important and when MLL translocations first become transforming.