GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

实验室正在进行的一项主要工作是使用斑马鱼作为体内模型来验证和研究小分子的生物效应，这些小分子是通过我们的另一个项目(HG000030-18)产生的，该项目旨在开发针对CBF白血病的新化合物，这些化合物具有影响CBF基因的融合基因，即RUNX1和Cbfb。这两个基因都是建立确定的造血所必需的，所以设计用来干扰它们在白血病中的功能的化合物将有望在胚胎发育期间影响确定的造血。斑马鱼还具有这些化学物质的毒性研究功能。事实证明，该分析系统是一种灵敏、快速和可靠的系统，可以在其他系统(如小鼠模型)进一步表征潜在化合物之前筛选它们。我们最近发表的论文(Cunningham等人，PNAS109：14592,2012年)报告了一些发现。 几年前，我们在斑马鱼中进行了ENU(N-乙基-N-亚硝脲)突变筛选，以确定参与早期造血的新基因。对筛选出的突变鱼线木乃伊(Mmy)进行了详细的研究，因为它存在多个造血缺陷。纯合子mmy胚胎缺乏循环血细胞，在受精后30小时死亡。Mmy突变体不表达髓系细胞标志，原始造血中祖细胞和红系标志的表达显著降低。通过定位克隆，我们在mmy鱼中发现了dhx8基因的一个截断突变。编码的dhx8蛋白是酵母剪接因子prp22的斑马鱼同源基因，它是一种Deah-box RNA解旋酶。Mmy突变体在许多基因上存在剪接缺陷，包括几个造血基因。Mmy胚胎也表现出细胞分裂缺陷，其特征是有丝分裂细胞中有丝分裂纺锤体无序和形成多个纺锤体极。在HeLa细胞中，DHX8基因敲除证实了这些细胞分裂缺陷。综上所述，我们的结果证实了dhx8参与了mRNA的剪接，并表明它在有丝分裂过程中对细胞分裂也是重要的。这是第一个dhx8的脊椎动物模型，它的功能对于发育中的胚胎的原始造血是必不可少的。这项工作于去年完成，结果于最近发表(English等人，《发展动力学》，第241：879889,2012年)。 尽管体内对糖酵解烯醇化酶和许多细菌非烯醇化酶超家族成员有详细的了解，但对脊椎动物非烯醇化酶超家族成员(ENOSF1s)的体内功能知之甚少。先前的研究结果表明ENOSF1的剪接形式与乳腺癌和结肠癌有关。因此，我们使用斑马鱼(Danio Rerio)作为脊椎动物模型来研究ENOSF1&946；功能。Wish表明斑马鱼ENOSF1&946；(Enosf1b)是受精卵，并通过前24个HPF普遍表达。24小时后，enosf1b的表达仅限于脊索。注射enosf1b-EGFP mRNA的胚胎生长慢于注射EGFP的胚胎，但在48hpf时赶上了注射EGFP的胚胎。注射ATG或外显子10 enosf1b mRNA靶向吗啡的胚胎出现脊索扭曲、前后轴缩短和循环水肿。对NTL或pax2a表达的愿望表明，注射任何一种吗啡的胚胎都有畸形的脊索和前肾。TUNEL染色显示脊索周围区细胞凋亡增加。这项研究是关于ENOSF1在脊椎动物中功能的第一次报道，并表明ENOSF1是胚胎发育所必需的。在enosf1b基因敲除后细胞凋亡率增加，提示在人类癌症中增加ENOSF1和ENOSF1的表达是一个潜在的生存优势。这项研究于去年完成，最近发表(Finckbeiner等人，细胞与生物科学，1：32,2011)。

An ongoing major effort in the lab is to use the zebrafish as an in vivo model to validate and study the biological effects of small molecules, which are being generated through our other project (HG000030-18) for the development of novel compounds that target CBF leukemia, those with fusion genes affecting CBF genes, i.e., RUNX1 and CBFB. Both of these genes are required for the establishment of definitive hematopoiesis, so compounds designed to interrupt their function in leukemia would be expected to affect definitive hematopoiesis during embryo development. The zebrafish also serve the function of toxicity studies for these chemicals. This assay system has turned out to be a sensitive, fast and reliable system to screen potential compounds before they are characterized further in other systems, such as mouse models. Some of the findings have been reported in our recently published paper (Cunningham et al., PNAS 109:14592, 2012). A few years ago we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in the zebrafish to identify novel genes involved in early hematopoiesis. The mutant fish line mummy (mmy) from the screen was investigated in detail because of its multiple hematopoietic defects. Homozygous mmy embryos lacked circulating blood cells and were dead by 30 hours post-fertilization (hpf). The mmy mutants did not express myeloid cell markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation of the dhx8 gene in the mmy fish. The encoded dhx8 protein is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. Mmy mutants had splicing defects in many genes, including several hematopoietic genes. Mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos. This work was finished last year and the results were published recently (English et al., Developmental Dynamics 241:879889, 2012). Despite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s). Results of previous studies suggest involvement of the β splice form of ENOSF1 in breast and colon cancers. We therefore used the zebrafish (Danio rerio) as a vertebrate model to study ENOSF1β function. WISH showed that zebrafish ENOSF1β (enosf1b) is zygotic and expressed ubiquitously through the first 24 hpf. After 24 hpf, enosf1b expression is restricted to the notochord. Embryos injected with enosf1b-EGFP mRNA grew slower than EGFP mRNA-injected embryos but caught up to the EGFP-injected embryos by 48 hpf. Embryos injected with ATG or exon 10 enosf1b mRNA-targeting morpholinos had kinked notochords, shortened anterior-posterior axes, and circulatory edema. WISH for ntl or pax2a expression showed that embryos injected with either morpholino have deformed notochord and pronephros. TUNEL staining revealed increased apoptosis in the peri-notochord region. This study is the first report of ENOSF1 function in a vertebrate and shows that ENOSF1 is required for embryonic development. Increased apoptosis following enosf1b knockdown suggests a potential survival advantage for increased ENOSF1β expression in human cancers. This study was finished last year and was published recently (Finckbeiner et al., Cell & Bioscience 1:32, 2011).