GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

斑马鱼胚胎发育的遗传分析

• 批准号: 7315993
• 负责人: PU PAUL LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7315993
• 关键词: GENETIC; ANALYSIS; ZEBRAFISH; EMBRYO; DEVELOPMENT

Zebrafish (Danio rerio) has emerged as ?Drosophila of the vertebrates? to study genetic controls of development and disease. It has several advantages over mice due to its small size, large progeny, and short reproduction cycle. Transparent embryos and ex vivo development allows noninvasive observations of embryonic development. Furthermore, the ability to do saturation mutagenesis using ENU (N-ethyl-N-nitrosourea) followed by forward and reverse genetic technologies to isolate mutants has a great potential in functional genomics. In order to identify novel genes required for embryonic hematopoiesis, particularly those affecting early stages as well as myelopoiesis, we conducted a whole mount RNA in situ hybridization screen of ENU-mutagenized F2 haploid embryos with antisense probes for cbfb and l-plastin, genes expressed in the hematopoietic stem cells and myeloid lineage cells, respectively. One mutant line, mummy, was identified from the screen that showed defects at or above the level of hematopoietic stem cells. Genetic mapping and positional cloning identified the mutated gene as dhx8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing. Our results demonstrate that mutations in an RNA helicase involved in splicing may show tissue specific developmental defects. In addition, we have identified two other mutant lines from the screen that have similar hematopoietic defects. Genetic mapping indicated that the mutated genes in these two mutants are not dhx8 and are not one of the genes known to be involved in hematopoiesis. Strong efforts are ongoing in the lab to clone these two mutated genes. Gene knockout technology has yet to be developed in the zebrafish, mainly due to the fact that embryonic stem cells have not been isolated. As an alternative, we have developed the approach of sequencing a large number of ENU-mutated fish genomes to identify mutations in the genes of interest. This approach has proven to be feasible and is amendable to high-throughput development of a large number of fish lines with a series of mutations in any gene of interest. We have generated a mutant library archived as a sperm bank and a DNA bank from ~3400 ENU-mutagenized F1 males. DNA samples are used to screen for mutations by sequencing or other heteroduplex screening methods. The corresponding sperm samples are used to revive the mutant fish by in vitro fertilization. We have screened this library for mutations in runx1 and gata1, two genes playing important roles in mammalian hematopoiesis and in human leukemia. We identified one point mutation in runx1 that results in an early truncation of the encoded protein, and two point mutations in the gata1 gene that substitutes conserved amino acid residues in the DNA binding domain of the encoded protein. We have recovered fish lines carrying these three mutations and have been analyzing their phenotypes. Preliminary data suggest that the mutations affect hematopoiesis as would be predicted from the functions of the genes, and at least one of the gata1 mutations is a hypomorphic one, i.e., it encodes a protein with reduced function. We plan to continue our analysis of these mutant lines and use them in additional mutagenesis screens to identify genetic modifiers.

斑马鱼（Danio rerio）已经成为？脊椎动物中的果蝇？研究发育和疾病的遗传控制。由于其体积小，后代大，繁殖周期短，它比小鼠有几个优势。透明胚胎和离体发育允许对胚胎发育进行非侵入性观察。此外，使用ENU（N-乙基-N-亚硝基脲）进行饱和诱变，然后通过正向和反向遗传技术分离突变体的能力在功能基因组学中具有很大的潜力。 为了确定新的基因所需的胚胎造血，特别是那些影响早期阶段以及骨髓，我们进行了一个完整的安装RNA原位杂交筛选ENU诱变的F2单倍体胚胎与cbfb和l-plastin，基因表达的造血干细胞和髓系细胞，分别反义探针。从筛选中鉴定出一个突变株系，木乃伊，它显示出造血干细胞水平或以上的缺陷。遗传作图和定位克隆将突变基因鉴定为dhx 8，其编码RNA解旋酶。dhx 8的酵母同源物prp 22在mRNA剪接期间起作用。我们的研究结果表明，在RNA解旋酶参与剪接突变可能会显示组织特异性发育缺陷。此外，我们还从筛选中鉴定出另外两个具有类似造血缺陷的突变株系。遗传作图表明，这两个突变体中的突变基因不是dhx 8，也不是已知参与造血的基因之一。实验室正在努力克隆这两个突变基因。 基因敲除技术尚未在斑马鱼中开发，主要是由于胚胎干细胞尚未分离。作为替代方案，我们已经开发了对大量ENU突变的鱼类基因组进行测序的方法，以识别感兴趣的基因中的突变。这种方法已被证明是可行的，并且可用于高通量开发大量在任何感兴趣的基因中具有一系列突变的鱼线。我们已经从约3400个ENU诱变的F1雄性中产生了一个突变体库，作为精子库和DNA库存档。DNA样品用于通过测序或其他异源双链筛选方法筛选突变。相应的精子样品用于通过体外受精使突变鱼复活。我们已经筛选了这个库中的突变runx 1和gata 1，两个基因在哺乳动物造血和人类白血病中发挥重要作用。我们确定了一个点突变的runx 1，导致在编码的蛋白质的早期截短，和两个点突变的gata 1基因，取代保守的氨基酸残基的DNA结合结构域的编码蛋白质。我们已经恢复了携带这三种突变的鱼线，并一直在分析它们的表型。初步数据表明，这些突变影响造血，正如从基因功能预测的那样，并且至少一个gata 1突变是亚型突变，即，它编码一种功能减弱的蛋白我们计划继续分析这些突变株系，并将其用于额外的诱变筛选以鉴定遗传修饰剂。