GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

斑马鱼胚胎发育的遗传分析

• 批准号: 6290314
• 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/6290314
• 关键词: animal genetic material tag; developmental genetics; gene expression; genetic library; genetic promoter element; genetic regulation; green fluorescent proteins; in situ hybridization; microinjections; molecular cloning; vertebrate embryology; zebrafish

Zebrafish offers a powerful model system in which to study hematopoietic development due to its external fertilization, transparent embryos and ease of genetic manipulation. These characteristics allow mutagenesis screens to be performed in zebrafish that are not currently feasible in other vertebrate systems. Over 30 hematopoietic mutants have already been isolated through two large- scale chemical (N-nitroso-N-ethylurea or ENU) mutagenesis screens. We are characterizing and mapping one of these recessive mutants, vlad tepes, which has few or no blood cells. Using RNA in situ hybridization with markers along the hematopoietic pathway, we have demonstrated that vlad tepes has a block in the erythroid pathway with preservation of the myeloid lineage. We have mapped the mutation to linkage group 11 using half-tetrad analysis, and have determined its approximate distance from the centromere to be 2.2-3.2 cM. While GATA-1 maps to this location and is a likely candidate gene, analysis by RT-PCR, sequencing, and Northern analysis have not revealed significant alterations. Further mapping now suggests that GATA-1 is not the involved gene based on recombinants found for the GATA-1 locus. Additional informative markers are being used to refine the map position of the mutation. We are now performing an ENU mutagenesis to screen for defects in the myeloid pathway with RNA in situ hybridization using myeloid-specific markers. We have generated over 450 F1 progeny that can be screened for recessive phenotypes through haploid screening. Future studies will focus on identifying zebrafish mutants that demonstrate loss of expression of one or more myeloid markers. We have also isolated a zebrafish cDNA clone for CBFB (named zCBFB), a gene that regulates hematopoiesis and is involved in human leukemias. In biochemical analyses, zCBFb binds human CBFa2 and enhances its DNA binding. Expression of zCBFB in normal zebrafish embryos is observed in the posterior intermediate cell mass (ICM, the location of hematopoietic progenitor cells), Rohon Beard cells (sensory neurons roughly similar to dorsal root ganglia), cranial nerves, retina, branchial arches, hind brain, and jaw. This expression pattern is very similar to that in the mouse embryos. Expression of zCBFB in hematopoietic mutants (bloodless) is decreased or absent in the ICM and Rohon Beard cells. We have also analyzed the expression of zSCL and zGATA-1 in the same blood mutants to ascertain the relative order of these transcription factors to zCBFB in zebrafish hematopoiesis. Most of our results indicate that the three transcription factors function in the same hierarchical order as in mice: SCL, CBFB, and GATA-1. The exception to this rule is the cloche mutant, in which zSCL expression is lost while zCBFB expression remains in the ICM region. This result suggests that CBFB and SCL may function in independent pathways. - genetics, hematology, bone marrow, leukemia, gene mapping (non-human), stem cell research

斑马鱼提供了一个强大的模型系统，在其中研究造血发育，由于其外部受精，透明的胚胎和易于遗传操作。这些特征允许在斑马鱼中进行诱变筛选，这在其他脊椎动物系统中目前是不可行的。已经通过两次大规模化学（N-亚硝基-N-乙基脲或ENU）诱变筛选分离了超过30种造血突变体。我们正在对这些隐性突变体之一弗拉德特佩斯进行特征描述和绘图，它几乎没有血细胞。使用RNA原位杂交标记沿着造血途径，我们已经证明，弗拉德tepes有一个块在红系途径与保存髓系。我们已经映射到连锁群11使用半四分体分析的突变，并已确定其距离着丝粒的近似距离为2.2-3.2厘米。虽然加塔-1定位于该位置并且是可能的候选基因，但是通过RT-PCR、测序和北方分析的分析没有揭示显著的改变。进一步的作图表明，加塔-1不是基于发现的加塔-1基因座的重组体的相关基因。正在使用额外的信息标记来细化突变的地图位置。我们现在正在进行ENU诱变筛选缺陷的骨髓途径与RNA原位杂交使用骨髓特异性标记。我们已经产生了超过450个F1后代，可以通过单倍体筛选隐性表型。未来的研究将集中在识别斑马鱼突变体，证明一个或多个髓系标记物的表达丢失。我们还分离了一个斑马鱼CBFB（命名为zCBFB）的cDNA克隆，CBFB是一种调节造血的基因，与人类白血病有关。在生物化学分析中，zCBFb结合人CBFa 2并增强其DNA结合。在正常斑马鱼胚胎中，在后中间细胞团（ICM，造血祖细胞的位置）、Rohon Beard细胞（大致类似于背根神经节的感觉神经元）、颅神经、视网膜、鳃弓、后脑和颌中观察到zCBFB的表达。这种表达模式与小鼠胚胎中的表达模式非常相似。在ICM和Rohon Beard细胞中，造血突变体（无血）中zCBFB的表达降低或缺失。我们还分析了zSCL和zGATA-1在相同血液突变体中的表达，以确定这些转录因子在斑马鱼造血中与zCBFB的相对顺序。我们的大多数结果表明，三个转录因子的功能在相同的层次顺序在小鼠：SCL，CBFB，和加塔-1。该规则的例外是钟形突变体，其中zSCL表达丢失，而zCBFB表达保留在ICM区域中。这一结果提示CBFB和SCL可能通过独立的通路发挥作用。- 遗传学、血液学、骨髓、白血病、基因图谱（非人类）、干细胞研究