Neuroblastoma Tumor Suppressor Genes in Zebrafish

斑马鱼神经母细胞瘤抑癌基因

• 批准号: 6710512
• 负责人: A. THOMAS LOOK
• 金额: $ 35.06万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-02 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6710512
• 关键词: cell differentiation; cell proliferation; chromosome translocation; clinical research; disease /disorder onset; embryogenesis; gene expression; gene mutation; genetic screening; genetic susceptibility; genetically modified animals; human tissue; in situ hybridization; loss of heterozygosity; molecular cloning; neuroblastoma; neurogenetics; peripheral nervous system; polymerase chain reaction; terminal nick end labeling; transcription factor; tumor suppressor genes; tyrosine 3 monooxygenase; zebrafish

DESCRIPTION (provided by applicant): Forward genetic screening in the zebrafish affords an unparalleled opportunity to discover previously elusive tumor suppressor genes whose alteration leads to disruption of the developing peripheral sympathetic nervous system (PSNS) and ultimately to overt neuroblastoma (NB), a biologically enigmatic, often fatal tumor in young children. This proposal tests two linked hypotheses: (i) genome-wide ethylnitrosourea (ENU) mutagenesis screens in the zebrafish can be used to identify dominant and recessive mutations that cause a deficiency or abnormal distribution of the PSNS, implicating genes important in NB pathogenesis and normal PSNS development; and (ii) transgenic zebrafish overexpressing the human MYCN oncogene in the developing PSNS can be used to determine the role of MYCN amplification in aberrant sympathetic neuroblast proliferation, survival and differentiation. These transgenic fish can also be mated to fish carrying candidate NB tumor suppressor genes identified in the ENU screen, to define the genetic pathways that cooperate with MYCN overexpression to induce early onset neuroblastoma. The zebrafish tyrosine hydroxylase (zTH) gene has already been cloned for use as a PSNS developmental marker in these screens, and its specificity for cells of the PSNS lineage was demonstrated by RNA in situ analysis. In Aim 1, mutant fish identified by in situ hybridization following ENU mutagenesis will be analyzed to determine the developmental stage at which the mutation occurred (neural crest vs. sympathetic neuroblast development). Next, the chromosomal location of each mutation will be mapped on the zebrafish genome, and examined for synteny with known regions of loss of heterozygosity (LOH) and deletion in human NB samples. Positional cloning will focus on genes most likely to have deleted or mutated counterparts in human NB. In Aim 2, transgenic zebrafish lines will be generated by using the zebrafish tyrosine hydroxylase (TH) promoter to drive expression of human MYCN in sympathetic neuroblasts. These lines will then be analyzed for their predisposition toward spontaneous tumorigenesis and crossed to the mutants isolated in Aim 1, to identify the combinations of genetic lesions leading to accelerated NB formation. Mutant zebrafish lines that harbor mutations in homologues of human tumor suppressor genes should provide reliable animal models for elucidating the molecular pathways leading to NB. A long-range goal is to use these models as a starting point for second-generation modifier screens to identify suppressors and enhancers of the genes causing PSNS defects, which may then be exploited as targets for therapeutic interventions in human NB.

描述（由申请人提供）：斑马鱼中的正向基因筛查提供了一个无与伦比的机会，可以发现以前难以捉摸的肿瘤抑制基因，其改变会导致破坏发展中的周围性交感神经系统（PSN），并最终导致明显的神经母细胞瘤（NB）（NB），一个生物学上神秘的，一个生物学上神秘的年轻儿童。 该提案检验了两个关联的假设：（i）斑马鱼中全基因组乙基硝基库（ENU）诱变筛选可用于识别引起PSN缺乏或异常分布的显性和隐性突变，这对NB病原体和正常PSN的发展含义很重要； （ii）在发育中的PSN中过表达人Mycn癌基因的转基因斑马鱼可用于确定MYCN扩增在异常的交感神经细胞增殖，生存和分化中的作用。 这些转基因鱼也可以与携带候选NB肿瘤抑制基因的鱼类配对，以定义与MYCN过表达合作的遗传途径，以诱导早期发作神经母细胞瘤。 斑马鱼酪氨酸羟化酶（ZTH）基因已经被克隆来用作这些筛选中的PSNS发育标记，并且它对PSNS谱系细胞的特异性通过RNA的原位分析证明。 在AIM 1中，将分析通过原位杂交识别的突变鱼，以确定发生突变发生的发育阶段（神经rest vs.交感神经细胞发育）。 接下来，每个突变的染色体位置将在斑马鱼基因组上映射，并检查与人NB样品中杂合性损失（LOH）和缺失的已知区域。 位置克隆将集中在最有可能删除或突变的人NB中的基因上。 在AIM 2中，通过使用斑马鱼酪氨酸羟化酶（Th）启动子在交感神经细胞中驱动人MYCN的表达来产生转基因斑马线。 然后，将分析这些线，以分析它们对自发性肿瘤发生的倾向，并越过AIM 1中分离的突变体，以鉴定导致加速NB形成的遗传病变的组合。 在人类肿瘤抑制基因同源物中含有突变的突变斑马鱼应提供可靠的动物模型，以阐明导致NB的分子途径。 一个远距离的目标是将这些模型用作第二代修饰符屏幕的起点，以识别引起PSN缺陷的基因的抑制剂和增强子，然后可以将其作为人类NB治疗干预的靶标。