In vivo studies of neural system in zebrafish

斑马鱼神经系统的体内研究

• 批准号: 8941394
• 负责人: Fumihito Ono
• 金额: $ 72.52万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 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/8941394
• 关键词: Adult; Affect; Age; Animal Model; Animals; Antibodies; Behavior; Biological Models; C-terminal; Cell physiology; Collaborations; Congenital Myasthenic Syndromes; DNA Sequence Rearrangement; Data; Deltastab; Development; Disease model; Drosophila genus; Drosophila melanogaster; Embryo; Exhibits; Festival; Fishes; GTP-Binding Proteins; Genes; Genetic; Genome; Homologous Protein; Human; Human Resources; International; Journals; Larva; Movement; Muscle; Muscle Fibers; Mutation; Myofibrils; National Eye Institute; National Institute of Child Health and Human Development; National Institute on Alcohol Abuse and Alcoholism; Nervous System Physiology; Nervous system structure; Neuromuscular Junction; Neurons; Neurosciences; Optics; Organism; Paper; Paralysed; Phenotype; Phosphoric Monoester Hydrolases; Phylogenetic Analysis; Point Mutation; Proteins; Publishing; Reporting; Research; Role; Sequence Alignment; Specificity; Staging; Structure; Synapses; System; Techniques; Testing; Touch sensation; Transgenes; Transgenic Organisms; Triad Acrylic Resin; United States National Institutes of Health; Universities; Vertebrates; Work; Zebrafish; alcohol response; cell motility; density; in vivo; medical schools; meetings; member; mutant; prevent; receptor; relating to nervous system; response; somitogenesis; symposium; synaptogenesis; voltage; zebrafish development

In 2014, three papers were published from the lab. A paper by Fero et al., a collaboration with Dr. Burgess at NICHD, was published in Disease Models and Mechanisms. In this paper, we identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. Dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly. Another paper by Won et al. , in collaboration with Dr. Ikeda at NIAAA, was published in PLoS ONE. In this paper, RGK proteins were studied. RGK proteins are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel β subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein-Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein--Cavβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggested that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago. A third paper by Park et al. was published in the Journal of Neuroscience. We reported in this paper a zebrafish mutant of the AChR δ subunit that exhibits two distinct NMJ phenotypes specific to two muscle fiber types: slow or fast. Mutations in AChR subunits, expressed as pentamers in neuromuscular junctions (NMJs), cause various types of congenital myasthenic syndromes. In AChR pentamers, the adult ε subunit gradually replaces the embryonic γ subunit as the animal develops. Because of this switch in subunit composition, mutations in specific subunits result in synaptic phenotypes that change with developmental age. However, a mutation in any AChR subunit is considered to affect the NMJs of all muscle fibers equally. Homozygous fish harboring a newly identified point mutation in the δ subunit formed functional AChRs in slow muscles, whereas receptors in fast muscles were nonfunctional. To test the hypothesis that different subunit compositions in slow and fast muscles underlie distinct phenotypes, we examined the presence of ε/γ subunits in NMJs using specific antibodies. Both wild-type and mutant larvae lacked ε/γ subunits in slow muscle synapses. These findings in zebrafish suggested that some mutations in human congenital myasthenic syndromes may affect slow and fast muscle fibers differently. The lab will move to the Osaka Medical College in 2015, and in 2014 we prepared for the transfer. Several projects, some mainly performed in the lab and others in collaboration with multiple labs at NIH, will be completed soon or will be continued in a new lab at Osaka. Personnel in the lab presented on-going works in several meetings, including the Mid-Atlantic Regional Zebrafish Meeting and the 11th International Conference on Zebrafish Development and Genetics. Fumihito Ono gave invited talks at the University of Osaka, National Eye Institute and the NIH research festival symposium.

2014年，实验室发表了三篇论文。与NICHD的Burgess博士合作Fero等人的论文发表在疾病模型和机制上。 在本文中，我们鉴定了斑马鱼中的早期胚胎运动突变体，这是由于将转基因整合到伪磷酸酶双重特异性磷酸酶27（DUSP27）基因引起的。 DUSP27突变体在胚胎和幼虫阶段几乎完全瘫痪，产生了极低水平的自发盘绕运动，并且触摸反应大大减少。 DUSP27的损失不能阻止体重病，但会导致肌肉纤维中收缩仪的严重混乱。突变体中的肉类结构几乎完全不存在，只有罕见的三合会。这些发现是第一个将DUSP27作为肌纤维成熟所需的基因的功能作用，并提供了一种动物模型来分析控制肌纤维组装的机制。 Won等人的另一篇论文。 ，与NIAAA的Ikeda博士合作，发表在PLOS One上。在本文中，研究了RGK蛋白。 RGK蛋白是与Ca2+通道β亚基相互作用的小GTP结合蛋白的RAS超家族的成员，以改变电压门控Ca2+通道功能。此外，RGK蛋白会影响几个细胞过程，例如细胞骨架重排，神经元树突复杂性和突触形成。为了探测RGK蛋白-CA2+通道相互作用的系统发育起源，我们确定了来自氘代表和原始动物超级球的遗传多样生物的基因组中潜在的RGK样蛋白同源物。从哺乳动物交感神经元中表达的danio rerio（斑马鱼）和果蝇果蝇（果蝇）和果蝇果蝇（果蝇）克隆的RGK样蛋白同源物降低了Ca2+电流密度，因为哺乳动物RGK蛋白的表达据报道。跨越原始的生物体/氘代表师分裂的进化多样的生物的序列比对揭示了与RGK蛋白-CAVβ亚基相互作用相关的RGK G-domain中残基的保存。此外，C末端11个残基是高度保守的，并且构成了RGK蛋白独有的签名序列，但功能未知。综上所述，这些数据表明，RGK蛋白以及修改Ca2+通道功能的能力是由大约5.5亿年前从氘代表分裂的祖先提出的。 Park等人的第三篇论文。发表在《神经科学杂志》上。我们在本文中报道了ACHRδ亚基的斑马鱼突变体，该突变体表现出对两种肌肉纤维类型的两种不同的NMJ表型：缓慢或快速。 ACHR亚基中的突变，在神经肌肉连接（NMJS）中表示为五聚体，引起各种类型的先天性肌无力综合征。在ACHR五聚体中，随着动物的发展，成年ε亚基逐渐取代了胚胎γ亚基。由于亚基组成的这种转换，特定亚基的突变会导致突触表型随发育年龄的变化。但是，任何ACHR亚基中的突变都被认为会平均影响所有肌肉纤维的NMJ。在缓慢的肌肉中形成功能性ACHR的纯合鱼，具有新鉴定的点突变，而快速肌肉中的受体则是无功能的。为了检验以下假设：慢速和快速肌肉中不同的亚基组成是不同的表型，我们使用特定的抗体检查了NMJ中ε/γ亚基的存在。野生型和突变幼虫都缺乏慢肌突触中的ε/γ亚基。斑马鱼中的这些发现表明，人类先天性疗法综合征中的某些突变可能会对缓慢和快速的肌肉纤维产生不同的影响。 该实验室将于2015年搬到大阪医学院，并于2014年为转会做准备。一些项目，一些项目主要在实验室中执行，另一些项目将与NIH的多个实验室合作，或者将很快完成，或者将继续在大阪的新实验室中继续。实验室的人员在几次会议上介绍了正在进行的著作，包括中大西洋地区斑马鱼会议和第11届斑马鱼发展与遗传学国际会议。 Fumihito Ono在大阪大学，国家眼科研究所和NIH研究节研讨会上进行了邀请的会谈。