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，实验室发表论文三篇。 Fero 等人与 NICHD 的 Burgess 博士合作发表的一篇论文发表在《疾病模型和机制》上。 在本文中，我们鉴定了斑马鱼的早期胚胎运动突变体，该突变体是由转基因整合到假磷酸酶双特异性磷酸酶 27 (dusp27) 基因中引起的。 Dusp27 突变体在胚胎和幼虫阶段表现出近乎完全瘫痪，产生极低水平的自发卷曲运动和大大减弱的触摸反应。 dusp27 的缺失不会阻止体节发生，但会导致肌纤维收缩装置的严重瓦解。突变体中的肌节结构几乎完全不存在，仅观察到罕见的三联体。这些发现首次表明 dusp27 作为肌纤维成熟所需基因的功能作用，并为分析控制肌原纤维组装的机制提供了动物模型。 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 蛋白是小 GTP 结合蛋白 Ras 超家族的成员，可与 Ca2+ 通道 β 亚基相互作用，从而改变电压门控 Ca2+ 通道功能。此外，RGK 蛋白影响多种细胞过程，例如细胞骨架重排、神经元树突复杂性和突触形成。为了探究 RGK 蛋白-Ca2+ 通道相互作用的系统发育起源，我们在后口动物和原口动物超门的遗传多样性生物体的基因组中鉴定了潜在的 RGK 样蛋白同源物。根据哺乳动物 RGK 蛋白表达的报道，从斑马鱼和果蝇中克隆的 RGK 样蛋白同源物在哺乳动物交感神经元中表达，可降低 Ca2+ 电流密度。来自跨越原口动物/后口动物分歧的进化多样性生物体的序列比对揭示了参与 RGK 蛋白-Cavβ 亚基相互作用的 RGK G 结构域内残基的保守性。此外，C端11个残基高度保守，构成RGK蛋白特有但功能未知的特征序列。总而言之，这些数据表明 RGK 蛋白以及改变 Ca2+ 通道功能的能力，起源于大约 5.5 亿年前原口动物与后口动物分裂之前的祖先。 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一种斑马鱼 AChR δ 亚基突变体，它表现出两种不同的 NMJ 表型，分别针对两种肌纤维类型：慢肌纤维或快肌纤维。 AChR 亚基的突变（在神经肌肉接头 (NMJ) 中表达为五聚体）会导致各种类型的先天性肌无力综合征。在AChR五聚体中，随着动物的发育，成年ε亚基逐渐取代胚胎γ亚基。由于亚基组成的这种转变，特定亚基的突变会导致突触表型随着发育年龄而变化。然而，任何 AChR 亚基的突变都被认为同等地影响所有肌纤维的 NMJ。 δ亚基中含有新发现的点突变的纯合鱼在慢肌中形成有功能的AChR，而快肌中的受体则无功能。为了检验慢肌和快肌中不同亚基组成构成不同表型的假设，我们使用特异性抗体检查了 NMJ 中 ε/γ 亚基的存在。野生型和突变型幼虫的慢肌突触均缺乏 ε/γ 亚基。这些在斑马鱼中的发现表明，人类先天性肌无力综合征的一些突变可能对慢肌纤维和快肌纤维产生不同的影响。 实验室将于2015年搬迁至大阪医科大学，2014年我们就做好了搬迁的准备。几个项目，其中一些主要在实验室进行，另一些与 NIH 的多个实验室合作，将很快完成或将在大阪的一个新实验室继续进行。实验室人员在多次会议上介绍了正在进行的工作，包括大西洋中部区域斑马鱼会议和第11届国际斑马鱼发育与遗传学会议。小野文仁受邀在大阪大学、国立眼科研究所和 NIH 研究节研讨会上发表演讲。