In vivo studies of neural system in zebrafish

斑马鱼神经系统的体内研究

基本信息

批准号：
7963854
负责人：
Fumihito Ono
金额：
$ 90.52万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
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/7963854
关键词：
American Animals Behavior Biological Models Brain Breeding Cell Lineage Cell membrane Cell physiology Cells Cessation of life Characteristics Chimeric Proteins Cholinergic Receptors Collaborations Complex Confocal Microscopy Development District of Columbia Dorsal Embryo Embryo Deaths Ethanol European Experimental Models Fertilization Festival First Pregnancy Trimester Fishes Genes Genetic Germ Lines Health Sciences Human Institutes Interneurons Italy Japan Journals Knowledge Left Locomotion Manuscripts Molecular Biology Motor Neurons National Institute of Child Health and Human Development National Institute of Neurological Disorders and Stroke Nervous System Physiology Nervous system structure Neuromuscular Junction Neurosciences Optics Oregon Paper Positioning Attribute Potato Proteins Publishing Research Role Rome Sexual Maturation Siblings Skeletal Muscle Spinal Spinal Cord Swimming Synapses Synaptic Transmission System Techniques Time Transgenes Transgenic Organisms United States National Institutes of Health Universities Vertebrates Visit Work Zebrafish abstracting alcohol research alcohol response cell type feeding fetal gene therapy in vivo meetings mutant null mutation posters professor red fluorescent protein relating to nervous system research study transcription factor transmission process

项目摘要

In FY2009, our section continued projects focused on the nervous system. All experiments use the nervous system of zebrafish as the experimental model. Some experiments focus on the neuromuscular junction, and others directly examine the brain. With regard to the alcohol research, we started to examine the change of nervous system in ethanol-treated zebrafish embryos in collaboration with Dr. Harold Burgess at NICHD. All projects combine genetics, confocal microscopy, molecular biology and cellular physiology. Through most of FY09, the section had four people including the PI. Dr.Takanori Ikenaga, who worked as a visiting fellow, obtained the position of assistant professor in the University of Hyogo, Japan, and left in 03/2009. A new visiting fellow, Dr. Jee-Young Park, joined the lab in 05/2009. We presented three posters in the Neuroscience Meeting held at Washington DC, one poster in the ARVO meeting held in Fort Lauderdale, FL, a poster and two talks in the 6th European Zebrafish meeting held in Rome, Italy, and a poster in the NIH research festival. These presentations include results of collaborations with Dr. Paul Brehm at the Oregon Health and Science University, Dr.Alan Davidson at Harvard University, Dr.Koichi Kawakami at the National institute of genetics in Japan, Dr.Ralph Nelson at NINDS, and Dr. Victoria Connaughton at the American University. In 12/2008, we published a paper in the Journal of Neuroscience entitled: A modified acetylcholine receptor subunit enables a null mutant to survive beyond the sexual maturation. The abstract is as follows: The contraction of skeletal muscle is dependent upon the synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester with characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the delta-subunit in zebrafish (sofa potato; sop-/-) leads to the death of embryos around 5 days post-fertilization (dpf). In sop-/- mutants, we expressed modified delta-subunits, with one (1YFP) or two yellow fluorescent protein (2YFP) molecules fused at the intracellular loop. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a stably-rescued line of sop-/-. These 2YFP/sop-/- embryos can mount escape behavior close to wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display normal locomotion, including such complex behaviors as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. Altered synaptic strength resulting from the transgene delineates requirements for gene therapies of NMJ. Another manuscript currently under review is entitled: Formation of spinal network in zebrafish determined by domain-specific Pax genes. The abstract is as follows: In the formation of the spinal network, various transcription factors interact to develop specific cell types. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) trapped the pax8 gene. RFP insertion marked pax8-lineage cells and abolished pax8 expression in homozygous embryos. Pax8 and pax2a are expressed in dorsal regions of the spinal cord. We examined the effect of pax genes on interneuron development. In pax8 mutants, the pax8-lineage cells undergo normal differentiation and the swimming behavior remained intact. In contrast, in pax2a/pax8 double mutants, the pax8-lineage cells displayed altered cell fates. Circumferential Descending (CiD) interneurons, which never express pax8 in normal embryos, arose from the pax8-lineage. The use of newly identified gene trap line revealed roles of transcription factors in the network formation of the spinal cord.

2009财年，我们部门继续开展以神经系统为重点的项目。所有实验均使用斑马鱼的神经系统作为实验模型。一些实验侧重于神经肌肉接头，另一些则直接检查大脑。关于酒精研究，我们与 NICHD 的 Harold Burgess 博士合作，开始研究乙醇处理的斑马鱼胚胎中神经系统的变化。所有项目都结合了遗传学、共焦显微镜、分子生物学和细胞生理学。在 2009 财年的大部分时间里，该部门共有 4 名员工，其中包括 PI。 Takanori Ikenaga博士以访问学者身份获得日本兵库大学助理教授职位，并于03/2009离开。一位新的客座研究员 Jee-Young Park 博士于 05/2009 年加入该实验室。我们在华盛顿特区举行的神经科学会议上展示了三张海报，在佛罗里达州劳德代尔堡举行的 ARVO 会议上展示了一张海报，在意大利罗马举行的第六届欧洲斑马鱼会议上展示了一张海报和两场演讲，在 NIH 研究中展示了一张海报节日。这些演讲包括与俄勒冈健康与科学大学的 Paul Brehm 博士、哈佛大学的 Alan Davidson 博士、日本国立遗传学研究所的 Koichi Kawakami 博士、NINDS 的 Ralph Nelson 博士和维多利亚·康诺顿在美国大学。 2008 年 12 月，我们在《神经科学杂志》上发表了一篇论文，题为：修饰的乙酰胆碱受体亚基使无效突变体能够在性成熟之后存活。摘要如下：骨骼肌的收缩依赖于神经肌肉接头（NMJ）处乙酰胆碱受体（AChR）的突触传递。缺乏 AChR 亚基会导致人类胎儿运动不能，导致妊娠早期死亡，并具有胎儿运动不能变形序列 (FADS) 的特征。斑马鱼（沙发马铃薯；sop-/-）中 δ 亚基的相应无效突变会导致胚胎在受精后 5 天左右死亡 (dpf)。在sop-/-突变体中，我们表达了修饰的δ亚基，其中一个（1YFP）或两个黄色荧光蛋白（2YFP）分子融合在细胞内环处。含有这些融合蛋白的 AChR 会发出荧光，在质膜上组装，在运动神经元末梢下形成簇，并产生突触电流。我们筛选了转基因的种系传播，并建立了稳定挽救的 sop-/- 系。这些 2YFP/sop-/- 胚胎可以产生接近野生型兄弟姐妹的逃逸行为。与野生型鱼相比，这些胚胎中的突触电流幅度更小，上升时间更慢，衰减更慢。值得注意的是，这些胚胎生长到成年并表现出正常的运动，包括进食和繁殖等复杂的行为。据我们所知，这是第一例相当于人类妊娠早期致死率的突变动物被转基因拯救并存活至成年。转基因引起的突触强度改变描述了 NMJ 基因治疗的要求。目前正在审查的另一篇手稿的标题是：由特定领域的 Pax 基因决定的斑马鱼脊柱网络的形成。摘要如下：在脊髓网络的形成过程中，各种转录因子相互作用以发育出特定的细胞类型。使用基因捕获技术，我们建立了一个稳定的斑马鱼品系，其中红色荧光蛋白（RFP）捕获了 pax8 基因。 RFP 插入标记了 pax8 谱系细胞并消除了纯合胚胎中的 pax8 表达。 Pax8 和 pax2a 在脊髓的背侧区域表达。我们检查了 pax 基因对中间神经元发育的影响。在 pax8 突变体中，pax8 谱系细胞经历正常分化，并且游泳行为保持完整。相比之下，在 pax2a/pax8 双突变体中，pax8 谱系细胞表现出改变的细胞命运。周向降序（CiD）中间神经元在正常胚胎中从不表达 pax8，源自 pax8 谱系。新鉴定的基因捕获线的使用揭示了转录因子在脊髓网络形成中的作用。