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：
7732131
负责人：
Fumihito Ono
金额：
$ 104.91万
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/7732131
关键词：
Acetylcholine Agrin Animals Behavior Biological Models Breeding Cell membrane Cessation of life Characteristics Chemical Synapse Chimeric Proteins Cholinergic Receptors Complex Embryo Embryo Deaths Equilibrium Fertilization First Pregnancy Trimester Fishes Genetic Germ Lines Human Journals Knowledge Locomotion Manuscripts Molecular Motor Neurons Nervous System Physiology Nervous system structure Neuromuscular Junction Neurosciences Optics Paper Pathway interactions Potato Process Protein Tyrosine Kinase Proteins Publishing Science Sexual Maturation Siblings Side Signal Transduction Skeletal Muscle Synapses Synaptic Transmission System Techniques Time Transgenes Transgenic Organisms Vertebrates Work Zebrafish abstracting alcohol response day density feeding fetal gene therapy in vivo mutant null mutation posters postsynaptic presynaptic receptor relating to nervous system synaptogenesis transmission process

项目摘要

In FY2008, the lab setup was completed, and the section now has four people including the PI. We presented three posters in two meetings: Two in the Neuroscience Meeting held at San Deigo CA, and one in the zebrafish meeting held at Madison WI. We published one review article entitled "Emerging picture of synapse formation: A balance of two opposing pathways" in the journal "Science Signaling". The abstract is as follows: The neuromuscular junction (NMJ) is a well-studied chemical synapse and has served as a tractable model system to clarify how synapse formation occurs. Proteins on both the presynaptic and postsynaptic side collaborate to induce the high-density accumulation of acetylcholine receptors (AChRs) at the NMJ. Two opposing pathways work in this process: A dispersing pathway works through acetylcholine and the AChR and a clustering pathway works through agrin and the transmembrane tyrosine kinase MuSK. The molecular mechanisms underlying these two signaling cascades are beginning to be understood. We also submitted a paper entitled "Modified acetylcholine receptor subunit enables a null mutant to survive beyond the sexual maturation" to the Journal of Neuroscience. The manuscript received a favorable review and is under revision now. 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 -subunit in zebrafish (sofa potato; sop-/-) leads to the death of embryos around 5 days post-fertilization (dpf). In sop-/- mutants, we expressed modified -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.

2008财年，实验室建设完成，该部门目前包括PI在内共有四人。我们在两次会议上展示了三张海报：两张是在加利福尼亚州圣代戈举行的神经科学会议上，一张是在威斯康星州麦迪逊举行的斑马鱼会议上。我们在《科学信号》杂志上发表了一篇题为“突触形成的新景象：两种相反途径的平衡”的评论文章。摘要如下：神经肌肉接头（NMJ）是一种经过充分研究的化学突触，并作为一个易于处理的模型系统来阐明突触形成是如何发生的。突触前和突触后侧的蛋白质协同诱导 NMJ 处乙酰胆碱受体 (AChR) 的高密度积累。此过程中有两条相反的途径起作用：分散途径通过乙酰胆碱和 AChR 发挥作用，聚集途径通过集聚蛋白和跨膜酪氨酸激酶 MuSK 发挥作用。人们开始了解这两种信号级联背后的分子机制。我们还向《神经科学杂志》提交了一篇题为“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生存”的论文。该手稿获得了好评，目前正在修改中。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-/-）中相应的 β 亚基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β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。