Genetics of neuromuscular synaptogenesis in zebrafish

斑马鱼神经肌肉突触发生的遗传学

• 批准号: 6984941
• 负责人: RITA J. BALICE-GORDON
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-18 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6984941
• 关键词: acetylcholine; alternatives to animals in research; cholinergic receptors; confocal scanning microscopy; developmental genetics; electrophysiology; gene expression; gene mutation; genetically modified animals; green fluorescent proteins; in situ hybridization; molecular cloning; molecular genetics; neuromuscular system; polymerase chain reaction; synaptogenesis; terminal nick end labeling; zebrafish

DESCRIPTION (provided by applicant): The goal of this revised proposal is to characterize vertebrate genes that play a role in neuromuscular synapse formation and maintenance, using zebrafish as a model system. We participated in a small-scale mutagenesis screen conducted by Drs. Mary Mullins and Michael Granato in the Dept. of Cell and Developmental Biology at the University of Pennsylvania. Using antibodies against synaptic vesicles to label presynaptic terminals, fluorescently conjugated abungarotoxin to label acetylcholine receptor (AChR) clusters, and light microscopy in intact fish, my lab identified eight mutants with defects in different aspects of neuromuscular synaptogenesis at 48 hours post fertilization (hpf). These include mutants with reduced pre- and/or postsynaptic specializations, abnormal size, alignment or clustering of pre- or postsynaptic specializations, and aberrant primary and/or secondary motor axon branching within the body wall musculature, resulting in aberrant synaptic localization. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Here I propose to: 1) Study neuromuscular synaptogenesis using in vivo imaging in wild type and transgenic fish in which presynaptic axons and terminals are labeled with GFP, and other approaches; 2) Determine the cellular and molecular phenotype of bossu, pongo, slytherin and xavier; 3) Determine a fine map location for bossu, xavier, pongo and slytherin; and 4) Determine the identity of the mutant gene in bossu and xavier. Together, these approaches will allow us to study the genetic, molecular and cellular mechanisms underlying synaptogenesis in vertebrates. Identification of the underlying genetic defects in these mutants will expand our understanding of the molecular mechanisms that mediate the formation and function of neuromuscular and other synapses.

描述（由申请人提供）：本修订提案的目标是使用斑马鱼作为模型系统来表征在神经肌肉突触形成和维持中发挥作用的脊椎动物基因。我们参加了由博士进行的小规模诱变筛选。宾夕法尼亚大学细胞与发育生物学系的玛丽·穆林斯 (Mary Mullins) 和迈克尔·格拉纳托 (Michael Granato)。我的实验室使用针对突触小泡的抗体来标记突触前末端，使用荧光结合的阿布加罗毒素来标记乙酰胆碱受体 (AChR) 簇，并在完整的鱼中使用光学显微镜，在受精后 48 小时 (hpf) 时，鉴定出 8 个在神经肌肉突触发生的不同方面存在缺陷的突变体。 。这些包括突触前和/或突触后特化减少的突变体，突触前或突触后特化的大小、排列或聚集异常，以及体壁肌肉组织内异常的初级和/或次级运动轴突分支，导致异常的突触定位。其中许多突变表型以前从未在斑马鱼中报道过，并且与其他生物体中描述的表型不同。在这里我建议：1）使用野生型和转基因鱼的体内成像研究神经肌肉突触发生，其中突触前轴突和末端用GFP标记，以及其他方法； 2）确定bossu、pongo、slytherin和xavier的细胞和分子表型； 3）确定bossu、xavier、pongo和slytherin的精细地图位置； 4)确定bossu和xavier突变基因的身份。总之，这些方法将使我们能够研究脊椎动物突触发生的遗传、分子和细胞机制。识别这些突变体中潜在的遗传缺陷将扩大我们对介导神经肌肉和其他突触的形成和功能的分子机制的理解。