Genetics of neuromuscular synaptogenesis in zebrafish

描述（由申请人提供）：本修订提案的目标是以斑马鱼为模型系统，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。我们参与了由该部门的玛丽穆林斯和迈克尔格拉纳托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教授。使用突触囊泡的抗体标记突触前末梢，荧光标记abungarotoxin标记乙酰胆碱受体（AChR）簇，并在完整的鱼光学显微镜，我的实验室确定了8个突变体的缺陷在不同方面的神经肌肉突触在受精后48小时（hpf）。这些包括具有减少的突触前和/或突触后特化的突变体、突触前或突触后特化的异常大小、对齐或聚类，以及体壁肌肉组织内的异常初级和/或次级运动轴突分支，导致异常突触定位。这些突变表型中的许多在斑马鱼中以前没有报道过，并且与其他生物中描述的那些不同。我在此提议：1）在野生型和转基因鱼中使用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、斯莱特林和xavier的细胞和分子表型; 3）确定bossu、xavier、pongo和斯莱特林的精细图谱位置;以及4）确定bossu和xavier中突变基因的身份。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。这些突变体中潜在遗传缺陷的鉴定将扩大我们对介导神经肌肉和其他突触形成和功能的分子机制的理解。

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.