Motor neuron disease modeling in Zebrafish

描述(由申请人提供)：1我们的最终目标是发现如何从成人干细胞再生出在退行性疾病期间被破坏的细胞。这项提议的具体目标是创造3条转基因斑马鱼，可以用来模拟运动神经元(MN)细胞的再生，运动神经元(MN)细胞是所有运动神经元疾病(MND)中丢失的4种细胞类型。斑马鱼具有惊人的细胞再生能力，甚至延伸到神经系统，包括成年斑马鱼的MN细胞。斑马鱼也是大规模正向遗传筛选的既定模型系统，通过随机突变基因组来识别特定生物过程所需的基因。为了结合这些属性，我们围绕一个可诱导的细胞消融平台开发了9种简单的筛选方法，可以用于识别再生-10缺陷突变，在这种情况下，是MN再生所需的基因。具体地说，将使用转基因11种方法将前药物转换酶硝基还原酶12(NTR)的表达靶向MN亚群。NTR的功能是将水溶性前体药物转化为细胞毒素，从而破坏特定表达该酶的MN。NTR和14个荧光报告基因(NTR-FP)之间的融合使得可以很容易地监测活着的斑马鱼中靶细胞的存在或不存在。在该系统中，FP丢失将指示MN退化16，而FP信号的后续增益提供MN再生的证据。通过使用高通量平板阅读器定量检测活斑马鱼中的荧光报告基因，可以进行18次大规模的遗传筛选(第二阶段)，以确定19MN再生所需的多个基因。因此，在像斑马鱼这样的脊椎动物模型系统中识别促进MN再生的分子因素应该为探索人类MND再生疗法的可能性提供一种手段。 与公共卫生相关：运动神经元是一种在许多衰弱的退行性疾病中失去的细胞类型，包括卢·格里克病。这项提议的目标是通过识别扰乱小型模式生物斑马鱼运动神经元再生的突变来发现运动神经元再生所需的基因。斑马鱼是一种具有再生能力的物种，甚至延伸到神经系统。在这些研究中产生的运动神经元疾病模型和获得的遗传学见解将有助于努力发现促进运动神经元再生的药物，从而为人类运动神经元疾病的再生治疗提供可能的途径。

DESCRIPTION (provided by applicant): 1 Our ultimate goal is to discover how cells targeted for destruction during degenerative disease 2 can be regenerated from adult stem cells. The specific goal of this proposal is to create 3 transgenic zebrafish that can be used to model the regeneration of motor neuron (MN) cells, the 4 cell type lost in all motor neuron diseases (MND). Zebrafish have a remarkable capacity for 5 cellular regeneration that extends even to the nervous system, including MN cells in adult 6 zebrafish. Zebrafish are also an established model system for large-scale forward genetic 7 screens, whereby the genome is randomly mutated to identify genes which are required for a 8 specific biological process. To combine these attributes, we have developed simple screening 9 methods around an inducible cellular ablation platform that can be used to identify regeneration- 10 deficient mutants, in this case, genes required for MN regeneration. Specifically, transgenic 11 methods will be used to target the expression of a pro-drug converting enzyme, nitroreductase 12 (NTR), to MN subpopulations. NTR functions to convert water soluble pro-drugs into cellular 13 toxins, thereby ablating the MN specifically expressing the enzyme. A fusion between NTR and 14 a fluorescent reporter (NTR-FP) allows the presence or absence of targeted cells to be easily 15 monitored over time in living zebrafish. In this system FP loss would indicate MN degeneration 16 while subsequent gains in FP signal provide evidence of MN regeneration. By using high- 17 throughput plate readers for quantitative detection of fluorescent reporters in living zebrafish, a 18 large-scale genetic screen could be performed (Phase II) to identify multiple genes required for 19 MN regeneration. Thus, the identification of molecular factors which promote MN regeneration 20 in a vertebrate model system such as zebrafish should provide a means to explore the 21 possibility of regenerative therapies for human MND. PUBLIC HEALTH RELEVANCE: Motor neurons are the cell type lost in a number of debilitating degenerative diseases, including Lou Gehrig's disease. The goal of this proposal is to discover genes required for motor neuron regeneration by identifying mutations that disrupt motor neuron regeneration in a small model organism, the zebrafish - a species with a regenerative capacity that extends even to the nervous system. The motor neuron disease models produced and genetic insights gained during these studies will facilitate efforts to discover drugs that promote motor neuron regeneration, thus suggesting possible avenues of regenerative therapies for human motor neuron diseases.