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体干细胞再生在退行性疾病2期间靶向破坏的细胞。该提案的具体目标是创建3种转基因斑马鱼，可用于模拟运动神经元（MN）细胞的再生，这是所有运动神经元疾病（MND）中丢失的4种细胞类型。斑马鱼具有非凡的细胞再生能力，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已建立的模型系统，其中基因组随机突变以鉴定特定生物过程所需的基因。为了将这些属性联合收割机结合起来，我们已经开发了围绕可诱导细胞消融平台的简单筛选9种方法，该方法可用于鉴定再生缺陷突变体，在这种情况下，MN再生所需的基因。具体地，转基因方法将用于将前药转化酶硝基还原酶12（NTR）的表达靶向MN亚群。NTR的功能是将水溶性前药转化为细胞毒素，从而消除特异性表达该酶的MN。NTR和荧光报告分子（NTR-FP）之间的融合允许在活斑马鱼中随时间容易地监测靶细胞的存在或不存在。在该系统中，FP损失将指示MN变性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17板读数器定量检测活斑马鱼中的荧光报告基因，可以进行18大规模遗传筛选（第II阶段）以鉴定19 MN再生所需的多个基因。因此，在脊椎动物模型系统如斑马鱼中鉴定促进MN再生的分子因子20应该提供一种探索人类MND再生疗法的可能性的手段。 公共卫生相关性：运动神经元是一种细胞类型，在许多衰弱性退行性疾病中丢失，包括Lou Gehrig病。这项提案的目标是通过识别破坏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甚至延伸到神经系统的物种。在这些研究中产生的运动神经元疾病模型和获得的遗传见解将有助于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疗法提供可能的途径。

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.