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退行性疾病中被破坏的细胞2。本提案的具体目标是创造3种转基因斑马鱼，可用于模拟运动神经元（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。斑马鱼也是一个成熟的模型系统，用于大规模的正向遗传筛选，通过随机突变基因组来识别特定生物过程所需的基因。为了结合这些特性，我们围绕诱导细胞消融平台开发了简单的筛选方法，可用于识别再生缺陷突变体，在这种情况下，是MN再生所需的基因。具体而言，转基因方法将用于靶向MN亚群中药物前转化酶硝基还原酶12 （NTR）的表达。NTR的功能是将水溶性前药转化为细胞毒素，从而吞噬特异性表达该酶的MN。NTR和荧光报告基因（NTR- fp）之间的融合使得在活斑马鱼中很容易监测目标细胞的存在或缺失。在这个系统中，FP信号的丢失表明MN退化，而随后FP信号的增加则证明MN再生。利用高通量读板仪对活斑马鱼荧光报告基因进行定量检测，可以进行大规模基因筛选（II期），以鉴定19 MN再生所需的多个基因。因此，在斑马鱼等脊椎动物模型系统中发现促进MN再生的分子因子将为探索人类MND再生治疗的可能性提供一种手段。

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.