Multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation.
Multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation.
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DOI：
10.3791/2093
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发表时间：
2010-09-20
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影响因子：
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通讯作者：
Mumm, Jeff S
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作者：
Ariga, Junko;Walker, Steven L;Mumm, Jeff S
活体斑马鱼幼鱼的高分辨率延时成像可用于可视化生物过程如何展开（综述见（1））。在相邻细胞类型中表达不同荧光报告基因的复合转基因鱼提供了一种随时间推移跟踪细胞相互作用（2）和/或组织水平对实验操作的响应的方法。在这个视频中，我们展示了可以用于成像多种转基因标记的细胞类型连续在个别鱼随着时间的推移，可以跨越几分钟到几天的方法。所描述的技术适用于寻求使相邻细胞类型随时间的“行为”相关的任何研究，包括：1）用于在连续几天内对大量鱼进行成像的连续“捕获和释放”方法，2）用于分离具有重叠激发/发射分布的荧光团的简化方法（例如，GFP和YFP），3）使用色素减退的突变株系以将可用于高分辨率成像的时间窗延长到发育的晚期幼虫阶段，4）使用膜靶向荧光报告物以揭示单个细胞的精细形态细节以及较大细胞群体中的细胞细节，和5）先前描述的用于转基因靶向细胞类型的化学诱导消融的方法;即，硝基还原酶（NTR）介导的前药底物如甲硝唑（MTZ）转化为细胞毒性衍生物（3，5）。作为这些方法的一个例子，我们将在几天内可视化单个鱼内视网膜双极神经元亚型的消融和再生。同时，我们将监测其他几种视网膜细胞类型，包括邻近的非靶向双极细胞和潜在的变性刺激的视网膜干细胞（即，M？ller glia）。这种策略正在我们的实验室中应用，以表征细胞和组织水平（例如，干细胞小生境）对靶向神经元细胞类型的选择性丧失和再生的响应。
High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see (1)). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions (2) and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives (3,5). As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., Mϋller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.