Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons.
Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons.
复制标题
红外激光诱导的基因表达，用于跟踪单一秀丽隐杆线虫胚胎神经元的发育和功能。
DOI：
10.1038/ncomms14100
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发表时间：
2017-01-18
影响因子：
16.6
通讯作者：
Shaham S
中科院分区：
文献类型：
--
作者：
Singhal A;Shaham S
在活体中可视化神经回路组装需要跟踪光学可分辨神经突起的生长。秀丽线虫胚胎神经系统由222个神经元和56个胶质细胞组成，对发育的综合研究很有吸引力；然而，胚胎报告基因被广泛表达，这使得单一神经突起的跟踪/操作具有挑战性。我们提出了一种方法，使用红外激光，在小的亚系(一到四个细胞)中进行可重复的热依赖基因表达，而不会造成辐射损伤。我们超越了原则证明，使用我们的系统来标记和跟踪早期神经系统组装过程中的单个神经元。我们揭示了轴突生长的逆行延伸机制，并揭示了SAX-3/Robo和VAB-1/EphR突变体中轴突导向缺陷的病因。我们还进行特定细胞的抢救，确定DAF-6/补丁相关的作用部位在感官发育过程中。使用我们的系统同时对细胞进行消融和标记，揭示了胶质细胞在树突延伸中的作用。我们的方法可以应用于其他光学/红外透明生物，并为高分辨率系统分析线虫的形态发生打开了大门。一直缺乏标记单个神经元并跟踪它们在线虫中的发育的工具。Singhal等人。优化了一种利用红外激光在线虫胚胎发育过程中诱导细胞分辨率的热依赖基因表达的方法，并表明它可以揭示胚胎形态发生的新方面。
Visualizing neural-circuit assembly in vivo requires tracking growth of optically resolvable neurites. The Caenorhabditis elegans embryonic nervous system, comprising 222 neurons and 56 glia, is attractive for comprehensive studies of development; however, embryonic reporters are broadly expressed, making single-neurite tracking/manipulation challenging. We present a method, using an infrared laser, for reproducible heat-dependent gene expression in small sublineages (one to four cells) without radiation damage. We go beyond proof-of-principle, and use our system to label and track single neurons during early nervous-system assembly. We uncover a retrograde extension mechanism for axon growth, and reveal the aetiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants. We also perform cell-specific rescues, determining DAF-6/patched-related site of action during sensory-organ development. Simultaneous ablation and labelling of cells using our system reveals roles for glia in dendrite extension. Our method can be applied to other optically/IR-transparent organisms, and opens the door to high-resolution systematic analyses of C. elegans morphogenesis. Tools to label single neurons and track their development in C. elegans have been lacking. Singhal et al. optimized a method, which applies infrared laser to induce heat-dependent gene expression at cellular resolution in developing C. elegans embryos, and show that it can uncover new aspects of embryo morphogenesis.