Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development
Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development
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秀丽隐杆线虫胚胎后发育过程中细胞动力学的实时成像
DOI：
10.1038/nprot.2012.128
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发表时间：
2012-12-01
期刊：
影响因子：
14.8
通讯作者：
Ou, Guangshuo
中科院分区：
文献类型：
--
作者：
Chai, Yongping;Li, Wei;Ou, Guangshuo
胚后发育是生物体在胚胎生长之后成熟的一个重要过程。尽管近年来通过荧光延时显微镜在理解胚胎发育方面取得了关键进展，但针对胚后发育的活细胞成像研究相对较少。在此，我们介绍一种对线虫秀丽隐杆线虫幼虫发育进行成像的实验方案。我们的方案阐述了荧光转基因秀丽隐杆线虫的构建、线虫幼虫的固定以及延时显微镜分析。为提高成像通量，我们开发了一种针对秀丽隐杆线虫的三荧光成像方法，采用了经线虫优化的蓝色荧光蛋白（TagBFP）、绿色荧光蛋白（GFP）和mCherry。此前，该方案已应用于Q神经母细胞不对称分裂、迁移和凋亡的延时成像分析，而我们在此表明它也可用于对L1期幼虫的神经突发生进行成像。该方案还可能有其他应用。整个方案可在3小时内完成，或许能为理解胚后发育提供新的视角。
Postembryonic development is an important process of organismal maturation after embryonic growth. Despite key progress in recent years in understanding embryonic development via fluorescence time-lapse microscopy, comparatively less live-cell imaging of postembryonic development has been done. Here we describe a protocol to image larval development in the nematode Caenorhabditis elegans. Our protocol describes the construction of fluorescent transgenic C. elegans, immobilization of worm larvae and time-lapse microscopy analysis. To improve the throughput of imaging, we developed a C. elegans triple-fluorescence imaging approach with a worm-optimized blue fluorescent protein (TagBFP), green fluorescent protein (GFP) and mCherry. This protocol has been previously applied to time-lapse imaging analysis of Q neuroblast asymmetric division, migration and apoptosis, and we show here that it can also be used to image neuritogenesis in the L1 larvae. Other applications are also possible. The protocol can be completed within 3 h and may provide insights into understanding postembryonic development.