中國科學(xué)院生物物理研究所歐光朔研究組在2012年12月期的Nature Protocols上發(fā)表題為Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development的文章,，介紹他們發(fā)展的研究線蟲胚胎后發(fā)育的熒光活體顯微成像方法。
胚胎后發(fā)育是生命體一個(gè)重要的發(fā)育時(shí)期,。例如,，線蟲的959個(gè)體細(xì)胞中有400多個(gè)是在胚胎后時(shí)期產(chǎn)生的。觀察線蟲胚胎時(shí)期發(fā)育的顯微成像技術(shù)相對成熟,，而研究線蟲胚胎后發(fā)育的活體熒光顯微成像方法缺乏。
該文章系統(tǒng)介紹了觀察活體線蟲胚胎后發(fā)育時(shí)期細(xì)胞動態(tài)的方法,，并對可能的技術(shù)難點(diǎn)進(jìn)行了討論,。歐光朔研究組將這項(xiàng)成像技術(shù)與線蟲遺傳學(xué)的結(jié)合，發(fā)現(xiàn)了遷移細(xì)胞的分子標(biāo)識(Ou & Vale, Journal of Cell Biology, 2009)、 一種新的細(xì)胞不對稱分裂方式(Ou et al., Science, 2010) ,、自體吞噬基因在凋亡細(xì)胞降解中的作用(Li et al., Journal of Cell Biology, 2012)等,。
該項(xiàng)工作得到科技部、國家自然科學(xué)基金委和“青年千人計(jì)劃”的資助,。(生物谷Bioon.com)
doi:10.1038/nprot.2012.128
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Live imaging of cellular dynamics duringCaenorhabditis elegans postembryonic development
Yongping Chai Wei Li Guoxin Feng Yihong Yang Xiangming Wang Guangshuo Ou
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.
