Keil Wolfgang, Kutscher Lena M, Shaham Shai, Siggia Eric D
Center for Physics and Biology, The Rockefeller University, New York, NY 10065, USA; Laboratory of Developmental Genetics, The Rockefeller University, New York, NY 10065, USA.
Laboratory of Developmental Genetics, The Rockefeller University, New York, NY 10065, USA.
Dev Cell. 2017 Jan 23;40(2):202-214. doi: 10.1016/j.devcel.2016.11.022. Epub 2016 Dec 29.
Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.
传统上，秀丽隐杆线虫幼虫发育的长期研究需要繁琐的人工观察，因为幼虫必须移动才能发育，而现有的固定技术要么会干扰发育，要么不适用于幼虫。在此，我们展示了一种简单的微流控装置，可在高时空分辨率下同时跟踪十只秀丽隐杆线虫幼虫从孵化到成虫（约3天）的发育过程。在微腔中生长的动物通过压缩定期固定，以便对即使是微弱的荧光信号也能进行高质量成像。使用该装置，我们获得了秀丽隐杆线虫外阴发育的细胞周期统计数据，这是器官发生的一个范例。我们结合微分干涉差显微镜和多通道荧光显微镜来研究胚胎后发育过程中的细胞命运特化、细胞死亡和转分化等过程。最后，我们通过自动图像配准生成了复杂神经分支的延时电影。我们的技术为定量分析秀丽隐杆线虫幼虫发育过程中控制细胞行为的时间依赖性现象打开了大门。
