Kurihara Daisuke, Kimata Yusuke, Higashiyama Tetsuya, Ueda Minako
Division of Biological Science, Graduate School of Science, Nagoya University; Higashiyama Live-Holonics Project, JST-ERATO, Nagoya University;
Division of Biological Science, Graduate School of Science, Nagoya University.
J Vis Exp. 2017 Sep 11(127):55975. doi: 10.3791/55975.
In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.
在大多数开花植物中，合子和胚胎深藏于母体组织之中，因此它们如何动态发育长期以来一直是个谜；例如，合子如何极化以确立体轴，以及胚胎在器官形成过程中如何确定各种细胞命运。本手稿描述了一种体外胚珠培养方法，用于对拟南芥发育中的合子和胚胎进行活细胞成像。优化后的培养基能使合子或早期胚胎发育成可育植株。通过将其与聚二甲基硅氧烷（PDMS）微柱阵列装置相结合，胚珠可在液体培养基中保持在同一位置。这种固定对于从合子分裂到胚胎后期阶段在显微镜下连续数天观察同一个胚珠至关重要。由此产生的活细胞成像可用于监测合子极化的实时动态，如核迁移和细胞骨架重排，以及胚胎模式形成过程中的细胞分裂时间和细胞命运确定。此外，这种胚珠培养系统可与抑制剂处理相结合，以分析各种因素对胚胎发育的影响，还可与激光破坏等光学操作相结合，以研究细胞间通讯的作用。
