Hatta Kohei, Tsujii Hitomi, Omura Tomomi
Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Nat Protoc. 2006;1(2):960-7. doi: 10.1038/nprot.2006.96.
The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.
细胞命运、形态和迁移的追踪是多细胞生物发育研究的重要组成部分。在此，我们报告一种使用蛋白质Kaede的方法，该蛋白质合成后呈绿色荧光，但可被紫光或紫外光光转化为红色。我们将Kaede与共聚焦激光扫描显微镜结合使用，以追踪斑马鱼胚胎中感兴趣模式下的标记细胞。这项技术能够可视化细胞运动并追踪神经元形态。我们提供了通过mRNA注射进行表达、通过DNA注射进行镶嵌表达以及利用UAS-Gal4系统创建永久性转基因鱼的示例，以可视化诸如神经胚形成、基板形成和后脑早期连合轴突导航等形态发生过程。该程序可适用于其他光转化和可逆荧光分子，包括KikGR和Dronpa；这些分子可与双光子共聚焦显微镜结合使用，以特异性突出埋在组织中的细胞。通过注射mRNA或DNA进行Kaede瞬时表达、光转化和成像的整个流程所需时间为2至8天。
