Zou Jian, Wei Xiangyun
Department of Ophthalmology, University of Pittsburgh, PA, USA.
J Vis Exp. 2010 Jul 19(41):1924. doi: 10.3791/1924.
Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.
在胚胎发育过程中，细胞的位置和特性会发生广泛变化。这些变化受细胞与其环境之间相互作用的调控。由不同遗传背景的细胞组成的嵌合胚胎是研究由感兴趣基因介导的细胞间相互作用的绝佳工具。斑马鱼在早期发育阶段的胚胎透明度允许在细胞水平直接观察组织和器官的形态发生。在此，我们展示了一种在斑马鱼胚胎中生成嵌合视网膜和大脑并对供体细胞进行实时成像的方案。该方案涵盖移植针的制备、将表达绿色荧光蛋白（GFP）的供体卵裂球移植到GFP阴性宿主中，以及在共聚焦显微镜实时观察下检查供体细胞行为。稍作修改后，该方案也可用于研究斑马鱼其他组织和器官的胚胎发育。还讨论了使用GFP标记供体细胞的优势。
