Joshi Sagar D, Kim Hye Young, Davidson Lance A
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA.
Methods Mol Biol. 2012;917:477-93. doi: 10.1007/978-1-61779-992-1_27.
Early Xenopus embryos, and embryonic tissues isolated from them, are excellent model systems to study morphogenesis. Cells migrate, change shape, and differentiate to form new tissues as embryos mature and recapitulate those same processes in tissue isolates. Both large-scale and small-scale cell and tissue movements can be visualized with a range of microscopy techniques. Furthermore, protein dynamics, fine-scale cell movements, and changes in cell morphology can be observed simultaneously as multicellular structures are sculpted. We provide an overview of complementary methods for visualizing macroscopic tissue movements, cell shape changes, and subcellular protein dynamics. Time-lapse imaging followed by quantitative image analysis aims to provide answers to some of the long-standing questions in developmental biology: How do tissues form? How do cells acquire specific shapes? How do proteins localize to specific positions? To address these questions we suggest strategies (1) to visualize whole embryos and tissue isolates using stereoscopes and epifluorescence imaging techniques, and (2) to visualize cell shapes and protein expression using high-resolution live imaging using confocal microscopy. These imaging approaches along with simple image analysis tools provide us with ways to understand the complex biology underlying morphogenesis.
早期非洲爪蟾胚胎以及从它们中分离出的胚胎组织,是研究形态发生的优秀模型系统。随着胚胎成熟,细胞迁移、改变形状并分化形成新组织,并且在组织分离物中重现相同的过程。一系列显微镜技术可用于观察大规模和小规模的细胞及组织运动。此外,在塑造多细胞结构时,可以同时观察蛋白质动态、精细尺度的细胞运动以及细胞形态的变化。我们概述了用于可视化宏观组织运动、细胞形状变化和亚细胞蛋白质动态的互补方法。延时成像随后进行定量图像分析旨在为发育生物学中的一些长期问题提供答案:组织如何形成?细胞如何获得特定形状?蛋白质如何定位到特定位置?为了解决这些问题,我们建议采用以下策略:(1)使用体视镜和落射荧光成像技术观察整个胚胎和组织分离物;(2)使用共聚焦显微镜进行高分辨率实时成像来观察细胞形状和蛋白质表达。这些成像方法以及简单的图像分析工具为我们提供了理解形态发生背后复杂生物学的途径。