Joshi Sagar D, Davidson Lance A
Bioengineering, University of Pittsburgh, USA.
J Vis Exp. 2010 May 23(39):1949. doi: 10.3791/1949.
Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues(1). A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.
胚胎上皮细胞是研究形态发生的理想模型,在形态发生过程中,多细胞组织会发生几何形状的变化,如细胞表面积和细胞高度的变化,同时细胞会进行有丝分裂和迁移。此外,上皮细胞还可以调节相邻组织中的形态发生运动(1)。研究上皮细胞和组织的传统方法包括化学固定和组织学方法,以确定细胞形态或特定感兴趣蛋白质的定位。这些方法仍然有用,并能提供细胞形状和组织结构的“快照”,然而,关于细胞如何获得特定形状、各种蛋白质如何移动或定位到特定位置以及细胞沿着什么路径走向最终分化命运,仍有许多有待了解。高分辨率实时成像补充了传统方法,还能更直接地研究多细胞上皮片形成、维持和形态发生过程中涉及的动态细胞过程。在这里,我们展示了从非洲爪蟾胚胎中分离动物帽组织到上皮细胞共聚焦成像以及简单测量方法的实验方法,这些方法共同可以加强上皮形态发生的分子和细胞研究。