The apical surface is established at the first cell division and maintained at the center of the growing structure. Cells were plated as a single-cell suspension in three dimensions, fixed, stained, and analyzed by confocal microscopy. (A) A two-cell structure stained for DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red). A three-dimensional reconstruction of the structure is shown in Video 5 (available). Note that apical and basolateral membrane domains (defined by ZO-1 and E-cadherin, respectively) are segregated and readily visible. (B) Caco-2 structures at different stages of development were stained for DNA (blue), aPKC (green), and filamentous actin (F-actin; red). Note that single cells (top) are unpolarized in three dimensions, whereas apical polarity is readily observed in the two-cell structure (second row). (C) Caco-2 structures at different stages of development stained for DNA (blue), tubulin (green), and filamentous actin (red). Note that the mitotic spindle is oriented to produce daughter cells that preserve the spherical monolayered structure of the cyst. (A–C) Single confocal sections through the middle of each structure are shown. Bars, 10 μm.