Tight junctions in early amphibian development: detection of junctional cingulin from the 2-cell stage and its localization at the boundary of distinct membrane domains in dividing blastomeres in low calcium

Abstract

Although functional studies indicate that tight junctions (TJ) are present in Xenopus laevis embryos from the 2-cell stage onward, morphological studies have failed to identify typical TJ structures before the 32-cell stage. Nothing is known about the expression and localization of TJ proteins in early Xenopus development. Here we have investigated the formation and composition of TJ in developing Xenopus embryos by whole-mount immunoperoxidase staining of eggs/embryos and immunoblotting of extracts with an antiserum against the TJ protein cingulin. Immunoblot analysis of eggs and embryo extracts showed that the antiserum labeled a major polypeptide of apparent M(r) 160 kD. Maternal cingulin was distributed diffusely in the cytocortical region of eggs and early embryos. Intense cingulin labeling became localized in the junctional region starting from the first cell division (2-cell stage). During cytokinesis, cingulin labeling was accumulated into new junctions in a precise spatial/temporal relationship with the deepening of the cleavage furrow. In semithin sections of stained embryos, labeling was detected in the most apical portion of the region of cell-cell contact. In embryos incubated in low calcium medium for 30 min, newly divided blastomeres failed to completely adhere to each other. However, cingulin labeling was accumulated along a linear structure that was at the border between distinct membrane domains (apical and lateral). These observations provide the first description of assembly of a TJ protein at the earliest stages of Xenopus development and suggest that TJ occur from the 2-cell stage onward and are assembled with maternal stores of protein. The formation of cingulin-containing structures even when lateral cell-cell adhesion is greatly reduced suggests that the apical cytocortex may have a determinative influence on TJ assembly and establishment of cell polarity.