Embryonic stem (ES) cells differentiating into embryoid bodies (EBs) have been shown to mimic events of very early development and have become a convenient system in which to identify and study early epithelial specific genes. We describe here the primary structure of a mouse epithelial-specific tight junction gene and its expression patterns in differentiating ES cell-derived EBs in vitro. Sequencing of a clone identified by differential display of 4- vs. 6-day-old EB cells revealed it to overlap exactly with a larger cDNA clone (20M24) that had been isolated, but not characterised, in a screen of an ectodermal library. Complete sequencing and analysis of 20M24 revealed an open reading frame for a 219-amino acid protein with structural features of a transmembrane protein. In cell-free reticulocyte lysates, a 20M24 cDNA corresponding to the open reading frame (660 bp) directed the synthesis of a approximately 23-kDa protein that was localized to cell membranes at cell-cell junctions in transfected HEK-293 cells. Database searches indicated that the cDNA was identical to a recently identified member of the Claudin tight junction family, namely Claudin-6. ES cell cultures were used to further examine the expression pattern of Claudin-6 by whole mount in situ hybridisation during aggregation-induced commitment to epithelial differentiation in vitro. The results indicate that Claudin-6 is one of the earliest molecules to be expressed in ES cells committed to the epithelial fate, and the onset of its expression coincides with the expression of the early epithelial marker, keratin 8 (K8). The initiation of expression of Claudin-6 in vitro is dependent upon plating density as well as serum components. In addition, it was found that Claudin-6 expression is inhibited by Noggin, the Bone Morphogenic Protein (BMP)-signalling pathway inhibitor, suggesting that BMPs may be involved in Claudin-6 expression and epithelialization. These studies establish Claudin-6 as a very early marker of epithelialization and provide evidence that the BMP signalling pathway may be one of the ways that its expression is regulated. These studies also support the power of in vitro ES cell technology to identify and screen novel molecules involved in the early epithelialization of the mouse embryo.
Copyright 2001 Wiley-Liss, Inc.