AbstractIn the adult gastrointestinal tract, the morphologic borders between esophagus and stomach and between stomach and small intestine are literally one cell thick. The patterning mechanisms that underlie the development of these sharp regional divisions from a once continuous endodermal tube are still obscure. In the embryonic endoderm of the developing gut, region‐specific expression of certain genes (e.g., intestine‐specific expression of the actin bundling protein villin) can be detected as early as 9.0 days post coitum, although the morphologic differentiation of the gut epithelium proper does not begin until 4 to 5 days later. By using a mouse model in which a β‐galactosidase marker has been inserted into the endogenous villin locus, we examined the development of the stomach/intestinal (pyloric) border during gut organogenesis. The data indicate that the border is not sharp from the outset. Rather, the initial border region is characterized by a decreasing gradient of villin/β‐galactosidase expression that extends into the distal stomach. A sharp epithelial border of villin/β‐galactosidase expression appears abruptly at day 16 and is further refined over the next 3 weeks to form the distinct one‐cell‐thick border characteristic of the adult. These results indicate that an important previously unrecognized patterning event occurs in the gut epithelium at 16 days; this event may define an epithelial compartment boundary between the stomach and the intestine. The villin/β‐galactosidase mouse model characterized here provides an excellent substrate with which to further dissect the mechanisms involved in this patterning process. © 2002 Wiley‐Liss, Inc.