Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development. No known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1). As Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they interact. The first suggests that the two interact transiently at the membrane. The second postulates that Notch is cleaved by a protease, enabling the cleaved fragment to enter the nucleus. Here we show that signalling by a constitutively active membrane-bound Notch-1 protein requires the proteolytic release of the Notch intracellular domain (NICD), which interacts preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. We also show that it is ligand binding that induces release of NICD.

The enteroendocrine cell system of the mammalian gastrointestinal tract is comprised of at least 16 different subpopulations. Each subpopulation shows a characteristic distribution along both the crypt-villus and cephalocaudal axes. In both the small intestine and colon of adult mice, multilabel immunohistochemistry has demonstrated that two or more neuroendocrine products can be coexpressed in various combinations in single cells along the crypt-villus axis, suggesting that enteroendocrine phenotypes may be actively regulated. Using bromodeoxyuridine (BrdU) incorporation and multilabel immunohistochcmistry, we have previously demonstrated an enteroendocrine cell differentiation pathway consisting of two subpopulations of cells in the mouse proximal small intestinmne involving the sequential expression of substance P, serotonin, and secretin in cells migrating out of the crypts into the villi, and a second involving the expression of substance P and serotonin in cells which remain in the crypts. In this report, we use double label immunohistochemistry and BrdU incorporation to define the temporal and spatial interrelationships between gastrin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-I), and gastric inhibitory peptide (GIP) immunoreactive cells in the mouse proximal small intestine. The expression of these products was compared with that of substance P, serotonin, and secretin. Minimal overlap of expression was found in cells immunoreactive for substance P or serotonin with gastrin, CCK, GLP-1, or GIP; however, secretin was found colocalized in villus-associated gastrin, CCK, and GLP-1 containing cells. We demonstrate that, similar to the bidirectionally migrating substance P and serotonin expressing cells, gastrin, CCK, GLP-1, and secretin are expressed in upwardly migrating cells, and gastrin, CCK, and GLP-1 are expressed in downwardly migrating cells that fail to express secretin. GIP containing cells only rarely coexpressed any of the products examined, but were found both in the villi and the crypts, suggesting both upwardly and downwardly migrating populations. These findings demonstrate several novel enteroendocrine cell differentiation pathways. In addition, the expression of secretin 0 1994 WILEY-LISS, INC.in the villi, but not in the crypts, by two otherwise distinct differentiation pathways, and the lack of secretin expression by villus-associated GIP expressing cells, suggests that local factors present in the crypts and/or on the villi are necessary, but not sufficient, for secretin expression.