Genetic evidence that intestinal Notch functions vary regionally and operate through a common mechanism of Math1 repression

Abstract

Notch signaling is active in many sites, and its diverse activities must require tissue-specific intermediaries, which are largely unknown. In the intestinal epithelium, Notch promotes crypt cell proliferation and inhibits goblet cell differentiation. Pharmacologic studies suggest that the latter effect occurs through the transcription factor Math1/Atoh1, which specifies all intestinal secretory cells. We tested this hypothesis using mouse mutants. Genetic loss of the Notch effector RBP-Jκ alone increases all intestinal secretory lineages, with variation between proximal and distal gut segments. This secretory cell excess observed with RBP-Jκ loss was blocked in the absence of Math1 in RBP-Jκ(Fl/Fl);Math1(Fl/Fl);Villin-Cre((ER-T2)) mice. Loss of both factors also restored progenitor replication, proving that Math1 is epistatic to Notch signaling in both secretory cell differentiation and crypt cell proliferation. Investigating mechanisms downstream of Math1, we found that expression of the known Notch effector protein Hes1 was predictably lost in RBP-Jκ(-/-) mice but surprisingly recovered in RBP-Jκ;Math1 compound conditional mutants. Furthermore, the cell cycle inhibitors p27(Kip1) and p57(Kip2) were selectively overexpressed in duodenal and ileal crypts, respectively, in RBP-Jκ-deficient mice. Regional activation of these products was completely abrogated in the absence of Math1. Thus, all intestinal Notch effects channel through the tissue-restricted factor Math1, which promotes secretory differentiation and cell cycle exit by regionally distinct mechanisms. Our data further suggest that, besides transmitting Notch signals, the transcription factor Hes1 acts downstream of Math1 to regulate expression of cell cycle inhibitors and intestinal crypt cell replication.

FIGURE 1.

Role for Notch signaling in all three intestinal secretory cell lineages. Tamoxifen treatment increased the numbers of ChromograninA-positive EE cells (top row) as well as Paneth cells expressing the cryptdin Crs4c1 (bottom row) in RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice, compared with wild-type littermates. Insets in the duodenal panels show high-magnification images of the boxed areas, and quantitation of immunohistochemical data is shown (right). The duodenum showed a larger increase in EE cells, whereas Paneth cells were more significantly increased in the ileum. Scale bars = 50 μm.

FIGURE 2.

Requirement for Math1 in secretory cell metaplasia induced by loss of Notch activity. A, Alcian blue-positive goblet cells increased substantially after tamoxifen treatment of RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice but not of Math1^Fl/Fl;Villin-Cre^(ER-T2) (top) or RBP-Jκ^Fl/Fl;Math1^Fl/Fl;Villin-Cre^(ER-T2) (bottom) mice. Similarly, Math1^Fl/Fl;Villin-Cre^(ER-T2), and RBP-Jκ^Fl/Fl;Math1^Fl/Fl;Villin-Cre^(ER-T2) small intestines lacked all Crs4c1-expressing Paneth and ChromograninA (ChgA)-positive EE cells, indicating loss of all secretory lineages with lone loss of Math1 or combined absence of Math1 and RBP-Jκ. Failure of secretory cell metaplasia was equally evident in the duodenum (top rows) and ileum (bottom rows). B, expression of intestinal alkaline phosphatase (AP) and the microvillus brush border marker phospho-Ezrin/Radixin/Moesin (p-ERM) was notably reduced in RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice after tamoxifen treatment, reflecting significant enterocyte loss, but restored in RBP-Jκ^Fl/Fl;Math1^Fl/Fl;Villin-Cre^(ER-T2) mice and preserved in Math1^Fl/Fl;Villin-Cre^(ER-T2) mice. The results indicate that Notch signaling is dispensable for enterocyte maturation, at least when Math1 is absent. Scale bars = 50 μm.

FIGURE 3.

Math1 acts downstream of Notch signaling as an obligate regulator of intestinal crypt cell replication. Assessment of crypt cell replication by incorporation of the S-phase marker BrdU. Tamoxifen-induced arrest of cell proliferation in RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice was fully rescued in the absence of Math1, in both duodenum (top row) and ileum (bottom row). Absence of Math1 alone modestly increased cell proliferation in both regions, and this degree of increase persisted in RBP-Jκ^Fl/Fl;Math1^Fl/Fl;Villin-Cre^(ER-T2) mice. Immunohistochemical data are quantified (top right). Scale bars = 50 μm.

FIGURE 4.

Recovery of Hes1 expression in the combined absence of RBP-Jκ and Math1. A, Hes1 immunohistochemistry reveals that its expression is abolished after tamoxifen treatment of RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) intestines, preserved or slightly increased in Math1^Fl/Fl;Villin-Cre^(ER-T2) intestines, and restored in RBP-Jκ^Fl/Fl;Math1^Fl/Fl;Villin-Cre^(ER-T2) duodenum and ileum. Insets in each panel show high-magnification images of the boxed crypts. Scale bars = 50 μm. B, schematic diagram illustrating that, in addition to its known role as a direct Notch target and effector protein, Hes1 is also regulated by Math1. Previously known relationships are indicated with solid black lines and the relationship uncovered in the present study with a solid blue line. The dashed lines reflect uncertainty whether RBP-Jκ represses Math1 directly or through Hes1.

FIGURE 5.

Differential Notch-induced repression of cell cycle inhibitors in the proximal and distal small intestine. Expression of the endogenous cell cycle inhibitor p27^Kip1, normally confined to a few crypt cells in tamoxifen-treated wild-type and Math1^Fl/Fl;Villin-Cre^(ER-T2) mice, was significantly increased in duodenal (top row) but not ileal (bottom row) crypts in RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice. This abnormal increase in p27^kip1 expression was not observed in the absence of Math1. Expression of the alternative cell cycle inhibitor p57^Kip2, also largely absent from tamoxifen-treated wild-type and Math1^Fl/Fl;Villin-Cre^(ER-T2) crypts, increased significantly in ileal (bottom row) but not duodenal (top row) crypts in RBP-Jκ^Fl/Fl;Villin-Cre^(ER-T2) mice. Ileal p57^Kip2 expression did not increase in the absence of Math1. Insets in each panel show high-magnification images of the boxed crypts. Scale bars = 50 μm.