A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal

Abstract

Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1-/-, Tcf3(flox), and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.

Fig 1

Absence of proliferating crypts in the small intestine derived from 17.5-dpc Tcf4^LoxP/LoxP_PGK^Cre mouse embryos. Histological analysis of the small intestine derived from 17.5-dpc mouse embryos showed the presence of proliferating/Ki67⁺ cells in the control tissue derived from PGK^Cre_Tcf4^WT/WT (A) and PGK^Cre_Tcf4^LoxP/WT (B) mice, while the proliferating/Ki67⁺ cells in the Tcf4^Hyg/Hyg (C) and the PGK^Cre_Tcf4^LoxP/LoxP (D) mice were completely abolished.

Fig 2

Lack of proliferating cells in the adult colon upon ablation of Tcf4. Histological analysis of the colon showed the presence of proliferating/Ki67⁺ cells in the control tissue derived from Tcf4^LoxP/LoxP mice (A) or Villin^Creert2_Tcf4^LoxP/LoxP mice (data not shown), while the proliferating/Ki67⁺ cells in the colon derived from the Villin^Creert2_Tcf4^LoxP/LoxP mice (B) were abolished 7 days after cre induction.

Fig 3

Lack of proliferating and stem cells in the adult small intestine upon ablation of Tcf4. Histological analysis at 1 day (B), 3 days (C), and 7 days (D) after tamoxifen-induced cre induction revealed, in comparison to a control Tcf4^LoxP/LoxP mouse (A), the disappearance of proliferating/Ki67⁺ cells over time in Villin^Creert2_Tcf4^LoxP/LoxP mice. Occasionally, large hyperplastic, proliferative Ki67⁺ escaper crypts (D, inset) could be detected. Histological analysis at 1 day (F), 3 days (G), and 7 days (H) after tamoxifen-induced cre induction revealed, in comparison to a control Tcf4^LoxP/LoxP mouse (E), the disappearance of intestinal stem cell/Olfm4⁺ cells over time in Villin^Creert2_Tcf4^LoxP/LoxP mice. Occasionally, large hyperplastic, proliferative Olfm4⁺ escaper crypts (H, inset) could be detected.

Fig 4

Lgr5 and Bmi1 expression in Tcf4-depleted crypt using two-color single-molecule fluorescent in situ hybridization. Tissue sections were simultaneously hybridized with two differentially labeled probe libraries (green, Lgr5-TMR; red, Bmi1-Cy5). Analysis at 1 day (D) and 3 days (G) after cre induction in the Villin^Creert2_Tcf4^LoxP/LoxP mice revealed, in comparison to a control mouse (A), the disappearance of Lgr5⁺ cells over time, while Bmi1, scattered in the crypt, remained present. Magnification of representative areas is shown (B, E, and H). The E-cadherin stainings are used to show the cell borders (C, F, and I), while DAPI staining (A, D, and G) showed nuclear staining.

Fig 5

Lack of crypt-based Paneth cells in the adult small intestine upon ablation of Tcf4. Histological analysis via lysozyme staining at 1 day (B), 3 days (C), and 7 days (D) after cre induction revealed, in comparison to a control mouse (A), the elimination of Paneth cells from the crypt of Lieberkühn and the aberrant location of these Paneth cells on the villi (see arrows in panel C and a magnified view in the inset). In situ hybridization analysis using cryptdin-1 as probe at 1 day (F), 3 days (G), and 7 days (H) after cre induction revealed, in comparison to a control mouse (E), the disappearance of cryptdin-1⁺ Paneth cells at the bottom of the crypt of Lieberkühn over time. At day 3, cryptdin-1⁺ cells Paneth and non-Paneth (intermediate) cells are scattered through the crypt and villi.

Fig 6

Generation of large numbers of intermediate cells upon Tcf4 ablation. Histological double staining showed generation of PAS⁺/cryptdin-1⁺ intermediate cells in the intestine in the Villin^Creert2_Tcf4^LoxP/LoxP mice on day 3 after cre induction (panel B versus A, arrows). cryptdin-1⁺ cells could never be detected in combination with enteroendocrine cells (D versus C, arrows) or tuft cells (panel F versus E, arrows).

Fig 7

EM analysis of Tcf4-ablated small intestines. EM analysis of Tcf4-depleted intestine derived from the Villin^Creert2_Tcf4^LoxP/LoxP mice at 1 day (C and D), 3 days (E and F), and 7 days (G and H) after cre induction revealed the gradual disappearance of intestinal stem cells, Paneth cells, and, ultimately, the complete proliferating crypt. Arrows indicate escaping Paneth cells on day 3 after cre induction (E). Bar, 10 μm.