Figure 4.

Cldn7 deletion in adult cKO SIs led to the loss of active crypt stem cells, increased epithelial cell proliferation, and aberrant positioning of Paneth cells. (A) The qRT-PCR analysis of Cldn7, Lgr5, Olfm4, and Hopx mRNA in 3-month-old control and cKO SIs. n = 4–5; 2-tailed, paired t test; *P < .05, **P < .01. (B) Active IESCs were labeled using FISH (left) and quantified (right) in 3-month-old control and cKO SIs. Each point represents the calculation from 50 crypts in each mouse. n = 5; 2-tailed, unpaired t test, ****P < .0001. (C) The control and cKO SIs were triple-labeled with lysozyme/PCNA/Hoechst to show the positions of Paneth and proliferating cells (left). Arrowheads and circles highlight the epithelial cells with positive PCNA and lysozyme signals. The statistical analysis of PCNA-positive cells is shown (right). Each point represents the calculation from 50 crypt–villus in each mouse. n = 5; 2-tailed, unpaired t test, ****P < .0001. (D) Paneth cells were labeled using antilysozyme antibody by immunohistochemistry to compare the position of the Paneth cells between control and cKO SIs (left, images in the square were enlarged as indicated by corresponding lines). The statistical analysis of Paneth cells is shown (right). Each point represents the calculation from 50 crypt–villus in each mouse. n = 5; 2-tailed, unpaired t test; ****P < .0001. Data are represented as means ± SEM. (E) Parallel labeling of lysozyme and mucin 2 (muc2) on the neighboring control and cKO SI sections. Arrowheads highlight the epithelial cells with positive lysozyme and muc2 signals. The images in the square were enlarged as indicated by corresponding lines. (F) Co-labeling of Alcian blue (blue) and antilysozyme antibody by immunohistochemistry (brown). Arrowheads indicate the epithelial cells with positive Alcian blue and lysozyme signals (n = 4). Scale bars: 100 μm.