Figure 6.

Loss of Sox9 in gastric chief cells prevents the acquisition of key metaplasia features. (A–H) Eight-week-old control and Fgf20^Cre.GFP/+; Sox9^fl/fl mice after high-dose tamoxifen treatment stained for markers of SPEM formation. (A) In control animals, SOX9 expression expands to the base regions of corpus units after injury. (C) GS-II lectin and (E) TFF2 expand in the base regions marking SPEM, and the entire gland gets recruited to the cell cycle, (G) as seen by the broad expression of Ki67 after injury. (B) In Fgf20^Cre.GFP/+; Sox9^fl/fl mice after injury, a subset of units completely lack SOX9. These Sox9^Δ/Δ units have significantly reduced expression of (D) GS-II and (F) TFF2 in the neck and base regions. (H) Although the neck region of Sox9^Δ/Δ units are recruited to the cell cycle, base regions show reduced Ki67 positivity. Representative histology of (I) Mist1^CreER/+; Sox9^fl/+ and (J) Mist1^CreER/+; Sox9^fl/fl corpus tissue stained with H&E after 1 week of LD tamoxifen treatment. LD tamoxifen treatment does not lead to atrophy or metaplasia induction. (K) Immunofluorescence staining for nuclei (DRAQ5, blue), tdTomato (mCherry antibody, white; ROSA26^tdTomatO), GS-II lectin (mucous neck cell marker, green), and GIF (chief cell marker, red) in Mist1^CreER/+; ROSA26^tdTomato/+ mice after LD treatment only. After only low-dose treatment, recombination of the ROSA26^tdTomato allele in Mist1^CreER/+; ROSA26^tdTomato/+ mice was restricted to mature chief cells at the base of corpus units. (L) To induce acute injury after gene deletion, mice were first treated with low-dose tamoxifen for 7 consecutive days followed by a week of rest. Mice then were treated with 2 doses of high-dose tamoxifen to induce injury. Recombination of the ROSA26^tdTomato allele in Mist1^CreER/+; ROSA26^tdTomato/+ mice is still enriched specifically in the base region of injured corpus units. (M-R) Eight-week-old control and Mist1^CreER/+; Sox9^fl/fl mice after low-dose/high-dose tamoxifen treatment. In control animals, tamoxifen-induced injury causes the expansion of (M) SOX9, (O) GS-II lectin, and (Q) Ki67 in the base region of corpus units. In Mist1^CreER/+; Sox9^fl/fl mice, after low-dose/high-dose tamoxifen treatment, have corpus bases with reduced (N) SOX9 expression and (P) GS-II lectin reactivity. These bases retain cells with larger, complex architecture and overall have a morphology more similar to control pre-injury bases. (Q) Base regions also have reduced cell-cycle entry, as shown by the lack of Ki67 positivity in corpus base regions. To quantify the reduction in cell-cycle entry, we counted Ki67-negative cells in the bottom 50 um of at least 60 units in control and treated Mist1^CreER/+; Sox9^fl/fl animals. Mist1^CreER/+; Sox9^fl/fl animals have significantly increased numbers of Ki67-negative cells compared with control animals. Smaller dots are individual counts (glands) per color-coded mouse replicate. Bold dots are the means of the individual mice, and error bars are the SEM. ∗∗∗P < .001, ∗∗∗∗P < .0001. Scale bars: 20 μm. TAM, Tamoxifen.