Effects of TNFR2 deletion on the development of mouse colonic epithelial organoids

The early phase of colonic epithelial wound healing involves cellular reprogramming to a fetal-like state and reorganization of discrete crypt units into merged wound channels. After re-epithelialization is complete, a latter phase restoring homeostatic signaling and crypt patterning must occur. However, the signals that mediate this regenerative transition are unknown. Here we show that injury-associated upregulation of a cytokine receptor, tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b), suppresses fetal-like signaling and promotes crypt production. We used tissue clearing and whole-mount imaging, RNA-Seq, and organoid cultures to characterize mice with a colonic epithelial-specific deletion of TNFR2. These mice exhibited increased crypt size and reduced fission in adult homeostasis and after colitis, abnormal persistence of fetal-like molecular markers in organoids and after injury, reduced terminal differentiation, and increased proliferative potential. These results demonstrate how epithelial cells can adapt to inflammatory cues to regulate wound healing morphogenesis and signaling. A total of 20 samples were analyzed. Each sample is composed of total RNA isolated from wildtype or Tnfr2-/- mouse colonic organoids grown for either 1, 2, 3, 5, 7 days after passage. Two biological replicates (i.e., mice) per genotype were studied.