BulkRNA sequencing of small intestinal IEC epithelial cells of Casp3/7?IEC mice and Casp3/7FL/FL littermates

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction during homeostasis. To experimentally address this concept, we generated intestinal epithelial cell (IEC)-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7?IEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape and proliferation rate remained unchanged in these mice. However, non-apoptotic cell extrusion was increased in Casp3/7?IEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single cell RNA sequences analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome composition and immune regulation. Overall design: To examine possible other compensatory signaling pathways in Casp3/7?IEC mice that could mediate an adaptive alternative developmental reprogramming in the gut in the absence of IEC apoptosis, we performed a transcriptomic analysis of bulk IEC material isolated and sorted live EPCAM+/CD45- epithelial cells from both WT and Casp3/7?IEC mice. We used for each genotype four biological replicates (4 times WT and 4 times DKO)