LGR5? Stem Cells Maintain Apex Position in Regenerative Hierarchy of the Intestinal Epithelium During Homeostasis and Injury

The cellular origin of intestinal epithelial regeneration has been a subject of continued debate, with recent models challenging the primacy of WNT-dependent LGR5? crypt base columnar (CBC) cells as the central regenerative population. Here, we revisit this question through quantitative integration of single-cell transcriptomic, chromatin accessibility, spatial, and lineage-tracing analyses across the proximal-to-distal axis of the small intestinal epithelium. Our data show that under homeostatic conditions, LGR5? cells exclusively sustain epithelial self-renewal in nearly all crypt–villus units along the entire length of the small intestine. Following irradiation or chemotoxic injury, surviving LGR5? CBC cells and their immediate progeny undergo transcriptional and epigenetic reprogramming into transient fetal-like cell states that initiate epithelial repair. Crucially, successful regeneration depends on the reactivation of canonical WNT/ß-catenin signaling, as evidenced by increased TCF motif accessibility and upregulation of WNT target genes, including Lgr5. Accordingly, pharmacological inhibition of WNT signaling blocks the reconstitution of LGR5? cells and crypt regeneration, leading to epithelial collapse. These findings reconcile prior controversies by demonstrating that epithelial regeneration throughout the small intestine, even following injury, is ultimately driven by LGR5? CBC cells in a WNT-dependent manner. Overall design: Mouse intestines were extracted at day 1 and 4 following 12Gy of irradiation and nuclei were isolated.