Single-Cell Transcriptome Profiles of Foxl1-Lineage Cells Along the Intestinal Crypt–Villus Axis

Background & Aims Intestinal epithelial cells rely on a complex array of stromal signals to determine their fate and function along the crypt–villus axis, but the precise cellular sources and combinations of signals at each position remain poorly defined. Methods We generated an atlas of Foxl1-lineage cells along the crypt–villus axis using single-cell RNA sequencing integrated with in situ hybridization, immunofluorescence, and reporter mouse models. Results We identify 4 spatially distinct Foxl1-lineage subepithelial populations: crypt, villus–base, villus–mid and villus–tip, that segregate into 2 related pairs. Crypt and villus–mid Foxl1-lineage cells share a low platelet-derived growth factor receptor α transcriptional program enriched for canonical Wnts and R-spondins, consistent with regenerative signaling functions. In contrast, villus–base and villus–tip Foxl1-lineage cells are platelet-derived growth factor receptor α–high and express noncanonical Wnt5a and bone morphogenetic protein ligands. Crypt Foxl1-lineage cells are additionally enriched for extracellular matrix proteins and complement components, whereas villus–base Foxl1-lineage cells express contractility-associated genes, suggesting a role in villus architecture. Villus–mid Foxl1-lineage cells display signatures of immune regulation and inflammation, whereas villus–tip Foxl1-lineage cells are specialized for regulation of ribonucleoprotein complexes and nutrient sensing. Conclusions This atlas provides a foundational resource for understanding how diverse stromal populations coordinate signaling niches and influence intestinal epithelial homeostasis.