Self-Organization Through Local Cell-Cell Communication Drives Intestinal Epithelial Zonation

The intestinal epithelium exhibits zonated gene expression along the crypt-villus axis, with distinct transcriptional programs in enterocytes at the villus top versus bottom. However, the mechanisms establishing these spatial patterns remain unclear. Three models could explain zonation: external gradients, cell-intrinsic temporal programs, or local self-organization. Using spatial transcriptomics and perturbations of two-dimensional intestinal organoids, we show that zonation emerges via spontaneous self-organization without mesenchymal, neural, or vascular inputs. Cell-intrinsic models were eliminated by transplanting cells into established monolayers; transplanted cells progressively adopted zonation profiles matching their new location, with strongly zonated genes showing the greatest adaptive responses. Pharmacological inhibition of EphA2 receptors disrupted zonation, revealing a previously unknown role for epithelial EphA-ephrin-A signaling in regulating enterocyte zonation. These findings demonstrate that self-organization through local epithelial cell-cell communication generates spatial patterns independently of external positional cues or cell-autonomous programs.