Proliferation and differentiation in intestinal organoids are balanced by ligand-modulated EGFR trafficking

Epidermal growth factor (EGF) signaling is associated with proliferation and tumorigenesis. Conversely, EGF-family ligands can also trigger a differentiation program, an effect attributed to ligand affinity and EGF receptor (EGFR) activity. Most of these observations have been made in immortalized cell culture experiments, whereas the mechanisms underlying EGF/EGFR-driven proliferation-differentiation dynamics in complex development and tissue self-renewal have not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and basal phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or Epiregulin (EREG) triggered receptor endocytosis, reducing cell-surface levels and expression. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared with untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to the constant presence of ligands over 96 h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle regulates its plasma membrane localization shaping the balance of proliferation and differentiation in mSIOs.