Extended Data Fig. 4. Multi-lineage differentiation upon LGR5, Rnf43 or Znrf3 ECD treatment.

a, Enterocyte, goblet and enteroendocrine differentiation are preserved upon LGR5, Rnf43 or Znrf3 ECD treatment. Adult mice received single i.v. injection of the indicated adenoviruses encoding soluble ECDs of LGR5, Rnf43 (Fc fusion) or Znrf3 (Fc fusion), or Rspo1 (Fc fusion) and the jejunum was analyzed at d7 after injection by H&E or histology with anti-Fabp1 (enterocyte), PAS (goblet) or anti-ChgA (enteroendocrine). Multi-lineage differentiation was maintained. b, LGR5 ECD but not Rnf43 ECD or Znrf3 ECD induces Paneth cell loss (d7 post-infection). Bars = 50 mm. c, LGR5 ECD induces transient Paneth cell loss. Time course of lysozyme expression in jejunum following single i.v. injection of Ad LGR5 ECD into mice. Bar = 50 mm. Note ballooning degeneration and upward migration of lysozyme⁺ Paneth cells at d3–4 (yellow brackets), followed by near-total Paneth cell loss at d7–10 and return of Paneth cells at d14 post-injection. The Paneth cell loss (after d3) occurs after the loss of Lgr5-eGFP signal (d2, Extended Data Fig. 2) and Paneth cell return correlates with the time course of disappearance of adenoviral LGR5 ECD serum expression (Extended Data Fig. 1g). d, Quantitation of Paneth cell loss in small intestine, d7, n=3 animals/condition, Error bars represent S.E.M., *= P<0.05. e, Ad LGR5 ECD induces loss of anti-Cd166 immunofluorescence (CBC/Paneth marker), jejunum, d7 after adenovirus treatment. f, H&E reveals ballooning degeneration and upward migration of lysozyme⁺ Paneth cells after Ad LGR5 ECD treatment (yellow brackets and arrows), consistent with an intermediate cell phenotype. g, Electron microscopy analysis of intestinal crypts after Ad LGR5 ECD i.v. injection reveals ballooning degeneration at d3–4 followed by Paneth cell loss by d7.