E-cad⁻ cells displayed reductions in multiple classes of intercellular junctions. Cre-ER;E-cad^fl/+ and Cre-ER;E-cad^fl/fl organoids were isolated, and widespread recombination was induced with tamoxifen. (A) Control organoids (E-cad⁺) maintained a smooth basal epithelial border. (B) E-cad⁻ organoids collectively migrated into Matrigel as single file columns (B’, red arrowhead). (C) Basally positioned E-cad⁻ cells (white arrowhead) were observed to round up and initiate single file cell columns. (D) Adeno-Cre was used to generate genetic mosaic organoids with a mixture of E-cad⁺ and E-cad⁻ cells. (E and F) Green, Cre⁺ cells reliably lacked E-cad (E and E’) and β-catenin (F and F’). (G) Basally positioned E-cad⁻ cells were located beyond the basement membrane protein laminin 332. Arrows in G′ indicate a single file column. (H) TEM was used to quantify desmosomes in Adeno-Cre–transduced E-cad^fl/+ (control) and E-cad^fl/fl organoids. Red outline indicates a representative region used for analysis. (I) E-cad⁻ epithelium had significantly fewer desmosomes compared with control E-cad⁺ epithelium. Error bars indicate SD. ***, P = 0.0008; two-tailed Student’s t test with equal variance. (J) Small desmosomes (J’, yellow arrowheads) were detected connecting cells in single file columns. Gamma adjustments were performed in E and F to improve image clarity. Bars: (A and B) 20 µm; (C and E–G) 10 µm; (H) 5 µm; (J) 0.5 µm. The black rectangle in the top right corner of J is a region of the image mosaic where no pixels were collected.