Requirements of nectin and cadherin activities for mosaic cellular patterning. (A) Mosaic-forming assay using HEK293 (293) cells expressing different combinations of cadherins. Boundaries between two colonies of cells, which were transfected with E-cadherin-EGFP (Ecad-293, green), E-cadherin-mCherry (Ecad-293, red), N-cadherin-EGFP (Ncad-293, green), N-cadherin-mCherry (Ncad-293, red), or mCherry (mCherry-293, red) in the indicated combinations. The graph shows the number of singly isolated cells in the mixtures of the two cell populations in each field. The results shown are the mean ± SD; n.s., not significant. n = 6. (B) Mosaic-forming assay using 293 cells expressing different combinations of nectins. Boundaries between two colonies of 293 transfectants are shown. The cells were doubly transfected with a nectin or truncated nectin (nectinΔ) and EGFP or mCherry. n2-293, 293 nectin-2 transfectants; n3-293, nectin-3 transfectants; n2Δ-293, nectin-2Δ transfectants; n3Δ-293, nectin-3Δ transfectants. The graph shows the number of singly isolated cells in the mixtures of the two cell populations in each field. The results shown are the mean ± SD; *, P < 0.001. n = 6. (C) Distributions of nectins and β-catenin in a mixed culture of nectin transfectants; 293 transfectants were mixed and triple stained for β-catenin (green), nectin-2 or nectin-2Δ (red), and nectin-3 or nectin-3Δ (blue). The panels show immunostaining in the mixed culture of n2- and n3-293 cells (top) or n2Δ- and n3Δ-293 cells (bottom). Arrows, heterotypic junctions; arrowheads, homotypic junctions between n2–n2 or n2Δ–n2Δ; double arrowheads, homotypic junctions between n3–n3 or n3Δ–n3Δ.