Delta localization at the signaling interface of the pIIa/pIIb cells is impaired in the absence of dEHBP1. (a) Schematic representation of the Delta pulse-chase assays. R.T., room temperature. (b–c′′′) Delta is reduced in dEHBP1^−/− pIIa and pIIb cells at 0-min time points (b–b′′′) and at the 60-min time point (c–c′′′) in pulse-chase assays, as shown in xy projections. In b′′, b′′′, c′′, and c′′′, arrows point to the interface of mutant and wild-type two-cell ESO clusters, respectively. (d–f′′′) Extracellular Delta is reduced in dEHBP1^−/− pIIa and pIIb cells, as shown in xy projections in d–d′′′, where arrows and arrowheads point to the interface of mutant and wild-type two-cell ESO clusters, respectively. Analysis of extracellular Delta in projections along the z axis of wild-type thoracic (e–e′′′′) and mutant (f–f′′′) thoracic clusters, respectively, reveal that Delta is severely reduced at the interface of the pIIa and pIIb cells and that it is mainly localized basally. (g) Normalized quantification of pulse-chased and total Delta at 0- and 60-min time points in wild-type (green bars) and mutant (black bars) ESO two-cell clusters. (h) Normalized quantifications of extracellular Delta at the interphase of wild-type (green bars) and mutant (black bars) pII cells, as well as total extracellular Delta throughout wild-type (green bars) and mutant (black bars) thoracic epithelia. The numbers at base of the bars in g and h represent the total number of clusters used for quantification of Delta. n.s., not significant; *, P < 0.05. Bars, 10 µm.