Ft and Crb regulate Ex independently. (A–B′) A portion of Ex::GFP remains at the apical membrane despite loss of Crb. XY confocal micrographs of Ex::GFP third instar wing imaginal discs containing crb^11A22 (A and A′) or crb^82-04 (B and B′) mutant clones (marked by absence of RFP shown in red), showing direct GFP fluorescence representing Ex protein (shown in gray). (C–D′) Validation of crb^11A22 as a protein null. XY confocal micrographs of third instar wing imaginal discs containing crb^11A22 mutant clones (marked by absence of RFP shown in red) with immunostaining to the extracellular domain (ECD) of Crb (C and C′—shown in gray) or the ICD of Crb (D and D′—shown in grandy). (E and E′) Validation of Ex immunostaining. XY confocal micrographs of third instar wing imaginal discs containing ex^e1 mutant clones (marked by absence of RFP shown in red) with Ex staining (shown in gray). (F and F′) Loss of Ft causes loss of apical Ex. XY confocal micrographs of third instar wing imaginal discs containing ft^5-5 mutant clones (marked by absence of RFP shown in red) with Ex staining (shown in gray). ft^5-5 is a remake of ft^fd and is a null allele. (G–G′′′) Colocalization of Ex, Ft, and Crb at apical junctions. Transverse confocal micrograph of third instar wing imaginal discs with direct fluorescence of Ex::GFP (green in G and G′′′), stained with Ft (mauve in G′ and G′′′), Crb (red in G′′ and G′′′), and Hoechst (cyan in merge) to mark nuclei. All images are orientated dorsal up. Clonal boundaries are marked by yellow dotted lines. Scale bars are 10 µm. (H–I′′) High-resolution localization patterns of Ex and Ft with even distribution (H–H′′) or punctate (I–I′′). High-resolution XY confocal micrographs of third instar wing imaginal discs stained for Ft (green in H′′ and I′′) and Ex (mauve in H′′ and I′′). The yellow arrowheads in I–I′′ highlight punctate colocalization. Images were denoised, scale bar is 2 µm. (J and J′) Loss of Ft does not affect Crb. XY confocal micrographs of third instar wing imaginal discs containing ft^5-5 mutant clones (marked by absence of RFP shown in red) with Crb staining (shown in gray). ft^5-5 is a remake of ft^fd and is a null allele. Images are oriented dorsal up. Scale bar is 10 µm. (K) Quantification of the ratio between basal Ex inside versus outside the MARCM clone normalized to the wild-type (wt) tissue. Data points represent an average of a single disc with the mean and SD indicated. No significance calculated using an unpaired T test.