not is required for the normal level and/or distribution of Hippo signaling components and polarity determinants in BCs. (A and B) Confocal micrographs showing stage 10 egg chambers, except where indicated, with either WT or not¹ GFP or lacZ⁺-labeled clones (green) stained with antibodies against β-gal to detect ex-lacZ expression. (D–R) Mer (D and E), YFP-Mer (F and G), Ena (I and J), Crb (L and M), aPKC (N and O), or Arm (Q and R) are shown in red, with the exception of YFP-Mer, which is shown in green. Nuclei are stained with TO-PRO-3 (blue). Scale bars are 25 µm (insets, 10 µm). Arrows indicate BCs, insets are magnified images with regions of interest, and yellow lines through clusters mark the position of line scans used for quantitation. (A) Mosaic BC clusters, showing the normal expression of ex-lacZ in GFP-labeled control clones (green outline) and their siblings (white outline). (B) Notably, there is a reduction in ex-lacZ expression in not¹ clones (green outline) compared with control sibling cells (white outline). (C) Quantitation of ex-lacZ expression; plots show overall mean ± SEM derived from means of n ≥ 6 egg chambers/replicate superimposed on plots of the individual measurements. (D and E) Mer staining is weak but clearly detectable at the inner-BC junctions in control clones (green outline) but in E, is lost in GFP-labeled not¹ cells (green outline) and not adjacent control cells of the same cluster. (F and G) A similar effect was observed with YFP-Mer in lacZ⁺-labeled clones (β-gal in red). Inset shows regions used to measure the ratio of BC junctional staining inside the cluster (blue dashed line) to nurse cell junctional staining outside the cluster (yellow dashed line). (H) Quantitation of YFP-Mer expression (n ≥ 6 egg chambers/replicate). (I and J) Ena is predominantly located at cell junctions around the polar cells and at inner and outer BC membranes in both control (I) and not¹ (J) clones. (K) Dot plots of area under curve for back, middle, and front (BMF) of the cluster derived from line scans of signal intensity for Ena and Crb taken from multiple egg chambers, showing mean ± SEM of replicates. (L and M) Crb is distributed at inner BC junctions in control BC clusters but in M, is strikingly redistributed to the cortex of not¹ BC clusters. (N and O) aPKC is distributed at inner BC junctions in control BC clusters, but in O, this distribution is disrupted in not¹ clones, with some loss of aPKC at the inner membranes and a more cytoplasmic distribution in the BCs. (P) Dot plots of intensity measurements (as in K) for aPKC and Arm. Sample size for dot plots was n ≥ 4/replicate. (Q and R) The adherens junction protein Arm is apically localized at inner junctions in controls and in not¹ BC clusters. **, P < 0.01; ****, P < 0.001 by unpaired two-tailed Student’s t test.