not regulates polar cell number and cluster size. (A) Graph of number of upd-lacZ⁺ polar cells versus BCs in individual not¹ BC clusters colored according to polar cell number: two, purple (n = 36); three, red (n = 12); four, green (n = 3); five, blue (n = 1); six, white (n = 1). (B) Quantitation of polar and BC numbers reveals a significant increase in numbers of both upd-lacZ⁺ polar cells and BCs in not¹ clusters compared with controls. (C) Stage 10 egg chamber with not¹ BCs labeled by MARCM with GFP (green), surrounding WT polar cells labeled with anti-Fasciclin 3 staining (Fas3; red). Lagging not¹ BCs can be seen (arrows). Insets show magnified image of BC with three polar cells (C’, nuclei indicated with dotted red lines; C”, Fas3 staining in red). (D)not¹ BC cluster containing six upd-lacZ⁺–labeled nuclei. Insets shows magnified image segmented in 3D into upd-lacZ⁺ nuclei (red spots) and GFP not¹ cells (green; D’) or upd-lacZ expression alone (grayscale; D”). (E) Graph showing relationship between percent not¹ clones/cluster, number of cells in the cluster, and percent migration for individual sets of measurements. (F) Graph showing relationship between proportion of not¹ clones/cluster, number of cells in the cluster, and frequency of splitting. In E and F, color coding refers to percentage of the cluster that was mutant. (G) Control egg chamber at mid-stage 9 showing slow BC expression with the slbo-lacZ reporter (red) in migrating BCs. Inset shows magnified image of slbo-lacZ alone (grayscale). (H) Stage 10 egg chamber with not¹ BCs labeled by MARCM with GFP (green). Inset shows that the normal pattern of slbo-lacZ is detected in GFP mutant cells (green outline) compared with WT sibling (white outline). (I) Quantitation of relative slbo-lacZ signal intensity (GFP⁻, internal control: GFP⁺ homozygous sibling cell; see Materials and methods), showing no significant difference in slbo expression between WT and not¹ cells. Plots show overall mean ± SEM derived from means of n > 7 egg chambers/replicate superimposed on Beeswarm plots of individual measurements for each of the indicated genotypes. (J) Control stage 10 egg chamber showing anti-Eyes Absent antibody staining (Eya, red). Inset shows magnified image of Eya alone (grayscale). (K) Stage 10 egg chamber with GFP-labeled not¹ BCs (green). In both control and not¹ cluster cells, Eya is restricted to outer BCs. Nuclei are labeled with TO-PRO-3 (blue) in all images. Scale bars in confocal images are 25 µm (insets, 10 µm). st., stage.