Figure 5. Analysis of tissue overgrowth and cell proliferation in lace and ACC mutant cells.

Confocal images of wing disc pouches corresponding to (A) wildtype, (B) ACC¹ mutant clones, (C) lace² mutant clones (arrows indicate tissue overgrowth regions in B and C), (D) lace² aph-1^D35 double mutant clones, and (E) lace² Su(H)^k07904 double mutant clones, with confocal signals for Hoechst (blue), Phalloidin (red) and Myc (green) staining used to reveal tissue architecture and mutant vs. wildtype cell territories in clone-bearing discs. (F) Confocal section of a lace² aph-1^D35 clone, showing high vesicular Notch accumulation (red). Hoechst-stained nuclei are shown in blue; faint green signal represents Myc antibody background staining that was used to identify the Myc-negative clones. (G–S) Mutant clones encompassing the wing posterior compartment were induced using the hh-GAL4; UAS-FLP system for (G) the control wildtype genotype (FRT40A FRTG13), (H, N, O, R) lace², (I) lace¹⁸, (J) lace¹⁹, (K, P, Q, S) ACC¹, (L) ACC², and (M) lace² Su(H)^k07904. (N–Q) UAS-cDNA constructs encoding wildtype LaceHA (N, Q) or ACC (O, P) were expressed in either lace² (N, O) or ACC¹ (P, Q) mutant clones as indicated. Discs in G–Q were examined for Myc expression to identify Myc-negative clone regions (lack of green signal), Phalloidin (red), and Hoechst (blue). Genotypes of each panel correspond to those listed in Supplemental Table S3. (R, S) Wing imaginal discs with lace² (R) or ACC¹ (S) posterior compartment clones were analyzed with anti-phosphohistone H3 antibody (pH 3; red), anti-Myc (green; clone marker as above), and Hoechst (blue). Genotypes of (R) and (S) are the same as (H) and (K), respectively. (T) The percentages of pH 3-positive nuclei in lace² or ACC¹ homozygous mutant cells compared to control heterozygous cells were determined by analyzing Myc-negative and Myc-positive 127 µm×127 µm sectors, respectively, for ten discs of each genotype (**; P<0.01 by t-test). Scale bars, 50 µm in A–E, G–S; 10 µm in F.