Analysis of cortical α-Spec level in different genetic background and the importance of the PH domain of β-Spec in cell shape determination. All images are z-projections at lateral position. (A and A’) A pupal eye disc containing GFP-negative cora⁵ mutant clones was stained for α-Spec. Note the increased cortical α-Spec level in the mutant PECs. Quantification of α-Spec membrane intensity is shown in E. (B and B’) A pupal eye disc containing GFP-positive MARCM clones with ank1 RNAi was stained for α-Spec. Note the normal cortical α-Spec level in ank1 mutant PECs. Quantification of α-Spec membrane intensity is shown in F. (C–D’) Pupal eye discs containing GFP-positive β-spec^C mutant MARCM clones expressing β-SpecΔPH (C and C’) or Myr-β-SpecΔPH (D and D’) were stained for Dlg. Myr-β-SpecΔPH, but not β-SpecΔPH, rescued the cell expansion phenotype of β-spec^C mutant PECs. Arrows mark wild-type PECs and arrowheads mark β-spec mutant PECs expressing the indicated β-Spec transgene. Quantification of ommatidium lateral size is shown in G. (E and F) Mean α-Spec membrane intensity in PECs analyzed in A–B. Data are means ± SEM (n ≥ 20 cells, representative of five animals). ****, P < 0.0001. N.S., no significance. (G) Normalized mean ommatidium lateral size analyzed in C–D. Data are means ± SEM (n ≥ 15 ommatidia, representative of five animals). ****, P < 0.0001. N.S., no significance. (H–K) Pupal eye discs containing GFP-positive MARCM clones with cpa RNAi (H–H”) or Dia^CA overexpression (J–J”) were stained for α-Spec (red). Cortical α-Spec level remained unchanged in cpa RNAi clones or decreased in Dia^CA overexpression clones. Mean α-Spec membrane intensity is shown in I–K. Data are means ± SEM (n ≥ 20 cells, representative of five animals). ****, P < 0.0001. Scale bars, 5 µm. N.S., no significance.