Mitochondrial morphology differences in PFCs and MBCs established by EGFR signaling. (A and B, left) Sagittal view (A) and surface view (B) schematic of postmitotic wild-type egg chambers. Blue lines demarcate the boundary between PFC and MBC regions, and orange indicates patterning. (right) Enlarged areas correspond to the boxes in MBC and PFC regions. Low magnification images are shown in Fig. S2 B. Mitochondria were labeled by mito-GFP (A) or HSP-60 (B), nuclei was labeled with Hoechst, PKC marks apical membranes, and Discs large (Dlg) marks lateral membranes of the follicle cells. (C) Mitochondrial matrix continuity examined by a FLIP assay (see Materials and methods). MBCs or PFCs expressing mito-GFP (pre-FLIP image) were photobleached repeatedly in the white boxes and monitored for 200 s. Cell boundaries are shown in red. (D) Quantification of FLIP experiment. Total loss of fluorescence was quantified from eight MBCs and eight PFCs from four different egg chambers. Error bars signify standard deviation. (E) Microirradiation (green spots) of TMRE-labeled mitochondria in MBCs versus PFCs in the S8 egg chamber reveals faster loss of TMRE signal in MBCs. Cell boundaries are shown in red. (F) Down-regulation of EGFR in egfr^t1/egfr^t1 S8 egg chambers causes PFC mitochondrial clustering relative to that in egfr^t1/+ chambers. Magnified regions from the boxes are shown on the right. (G) EGFR-DN PFC clones (marked by CD8GFP) in S8 chambers have more tightly clustered mitochondria (labeled with HSP-60) than in surrounding nonclonal cells. (H, middle) drp1^KG + EGFR-DN PFC clones (CD8GFP label) show patches of Hnt-positive cells. (bottom) EGFR-DN clones alone (CD8GFP label) show Hnt labeling. Hoechst stains DNA. (I, top) drp1^KG + EGFR-Act PFC clones (CD8GFP label) do not show patches of Hnt-positive cells. (bottom) EGFR-Act clones alone (CD8GFP label) show Hnt labeling. Hoechst stains DNA. The white lines define the clone boundary, and the dotted lines outline the egg chambers. a.u., arbitrary unit. Bars, 5 µm.