Precluster formation as revealed by live imaging and fixed tissue staining. Precluster formation as revealed by live imaging and fixed tissue staining. (A,A′) Low magnification view of a mosaic eye disc displaying precluster formation; flrEY-P2 mutant clones are outlined (white dotted line). (B-C″) High magnification view of wild-type tissue (B-B″) and a nearby mutant clone (C-C″) as outlined by the blue and yellow boxes, respectively, in A′. A strong increase in F-actin levels occurs in the precluster primordia and appears to be cortical, underlying Arm-labeled AJs (C″). 1, 2, 3 indicate the column in which each precluster is located. (D-F″) Enlarged view of wild-type (E-E″) and mutant (F-F″) preclusters as numbered in B′ and C′, respectively. (D-D″) Schematic interpretations of wild-type AJ staining in E-E″ showing progressive stages during precluster formation. (G) Low magnification view of the MF and preclusters of a cultured eye disc during live imaging; Arm-GFP provides labeling of AJs. A, anterior direction; E, equatorial direction. (H) Diagram of precluster formation based on lead sulfide staining of the membrane by Wolff and Ready (Wolff and Ready, 1991). A large rosette (column 0) is shown emerging from the MF and developing into an arc of various shapes (in columns 1, 2 and 3), finally forming a five-cell precluster (column 4). (I,I′) A time-lapse series of images showing dynamic remodeling of AJs during the formation of a five-cell precluster. A large rosette (from the yellow box in G) and subsequent clusters of cells derived from it are outlined with yellow dotted lines. Schematic interpretations of the time-lapse series are shown beneath. The green line or dot indicates the AJ contact between R3 and R4. Scale bars: 10 μm in A-C″; 1 μm in E-F″,I; 5 μm in G. Dandan Chu et al. Development 2012;139:3561-3571 © 2012.