Fig. 3.
DAAM and FRL are required for lateral attachment of the IOCs and patterning of the retinal floor. (A-H′) Confocal z-sections of eyes with 54C-Gal4/UAS-mCD8-GFP in a wild-type (control) or a DAAMEx4; frl59 mutant background at 48 h APF stained for Dlg and Cut (A-B′) and actin (C-H′), which were used to trace individual IOCs across the apical-basal axis of the eye (note that GFP is not shown here). Schematic drawings on the left indicate the wild-type ommatidial cell pattern at four positions along the apical-basal axis. Note that each cell is color coded, and that the IOCs [the six secondary pigment cells (SPCs), three tertiary pigment cells (TPCs) and three bristle cells (BCs)] at the apical (top row) and lateral (second row) layers of the eye appear as thin walls in between the clusters. At the basal layers (bottom rows), flattened feet of the IOCs become apparent, forming a flower petal pattern where the six SPCs link the axonal exit sites (located in the central position in each cluster) and separate the TPCs and BCs, located at alternating positions in between the petals. Panels A′-H′ show the same images as A-H, and in A′-H′, a group of IOCs, surrounding four ommatidia, are contoured using the same color code shown in the schematics on the left. In addition, the contoured SPCs are coded individually as well with colored dots and crosses. Note that at the septate junctions (marked by Dlg), the IOCs form a hexagonal lattice that is evident in the wild-type (A,A′) and formin mutant (B,B′) eye as well, although many of the horizontal SPCs appear shorter than normal in the mutant eye. Compared with this, at the lateral layer (C-D′), the IOCs often lose connection with each other (yellow arrows) and they often exhibit altered cell shapes, resulting in the formation of a broken lattice in the mutant eye. In the basal layers (E-H′), the shape of the IOCs is highly irregular in the formin mutants (F,F′,H,H′), and the TPCs and BCs occasionally make aberrant contacts with each other (blue arrows in H′), which is not observed in the wild-type eyes. The green arrow in H points to axonal exit sites erroneously contacting each other. Images are representative of ten animals per genotype. Scale bars: 10 µm. CC, cone cell; PPC, primary pigment cell; RC, photoreceptor cell. (I) Graphical 3D reconstructions of a group of IOCs consisting of three SPCs (in purple), one TPC (in red) and one BC (in yellow) illustrate the alterations in cell shape and cell contacts in wild-type versus formin mutant eyes. Note the discontinuities in the lattice wall (formed by the SPCs) due to impaired lateral adhesion between the SPCs and the corner cells, and the cell shape changes in the endfeet region. Reconstruction in the case of the mutant is based on the confocal z-sections of the cells numbered from 1 to 5 in H′.