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First published online 23 March 2005
doi: 10.1242/dev.01800

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IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin

Institut für Genetik, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany

View larger version (147K): [in a new window]   Fig. 1. Overexpression of Crbintra causes a rough eye phenotype. Scanning electron micrographs of adult eyes. (A,A') Wild-type eyes show a regular, hexagonal arrangement of ommatidia. Bristles are found at alternating vertices of the hexagonal array. (B,B') sev-Gal4>UAS-crbintra. The roughening of the eye is induced by disorganisation of the array of facets; bristles are often doubled (arrowhead) and some lenses adopt a square shape (asterisk). Anterior is towards the left and dorsal towards the top.

View larger version (113K): [in a new window]   Fig. 2. Interommatidial cells in pupal eye imaginal discs overexpressing crbintra or mutant for IrreC-rst are not properly sorted and surplus cells fail to be eliminated. (A-C) Wild-type; (D) GMR-Gal4>UAS-crbintra; (E) irreCCT. Anti-DE-cadherin antibody was used to visualise the plasma membranes. (A) At the onset of cell sorting (16% p.d.), formation of the primary pigment cells (1°; blue) around the cone cell quartet (C) is complete. Sorting of the interommatidial cells (IOC; brown) is in progress. Most ommatidial clusters are still separated by more than one row of IOCs (arrowheads). (B) At 21% p.d., cell sorting has been completed. The IOCs (brown) now form single rings of lattice cells, aligned head-to-tail, separating the ommatidial clusters from each other. b, bristle. (C) At 42% p.d. all surplus cells have been eliminated by apoptosis. The remaining cells are now arranged in a regular hexagonal pattern, in which secondary (2°) and tertiary (3°) pigment cells, as well as bristles (b), can be distinguished. (D) Discs overexpressing Crbintra, examined at 42% p.d., after the time at which cell death occurs in the wild type. Cells are not properly rearranged and not all surplus cells have been eliminated; occasionally two rows of cells are left between ommatidial clusters (see also Fig. 5G,H). In several instances an `astral' arrangement of the 2° and 3° pigment cells can be observed, in which four cells, instead of the usual three, surround a bristle (arrowheads, compare with C). (E) An irreC-rstCT retina at 40% p.d. Here, none of the surplus cells has been eliminated, leaving the ommatidial clusters surrounded by two or three rows of cells. The arrangement is similar of that seen in discs overexpressing Crbintra, although the phenotype is stronger, because the cell sorting is now disrupted in all instances (compare with D).

View larger version (148K): [in a new window]   Fig. 3. Defects in IrreC-rst distribution in pupal eyes discs overexpressing crbintra or mutant for irreC-rst. (A-C,J) Wild-type; (D-F,K) irreCCT; (G-I,L) GMR-Gal4>UAS-crbintra. (A-C) At 21% p.d., after completion of cell sorting, the IOCs form a single layer of lattice cells between the ommatidia. DE-cadherin (green) marks the apical regions of all cells. By contrast, IrreC-rst (violet) accumulates almost exclusively at the borders between primary pigment cells and IOCs (1°/IOC), where it colocalises with DE-cadherin (C). (D-F) In an irreC-rstCT retina at 18% p.d., staining of IrreC-rst protein at the apical membranes of the 1°/IOC cell border is discontinuous. The protein localises in small patches along the membrane of the IOCs. The ommatidial clusters are surrounded by two or three rows of IOCs, outlined by staining for DE-cadherin. The distribution of DE-cadherin is unaffected by the mislocalisation of IrreC-rst (violet) and the protein still appears at the apex of all cells (green). (G-I) In a retina overexpressing Crbintra at 22% p.d., DE-cadherin (green) and IrreC-rst (purple) are discontinuously scattered in the apical cell membranes where they mostly colocalise. In addition, IrreC-rst also is also found diffusely distributed and in vesicles in the cytoplasm. (J,K) Crbintra-overexpressing pupal retina at 42% p.d. (L), after the phase of cell death. The distribution of IrreC-rst now resembles the wild-type pattern (compare L with J). IrreC-rst accumulates, as in wild-type (42% p.d.), at the 1°/IOC border and around the bristles. In an irreC-rstCT retina at 40% p.d. (K) there is no continuous zone of IrreC-rst protein at the apical membranes that form the 1°/IOC border. Instead, the protein is localised in vesicles in the cytoplasm.

View larger version (111K): [in a new window]   Fig. 4. IrreC-rst colocalises with DE-cadherin in the zonula adherens. (A-C) In the wild-type retina at 30% p.d., DE-cadherin (green) marks the apical region of all accessory cells, cone cells, primary pigment cells, IOCs and bristles (see also Fig. 3B). At this stage and later (Fig. 3J), IrreC-rst (violet) accumulates almost exclusively at the borders between primary pigment cells and IOCs (1°/IOC). DE-cadherin and IrreC-rst colocalise at the border between IOCs and primary pigment cells. (D-F) Longitudinal sections through a wild-type retina at 35% p.d. confirm the colocalisation in the apical region of the pigment cells, at the site of the adherens junctions. DE-cadherin is also found at the adherens junctions of the photoreceptor cells (arrowhead), which have not yet begun to elongate. (G-I) Retina overexpressing Crbintra at 30% p.d. DE-cadherin (green) and IrreC-rst (violet) colocalise at the border between IOCs and primary pigment cells. The staining pattern is nearly the same as in wild type, showing only a few interruptions. (J-L) Longitudinal sections through a Crbintra-overexpressing retina at 38% p.d. confirm the colocalisation of IrreC-rst and DE-cadherin in the apical region of the pigment cells, at the site of the adherens junctions. The structure of the adherens junction of photoreceptor cells (arrowhead) is less regular than in wild type.

View larger version (153K): [in a new window]   Fig. 5. Crbintra expression prevents programmed cell death. (A-C) Wild-type, (D-H) GMR-Gal4>UAS-crbintra. (A-F) Apoptotic cells visualised by Acridine Orange staining. (A-C) In the wild-type third-instar eye disc (A; eye, posterior to the left), dying cells appear anterior and posterior to the morphogenetic furrow (arrowheads). Cell death is also observed in the antennal region of the disc (ant). In pupae, fluorescent fragments of dying cells are visible throughout the disc at 31% p.d. (B) and in the perimeter clusters at 40% p.d. (C). (D-F) Cell death in GMR-Gal4>UAS-crbintra larval discs (D) is strongly reduced posterior to the morphogenetic furrow (arrowhead), but still present anterior to the furrow and in the antennal disc (where GMR-Gal4 is not expressed). In pupal discs at 31% p.d. (E) cell death is locally suppressed (arrowheads), while at 40% p.d. cell death in perimeter clusters is not affected (F). (G,H) Elimination of perimeter clusters in pupal imaginal discs overexpressing Crbintra, visualised by immunostaining for IrreC-rst (violet) and DE-cadherin (green). At 38% p.d. (G), the perimeter clusters can still be identified (arrowheads). At 42% p.d. (H), the perimeter clusters have been eliminated.

View larger version (155K): [in a new window]   Fig. 6. Disruption of the adherens belt perturbs IrreC-rst localisation. (A-C) Clones homozygous mutant for shgIH in a retina at 26% p.d., identified by the absence of DE-cadherin staining (green). IrreC-rst (violet) is not localised and appears as diffuse staining in the cytoplasm, while in the region heterozygous for shgIH it colocalises with DE-cadherin at the 1°/IOC border. (D-F) GMR-Gal4<UAS-CAD retina at 22% p.d. exhibits a patchy distribution of -catenin (green) in the apical regions of the cells and diffuse staining in the cytoplasm. IrreC-rst (violet) is colocalised with -catenin at the 1°/IOC borders, but is also found at IOC/IOC borders. (G-I) In a GMR-Gal4<UAS-RhoN19 retina at 18% p.d. the continuous adherens junctions, as revealed by staining for DE-cadherin (green), are eliminated. Single cells or ommatidia are difficult to discern and only minor speckles of immunoreactivity can be detected in the plasma membranes of IOCs and primary pigment cells. The majority of the protein appears as diffuse staining in the cytoplasm. IrreC-rst (violet) adopts an identical pattern. [Flies raised at 18 (shown here) or 25°C die as pharate adults with severe head defects and very rough eyes that are only one-third the size of wild-type eyes.] (J-L) Notchts1 discs heat-shocked at 19% p.d. In the absence of Notch function during cell sorting, IrreC-rst (violet) becomes ubiquitously distributed on all plasma membranes. Only partial cell sorting has taken place, leaving two rows of IOCs between ommatidia in many cases. DE-cadherin (green) is localised in a continuous apical belt but now also colocalises with IrreC-rst on all plasma membranes.

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