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First published online 3 August 2006
doi: 10.1242/dev.02468

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Cooperative activities of Drosophila DE-Cadherin and DN-Cadherin regulate the cell motility process of ommatidial rotation

Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY, USA.

View larger version (120K): [in a new window]   Fig. 1. Ommatidial rotation during PCP establishment in Drosophila eye. (A) Schematic presentation of ommatidial rotation in 3rd instar larval eye disc. Photoreceptors differentiate behind the morphogenetic furrow (MF, vertical yellow line). Five-cell preclusters are first organized in the AP axis. As photoreceptors become specified in the precluster, the group of cells simultaneously undergoes a 90° rotation towards the equator (horizontal yellow line). Green cells highlight R3/R4 precursors, and red marks future R1/R6 (stained with anti-Bar in C,D). (B) Tangential section of adult eye with ommatidia having completed 90° rotation (equator: yellow line). Right panel shows schematic presentation with dorsal and ventral chiral forms indicated with black and red arrows, respectively. (C,D) Larval eye disc with differentiating ommatidial preclusters posterior to MF, stained with neuronal marker Elav (blue; labeling all photoreceptor nuclei), Bar (red; R1and R6; also indicated in red in A) and Boss (green; labeling R8 in the center of the cluster). (C) Whole disc; (D) a higher magnification of a ventral area. White bars in D indicate the degree of rotation of the respective cluster. Anti-Bar-staining is detected from about a 30° angle to the posterior margin of disc, where all clusters acquire a 90° angle from their original position.

View larger version (112K): [in a new window]   Fig. 2. DE-cadherin promotes rotation. (A,B) Tangential sections of adult eyes (dorsal is upwards, anterior leftwards) with a schematic representation shown in bottom panels (arrows as in Fig. 1). (A) Eye of homozygous hypomorphic shgR6 escaper, showing rotation defects with ommatidia often rotating less than 90°. (B) Clone of an intermediate allele, shgP34-1 (marked by increased pigment levels; grey area in schematic) show rotation defects in addition to severe adhesion defects causing photoreceptor cell loss. (C-F) Confocal microscopy images of ventral regions of 3rd instar eye discs (anterior is leftwards, dorsal upwards). (C-C'') Clones of intermediate shgP34-1 allele. Blue: Elav, all photoreceptors. Red: Bar, labeling R1/R6 and highlighting rotation angle of each cluster. Green: GFP labeling wild-type tissue, mutant clones marked by absence of GFP. Non-rotating ommatidia are present, as visualized by Bar-staining (red in C,C') in mutant areas. (C'') Semi-schematic version of C', white bars indicate orientation of wild type and yellow bars indicate orientation of mutant clusters. (D-D'') Clones of shgP34-1 allele. Rotation is abnormal from its onset: anti-Spalt (Sal in red) is the earliest R3/R4 marker. R3/R4 fate is correctly specified in shgP34-1 clone, but many ommatidia do not initiate rotation (Elav, blue; GFP, green, marking wild-type tissue). White and yellow bars in D'' are as in C'' (white representing wild-type and yellow mutant clusters). (D',D'') Orange arrowheads mark preclusters composed of wild-type cells adjacent to mutant tissue that are misrotated, suggesting that shg/DE-cad is also required in interommatidial cells for rotation. (E-E') sev>DEcadWT partially rescues rotation in shgP34-1 clones (Bar, red; GFP, green, marking wild-type tissue). Many mutant preclusters have a `rescued' rotation angle. (F,F') Markers of apicobasal polarity are normally localized within shgP34-1 clones. Discs Large (Dlg; red), apical F-actin (phalloidin, green) and GFP (blue; marking wild-type tissue) are shown. Apical to epithelial disc layer are the squamous cells of the peripodial membrane (yellow arrowhead).

View larger version (71K): [in a new window]   Fig. 3. DE-cad requirements for ommatidial rotation. Tangential sections of adult eyes are shown (dorsal is upwards, anterior leftwards); associated bottom panels show schematic representations (arrows as in Fig. 1). (A) sevGal4; UAS-DE-cadDN (sev>DE-cadDN), a dominant-negative construct with two internally deleted CAD repeats, causes under-rotation. The under-rotation is enhanced by decreasing endogenous DE-cad/shg (B; sev>DEcadDN; shg-/+), and is suppressed by co-expression of full-length DE-cad (C; sev>DEcadDN;sev>DE-cadWT). (D) Decreasing endogenous Arm (armYD35/+, null allele) enhances under-rotation and adhesion defects of sev>DE-cadDN. (E) armXM19, an arm allele defective for Wg signaling, but functional for cell adhesion, does not modify the sev>DE-cadDN phenotype.

View larger version (126K): [in a new window]   Fig. 4. Complementary distribution of DE- and DN-cadherins during ommatidial rotation. All panels show confocal microscopy images of 3rd instar eye discs (anterior is leftwards, dorsal is upwards). (A) DE-cad (green) and Arm (red). Enriched staining is found in morphogenetic furrow (MF, white arrowhead) and within precluster cells (see also D and E for high-magnification images). Arm and DE-cad overlap, except at R3/R4 border, where DE-cad is almost absent (examples indicated by arrows). (B) Arm (red), DN-cad1 (blue). (C) Enriched DN-cad staining is present at the R3/R4 cell border (arrows in B,C; see also E for high magnification.) (D,E) High magnification of preclusters stained for DE-cad (green) and Arm (red) in D-D'', and DE-cadGFP (green), DN-cad (blue) and Fmi (red) in E-E''. DE-cad and DN-cad1 distribution is largely non-overlapping (E''). (F-I) DE-cad and Arm localization analyses in mutant backgrounds. (F) Arm (red) localization in N-cad14 clones (marked by absence of LacZ). Arm is reduced only at the R3/R4 border membrane (examples marked by arrowheads; compare with wild type marked by arrow). (G) Arm (red) and DN-cad (blue and G') in shgP34-1 mutant tissue. Arm is almost completely lost from apicolateral membranes, except where it co-localizes with DN-cad (which is not affected). (H). Example of DN-cad1 distribution in shgP34-1 clone (wild type marked by GFP: green): accumulation at R3/R4 border is unchanged (see arrowheads). (I-I'') Overexpression of DN-cad1 (marked by GFP; green) reduces DE-cad (blue and I'), but not Fmi (red and I'') levels.

View larger version (113K): [in a new window]   Fig. 5. DN-cad1 and DN-cad2 are required for normal ommatidial rotation. (A,B) N-cad14 double mutant clones in adult eyes (A) and eye discs (B-B''). (A) A mosaic eye containing N-cad14 clones (owing to the nature of the allele, the clone cannot be marked). Several clusters with rotation defects are present (dots in schematic indicate ommatidia with photoreceptor loss that cannot be scored for rotation). (B-B") Ventral part of eye disc with N-cad14 clone marked by absence of ßGal (green), stained for Elav (blue) and Bar (red); in semi-schematic (B''), the rotation angles are indicated with white (wild-type clusters) or yellow (mutant or mosaic clusters) bars. Most mutant clusters are over-rotated relative to their stage. (C) sev>DN-cad1 eye disc, Bar staining highlights random rotation throughout the disc (D, Bar-staining in wild-type disc for comparison). (E) Clone of cells overexpressing DN-cad1 (GFP). Localization of Arm (red and E') to plasma membranes and cytoplasmic membrane compartments is increased in the clone. (F-H) sev>DN-cad1 is suppressed by gene dose reduction in arm (sev>DN-cad1/arm4; compare G with F). Gaps between ommatidia resulting from decreased adhesion (although rotation defects are not enhanced by Arm decrease) and enhanced by Arm co-overexpression (sev>DN-cad1;>ArmS2; H).

View larger version (116K): [in a new window]   Fig. 6. RhoA affects DE-cad function in rotation and localization. (A,B) Tangential sections of adult eyes of the following genotypes (anterior is leftwards and dorsal upwards). (A) Co-expression of sev>DEcadDN with UAS-GFP (control). (B) Co-expression of sev>DEcadDN with UAS-RhoAIR. UAS-RhoAIR enhances under-rotation associated with sev>DEcadDN (compare A with B). Arrows are as in Fig. 1. (C) Confocal microscopy image of DE-cad (magenta and C') localization in rhoA72R clones (hypomorphic allele). DE-cad distribution within nascent ommatidial preclusters is disorganized, the overall DE-cad levels are reduced posterior to MF. Green: GFP labeling wild-type tissue.

View larger version (16K): [in a new window]   Fig. 7. Model for signaling input into cadherin function in cell motility. DE-cadherin appears to receive positive input from PCP and RTK (e.g. Egfr) signaling, which it translates into directed cell movement. The Egfr input could be either direct (as suggested from cell biology literature) or via nuclear signaling input, as suggested by the observed interaction with the ETS-factor Pointed. DN-cadherin serves as a `brake' to this movement, and might directly affect DE-cadherin expression. See text for details.