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First published online April 16, 2004
doi: 10.1242/10.1242/jcs.01034

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Genetic characterization of the Drosophila homologue of coronin

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India 500 007

View larger version (33K): [in a new window]   Fig. 1. (A) Genomic organization of the coro gene and the sequence of its protein product. Its cytological location is 42C5-D6. coro transcriptional length is 10443 bp, whereas the mRNA is 2610 nucleotides long. The P-element insertion in 409-GAL4 was in the 5' UTR (within the first exon) of CG9446. (B) Northern blot showing a single Coro transcript of 2.5 kb. (C) The predicted protein is 528 amino acids long, with 2 distinct WD40 repeats, shown in red. (D) Predicted coiled coil domain in the C-terminal of Coro protein. Predictions were carried out by submitting the protein sequence to BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html).

View larger version (36K): [in a new window]   Fig. 2. Expression patterns of Coro transcripts. (A) The expression pattern of 409-GAL4 in wing discs revealed by UAS-nuclear lacZ (green). Note that 409-GAL4 is expressed only in the peripodial membrane. The disc proper is stained for Wingless (red). (B-D) Embryonic expression patterns of Coro. It is ubiquitously expressed in cellular blastoderm (B), extended germ band (C) and retracted germ band (D) stages. RNA in situ hybridization was done using antisense probes for LD32977. (E,F) Embryos of cellular blastoderm (E) and extended germ band (F) stages probed with sense RNA used as a negative control. (G-H) Post-embryonic expression patterns of Coro. It is expressed at high levels in eye, leg (G) and wing (H) imaginal discs. Note that it is expressed at very low levels in the larval CNS (G).

View larger version (90K): [in a new window]   Fig. 3. Leg and wing phenotypes displayed by homozygous coroex11 flies. (A) Molecular lesions in coroex11 and coroex6 alleles. coroex11 carries a P-element insertion at the 5'UTR. coroex6 has a 100 bp deletion in the upstream regulatory region and a translocation within the third exon. (B) Wild-type leg. (C-E) coroex11/coroex11 legs. Note shortening and thickening of legs (C) and duplication of sex combs (D-E). (F) Wild-type wing blade. (G-H) Wing margin phenotypes seen in coroex11/coroex11 flies. (G) Both anterior and posterior margins are affected. (H) The anterior margin defect at higher magnification. (I) Wild-type wing blade showing distal most region at higher magnification. (J) coroex11/coroex11 wing blade. Note thickening of veins, which resembles the Delta phenotype, and small necrotic patches. (K) Adult coroex11/coroex11 fly showing highly reduced wing blade.

View larger version (116K): [in a new window]   Fig. 4. Eye phenotypes of coro mutants. (A) Adult eye of w1118 flies. (B) Adult eye of a coroex11/coroex11 fly. Note rough-eye phenotype. (C) Total loss of eyes is often seen in coroex11/coroex11 flies. (D) coroex11/coroex11 fly showing loosely attached ommatidia, which are protruding anteriorly. (E) SEM image of wild-type eye showing regular array of ommatidia. (F,G) SEMs of coroex11/coroex11 eyes showing loss of ommatidia, fusion of ommatidia (arrows) and loss of inter-ommatidial bristles.

View larger version (83K): [in a new window]   Fig. 5. Disruption of actin cytoskeleton and reduction in Arm and FASIII patterns along the apicobasal axis. (A-C) FITC-conjugated actin-phalloidin staining of wild-type (A), coroex11/coroex11 (B) and coroex6/coroex6 (C) wing discs. A' and A'' show disc in A at higher magnifications. Similarly B' and C' are higher magnification images of discs shown in B and C, respectively. Note moderate to severe disruption of fine network of the actin cytoskeleton in B and C, respectively. coroex6/coroex6 discs show a large number of darkly stained actin foci. Actin-phalloidin is normally localized to the apical surface of epithelial cells, which is reflected in higher intensity of staining at the DV boundary and at the presumptive hinge regions (A). In coroex6/coroex6 discs, such intense staining of actin-phalloidin is lost (C) suggesting loss of actin-phalloidin from the apical surface. (D,E) Wild-type (D) and coroex6/coroex6 (E) wing discs stained for Arm (green; D,E) and FASIII (red; D',E'). D'' and E'' are the merged images of D and D' and E and E', respectively. Note that in coroex6/coroex6 discs levels of both Arm and FASIII are very low and the staining is more diffused. (F-H) X-Z sections of wild-type (F), coroex11/coroex11 (G) and coroex6/coroex6 (H) wing discs stained for Arm (green) and FASIII (red). Note that normally Arm is apical and FASIII is basolateral. In wild-type wing discs both are expressed at very high levels. Both coroex11/coroex11 and coroex6/coroex6 show significant reduction in Arm and marginal reduction in FASIII staining indicating a possible defect in apicobasal polarity. In addition, cells are loosely arranged in coro- discs compared to wild-type disc, wherein cells are more compact. Normally coroex6/coroex6 discs show severe loss of FASIII and Arm staining (as shown in E) and general loss of cell and disc morphology (as shown in C). To ascertain specific effects of loss of coro on apicobasal polarity of wing discs, we have chosen those coroex6 wing discs, which show milder phenotype.

View larger version (146K): [in a new window]   Fig. 6. coro mutant wing discs show defective AP boundary and reduced Dpp signaling to distant cells. (A) Wild-type wing disc. (B) coroex11/coroex11 wing disc showing separation of anterior and posterior compartments. (B') Higher magnification image of the disc shown in B. (C) coroex6/coroex6 wing disc. Note that the disc is very small and no specific defect in the AP or DV boundary is seen. (D) coroex6/coroex11 wing disc. Note both small-disc phenotype as in coroex6/coroex6 discs and separation of dorsal and ventral compartments along the DV boundary. (E,E'') Optical sections of anti-Arm-stained wild-type wing disc to show that the epithelium is contiguous in all focal planes. (F,F'') Optical sections of anti-Arm-stained coroex11/coroex11 wing disc to show that peripodial membrane is contiguous and large squamous epithelial cells of the peripodial membrane are normal. Also in the disc proper, the columnar epithelium is contiguous, although mainly at the basal end of the cells. Images in E,E'' and F,F'' were generated by taking nearly 60 optical sections each of 0.5 µm thickness, which were then used to reconstruct disc images at three equal focal planes.

View larger version (77K): [in a new window]   Fig. 7. Down regulation of Dpp signaling in coro mutants. (A,B) Ci expression patterns of wild-type (A) and coroex11/coroex11 (B) wing discs. Expression of Ci in coroex11 wing discs is normal, although they show defective AP boundary (arrows). (C,D) Expression pattern of Dpp in wild-type (C) and coroex11/coroex11 (D) wing discs. Dpp is visualized with the help of dpp-GAL4/UAS-GFP. In coroex11/coroex11 wing disc Dpp expression is normal despite separation of anterior and posterior compartments along the AP boundary (arrows). GFP seen in cells outside the AP boundary is mainly because of residual GAL4 activity of coroex11. (E,F) Wg expression patterns of wild-type (E) and coroex11/coroex11 (F) wing discs. Wg expression along the DV boundary is normal in the coroex11 wing disc. (G-I) Wild-type (G), coroex11/coroex11 (H) and coroex6/coroex6 (I) wing discs stained for Sal expression. Note reduced levels of Sal in coroex11 and coroex6 discs, particularly in the posterior compartment. Here too we have chosen those coroex6 wing discs that show milder phenotype, to examine the effect on Dpp signaling. (J-K) Wild-type (J) and coroex11/coroex11 (K) wing discs stained for Omb expression. Note that Omb expression in the posterior compartment is nearly absent.

View larger version (98K): [in a new window]   Fig. 8. Enhanced response to Dpp over-expression in coro mutants. (A,B) Low magnification images of dpp;GAL4/UAS-Dpp::GFP (A) and coroex11/coroex11; dpp;GAL4/UAS-Dpp::GFP (B) wing discs. Note differences in the size of the two discs. coroex11 and coroex6 heterozygous and coroex6/coroex6 and coroex6/coroex11 flies too showed enhanced growth response to Dpp-overexpression (data not shown). (C-E) Dpp::GFP-expressing wild-type (C), coroex11/coroex11 (D) and coroex6/coroex6 (E) wing discs stained for both GFP (green) and Arm (red). In the wild-type background, bright punctate spots of over-expressed Dpp::GFP are visible only in cells close to the AP boundary, the source of the morphogen. In coro mutant discs, bright spots of Dpp::GFP are visible in all cells of the disc, even in cells farthest from the AP boundary. (F,G) Dpp::GFP-expressing wild-type (F) and coroex11/coroex11 (G) wing discs shown at higher magnification. Note that in the coroex11 disc, bright spots of Dpp::GFP are larger, more numerous and mostly associated with the plasma membrane. (H,I) Intensity profile of wing discs shown in C and D, respectively. Posterior compartment cells immediately adjacent and parallel to the AP boundary were used for intensity profiling in both the discs. Note large number of Dpp::GFP peaks (green) outside Arm peaks (red) in wild-type background. However, nearly all DPP::GFP peaks coincide with Arm peaks in coroex11/coroex11 background suggesting that Dpp containing-vesicles are on the membrane. (J) coroex6/coroex6; dpp;GAL4/UAS-Dpp::GFP wing disc stained with FITC-conjugated actin-phalloidin. Note considerable rescue of actin cytoskeleton. Compare this disc with that of coroex6/coroex6 shown in Fig. 5C. (K) coroex6/coroex6; dpp;GAL4/UAS-Dpp::GFP wing disc stained with Arm (green) and FASIII (red). Note that the levels of both Arm and FASIII are now comparable to the wild-type (shown in Fig. 5D).

View larger version (129K): [in a new window]   Fig. 9. Over-expression of activated Tkv mimics coro phenotypes. (A-C) dpp;GAL4/UAS-Tkv* (A), coroex11/+; dpp;GAL4/UAS-Tkv* (B) and coroex6/+; dpp;GAL4/UAS-Tkv* (C) wing discs stained for Arm to outline the cells. Over-expression of Tkv* in the AP boundary causes cleft formation similar to that in coro- discs (Fig. 6B), although the phenotype is milder. Over-expression of Tkv* in coroex6 and coroex11 heterozygous backgrounds, however, results in the formation of a deep cleft, which is normally seen in coroex11 homozygous background (Fig. 6B). Over-expression of Tkv* in coroex6 and coroex11 homozygous backgrounds caused very high levels of lethality in early larval stages. (D,E) Adult wing blades of dpp;GAL4/UAS-Tkv* (D) and coroex11/+; dpp;GAL4/UAS-Tkv* (E) flies. Note severe notching of the wing blade in E. coroex6/+; dpp;GAL4/UAS-Tkv* also showed similar phenotypes. (F,G) dpp-GAL4/UAS-DNSyx1A (F) and coroex11/coroex11; dpp-GAL4/UAS-DNSyx1A (G) wing discs stained for Arm to outline the cells. Over-expression of DN-Syx1A in the AP boundary does not cause any significant phenotype. The severity and penetrance of cleft phenotype is enhanced when DN-Syx1A is expressed in coroex11 background. G and G' show two different levels of optical sections. coroex6 over-expressing DN-Syx1A were lethal at early-third instar larval stages and therefore we were unable to examine third instar wing discs. (H,I) dpp;GAL4/UAS-Tkv* (H), coroex11/+; dpp;GAL4/UAS-Tkv* (I) and coroex6/+; dpp;GAL4/UAS-Tkv* (J) wing discs stained for Sal. Over-expression of Tkv* causes down regulation of Sal in distant cells, which is more pronounced in the posterior cells (Lecuit and Cohen, 1998). Sal levels are severely reduced in both anterior and posterior cells when Tkv* is over-expressed in coro heterozygous backgrounds.

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