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First published online 14 July 2004
doi: 10.1242/dev.01278

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Functional divergence between eyeless and twin of eyeless in Drosophila melanogaster

Claudio Punzo^*, Serge Plaza, Makiko Seimiya, Pamela Schnupf, Shoichiro Kurata, Johannes Jaeger^¶ and Walter J. Gehring^**

Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland

View larger version (52K): [in a new window]   Fig. 7. ey mutant clones in the eye disc express DLL. (A) Genomic organization of the ey region. The nine exons of the ey gene are indicated as turquoise boxes. The areas coding for the PD are highlighted in blue and the areas coding for the HD in red. The extent of the eyJ5.71 deletion is indicated by a gray box. The DOC-element insertion of the ey2 mutant into the second intron of the ey gene is indicated by a triangle. The two genomic P-element clones and the respective genomic areas they cover are marked below. (B) Flies generated to perform the cross for the clonal analysis. Larvae that were y– (25%) were dissected for antibody staining. (C-F) Antibody staining on eye discs where mutant clones were induced. DLL expression is shown in red, GFP expression in green recapitulates the smo enhancer expression domain in the posterior part of the disc. Posterior mutant clones are marked by a lack of GFP expression. Only clones visualized in the posterior domain of the eye disc are shown. DLL expression was never detected in cells present in the ey domain (anterior part of the eye disc) in ey–/– cells. Anterior clones were detected by the use of an ey antibody. (C) Shows the entire eye-antennal disc with DLL expression in the antenna and a clone of DLL-expressing cells in the eye disc. (D-F) Magnification of the image shown in C. (D) Merged image of GFP (E) and DLL (F) expression. (G) Antibody staining with the rabbit -EY antibody where clones were randomly induced. Lack of EY protein is only visible in the anterior part of the eye disc (where EY is expressed). Brighter twin clones have two copies of the ey transgene (arrows).

View larger version (59K): [in a new window]   Fig. 1. Ectopic eyes in an ey2 mutant background. For each panel (A-L), the UAS transgene that is driven by the dppblink-Gal4 transgene in an ey2 mutant background is indicated in the lower right of the panel. (A-D) Phenotype of adult flies carrying the UAS transgene indicated. Misexpression of toy (A), toyHD (B), sey (C) and seyHD (D) leads to the induction of ectopic eyes in an ey2 mutant background. (E-L) Early and late marker genes are induced during ectopic eye development in the absence of endogenous ey. (E-H) lacZ expression of third instar leg discs carrying the so-enhancer trap, in addition to the dppblink-Gal4 transgene and the UAS-construct indicated in an ey2 mutant background. All four transgenes were able to induce lacZ expression. (I-L) Rhodopsin 1 expression in ectopic eyes. Rhodopsin 1 was monitored with an -Rhodopsin 1 antibody on cryosections of ectopic eyes induced by the UAS transgene indicated driven by dppblink-Gal4 in an ey2 mutant background. Rhodopsin 1 was found to be expressed in ectopic eyes induced by all four transgenes. Arrows indicate the retina.

View larger version (54K): [in a new window]   Fig. 2. Dosage dependence of the phenotype. (A,B) Flies carrying a UAS-eyHD transgene in addition to the dppblink-Gal4 transgene. A and B are two independent transgenic lines of the same transgene showing ectopic eyes (arrowheads) obtained to a different degree of strength. (C) Western blot analysis using a rabbit -EY antibody on third instar leg discs expressing the transgenes used for the phenotypes obtained in panels A and B. The strength of the phenotype correlates with the amount of ectopically induced protein. Asterisk marks the protein expressed at lower level corresponding to the ectopic eyes in panel A. Molecular weight marker is indicated at the right. Lanes 1 and 2, yw control wing and leg discs, respectively; lanes 3-5, leg discs expressing the various ey constructs by dppblink-Gal4. (Lane 3) Misexpression of eyHD, transgenic line of panel A. (Lane 4) Misexpression of eyHD, transgenic line of panel B. (Lane 5) Misexpression of full-length ey. (D-F) Temperature dependence of the Gal4 system. (D,E) Flies carrying the UAS-toy transgene and the dppblink-Gal4 transgene in an ey2 mutant background raised either at 18°C (D) or at 25°C (E). Ectopic eyes were never observed on flies raised at 18°C, in contrast to flies raised at 25°C. (F) Western blot analysis with a rabbit -quail-PD antibody of third instar leg discs expressing the various Pax6 transgenes under the control of the dppblink-Gal4 transgene in a wild-type background. Pax6 proteins are marked by asterisks. Molecular weight marker is indicated at the right. (Lanes 6-11) Misexpression of ey (lane 6), eyHD (lane 7), toy (lane 8), toyHD (lane 9), seyHD (lane 10) and seyHD (lane 11).

View larger version (50K): [in a new window]   Fig. 3. Ectopic eyes on the antenna depend on the C terminus of EY. (A) Column 1 shows the head region, including the antenna of flies carrying the dppblink-Gal4 transgene and the corresponding transgene (marked on the left of each row) in a wild-type background. Ectopic eyes (arrows) on the antenna were induced only by transgenes containing the EY C terminus. Column 2 shows so10-lacZ expression on eye-antennal discs of third instar larval stage. In addition to the so10-lacZ transgene, they express the same transgenes as the flies in column 1. ß-Galactosidase expression was detected only in antennal discs where ectopic eyes on the adult antenna were also observed. Asterisks mark the area of ectopic staining. (B) Western blot analysis of third instar leg discs carrying the so10-lacZ, the dppblink-Gal4 transgenes and the different ey/toy chimeras in a wild-type background. Molecular weight marker is indicated at the right. The western blot was probed once with a rabbit -quail-PD antibody (upper) and once with an anti-ß-Galactosidase antibody (lower). The various Pax6 proteins are indicated by an asterisk. (Lanes 1-6) Misexpression of toy (lane 1), ey (lane 2), toyPD-EYBB (lane 3), EYBB-toyCT (lane 4), TOYBB-eyCT (lane 5) and eyPD-TOYBB (lane 6). Lanes 3 and 5 show a higher amount of ß-Galalactosidase protein, consistent with the fact that both transgenes inducing ß-Galalactosidase contain the ey C terminus.

View larger version (72K): [in a new window]   Fig. 4. Antenna duplication by DLL induction. (A-F) Eye discs stained with an -DLL antibody (B,D,F) expressing UAS-P35 under the control of ey-Gal4 in an ey2 mutant background. (A,C,E) Brightfield microscopy pictures of eye discs shown in B,D and F, respectively. (A,B) Late second instar, (C,D) early third and (E,F) late third instar eye discs. DLL is gradually induced (D), independently of endogenous DLL expression in the antenna, and leads to a transformation of the eye disc into an antennal disc (F). (G,H) The duplication of the antennal disc leads to a duplication of the adult antenna with all segments present, and the duplicated antenna in the location of the missing eye (arrows in G,H).

View larger version (98K): [in a new window]   Fig. 5. Induction of Dll expression in the eye disc. (A-C) Antibody staining with -DLL antibody on late third instar eye discs carrying the ey-Gal4 and the UAS-ey (A), the UAS-eyPD (B) and the UAS-eyHD (C) transgenes in an ey2 mutant background. DLL is induced in small patches of cells only in the absence of the EY-HD (arrows in C). (D) Antennal-like outgrowth from the adult eye of a fly carrying the ey-Gal4 and the UAS-eyHD transgene in an ey2 mutant background. The arrow indicates the regular antenna and the arrowhead the ectopic antenna.

View larger version (128K): [in a new window]   Fig. 6. Repression of antennal identity by the EY-HD. (A-F) Flies carrying the ey-Gal4, the UAS-P35 and the UAS-ey (A,D), the UAS-eyPD (B,E) or the UAS-eyHD (C,F) transgenes in an ey2 mutant background. A and C show a full rescue of the ey2 mutant eye phenotype by a PD containing EY protein. The percentage of flies rescued by ey or eyHD are 50% or 79%, respectively, for a rescue of at least 80% of the wild-type eye size (Punzo et al., 2001). D and F show the cases where the rescue did not work at all, even though the PD was present. In those cases, the co-expression of P35 led to a duplication of the antenna (arrowhead, duplicated antenna; arrow, wild-type antenna) only in the absence of the EY-HD, indicating that the HD is able to repress antennal identity. B and E show two different eye sizes of an ey2 mutant, where no rescue is observed because of the expression of a PD-deleted EY protein, but, in addition, no duplication of the antenna is observed because of the presence of the HD.