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Regulation of Apterous activity in Drosophila wing development

Ulrich Weihe, Marco Milán and Stephen M. Cohen*

European Molecular Biology Laboratory, Meyerhofstr 1, 69117 Heidelberg, Germany



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  		Fig. 1. Ap protein levels differ between the dorsal wing pouch and the hinge region. (A) ap mRNA levels visualized in a wild-type disc by in situ hybridization with an ap antisense RNA probe. (B) Third instar wing imaginal disc labeled with anti-Ap. Ap levels were higher in the hinge region than in the dorsal wing pouch. The difference in Ap protein levels between pouch and hinge is not reflected by a difference in transcript levels. (C) Anti-dLMO staining of the disc in B. (D) Overlay of B and C. Low Ap levels coincide with high dLMO levels.

 


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  		Fig. 2. dLMO decreases Ap levels in the dorsal wing pouch. (A) Wing disc with a clone of dLMO{Delta}39 mutant cells. dLMO protein was not detectable in the mutant clone (center). Ap levels were higher in the dLMO mutant cells (left ). Overlay of Ap and dLMO staining (right). (B) Region of an ap-lacZ wing disc with a large clone of dLMO{Delta}39 mutant cells (arrow). The clone was labeled by the absence of GFP (right). Ap protein increased in the clone (left), but ap-lacZ transcript levels reflected by anti-ß-gal did not differ between the clone and the surrounding tissue (center). (C) Disc with clones of dLMO-expressing cells (labeled by co-expression of GFP, green). The reduction of Ap is most obvious in the hinge where the endogenous protein level is higher (long arrow). dLMO also reduced Ap levels in the wing pouch (note the loss of nuclear label in the clone near the short arrow). The residual signal in the clone reflects nonspecific background produced by the antibody (as in the ventral compartment in B).

 


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  		Fig. 3. Ap is degraded by a proteasome-mediated mechanism in the wing pouch. (A,B) Wild-type, DMSO-treated discs stained with anti-Ap (A) and anti-Ci (B) antibodies. (C,D) Wild-type discs treated with MG-132 for 3 hours. Ap staining was more intense in the dorsal wing pouch after treatment with the proteasome-inhibitor (D) when compared with the hinge (C). In D, the previously reported stabilization of Ci is evident. (E) Ratio of Ap staining intensity in the pouch and hinge region (averaged over three areas of the wing pouch in the discs in A,C). The level of Ap in the pouch and hinge are more similar in the MG132-treated disc.

 


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  		Fig. 4. Complex formation with Chip is required to stabilize Ap protein. (A) Wing imaginal disc with homozygous Chipe5.5 mutant clones. Clones were marked by the absence of ß-gal (right). Ap levels decreased in mutant clones, compared with the surrounding tissue (left panel, arrows). Ap levels were higher in homozygous wild-type twin spots, which contain two copies of the Chip gene, than in the tissue heterozygous for Chip (arrowheads). (B) ptcGal4 uas-EGFP; uas-Chip{Delta}LID wing disc. Chip{Delta}LID overexpression was visualized by co-expression of GFP (left panel). Ap expression was reduced (anti-Ap shown in white, center). ap-lacZ transcript levels reflected by anti-ß-Gal did not differ (right). (C) dppGal4 uas-EGFP; uas-ChAp wing disc. ChAp and GFP were co-expressed (left; GFP shown in green). Endogenous Ap protein was reduced to background levels in these cells (right panel, arrow). Note the anti-Ap antibody does not recognize ChAp.

 


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  		Fig. 5. Complex formation with DNA and Chip stabilizes Ap protein. (A) Immunoblot of S2 cell lysates transfected with constructs to express myc-tagged Ap and myc-tagged dLMO. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of dLMO-myc as indicated. Total levels of transfected DNA were held constant using empty vector to compensate for alteration in the level of dLMO-myc plasmid. Both proteins were visualized with anti-myc. The arrowhead indicates a nonspecific band to serve as a loading control. (B) Immunoblot of S2 cell lysates transfected with a plasmid to express myc-tagged Ap and with plasmids containing additional Ap-binding sites. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of plasmid containing additional Ap-binding sites (competitor). Total DNA levels were held constant in the transfection by addition of empty vector. Ap protein levels increased with increasing copies of the binding site construct. (C) Immunoblot of S2 cell lysates transfected with constructs to express myc-tagged Ap and myc-tagged ChAp. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of ChAp-myc as indicated. Both proteins were visualized with anti-myc. Increasing amounts of ChAp decreased levels of Ap-myc protein. We tested whether the endogenous dLMO gene was induced in ChAp transfected cells by immunoblotting with anti-dLMO, and were unable to detect it (not shown). The level of dLMO that was needed to reduce Ap-myc protein in S2 cells was readily detectable by immunoblotting, so we conclude that the effect of ChAp-myc was not due to increased levels of dLMO.

 


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  		Fig. 6. The Ap-dLMO fusion protein dLAp is functional. (A) Domain organization of Apterous, dLMO and dLAp-flag. For the fusion protein dLAp-flag, the LIM-domains of Apterous were replaced by the LIM-domains of dLMO. In addition, dLAp contains a C-terminal flag-tag to make it distinguishable from the endogenous Ap and dLMO proteins. (B) Confocal image of a third instar wing imaginal disc of the genotype dppGal4; uas-dLAp-flag stained with anti-Ap (red) and anti-Wg (green). Endogenous Ap protein barely detectable as faint red label in the dorsal compartment. The intense red stripe reflects overexpression of dLAp in the dppGal4 domain. The Wg stripe follows the border between dLAp-expressing and non-expressing ventral cells. (C) Cuticle preparation of an apGal4/apUGO35wing. The heteroallelic combination apGal4/apUGO35 shows strongly reduced Ap activity but retains Gal4 expression in the Ap domain. (D) Cuticle preparation of an apGal4/apUGO35; uas-dLAp-flag wing. Expression of the uas-dLAp-flag transgene in this domain is able to support wing development, indicating that it can provide Ap function. (E) Cuticle preparation of a dppGal4; uas-dLAp-flag wing. Overexpression of dLAp along the AP boundary by dppGal4 leads to the formation of ectopic wing margin in the ventral compartment (arrow).

 


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  		Fig. 7. Overexpression of dLAp-flag causes an ap gain-of-function phenotype. (A) Cuticle preparation of an apGal4; uas-dLAp-flag wing. Overexpression of dLAp-flag caused extra vein tissue between veins 4 and 5 and other vein defects. (B) apGal4; uas-dLAp-flag fly showing the abnormal wing posture and upward curvature of the wing caused by the reduced size of the D compartment. (C) Serrate protein expression (green) in a disc overexpressing uas-ap in the apGal4domain. The pattern of Serrate staining is the same as in wild-type discs and shows elevated expression along veins 3 and 4 and on both sides of the DV boundary. (D) Expression of uas-dLAp-flag caused overexpression of Serrate in the D compartment. Ectopic Serrate staining can be seen in intervein regions (e.g. arrow). Note the reduced size of the D compartment. (E) dLMO and Wg protein expression in an apGal4; uas-dLMO; uas-ap third instar wing disc. Anti-dLMO (green) and Anti-Wg (blue). Note the small wing pouch (arrow) and the absence of Wg expression at the interface between D (green) and V (not green) cells. (F) dLMO and Wg protein expression in an apGal4; uas-dLMO; uas-dLAp third instar wing disc. Note that Wg expression is restored along the DV boundary and growth of the wing pouch is restored.

 


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  		Fig. 8. Model. (A) Ap activity regulation under wild-type conditions. Once dLMO is expressed, Ap detaches from Chip and is degraded. Transcriptional activation of its target genes is abolished. For simplicity we depict degradation of Ap monomer. It is also possible that any form of incomplete Ap;Chip complex is degraded. (B) Inhibition of Ap:Chip tetramer formation by Chip{Delta}LID. (C) ChAp binds to Ap target sites on DNA and displaces endogenous Ap. dLMO cannot interfere. Ap is degraded. (D) Inhibition of Ap activity by dLMO can be reverted by providing the fusion protein dLAp. dLAp competes with dLMO and activates Ap-dependent transcription.

 

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© The Company of Biologists Ltd 2001