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First published online 2 September 2008
doi: 10.1242/jcs.035014

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Hairless induces cell death by downregulation of EGFR signalling activity

University of Hohenheim, Institute of Genetics (240), 70599 Stuttgart, Germany

View larger version (29K): [in this window] [in a new window]   Fig. 1. H induces apoptosis in S2 cell culture. (A) TUNEL measurements on cell lines that express the indicated constructs. Stably transfected S2 cells contain the copper-inducible pMT-Gal4 plasmid and the indicated UAS constructs. For controls, S2 cells and stably transfected S2 cells with pMT-Gal4 and the pMT-empty UAS vector (mock) were used. Forty-eight hours after induction, TUNEL staining was performed and the number of TUNEL-positive cells was determined. y-axis, percentage of dead cells. Values were derived from three independent experiments. Error bars indicate s.d.; brackets indicate genotypes compared for significant differences by Student's t-test (*P<0.001, **P>0.05). (B) Examples of S2 cells stained with TUNEL reagent and visualised by confocal imaging. The sequence of the cells is according to A. (C) Hairless full-length and mutant proteins are expressed at similar levels and at the expected molecular weight upon induction. Western blots were probed with anti-NTH [-H(150kDa)] to detect the long HP150 isoform (left panel), and with anti-Actin [-Act(42kDa)] to detect the overall protein levels. Note the low levels of endogenous H protein in untransfected S2 cells.

View larger version (84K): [in this window] [in a new window]   Fig. 2. H-mediated cell death in the developing eye depends on the binding of Gro and CtBP. (A) Drosophila compound eye with the typical regular hexagonal arrangement of ommatidia. As a control, Gmr-Gal4/+; UAS-lacZ/+ is shown (Gmr, glass multimer reporter; expression domain is restricted to the differentiating eye field posterior to the morphogenetic furrow). (B,D) Overexpression of full-length H in the Gmr pattern causes a small, rough eye phenotype (Gmr-Gal4>UAS-HFL/+; UAS-lacZ/+). UAS-lacZ was included in the genotype shown in B to illustrate the titration effect of an additional UAS-copy. (C) The small eye phenotype is rescued by combined overexpression with the anti-apoptotic factor p35; UAS-p35/+; Gmr-Gal4>UAS-HFL/+. Note, however, that the arrangement of the ommatidia is somewhat irregular owing to underlying differentiation defects. (E,F) Overexpression of H*G (E, Gmr-Gal4/+; UAS-H*G/+) or H*C (F, Gmr-Gal4/+; UAS-H*C/+) has significantly milder effects on eye size and roughness than the wild-type HFL (D). (G) Gmr-Gal4/+; UAS-H*GC/+ flies have eyes that very closely resemble those of wild type. (A'-G') Cell outlines were stained in pupal retinae to visualise the number and arrangement of pigment and cone cells. Note the normal complement of four cone cells in the control (A') and upon suppression of apoptosis by p35 (C'). Cone cells are marked with an asterisk. Overexpression of HFL induces cone cell death, such that two to three cone cells typically remain per ommatidium (B',D', arrowheads). Upon overexpression of H mutant constructs that lack Gro (E') or CtBP (F') co-repressor binding sites, most cone cells of the eye develop. The double mutant, H*GC (G'), has little effect and the retina closely resembles that of the wild type with regard to cone and pigment cell numbers. (H) Determination of eye size by quantification of facet numbers. Bars represent eye size, mean facet number is given. Error bars indicate s.d. (I) Quantification of cone cell numbers for the given genotypes; error bars indicate s.d.

View larger version (115K): [in this window] [in a new window]   Fig. 3. H overexpression results in Caspase 3 activation. Cell clones overexpressing HFL, H*GC, or GFP as control, were generated in eye imaginal discs and stained with an antibody against activated Caspase 3, thereby assessing cell death induction in vivo. HFL clones induce a strong Caspase 3 activity within the clone (red; middle row). By contrast, H*GC, which is defective in co-repressor binding, does not induce Caspase 3 activation (lower row), thereby resembling the control (upper row).

View larger version (65K): [in this window] [in a new window]   Fig. 4. Negative relationship between EGFR signalling and Hairless with regard to cell death in the developing eye. (A,B) Heads of adult female flies carrying Gmr-Gal4>UAS-HFL/+ combined with different mutants that affect components of the EGFR signalling pathway (A); control flies (B). The upper row shows scanning electron micrographs that were used for facet counts (see C); the lower row shows live specimens at low magnification that were used to measure the surface area. Images within each row are at the same magnification. (1) Gmr-Gal4>UAS-HFL/+; (2) Gmr-Gal4>UAS-HFL/+, argosrlt/+; (3) Gmr-Gal4>UAS-HFL/aop1; (4) Gmr-Gal4>UAS-HFL/StarIIN; (5) argosrlt/+; (6) aop1/+; (7) StarIIN/+. (C) Bar chart of facet counts for the different genotypes; mean facet number is given. Error bars indicate s.d. Numbers beneath the bars refer to genotypes shown in A,B. (D) Quantification of eye size by area measurements in pixels (x1000). Error bars indicate s.d. Numbers beneath the bars refer to genotypes shown in A,B.

View larger version (54K): [in this window] [in a new window]   Fig. 5. H gain-of-function clones show reduced EGFR signalling activity along the morphogenetic furrow in eye imaginal discs. Cell clones overexpressing full-length HFL or the double mutant H*GC were induced in eye imaginal discs; they are marked by the expression of GFP (green). (A-A'') rho-lacZ expression (red in A',A'') is reduced in HFL gain-of-function clones. (B-B'') H*GC gain-of-function clones do not cause any apparent alterations in rho-lacZ expression (red in B',B''). (C-D'') The level of diP-ERK (red in C',C'' and D',D''), a direct readout of EGFR signalling activity, is reduced in HFL clones (arrows, green in C,C''), but not in H*GC clones (arrows, D,D''). Note that the effect of H on EGFR signalling activity is primarily observed along the morphogenetic furrow. In all panels, third instar eye discs are shown with anterior towards the right and dorsal upwards.

View larger version (87K): [in this window] [in a new window]   Fig. 6. H genetically interacts with lozenge and klumpfuss. (A,B) Heads of adult female flies carrying Gmr-Gal4>UAS-HFL combined with lozenge and klumpfuss heterozygous mutants (A), and the respective controls (B). The upper row shows scanning electron micrographs that were used to determine facet numbers (see C); the lower row shows live specimens at low magnification as used to determine surface size (see D). Images within each row are at the same magnification. (1) Gmr-Gal4>UAS-HFL/+; (2) lz1/+, Gmr-Gal4>UAS-HFL/+; (3) Gmr-Gal4>UAS-HFL/+, klu09036/+; (4) lz1/+; (5) klu09036/+. (C) Facet counts for genotypes shown in A,B. Numbers refer to average facet counts. Error bars indicate s.d. *P<0.001 by Student's t-test; brackets indicate genotypes compared. (D) Quantification of eye size by area measurements in pixels (x1000). Error bars indicate s.d. *P<0.001 by Student's t-test; brackets indicate genotypes compared. Numbers beneath the bars refer to genotypes shown in A,B.

View larger version (74K): [in this window] [in a new window]   Fig. 7. H induces the expression of lozenge, klumpfuss and argos. Cells overexpressing full-length HFL or the double mutant H*GC were generated in eye imaginal discs. They are marked by GFP expression (green). Expression of Lozenge (A-B''), klu-lacZ (C-D'') and Argos (E-F'') (all in red) is shown. All three are activated by HFL, which is best seen in areas in front of the morphogenetic furrow. These effects are not observed in H*GC gain-of-function clones (B,B'',D,D'',F,F''). In all panels, third instar eye discs are shown with posterior towards the right and dorsal upwards.

View larger version (21K): [in this window] [in a new window]   Fig. 8. H mediates apoptosis by downregulation of EGFR signalling activity. H, together with the co-repressors Gro and CtBP, impedes EGFR signalling activity at various levels. First, it inhibits rho expression, presumably by direct transcriptional silencing. It thereby interferes with the production of the EGFR ligand and subsequent activation of the EGFR signalling pathway. This results in a diminished degree of phosphorylation and reduced activation of the MAPK. Second, H causes activation of negative regulators of the EGFR signalling pathway, including lozenge (lz), argos (aos) and klumpfuss (klu). Since Lz activates transcription of the other two genes, the effect of H may be explained solely through its positive regulation of lz. Since H acts as a repressor, we favour a model in which activation of lz, aos and klu is a result of the inhibition of an as yet unknown repressor(s) that normally restricts their activity. As these three factors have been shown to induce apoptosis in the Drosophila developing retina, induction of apoptosis is to be expected for H.

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