JCS035014 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Hairless wild-type and mutant forms induce apoptosis in cell culture. A total of 1×10⁶ S2 cells each were transiently transfected with pMT-Gal4 plus pUAST-reaper, pMT-HFL, pMT-HΔS, pMT-HΔG, pMT-HΔC or pMT-Gal4 (mock) along with the hs-lacZ reporter. lacZ activity was determined as a measure of cell viability. y-axis represents percentage of dying cells determined by the drop of lacZ activity in induced versus uninduced cultures. Values were gained from two independent experiments. Error bars represent s.d.; brackets indicate genotypes compared with pMT-HFL for statistical significance by Student's t-test. *P<0.001. Note that H constructs lacking the binding sites for either Su(H) (HΔS), Gro (HΔG) or CtBP (HΔC) fail to induce apoptosis, just like the control (mock).

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Failure of co-repressor binding restores eye size in H gain-of-function flies. (A-G) Documentation of adult female eyes. (A) Gmr-Gal4/+; UAS-lacZ/+. (B) Gmr-Gal4>UAS-HFL/+; UAS-lacZ/+. (C) UAS-p35/+; Gmr-Gal4>UAS-HFL/+. (D) Gmr-Gal4>UAS-HFL/+. (E) Gmr-Gal4/+; UAS-H*G/+. (F) Gmr-Gal4/+; UAS-H*C/+. (G) Gmr-Gal4/+; UAS-H*GC/+. (H) Quantification of eye size. The surface area of at least 20 eyes of each genotype was measured using ImageJ software and the mean value was determined. Error bars represent s.d. *Improper size measurement due to bulging of the eye.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Ectopic H signalling influences cone cell fate. (A-A′′) Overexpression of HFL using Gmr-Gal4 results in cone cell loss during early pupal development. Pupal retinae were stained with anti-Cut to show cone cell fate (green in A,A′′, arrows) and anti-Elav (red in A′,A′′). (B-B′′) Late pupal retinae show increased signs of cell death making cell type characterisation impossible; anti-Cut (green in B,B′′), anti-Elav (red in B′,B′′). Genotype in A-B′′: Gmr-Gal4>UAS-HFL/+. (C-C′′) The wild-type complement of four cone cells (anti-Cut green in C,C′′) is detectable upon overexpression of H*GC (Gmr-Gal4/+; UAS-H*GC/+); anti-Elav red in C′,C′′.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. HFL induction causes Caspase 3 activity and cone cell loss. (A-D′′) HFL clones (marked with GFP in A′′,B′′,C′′,D′′) induced during eye development results in increased Caspase 3 activity (anti-Caspase 3, red in A,B,C,D) and reduced cone cell number (anti-Cut, blue in A′,B′,C′,D′). (A-A′′) Overview of a pupal eye disc where different HFL clones were induced. Three areas are boxed (1-3), the magnifications of which are shown in B-B′′′ (1), C-C′′′ (2) and D-D′′′ (3). (B-B′′′) Magnification of HFL clone (GFP in B′′,B′′′) lying most anteriorly (box 1). Although within the HFL clone Caspase 3 activity is induced (red in B,B′′′), the normal complement of four cone cells is still traceable (blue in B′,B′′′). (C-C′′′) Within a HFL clone located more posteriorly (box 2) (GFP in C′′,C′′′), Caspase 3 activation (red in C,C′′′) causes a reduced Cut signal (blue in C′,C′′′, arrows). (D-D′′′) HFL clone located most posteriorly (box 3), where cell type specification has proceeded most, reveals a loss of cone cells (blue in D′,D′′′, asterisk) and of Caspase 3 signal (red in D,D′′′) due to cell type loss. (E-E′′′) No signs of Caspase 3 activity (red in E,E′′′) and cone cell loss (blue in E′,E′′′) are detectable in H*GC clones (green in E′′,E′′′) induced during eye development. Clones are framed in yellow.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Ectopic H signalling does not impair Diap1, eyg-lacZ or E2F expression. (A-A′′) Ectopic expression of HFL in the posterior compartment of the eye using Gmr-Gal4 does not affect Diap1 protein levels. The ectopic expression of HFL is visualised with anti-H (green in A′,A′′). Diap1 was detected by an anti-Diap1 antibody (red in A,A′′). Genotype is Gmr-Gal4>UAS-HFL/+. (B-B′′) HFL gain-of-function clones induced during early larval development show no modification of eyg-lacZ expression (red in B,B′′). HFL clones are marked with anti-H (green in B′,B′′). (C-C′′) HFL gain-of-function clones do not interfere with wild-type E2F expression in the eye imaginal disc. HFL clones are marked with GFP (green in C′,C′′), whereas E2F expression was followed by anti-E2F staining (red in C,C′′). Third instar eye discs are shown; posterior is towards the left.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. Overexpression of EGFR factors enhances Gmr-HFL eye phenotype. (A,B) Heads of adult female flies carrying Gmr-Gal4>UAS-HFL/+ (A) or Gmr-Gal4/+ (B) combined with different EGFR UAS-transgenes raised at 25°C as well as controls are shown in SEM pictures (upper panels) and Pixera pictures (lower panels). (1) Gmr-Gal4>UAS-HFL/+, UAS-lacZ/+; (2) Gmr-Gal4>UAS-HFL/+, UAS-argos/+; (3) Gmr-Gal4>UAS-HFL/UAS-DERDN; (4) Gmr-Gal4>UAS-HFL/+, UAS-klu/+; (5) Gmr-Gal4>UAS-HFL/+, EP-rho; (6) Gmr-Gal4>UAS-lacZ; (7) Gmr-Gal4>UAS-argos/+; (8) Gmr-Gal4>UAS-DERDN; (9) Gmr-Gal4>UAS-klu/+; (10) Gmr-Gal4>EP-rho/+. (C) Quantification of eye size by area measurements of documented eyes. At least 20 eyes of each genotype were measured and the mean value was determined. Error bars represent s.d. Numbers below the bars refer to panels of the same genotype as shown in A and B. *Improper size measurement due to bulging of the eye.