DEV011577 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Cos2-572D displays a decreased co-localisation with Ci and Smo compared to the other Cos2 variants. (A,B) Wild-type embryo stained for Cos2 (green), Ci (red) and Nrt (blue). (C-H) Embryos over-expressing Cos2-WT-myc (C and D) Cos2-572A-myc (E and F) and Cos2-572D-myc (G and H) with the armGal4 driver. Embryos were stained for Myc (green), Ci (red) and Nrt (blue). (B′,D′,F′ and H′) are images showing only pixels of Ci-Cos or Ci-Myc co-localisation superposed to the Nrt channel of images shown in B, D, F and H, respectively. (I-K) embryos over-expressing Cos2-WT-myc (I), Cos2-572A-myc (J) and Cos2-57D-myc (K) were stained for Myc (green) and Smo (red). (I′,J′ and K′) Images showing common pixels between Smo and Myc labelling. Cells expressing Hh are indicated by thin lines in B, D, F, H, I, J and K. (L,M) Graphs showing the number of pixels that co-localise between Ci-Cos2-myc (L) or Smo-Cos2-myc (M) staining. Cos2-WT and Cos2-572A proteins behaved similarly to endogenous wild-type Cos2 protein, with a characteristic alternate pattern of stabilisation (A, C and E). The repeated segmental stabilisation/destabilisation pattern shown in C and E was observed with a frequency of 83% and 75% in Cos2-WT and Cos2-572A-myc expressing embryos, respectively, and was never observed in embryos expressing Cos2-572D-myc (n=12-15 embryos of stage 11). Note that Cos2^572D showed a lower level of co-localisation with Ci and Smo than did the other constructs (D-D', F-F', H-H', I-K’, L-M). A stronger cytoplasmic localisation was observed with Cos2^572D.

• Supplemental Figure S2 -

  Fig. S2. Repression of endogenous Ptc expression by the Cos2 variants. (A) 71BGAL4/UAS cos2-WT-myc, (B) 71BGAL4/ cos2-572A-myc, (C) 71BGAL4/cos2-572D-myc imaginal discs are stained for myc (in red) and Ptc in blue. (A′-C′) show Ptc staining in white. The 71B line drove the expression of Gal4 in a large domain centred in the wing pouch but often showed a very patchy expression of the transgenes as visualised by myc staining (A-C). (A) Cos2 WT strongly inhibited the endogenous expression of ptc. (B,C) Mutant Cos2 transgenes displayed a weaker repression as inhibition of endogenous ptc expression is only observed in cells with strongest expression of the driver.

• Supplemental Figure S3 -

  Fig. S3. Ectopic Ptc expression is repressed by the Cos2 variants. (A) Wild-type, (B) yw,hs-flp; FRT42D cos2^W1/FRT42D armLacZ,(C) yw,hs- flp; FRT42D cos2^W1/FRT42D armLacZ; 71BGAL4/UAS cos2-WT-myc, (D) yw,hs-flp; FRT42D cos2^W1/FRT42D armLacZ; 71BGAL4/ cos2-572A-myc and (E) yw,hs-flp; FRT42D cos2^W1/FRT42D armLacZ; 71BGAL4/cos2-572D-myc imaginal discs are stained for myc in red, Ptc in blue and β-galactosidase in green, except in A and B where Ptc is in red. (A) Ptc pattern: Ptc is overexpressed in 3 to 4 cells in the anterior compartment near the AP border. (B) Cos2 inhibits the expression of ptc in cells far from Hh source. In cos2^W1 clones, ptc is ectopically expressed as revealed by the 5E11 antibody (arrow). On the contrary, Cos2 is necessary for strong ptc expression near the AP boundary. In cos2^W1 clones, Ptc expression is diminished (indicated with a star). (C-E′′) Examples of rescue experiments with UAS-Cos2-WT (C), UAS-Cos2 572A (D) and UAS-Cos2 572D (E) driven by 71BGal4, illustrating the inhibition efficiency of the three transgenes on Ptc ectopic expression induced in cos2^W1 clones. The rescued domain is visualised by myc staining and encircled with dotted lines (C-E). cos^WI loss of function clones are identified by a loss of β-galactosidase staining, and all anterior clones are encircled by an unbroken line (C′-E′) or indicated with arrows (C′′-E′′). Ptc expression in anterior clones situated inside the 71B domain was observed in different discs from distinct experiments (5 to 7 for each Cos2 variants). Ptc and Collier (data not shown) expressions were consistently repressed by the Cos2 variants even when larvae were grown at 18°C to reduce Gal4 activity as much as possible. All of the other tested drivers were stronger expressers than 71Bgal4 (data not shown). Note that in some clones, heterogenous expression of Ptc is observable but correspond to cells in which the driver is not expressed (or very reduced).