DEV02145 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression levels of Frizzled2 and FrizzledGPI in imaginal disc.  Imaginal discs were stained with anti-Frizzled2 to gauge the amount of receptor expressed. (A) dpp-Gal4 UAS-D-Frizzled2, (B) dpp-Gal4 with two copies of UAS-D-Frizzled2 and (C) dpp-Gal4/ UAS- Frizzled2GPI. Discs shown in A and C are of the same genotypes as those shown in Fig. 2A and B, respectively. Staining and imaging were performed under identical conditions in the three genotypes. Expression of Frizzled2GPI is much lower than that of Frizzled2, even though it leads to more extensive Wingless stabilisation.

• Supplemental Figure 2 -

  Fig. S2. Wingless accumulation in arrow mutant clones is not due to ectopic transcription. (A) Wingless protein (red; detected with the traditional staining protocol) accumulates within arrow mutant clone (absence of GFP), even far from the source (up to 10 cell diameters away). (B,B¢) Although wingless transcription is affected near the source of Wingless in arrow mutant clones (the white line marks the clone outline determined from the absence of GFP in B¢), no ectopic transcription is seen more than a few cell diameters away.

• Supplemental Figure 3 -

  Fig. S3. Wingless endocytosis in arrow mutant clones and arrow RNAi-treated cells. (A,A¢) Disc carrying an arrow mutant clone and labeled with dextran as described in the Materials and methods. Accumulated Wingless (green) in the clone (outlined) is both at the cell surface and in Dextran-positive vesicles (red). (B,B¢) S2R+ cells treated with arrow RNAi and transfected with Frizzled2 internalize exogenously applied GFP-Wingless. Anti-FLAG (to reveal transfected D-Frizzled2) is shown in red and Wingless is in green. To verify that Arrow was knocked down by the RNAi treatment, a signaling assay was performed in parallel using cells transfected with a TOPFLASH construct, which produces luciferase in response to Wingless signaling (see Materials and methods). As expected, arrow RNAi reduces luciferase activity from a 105-fold induction by Wingless to a 1.3-fold induction.