Figure 1. Arm^34–87 peptide interferes with Arm/β-catenin signaling in Drosophila wing development.

(A) Drosophila Arm and human β-catenin N-terminal sequence with Arm^34–87 and β-cat^24–79 peptides marked in yellow. Arm^34–87 or β-cat^24–79 is highly conserved between Drosophila and mammals. Numbered red residues are phosphorylation sites of GSK3β (residues 33, 37, and 45 in human) and CK1 α (residue 41 in human) within this region of β-catenin.

(B and C) Arm^34–87 and IFT140 are physically associated. (B) Direct binding of Arm^34–87 and IFT140. IFT140 (MBP fusion, lane 3: input 10%) was pulled down by GST-Arm^34–87 (lane 4), whereas full-length MBP-Kap3 and MBP-Klp64D (lanes 1 and 2 from left: input 10%) were not pulled down (lanes 5 and 6). (C) In vivo co-immunoprecipitation assay of IFT144 and IFT140 with Arm^34–87 from nub>IFT144myc; Arm^34–87GFP (negative control, lane 1) and nub>IFT140myc; Arm^34–87GFP (lane 2) wing imaginal discs. Protein extracts from wing discs were co-immunoprecipitated with anti-GFP. Co-immunoprecipitations and input (15% of wing disc lysates used in co-immunoprecipitation) were analyzed by blotting with anti-myc to IFT144 or IFT140.

(D–K″) All wings and wing discs shown use the C96-Gal4 driver (Figure S1C shows expression pattern: C96 is expressed at a slightly higher level in the posterior region of wing discs, right half in picture). Adult wings: anterior is up and distal right; wing discs: dorsal is up and anterior left.

(D–G″) Interaction between Arm^34–87 and endogenous Arm. All genotypes were reared at 25°C. (D–D″) UAS-GFP (C96>GFP) control wing and control wing disc with wild-type Sens (green) (D′) and Dll (red) (D″) expression near D/V boundary. (E–E″) C96>Arm^34–87 wing and wing disc. Note partial loss of margin (E) and partial loss of Sens (green, E′) and reduction of Dll (red, E″), consistent with adult wing defects. (F–F″) C96>Arm^34–87; UAS-arm^RNAi. Margin loss caused by Arm^34–87 was enhanced, and Sens (F′) and Dll (F″) expression was further reduced (cf. to E–E″). (G–G″) C96>Arm^34–87 >Arm^wt: note that phenotype caused by Arm^34–87 is suppressed by co-expression of wild-type Arm, and Sens (green, G′) and Dll expression (red, G″) were restored.

(H–K″) Interaction between IFT140 and Arm^34–87 (all genotypes reared at 29°C). (H–H″) C96>Arm^34–87: note increased margin loss at 29°C (cf. to E) and reduced Sens (H′) and Dll expression (H″). (I–I″) C96>Arm^34–87; >Ift140^RNAi: wing margin defects, and expression of Sens (I′) and Dll (I″) were reduced, compared to C96>Arm^34–87 (cf. to H–H″). (J–J″) C96>Arm^34–87; >IFT140myc: co-expression of IFT140myc in C96>Arm^34–87 wings suppressed phenotype and restored Sens (J′) and Dll (J″) expression. (K–K″) C96>IFT140myc control: no effect of increased IFT140 levels by itself (wild-type margin and normal Sens and Dll expression in imaginal discs). Scale bar represents 100 μm in adult wings (D–K) and 50 μm in imaginal discs (D′–K″). Figure S1 shows quantification of Sens staining, as well as Figures S1 and S2.