DEV02197 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Distribution of Rab5 and Rab7 endosomes in disc cells and presence of Wg in basal endosomes. (A) The fraction of endosomes positive for Rab5 only, Rab7 only or both Rab5 and Rab7 plotted as a function of distance from the apical (upper) or basal (lower) membranes of a single wing disc. (B) A single basal section of a disc expressing CFPRab5 and YFPRab7 stained for Wingless. Images are merged pairwise as indicated at upper left of each panel.

• Supplemental Figure 2 -

  Fig. S2. Effects of Rab5SN expression on endocytosis, apoptosis and wg expression. (A) Transgene induction in wing imaginal discs. (B,C) Wing discs in which Rab5SN expression was initiated in the dorsal compartment by excision of an HcRed-expressing FLP cassette 2 hours previously. (B) Residual HcRed fluorescence marks the dorsal compartment. (C) Fluid phase uptake after a 15-minute incubation with Alexa-Fluor 488-labeled dextran. Uptake is inhibited in the Rab5SN-expressing dorsal cells compared with wild-type ventral cells. Residual dextran uptake may reflect activity of Rab5-independent uptake pathways. Scale bar: 20 mm. (D-G) Projected images of wing discs stained for activated Caspase to reveal apoptosis 1 hour (D), 4 hours (E), 6 hours (F) or 8 hours (G) after induction of Rab5SN in the dorsal compartment. Nuclear staining indicates activated caspase. Diffuse residual HcRed fluorescence collected in same channel marks dorsal compartment. Projection comprises the entire disc epithelium, including extruded dead cells accumulating basally. The number of dying cells still within the epithelium represents a small fraction of positive cells. (H) b-Galactosidase staining, wing disc expressed Rab5SN for 5.5 hours in the dorsal compartment (indicated) and a wg promoter:LacZ reporter construct. No ectopic wg promoter activity is detected.

• Supplemental Figure 3 -

  Fig. S3. Quantification of Wg protein on the apical and basal surfaces of wing discs expressing Rab5SN in the dorsal compartment. Quantification of Wingless staining intensity on the apical and basal membranes corresponding to images shown in Fig. 2A,B. Black vertical lines indicate the DV boundary. Wingless staining extends further from the DV boundary in Rab5SN expressing tissue than in wild-type tissue by 4 hours (blue) after Rab5SN induction. Its range is extended even further after 5.5 hours (pink).

• Supplemental Figure 4 -

  Fig. S4. Effects of perturbing Rab7 function. (A-D) Wing disc expressing YFP-Rab7 and CFP-Rab5 ubiquitously and the Rab7TN mutant for 48 hours in the dorsal compartment (indicated by bracket). Disc was stained for Wg. Scale bar: 20 mm. (A) YFP-Rab7 shifts from a punctate to a cytoplasmic localization in dorsal Rab7TN-expressing cells. (B) CFP-Rab5 endosomes are unaffected by Rab7TN expression. (C) Rab7TN expression causes mild Wg accumulation. (D) Overlay of Wg, CFP-Rab5 and YFP-Rab7. Rab7TN expression causes Wg accumulation in Rab5 endosomes. (E) Wing disc after 22 hours of Rab7TN expression in the dorsal compartment (under broken line). Disc also expressed ubiquitous Rab11-YFP. The Rab11 compartment becomes more intense in Rab7TN-expressing tissue. (F) Upper panel shows in situ detection of rab7 RNA in a disc expressing rab7 RNAi in the posterior wing disc compartment (to the right). Lower panel shows wing disc expressing rab7 RNAi in the dorsal compartment (bottom half) accumulates more Wg.

• Supplemental Figure 5 -

  Fig. S5. Effects of perturbing Rab11 and Rab4 functions. (A-C�) Discs after 4.5 hours Rab11SN expression in the dorsal compartment (below broken lines). Disc expressing ubiquitous YFP-Rab11. Rab11SN disperses the YFP-Rab11 fluorescence. Disc expressing ubiquitous YFP-Rab7. Rab11SN enlarges the Rab7 compartment. (C,C�) Disc stained for the EGF receptor. (C) Apical section: EGF receptor no longer localizes to cell membranes in Rab11SN-expressing domain. (C�) Middle section: elevated EGF receptor is found in more basal structures. (D) Disc after 15h Rab4SN expression in the dorsal compartment. Ubiquitous YFP-Rab4 becomes dispersed in expressing domain. (E) In situ hybridization to rab11 mRNA. RNAi against rab11 was induced in the dorsal compartment 60 hours before. rab11 transcript is depleted from the dorsal compartment. (F,F�), Disc subjected to Rab11 RNAi in the dorsal compartment. Unlike Rab11SN, Rab11 RNAi does not induce apoptosis (inset). Its depletion has no effect on the Wg gradient observed by conventional (F) or extracellular (F�) staining.

• Supplemental Figure 6 -

  Fig. S6. Effects of Arrow and Dlp RNAi. (A) Quantification of Wg-containing endosomes in the non-expressing ventral compartment (wild-type) versus the arrow RNAi-expressing dorsal compartment of a wing disc. Wg endosomes are more numerous and their distribution is shifted apically in cells lacking Arrow. (B) Wing disc subjected to arrow RNAi in the dorsal compartment for 72 hours and stained for Dlp. Dlp protein levels are normally low near the DV boundary but are elevated in this region by Arrow RNAi in the dorsal compartment. (C) XZ section perpendicular to the DV boundary of a wing disc expressing Rab5SN in the dorsal compartment (right). The disc was stained for Dfz2 (red, upper panel), Arrow (red, lower panel) and Armadillo (green). Yellow arrowheads indicate the apical side of cells in the fold at the edge of the wing pouch where Fz2 and Arrow expression is highest. Both Fz2 and Arrow accumulate apical to Armadillo in tissue expressing Rab5SN but not wild-type tissue. (D) Quantification of apical and basolateral Dlp staining shown in Fig. 6A,B.