Figure 5.

The Tnks and APC binding domains of Axin are important for promoting Wingless signaling. (A) Schematic representation of Axin-V5, AxinΔTBD-V5, and AxinΔRGS-V5. (B) Lysates of S2R+ cells transfected with the indicated plasmids (250 ng) were analyzed by immunoblotting using a V5 antibody. Deletion of the Tnks binding domain of Axin (AxinΔTBD-V5) or the APC binding domain of Axin (AxinΔRGS-V5) resulted in aberrant stabilization of Axin compared to wild-type controls (Axin-V5). (C) Lysates of third instar larvae expressing the indicated transgenes with C765-Gal4 driver were analyzed by immunoblotting. AxinΔTBD-V5 and AxinΔRGS-V5 were stabilized compared with Axin-V5 protein. Confocal images of third instar larval wing discs expressing Axin-V5 (D and E), AxinΔTBD-V5 (H and I), or AxinΔRGS-V5 (L and M) with the C765-Gal4 driver. Staining with Wingless and Senseless antibodies shows that expression of Axin-V5 did not disrupt expression of the Wingless pathway target Senseless (D) or of the number of cells expressing Wingless (E, arrowheads), indicating that Axin-V5 was expressed at a level that is compatible with physiological regulation. In contrast, expression of AxinΔTBD-V5 resulted in loss of Senseless (H, arrows) and expansion in the number of cells expressing Wingless (I, arrowheads), indicating that Wingless signaling is inhibited by AxinΔTBD-V5. Expression of AxinΔRGS-V5 results in loss of Senseless (L, arrows), indicating that AxinΔRGS-V5 inhibits Wnt signaling. Adult wings expressing Axin-V5 (F and G), AxinΔTBD-V5 (J and K), or AxinΔRGS-V5 (N and O) using the C765-Gal4 driver are shown. A total of 95% of wings expressing Axin-V5 (F and G) have normal morphology (n = 136), whereas 92% of wings expressing AxinΔTBD-V5 (J and K) (n = 127) and 30% of wings expressing AxinΔRGS-V5 (N and O) (n = 135) display loss of sensory bristles and tissue at the wing margin (K and O, arrow), as well as extra bristles in the wing blade (K and O, arrowhead), which indicate inhibition of Wingless signaling.