Fig. 3. DSL signaling depends on endocytosis, ubiquitination and Lqf. (A) Wing disc with clones of cells overexpressing Myc-tagged Dl^LDL+ (green) stained for Cut (red). Clones in the D compartment located close to the DV boundary activate Cut, indicating that Dl^LDL+ has signaling activity. The white box marks a clone that is shown at higher magnification in apical and sub-apical planes of focus in A'' and A''. Note the presence of cytosolic puncta in A''. In this, and all other images in this figure, the various Dl chimeric proteins were detected with guinea pig -Dl antisera to allow us to visualize Cut expression (which requires a mouse -Cut antisera). Hence, both endogenous Dl and the exogenous Dl chimera are detected. However, the exogenous Myc-tagged Dl chimeras are expressed at several-fold higher levels than the endogenous protein, so the contribution of the endogenous protein to the Dl stain is negligible. All of these experiments were also performed using mouse -Myc antisera, and identical results were obtained for the subcellular distribution of the various Myc-tagged Dl chimeras. (B) lqf^- clones overexpressing Myc-tagged Dl^LDL+ located close to the DV boundary can induce Cut, indicating that the presence of the LDL internalization signal allows the chimeric Dl protein to bypass the requirement for Lqf. The subcellular distribution of Myc-tagged Dl^LDL+ appears to be unaffected by the absence of Lqf. (C) Clones of cells overexpressing Myc-tagged Dl^LDLm fail to activate Cut ectopically, and also block endogenous Cut expression when they abut the DV boundary. Myc-tagged Dl^LDLm accumulates on the apical surface, but not in cytosolic puncta, indicating that mutation of the LDL internalization signal blocks endocytosis as well as signaling activity. (D) Clones of cells overexpressing Myc-tagged Dl^R+ induce Cut; Myc-tagged Dl^R+ accumulates both apically and in cytosolic punta. (E) Clones of lqf^- cells overexpressing Myc-tagged Dl^R+ do not induce ectopic Cut, and block normal Cut expression when they abut the DV boundary; nevertheless, the subcellular distribution of Myc-tagged Dl^R+ is not detectably altered by the absence of Lqf. (F) Clones of cells overexpressing Myc-tagged Dl^Rm fail to activate Cut, and block normal Cut expression at the DV boundary. Myc-tagged Dl^Rm accumulates on the apical surface, but not in cytosolic puncta. (G) Clones of cells overexpressing Myc-tagged Dl^Ubi+ upregulate expression of the vg-boundary enhancer-lacZ reporter gene (red), although they do not activate Cut (not shown), indicating detectable, but weak, signaling activity. Myc-tagged Dl^Ubi+ accumulates apically, as well as in cytosolic puncta. (H) Clones of cells overexpressing Myc-tagged Dl^Ubim fail to ectopically upregulate the vg-boundary enhancer-lacZ reporter gene and block its normal expression when they abut the DV boundary. Myc-tagged Dl^Ubim accumulates apically; however, accumulation in cytosolic puncta is greatly reduced when compared with Myc-tagged Dl^Ubi+.