EM analysis of lgd cells. (A) Possible scenario in lgd cells. Most endosomal markers are localised to distinct subdomains of the limiting membrane of the ME. Loss of lgd function could lead to a mis-distribution of the marker throughout the LM. This would fake an enlargement of the endosome if the cells were monitored with the fluorescence microscope, although the morphology of the endosome has not changed. (B–F) Strategy to generate wild-type and lgd cells within one disc at distinct regions. (B) Expression of hhGal4 and Wg in a wild-type wing imaginal disc. The arrowhead points to the expression of Wg along the D/V compartment boundary. (C,D) Depletion of the function of lgd causes the expansion of expression of Wg (arrows) in a similar manner as in the lgd mutant discs. (E) The depletion causes the formation of enlarged Notch-positive endosomes. (F) Transverse semi-section through wing discs at the level indicated by the white line in B. The arrowheads point to the region of the peripoidial membrane, whose normally cuboidal cells have adopted a more columnar morphology of cells of the imaginal disc (asterisk). (G,H) MVBs in wild-type (G) and lgd-depleted cells (H) highlighted in yellow. The arrows point to lysosomes. (I,J) Statistical analysis of the endosomes in the two genotypes. Results reveals that lgd cells contain a class of MVBs that is larger than in wild-type cells (red bar in J). This class includes 23% of all MVBs (Ji).

lgd cells are defective in degradation of transmembrane proteins that are residents of the plasma membrane. (A–E) Design of the degradation assay and control experiment. The tested protein is expressed in the posterior compartment using hhGal4, and lgd^d7 mutant clones are induced throughout the disc. (A) Overview of the disc. The arrow points to a posterior region that contains a lgd clone. This region is shown in B–E at higher magnification. (B) Outline of the lgd clone labelled by loss of lacZ. (C) Distribution of GFP–GPi. (D) lgd cells contain enlarged Notch-positive endosomes. (E) Merge of B–D. This control experiment reveals that the distribution and concentration of GFP–GPi is the same in wild-type and lgd cells. The arrow in B–E highlights the clone. (F–N) By contrast, the levels of CEN and endogenous Notch are elevated in lgd cells (F–H). The antibody is directed against NECD, which is absent in CEN. (I–N) The same accumulation is observed for T48–GFP and tkv-GFP. (O–Q) Expression of CEN, tkv and T48 in the posterior with hhGal4 in lgd mutant disc suppresses the ectopic activation of Notch compartment (arrow). Note that the ligand-dependent activation along the D/V boundary is not affected. (R) Expression of a full-length Notch construct results in a dramatic enhancement of Wg expression (arrow) and overproliferation. (S) Expression of lamp1-GFP has no effect.

Activation of Notch pathway in lgd cells is independent of the activity of kuz, but depends on the activity of the γ-secretase complex. (A–M) The activity of the Notch pathway is detected using a Gbe+Su(H)-nlsGFP reporter. (A) Wild-type wing disc. (B–M) Clones are labelled by the loss of lacZ (B,E,H) or RFP (K) staining. In lgd clones all cells activate the reporter (B,D). Ectopic activation is suppressed in hrs lgd cells (E–G) but not in lgd kuz clones (K–M). By contrast, the ectopic activation is lost in lgd aph-1 clones (H–J). (N,O) Loss of one copy of aph-1 reduces the extent of the ectopic expression of Wg in lgd wing imaginal discs.

Requirements for the ligand-independent activation of Notch in lgd cells. (A–D) Constructs are expressed with hhGal4 in the posterior compartment. The activity of the Notch pathway is measured through the expression of the target gene wg. (A) Depletion of the activity of vATPase results in suppression of the ectopic activity. Note, that the ligand-dependent activation along the D/V boundary (arrow) is not affected. (B) Expression of N^ΔEGF in lgd mutant discs results in an enhanced activation (arrow), similar to full-length Notch (compare with Fig. 3R). (C) Expression of N^ΔEGF in a wild-type disc weakly suppresses the ligand-dependent activation of Notch along the D/V boundary (arrow). (D) Expression of N^BC, which lacks the Furine cleavage site, suppresses ectopic and ligand-dependent activation of Notch in lgd discs (arrow). (E) Expression of N-BC during normal wing development results in a weak suppression of expression of Wg at the D/V boundary (arrow).

Activation of Notch in lgd cells occurs in the lysosome. (A–E) Association of Rab7 with the MEs in lgd cells is weakened upon reduction of shrub activity. (A) Overview of a wing disc that bears lgd shrub/lgd + clones. The clones are labelled by two copies of GFP. The arrow points to a clone that is shown in B–E at higher magnification. Compare with Fig. 1A–G. The arrowheads in B–E highlight the clone boundary, the arrows in C,E point to one of the Notch endosomes that are not associated with Rab7. (F–J) Depletion of Rab7 in the posterior compartment of wild-type (F–I) and lgd discs (J). The depletion in the wild-type results in the formation of enlarged Notch-positive endosomes and loss of Rab7 protein (F–H, arrow). (I) The expression of Wg along the D/V boundary (arrow) is not disturbed, indicating that ligand-dependent activation is unaffected. (J) Depletion of Rab7 in lgd discs suppresses the ectopic ligand-independent activation. (K,L) Loss of car function results in a suppression of the ectopic activation of Notch in lgd discs. (M) The same effect is achieved upon depletion of Vps39 in the posterior compartment. The arrows in I,J,L,M point to the remaining ligand-dependent expression along the D/V boundary, which is not affected. Thus, preventing the fusion of the endosome with the lysosome suppresses specifically the ectopic activation of Notch in lgd discs.

Consequences of loss of lgd function in the follicular epithelium. (A–L) lgd clones are labelled by the absence of lacZ staining. (A–D) Ectopic activation of Notch in follicle cells revealed by ectopic activation of the Gbe+Su(H) reporter and Hnt. The panels show a wild-type (wt) and lgd clones bearing ovariole. The red arrows point to two egg chambers of stages 6 and 7, which bear the clones. The yellow arrows highlight the correspondent wild-type counterparts. Arrowheads point to the clone regions. The cells of the lgd clones precociously express Gbe+Su(H) and Hnt in stage 6 egg chambers. In addition, the mutant cells of the older stage 7 egg chamber express these markers stronger than their wild-type counterparts. (E–H) Early stages of oogenesis in an ovariole where the whole epithelium is mutant. All follicle cells express Gbe+Su(H), indicating that the Notch pathway is activated immediately after birth of these cells at positions highlighted by the arrowheads. At these early stages, Hnt is not expressed. The arrow in E points to the elongated stalk. (I–L) Loss of lgd fuction results in a suppression of Cut expression. The arrowheads point to the clone and the arrow in L to an enlarged nucleus. (M,N) Hoechst staining of the anterior region of a wild-type ovariole and a counterpart where the whole follicular epithelium is mutant. The arrows point to the stalk. (O–R) MARCM clones mutant for lgs and depleted of Rab7. The arrowheads highlight the clone that lacks Rab7 (P) and fails to ectopically express Gbe+Su(H) (Q).

Analysis of the phenotype of lgd and shrub follicle cells. (A–E) The enlarged endosomes of lgd cells contain Notch, Dl. (F,G) Endosomes contain NICD as well as NECD. (H–J) They are further positive for Rab5 (H,I) and Rab7 (J). (K–M) Loss of shrub function causes the activation of expression of Hnt and Gbe+Su(H). (N–P) Depletion of Rab7 does not suppress the expression of Hnt in shrub cells. Arrows point to the lgd clone.