Fig. 3.
V-ATPase activity is required to activate the Notch receptor downstream of ligand binding in epithelial tissue. (A-C′) Drosophila eye discs stained to detect the Notch reporter mβ-lacZ and Notch (single mβ-lacZ channel is shown in A′-C′). Compared with WT (A), mβ-lacZ expression is reduced in predominantly mutant Vha68-2R6 discs (B) and in clones of Vha68-2R6 mutant cells located posterior to the morphogenetic furrow in mosaic discs (C). Mutant tissue is outlined in B′,C′. The regions of the tissue that accumulate Notch the most appear to express mβ-lacZ the least (compare B with B′). In C, mβ-lacZ is expressed at high level in WT developing photoreceptors but is absent in clones of mutant cells. (D-G) Egg chambers at stages 5-7 of oogenesis stained for Cut (D,E) and Hnt (F,G). In WT cells, Cut expression is subjected to Notch-dependent downregulation after stage 5 (D), whereas Hnt is expressed in a Notch-dependent manner from stage 6 onwards (F). In Vha68-2 mutant FCs, Cut expression is maintained after stage 5 (E; for GFP channel see Fig. S4C in the supplementary material), whereas in Vha55 mutant FCs, Hnt expression is reduced after stage 5 (G; for GFP channel see Fig. S4D in the supplementary material), in both cases indicating impairment in Notch signaling activation in V-ATPase mutant FCs. (H) Schematic of the Notch cleavage events preceding signaling activation. The full-length Notch is cleaved at the S2 site to generate the γ-secretase cleavage substrate NEXT. NEXT is subsequently cleaved by γ-secretase at the S3 site to generate the active cytoplasmic NICD fragment. (I-K) Mosaic eye discs generated using the MARCM system stained to detect NICD. GFP-positive cells expressing NEXT overproliferate to form very large clones (I). By contrast, GFP-positive cells expressing NEXT that lack V-ATPase activity underproliferate and form small clones (J). In GFP-positive cells that express NICD and lack V-ATPase activity, normal proliferation is restored (K). (L) Quantification of the experiment shown in I-K. Scale bars: 100 μm in A-B′,I-K; 10 μm in C-G.