NC acidification is dependent on their envelopment by the overlying AFCs. (A 1 –C 3 ) Stagewise expression pattern of BB127-lacz and LysoTracker during late oogenesis: stage 11 (A1–A3), stage 12 (B1–B3), stage 13 (C1–C3) egg chambers. White arrowheads mark the presence of BB127-lacz (in A2, B2, and C2), and yellow arrowheads mark the LysoTracker-positive NCs in stage 13 egg chambers (C3). Egg chambers were costained with LysoTracker in red, BB127-lacz in green, and DAPI in white. (D1–E2) Depletion of EcR does not induce Dcp-1 expression in stage 11 egg chambers. DAPI is in red, and Dcp-1 is in green. The merged image is shown in the inset. (F) Quantification of the number of AFCs in stage 11 egg chambers captured from the superficial layer to the midplane. n indicates the number of egg chambers analyzed. The mean number of AFC nuclei is provided in the graph. (G1–G6) Montage of control egg chambers of genotype Dicer2; GR1-Gal4, UAS mCD8GFP. AFCs marked in green by GFP are trying to surround the NCs. Acidification of NCs is indicated by the uptake of LysoTracker DYE in red. Yellow arrowheads and white arrowheads mark acidified and nonacidified NCs, respectively. (H1–H6) Montage of EcR-depleted egg chambers. AFCs exhibit a lack of envelopment of NCs. AFCs are marked by GFP in green. White arrowheads mark NCs that are either incompletely acidified or nonacidified.