Loss of atp6ap1b is causative of aa24.2^pd1209 mutant phenotypes. (A) Survival curve of aa24.2^pd1209 mutants. n = 82 mutants and 109 WT siblings. (B–G) Rescue experiments for aa24.2^pd1209 mutants. (B)aa24.2^pd1209 heterozygotes were crossed, and one-cell stage embryos were injected with atp6ap1b cRNA with a portion of embryos kept as noninjected controls (NIC) and analyzed at 4 dpf for craniofacial dysmorphology (arrowheads) and hypopigmentation (arrows). Scale bar is 500 µm. (C) Quantitation of hypopigmentation. Severe is denoted as the aa24.2^pd1209 NIC phenotype shown in A, which is fully penetrant in control mutants. (D) Complementation analysis of the aa24.2^pd1209 allele with the atp6ap1b^hi112Tg allele previously described (Nuckels et al., 2009). n values are shown in C and D. (E) Mosaic expression of HA-tagged Atp6ap1b in enterocytes cell-autonomously rescues p75-GFP localization in aa24.2^pd1209 mutants. Magenta lines indicate HA-positive cells and arrows point to intracellular accumulation. Scale bars are 10 µm. n > 10 mutants in two independent experiments. (F and G)aa24.2^pd1209 heterozygotes were crossed, and one-cell stage embryos were injected with cRNA encoding untagged or GFP-tagged Atp6ap1b, and 5-dpf larvae were live imaged for craniofacial dysmorphology (arrows) and body length quantification (G). Scale bar is 500 µm. Error bars are SD. ****, P < 0.0001; ***, P < 0.001; two-way ANOVA with Tukey’s multiple comparison test. n ≥ 29 mutants per condition. (H) Localization of Anpep in different V₀ and V₁ sector V-ATPase mutants. Arrows point to vacuolar accumulation, and arrowheads show basolateral missorting. Scale bars are 10 µm and 2 µm in magnified insets. n ≥ 10 mutants in two independent experiments per allele.