Extended Data Fig. 4: Hodor sustains luminal acidity and maintains luminal and cell volume.

a, The copper cell region (#) of Drosophila larvae is normally acidic (bromophenol blue dye appears yellow/orange) (Methods), but becomes less acidic (purple/blue) upon expression of hodor RNAi in interstitial cells (hodor-gal4) or in hodor mutants. The reduced acidity of hodor mutant midguts can be rescued by re-expressing hodor in hodor-Gal4-expressing cells. Intestinal acidity is also lost by downregulating the gene that encodes the Vha16-1 subunit of the V-ATPase proton pump in copper cells using lab-Gal4. b, Quantification of intestinal acidity. Depletion (by RNAi) or loss of hodor results in a reduction in the number of larvae with acidic middle midguts, as does depletion of the V-ATPase subunit Vha16-1 in copper cells using lab-gal4. c, Larval developmental rate is unaffected when acidity is lost by reducing the activity of V-ATPase within copper cells (using lab-Gal4). d, Electron micrographs of interstitial cells of first-instar larvae, showing a reduction in their characteristic basal infoldings (arrows) in hodor mutants (* denotes basal lamina) relative to control cells. e, hodor-Gal4 driven expression of mCD8-GFP in interstitial cells of control and hodor mutant larvae reveals an increase in luminal volume (*) and interstitial cell volume (insets with quantifications to the right) in first-instar mutant larvae when compared to controls (all raised on a low-yeast diet). See Methods for details of volume quantifications. f, Overexpression of the dominant-negative Shibire Shi^K44A in hodor-expressing cells (using hodor-Gal4) reveals an increase in interstitial cell volume in hodor second-instar mutant larvae relative to controls (all raised on a low-yeast diet). LysoTracker staining in green was used to reveal the cytoplasm. Quantifications are shown to the right. Second-instar larvae raised on a low-yeast diet were used for all experiments involving Shi^K44A expression. g, This genetic manipulation also results in an increase in the width of the copper cell region (#) but does not affect the subcellular localization of Hodor in interstitial cells (insets). h, Quantification of the copper cell region width in controls, hodor mutant larvae and larvae expressing Shi^K44A from hodor-Gal4. i, Expression of Shi^K44A in hodor-expressing cells (hodor>Shi^K44A) does not alter developmental rate. See Supplementary Information for sample sizes and full genotypes. Scale bars, 500 μm (a); 500 nm (d); 10 μm (e, f); 250 μm (g). For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. For cases in which more than two groups were compared, an ordinary one-way ANOVA test was performed with a Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Box plots: line, median; box, 75th–25th percentiles; whiskers, minimum to maximum.