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

a, The copper cell region (#) of Drosophila larvae is normally acidic (bromophenol blue dye appears yellow/orange, see Methods), but becomes less acidic (purple/blue) when using hodor RNAi in interstitial cells (hodor-gal4) or in hodor mutants. The latter phenotype can be rescued by re-expressing hodor in hodor-Gal4-expressing cells. Intestinal acidity is also lost by downregulating the gene coding for the Vha16–1 subunit of the V-ATPase proton pump in copper cells using lab-Gal4. b, Quantifications 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 due to reducing V-ATPase activity 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 mCD8-GFP expression 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 low-yeast diet). Lysotracker staining in green was used to reveal their 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 localisation of Hodor in interstitial cells (insets). h, Quantifications of copper cell region width in controls, hodor mutant larvae and larvae expressing Shi^K44A from hodor-Gal4. i, Expression of Shik^k44A in hodor-expressing cells (hodor> Shi^K44A) does not alter developmental rate. See Supplementary information for sample sizes and full genotypes. Scale bars: a, 500μm; d, 500nm; e and f, 10μm; g: 250μm. For comparisons involving two groups, a non-parametric Mann Whitney U test was used. Where more than two groups were compared, an ordinary one-way ANOVA test was performed with a Tukey post-hoc test. Significance values are denoted as follows: p< 0.05 *, p< 0.01 **, p< 0.001 ***. Box plots: line, median; box, 75th–25th percentiles; whiskers, minimum to maximum.