ERM-1 T544 phosphorylation is required for excretory canal lumenogenesis. (A) Schematic drawing of the excretory canal system in an L4 animal. Red dashed rectangle and blue dashed circle indicate areas shown in D by fluorescence and electron microscopy, respectively. (B) Quantification of excretory canal outgrowth in L4 animals. All four canal branches were measured per animal, and each data point represents one branch (n=25, 25, 22, 25 and 14 animals from top to bottom). Error bars are the median±95% CI. (C) Frequency of anterior and posterior canals from B extending <35% of the distance between cell body and tips. (D) Lumen morphology in L4 animals visualized by using a VHA-5::GFP transgene (top panels) or in cross-section by transmission electron microscopy (bottom panels). (E) Quantification of canal width in L4 animals. Each data point represents the average of three measurements at the three widest points in a single posterior canal (n=15, 17, 13, 18 and 13). Error bars are the median±95% CI. Tests of significance: Dunn's multiple comparisons test for B and E; Fisher's exact test for C. ns, not significant. *P≤0.05, **P≤0.01, ****P≤0.0001. Outgrowth and width were measured by fluorescence microscopy using VHA-5::GFP as a marker. Unless indicated otherwise, statistical comparisons are with the VHA-5::GFP control line.