Fig. 5. Wnt signaling directs tube orientation through the Fzd1/2/7 receptor.

a, b Wnt inhibition with ETC159 from late gastrula to L2 caused a change in orientation of the tube outgrowth from anterior-posterior to anterior-dorsal. Confocal z-stacks are dorsal views and lateral views obtained from orthogonal projections of the same larvae. c qPCR shows Wnt8 gene expression decreases when ETC159 (that block secretion of Wnts) is used. Bars represent mean with standard error of the mean (SEM). n = 3 biologically independent experiments. d FISH showing that the Wnt receptor Fzd1/2/7 is expressed in the tube cells in early larvae. e, f In control larvae tubes grow parallel to the anterior-posterior axis, while in Fzd1/2/7 knock out larvae the tubes grow towards the dorsal side and recapitulate the same tube orientation defects seen with ETC159 treatment. Percentage of the larvae showing the Fz1/2/7 KO phenotype was 86%; for genomic mutation see Supplementary Fig.13. g Tube length measured as a ratio of tube length/esophagus length; n = 24 larvae for controls, n = 36 larvae for Fz1/2/7 KO. h Tube orientation measured as the angle that the tube makes with respect to the anterior-posterior axis of the larva. Number of tubes measured is n = 30 for controls, n = 30 for ETC159 treated and n = 23 for Fzd1/2/7 KO. Same data shown as a rose plot to indicate difference in orientation. i Schematic summarizes the observed phenotypes. All experiments are representative of 3 biological replicates. a, b, d–f scale bar is 50 μm. a and b lateral views are 20 μm. Statistical significance was assessed by a two-sided Student’s t-test (****p < 0.0001; ns = not significant). In box plots the median is the middle line, box represents 25th and 75th percentiles, whiskers indicate the minimum and maximum data range. Source data are provided as a Source Data file.