Ordered membrane environments are essential for Wnt3a‐mediated β‐catenin signaling. (A) Average and standard deviation (error bars, three independent experiments) values of pBAR luciferase reporter activity monitoring Wnt/β‐catenin signaling activity (normalized to renilla luciferase activity) in HEK293T cells treated with COase, myriocin, or oseltamivir and induced with the control‐ or Wnt3a‐conditioned media. (B, C) GFP or mCherry whole‐mount in situ hybridization showing downregulation of signaling in the two transgenic zebrafish embryos TOPdGFP (B) and 7xTcf:mCherry (C) after treatment with COase, myriocin, and oseltamivir. Arrows highlight the regions where reduction in Wnt/β‐catenin signaling activity is observed. All expression domains detected in the control embryos are downregulated after drug treatment. Note that the drugs appear to influence these domains differently. amd, anterior‐most domain; mhb, midbrain–hindbrain boundary; otv, otic vesicle; llp, lateral line primordium; pmes, posterior mesoderm. (D) Morphological phenotypes at 24 hpf of embryos treated with cholesterol oxidase (3 U·mL⁻¹, 23/24 embryos), myriocin (187.5 μm, 19/21 embryos), or oseltamivir (150 μm, 19/19 embryos) for 19 h. Arrows and the dashed line show the reduction of size in trunk and tail. (E) FCS diffusion law of Wnt3‐EGFP in live transgenic embryos treated with COase, myriocin, or oseltamivir. COase completely diminishes the domain diffusion while myriocin or oseltamivir significantly reduces it. Error bars represent the standard error of the mean at each binned area (number of data points is 36 for 1 × 1 binning, 25 for 2 × 2 binning, and 16 for 3 × 3 binning). Statistical significance is evaluated by unpaired t‐test for luciferase assay (panel A) and by a Kolmogorov‐Smirnov (KS) test, for SPIM‐FCS diffusion data (panel E). ***P < 0.001, **P < 0.01, *P < 0.05.