Fig. 9: In vivo effect of cholesterol modulation on Wnt receptor organization and βcat activation.

A Drosophila midgut-hindgut intestinal tissue model. ISCs/progenitor cells express humanized hFzd7 or hLRP6 under control of the UAS and esg-Gal4^TS. B Filipin III-stained midgut from Drosophila fed various cholesterol diets (red arrow, intestinal lumen). C Quantification of total cholesterol from Drosophila midgut. Cholesterol was calculated from luciferase luminescence data using the Amplex™ Red cholesterol assay and normalized to total protein (mean ± SD, from n = 3 independent biological replicates). D Filipin III fluorescence distribution of Drosophila intestinal epithelium (red arrow, ISCs). Effects of cholesterol on Wnt receptor organization. Flies co-expressing EGFP- and mCherry-tagged E LRP6 or F Fzd7 or G EGFP-LRP6 and mCherry-Fzd7 were used to perform FLIM-FRET in flies fed various cholesterol diets. FRET efficiency was calculated from FLIM data (mean ± SD, from n = 5–10 guts, ROIs analyzed provided below each bar, n value is shown in each graph). H Quantitative analysis of free cholesterol-induced βcat activation. 3T3 LL cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), and MβCD-cholesterol (cholesterol) (10 mM, 30 min), and incubated with control or Wnt3a-conditioned media for 24 h. Luciferase luminescence was measured using a Luciferase Assay System kit. Luciferase luminescence fold change was normalized to total protein (mean ± SD, from n = 3 independent biological replicates). I Quantitative analysis of Wnt signaling activation. The percentage of TCF-LacZ+ cells is shown. Error bars represent n = 5 independent biological replicates (mean ± SD, ~100 cells analyzed per group). J Qualitative analysis of cholesterol-induced Wnt activation. TCF-LacZ+ cells activity in Drosophila posterior midguts from control (w¹¹¹⁸; esgGal4^TS, UAS-GFP; TCF-LacZ) and hLRP6-expressing ISCs (w¹¹¹⁸; esgGal4^TS, UAS-GFP, UAS-hLRP6; TCF-LacZ). Flies were feed a cholesterol free, standard (Std. Diet) or high cholesterol diet for 5 days. ISCs, GFP; nuclei, DAPI. Statistical significance determined by C two-way ANOVA or E–I one-way ANOVA and post hoc Tukey’s test. Different letters indicate significant differences between treatment groups (P < 0.05). Representative images and scale bars are provided for microscopy data. Enterocytes, ECs; visceral muscle cells, muscle; intestinal stem cells, ISCs; enteroblasts, EB. Source data file provided.