Fig. 3: Rnf43/Znrf3^del livers present altered lipid composition and zonation and cell-autonomous accumulation of lipid droplets.

a–e Lipidomics analysis in the mutant (R&Z^del) and control (R&Z^flox) mice at 3 (3M) and 7 months (7M) post-deletion. a Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) model of hepatic lipid profiles discriminates between genotypes. R² = 0.85, Q² = 0.80, p = 0.02. b Volcano plot highlights significantly changed lipids; horizontal line shows p = 0.05 as determined by unpaired t test. c Heatmap analysis of lipid features across the two genotypes and time points. The top 20 features (based on ANOVA) are shown. d PC(36:4) abundance. Unpaired two-tail t test. 3M, **p = 0.0042; 7M, ***p = 0.0005. e PC Fads2 index and RNA correlation. Left, Fads2 index [PC(36:4) + PC(38:4)/PC(34:2) + PC(36:2)]. Data are represented as mean + /− SD of n = 3 (3M) and n = 4–5 (7M) mice per group. Unpaired two-tail t test. 3M, *p = 0.0103; 7M, **p = 0.0059. Right, the correlation between PC Fads2 index and Fads2 RNA expression. Pearson correlation, r = 0.69, CI 95% (0.2597–0.8952), two-tailed p = 0.005. f Mass spectrometric imaging analysis shows increased intensity and expansion of area covered by PC(36:4) relative to PC(32:1) and PC(34:2) in R&Z^del liver. g–k Isolated adult hepatocytes derived from either AlbCreERT-R&Z^flox or R&Z^flox livers were expanded and treated or not with hydroxy-tamoxifen (OH-Tam) or AAV8-TGB-Cre infection to induce Rnf43/Znrf3 deletion in vitro. The derived organoid lines and parental non-recombined lines were treated with Wnt inhibitors (Wnti, h, i) or WNT3A conditioned media (Wnt3a, j, k) and analysed for lipid droplet content by Bodipy staining. g Experimental design. h–k Bodipy staining and quantification of R&Z^flox and R&Z^del hepatocyte organoids grown in control medium or medium supplemented with Wnti. h Representative pictures from n = 2 independent experiments. Scale bar, 100 μm. i Image analysis quantification of the intracellular lipid droplets size and content (n  = 5). Two-way ANOVA with Tukey’s multiple comparisons. Small particles, R&Z^floxvsR&Z^del ***p = 0.0001; R&Z^delvsR&Z^del + Wnti ***p = 0.0001. Big particles, R&Z^delvsR&Z^del + Wnti *p = 0.0198. j, k Bodipy staining and quantification of R&Z^flox wild-type hepatocyte organoids grown in control or WNT3A media. j Representative pictures from n = 2 independent experiments. Scale bar, 100 μm. k Image analysis quantification of the intracellular lipid droplets size and content (n  = 5), two-way ANOVA, *p = 0.0000. Source data provided in Source data file.