Fig. 6

PORCN inhibitors alter endochondral ossification in tibias. Tibias isolated from E18 mouse embryos were cultured with Wnt inhibitors (C59, LGK974, PF670462) at concentrations of 0.5 or 1 μM. (A) Schema of individual zones during endochondral ossification and their markers selected for further analyses. (B) Differences in tibia length before and after 8 d of cultivation (data are displayed as subtraction of initial length from final bone length). At least 4 tibias were evaluated for each experimental condition. Two-tailed Student’s t-test or Mann–Whitney was used for the statistical analyses performed on samples from 3 independent experiments (see Supplementary material for other repetitions). (C) Expression of Ptch1 (marker of proliferating zone) detected on histological sections of cultivated tibias by RNAscope. The treatment with C59 (0.5 or 1 μM) downregulated Ptch1 expression in the tibia growth plate. Scale bar = 100 μm. (D) The overall appearance of tibias treated with PF670462 at the end of the experiment, where the shown tibias are representatives selected from 1 independent experiment after 8 d of cultivation. Histological sections stained by H&E reveal an increase in the hypertrophic zone (HZ) and a decrease of collagen type 10a1 (a marker of the HZ, DAPI counterstain nuclei). Scale bar = 100 μm. (E) The overall appearance of tibias treated with C59 at the end of the experiment. C59 treatment elongated tibias with a more considerable difference observed when using 1 μM concentration after 8 d of cultivation. An expanded HZ was observed on histological sections stained by H&E even at the lowest-used concentration (0.5 μM) of C59. Col10a1 (DAPI counterstain nuclei) expression slightly increased in tibias treated with C59 inhibitor. Scale bar = 100 μm. (F) Overall appearance of the tibia treated by LGK974 at the end of the experiment. LGK974 treatment noticeably elongated cultured tibias after 8 d of cultivation, especially when using 1 μM concentration. An expanded HZ was visible on histological sections stained with H&E at both analyzed concentrations. An increase of Col10a (red signal, DAPI counterstain nuclei) expression was observed in tibias treated with LGK974 inhibitor. Scale bar = 100 μm. Abbrevation: PORCN, Porcupine.