Fig. 2

Effect of PORCN inhibitors on mesenchymal condensation formation and production of cartilaginous ECM. (A) Micromass cultures established from chicken forelimb buds (stage HH20) were cultivated for 3 d to evaluate the effect of Wnt inhibitors on early chondrogenesis. Alcian blue, hematoxylin, and rhodamine-conjugated PNA staining revealed an increased amount of cartilaginous nodules after C59 and LGK974 treatment. In contrast, the treatment with PF670462 reduced the amount of cartilaginous nodules (1 μM concentration of inhibitors was used). Scale bar = 1 mm. (B) Schema of experimental design. Micromasses were established from chicken forelimb buds (stage HH20) and were collected for gene expression analyses evaluated by qPCR or stained for the assessment of ECM production. (C) Micromass cultures established from chicken forelimb buds (stage HH20) cultivated for 6 d. Production of the ECM was evaluated using Alcian blue staining. An increased amount of ECM was produced after C59 and LGK974 treatment, while a low production level was observed after the PF670462 application. Three different concentrations of inhibitors were used (0.1 μM, 0.3 μM, or 1 μM), and progression in ECM production was observed with increased concentrations of PORCN inhibitors. Scale bar = 1 mm. (D) Graphs displaying analyses of blue pixels in Alcian blue stained micromasses treated with individual Wnt inhibitors after 6-d cultivations. Data is shown as a number of blue pixels. Two-tailed Student’s t-test or Welch’s t-test was used for statistical analyses (n = 4; spots evaluated per cultivated conditions) performed on samples from three independent experiments (see Supplementary material for other repetitions). Abbrevations: ECM, extracellular matrix; PNA, peanut agglutinin; PORCN, Porcupine.