Fig. 1

PORCN expression during endochondral ossification in limbs and early chondrogenesis in micromasses. (A-D) PORCN and Sox9 expression detected in differentiated chondrocytes by immunohistochemistry. (A, B) PORCN signal was located in Sox9-positive cells during early endochondral ossification as observed at E13.5 of mice hindlimb. (C, D) Later at E18, weaker PORCN signal was observed in proliferating and prehypertrophic zone in the tibia growth plate while Sox9 expression was still strong. Nuclei are counterstained by DAPI. Scale bar = 100 μm. (E) Micromass cultures established from chicken forelimb buds (stage HH20) cultivated for 1, 3, and 6 d. Alcian blue staining uncovered the production of ECM from day 3 with the progression of its deposition at day 6. Small cartilaginous nodules were produced and observed from day 1 when visualized by hematoxylin staining. Scale bar = 1 mm. (F) PORCN expression analyzed by qPCR uncovered the highest expression in 1-d cultures and significantly decreased in micromasses cultivated for 3 and 6 d. (G) At the same time points, Sox9 and Col2a1 were evaluated to determine progress of chondrogenesis in micromass cultures. Gene expression was evaluated as a relative expression to Hprt1 gene expression (housekeeping). Four spots were seeded in each well, four wells were analyzed as biological replicates per cultivated conditions and each sample was run in 3 technical replicates in each qPCR reaction. Data are displayed as FC as the ratio to gene expression on day 1. Two-tailed Student’s t-test or Welch’s t-test was used for the statistical analyses performed on samples from three independent experiments (see Supplementary material for other repetitions). Abbrevations: ECM, extracellular matrix; FC, fold changes; PORCN, Porcupine.