Fig. 1

The Xenopus tropicalis humerus mostly followed the standard process of cartilage maturation. (A,G) Compared to the NF55 humerus (A), Safranin O staining of the NF57 humerus (G) showed that mature chondrocytes (dashed black lines) in the diaphysis had become overtly hypertrophic. (B,H) Compared to the NF55 humerus (B), sox9 expression was eventually down-regulated in mature chondrocytes of the NF57 humerus (H, red arrow). (C,I) Similarly, col2a1 was down-regulated in mature chondrocytes of the NF57 humerus (I, red arrow), compared to the NF55 humerus (C). (D,J) Expression of col10a1 was undetectable in the humerus at both NF55 (D, translucent dashes outline the humerus) and even in hypertrophic chondrocytes at NF57 (J, dashed black lines). (E,F,K) Immunostaining showed expression of Col2 protein in cross-sections of the epiphysis (E, red) and diaphysis (F, green) of the NF55 humerus and immature and mature cartilage of the NF57 humerus (K). (L-N) Histological staining of an NF59 tooth with Safranin O (L) and Trichrome (M) helps to identify col10a1 expression in odontoblasts and dentin ECM (N), which has properties similar to osteoid found in bone.103 (O) Cross-sectional areas of chondrocytes from NF55 and NF57 humeri quantitating the increase in cell size (hypertrophy) from immature to mature chondrocytes. Asterisks indicate significance at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 and error bars indicate a 95% confidence interval. Abbreviations: ECM, extracellular matrix; N, total number of cells measured from three biological replicates; Tri, Trichrome; Saf O, Safranin O. Scale bars = 100 μm for panels A to K; Scale bars = 50 μm for panels L to N