Fig. 4

Development of growth plates in juvenile zebrafish. (A) Trichrome staining of a coronal section through the juvenile fish jaw. (B) Coronal section of a 28dpf juvenile jaw shows osteoblast precursors labeled by sp7:GFP, col10a1-expressing hypertrophic chondrocytes and BrdU⁺ proliferating cells. Hoechst labels nuclei in white. The ceratohyal cartilage (yellow box) displays a mammalian-like growth plate architecture. BrdU⁺ and col10a1-expressing zones are also observed in other cartilages, e.g. Meckel′s (arrowhead). (C) Double fluorescent in situ hybridization at 14dpf shows sox9a or col2a1a expression (green) in growth plate chondrocytes at either end of the ceratohyal cartilage and col1a1a expression (red) in the periosteum and a subset of chondrocytes at the hypertrophic borders. spp1 expression (red) is confined to the periosteum. At 21dpf, col2a1a continues to be expressed in two zones of chondrocytes and col1a1a is now largely confined to the periosteum. At 28dpf, bglap is expressed exclusively in the periosteum, and col10a1 is expressed in two stripes of hypertrophic chondrocytes surrounding each zone of col2a1a⁺ chondrocytes. A comparison of col2a1a expression with BrdU reactivity shows that col2a1a labels proliferating chondrocytes. (D) At 16dpr, mineralization labeled by Alizarin Red occurs exclusively within the periosteum. By 49dpf, two growth zones of col2a1a^BAC:GFP⁺ chondrocytes persist, yet Alizarin Red⁺ mineralization remains confined to a layer covering the ceratohyal cartilage. Scale bars: 100µm.