The osterix-expressing cells with chondrocyte morphology express markers of chondrocyte and osteoblast differentiation. (A) Confocal stacks taken of the ceratohyal of 5 dpf embryos stained for collagen II in red, GFP (osterix) in green and DAPI in blue. In wild-type embryos osteoblasts can be seen lying on the surface of the collagen II-positive matrix (white arrows). In ptc1 mutant embryos, similar cells can be seen (white arrows), and also osterix-positive cells, which are completely surrounded by collagen II-positive matrix can be seen (blue arrows). In ptc1/2 double mutants the cartilage structure is disorganised, but all cells surrounded by collagen II-positive matrix are also positive for osterix (blue arrows). In the ihha-/- mutants, neither population of osterix-positive cells are seen, despite the normal appearance of the collagen II-positive cartilage matrix. (B) Confocal stack from a 4 dpf ptc2 mutant osterix-nuGFP larva, injected with a BAC containing mCherry inserted after the ATG of collagen2a1, leading to mosaic expression through the cartilage. White arrows mark cells expressing osterix, which do not contain col2 mCherry. Yellow arrow marks a cell expressing col2 which is negative for osterix, whereas blue arrows mark cells expressing both mCherry and GFP. (C) Confocal stacks from the ceratohyal of collagen 2 (red) and anti-GFP osterix (green) antibody stained 6 dpf larvae treated from 2-6 dpf with purmorphamine. White arrows indicate bone collar osteoblasts overlying the cartilage matrix. Blue arrows mark cells expressing osterix, which are surrounded by cartilage matrix. (D) Col1a2 in situ hybridisation in 9 dpf wild-type embryo, showing the palatoquadrate. Two populations of col1a2 expressing-osteoblasts can be seen. One population is located outside but adjacent to the cartilage matrix (black arrows), whereas the other comprises cells located within the cartilage matrix (red arrows). (E) Ventral views of 5 dpf embryos in situ hybridised for osteocalcin, anterior to right. In all genotypes osteocalcin staining can be seen in the teeth (black arrows). Particularly strong staining in the osteoblasts of the bone collar (red arrowheads) is present in wild-type and ptc2-/-, embryos but is absent for ihha-/-. In ptc2 mutant embryos weaker osteocalcin expression can be seen in cells within the cartilage (blue arrows), which are not seen in wild-type embryos at this stage. (F) High magnification image of Meckel′s cartilage from a ptc2-/- 5 dpf embryo showing osteocalcin expression in cells within the cartilage element (blue arrows). wt, wild type.
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