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Fig. 3 There are two populations of endochondral osteoblasts with differential sensitivity to Hh signalling. (A) Confocal stacks from wild-type, ptc1-/- and ptc2-/- 6 dpf larvae in an osterix-nuclear GFP background, imaged for GFP expression. The far right image is a single confocal plane to allow distinstintion of the cell morphology. At this stage in both ptc mutants and wild-type larvae, similar numbers of bone collar osteoblasts are present (red arrowheads). However, in ptc1-/- and ptc2-/- larvae, there is a significant increase in the number of chondrocytes that express osterix (blue arrows). A single chondrocyte is outlined in black in the image of the ptc1 mutant. (B) A ventral view of one side of the Meckel′s cartilage and ceratohyal of a 6 dpf wild-type larva stained with Alcian Blue and Alizarin Red; a single chondrocyte is outlined in black to demonstrate the morphology of the chondrocyte cells. (C) Confocal stacks of the ceratohyal from ptc1-/- and wild-type embryos at 3 dpf. In ptc1-/- embryos (but not in wild-type siblings) cells can be found that retain the shape of chondrocytes but that express osterix (blue arrow). (D) Confocal stack of the Meckel′s cartilage of a 9 dpf wild type. Expression of osterix is visible in cells within the cartilage element (blue arrows) and outside (red arrowhead). (E) Confocal stacks of the Meckel′s cartilage of wild-type and ihha-/- 9 dpf larvae. By this stage the wild-type embryo has osterix- expressing bone collar cells (red arrowhead) and transdifferentiating chondrocytes (blue arrows), while the ihha-/- larvae has neither. White arrowheads mark the maxilla, a dermal bone, which forms adjacent to the Meckel′s cartilage. (F) Quantification of number of osteoblasts at 6 dpf showing number of osteoblasts in the bone collar of the Meckle′s cartilage and the number of chondrocytes differentiating as osteoblasts in the Meckel′s cartilage. Data are presented as means±s.d. taken from at least six fish per genotype; asterisk represents P<0.05 in paired Student′s t-tests of mutant versus wild type. (G) Quantification of osteoblast number at 6 dpf in the Meckel′s cartilage of embryos treated with 20 μm purmorphamine or controls. Data are presented as means±s.d. taken from at least four fish per genotype; * represents P<0.05 in paired Student′s t-tests of treated versus untreated. (H) Quantification of osteoblast number at 6 dpf in the Meckel′s cartilage of embryos treated with 75 μM cyclopamine or controls. Data are presented as means±s.d. taken from at least four fish per genotype; * represents P<0.05 in paired Student′s t-tests of treated versus untreated. wt, wild type.