Cells expressing osteoclastic markers appear at ectopic mineralization sites in dgf mutants. Staining of Trap in a wild-type sibling (A) and dgf embryo (B). Trap staining (red) was visible in a dgf embryo at the region of the heart and yolk sac (B, arrowhead). Ventral view of sibling (C) and mutant (D) embryo with ectopic mineralization in the heart and yolk sac region. (C) No cathepsinK-positive cells were visible in siblings at this timepoint; (D) mutants showed colocalization of ectopic soft tissue mineralization and cathepsinK-positive cells, but no cathepsinK-positive cells were aligned to skeletal elements, such as the cleithrum (cl). (E,F) Ventral view of the yolk sac area of embryos that had been stained for Trap and with an antibody against GFP (α GFP). (F) Trap staining appeared in association with cathepsinK-positive cells. White arrows indicate loci of osteoclasts with high Trap activity. (G) Higher-magnification image of cathepsinK and Trap colocalization in a dgf mutant. Scale bar: 20μm. The staining of Trap in E–G is pseudo-colored for improved visibility. In the axial skeleton, cathepsinK-positive cells were not visible in siblings (H); however, in dgf mutants (I), cathepsinK-positive cells appeared from 9 dpf onwards and colocalized with mineralized vertebral bodies. White arrows indicate osteoclasts aligning with vertebral elements. (J) Ventral view of accumulating cathepsinK-positive cells in the skin of the heart region. These cells also express the macrophage marker mpeg1.
|
