Zebrafish elasmoid scale structure and bone cell types. (A) Single scale from the flank of a 3 months old fish carrying the sp7:gfp osteoblast reporter transgene (green) and stained for Alizarin Red S (AR, red). Whole scale is shown in bright field (i) and gray scale images for AR (ii) and GFP (iii) in the top panels. The brightfield image (i) depicts the anterior anchor region (A, black dotted line boundaries), the lateral circuli (L, green dotted line boundaries, white arrow), central region (C, surrounded by black, green, and light blue dotted lines), and central region covered by epidermis (C+E, light blue dotted line, with grooves by green arrow) with enhanced mineralization. (B) Confocal images showing a merge image of osteoblasts (sp7:gfp transgenic fish, green) abundantly distributed over the freshly harvested scale and AR staining (red). Individual channels are depicted in gray scale images. Note increased mineralization at the edge of the scale corresponding increased GFP presence (blue arrows). Insets focus on the lateral circulus and note osteoblast cytoplasmic protrusions (pink arrows). (C) Confocal images visualizing osteoclasts with cathepsin K (ctsk) YFP reporter expression (green), mineralization by AR (red), and brightfield (gray). Note that YFP positive cells were predominantly seen in the central region with epidermis (C+E) and distal edges of the central region (C). (D) Multiphoton forward scattering (second harmonic generation (SHG), 880 nm wavelength) visualizes collagen fibrils in an ethanol fixed scale. Inset (i) shows the organization of collagen fibrils in a plywood structure. Wildtype strains (panel): TL/EKK (A), TL (B), AB/TL (C). Scale bars 100 μm.
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