Fig. 4

(A, B) Ventral views of col2a1a:CreERT2; bactin2:tagBFP >DsRed animals treated at 5 dpf with 4-OHT and imaged at 12 dpf. Confocal sections and projections as indicated demonstrate specific conversion (red) throughout cartilage, as shown by co-localization with the chondrocyte-specific marker col2a1a-BAC:GFP (A) and lack of co-localization with the osteoblast and periosteum marker sp7:GFP (B). Boxed areas are magnified in the top right insets. n = 6 for each. (C) After conversion of col2a1a:CreERT2; bactin2:tagBFP>DsRed animals at five dpf, a confocal projection through the dissected Ch of an adult (30 mm SL) shows extensive DsRed+ cells in the growth plates (GP) and throughout the bone. A higher magnification view of the boxed region, along with brightfield, shows DsRed fluorescence in the thin cytoplasm surrounding the prominent lipid vesicles indicative of marrow adipocytes, as well as in osteoblasts (osteo) of cortical bone and mesenchymal cells (mes) within the marrow cavity. The dashed line in the x-y slice shows the position of the x-z slice above. n = 10. (D) In this example of a col2a1a:CreERT2; bactin2:tagBFP>DsRed animal converted at five dpf and imaged as an adult, a prominent clone of DsRed+ cells are evident at the bottom of the growth plate, consisting of GP chondrocytes, adipocytes (Ac), and osteoblasts (Ob) associated with Calcein Green+ cortical bone. Similar clonal contributions were seen in four independently converted animals. (E) Pentachrome staining of a portion of the Ch growth plate at 19 mm SL shows the endosteum and periosteum. Note that zebrafish have osteocytes embedded in their cortical bone (the dark nuclei in the reddish-brown matrix). (F, G) High-magnification images of a section of Ch cortical bone from an animal converted at five dpf and imaged at 28 mm SL. DsRed+ osteoblasts/osteocytes are seen in the endosteal surface (arrows), periosteal surface (arrowheads), and embedded in bone (double arrowhead). The merged brightfield and fluorescence image from a different example (G) shows a DsRed+ cell with cellular processes characteristic of osteocytes. Scale bars = 100 μm (A,B), 200 μm (C,D), 50 μm (F), 20 μm (G).