Fig. 5

NC contribution to mineralized tissues of the adult skull.

A) Bones derived by ossification of the pharyngeal arch cartilages are also NC-derived, seen in a horizontal section through a 44 dpf fish stained for GFP immunoreactivity. B) The odontoblasts of the pharyngeal teeth on the fifth ceratobranchial express the RUNX2:egfp transgene (B′) and are also NC-derived, seen by nucCh+ nuclei (B′′). C, D) The scleral cartilages are NC-derived, shown by GFP immunohistochemistry (C), as are the ossicles derived by their ossification, seen as GFP+ in freshly dissected tissue (D). E) The parasphenoid shows no NC contribution, as seen in freshly dissected tissue. The scattered nucCh+ cells (E′, E′′) are in associated soft tissue. The kinethmoid (F) also shows no NC contribution, although some of the associated soft tissue is NC-derived (nucCh+ in F′, F′′). G-G′′) Sections through the kinethmoid cartilage at 7 weeks show no GFP expression (G2) by immunohistochemistry (G is DAPI counterstain, G′′ shows overlay). H, I) Dissections were used to determine the status of unresolved bones throughout the skull, e.g. the pterosphenoid is nucCh+ in freshly dissected tissue (I) and the supratemporal is not. J, K) The anterior portion of the frontal bones are NC-derived, seen as GFP+ cells in a live, 6-week-old fish (J), and also as nucCh+ cells in freshly dissected tissue (K). Dotted lines indicate the location of the coronal suture. The more posterior portion of the frontal bones, and the other flat bones of the skull, show no evidence of NC contribution. L-M′′) Sections through the anterior frontal bone of a 7-week fish (L-L′′) show GFP+ osteoblasts by immunohistochemistry (arrowheads in L2) aligned under the acellular bone matrix (bracket in L), as well as GFP+ cartilage cells in the underlying epiphyseal bar. A similar section through the posterior frontal bone (M-M′′) shows no GFP expression in the osteoblasts (M′). In each set of panels, the first is the DAPI counterstain, the second the GFP immunohistochemistry, and the third the overlay.