Osteoblasts in dermal bone of the skull dedifferentiate following injury. (A) Scheme illustrating the bone injury (500µm hole) in the os frontale (purple filled circle) or at the boundary between the os frontale and os parietale (empty circle) of the dorsal skull. F, os frontale; N, os nasale; P, os parietale; Pmx, os praemaxillare; Soc, os supraoccipitale. (B) Whole-mount view of uninjured and injured skulls at different times post injury stained with Alizarin Red. Arrowhead, islands of bone matrix. (C) Calcein staining of whole-mount injured skulls at different times post injury. (D) Reduction of GFP signal intensity in osteocalcin:GFP transgenic fish in proximity to the drill injury (asterisk), revealed by repeated live imaging of the same fish. (E) Quantification of experiment shown in D. Error bars, s.e.m. (F,G) Immunofluorescence of transverse sections of the dorsal skull stained for GFP and Zns5. Bracket, injury site; dashed line, injury boundary; arrowhead, osteoblasts in uninjured area; arrow, osteoblasts lining newly formed matrix in injury area. (F) osteocalcin:GFP fish. (G) runx2:GFP fish. Arrowhead, GFP+ osteoblasts lining newly formed matrix in injury area. (H) Skull of an osterix:nGFP fish before and after injury. (I) osterix+ osteoblasts contribute to repair of skull bone after injury. In osterix:CreERT2-p2a-mCherry×hs:R to nG double transgenic fish genetically labeled osteoblasts (bright nuclear signals) cover the injury site at 7dpi (arrowhead). Only background fluorescence is detected in vehicle (ethanol)-treated fish. Scale bars: 200µm in B,C,H,I; 500µm in D; 50µm in F,G.
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