The wnt16−/− zebrafish display precocious activation of the canonical Wnt pathway during preosteoblast proliferation and differentiation postfracture. (A) Fractures were induced in Wnt:GFP transgenic zebrafish that express GFP in cells responding to activation of the canonical Wnt signaling pathway. Representative images are shown from 0 to 7 days postfracture (dpi). (B) Levels of Wnt pathway activation throughout fracture repair were quantified by measuring the fluorescence intensity of Wnt:GFP within the fracture site normalized to control bone in the same fin (intensity ratio). Gray dotted line indicates where canonical Wnt activity at the fracture site = uninjured bone. The wnt16−/− zebrafish displayed significantly higher levels of canonical Wnt activity at 2 dpi compared with WT fractures. High levels of Wnt:GFP at the fracture site were sustained through to 4 dpi in wnt16 mutants, where they became comparable with WT. n ≥ 6 per genotype. (C) In situ hybridization of runx2a in WT uninjured and fractured fins at 4 dpi shows coexpression of runx2a and Wnt:GFP, both peaking at 4 dpi (i). (D) Expression of runx2a (measured as above) increased significantly by 2 dpi, peaking at 4 dpi, before decreasing at 7 dpi. Un = Uninjured control, n ≥ 8 per time point. (E) In situ hybridization of fractures at 7 dpi showed the colocalization of runx2a, wnt16 with low levels of Wnt:GFP (ii). ****p < 0.0001, ***p < 0.001, *p < 0.05. Dotted lines = bone outline; white asterisk = center of fracture. Scale bar C, D = 200 μm; scale bar i, ii = 20 μm.
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