Bone mineralization and osteoblast recruitment is significantly delayed postfracture in wnt16−/− zebrafish. (A) Schematic illustrating fracture induction assay and labeling of old bone (Alizarin Red) and new bone (calcein green). (B) Callus formation was quantified by measuring the calcein intensity ratio between the fracture site and uninjured bone. Callus formation was significantly reduced from 2 to 7 days postinjury (dpi) in wnt16 mutant compared with WT fractures. N ≥ 5 per condition. Gray dotted line indicates where calcein intensity at the fracture site = uninjured bone. (C) Representative images of WT and wnt16−/− fish at selected time points postinjury show old bone labeled by Alizarin Red (gray) and callus formation labeled by calcein. White asterisk = center of fracture. Scale = 200 μm. (D) Representative images of calcified bone (Alizarin Red) and osteoblasts (osx:GFP) at fracture site in WT and wnt16−/− throughout fracture repair. White asterisk = center of fracture. Scale bar = 100 μm. (E) Osteoblast density was quantified by measuring the fluorescence intensity of osx:GFP within the fracture site normalized to control bone in the same fin (intensity ratio). Gray dotted line indicates where osx:GFP intensity at the fracture site = uninjured bone. Osteoblast recruitment was delayed in wnt16 mutants, which had a significantly lower osx:GFP intensity ratio at the fracture site 4 dpi, but significantly higher osx:GFP intensity ratio at 10 dpi compared with WT zebrafish. (F) Confocal imaging of bone in amputated fins at the end of the time course (15 dpi) shows complete union of fractures in both WT and wnt16−/− zebrafish. Scale bar = 100 μm. (B & E): N.s = no significant difference, *p < 0.05, **p < 0.01, ****p < 0.0001. N ≥ 6 per genotype.