The Wnt16 mutants are susceptible to spontaneous fractures while wnt16 expression is upregulated in injured bone. (A) μCT images indicate lower and more variable tissue mineral density (TMD) and the presence of bone calluses (arrowheads) in the fins of wnt16−/− zebrafish. (B) Violin plots show distribution around mean (black line) TMD in WT and wnt16−/− fins. N = 3; scale bar = 1 mm. (C) Uninjured WT and wnt16−/− zebrafish were live‐stained with Alizarin Red at ages 6, 20, or 30 months (mo). Scale = 1 mm. (D) Higher magnification of fins (from C) shows the presence of bone calluses (arrowheads) resulting from bone repair in 6‐month‐old wnt16−/− and 30‐month‐old WT zebrafish but not 6‐month‐old zebrafish. Scale = 200 μm. (E) Quantification of bone calluses per fin shows that young wnt16 mutants display a significantly higher number of calluses compared with WT fish at the same age but no significant difference compared with aged WT zebrafish. N ≥ 5 per condition. (F) Representative images showing fluorescent in situ hybridization of wnt16 performed on WT uninjured bone and fractures between 2 and 14 dpi. (G) wnt16 expression within the fracture site was quantified relative to uninjured bone (un) in the same fin (intensity ratio). The expression of wnt16 increased significantly postfracture between 4 and 7 dpi. *p < 0.05, ****p < 0.0001; N ≥ 8 per time point.
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