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.

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.

Loss of Wnt16 does not perturb leukocyte recruitment to bone postfracture. Fractures were induced in WT and wnt16^−/− zebrafish carrying lyzC:DsRed and mpeg1:mCherry transgenes to measure the recruitment of neutrophils and macrophages, respectively. (A) Schematic depicting regions of interest around the fracture site where leukocyte recruitment was quantified. (B) Representative images of from WT and wnt16^−/− zebrafish show neutrophil (lyzC⁺ cells) recruitment to fractured bone at 0, 8, and 24 hours postinjury (hpi) and 7 days postinjury (dpi). Scale bar = 100 μm. (C,D) The number of neutrophils within 100 μm (C) and 300 μm (D) of the fractures were quantified in an automated manner using modular image analysis (MIA) from 0 hpi to 14 dpi. WT and wnt16 mutants displayed comparable numbers of neutrophils at the fracture site at all time points post injury. N ≥ 5 per genotype. (E) Representative images of from WT and wnt16^−/− zebrafish show macrophage (mpeg1⁺ cells) recruitment to fractured bone at selected time points from 0 to 14 dpi. Scale bar = 100 μm. (F,G) The number of neutrophils within 100 μm (F) and 300 μm (G) of the fractures were quantified using MIA from 0 hpi to 14 dpi. WT and wnt16 mutants displayed comparable numbers of macrophages at all time points postinjury, with the exception of 8 hpi when wnt16 mutants had recruited significantly more macrophages to within 100 μm of the fracture site (F). *p < 0.05; N ≥ 5 per genotype.

TRAP⁺ punctae accumulate near to fractures in wnt16^−/− zebrafish postinjury. Fins from WT and wnt16^−/− zebrafish were amputated at 0 hours postfracture (hpi), 24 hpi, 4 days postfracture (dpi) and 7 dpi before undergoing staining to detect the presence of tartrate‐resistant acid phosphatase (TRAP). (A) Representative images of fractures stained for TRAP. Scale bar = 100 μm. (B) Overall coverage of TRAP was measured by calculating the total % area stained within 300 μm of the fracture site. No significant difference in the amount of TRAP⁺ stained area between WT and wnt16^−/− fractures was found. (C) The number of TRAP⁺ punctae present within 300 μm of the fracture site were quantified and showed a significantly higher number of punctae at 24 hpi and 4 dpi in the fractures of wnt16^−/− zebrafish compared with WT. *p < 0.05; N ≥ 6 per genotype.

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.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.