Wnt16 Elicits a Protective Effect Against Fractures and Supports Bone Repair in Zebrafish

Bone homeostasis is a dynamic, multicellular process that is required throughout life to maintain bone integrity, prevent fracture, and respond to skeletal damage. WNT16 has been linked to bone fragility and osteoporosis in human genome wide-association studies, as well as the functional hematopoiesis of leukocytes in vivo. However, the mechanisms by which WNT16 promotes bone health and repair are not fully understood. In this study, CRISPR-Cas9 was used to generate mutant zebrafish lacking Wnt16 (wnt16^-/- ) to study its effect on bone dynamically. The wnt16 mutants displayed variable tissue mineral density (TMD) and were susceptible to spontaneous fractures and the accumulation of bone calluses at an early age. Fractures were induced in the lepidotrichia of the caudal fins of wnt16^-/- and WT zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell dynamics in vivo. In WT fins, wnt16 expression increased significantly during the early stages for bone repair. Mineralization of bone during fracture repair was significantly delayed in wnt16 mutants compared with WT zebrafish. Surprisingly, there was no evidence that the recruitment of innate immune cells to fractures or soft callus formation was altered in wnt16 mutants. However, osteoblast recruitment was significantly delayed in wnt16 mutants postfracture, coinciding with precocious activation of the canonical Wnt signaling pathway. In situ hybridization suggests that canonical Wnt-responsive cells within fractures are osteoblast progenitors, and that osteoblast differentiation during bone repair is coordinated by the dynamic expression of runx2a and wnt16. This study highlights zebrafish as an emerging model for functionally validating osteoporosis-associated genes and investigating fracture repair dynamically in vivo. Using this model, it was found that Wnt16 protects against fracture and supports bone repair, likely by modulating canonical Wnt activity via runx2a to facilitate osteoblast differentiation and bone matrix deposition. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.