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

Lucy M Mcgowan,Stephen Cross,Erika Kague,Elis Newham,Chrissy L Hammond,Alistair Vorster

doi:10.1002/jbm4.10461

Abstract

ABSTRACTBone 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.

Highlights

The maintenance of skeletal health is central to many essential processes in the body
Young wnt16 mutant zebrafish are susceptible to spontaneous fractures that heal more slowly compared with WT fish
Osteoblasts differentiate from mesenchymal stem cell (MSC) precursors, initially expressing runx2 before downregulating runx2 and expressing the transcription factor osterix. osx+ osteoblasts synthesize bone matrix within the initial soft callus; the callus hardens as it mineralizes and is remodeled to restore the bone to a healthy state.[40] transcriptomic analysis of osteoblast-prone clones isolated from tonsil–derived MSCs showed that upregulation of WNT16 is predictive of osteogenic differentiation.[41]. In zebrafish, osteoblasts dedifferentiate and proliferate in response to bone injury, migrating to the damaged tissue where they initiate bone repair.[42] we investigated whether osteoblast activity impaired postfracture repair in wnt16−/− zebrafish

Summary

Introduction

The maintenance of skeletal health is central to many essential processes in the body. Wnt signaling pathways are highly conserved, central regulators of skeletal development and homeostasis, which act on bone throughout the lifetime of vertebrate organisms.[7] Canonical Wnt pathway activation leads to the stabilization of β-catenin and activation of transcription factors, whereas the calciumdependent and planar cell polarity noncanonical Wnt signaling pathways regulate intracellular calcium levels and Jun Nterminal kinase (JNK) activity, respectively.[8] Wnt ligands are a 1 of 14 n family of secreted glycoproteins that influence cell stemness, proliferation, differentiation, and migration via Wnt signaling pathways. WNT16 is one such ligand that can influence the activity of canonical and noncanonical Wnt pathways.[9,10] Recently, WNT16 has emerged as a regulator of cortical bone thickness and BMD, with mutations in WNT16 linked to osteoporosis susceptibility in human genome wide-association studies (GWASs).(11,12)

Methods

Results

Conclusion