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

Abstract

SummaryBone homeostasis is a dynamic, multicellular process which 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 humans, as well as functional haematopoiesis of leukocytes in vivo, but the mechanisms by which it promotes bone health and repair are not fully understood. We used CRISPR-Cas9 to generate mutant zebrafish lacking Wnt16 (wnt16-/-) to study its effect on bone dynamically. wnt16 mutants displayed variable tissue mineral density 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 wild type (WT) zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell-dynamics in vivo. wnt16 mutants repaired fractures more slowly compared to WT zebrafish. Osteoblast cell number was reduced at the fracture site 4 days post-injury in wnt16 mutants, coinciding with prolonged activation of the canonical Wnt signalling pathway. Surprisingly, we found no evidence that the recruitment of innate immune cells to fractures was altered in wnt16 mutants. This study highlights zebrafish as an emerging model for functionally validating osteoporosis-associated genes and investigating fracture repair dynamically in vivo. Using this model, we demonstrate that Wnt16 protects against fracture and is likely to support bone repair by attenuating the activation of the canonical Wnt signalling pathway to facilitate osteoblast recruitment and bone matrix deposition.

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome

“…In our study, phenotypes in wnt16 -/- mutant larvae were indistinguishable for three different alleles. Moreover, wnt16 -/- mutants did not exhibit loss of caudal fin rays, in agreement with a wnt16 presumptive null allele generated by McGowan et al (McGowan et al, 2021), and contrasting with the allele of Qu et al (Qu et al, 2021). The allele of Qu et al comprised a 11bp deletion in exon 3, which was generated by two gRNAs with different target sites.…”

“…Previous studies have reported disparate skeletal phenotypes in zebrafish homozygous for presumptive wnt16 null alleles; for example, Qu et al reported loss of caudal fin rays, whereas caudal fin rays in mutants described by McGowan et al are intact (McGowan et al, 2021; Qu et al, 2021). To help resolve these discrepancies, we generated multiple wnt16 loss-of-function alleles using CRISPR/Cas9-based gene editing (Supplemental Fig 2A).…”

“…Expression of wnt16 in anterior somitic cells has been shown to mediate the specification of hematopoietic stem cells (Clements et al, 2011). Moreover, wnt16 has been implicated in mediating spine bone mineral density and bone fragility in fin rays in zebrafish (McGowan et al, 2021). However, the function of wnt16 in the development of skeletal muscle and lean mass is unknown.…”

“…Moreover, WNT16 suppresses osteoclastogenesis (Moverare-Skrtic et al, 2014). Zebrafish with mutations in wnt16 exhibit decreased bone mineral density (Qu et al, 2021) and bone fragility in fin rays (McGowan et al, 2021). While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at the CPED1-WNT16 locus.…”

Skeletal Biology and Disease Modeling in Zebrafish

“…4B). ( 151 ) Emerging phenomics‐based deletion approaches in mosaic zebrafish (crispants) now present an efficient model for the rapid validation of novel GWAS‐derived genes related to bone disease ( 29,40 ) (Fig. 4B, C, Table 1).…”