Loss of Wnt16 Leads to Skeletal Deformities and Downregulation of Bone Developmental Pathway in Zebrafish

Abstract

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein–protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.

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).…”

“…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.…”

Functional Validation of Osteoporosis Genetic Findings Using Small Fish Models

“…They demonstrated that despite normal bone healing, the recruitment of osteoblasts was compromised in mutants during the early stages of repair [ 95 ]. While McGowan et al focused on bone fractures, Qu et al studied the skeletal structure of a second wnt16 mutant, reporting reduced bone density, curved spines and abnormal jaw [ 96 ]. The same group performed RNA-seq, identifying differentially expressed genes associated with mTOR, FoxO and VEGF pathways [ 96 ].…”

“…While McGowan et al focused on bone fractures, Qu et al studied the skeletal structure of a second wnt16 mutant, reporting reduced bone density, curved spines and abnormal jaw [ 96 ]. The same group performed RNA-seq, identifying differentially expressed genes associated with mTOR, FoxO and VEGF pathways [ 96 ]. Finally, recent work by Watson et al interrogated the biology behind the pleiotropy at the locus (which was associated with BMD and lean mass) [ 97 ].…”