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

Xiaochao Qu,Lijun Tan,Xiangding Chen,Jianmei Long,Qiaorong Yi,Mei Liao,Yun Deng,Yisheng Cai,Weiwei Liu,Hongwen Deng

doi:10.3390/ijms22136673

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.

Highlights

Introduction published maps and institutional affilOsteoporosis is a skeletal disease that affects millions of people around the world, postmenopausal women and older men [1]
As a ligand of Wnt signaling pathway, wnt16 is related to bone mineral density, cortical thickness, bone strength, and fracture risk, and this molecule might be an attractive target for pharmacologic intervention in treating osteoporosis [7,8,9,10]
We found that the skeletal structure of the wnt16 knockout zebrafish had a distinct deformation and bone mineral density, and the number of bone trabecula decreased

Summary

Introduction

Introduction published maps and institutional affilOsteoporosis is a skeletal disease that affects millions of people around the world, postmenopausal women and older men [1]. It is estimated that 50% of women over the age of 50 have low bone quality, and about 25% of women over the age of 60 suffer from osteoporosis disease [2]. It is mainly low bone mass and the degradation of bone tissue microstructure that result in increased bone fragility and increased bone risk [3,4]. Wnt signaling plays an important role in maintaining bone mass and bone metabolism, and causes bone diseases including osteoporosis and osteoarthritis [5,6].

Methods

Results

Conclusion