PUBLICATION

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

Authors

Qu, X., Liao, M., Liu, W., Cai, Y., Yi, Q., Long, J., Tan, L., Deng, Y., Deng, H., Chen, X.

ID

ZDB-PUB-210703-25

Date

2021

Source

International Journal of Molecular Sciences 22(13): (Journal)

Registered Authors

Keywords

CRISPR-Cas9, RNA sequencing, skeletal development, wnt16, zebrafish

MeSH Terms

Animals
Zebrafish/genetics*
Animals, Genetically Modified
Bone and Bones/abnormalities*
Zebrafish Proteins/chemistry
Zebrafish Proteins/deficiency*
Zebrafish Proteins/metabolism
Molecular Sequence Annotation
Osteogenesis/genetics*
Wnt Proteins/chemistry
Wnt Proteins/deficiency*
Wnt Proteins/metabolism
Gene Ontology
Gene Expression Profiling
Transcriptome
Disease Models, Animal
Gene Knockout Techniques
Musculoskeletal Abnormalities/diagnosis
Musculoskeletal Abnormalities/genetics*
Musculoskeletal Abnormalities/metabolism*
Phenotype
Computational Biology/methods

(all 22)

PubMed

34206401 Full text @ Int. J. Mol. Sci.

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.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
akt1	AB	standard conditions	4-cell	whole organism	RTPCR	Figure 6
akt1	AB	standard conditions	1-cell	whole organism	RTPCR	Figure 6
akt1	wnt16^{zf3561/zf3561} (AB)	standard conditions	4-cell	whole organism	RTPCR	Figure 6
akt1	wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism	RTPCR	Figure 6
bnip4	AB	standard conditions	4-cell	whole organism	RTPCR	Figure 6
bnip4	AB	standard conditions	1-cell	whole organism	RTPCR	Figure 6
bnip4	wnt16^{zf3561/zf3561} (AB)	standard conditions	4-cell	whole organism	RTPCR	Figure 6
bnip4	wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism	RTPCR	Figure 6
pla2g4f.2	AB	standard conditions	4-cell	whole organism	RTPCR	Figure 6
pla2g4f.2	AB	standard conditions	1-cell	whole organism	RTPCR	Figure 6

1 - 10 of 33

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism akt1 expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism bnip4 expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism pla2g4f.2 expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism prkab1a expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism ptena expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism twsg1b expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	1-cell	whole organism vegfaa expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	4-cell	whole organism akt1 expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	4-cell	whole organism bnip4 expression decreased amount, abnormal	Figure 6
wnt16^{zf3561/zf3561} (AB)	standard conditions	4-cell	whole organism pla2g4f.2 expression decreased amount, abnormal	Figure 6

1 - 10 of 24

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
zf3561		Complex	wnt16

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
wnt16	CRISPR3-wnt16	CRISPR
wnt16	CRISPR4-wnt16	CRISPR

Fish

Fish
AB
wnt16^{zf3561/zf3561} (AB)

Antibodies

Orthology

Engineered Foreign Genes

Mapping

Qu et al., 2021 ZDB-PUB-210703-25 PMID:34206401

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

Qu et al., 2021

ZDB-PUB-210703-25
PMID:34206401