PUBLICATION

vwa1 Knockout in Zebrafish Causes Abnormal Craniofacial Chondrogenesis by Regulating FGF Pathway

Authors

Niu, X., Zhang, F., Ping, L., Wang, Y., Zhang, B., Wang, J., Chen, X.

ID

ZDB-PUB-230429-51

Date

2023

Source

Genes 14(4): (Journal)

Registered Authors

Chen, Xiaowei, Zhang, Bo, Zhang, Fuyu

Keywords

FGF pathway, VWA1, cranial neural crest cells, hemifacial microsomia

MeSH Terms

Zebrafish*/genetics
Zebrafish*/metabolism
Chondrocytes/metabolism
Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism
Cartilage/metabolism
Chondrogenesis*/genetics
Animals
Receptor, Fibroblast Growth Factor, Type 4/metabolism
Receptor, Fibroblast Growth Factor, Type 3

(all 10)

PubMed

37107596 Full text @ Genes (Basel)

Abstract

Hemifacial microsomia (HFM), a rare disorder of first- and second-pharyngeal arch development, has been linked to a point mutation in VWA1 (von Willebrand factor A domain containing 1), encoding the protein WARP in a five-generation pedigree. However, how the VWA1 mutation relates to the pathogenesis of HFM is largely unknown. Here, we sought to elucidate the effects of the VWA1 mutation at the molecular level by generating a vwa1-knockout zebrafish line using CRISPR/Cas9. Mutants and crispants showed cartilage dysmorphologies, including hypoplastic Meckel's cartilage and palatoquadrate cartilage, malformed ceratohyal with widened angle, and deformed or absent ceratobranchial cartilages. Chondrocytes exhibited a smaller size and aspect ratio and were aligned irregularly. In situ hybridization and RT-qPCR showed a decrease in barx1 and col2a1a expression, indicating abnormal cranial neural crest cell (CNCC) condensation and differentiation. CNCC proliferation and survival were also impaired in the mutants. Expression of FGF pathway components, including fgf8a, fgfr1, fgfr2, fgfr3, fgfr4, and runx2a, was decreased, implying a role for VWA1 in regulating FGF signaling. Our results demonstrate that VWA1 is essential for zebrafish chondrogenesis through effects on condensation, differentiation, proliferation, and apoptosis of CNCCs, and likely impacts chondrogenesis through regulation of the FGF pathway.

Genes / Markers

Figures

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Expression

Fish	Conditions	Stage	Anatomy	Assay	Figure
TU	standard conditions	Long-pec	pharyngeal arch	ISH	Figure 4
vwa1^{pkm101/pkm101} (TU)	standard conditions	Long-pec	pharyngeal arch	ISH	Figure 4
TU	standard conditions	Protruding-mouth	pharyngeal arch	ISH	Figure 4
vwa1^{pkm101/pkm101} (TU)	standard conditions	Protruding-mouth	pharyngeal arch	ISH	Figure 4
TU	standard conditions	Prim-5	neural crest	ISH	Figure 4
vwa1^{pkm101/pkm101} (TU)	standard conditions	Prim-5	neural crest	ISH	Figure 4
TU	standard conditions	Prim-15	pharyngeal arch	ISH	Figure 4
vwa1^{pkm101/pkm101} (TU)	standard conditions	Prim-15	pharyngeal arch	ISH	Figure 4
ba2Tg (TU)	standard conditions	Day 4	cranial cartilage chondrocyte	IFL	Figure 3
ba2Tg (TU)	standard conditions	Prim-5	cranial cartilage chondrocyte	IFL	Figure 5

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
TU + CRISPR4-vwa1	control	Day 5	ceratobranchial cartilage decreased amount, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	ceratobranchial cartilage hypoplastic, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	ceratohyal cartilage increased angle to ceratohyal cartilage, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	Meckel's cartilage hypoplastic, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	palatoquadrate cartilage hypoplastic, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	pericardium edematous, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	swim bladder uninflated, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	ventral mandibular arch dysplastic, abnormal	Figure 2
TU + CRISPR4-vwa1	control	Day 5	whole organism vwa1 expression decreased amount, abnormal	Figure 2
vwa1^{pkm101/pkm101} (TU)	control	Day 5	ceratobranchial cartilage decreased amount, abnormal	Figure 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
ba2Tg	Tg(-4.9sox10:EGFP)	Transgenic Insertion
pkm101		Small Deletion	vwa1

Human Disease / Model

Human Disease	Fish	Conditions	Evidence
Goldenhar syndrome			TAS

Sequence Targeting Reagents

Target	Reagent	Reagent Type
vwa1	CRISPR1-vwa1	CRISPR
vwa1	CRISPR2-vwa1	CRISPR
vwa1	CRISPR3-vwa1	CRISPR
vwa1	CRISPR4-vwa1	CRISPR

Fish

Fish
ba2Tg (TU)
TU
TU + CRISPR4-vwa1
vwa1^{pkm101/pkm101}; ba2Tg (TU)
vwa1^{pkm101/pkm101} (TU)

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab47-h3	monoclonal		IgG2b	Mouse

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP

Mapping

Niu et al., 2023 ZDB-PUB-230429-51 PMID:37107596

vwa1 Knockout in Zebrafish Causes Abnormal Craniofacial Chondrogenesis by Regulating FGF Pathway

Niu et al., 2023

ZDB-PUB-230429-51
PMID:37107596