PUBLICATION

Zebrafish scube1 and scube2 cooperate in promoting Vegfa signaling during embryonic vascularization

Authors

Tsao, K.C., Lin, Y.C., Chen, Y.T., Lai, S.L., Yang, R.B.

ID

ZDB-PUB-210401-11

Date

2021

Source

Cardiovascular research 118(4): 1074-1087 (Journal)

Registered Authors

Lai, Shih-Lei (Ben), Yang, Ruey-Bing (Ray)

Keywords

vegfa, angiogenesis, embryonic vascularization, genome editing, vasculogenesis, zebrafish

MeSH Terms

Zebrafish*/genetics
Zebrafish*/metabolism
Membrane Proteins/genetics
Membrane Proteins/metabolism
Endothelial Cells/metabolism
Adaptor Proteins, Signal Transducing/metabolism
Neovascularization, Physiologic
Animals
Vascular Endothelial Growth Factor A*/genetics
Vascular Endothelial Growth Factor A*/metabolism
Calcium-Binding Proteins/genetics
Calcium-Binding Proteins/metabolism
Neovascularization, Pathologic/metabolism

(all 13)

PubMed

33788916 Full text @ Cardiovasc. Res.

Abstract

Aims The secreted and membrane-anchored SCUBE (signal peptide-CUB-EGF domain-containing proteins) gene family composed of 3 members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor A (VEGFA) and acts as a co-receptor for VEGF receptor 2 (VEGFR2) to modulate EC migration, proliferation and tube formation during postnatal and tumor angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signaling during embryonic vascular development remains unknown.

Methods and results To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signaling during zebrafish embryonic vascularization.

Conclusions We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviors and formation of functional vessels during zebrafish embryonic development.

Translational perspective Our studies indicate that targeting SCUBE1 and/or SCUBE2 on modulating VEGF signaling might provide potential therapeutic treatments or VEGF-mediated proliferative pathological vascular diseases.

Genes / Markers

Figures

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Expression

Fish	Conditions	Stage	Anatomy	Assay	Figure
sd2Tg; y1Tg (AB)	standard conditions	Protruding-mouth	nucleate erythrocyte	IFL	Fig. 1
sd2Tg; y1Tg (AB)	standard conditions	Protruding-mouth	endothelial cell	IFL	Fig. 1
sd2Tg; y1Tg (AB)	standard conditions	Protruding-mouth	endothelial cell	RTPCR	Fig. 1
AB	standard conditions	Prim-15	whole organism	RTPCR	Fig. S2
AB	standard conditions	Protruding-mouth	whole organism	RTPCR	Fig. S2
AB	standard conditions	Long-pec	whole organism	RTPCR	Fig. S2
scube1^as401/as401 (AB)	standard conditions	Long-pec	whole organism	RTPCR	Fig. S2
scube2^as403/as403 (AB)	standard conditions	Protruding-mouth	whole organism	RTPCR	Fig. S2
scube3^as405/as405 (AB)	standard conditions	Prim-15	whole organism	RTPCR	Fig. S2
sd2Tg; y1Tg (AB)	standard conditions	Protruding-mouth	endothelial cell	RTPCR	Fig. 1

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
ci5Tg; y7Tg (AB)	standard conditions	Prim-5	intersegmental vein endothelial tip cell normal speed, normal	Fig. 4
ci5Tg; y7Tg (AB)	standard conditions	Prim-5	intersegmental vein filopodium normal amount, normal	Fig. 4
ci5Tg; y7Tg (AB)	standard conditions	Long-pec	dorsal aorta normal width, normal	Fig. 3
ci5Tg; y7Tg (AB)	standard conditions	Long-pec	intersegmental vein endothelial cell normal amount, normal	Fig. 3
ci5Tg; y7Tg (AB)	standard conditions	Long-pec	posterior cardinal vein normal width, normal	Fig. 3
ci5Tg; y7Tg (AB)	standard conditions	Protruding-mouth	subintestinal vein normal length, normal	Fig. 6
scube1^as401/as401 (AB)	standard conditions	Prim-5	whole organism morphology, normal	Fig. S3
scube1^as401/as401 (AB)	standard conditions	Prim-15	whole organism morphology, normal	Fig. S3
scube1^as401/as401 (AB)	standard conditions	Long-pec	whole organism scube2 expression increased amount, abnormal	Fig. S2
scube1^as401/as401 (AB)	standard conditions	Long-pec	whole organism scube3 expression increased amount, abnormal	Fig. S2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
as401		Small Deletion	scube1
as402		Small Deletion	scube1
as403		Indel	scube2
as404		Small Deletion	scube2
as405		Small Deletion	scube3
as406		Small Deletion	scube3
ci5Tg	Tg(-6.5kdrl:mCherry)	Transgenic Insertion
sd2Tg	Tg(gata1a:DsRed)	Transgenic Insertion
y1Tg	Tg(fli1:EGFP)	Transgenic Insertion
y7Tg	Tg(fli1:nEGFP)	Transgenic Insertion

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
kdrl	MO1-kdrl	MRPHLNO
scube1	TALEN1-scube1	TALEN
scube2	TALEN1-scube2	TALEN
scube3	TALEN1-scube3	TALEN
tnnt2a	MO1-tnnt2a	MRPHLNO
vegfaa	MO1-vegfaa	MRPHLNO

Fish

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed	EFG	DsRed
EGFP	EFG	EGFP
mCherry	EFG	mCherry

Mapping

Tsao et al., 2021 ZDB-PUB-210401-11 PMID:33788916

Zebrafish scube1 and scube2 cooperate in promoting Vegfa signaling during embryonic vascularization

Tsao et al., 2021

ZDB-PUB-210401-11
PMID:33788916