PUBLICATION

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume

Authors

Grassini, D.R., Lagendijk, A.K., De Angelis, J.E., Da Silva, J., Jeanes, A., Zettler, N., Bower, N.I., Hogan, B.M., Smith, K.A.

ID

ZDB-PUB-180513-7

Date

2018

Source

Development (Cambridge, England) 145(12): (Journal)

Registered Authors

Da Silva, Jason, De Angelis, Jessica, Grassini, Daniela, Hogan, Ben M., Jeanes, Angela, Smith, Kelly

Keywords

ANF, Cardiac development, Cardiac jelly, Nppa, Zebrafish

MeSH Terms

Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism
Animals, Genetically Modified
Bone Morphogenetic Protein 4/genetics
Bone Morphogenetic Protein 4/metabolism
Natriuretic Peptide, Brain/genetics*
Animals
Receptors, Atrial Natriuretic Factor/genetics*
Zebrafish/embryology*
Zebrafish/genetics
T-Box Domain Proteins/metabolism
Heart/embryology*
Versicans/metabolism
Hyaluronan Synthases/metabolism
Atrial Natriuretic Factor/genetics*
Heart Defects, Congenital/genetics
Gene Editing

(all 17)

PubMed

29752386 Full text @ Development

Abstract

Atrial natriuretic peptide (nppa/anf) and brain natriuretic peptide (nppb/bnp) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy; however, their genomic location in cis has impeded formal analysis. Using genome editing, we have generated mutants for nppa and nppb, and found that single mutants were indistinguishable from wild type, whereas nppa/nppb double mutants displayed heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4, tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirmed cardiac jelly expansion in nppa/nppb double mutants. Finally, bmp4 knockdown rescued the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.

Genes / Markers

Figures

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Expression

Gene	Antibody	Fish	Conditions	Stage	Anatomy	Assay	Figure
alcama	zn-8	nppa^uq19ks/+; nppb^uq20ks/+	standard conditions	Protruding-mouth	cardiac muscle cell plasma membrane	IHC	Fig. 2
alcama	zn-8	nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}	standard conditions	Protruding-mouth	cardiac muscle cell plasma membrane	IHC	Fig. 2
alcama	zn-8	WT	standard conditions	Protruding-mouth	cardiac muscle cell plasma membrane	IHC	Fig. 2
bmp4		nppa^uq19ks/+; nppb^uq20ks/+	standard conditions	Protruding-mouth	atrioventricular canal	ISH	Fig. 2
bmp4		nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}	standard conditions	Protruding-mouth	atrioventricular canal	ISH	Fig. 2
bmp4		nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}	standard conditions	Protruding-mouth	atrium	ISH	Fig. 2
bmp4		nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}	standard conditions	Protruding-mouth	cardiac muscle cell	ISH	Fig. 2
bmp4		WT	standard conditions	Protruding-mouth	atrioventricular canal	ISH	Fig. 2
DsRed2		f2Tg	standard conditions	Protruding-mouth	cardiac muscle cell nucleus	IFL	Fig. 2
DsRed2		nppa^uq19ks/+; nppb^uq20ks/+; f2Tg	standard conditions	Protruding-mouth	cardiac muscle cell nucleus	IFL	Fig. 2

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
nppa^uq19ks/+; nppb^uq20ks/+	standard conditions	Protruding-mouth	cardiac muscle cell normal size, normal	Fig. 2
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg	standard conditions	Protruding-mouth	atrium cardiac jelly development process quality, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg	standard conditions	Protruding-mouth	atrium cardiac jelly thickness, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg	standard conditions	Protruding-mouth	cardiac ventricle cardiac jelly development process quality, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg	standard conditions	Protruding-mouth	cardiac ventricle cardiac jelly thickness, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg + MO3-bmp4	standard conditions	Protruding-mouth	atrium cardiac jelly development process quality, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg + MO3-bmp4	standard conditions	Protruding-mouth	atrium cardiac jelly thickness, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg + MO3-bmp4	standard conditions	Protruding-mouth	cardiac ventricle cardiac jelly development process quality, normal	Fig. 4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg + MO3-bmp4	standard conditions	Protruding-mouth	cardiac ventricle cardiac jelly thickness, normal	Fig. 4
nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}	standard conditions	Long-pec	heart nppb expression decreased amount, abnormal	Fig. 1

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
f2Tg	Tg(-5.1myl7:DsRed2-NLS)	Transgenic Insertion
s916Tg	Tg(kdrl:Hsa.HRAS-mCherry)	Transgenic Insertion
uq7ksTg	Tg(ubb:SEC-EGFP)	Transgenic Insertion
uq19ks		Small Deletion	nppa
uq20ks		Insertion	nppb
uq21ks		Insertion	nppb
uq25bhTg	Tg(ubb:SEC-Rno.Ncan-EGFP)	Transgenic Insertion
y7Tg	Tg(fli1:nEGFP)	Transgenic Insertion

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
bmp4	MO1-bmp4	MRPHLNO
bmp4	MO3-bmp4	MRPHLNO
nppa	TALEN1-nppa	TALEN
nppb	CRISPR1-nppb	CRISPR
tp53	MO4-tp53	MRPHLNO

Fish

Fish
f2Tg
nppa^uq19ks/+; nppb^uq20ks/+
nppa^uq19ks/+; nppb^uq20ks/+; f2Tg
nppa^uq19ks/+; nppb^uq20ks/+ + MO3-bmp4
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg
nppa^uq19ks/+; nppb^uq20ks/+; uq25bhTg + MO3-bmp4
nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}
nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}; f2Tg
nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks} + MO3-bmp4
nppa^{uq19ks/uq19ks}; nppb^{uq20ks/uq20ks}; uq25bhTg

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Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed2	EFG	DsRed2
EGFP	EFG	EGFP
mCherry	EFG	mCherry

Mapping

Grassini et al., 2018 ZDB-PUB-180513-7 PMID:29752386

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume

Grassini et al., 2018

ZDB-PUB-180513-7
PMID:29752386