PUBLICATION

Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch.

Authors

Sugimoto, K., Hui, S.P., Sheng, D.Z., Kikuchi, K.

ID

ZDB-PUB-170518-2

Date

2017

Source

eLIFE 6: (Journal)

Registered Authors

Hui, Subhra Prakash, Kikuchi, Kazu, Sheng, Delicia

Keywords

cardiomyocyte, developmental biology, epicardium, heart development, heart regeneration, knockout animals, stem cells, zebrafish

MeSH Terms

Hedgehog Proteins/genetics*
Hedgehog Proteins/metabolism*
Zebrafish Proteins/genetics*
Zebrafish Proteins/metabolism*
Heart/embryology*
Gene Targeting*
Animals
Recombination, Genetic
Morphogenesis
Zebrafish/embryology*
Zebrafish/genetics*

PubMed

28513431 Full text @ Elife

Abstract

Despite the extensive use of zebrafish as a model organism in developmental biology and regeneration research, genetic techniques enabling conditional analysis of gene function are limited. In this study, we generated Zwitch, a Cre-dependent invertible gene-trap cassette, enabling the establishment of conditional alleles in zebrafish by generating intronic insertions via in vivo homologous recombination. To demonstrate the utility of Zwitch, we generated a conditional sonic hedgehog a (shha) allele. Homozygous shha mutants developed normally; however, shha mutant embryos globally expressing Cre exhibited strong reductions in endogenous shha and shha target gene mRNA levels and developmental defects associated with null shha mutations. Analyzing a conditional shha mutant generated using an epicardium-specific inducible Cre driver revealed unique roles for epicardium-derived Shha in myocardial proliferation during heart development and regeneration. Zwitch will extend the utility of zebrafish in organ development and regeneration research and might be applicable to other model organisms.

Genes / Markers

Figures

Show all Figures

Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
pd37Tg/+ (EKW)	amputation: cardiac ventricle	Adult	epicardium aldh1a2 expression increased amount, abnormal	Fig. 5
pd37Tg/+ (EKW)	amputation: cardiac ventricle	Adult	epicardium dhh expression increased amount, abnormal	Fig. 5
pd37Tg/+ (EKW)	amputation: cardiac ventricle	Adult	epicardium ihhb expression increased amount, abnormal	Fig. 5
pd37Tg/+ (EKW)	amputation: cardiac ventricle	Adult	epicardium shha expression increased amount, abnormal	Fig. 5
shha^vcc8Gt/+; vcc9Tg/+ (EKW)	control	Protruding-mouth	whole organism shha expression decreased amount, abnormal	Fig. 3 S2
shha^{vcc8Gt/vcc8Gt}; pd37Tg/+; pd42Tg/+ (EKW)	chemical treatment by environment: tamoxifen	Day 4	epicardium aldh1a2 expression absent, abnormal	Fig. 4
shha^{vcc8Gt/vcc8Gt}; pd37Tg/+; pd42Tg/+ (EKW)	amputation: cardiac ventricle, chemical treatment by environment: tamoxifen	Adult	epicardium shha expression absent, abnormal	Fig. 5
shha^{vcc8Gt/vcc8Gt}; pd42Tg/+ (EKW)	chemical treatment by environment: tamoxifen	Day 4	cardiac muscle cell decreased amount, abnormal	Fig. 4
shha^{vcc8Gt/vcc8Gt}; pd42Tg/+ (EKW)	chemical treatment by environment: tamoxifen	Day 4	heart aldh1a2 expression decreased amount, abnormal	Fig. 4
shha^{vcc8Gt/vcc8Gt}; pd42Tg/+ (EKW)	chemical treatment by environment: tamoxifen	Day 4	heart fgf16 expression decreased amount, abnormal	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
ae1Tg	Tg(-14.8gata4:GFP)	Transgenic Insertion
f1Tg	Tg(myl7:GFP)	Transgenic Insertion
pd15Tg	Tg(-5.1myl7:DsRed2)	Transgenic Insertion
pd25Tg	TgBAC(gata5:EGFP)	Transgenic Insertion
pd37Tg	TgBAC(tcf21:DsRed2)	Transgenic Insertion
pd42Tg	TgBAC(cryaa:EGFP,tcf21:Cre-ERT2)	Transgenic Insertion
vcc8Gt	Gt(FRT-cryaa:EGFP-FRT,LOXP-LOX5171-2A-TagRFP-LOXP-LOX5171)	Transgenic Insertion	shha
vcc9Tg	Tg(ubb:iCre-EGFP)	Transgenic Insertion

1 - 8 of 8

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
shha	TALEN1-shha	TALEN

Fish

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
Cre	EFG	Cre
DsRed2	EFG	DsRed2
EGFP	EFG	EGFP
GFP	EFG	GFP
TagRFP	EFG	TagRFP

Mapping

Sugimoto et al., 2017 ZDB-PUB-170518-2 PMID:28513431

Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch.

Sugimoto et al., 2017

ZDB-PUB-170518-2
PMID:28513431