PUBLICATION

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates

Authors

Ben, J., Elworthy, S., Ng, A.S., van Eeden, F., and Ingham, P.W.

ID

ZDB-PUB-111114-3

Date

2011

Source

Development (Cambridge, England) 138(22): 4969-4978 (Journal)

Registered Authors

Ben, Jin, Elworthy, Stone, Ingham, Philip, van Eeden, Freek

Keywords

Talpid3, zebrafish, primary cilium, cystic kidney, hedgehog signalling, Gli2 processing, ciliopathy

MeSH Terms

Animals, Genetically Modified
Female
Cilia/genetics*
Cilia/physiology
Zebrafish/embryology
Zebrafish/genetics*
Zebrafish/physiology
Chickens/genetics
Signal Transduction/genetics
Signal Transduction/physiology
Male
Organogenesis/genetics
Sequence Homology, Amino Acid
Zebrafish Proteins/genetics
Zebrafish Proteins/physiology*
Base Sequence
Vertebrates/embryology
Vertebrates/genetics*
Animals
Embryo, Nonmammalian
Molecular Sequence Data
Amino Acid Sequence
Hedgehog Proteins/genetics*
Hedgehog Proteins/metabolism
Hedgehog Proteins/physiology
Mutagenesis, Site-Directed
Conserved Sequence/physiology

(all 27)

PubMed

22028029 Full text @ Development

Abstract

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer’s vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.

Genes / Markers

Figures

Show all Figures

Expression

Gene	Antibody	Fish	Conditions	Stage	Anatomy	Assay	Figure
EGFP		si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; i233Tg	standard conditions	Prim-5	fast muscle cell	IFL	Fig. 4
EGFP		si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; i233Tg	standard conditions	Prim-5	slow muscle cell	IFL	Fig. 4
EGFP		si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; y1Tg	standard conditions	Long-pec	brain vasculature blood vessel endothelium	IFL	Fig. 5
EGFP		si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; i233Tg	standard conditions	Prim-5	slow muscle cell	IFL	Fig. 4
EGFP		si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; y1Tg	standard conditions	Long-pec	brain vasculature blood vessel endothelium	IFL	Fig. 5
fabp1a		WT	standard conditions	Long-pec	liver	ISH	Fig. 7
gli2a	ab1-gli2a	ptch1^tj222/tj222; ptch2^{hu1602/hu1602}	standard conditions	Prim-5	whole organism	WB	Fig. 8
gli2a	ab1-gli2a	si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	Prim-5	whole organism	WB	Fig. 8
gli2a	ab1-gli2a	si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	Prim-5	whole organism	WB	Fig. 8
gli2a	ab1-gli2a	WT	standard conditions	Prim-5	whole organism	WB	Fig. 8

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
ptch1^tj222/tj222; ptch2^{hu1602/hu1602}	standard conditions	Prim-5	smoothened signaling pathway process quality, abnormal	Fig. 8
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	1-4 somites to 5-9 somites	cilium assembly decreased process quality, abnormal	Fig. 7
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	1-4 somites to 5-9 somites	Kupffer's vesicle motile cilium absent, abnormal	Fig. 7
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	10-13 somites	cilium assembly decreased process quality, abnormal	Fig. 6
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	10-13 somites	neural tube cilium absent, abnormal	Fig. 6
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	14-19 somites	cilium assembly decreased process quality, abnormal	Fig. 6
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	14-19 somites	otic vesicle cilium absent, abnormal	Fig. 6
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	Prim-5	cilium assembly decreased process quality, abnormal	Fig. 6
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	Prim-5	lens mislocalised, abnormal	Fig. 4
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+	standard conditions	Prim-5	muscle pioneer decreased amount, abnormal	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
hu1602		Point Mutation	ptch2
i233Tg	Tg(-10en2a:EGFP)	Transgenic Insertion
i262		Indel	si:ch211-185a18.2
i263		Insertion	si:ch211-185a18.2
i264		Insertion	si:ch211-185a18.2
i265		Insertion	si:ch211-185a18.2
i266		Small Deletion	si:ch211-185a18.2
i267		Small Deletion	si:ch211-185a18.2
i268		Small Deletion	si:ch211-185a18.2
tj222		Point Mutation	ptch1

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
dnd1	MO1-dnd1	MRPHLNO
si:ch211-185a18.2	MO1-si:ch211-185a18.2	MRPHLNO
si:ch211-185a18.2	MO2-si:ch211-185a18.2	MRPHLNO
si:ch211-185a18.2	MO3-si:ch211-185a18.2	MRPHLNO

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Fish

Fish
dzip1^{ts294e/ts294e}
i233Tg
ptch1^tj222/tj222
ptch1^tj222/tj222; ptch2^{hu1602/hu1602}
ptch2^{hu1602/hu1602}
si:ch211-185a18.2^i263/+
si:ch211-185a18.2^i264/+; si:ch211-185a18.2^i265/+; si:ch211-185a18.2ⁱ²⁶²
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; i233Tg
si:ch211-185a18.2^i264/+; si:ch211-185a18.2ⁱ²⁶⁵; si:ch211-185a18.2^i262/+; y1Tg

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Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-eng	monoclonal	en1a en1b en2a en2b	IgG1	Mouse
Ab1-gli2a	polyclonal	gli2a		Rabbit
Ab1-tuba	monoclonal		IgG2b	Mouse
Ab1-tubg1	monoclonal		IgG1	Mouse
Ab2-Prox1	polyclonal	prox1a		Rabbit

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Orthology

Gene	Orthology
si:ch211-185a18.2

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP

Mapping

Ben et al., 2011 ZDB-PUB-111114-3 PMID:22028029

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates

Ben et al., 2011

ZDB-PUB-111114-3
PMID:22028029