PUBLICATION

Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish

Authors

Navis, A., Marjoram, L., and Bagnat, M.

ID

ZDB-PUB-130403-23

Date

2013

Source

Development (Cambridge, England) 140(8): 1703-1712 (Journal)

Registered Authors

Bagnat, Michel

Keywords

Cftr, fluid secretion, Kupffer's vesicle, zebrafish

MeSH Terms

Chlorocebus aethiops
HEK293 Cells
COS Cells
In Situ Hybridization
Chromosomes, Artificial, Bacterial
Fluorescent Antibody Technique
DNA Primers/genetics
Embryo, Nonmammalian/embryology*
Embryo, Nonmammalian/physiology
Humans
Zebrafish/embryology*
Zebrafish/genetics
Cystic Fibrosis Transmembrane Conductance Regulator/genetics
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism*
Gene Expression Regulation, Developmental/genetics
Gene Expression Regulation, Developmental/physiology*
Animals
Morphogenesis/physiology*
Body Patterning/physiology*
Mutagenesis

(all 20)

PubMed

23487313 Full text @ Development

Abstract

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer’s vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
cftr	WT	standard conditions	10-13 somites	Kupffer's vesicle	ISH	Fig. 5
cftr	WT	standard conditions	1-4 somites	Kupffer's vesicle	ISH	Fig. 5
cftr	WT	standard conditions	10-13 somites	notochord	ISH	Fig. 5
DsRed	cftr^{pd1049/pd1049}; gz15Tg	standard conditions	Day 4	liver	IFL	Fig. 2
DsRed	gz15Tg	standard conditions	Day 4	liver	IFL	Fig. 2
GFP	cftr^{pd1048/pd1048}; s870Tg	standard conditions	10-13 somites	Kupffer's vesicle	IFL	Fig. 3
GFP	cftr^{pd1049/pd1049}; pd1080Tg	heat shock	1-4 somites	Kupffer's vesicle apical plasma membrane	IFL	Fig. 4
GFP	cftr^{pd1049/pd1049}; s870Tg	standard conditions	5-9 somites	Kupffer's vesicle	IHC	Fig. 4
GFP	cftr^{pd1049/pd1049}; s870Tg	standard conditions	10-13 somites	Kupffer's vesicle	IFL	Fig. 3
GFP	pd1041Tg	standard conditions	14-19 somites	Kupffer's vesicle apical plasma membrane	IFL	Fig. 6

1 - 10 of 24

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment: 3-isobutyl-1-methylxanthine, chemical treatment: forskolin	10-13 somites	Kupffer's vesicle increased area, abnormal	Fig. 7
WT	chemical treatment: 3-isobutyl-1-methylxanthine, chemical treatment: forskolin	10-13 somites	protein kinase A signaling increased occurrence, abnormal	Fig. 7
WT	chemical treatment: ouabain	10-13 somites	Kupffer's vesicle decreased area, abnormal	Fig. 7
WT	chemical treatment: ouabain	10-13 somites	P-type sodium:potassium-exchanging transporter activity decreased occurrence, abnormal	Fig. 7
cftr^{pd1048/pd1048}	standard conditions	10-13 somites	Kupffer's vesicle decreased volume, abnormal	Fig. 3
cftr^{pd1048/pd1048}	standard conditions	10-13 somites	Kupffer's vesicle development decreased process quality, abnormal	Fig. 3
cftr^{pd1048/pd1048}	standard conditions	10-13 somites	Kupffer's vesicle unlumenized, abnormal	Fig. 3
cftr^{pd1048/pd1048}; s870Tg	standard conditions	10-13 somites	Kupffer's vesicle development decreased process quality, abnormal	Fig. 3
cftr^{pd1048/pd1048}; s870Tg	standard conditions	10-13 somites	Kupffer's vesicle unlumenized, abnormal	Fig. 3
cftr^{pd1049/pd1049}	standard conditions	5-9 somites	Kupffer's vesicle cilium assembly process quality, normal	Fig. 3

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
gz15Tg	Tg(ela3l:EGFP) Tg(fabp10a:DsRed)	Transgenic Insertion
pd1041Tg	TgBAC(cftr-GFP)	Transgenic Insertion
pd1042Tg	TgBAC(cftr-RFP)	Transgenic Insertion
pd1048		Indel	cftr
pd1049		Indel	cftr
pd1050		Indel	cftr
pd1080Tg	Tg(hsp70l:GFP-podxl)	Transgenic Insertion
s870Tg	Tg(sox17:GFP)	Transgenic Insertion

1 - 8 of 8

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
cftr	TALEN1-cftr	TALEN

Fish

Fish
cftr^{pd1048/pd1048}
cftr^{pd1048/pd1048}; s870Tg
cftr^{pd1049/pd1049}
cftr^{pd1049/pd1049}; gz15Tg
cftr^{pd1049/pd1049}; pd1041Tg
cftr^{pd1049/pd1049}; pd1080Tg
cftr^{pd1049/pd1049}; s870Tg
cftr^{pd1050/pd1050}
gz15Tg
pd1041Tg

1 - 10 of 15

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Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed	EFG	DsRed
EGFP	EFG	EGFP
GFP	EFG	GFP
RFP	EFG	RFP

Mapping

Navis et al., 2013 ZDB-PUB-130403-23 PMID:23487313

Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish

Navis et al., 2013

ZDB-PUB-130403-23
PMID:23487313