PUBLICATION

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis

Authors

Kenyon, E.J., Luijten, M.N., Gill, H., Li, N., Rawlings, M., Bull, J.C., Hadzhiev, Y., van Steensel, M.A., Maher, E., Mueller, F.

ID

ZDB-PUB-160709-3

Date

2016

Source

BMC Developmental Biology 16: 23 (Journal)

Registered Authors

Hadzhiev, Yavor, Kenyon, Emma, Li, Nan

Keywords

Birt-Hogg-Dubé syndrome (BHD), Brain, Cell cycle, Folliculin, Zebrafish, flcn, zFucci

MeSH Terms

Animals
Gene Expression Regulation, Developmental
Time-Lapse Imaging
Tumor Suppressor Proteins/genetics*
Tumor Suppressor Proteins/metabolism
Cell Cycle
Retina/growth & development
Retina/metabolism
Zebrafish Proteins/genetics*
Zebrafish Proteins/metabolism
Humans
Up-Regulation
Proto-Oncogene Proteins/genetics*
Proto-Oncogene Proteins/metabolism
Gene Knockdown Techniques
Somites/growth & development
Somites/metabolism
Zebrafish/embryology*
Zebrafish/genetics
Zebrafish/metabolism
Brain/growth & development*
Brain/metabolism

(all 22)

PubMed

27391801 Full text @ BMC Dev. Biol.

Abstract

Background Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish.

Results flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development.

Conclusions In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic brain development.

Genes / Markers

Figures

Show all Figures

Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
EGFP	li1Tg	standard conditions	Long-pec	pronephric glomerulus	IFL	Fig. S1
EGFP	li1Tg	standard conditions	Long-pec	pronephric tubule	IFL	Fig. S1
EGFP	li1Tg + MO3-flcn	standard conditions	Long-pec	pronephric glomerulus	IFL	Fig. S1
EGFP	li1Tg + MO3-flcn	standard conditions	Long-pec	pronephric tubule	IFL	Fig. S1
flcn	AB	standard conditions	Long-pec	brain	ISH	Fig. 1
flcn	AB	standard conditions	Prim-5	forebrain	ISH	Fig. 1
flcn	AB	standard conditions	Prim-5	hatching gland	ISH	Fig. 1
flcn	AB	standard conditions	Prim-5	hindbrain	ISH	Fig. 1
flcn	AB	standard conditions	Long-pec	optic tectum posterior region	ISH	Fig. 1
flcn	AB	standard conditions	Prim-5	optic tectum	ISH	Fig. 1

1 - 10 of 34

Show

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB + MO3-flcn	standard conditions	14-19 somites	embryo development delayed, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Prim-5	brain cell death increased occurrence, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Prim-5	central canal motile cilium assembly process quality, normal	Fig. S1
AB + MO3-flcn	standard conditions	Prim-5	embryo development delayed, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Prim-5	post-vent region notochord bent, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Prim-5	pronephric duct motile cilium assembly process quality, normal	Fig. S1
AB + MO3-flcn	standard conditions	Prim-5	somite U-shaped, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Prim-5	trunk cell death increased occurrence, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Long-pec	ball increased size, abnormal	Fig. 2
AB + MO3-flcn	standard conditions	Long-pec	brain edematous, abnormal	Fig. 2

1 - 10 of 28

Show

Mutations / Transgenics

Allele	Construct	Type
li1Tg	Tg(wt1b:EGFP)	Transgenic Insertion
ncv3Tg	Tg(kdrl:mVenus-gmnn)	Transgenic Insertion
ncv4Tg	Tg(kdrl:mCherry-cdt1)	Transgenic Insertion

1 - 3 of 3

Show

Human Disease / Model

Human Disease	Fish	Conditions	Evidence
Birt-Hogg-Dube syndrome	AB + MO3-flcn	standard conditions	TAS

Sequence Targeting Reagents

Target	Reagent	Reagent Type
flcn	MO1-flcn	MRPHLNO
flcn	MO2-flcn	MRPHLNO
flcn	MO3-flcn	MRPHLNO

Fish

Fish
AB
AB + MO3-flcn
li1Tg
li1Tg + MO3-flcn
ncv3Tg; ncv4Tg
ncv3Tg; ncv4Tg + MO3-flcn

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
mCherry	EFG	mCherry
Venus	EFG	Venus

Mapping

Kenyon et al., 2016 ZDB-PUB-160709-3 PMID:27391801

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis

Kenyon et al., 2016

ZDB-PUB-160709-3
PMID:27391801