PUBLICATION

Identification and requirements of enhancers that direct gene expression during zebrafish fin regeneration

Authors

Thompson, J.D., Ou, J., Lee, N., Shin, K., Cigliola, V., Song, L., Crawford, G.E., Kang, J., Poss, K.D.

ID

ZDB-PUB-200716-2

Date

2020

Source

Development (Cambridge, England) 147(14): (Journal)

Registered Authors

Cigliola, Valentina, Kang, Junsu, Lee, Nutishia, Ou, Jianhong, Poss, Kenneth D., Thompson, John D.

Keywords

Chromatin, Enhancers, Fin regeneration, Genome-wide profiling, Regeneration, Zebrafish

Datasets

GEO:GSE146960

MeSH Terms

Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism
Animals, Genetically Modified/metabolism
Regulatory Sequences, Nucleic Acid/genetics
Enhancer Elements, Genetic/genetics*
Chromatin/metabolism
Zebrafish/metabolism*
Connexin 43/genetics
Connexin 43/metabolism
Animal Fins/metabolism*
Animal Fins/physiology
Regeneration/physiology*
Animals
Midkine/genetics
Midkine/metabolism
Fibroblast Growth Factors/genetics
Fibroblast Growth Factors/metabolism
Chromatin Assembly and Disassembly
Gene Expression
Fibroblasts/cytology
Fibroblasts/metabolism
Leptin/genetics
Leptin/metabolism

(all 23)

PubMed

32665240 Full text @ Development

Abstract

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka, and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka, and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Qualifier	Anatomy	Assay	Figure
EGFP	nkhgn21aEt	standard conditions	Day 5		brain	IFL	Fig. 2
EGFP	nkhgn21aEt	standard conditions	Adult		caudal fin distal margin	IFL	Fig. 2
EGFP	nkhgn21aEt	amputation: caudal fin	Adult		caudal fin regeneration epithelium	IFL	Fig. 2
EGFP	nkhgn21aEt	standard conditions	Day 5		trunk musculature	IFL	Fig. 2
EGFP	pd320Tg	standard conditions	Day 5		brain	IFL	Fig. 2
EGFP	pd320Tg	standard conditions	Adult	Not Detected	caudal fin distal margin	IFL	Fig. 2
EGFP	pd320Tg	amputation: caudal fin	Adult		caudal fin regeneration epithelium	IFL	Fig. 2
EGFP	pd321Tg	standard conditions	Day 5		brain	IFL	Fig. 3
EGFP	pd321Tg	amputation: caudal fin	Adult		caudal fin regeneration epithelium	IFL	Fig. 3
EGFP	pd321Tg	standard conditions	Day 5		lens	IFL	Fig. 3

1 - 10 of 30

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
en.gja1be24^pd326/pd326	amputation: caudal fin	Adult	caudal fin gja1b expression decreased amount, abnormal	Fig. 5
en.gja1be24^pd326/pd326	amputation: caudal fin	Adult	caudal fin normal length, normal	Fig. 5
en.gja1be24^pd326/pd326	amputation: caudal fin	Adult	caudal fin principal ray decreased length, abnormal	Fig. 5
mdka^pd324/pd324	amputation: caudal fin	Adult	fin regeneration delayed, abnormal	Fig. 3

1 - 4 of 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
cz1701Tg	Tg(-3.5ubb:LOXP-EGFP-LOXP-mCherry)	Transgenic Insertion
nkhgn21aEt	Et(T2KHG)	Transgenic Insertion
pd250Tg	Tg(tph1b:Cre-ERT2,cryaa:EGFP)	Transgenic Insertion
pd281		Indel	en.len
pd320Tg	Tg(en.fgf20ae48-Mmu.Fos:EGFP)	Transgenic Insertion
pd321Tg	Tg(en.mdkae20-Mmu.Fos:EGFP)	Transgenic Insertion
pd322Tg	Tg(en.gja1be24-Mmu.Fos:EGFP)	Transgenic Insertion
pd323		Small Deletion	en.fgf20ae48
pd324		Small Deletion	mdka
pd325		Small Deletion	en.mdkae20

1 - 10 of 11

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
en.fgf20ae48	CRISPR1-en.fgf20ae48	CRISPR
en.fgf20ae48	CRISPR2-en.fgf20ae48	CRISPR
en.gja1be24	CRISPR1-en.gja1be24	CRISPR
en.gja1be24	CRISPR2-en.gja1be24	CRISPR
en.len	CRISPR1-en.len	CRISPR
en.len	CRISPR2-en.len	CRISPR
en.mdkae20	CRISPR1-en.mdkae20	CRISPR
en.mdkae20	CRISPR2-en.mdkae20	CRISPR
mdka	CRISPR7-mdka	CRISPR
mdka	CRISPR8-mdka	CRISPR

1 - 10 of 10

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Fish

Fish
en.fgf20ae48^pd323/pd323
en.gja1be24^pd326/pd326
mdka^pd324/pd324
nkhgn21aEt
pd320Tg
pd321Tg
pd322Tg
WT

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
Cre	EFG	Cre
EGFP	EFG	EGFP
mCherry	EFG	mCherry

Mapping

Thompson et al., 2020 ZDB-PUB-200716-2 PMID:32665240

Identification and requirements of enhancers that direct gene expression during zebrafish fin regeneration

Thompson et al., 2020

ZDB-PUB-200716-2
PMID:32665240