PUBLICATION

Loss of Mgat5a-mediated N-glycosylation stimulates regeneration in zebrafish.

Authors: Pei, W., Huang, S.C., Xu, L., Pettie, K., Ceci, M.L., Sánchez, M., Allende, M.L., Burgess, S.M.
ID: ZDB-PUB-161101-12
Date: 2016
Source: Cell regeneration (London, England) 5: 3 (Journal)
Registered Authors: Allende, Miguel L., Burgess, Shawn, Pei, Wuhong, Xu, Lisha
Keywords: CRISPR/Cas9, N-glycosylation, Regeneration, Zebrafish, mgat5a
MeSH Terms: none
PubMed: 27795824 Full text @ Cell Regen (Lond)

Abstract

Background We are using genetics to identify genes specifically involved in hearing regeneration. In a large-scale genetic screening, we identified mgat5a, a gene in the N-glycosylation biosynthesis pathway whose activity negatively impacts hair cell regeneration.

Methods We used a combination of mutant analysis in zebrafish and a hair cell regeneration assay to phenotype the loss of Mgat5a activity in zebrafish. We used pharmacological inhibition of N-glycosylation by swansonine. We also used over-expression analysis by mRNA injections to demonstrate how changes in N-glycosylation can alter cell signaling.

Results We found that mgat5a was expressed in multiple tissues during zebrafish embryo development, particularly enriched in neural tissues including the brain, retina, and lateral line neuromasts. An mgat5a insertional mutation and a CRISPR/Cas9-generated truncation mutation both caused an enhancement of hair cell regeneration which could be phenocopied by pharmacological inhibition with swansonine. In addition to hair cell regeneration, inhibition of the N-glycosylation pathway also enhanced the regeneration of lateral line axon and caudal fins. Further analysis showed that N-glycosylation altered the responsiveness of TGF-beta signaling.

Conclusions The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.

Genes / Markers

Figures

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Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment by diet: 1-DEOXYNOJIRIMYCIN, chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell differentiation involved in neuromast regeneration increased process quality, abnormal	Fig. 5
WT	chemical treatment by diet: swainsonine, chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell differentiation involved in neuromast regeneration increased process quality, abnormal	Fig. 5
WT	chemical treatment by diet: DAPT, chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell differentiation involved in neuromast regeneration increased process quality, abnormal	Fig. 7
WT	chemical treatment by diet: DAPT, amputation: caudal fin	Day 6	caudal fin fin regeneration decreased process quality, abnormal	Fig. 7
WT	chemical treatment by diet: swainsonine, amputation: caudal fin	Day 6	caudal fin fin regeneration increased process quality, abnormal	Fig. 6
gl23Tg; nl1Tg	surgical manipulation: posterior lateral line nerve, chemical treatment by diet: swainsonine	Day 4	lateral line nerve neural tissue regeneration increased process quality, abnormal	Fig. 6
mgat5^hg29/hg29	chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell differentiation involved in neuromast regeneration increased process quality, abnormal	Fig. 4
mgat5^{mn0157Gt/mn0157Gt}	chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell differentiation involved in neuromast regeneration increased process quality, abnormal	Fig. 1
mgat5^{mn0157Gt/mn0157Gt}; tnks1bp1^y229Gt	chemical ablation: neuromast hair cell	Days 7-13	neuromast hair cell mRFP1 expression decreased amount, abnormal	Fig. 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
gl23Tg	Tg(mpeg1:mCherry)	Transgenic Insertion
hg29		Small Deletion	mgat5
mn0157Gt	Gt(GBT-rp2)	Transgenic Insertion	mgat5
nl1Tg	TgBAC(neurod1:EGFP)	Transgenic Insertion
sqet20Et	Et(krt4:EGFP)	Transgenic Insertion
y229Gt	Gt(T2KSAG)	Transgenic Insertion	tnks1bp1

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
fut8a	CRISPR1-fut8a	CRISPR
fut8a	CRISPR2-fut8a	CRISPR
man1a1	CRISPR2-man1a1	CRISPR
man1a1	CRISPR3-man1a1	CRISPR
man1b1a	CRISPR1-man1b1a	CRISPR
man1b1a	CRISPR2-man1b1a	CRISPR
man2a1	CRISPR2-man2a1	CRISPR
man2a1	CRISPR3-man2a1	CRISPR
man2b1	CRISPR1-man2b1	CRISPR
man2b1	CRISPR3-man2b1	CRISPR

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Fish

Fish
gl23Tg; nl1Tg
mgat5^hg29/hg29
mgat5^{mn0157Gt/mn0157Gt}
mgat5^{mn0157Gt/mn0157Gt}; sqet20Et
mgat5^{mn0157Gt/mn0157Gt}; tnks1bp1^y229Gt
WT

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
GFP	EFG	GFP
mCherry	EFG	mCherry
mRFP1	EFG	mRFP1

Mapping

Pei et al., 2016 ZDB-PUB-161101-12 PMID:27795824

Loss of Mgat5a-mediated N-glycosylation stimulates regeneration in zebrafish.

Pei et al., 2016

ZDB-PUB-161101-12
PMID:27795824