PUBLICATION

Notochord vacuoles absorb compressive bone growth during zebrafish spine formation

Authors

Bagwell, J., Norman, J., Ellis, K.L., Peskin, B., Hwang, J., Ge, X., Nguyen, S., McMenamin, S.K., Stainier, D.Y., Bagnat, M.

ID

ZDB-PUB-200130-13

Date

2020

Source

eLIFE 9: (Journal)

Registered Authors

Bagnat, Michel, McMenamin, Sarah, Stainier, Didier

Keywords

cell biology, developmental biology, zebrafish

MeSH Terms

Notochord/metabolism*
Zebrafish/embryology*
Mutation
Receptor-Interacting Protein Serine-Threonine Kinases/genetics
Zebrafish Proteins/genetics
Animals
Spine/growth & development*
Gene Expression Regulation, Developmental
Vacuoles/metabolism*

(all 9)

PubMed

31995030 Full text @ Elife

Abstract

The vertebral column or spine assembles around the notochord rod which contains a core made of large vacuolated cells. Each vacuolated cell possesses a single ﬂuid-ﬁlled vacuole, and loss or fragmentation of these vacuoles in zebrafish leads to spine kinking. Here, we identified a mutation in the kinase gene dstyk that causes fragmentation of notochord vacuoles and a severe congenital scoliosis-like phenotype in zebrafish. Live imaging revealed that Dstyk regulates fusion of membranes with the vacuole. We find that localized disruption of notochord vacuoles causes vertebral malformation and curving of the spine axis at those sites. Accordingly, in dstyk mutants the spine curves increasingly over time as vertebral bone formation compresses the notochord asymmetrically, causing vertebral malformations and kinking of the axis. Together, our data show that notochord vacuoles function as a hydrostatic scaffold that guides symmetrical growth of vertebrae and spine formation.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Qualifier	Anatomy	Assay	Figure
dstyk	AB/TL	standard conditions	Prim-5		notochord posterior region	ISH	Fig. 3
dstyk	AB/TL	standard conditions	20-25 somites		notochord posterior region	ISH	Fig. 3
dstyk	AB/TL	standard conditions	10-13 somites		notochord	ISH	Fig. 3
dstyk	AB/TL	standard conditions	1-cell		whole organism	ISH	Fig. 3
EGFP	dstyk^s739/s739; mu286Tg; pd1083Tg	standard conditions	Long-pec		notochord inner cell vacuolar membrane	IFL	Fig. 5
EGFP	dstyk^s739/s739; mu286Tg; pd1083Tg	standard conditions	Pec-fin		notochord inner cell vacuolar membrane	IFL	Fig. 5
EGFP	dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg	standard conditions	Days 14-20		notochord inner cell cytoplasm	IFL	Fig. 8
EGFP	dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg	standard conditions	Days 14-20	Not Detected	notochord inner cell vacuolar lumen	IFL	Fig. 8
EGFP	dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg; pd46Tg	standard conditions	Days 21-29		notochord inner cell cytoplasm	IFL	Fig. 7
EGFP	dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg; pd46Tg	standard conditions	Days 14-20		notochord inner cell cytoplasm	IFL	Fig. 7

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
c264Tg; mu286Tg	chemical treatment by environment: all-trans-retinoic acid	Days 14-20	centrum bone mineralization increased process quality, abnormal	Fig. 11
c264Tg; mu286Tg	chemical treatment by environment: all-trans-retinoic acid	Days 14-20	notochord deformed, abnormal	Fig. 11
c264Tg; mu286Tg	chemical treatment by environment: all-trans-retinoic acid	Days 14-20	notochord inner cell irregular spatial pattern, abnormal	Fig. 11
c264Tg; mu286Tg	chemical treatment by environment: all-trans-retinoic acid	Days 14-20	notochord inner cell vacuole broken, abnormal	Fig. 11
dstyk^{pd1133/pd1133}	standard conditions	Long-pec	notochord decreased length, abnormal	Fig. 3
dstyk^{pd1133/pd1133}	standard conditions	Long-pec	notochord inner cell notochord cell vacuolation decreased process quality, abnormal	Fig. 3
dstyk^{pd1133/pd1133}	standard conditions	Long-pec	notochord inner cell vacuole broken, abnormal	Fig. 3
dstyk^s739/+; dstyk^pd1133/+	standard conditions	Long-pec	notochord decreased length, abnormal	Fig. 3
dstyk^s739/+; dstyk^pd1133/+	standard conditions	Long-pec	notochord inner cell notochord cell vacuolation decreased process quality, abnormal	Fig. 3
dstyk^s739/+; dstyk^pd1133/+	standard conditions	Long-pec	notochord inner cell vacuole broken, abnormal	Fig. 3

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
c264Tg	Tg(UAS-E1B:NTR-mCherry)	Transgenic Insertion
hi2499aTg	Tg(nLacz-GTvirus)	Transgenic Insertion	vps18
hu3718		Point Mutation	entpd5a
hu7478Tg	TgBAC(entpd5a:KillerRed)	Transgenic Insertion
mu286Tg	TgBAC(nfatc1:GAL4FF)	Transgenic Insertion
nksagff214aGt	Gt(GAL4FF)	Transgenic Insertion
nkuasgfp1aTg	Tg(5xUAS:EGFP)	Transgenic Insertion
pd46Tg	Tg(Ola.Sp7:mCherry-Eco.NfsB)	Transgenic Insertion
pd115Tg	Tg(actb2:LOXP-TagBFP-LOXP-STOP-LOXP-gata4)	Transgenic Insertion
pd1023Tg	Tg(rcn3:GAL4)	Transgenic Insertion

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
dstyk	CRISPR1-dstyk	CRISPR
dstyk	CRISPR2-dstyk	CRISPR

Fish

Fish
AB/TL
c264Tg; mu286Tg
dstyk^{pd1133/pd1133}
dstyk^s739/s739
dstyk^s739/s739; mu286Tg; pd1083Tg
dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg
dstyk^s739/s739; nksagff214aGt; nkuasgfp1aTg; pd46Tg
dstyk^s739/+; dstyk^pd1133/+
entpd5a^{hu3718/hu3718}
mu286Tg; pd1083Tg

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Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
GAL4	EFG	GAL4
GAL4FF	EFG	GAL4FF
GFP	EFG	GFP
KillerRed	EFG	KillerRed
mCherry	EFG	mCherry
NTR	EFG	NTR
TagBFP	EFG	TagBFP

Mapping

Bagwell et al., 2020 ZDB-PUB-200130-13 PMID:31995030

Notochord vacuoles absorb compressive bone growth during zebrafish spine formation

Bagwell et al., 2020

ZDB-PUB-200130-13
PMID:31995030