PUBLICATION

Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway

Authors

Sun, X., Zhou, Y., Zhang, R., Wang, Z., Xu, M., Zhang, D., Huang, J., Luo, F., Li, F., Ni, Z., Zhou, S., Chen, H., Chen, S., Chen, L., Du, X., Chen, B., Huang, H., Liu, P., Yin, L., Qiu, J., Chen, D., Deng, C., Xie, Y., Luo, L., Chen, L.

ID

ZDB-PUB-200128-2

Date

2020

Source

Nature communications 11: 479 (Journal)

Registered Authors

Chen, Lin, Li, Fangfang, Luo, Lingfei, Qiu, Juhui

Keywords

none

MeSH Terms

Active Transport, Cell Nucleus
Animals
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism
Disease Models, Animal
Gene Knockdown Techniques
Humans
Models, Biological
Mutation
Notochord/abnormalities
Notochord/metabolism
Notochord/ultrastructure
Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors
Receptor-Interacting Protein Serine-Threonine Kinases/genetics*
Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
Scoliosis/congenital
Scoliosis/genetics*
Scoliosis/metabolism
Signal Transduction
Spine/abnormalities
Spine/metabolism
Transcription Factors/metabolism
Vacuoles/metabolism
Zebrafish/abnormalities*
Zebrafish/genetics*
Zebrafish/metabolism
Zebrafish Proteins/antagonists & inhibitors
Zebrafish Proteins/genetics*
Zebrafish Proteins/metabolism

(all 28)

PubMed

31980602 Full text @ Nat. Commun.

Abstract

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.

Genes / Markers

Marker	Marker Type	Name
actb1	GENE	actin, beta 1
atp6v0e1	GENE	ATPase H+ transporting V0 subunit e1
clcn7	GENE	chloride channel 7
cmn	GENE	calymmin
col11a2	GENE	collagen, type XI, alpha 2
col2a1a	GENE	collagen, type II, alpha 1a
col8a1a	GENE	collagen, type VIII, alpha 1a
col9a1b	GENE	collagen, type IX, alpha 1b
cyb5r2	GENE	cytochrome b5 reductase 2
dstyk	GENE	dual serine/threonine and tyrosine protein kinase

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Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
atp6v0e1	AB	standard conditions	Prim-25	whole organism	RTPCR	Fig. S7
atp6v0e1	dstyk^cq75/cq75 (AB)	standard conditions	Prim-25	whole organism	RTPCR	Fig. S7
clcn7	AB	standard conditions	Prim-25	whole organism	RTPCR	Fig. S7
clcn7	dstyk^cq75/cq75 (AB)	standard conditions	Prim-25	whole organism	RTPCR	Fig. S7
cmn	AB	standard conditions	Prim-5	notochord	ISH	Fig. 5
cmn	dstyk^cq75/cq75 (AB)	standard conditions	Prim-5	notochord	ISH	Fig. 5
col11a2	AB	standard conditions	Prim-25	notochord	ISH	Fig. 5
col11a2	dstyk^cq75/cq75 (AB)	standard conditions	Prim-25	notochord	ISH	Fig. 5
col8a1a	AB	standard conditions	Prim-25	notochord	ISH	Fig. 5
col8a1a	dstyk^cq75/cq75 (AB)	standard conditions	Prim-25	notochord	ISH	Fig. 5

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
cq76Tg (AB)	standard conditions	Prim-25	notochord ab8-rps6 labeling increased amount, abnormal	Fig. S7
dstyk^cq75/cq75 (AB)	control	Long-pec	notochord decreased width, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Long-pec	notochord vacuole decreased area, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Day 6	whole organism decreased length, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 7-13	notochord vacuole decreased area, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 7-13	whole organism decreased length, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 30-44	centrum bone mineralization decreased process quality, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 30-44	vertebral column increased curvature, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 45-89	vertebral column increased curvature, abnormal	Fig. 9
dstyk^cq75/cq75 (AB)	control	Days 45-89	vertebral column kinked, abnormal	Fig. 9

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
cq75		Complex	dstyk
cq76Tg	Tg(actb1:ras-GFP)	Transgenic Insertion
cq77Tg	Tg(-1.7col2a1a:EGFP)	Transgenic Insertion
cq78Tg	Tg(hsp70l:dstyk-GFP)	Transgenic Insertion
cq79Tg	Tg(hsp70l:Hsa.DSTYK-GFP)	Transgenic Insertion
cq80Tg	Tg(hsp70l:Hsa.LAMP1-mCherry)	Transgenic Insertion
cq81Tg	TgBAC(cyb5r2:GFP)	Transgenic Insertion
pd43Tg	Tg2(Ola.Sp7:mCherry)	Transgenic Insertion
zf3306		Small Deletion	dstyk
zf3307		Indel	dstyk

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
dstyk	CRISPR3-dstyk	CRISPR
mtor	MO1-mtor	MRPHLNO

Fish

Fish
AB
cq76Tg (AB)
cq77Tg (AB)
cq77Tg; pd43Tg (AB)
cq78Tg; cq80Tg (AB)
cq79Tg; cq80Tg (AB)
cq81Tg (AB)
dstyk^cq75/cq75 (AB)
dstyk^cq75/cq75; cq76Tg (AB)
dstyk^cq75/cq75; cq77Tg (AB)

Antibodies

Name	Type	Isotypes	Host Organism
Ab1-col2a1			Mouse
Ab1-lamp3	polyclonal	IgG	Rabbit
Ab1-rab7a	polyclonal	IgG	Rabbit
Ab2-tfeb	polyclonal	IgG	Rabbit
Ab3-lamp1	monoclonal	IgG1	Mouse
Ab5-rps6	monoclonal	IgG	Rabbit
Ab7-mtor	monoclonal	IgG	Rabbit
Ab7-rps6k	monoclonal		Rabbit
Ab8-rps6	monoclonal	IgG	Rabbit
Ab11-rps6k	polyclonal		Rabbit

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Orthology

Engineered Foreign Genes

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

Mapping

Sun et al., 2020 ZDB-PUB-200128-2 PMID:31980602

Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway

Sun et al., 2020

ZDB-PUB-200128-2
PMID:31980602