PUBLICATION

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

Authors

Santos-Ledo, A., Garcia-Macia, M., Campbell, P.D., Gronska, M., Marlow, F.L.

ID

ZDB-PUB-170718-1

Date

2017

Source

PLoS Genetics 13: e1006918 (Journal)

Registered Authors

Campbell, Philip, Marlow, Florence

Keywords

Chondrocytes, Cartilage, Embryos, Secretion, Autophagic cell death, Phenotypes, Lysosomes, Zebrafish

MeSH Terms

Cartilage/growth & development*
Morphogenesis
Chondrocytes
Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism*
Animals
Kinesins/genetics
Kinesins/metabolism*
Cell Differentiation
Osteogenesis*
Zebrafish/embryology*
Zebrafish/genetics
Zebrafish/metabolism
Gene Expression Regulation, Developmental*

(all 14)

PubMed

28715414 Full text @ PLoS Genet.

Abstract

During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.

Errata / Notes

This article is corrected by ZDB-PUB-220906-86 .

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
col2a1a	kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Long-pec	mandibular arch skeleton chondrocyte	ISH	Fig. 1
col2a1a	WT	standard conditions	Long-pec	mandibular arch skeleton chondrocyte	ISH	Fig. 1
EGFP	ba2Tg	standard conditions	Day 6	chondrocyte	IFL	Fig. 3
EGFP	ba2Tg	standard conditions	Protruding-mouth	chondrocyte	IHC	Fig. 4
EGFP	ba2Tg	standard conditions	Days 7-13	chondrocyte	IFL	Fig. 3
EGFP	ba2Tg	standard conditions	Day 4	mandibular arch skeleton chondrocyte	IHC	Fig. 5
EGFP	ba2Tg	standard conditions	Pec-fin	mandibular arch skeleton chondrocyte	IHC	Fig. 2
EGFP	kif5ba^ae12/ae12; ba2Tg	standard conditions	Pec-fin	mandibular arch skeleton chondrocyte	IHC	Fig. 2
EGFP	kif5ba^ae12/ae12; kif5bb^ae24/ae24; ba2Tg	standard conditions	Day 6	chondrocyte	IFL	Fig. 3
EGFP	kif5ba^ae12/ae12; kif5bb^ae24/ae24; ba2Tg	standard conditions	Days 7-13	chondrocyte	IFL	Fig. 3

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment by environment: ammonium chloride	Day 4	chondrocyte ab3-map1lc3b labeling increased amount, abnormal	Fig. 5
WT	chemical treatment by environment: ammonium chloride	Day 4	whole organism ab3-map1lc3b labeling increased amount, abnormal	Fig. 5
gpc4^fr6/fr6	standard conditions	Day 5	mandibular arch skeleton chondrocyte morphology, abnormal	Fig. 4
gpc4^fr6/fr6	standard conditions	Day 5	mandibular arch skeleton chondrocyte position, abnormal	Fig. 4
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Day 5	chondrocyte extracellular matrix ab1-col2a labeling spatial pattern, abnormal	Fig. 4
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Day 5	chondrocyte secretion decreased occurrence, abnormal	Fig. 4
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Day 5	mandibular arch skeleton chondrocyte morphology, abnormal	Fig. 4
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Day 5	mandibular arch skeleton chondrocyte position, abnormal	Fig. 4
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24	standard conditions	Day 5	mandibular arch skeleton decreased size, abnormal	Fig. 4
kif5ba^ae12/ae12	standard conditions	Day 5	chondrocyte endoplasmic reticulum position, abnormal	Fig. 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
ae12		Small Deletion	kif5ba
ae21		Small Deletion	kif5bb
ae22		Small Deletion	kif5bb
ae23		Small Deletion	kif5bb
ae24		Insertion	kif5bb
ba2Tg	Tg(-4.9sox10:EGFP)	Transgenic Insertion
fr6		Point Mutation	gpc4
sq9Tg	Tg(sox10:GAL4-VP16)	Transgenic Insertion
ta98		Point Mutation	wnt5b

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
kif5bb	CRISPR1-kif5bb	CRISPR

Fish

Fish
ba2Tg
gpc4^fr6/fr6
gpc4^fr6/fr6; kif5ba^ae12/ae12; kif5bb^ae24/ae24
kif5ba^ae12/ae12
kif5ba^ae12/ae12; ba2Tg
kif5ba^ae12/ae12; kif5bb^ae24/ae24
kif5ba^ae12/ae12; kif5bb^ae24/ae24; ba2Tg
kif5ba^ae12/ae12; kif5bb^ae24/+
wnt5b^ta98/ta98
wnt5b^ta98/ta98; kif5ba^ae12/ae12; kif5bb^ae24/ae24

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Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-col2a	monoclonal		IgG1	Mouse
Ab1-tuba	monoclonal		IgG2b	Mouse
Ab1-tubg1	monoclonal		IgG1	Mouse
Ab2-mmp14	polyclonal	mmp14a	IgG	Rabbit
Ab3-map1lc3b	polyclonal	map1lc3b	IgG	Rabbit
Ab3-mtor	polyclonal			Rabbit
Ab4-mtor	polyclonal			Rabbit
Ab8-rps6	monoclonal		IgG	Rabbit
Ab13-gfap	polyclonal		IgG	Rabbit

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Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
GAL4	EFG	GAL4

Mapping

Santos-Ledo et al., 2017 ZDB-PUB-170718-1 PMID:28715414

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

Santos-Ledo et al., 2017

ZDB-PUB-170718-1
PMID:28715414