PUBLICATION

Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration

Authors

Govindan, J., Tun, K.M., Iovine, M.K.

ID

ZDB-PUB-160202-7

Date

2016

Source

PLoS One 11: e0148202 (Journal)

Registered Authors

Iovine, M. Kathryn

Keywords

Cell proliferation, Morpholino, Fishes, Epithelium, Alizarin staining, In situ hybridization, Zebrafish, Growth factors

MeSH Terms

Animal Fins/drug effects
Animal Fins/physiology*
Animals
Bone and Bones/drug effects
Bone and Bones/physiology*
Connexin 43/metabolism*
Extracellular Matrix/drug effects
Extracellular Matrix/metabolism*
Extracellular Matrix Proteins/metabolism*
Gene Expression Regulation, Developmental/drug effects
Gene Knockdown Techniques
Hyaluronic Acid/metabolism
In Situ Hybridization
Models, Biological
Morpholinos/pharmacology
Nerve Growth Factors/metabolism*
Phenotype
Protein Binding/drug effects
Protein Stability/drug effects
Proteoglycans/metabolism*
Regeneration*/drug effects
Regeneration*/genetics
Semaphorins/metabolism*
Zebrafish/genetics
Zebrafish/physiology*
Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism*

(all 27)

PubMed

26828861 Full text @ PLoS One

Abstract

Skeletal development is a tightly regulated process and requires proper communication between the cells for efficient exchange of information. Analysis of fin length mutants has revealed that the gap junction protein Connexin43 (Cx43) coordinates cell proliferation (growth) and joint formation (patterning) during zebrafish caudal fin regeneration. Previous studies have shown that the extra cellular matrix (ECM) protein Hyaluronan and Proteoglycan Link Protein1a (Hapln1a) is molecularly and functionally downstream of Cx43, and that hapln1a knockdown leads to reduction of the glycosaminoglycan hyaluronan. Here we find that the proteoglycan aggrecan is similarly reduced following Hapln1a knockdown. Notably, we demonstrate that both hyaluronan and aggrecan are required for growth and patterning. Moreover, we provide evidence that the Hapln1a-ECM stabilizes the secreted growth factor Semaphorin3d (Sema3d), which has been independently shown to mediate Cx43 dependent phenotypes during regeneration. Double knockdown of hapln1a and sema3d reveal synergistic interactions. Further, hapln1a knockdown phenotypes were rescued by Sema3d overexpression. Therefore, Hapln1a maintains the composition of specific components of the ECM, which appears to be required for the stabilization of at least one growth factor, Sema3d. We propose that the Hapln1a dependent ECM provides the required conditions for Sema3d stabilization and function. Interactions between the ECM and signaling molecules are complex and our study demonstrates the requirement for components of the Hapln1a-ECM for Sema3d signal transduction.

Genes / Markers

Marker	Marker Type	Name
acana	GENE	aggrecan a
acanb	GENE	aggrecan b
gja1b	GENE	gap junction protein alpha 1b
hapln1a	GENE	hyaluronan and proteoglycan link protein 1a
has1	GENE	hyaluronan synthase 1
has2	GENE	hyaluronan synthase 2
has3	GENE	hyaluronan synthase 3
hsp70l	GENE	heat shock cognate 70-kd protein, like
sema3d	GENE	sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3D

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Figures

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Expression

Gene	Antibody	Fish	Conditions	Stage	Anatomy	Assay	Figure
acanb		AB/C32	amputation: caudal fin	Adult	blastema	ISH	Fig. 1
acanb		AB/C32	amputation: caudal fin	Adult	regenerating fin mesenchyme	ISH	Fig. 1
acanb	ab1-acan	AB/C32	amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 5
acanb		AB/C32	amputation: caudal fin	Adult	regenerating fin	ISH	Fig. 1
acanb	ab1-acan	AB/C32	amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 6
acanb	ab1-acan	AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 6
acanb	ab1-acan	AB/C32 + MO2-acanb	amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 6
acanb	ab1-acan	AB/C32 + MO2-hapln1a	amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 5
GFP		uw14Tg	heat shock, amputation: caudal fin	Adult	fin	IHC	Fig. 9
GFP		uw14Tg	heat shock, amputation: caudal fin	Adult	regenerating fin	IHC	Fig. 9

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin acanb expression decreased amount, abnormal	Fig. 6
AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin bone mineralization decreased occurrence, abnormal	Fig. 6
AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin cell population proliferation decreased occurrence, abnormal	Fig. 6
AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin decreased length, abnormal	Fig. 6
AB/C32 + MO1-acanb	amputation: caudal fin	Adult	regenerating fin lepidotrichium segment decreased length, abnormal	Fig. 6
AB/C32 + MO1-has1	amputation: caudal fin	Adult	regenerating fin cell population proliferation decreased occurrence, abnormal	Fig. 3
AB/C32 + MO1-has1 + MO4-has2	amputation: caudal fin	Adult	regenerating fin cell population proliferation decreased occurrence, abnormal	Fig. 4
AB/C32 + MO1-has1 + MO4-has2	amputation: caudal fin	Adult	regenerating fin decreased length, abnormal	Fig. 4
AB/C32 + MO1-has1 + MO4-has2	amputation: caudal fin	Adult	regenerating fin lepidotrichium segment decreased length, abnormal	Fig. 4
AB/C32 + MO2-acanb	amputation: caudal fin	Adult	regenerating fin acanb expression decreased amount, abnormal	Fig. 6

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
b123		Unknown	gja1b
uw14Tg	Tg(hsp70l:sema3d-GFP)	Transgenic Insertion

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
acanb	MO1-acanb	MRPHLNO
acanb	MO2-acanb	MRPHLNO
hapln1a	MO2-hapln1a	MRPHLNO
has1	MO2-has1	MRPHLNO
has1	MO1-has1	MRPHLNO
has2	MO4-has2	MRPHLNO
has2	MO5-has2	MRPHLNO
sema3d	MO5-sema3d	MRPHLNO

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Fish

Fish
AB/C32
AB/C32 + MO1-acanb
AB/C32 + MO1-has1
AB/C32 + MO1-has1 + MO4-has2
AB/C32 + MO2-acanb
AB/C32 + MO2-hapln1a
AB/C32 + MO2-hapln1a + MO5-sema3d
AB/C32 + MO2-has1
AB/C32 + MO4-has2
AB/C32 + MO5-has2

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Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-acan	monoclonal	acanb	IgG1	Mouse
Ab1-sema3d	polyclonal	sema3d		Rabbit

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
GFP	EFG	GFP

Mapping

Govindan et al., 2016 ZDB-PUB-160202-7 PMID:26828861

Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration

Govindan et al., 2016

ZDB-PUB-160202-7
PMID:26828861