PUBLICATION

Quantitative proteomics identify DAB2 as a cardiac developmental regulator that inhibits WNT/β-catenin signaling

Authors

Hofsteen, P., Robitaille, A.M., Chapman, D.P., Moon, R.T., Murry, C.E.

ID

ZDB-PUB-160113-5

Date

2016

Source

Proceedings of the National Academy of Sciences of the United States of America 113(4): 1002-7 (Journal)

Registered Authors

Moon, Randall T.

Keywords

WNT/β-catenin, cardiomyocyte, embryonic stem cell, quantitative proteomics, zebrafish

MeSH Terms

Humans
beta Catenin/physiology*
Embryonic Stem Cells/cytology
Female
Myocytes, Cardiac/cytology
Cell Differentiation
Tumor Suppressor Proteins/physiology*
Intercellular Signaling Peptides and Proteins/physiology
Adaptor Proteins, Signal Transducing/physiology*
Cells, Cultured
Proteomics*
Heart/embryology*
Wnt Signaling Pathway/physiology*
Animals
Zebrafish/embryology

(all 15)

PubMed

26755607 Full text @ Proc. Natl. Acad. Sci. USA

Abstract

To reveal the molecular mechanisms involved in cardiac lineage determination and differentiation, we quantified the proteome of human embryonic stem cells (hESCs), cardiac progenitor cells (CPCs), and cardiomyocytes during a time course of directed differentiation by label-free quantitative proteomics. This approach correctly identified known stage-specific markers of cardiomyocyte differentiation, including SRY-box2 (SOX2), GATA binding protein 4 (GATA4), and myosin heavy chain 6 (MYH6). This led us to determine whether our proteomic screen could reveal previously unidentified mediators of heart development. We identified Disabled 2 (DAB2) as one of the most dynamically expressed proteins in hESCs, CPCs, and cardiomyocytes. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis in zebrafish to assess whether DAB2 plays a functional role during cardiomyocyte differentiation. We found that deletion of Dab2 in zebrafish embryos led to a significant reduction in cardiomyocyte number and increased endogenous WNT/β-catenin signaling. Furthermore, the Dab2-deficient defects in cardiomyocyte number could be suppressed by overexpression of dickkopf 1 (DKK1), an inhibitor of WNT/β-catenin signaling. Thus, inhibition of WNT/β-catenin signaling by DAB2 is essential for establishing the correct number of cardiomyocytes in the developing heart. Our work demonstrates that quantifying the proteome of human stem cells can identify previously unknown developmental regulators.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
axin2	AB	control	Prim-5	whole organism	RTPCR	Fig. 4
axin2	AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism	RTPCR	Fig. 4
dab2	AB	control	Prim-5	whole organism	RTPCR	Fig. 4
dab2	AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism	RTPCR	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+	control	Prim-5	forebrain	IFL	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+	control	Prim-5	hindbrain	IFL	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+	control	Prim-5	midbrain	IFL	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+	control	Prim-5	pronephros anterior region	IFL	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+	control	Prim-5	vasculature posterior region	IFL	Fig. 4
mCherry	ia5Tg/+; zn1Tg/+ + CRISPR1-dab2	standard conditions	Prim-5	forebrain	IFL	Fig. 4

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism axin2 expression increased amount, abnormal	Fig. 4
AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism dab2 expression decreased amount, abnormal	Fig. 4
AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism tcf3b expression increased amount, abnormal	Fig. 4
AB + CRISPR1-dab2	standard conditions	Prim-5	whole organism tcf7l1a expression increased amount, abnormal	Fig. 4
f2Tg/+ + CRISPR1-dab2	standard conditions	Long-pec	atrium cardiac muscle cell decreased amount, abnormal	Fig. 4
f2Tg/+ + CRISPR1-dab2	standard conditions	Long-pec	cardiac ventricle cardiac muscle cell decreased amount, abnormal	Fig. 4
f2Tg/+; w32Tg/+ + CRISPR1-dab2	control	Long-pec	cardiac muscle cell decreased amount, abnormal	Fig. 4
f2Tg/+; w32Tg/+ + CRISPR1-dab2	control	Long-pec	heart physical object quality, abnormal	Fig. 4
f2Tg/+; w32Tg/+ + CRISPR1-dab2	control	Long-pec	whole organism malformed, abnormal	Fig. 4
f2Tg/+; w32Tg/+ + CRISPR1-dab2	heat shock	Long-pec	cardiac muscle cell amount, ameliorated	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type
f2Tg	Tg(-5.1myl7:DsRed2-NLS)	Transgenic Insertion
ia5Tg	Tg(7xTCF-Xla.Sia:NLS-mCherry)	Transgenic Insertion
w32Tg	Tg(hsp70l:dkk1b-GFP)	Transgenic Insertion
zn1Tg	Tg(kdrl:grcfp)	Transgenic Insertion

1 - 4 of 4

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
dab2	CRISPR1-dab2	CRISPR

Fish

Fish
AB
AB + CRISPR1-dab2
f2Tg/+ + CRISPR1-dab2
f2Tg/+; w32Tg/+ + CRISPR1-dab2
ia5Tg/+; zn1Tg/+
ia5Tg/+; zn1Tg/+ + CRISPR1-dab2

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed2	EFG	DsRed2
GFP	EFG	GFP
grcfp	EFG	grcfp
mCherry	EFG	mCherry

Mapping

Hofsteen et al., 2016 ZDB-PUB-160113-5 PMID:26755607

Quantitative proteomics identify DAB2 as a cardiac developmental regulator that inhibits WNT/β-catenin signaling

Hofsteen et al., 2016

ZDB-PUB-160113-5
PMID:26755607