The role of Foxc1 during the differentiation of embryonic stem cells into cardiomyocytes

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons Dyrk1a Intro

The Role of Foxc1 During the Differen=a=on of Embryonic Stem Cells into

Cardiomyocytes

Monday December 1, 2014 CommiMee Mee=ng

Erin Lambers

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons Background

Cardiovascular Disease is the #1 Cause of Death Worldwide

World Health Organiza=on

2011:

17 million people died from cardiovascular

disease

U.S. Healthcare System $300 billion annually

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons

Myocyte Loss and Dysfunc=on

Background

Myocardial Infarc=on

Chronic hypertension

Aging

Embryonic Stem Cell Differen=a=on

Endothelial Fibroblast

Func?onal Cardiomyocytes

Molecular Circuitry ?

Role of Foxc1 in Heart Development

Smooth Muscle


FoxC1 muta=ons causes heart malforma=ons

Chr. 6 (6p25) Axenfeld-‐Rieger Anomaly:

Eye Malforma?ons: -‐Displacement of the pupil and -‐Hypoplasia of the iris

Palmer et al, 1992 Winnier et al, 1998

“Holes in the Heart”

Heart Malforma?ons: -‐Hypoplasia of Atrial and Ventricular Septums

Background


Foxc1 Muta=ons in Mice also causes heart malforma=ons

Wildtype Foxc1 Mutant Ventricular Septal

Defect

Reduced thickness

myocardium

Background

Winnier 1999


•  “Forkhead Box” Transcrip=on Factors

•  All contain Winged Helix Domains

FOXC1

Foxc1 structure and func=on

•  Core Consensus

Binding Site

Background

Foxc1 Targets in the heart?


Specifica=on of Cardiac Cell Lineages in Development Foxc1 Targets in the Second Heart Field

Cardiac''Mesoderm'

FHF'Progenitor' SHF'Progenitor'

Mesp1+'Flk1+'

Tbx5+'Nkx2.5+'Mef2c+'Gata4+'

Isl1+'Tbx1+'Nkx2.5+'Mef2c+'Gata4+'

Mesoderm'Brachyury+'

A'

B' E7.0'

Cardiac'Crescent'

LV'myocytes''

Atrial'myocytes''

ConducNon'cells''

E9.0'

Cardiac'Looping'

Adult'Heart'

Right'ventricular'myocytes'Atrial'myocytes'

RConducNon'Cells'

''RVascular'SMCs'

REndothelial'Cells'

FHF'

SHF' RV' LV' RA' LV'

RV'

LA'OFT'

Huansheng Xu, 2004

TBX1 +/+

TBX1 -‐/-‐

Olson Nature Review 2013

Seo, 2006 Developmental Biology

Background

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons Background

Summary of what is known and unknown

What is known: •  FoxC1 muta=ons are associated with heart malforma=ons in humans

and mice •  FoxC1 is known to directly regulate Tbx1, necessary for forma=on of

the OFT, which is restricted to the SHF •  FoxC1 mutants have more severe heart malforma=ons than TBX1

mutants including hypoplasia of both ventricles •  FoxC1 expression extends across both heart fields as early as the

cardiac crescent stage E7.5 What is unknown: •  Other transcrip=onal direct targets of Foxc1 during heart

development that could explain defects in both heart fields •  The specific cell types in the heart in which Foxc1 func=ons during

early cardiac development

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons Hypothesis

Central Hypothesis

Foxc1 is cri=cal for ESC differen=a=on in the func=onal cardiomyocytes through the direct regula=on of downstream gene networks

Func?onal Cardiomyocytes


Specific Aims

Hypothesis

Test the central hypothesis with 3 specific aims:

Specific Aim 1: To determine if Foxc1 is necessary for the differen=a=on of ESCs into func=onal cardiomyocyte

Specific Aim 3: To determine the gene targets that are directly regulated by Foxc1 during cardiac differen=a=on

Specific Aim 2: To determine if Foxc1 is sufficient to increase ESC differen=a=on into func=onal cardiomyocytes


Hanging Drop 48hrs 5,000 cell/ 20ul

8 day differen=a=on

.1% Gela=n coated plates

D0 D2 D4 D6 D8

ESC as a model to study cardiac differen=a=on: Hanging Drop method of Embryoid Body Forma=on

Assess the =ming of Foxc1 Expression

Hypothesis


0

5

10

15

D0 D2 D4 D6 D8

Rela?v

e Expression

to ESC D0

(normalized

to 18S)

Foxc1

0

0.5

1

1.5

D0 D2 D4 D6 D8

Rela?v

e Expression

to

ESC D0

(normalized

to 18S)

Oct4

0

10

20

30

40

D0 D2 D4 D6 D8 Rela?v

e Expression

to ESC D0

(normalized

to 18S) Brachury

0 1 2 3 4 5

D0 D2 D4 D6 D8 Rela?v

e Expression

to ESC D0

(normalized

to 18S) Nkx2.5

Hypothesis

ESC as a model to study cardiac differen=a=on: Timing of Foxc1 expression


Specific Aim 1: ESC Model and Differen=a=on Methods

Knockdown

Specific Aim 1: To determine if Foxc1 is necessary for the early differen=a=on of ESCs into func=onal cardiomyocyte lineages

Nkx2.5 GFP Reporter cells

NeoR Nkx2.5-‐EmGFP

Nkx2-‐5 locus on mouse Chromosome 13

Exon1 Exon1

Endogenous Nkx2-‐5 Promoter

Transduce with Len?viral Par?cles shRNA against Foxc1 or Scrambled shRNA Control

1) Nkx-‐ESC-‐KD

2) Nkx-‐ESC-‐ScrCrl


0

0.5

1

1.5

Nkx-‐ESC-‐ScrCtrl Nkx-‐ESC-‐KD

570n

m OD Fold

Chan

ge to

Nkx-‐ESC-‐

Scr C

trl

MTT Prolifera?on Assay

0 20 40 60 80

100 120

Nkx-‐ESC-‐ScrCtrl Nkx-‐ESC-‐KD Rela?v

e Expression

to Nkx-‐ESC Scr. C

trl

(normalized

to 18S )

FOXC1 FOXC2 Nkx-‐ESC-‐ScrCtrl

Nkx-‐ESC-‐ Foxc1 KD

N.S

*

Specific Aim 1: Preliminary Data Foxc1 KD does not effect pluripotent proper=es

Knockdown

Background Hypothesis Knockdown Overexpression Gene Targets Future Direc=ons SSC

FSC

Scr. Ctrl EB D6

FITC

(GFP)

PI (Live/Dead)

16.2

FITC

(GFP)

PI (Live./Dead)

Nkx-‐ESC-‐ScrCtrl Undifferen?ated 0.7

Specific Aim 1: Preliminary Data Foxc1 KD decreases number of nkx2.5+ cells

Knockdown

Foxc1-‐KD EB D6

FITC

(GFP)

PI (Live/Dead)

11.6

0

0.5

1

1.5

Scr Ctrl Foxc1-‐KD Rela?v

e Expression

to

Scr. Ctrl D6

(Normalized

to 18s)

Nkx2.5

*

0 0.2 0.4 0.6 0.8 1

1.2

Nkx2.5+ Live Ce

lls

Fold Cha

nge to Ctrl

**

*


•  Assess cardiomyocyte marker expression (Isl1, Gata4, Mef2c, CTT, alpha-‐MHC) •  Quan=fy the number of bea=ng EBs •  Assess if EB can respond to external s=muli and beat in synchrony using electrophysiological calcium handling studies

Specific Aim 1: Future Assessments

Knockdown


Specific Aims

Specific Aim 1: To determine if Foxc1 is necessary for the differen=a=on of ESCs into func=onal cardiomyocyte

Specific Aim 3: To determine the gene targets that are directly regulated by Foxc1 during early cardiac differen=a=on

Specific Aim 2: To determine if Foxc1 overexpression is sufficient to increase ESC differen?a?on into func?onal cardiomyocytes

Overexpression


Puro Foxc1 ORF

hCMV Promoter

ROSA26 Locus on mouse Chromosome 6

SA-‐tTA IRES Venus

Rosa26 Promoter

His6-‐FLAG

Tet-‐off System (+) Dox the tTA protein is sequestered and cannot bind the hCMV promoter

(-‐) Dox the tTA protein is binds the hCMV promoter thereby ac?ving hCMV and overexpressing exogenous Foxc1

Specific Aim 2: Foxc1 is sufficient to increase ESC differen=a=on into func=onal cardiomyocytes

Overexpression


0

5

10

ESC (+) DOX 48hr

ESC (-‐) DOX 48hr

Rela?v

e Expression

to (+

) Dox

Foxc1

Foxc1

Foxc2

Gapdh

* 0.6

FITC

(GFP)

PI (Alive/Dead)

(+) DOX 48hrs

91.8

PI (Alive/Dead) FITC

(GFP)

(-‐ )DOX 48hrs

(+ ) DOX 48hrs (-‐) DOX 48hrs

Specific Aim 2: Preliminary Data Valida=on of Foxc1 Tet-‐Off System

Overexpression


0 0.2 0.4 0.6 0.8 1

1.2 1.4

C57 Wt ESC Foxdox ESC

570n

m OD

Fold Cha

nge to (+

)DOX ESC

MTT Prolifera?on Assay (+)DOX 48hrs (-‐) DOX 48hrs

***

FoxDox-‐ESCs (+) DOX 48hrs

FoxDox-‐ESCs (-‐) DOX 48hrs

Specific Aim 2 Preliminary Data: Foxc1 OE decreases prolifera=on and induces differen=a=on

Overexpression


D6

10X

10X

D6

10X

10X

D1

D1

-‐ DOX

+ DOX

0 2 4 6 8

(+) Dox EB D6 (-‐) Dox EB D6 Rela?v

e Expression

to ESC D

0 +D

OX

(Normalized

to 18S) Foxc1

Overexpression


Specific Aim 2 Preliminary Data: Foxc1 OE Enhances early markers of cardiomyocyte commitment

Overexpression

D

0

1

2

3

4

(+) Dox EB D6 (-‐) Dox EB D6

Rela?v

e Expression

to

ESC D0 +D

OX Nkx2-‐5 *

0 2 4 6 8

10


e Expression

to ESC D

0 +D

OX

Gata4

*

0

20

40

60


e Expression

to ESC D

0 +D

OX

(Normalized

to 18S) Mef2c

*

0 10 20 30 40 50


e Expression

to ESC D

0 +D

OX

(Normalized

to 18S)

Isl1

*


Specific Aim 2 Preliminary Data: Foxc1 OE Enhances Markers of fully commiMed cardiomyocytes

Overexpression

0 10 20 30 40 50


e Expression

to

ESC D0 +D

OX

(Normalized

to 18S)

Alpha-‐MHC *

0 2 4 6 8

10

(+) Dox EB D6 (-‐) Dox EB D6 Re

la?v

e Expression

to

ESC D0 +D

OX

(Normalized

to 18S)

Cardiac Troponin T

*


Specific Aim 2 Preliminary Data: Embryoid Body Bea=ng with Endogenous Foxc1

Overexpression


0 2 4 6 8

10 12 14 16

(+) DOX (-‐) Dox Pe

rcen

t of B

ea?n

g

Specific Aim 2 Preliminary Data: Embryoid Body Bea=ng with Overexpression Foxc1

*

Overexpression


•  Assess cardiomyocyte marker expression at Day 10 to substan=ate bea=ng data

(Isl1, Gata4, Mef2c, CTT, alpha-‐MHC) •  Assess if EB can respond to external s=muli and beat in synchrony using calcium handling studies

Specific Aim 1: Future Assessments

Overexpression


Specific Aims

Test the central hypothesis with 3 specific aims:

Specific Aim 1: To determine if Foxc1 is necessary for the early differen=a=on of ESCs into func=onal cardiomyocyte lineages

Specific Aim 3: To determine the gene targets that are directly regulated by Foxc1 during early cardiac differen?a?on

Specific Aim 2: To determine if Foxc1 is sufficient to increase ESC differen?a?on into func?onal cardiomyocytes

Gene Targets


Mef2c

Nkx2.5 Cow

Chimpanzee Human

Cow

Chimpanzee

Human

Foxc1 Binding sites Found in gene upregulated aner Foxc1 Overexpression

Gene Targets

Searched 10KB upstream of genes found to be upregulate aner Foxc1 Overexpression


Preliminary RNA-‐Seq Results: Up-‐regula=on of genes involved in cardiomyocyte func=on

1543

1983

Differen?ally regulated gene in Foxc1

Overexpressing EBs at Day 6

Upregulatd Downregulated

42

22 20

19

17

16

Up-‐regulated Genes Involved in Cardiomyocyte func?on

Focal adhesion

Regula=on of ac=n cytoskeleton Cell adhesion molecules (CAMs) Calcium signaling pathway Gap junc=on

Tight junc=on

Gene Targets

RNA-‐sequencing to assess gene pathways that may be regulated by foxc1


Specific Aim 3: Experimental Design

Gene Targets

1) Differen=ate wt ESCs un=l Embryoid Body Day 6

2) Fix/crosslink cells for ChIP-‐Seq

3) Pull down Foxc1 using an=body

4) Analyze the genomic sequences that are pulled down

Goal: Gain a comprehensive list of genes directly regulated by Foxc1 Genome wide high through put ChIP-‐sequencing analysis


Specific Aim 3

1) I expect ChIP sequencing to come up with a long list of targets

•  Narrow Down targets ChIP sequencing results via func=on 1) Development of the heart 2) Cardiomyocyte func=on Pick the top ten and the RNA-‐seq (differen=al regula=on)

2) Demonstrate foxc1 regula=on through the puta=ve binding site with luciferase assays

Co-‐tranfec=on of Cells with Foxc1 and Luciferase constructs 1) Mutated binding site 2) Wild-‐type binding site

Assess luciferase ac=vity

Gene Targets

Specific Aim 3: Experimental Design


Transcrip?on ac?vator-‐like effector nucleases (TALENs) are ar=ficial restric=on enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain.

Talen Edi=ng: To test func=onal relevance of Foxc1 binding site in the puta=ve target gene

Future Direc?ons


Target Expression

FOXC 1

Target gene

To test func=onal relevance of target genes binding site

TALEN (Transcrip?on ac?vator-‐like

effector nucleases)

FOXC 1

FoxC1 Binding Site AAAAACA

Future Direc?ons


So What? 1) These studies will shed light on the molecular circuitry guiding ESC differen=a=on into cardiomyocytes 2) May lead us develop strategies for cardiac regenera=on with therapeu=c poten=al

Future Direc?ons


Thank you! Future Direc?ons

Ques=ons?

Documents

The role of Foxc1 during the differentiation of embryonic stem cells into cardiomyocytes