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S U P P L E M E N TA RY I N F O R M AT I O N

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Figure S1 CMV promoter activity of the MesP1 overexpression construct over time of ES cell differentiation: FACS analyses for EGFP expression reveal 89 % positive cells at day 0 and 12 % at day 4 of differentiation.

A

B

Error Bars: s.d.

n=16

n=6

n=12

n=5

n=4

n=8

Figure S2 (A) Examples of ventricle-like (“Ventr.”), atrial-like (“Atr.”), pacemaker-like (“SA/AV”) and “intermediate” action potentials recorded from cells isolated from MesP1 ES clones at day 6+12, demonstrating the occurrence of both correctly differentiated and immature cardiac cells. Time bars represent 100 ms. The chronotropic effects of isoproterenol and carbachol on the action potential (AP) rate is qualitatively given as strong (++), weak (+) or absent (-). (B) If measurements (in Ba/Cd-containing bath solution) were used in addition to AP recordings to discriminate between cells of the intermediate type and more differentiated pacemaker-like cells that have similarly stable spontaneous activity. Left panel: Representative traces of If elicited at –110 mV of MesP-cells showing a “SA/AV” AP, an intermediate AP and a ventricle-like AP. No If could be measured in ventricle-like cells. Right panel: SA/AV/pacemaker-like cells (both MesP and control cells as well as mouse embryonic cardiomyocytes (“Embryo”) show a much higher If density compared to intermediate-stage cells (data are mean ± s.d., numbers of experimental values are shown in the diagram).



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Figure S3 FACS analyses for VEGFR-2 during ES cell differentiation. Increased appearance of VEGFR-2 in MesP1 ES-cells is first observed at day 4.

UntransfectedControl

MesP1-IRES-EGFP

EGFP-Control

day 20.3% 0.3% 0.3%

day 40.6%0.7% 2.2%

Undiffer-entiated

0.25% 0.25% 0.2%

1.2%1.2% 3.5%day 6

EGFP

VE

GF

R-2




Figure S4 “Rescue” experiment using stably expressed human MesP1 specific shRNA in hMesP1 ES cell clones. (A) Functionality of the “rescue” shRNA verified via a decrease of EGFP expression from the bicistronic hMesP1-IRES-EGFP cassette with ~7% remaining EGFP activity of the

initial level (note: plotted on a logarhythmic axis). (B) Reduced incidence of beating areas comparable to control levels in shRNA expressing “rescued” ES cells. (data are mean ± s.d., n=12, P<0,0025) (C) Reduction of TnI expressing cells to control levels in shRNA expressing “rescued” ES cells.

g. S4

B

untransfected

shRNA rescue

MesP1-IRES-EGFP

A

EGFP fluorescence

0

0,5

1

1,5

2

2,5

3

1ControlControl hMesP1shRNA resc.

Bea

tin

gar

eas

at d

ay 1

6p

er 2

500

pla

ted

cells

n=12; Error bars: s.d.

P<0,0025

P<0,0025

n.s.

shRNA rescue

Tro

poni

nI 8%

hMesP1

Tro

poni

nI 28%

PE+

Control

CPE+

9%

Tro

poni

nI




Figure S5 (A) Comparison of the murine, human, chicken and zebrafish Dkk-1 promoter regions with respect to putative MesP1 responsive motifs. (B) Quantitative Real-Time-PCR: Injection of hMesP1 mRNA and hMesP1 expression plasmid in Two-Cell-Stage Xenopus embryos subsequently leads to increased Dkk-1 mRNA levels at early neurula stage (NF14) (data are mean ± s.e.m., n=4). (C) Schematic model for the induction of

cardiovasculogenic events in ES cells by MesP1: MesP1 driven upregulation of Dkk-1 expression blocks wnt-signalling and leads to increased endodermal Hex expression. This causes anteriorisation of the lateral plate mesoderm resulting in cardiovascular differentiation. Further downstream the augmented presence of cardiac tissue causes an enhanced neurogenesis and reduced epidermis formation in the ectoderm.

David et al., Fig. S5 A

agCATATGctmDkk-1 (-3,8kb)

caCATATGtgmDkk-1 (-2,2kb)

mDkk-1

ATG

hDkk-1

ATG

acCATATGgthDkk-1 (-11,6kb)

taCATATGtahDkk-1 (-9,5kb)

cDkk-1

ATGatCATATGcc

cDkk-1 (-17,6kb)taCATATGtg

cDkk-1 (-16,4kb)agCATATGta

cDkk-1 (-2,3kb)

zfDkk-1

ATGtgCATATGga

zfDkk-1 (-14,8kb)acCATATGtt

zfDkk-1 (-12,5kb)tcCATATGtc

zfDkk-1 (-10,9kb)

A




Re l

. am

ou

nt

Dk k

-1m

RN

A

100%

Control MesP1mRNA

MesP1Plasmid

n=4; Error bars: s.e.m.

P

IC Mesoderm

Lat. Plate

Neural Tissue

Epidermis

MesP1

Lateral Plate Mesoderm

Endoderm

Meso-derm

Ecto-derm

Endoderm

wnt

???

Dkk-1Dkk-1Dkk-1Dkk-1

Figure S5 continued

B

C




Figure S6 Full scans of key western and gel data shown in Figs. 2, 4 and 5. Sections irrelevant for the images shown in the respective

figures are crossed out. For PCR gels the 100bp ladder was used as MW standard.

MLC2v -RT

Figure 4A

Figure 2FRT-PCR d0: H4

Figure 5AFigure 5B

Figure 2E

Figure 5D

-RT




Supplementary movie legends

Movies S1, S2, S3 Ectopic contracting cardiac tissue in MesP1 overexpressing stage 45 Xenopus tadpoles previously injected at 2 cell stage.

Movie S4 Control injected Xenopus tadpole. The animals in Suppl. movies 1-4 were anaesthesized and, therefore, the skeletal musculature is quiescent.

Movie S5 Increased beating activity in MesP1 overexpressing ES cells at day 18 of ES cell differentiation.

Movie S6 Control transfected ES cells at day 18 of ES cell differentiation.


Table S1 AP parameters of (near-)terminally differentiated cardiomyocytes demonstrate correct development of cardiac cells. Action potential parameters of spontaneously beating cardiomyocytes, isolated from MesP1 and control embryoid bodies do not differ significantly. Of 28 cells from MesP1 clones and 19 from control clones, 12 and 15, respectively, could be classified as ventricular-like (ventr.), atrial-like (atr.) or pacemaker-like (SA/AV) types whereas the rest were classified as various intermediate types 4 with a fast, stable DDR of 30-60 mV/s but otherwise quite varying parameters and shapes (both MesP1 and control) (data are mean ± s.d., numbers of experimental values are indicated in the table).

MesP1 Controls

ventr.(n=7)

atr.(n=3)

SA/AV(n=2)

ventr.(n=10)

atr.(n=2)

SA/AV(n=3)

MDP -72.7 ± 7ö8 mV -71.9 ± 5.6 mV -58.5 ± 2.4 mV -75.8 ± 4.6 mV -76 ± 9.9 mV -56.2 ± 6.4 mV

DDR 2.34 ± 2.0 mV/s 2.7 ± 0.6 mV/s 103 ± 31.1 mV/s 3.29 ± 1.6 mV/s 4.5 ± 1.7 mV/s100.3 ± 23.8

mV/s

upstroke velocity

83.3 ± 27.1 V/s 45.9 ± 9.6 V/s 2.2 ± 0.7 V/s 67.9 ± 11.4 V/s 49.5 ± 40.3 V/s 2.9 ± 1.25 V/s

Overshoot +33.4 ± 8.7 mV +20.0 ± 3.1 mV +10.8 ± 1.8 mV +30 ± 4.6 mV +21.9 ± 5.3 mV +9.5 ± 3.8 mV

AP duration

216.9 ± 44.3 ms 133.7 ± 22.9 ms 132 ± 22.6 ms 224 ± 11 ms 135 ± 61.5 ms 136 ± 17 ms

Repol.velocity

24.9 ± 16.7 V/s 0.47 ± 0.28 V/s 0.4 ± 0.28 V/s 18.9 ± 6.1 V/s 0.87 ± 0.2 V/s 0.6 ± 0.3 V/s

Data are mean of n-cells ± SD.Abbreviations: AP, action potential; MDP, maximal diastolic potential; DDR, diastolic depolarisation rate


Supplementary Information

Suppl. Methods Isolation of cardiac cells from spontaneously beating embryoid bodies and

embryonic mouse hearts.

Single cardiomyocytes were prepared from 6+12 day-old embryoid bodies or

embryonic hearts, developmental day 10. Beating areas of the embryoid bodies

or the heart of mouse embryos were excised under a microscope and transferred

to 37°C-prewarmed isolation buffer, containing: 116 mM NaCl, 5 mM KCl, 0.8

mM MgSO4, 1 mM NaH2PO4, 20 mM HEPES and 5.5 mM glucose. ph was

adjusted to 7.3 with NaOH. For the enzymatic release of single cells, the tissue

pieces were incubated for 15 minutes at 37°C and slight shaking in isolation

buffer with the addition of 0.5 mg/ml Collagenase B (Roche) and 1 mg/ml

Pankreatin (Gibco/Invitrogen). Immediately after incubation, the tissue pieces

were mechanically dispersed in isolation buffer/medium (DMEM + 10 % fetal calf

serum, PAA) (1:1 vol/vol) and plated on polylysated glass coverslips. The cells

were allowed to settle for 5 minutes before medium was added. Cells were kept

at 37°C, 10% CO2 for 12-24 hours before electrophysiological recordings were

performed.

Electrophysiological characterization of spontaneously beating cells.

12-24 hours after isolation, spontaneous action potentials and currents were

recorded from beating cardiomyocytes at 37°C in the perforated patch

configuration using a MultiClamp 700B amplifier and pClamp9 software (both

Axon Instruments/Molecular Devices, Union City, USA). Offline data analysis was

done with Origin 6.0 software (Microcal, Northampton, USA). Patch pipettes were

pulled from borosilicate glass, heat polished and had a resistance of 2-5 MΩ

when filled with intracellular solution which contained: 10 mM NaCl, 130 mM

potassium aspartate, 0.04 mM CaCl2, 2 mM Mg-ATP, 6.6 mM creatine-

phosphate, 10 mM HEPES and 200 µg/ml Amphotericin B, pH adjusted to 7.2

with KOH. The extracellular (bath) solution contained: 140 mM NaCl, 5.4 mM


KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 5.5 mM Glucose, pH adjusted to

7.4 with NaOH. For If-recordings, 2 mM BaCl2 and 0.3 mM CdCl2 were added to

the bath solution to block IKI and ICa. If was measured by stepping from a holding

potential of –40 mV to test potentials between –120 mV and +20 mV. The current

amplitude after 2 seconds during the –110 mV pulse was divided by the cell

capacitance to calculate the current (If) density. Isoproterenol or carbachol

(Sigma, Taufkirchen, Germany) were directly dissolved in the bath solution on

the day of the experiment and applied to the cells via a fast exchange

superfusion system. APs were recorded with a 10 kHz sampling rate. Analyses

were done on the original traces; for display in Fig. S2, the data was reduced by

averaging 100 adjacent data points. The slope of the linear fit of the distance

from the MDP to the threshold potential is the DDR; the slope of the linear fit from

the threshold to the AP-peak represents the upstroke velocity. AP duration is the

time from threshold potential to MDP.

Whole-mount in situ hybridization

For whole-mount in situ hybridization and histology, embryos were processed as

described previously Suppl. Ref. 1. The murine MesP1 antisense probe

corresponded to bp 31-701 of the mMesP1 ORF, the murine Dkk-1 antisense

probe contained the full ORF.

shRNA knockdown

For stable knockdown of either the endogenous murine MesP1 mRNA as well as

the overexpressed human MesP1 mRNA the BLOCK-IT system (Invitrogen) was

used for Zeocin based ES cell clone selection according to the supplier`s

manual. Thereby the following oligonucleotides were used for initial plasmid

generation (loop sequences shown in bold):

murine MesP1, upper strand:

5`-caccGCGCCTGGCCATCCGCTACAtcgaaaTGTAGCGGATGGCCAGGCGC-

3`

murine MesP1, lower strand:


5`-aaaaGCGCCTGGCCATCCGCTACAtttcgaTGTAGCGGATGGCCAGGCGC-

3`

human MesP1, upper strand:

5`-caccGCACGAGCTGCGCCGCTTTCtcgaaaGAAAGCGGCGCAGCTCGTGC-

3`

human MesP1, lower strand:

5`-aaaaGCACGAGCTGCGCCGCTTTCtttcgaGAAAGCGGCGCAGCTCGTGC-

3`

MesP1 scrambled, upper strand:

5`-caccGCGGCTGCCCAACGGGTTCAtcgaaaTGAACCCGTTGGGCAGCCGC-

3`

MesP1 scrambled, lower strand:

5`-aaaaGCGGCTGCCCAACGGGTTCAtttcgaTGAACCCGTTGGGCAGCCGC-

3`

Real-Time-PCR

Quantitative Real-Time-PCR was performed using RNA isolated from 200 – 250

pooled EBs collected at day 3 of differentiation via the Rneasy-Kit (Qiagen). First-

strand cDNA was synthesized at 37°C from 2 µg RNA with AMV reverse

transcriptase (Amersham) and random hexamer primers. Real-time PCR was

performed with an iCycler and the MyiQ detection system (Biorad) using the IQ

Syber Green Super Mix kit (Biorad). Primers were designed using the DNA Star

software and the specificity of each primer pair was confirmed by agarose gel

electrophoresis. The annealing temperature was 57°C for all primer pairs and the

amplified murine cDNA fragments corresponded to bp 800-974 of MesP1, to bp

679-799 of Dkk-1, to bp 270-395 of Nkx2.5 and to bp 641-1058 of GATA4. All

samples were analyzed in triplicates and pooled total RNA from differentiated ES

cells as well as murine hearts was used as control and to generate a standard

curve for markers analyzed. The annealing temperature for the Xenopus specific

Dkk-1 primer pair was 60°C and the cDNA fragment corresponded to bp 656-


769. In each experiment two independent samples corresponding to three

embryos were used, which were additionally analyzed in duplicates.

As a negative control total RNA from each of the samples was run without

reverse transcriptase. No signal was detected after 40 PCR cycles in the

absence of reverse transcriptase, indicating that all samples were free of DNA

contamination. In addition, no signal was detected when reverse transcriptase

was added but RNA template was not, indicating that there was no contamination

from exogenous RNA or DNA. The standard curve for all genes displayed an

increase of one threshold cycle for each halving of template concentration.

Evaluation of relative gene expression levels was performed based on the � CT

method. Factors of changes in relative mRNA expression levels were calculated

using murine H4 (Fragment: bp 64-189, annealing temperature 57°C) or

Xenopus H4 (Fragment: bp 139-254, annealing temperature 60°C) as a

reference gene whereby the expression value in control ES-cells or control

embryos was defined as 100%.

Reporter Gene Assays

The human MesP1 response elements (-9,5kb: TACATATGTA; -11,6kb:

ACCATATGGT) were cloned 5´ of the minimal HSV-TK promoter in the HindIII

and BamHI sites of pTk-Luc to drive Firefly luciferase. A Twin DR0 oligo (2xDR0;

GGTGACCTTGACCTTTTCCCGGGatggtGACCTTGACCTTT), a known

response element for the nuclear orphan receptor GCNF served as a control

element which contains a MesP1 half site element (in lower case letters).

Transient transfections of control and MesP1 overexpressing ES cell lines with

these three reporter constructs were performed using Mirus LT-1 (Panvera).

Subsequently, reporter assays were performed using the Dual-Luciferase

Reporter System (Promega) according to the supplier`s manual. Renilla

luciferase served as transfection control for standardization.


Suppl. Reference 1. Lickert, H. et al. Formation of multiple hearts in mice following deletion of

beta-catenin in the embryonic endoderm. Dev Cell. 3, 171-81 (2002).

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