Supplemental Methods Mice Cre/+ lacZ +/GFPcircres.ahajournals.org/content/suppl/2009/02/17/CIRCRESAHA.108... · Unbiased Stereology. Three-dimensional Measurement in Three-dimensional

Supplement Material S1

Supplemental Methods

Mice

In order to generate double transgenic mice Tbx3Cre/+

, R26RlacZ

and Nppa::Cre3 mice were

crossed with Tbx18+/GFP

heterozygous mice. Genomic DNA prepared from amnion or toe

biopsies was used for genotyping by PCR. Following primers were used: Tbx18 wild type

allele (forward: GCG CGG AAA AGG GCT CGG and reverse: AGG AAG CTA CTG TCT

GGG G), Tbx18 mutant allele (forward: GAC AAC CAC TAC CTG AGC AC and reverse:

CCG GCT TTG GTG ATG ATC), Tbx3 wild type allele (forward: AGC GGA GCC AAG

CCA GCA and reverse: CCT TGG CCT CCA GGT GCA C), Tbx3 mutant allele (forward:

see wild type allele and reverse: see Cre reverse primer), EGFP (forward: CGA CGT AAA

CGG CCA CAA GTT and reverse: TTG ATG CCG TTC TTC TGC TTG T), Cre (forward:

GGT TCG CAA GAA CCT GAT GGA CAT and reverse: GCT AGA GCC TGT TTT GCA

CGT TCA) and lacZ (forward: CTG CGC TGC GGG ACG CGC GAA TTG AAT TAT and

reverse: GAC ACC AGA CCA ACT GGT AGC GAC).

Collection and preparation of embryos

For timed pregnancies, vaginal plugs were checked in the morning after mating, noon was

taken as embryonic day (E) 0.5. Embryos of developmental stages between E10.5 and 17.5

were isolated for analysis. They were dissected in PBS and fixed in 4% paraformaldehyde

overnight for in situ hybridization or immunohistochemistry (detection of GFP, cleaved

caspase-3, BrdU and TUNEL assay), respectively. Embryos used for beta-galactosidase

activity detection and immunohistochemistry (Hcn4, Connexin 40) were fixed in 4%

paraformaldehyde for 15 min on ice and then incubated in 10% sucrose overnight. Next day,

they were embedded in OCT Embedding medium and stored at -20°C.

Proliferation and apoptosis analysis

The proliferation (BrdU assay) and apoptosis (cleaved caspase-3 detection and TUNEL

assay) analyses were performed as described previously.1,2

ββββ-Galactosidase activity detection and immunohistochemistry

For detection of β-galactosidase activity 10 µm cryostat sections were fixed with 4%

paraformaldehyde for 10 minutes at room temperature, followed by X-gal staining. For

immunohistochemistry the following primary antibodies were used: rabbit polyclonal

antibodies against Hcn4 (1:250, Chemicon) and GFP (1:50, Santa Cruz Biotechnology) and

monoclonal antibodies against Cx40 (1:100, USBio) and MF20 (1:50, Hybridoma bank, Iowa

City, IA, USA). Immunohistochemical analysis of Hcn4 and Connexin 40 was performed on

10 µm cryostat sections. In order to block endogenous mouse IgG, sections used to detect

Cx40 were pre-incubation with an unconjugated Fab fragment goat anti-mouse IgG (H+L)

(1:10, Jackson ImmunoResearch Laboratories). Secondary antibodies were Alexa 568 goat

anti-rat, goat anti-rabbit (1:250, Molecular Probes) and Alexa 488 goat anti-mouse (1:400).

Nuclei were counterstained with SYTOX green / orange nucleic acid stain (Molecular

Probes) or DAPI (Molecular Probes), respectively. GFP expression was detected on 5 µm

paraplast sections. Non-fluorescent staining was performed using kits from Vector

Laboratories (ABC peroxidase kit (Rabbit IgG), DAB substrate kit).

Non-radioactive in situ hybridization

Non-radioactive in situ hybridization on sections was performed as described.3 RNA probes

were kindly provided for Nkx2-5 (R. Harvey, Victor Chang Cardiac Research Institute,

University of New South Wales), Hcn4 (B. Santoro, Center for Neurobiology and Behavior,

Supplement Material S2

Columbia University, New York), Tbx3 (V. Papaioannou, Department of Biological

Sciences, University of Pittsburgh), Shox2 (G. Rappold, Institute for Human Genetic,

University Heidelberg, Germany) and Lbh (K. J. Briegel, Institute for Cellular and Molecular

Biology, University of Texas, Austin). Other probes have been described previously.1,4-6

Three-dimensional reconstructions and quantification of sinus node volume

Three-dimensional visualization and geometry reconstruction of patterns of gene and protein

expression have been performed as described.7 The quantification of expression domains (e.g.

sinus node volume) has been described previously.6 Files with reconstructions are available

on request.

Determination of atrial wall thickness and cell density

The atrial wall thickness was determined using Scion Image as theoretically described

previously.8 To determine the cell density in the sinus node of E14.5 old wild type and Tbx18

mutant embryos, the single nuclei within the Tbx3 labels (“masks of Tbx3” produced in

Amira) were counted per each section using the program Image Pro. The area of the masks

was measured using Scion Image and the cell number per area was calculated. To determine

the number of cells per volume, a nucleus size of 10 µm was assumed (determined previously

from embryonic chicken myocytes) and a section thickness of 10 µm was considered. The

cell density is the number of cells per area divided by the nucleus diameter and the section

thickness. The cell density in the sinus node of E12.5 old Tbx3 mutant and wild type embryos

was estimated in a similar way but a fixed volume within the sinus node was used. The

section thickness was 7 µm.

Embryonic explant cultures

In order to isolate Tbx18-expressing mesenchyme in the region of the developing sinus horns,

both lateral parts of the EGFP-expressing area in heterozygous Tbx18 EGFP knock-in

embryos at E9.5 were micro-dissected. Explants were similar cultured as previously

described9. Directly after isolation and after cell culture, respectively, samples were fixed in

4% paraformaldehyde and used for immunocytochemistry.

Statistics

Results are expressed as mean ± SEM. Statistical significance was tested with unpaired, two-

tailed student’s t-test. (* for P < 0.05, ** for P< 0.005 and *** for P< 0.001).

References

1. Bussen M, Petry M, Schuster-Gossler K, Leitges M, Gossler A, Kispert A. The T-box

transcription factor Tbx18 maintains the separation of anterior and posterior somite

compartments. Genes Dev. 2004;18:1209-1221

2. Bakker ML, Boukens BJ, Mommersteeg MTM, Brons JF, Wakker V, Moorman AFM,

Christoffels VM. Transcription factor Tbx3 is required for the specification of the

atrioventricular conduction system. Circ Res. 2008;102:1340-1349

3. Moorman AFM, Houweling AC, de Boer PAJ, Christoffels VM. Sensitive

nonradioactive detection of mRNA in tissue sections: novel application of the whole-

mount in situ hybridization protocol. J Histochem Cytochem. 2001;49:1-8

Supplement Material S3

4. Kraus F, Haenig B, Kispert A. Cloning and expression analysis of the mouse T-box

gene Tbx18. Mech Dev. 2001;100:83-86

5. Delorme B, Dahl E, Jarry-Guichard T, Marics I, Briand JP, Willecke K, Gros D,

Théveniau-Ruissy M. Developmental regulation of connexin40 gene expression in

mouse heart correlates with the differentiation of the conduction system. Dev Dyn.

1995;204:358-371

6. Hoogaars WMH, Tessari A, Moorman AFM, de Boer PAJ, Hagoort J, Soufan AT,

Campione M, Christoffels VM. The transcriptional repressor Tbx3 delineates the

developing central conduction system of the heart. Cardiovasc Res. 2004;62:489-499

7. Soufan AT, Ruijter JM, van den Hoff MJB, de Boer PAJ, Hagoort J, Moorman AFM.

Three-dimensional reconstruction of gene expression patterns during cardiac

development. Physiol Genomics. 2003;13:187-195

8. Howard CV, Reed MG. Unbiased Stereology. Three-dimensional Measurement in

microscopy, ed1, Liverpool, UK, Bios Scientific Publishers, 1998, pp pp 246

9. Zaffran S, Kelly RG, Meilhac SM, Buckingham ME, Brown NA. Right ventricular

myocardium derives from the anterior heart field. Circ Res. 2004;95:261-268

Supplement Material S4

Online Figure I

Gene expression patterns of the SAN and adjacent tissues in Tbx18-deficient and control

(wild-type or heterozygous) embryos at different developmental stages. A, Optical section

through the SAN region of the 3D reconstruction (description see figure legend 1). B, C and

D, linear representations of gene expression patterns in heterozygous and homozygous

Tbx18-mutant embryos at E10.5, E12.5 and E17.5, respectively. Description and color code

in the model correspond to the description shown in A. Black bars indicate specific gene

expression in the corresponding tissue. E, Analysis of Shox2 gene expression in Tbx18

mutant embryos (E11.5). Shown are in situ hybridizations on serial transversal sections using

probes for cardiac troponin I (cTnI) to label the myocardium and Shox2. The black arrows

indicate the myocardial border in wild type and Tbx18 mutant embryos and red arrows

demonstrate the absence of myocardium and Shox2 expression in the left superior caval vein

of Tbx18 mutant embryo. rsc, right superior caval vein; ra, right atrium.

Online Figure II

Characterization of the sinus node in Tbx18 mutant embryos. A, Determination of the length

(longitudinal axis along the rscv) of the sinus node head at E14.5. B, Determination of the

BrdU labeling index in the Tbx18/GFP-positive area of the sinus node at E14.5 and E17.5 in

Tbx18 mutant and heterozygous embryos (n=3 and 4, respectively). C,

Immunohistochemistry on E14.5 old heterozygous and Tbx18 homozygous mutant embryos

using GFP (Tbx18-positive cells), BrdU (proliferating cells) and DAPI (cell nuclei). D,

Determination of the cell density in the sinus node of E14.5 old wild type and Tbx18 mutant

embryos (n=3).

Online Figure III

Analysis of the function of Tbx3 in sinus node development. A, Determination of the sinus

node length (longitudinal axis along rscv) (E12.5, n=5 for wild type; n=3 for Tbx3Cre/Cre

). B

and C, Proliferation analysis using BrdU incorporation (E12.5), B, Quantification of the

BrdU labeling index in the sinus node head and the venous valve part of the sinus node

separately (n=4). C, Immunofluorescence staining showing BrdU (red) and nuclei (sytox

green, green). D, Determination of the cell density in the sinus node of E12.5 old Tbx3

mutant and wild type embryos (n=4).

Online Figure IV

Analysis of the sinus node development in Tbx18-Tbx3 double homozygous embryos

(Tbx18+/GFP

;Tbx3+/Cre

) at E12.5. For description of the lower panel of each genotype see

Figure 7 description. Upper panel of each genotype shows the more distal part of the sinus

node head indicated by the black arrow. In Tbx18 mutant and Tbx18-Tbx3 double mutant

embryos the sinus node head (black arrow head in wild-type) is absent, whereas the tail

(white arrow head) is present.

Online Figure V

Collection of 10 sections through the sinus node of a wild type and Tbx18 mutant embryo at

E12.5 used for 3D reconstructions shown in Figure 4A. a-k, in situ hybridizations of Tbx3 in

a wild type sinus node, a’-k’ in situ hybridizations of Tbx3 in a Tbx18 mutant sinus node.

Supplement Material S5

Online Figure VI

Characterization of the sinus node tail. A, The sinus node tail, which expresses Tbx3,

(indicated by a white arrow head) is negative for Tbx18 (wild type) and GFP (Tbx18 mutant

embryo), respectively. Shown is an E12.5 old embryo.

B, In the sinus node tail, which is negative for connexin 40 (Cx40), Nkx2-5 is gradually up-

regulated at E17.5 in wild type and Tbx18 mutant embryo.

cTnI Shox2

wt

Tb

x18

GF

P/G

Fp

ra

rcv

rcv

ra

Online Figure I

Tb

x1

8

Nk

x2

-5

Nk

x2

-5

Hc

n4

E12.5

Tb

x3

Hc

n4

Cx

40

cT

nI

Tb

x3

Cx

40

cT

nI

GF

P

he

ad

Tbx18+/GFP

rash

me

s

tail

asvv

E10.5

Tb

x3

Hc

n4

Cx

40

cT

nI

Tb

x1

8

Nk

x2

-5

B

Nk

x2

-5

Tb

x3

Hc

n4

Cx

40

cT

nI

GF

P

Tbx18GFP/GFP

A

V

Cra

Cau

D

ra

asvvtail

head

sh

mes

E14.5-17.5

Tb

x3

Hc

n4

Cx

40

cT

nI

Tb

x1

8

Nk

x2

-5

Nk

x2

-5

Tb

x3

Hc

n4

Cx

40

cT

nI

GF

P

CTbx18

+/GFPTbx18

GFP/GFP

DTbx18

+/GFPTbx18

GFP/GFP

E

A

0

200

400

600

800

1000

1200

sin

us n

od

e h

ead

len

gth

(µµ µµ

m)

*

Tbx18GFP/GFP

Tbx18+/GFP

C

rscv

atrium

GFP

BrdU

Tb

x1

8G

FP

/GF

PT

bx

18

+/G

FP

DAPI

san

rscv

GFP

BrdU DAPI

san

atrium

B

0

2

4

6

8

10

12

14

Brd

U lab

elin

g in

dex (

%)

E14.5 E17.5

Tbx18GFP/GFP

Tbx18+/GFP

D

0

0.0001

0.0002

0.0003

0.0004

0.0005

cell d

en

sit

y (

cells/ µµ µµ

m3)

Tbx18GFP/GFP

Tbx18+/GFP

Online Figure II

0

100

200

300

400

500

600

sin

us n

od

e

len

gth

(µµ µµ

m)

A

C

rscvrscv

rara

sansan

wt

BrdU/Sytox.BrdU/Sytox.

Tbx3Cre/Cre

0

2

4

6

8

10

12

14

16

18

Brd

U lab

elin

g in

dex (

%)

sinus node

head

venous

valve (tail)

B

**

wt

Tbx3Cre/Cre

D

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006cell d

en

sit

y (

cells/ µµ µµ

m3)

wt

Tbx3Cre/Cre

wt

Tbx3Cre/Cre

Online Figure III

wt

Tb

x1

8G

FP

/GF

P;T

bx3

Cre

/Cre

Tb

x1

8G

FP

/GF

P

cTnI Hcn4 Tbx3/Cre Cx40 Tbx18/GFP

ra

ra

ra

ra

ra

ra

ra

rvv

rvv

rvv

rcv

rcv

rcv

rcv

rcv

Online Figure IV

Online Figure V

wt Tbx18GFP/GFP

E12.5

a

a

b

c

d

e

f

g

h

i

j

a'

b'

c'

d'

e'

f'

g'

h'

i'

j'

wt Tbx18GFP/GFP

E12.5

Online Figure VI

Tbx3 Tbx3

Tbx18 GFP

E17.5

wt Tbx18GFP/GFP

Nkx2-5 Nkx2-5

Cx40Cx40

B

A

ra

rcv

ra

rcv

san

san

san

san