Supplement Material. Supplemental Methods

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1 Supplement Material S1 Supplemental Methods Mice In order to genete double tnsgenic mice Cre/+, R26R lacz and Nppa::Cre3 mice were crossed with Tbx18 +/ 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), wild type allele (forward: AGC GGA GCC AAG CCA GCA and reverse: CCT TGG CCT CCA GGT GCA C), mutant allele (forward: see wild type allele and reverse: see Cre reverse primer), E (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 prepation of embryos For timed pregnancies, vaginal plugs were checked in the morning after mating, noon was taken as embryonic day (E).5. Embryos of developmental stages between E1.5 and 17.5 were isolated for analysis. They were dissected in PBS and fixed in 4% paformaldehyde overnight for in situ hybridization or immunohistochemistry (detection of, cleaved caspase-3, BrdU and TUNEL assay), respectively. Embryos used for beta-galactosidase activity detection and immunohistochemistry (, Connexin 4) were fixed in 4% paformaldehyde for 15 min on ice and then incubated in 1% sucrose overnight. Next day, they were embedded in OCT Embedding medium and stored at -2 C. Prolifetion and apoptosis analysis The prolifetion (BrdU assay) and apoptosis (cleaved caspase-3 detection and TUNEL assay) analyses were performed as described previously. 1,2 β-galactosidase activity detection and immunohistochemistry For detection of β-galactosidase activity 1 µm cryostat sections were fixed with 4% paformaldehyde for 1 minutes at room tempeture, followed by X-gal staining. For immunohistochemistry the following primary antibodies were used: bbit polyclonal antibodies against (1:25, Chemicon) and (1:5, Santa Cruz Biotechnology) and monoclonal antibodies against (1:1, USBio) and MF2 (1:5, Hybridoma bank, Iowa City, IA, USA). Immunohistochemical analysis of and Connexin 4 was performed on 1 µm cryostat sections. In order to block endogenous mouse IgG, sections used to detect were pre-incubation with an unconjugated Fab fgment goat anti-mouse IgG (H+L) (1:1, Jackson ImmunoResearch Labotories). Secondary antibodies were Alexa 568 goat anti-t, goat anti-bbit (1:25, Molecular Probes) and Alexa 488 goat anti-mouse (1:4). Nuclei were counterstained with SYTOX green / onge nucleic acid stain (Molecular Probes) or DAPI (Molecular Probes), respectively. expression was detected on 5 µm paplast sections. Non-fluorescent staining was performed using kits from Vector Labotories (ABC peroxidase kit (Rabbit IgG), DAB substte kit). Non-dioactive in situ hybridization Non-dioactive in situ hybridization on sections was performed as described. 3 RNA probes were kindly provided for (R. Harvey, Victor Chang Cardiac Research Institute, University of New South Wales), (B. Santoro, Center for Neurobiology and Behavior,

2 Supplement Material S2 Columbia University, New York), (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 labels ( masks of produced in Ami) were counted per each section using the progm 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 1 µm was assumed (determined previously from embryonic chicken myocytes) and a section thickness of 1 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 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 latel parts of the E-expressing area in heterozygous Tbx18 E knock-in embryos at E9.5 were micro-dissected. Explants were similar cultured as previously described 9. Directly after isolation and after cell culture, respectively, samples were fixed in 4% paformaldehyde and used for immunocytochemistry. Statistics Results are expressed as mean ± SEM. Statistical significance was tested with unpaired, twotailed student s t-test. (* for P <.5, ** for P<.5 and *** for P<.1). References 1. Bussen M, Petry M, Schuster-Gossler K, Leitges M, Gossler A, Kispert A. The T-box tnscription factor Tbx18 maintains the sepation of anterior and posterior somite compartments. Genes Dev. 24;18: Bakker ML, Boukens BJ, Mommersteeg MTM, Brons JF, Wakker V, Moorman AFM, Christoffels VM. Tnscription factor is required for the specification of the atrioventricular conduction system. Circ Res. 28;12: Moorman AFM, Houweling AC, de Boer PAJ, Christoffels VM. Sensitive nondioactive detection of mrna in tissue sections: novel application of the wholemount in situ hybridization protocol. J Histochem Cytochem. 21;49:1-8

3 Supplement Material S3 4. Kus F, Haenig B, Kispert A. Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev. 21;1: Delorme B, Dahl E, Jarry-Guichard T, Marics I, Briand JP, Willecke K, Gros D, Théveniau-Ruissy M. Developmental regulation of connexin4 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn. 1995;24: Hoogaars WMH, Tessari A, Moorman AFM, de Boer PAJ, Hagoort J, Soufan AT, Campione M, Christoffels VM. The tnscriptional repressor delineates the developing centl conduction system of the heart. Cardiovasc Res. 24;62: 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. 23;13: Howard CV, Reed MG. Unbiased Stereology. Three-dimensional Measurement in microscopy, ed1, Liverpool, UK, Bios Scientific Publishers, 1998, pp pp Zaffn S, Kelly RG, Meilhac SM, Buckingham ME, Brown NA. Right ventricular myocardium derives from the anterior heart field. Circ Res. 24;95:

4 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 E1.5, 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 tnsversal sections using probes for cardiac troponin I () to label the myocardium and Shox2. The black arrows indicate the myocardial border in wild type and Tbx18 mutant embryos and red arrows demonstte the absence of myocardium and Shox2 expression in the left superior caval vein of Tbx18 mutant embryo. rsc, right superior caval vein;, right atrium. Online Figure II Chacterization 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/-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 (Tbx18-positive cells), BrdU (prolifeting 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 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 Cre/Cre ). B and C, Prolifetion analysis using BrdU incorpotion (E12.5), B, Quantification of the BrdU labeling index in the sinus node head and the venous valve part of the sinus node sepately (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 mutant and wild type embryos (n=4). Online Figure IV Analysis of the sinus node development in Tbx18- double homozygous embryos (Tbx18 +/ ; +/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- 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 1 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 in a wild type sinus node, a -k in situ hybridizations of in a Tbx18 mutant sinus node.

5 Supplement Material S5 Online Figure VI Chacterization of the sinus node tail. A, The sinus node tail, which expresses, (indicated by a white arrow head) is negative for Tbx18 (wild type) and (Tbx18 mutant embryo), respectively. Shown is an E12.5 old embryo. B, In the sinus node tail, which is negative for connexin 4 (), is gdually upregulated at E17.5 in wild type and Tbx18 mutant embryo.

6 Online Figure I A mes sh head tail asvv C D V Cau B head asvv tail sh mes Tbx18 +/ Tbx18 E12.5 D Tbx18 +/ E1.5 C Tbx18 +/ E E Tbx18 Tbx18 Tbx18 /GFp Shox2

7 Online Figure II A Tbx18 +/ B 14 Tbx18 +/ sinus node head length (µm) * BrdU labeling index (%) E14.5 E17.5 C Tbx18 +/ rscv atrium atrium rscv BrdU DAPI D cell density (cells/µm 3 ) Tbx18 +/ BrdU DAPI

Online Figure III A C sinus node length (µm) 6 5 4 3 2 1

rscv Cre/Cre B BrdU labeling index (%) 18 16 14 12 1 8 6 4 2 D

8 Online Figure III A C sinus node length (µm) BrdU/Sytox. rscv Cre/Cre BrdU/Sytox. rscv Cre/Cre B BrdU labeling index (%) D cell density (cells/µm 3 ) Cre/Cre * * sinus node head venous valve (tail) Cre/Cre

9 Online Figure IV rvv ; Cre/Cre rvv rvv /Cre Tbx18/

10 Online Figure V E12.5 a f a' f' b g b' g' a c h c' h' d i d' i' e j e' j'

11 A Online Figure VI E12.5 Tbx18 B E17.5

a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53

1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1. Induction of p53 LOH by MADM. a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 mouse revealed increased p53 KO/KO (green,