University of Ulm Department of Internal Medicine I of the University Hospital Ulm Prof. Dr. med. Thomas Seufferlein

Tbx3 directs cell fate decision toward mesendoderm

Dissertation for obtaining the doctoral degree of Doctor of Medicine (Dr.med) of the Medical Faculty Ulm University

Submitted by Clair Elisabeth Hartmann (née Weidgang) born in Bonn, Germany 2015

Incumbent Dean : Prof. Dr. rer. nat. Thomas Wirth 1st Correspondence : apl. Prof. Dr. A. Kleger 2nd Correspondence : Prof. Dr. S. Liebau Day of Dissertation : 16.06.2016

This thesis is dedicated to my parents

Table of contents

Table of contents I

List of Abbreviations III

1. INTRODUCTION ...... - 1 -

Classes of stem cells and cell potency ...... - 2 -

Regulation of pluripotency ...... - 7 -

Early mouse embryonic development in vivo ...... - 13 -

The T-Box family ...... - 16 -

Aim of the thesis ...... - 22 -

2 MATERIAL AND METHODS ...... - 23 -

Material………………………………………………………………………... - 23 -

Methods ...... - 35 -

3 RESULTS ...... - 49 -

Tbx3 expression is vigorously regulated in mouse early embryonic development ...... - 49 -

Tbx3 drives mESC differentiation towards mesoderm and endoderm .... - 50 -

Tbx3 expression promotes mature mesendodermal-derived tissues ...... - 56 -

Tbx3 acts via a Nodal signalling dependent, non-cell-autonomous mechanism to drive mesendodermal cell fate...... - 58 -

Tbx3 directly regulates key mesendoderm inducting determinants ...... - 63 -

During early development Tbx3 is integrated in a complex regulatory mechanism with closely related T-Box factors ...... - 66 -

4 DISCUSSION ...... - 71 -

Impact of Tbx3 in sustaining and exiting pluripotency ...... - 71 -

I

Lineage fate of pluripotency factors ...... - 71 -

Tbx3 in vitro data matches it's in vivo counterpart ...... - 74 -

Tbx3 loss of function and compensating mechanisms ...... - 75 -

5 SUMMARY ...... - 78 -

6 REFERENCES ...... - 80 -

7 FIGURES AND TABLES ...... - 96 -

ACKNOWLEDGEMENTS…………………………………………………………..- 98 -

CURRICULUM VITAE………………………………………………………….… - 100 -

PUBLICATION LIST…………………………………………………………….... - 102 -

COMMENT (Licenses)………………………….…………………………………- 103-

II

List of Abbreviations

ALP alkaline phosphatase Alb Albumin APS Ammonium Persulfate ATG start codon Bp bFGF basic Growth Factor, FGF2, FGF  BMP morphogenetic BSA Bovine Serum Albumin CD31 Cluster of differentiation 31 (Pecam) Cdx2 Caudal type 2 Cer1 Cerberus ChIP Chromatin Immunoprecipitation Chrd Chordin CM conditioned medium CP crossing point CPC cardiac progenitor cells Cre cre recombinase DE definitive endoderm Dkk1 Dickkopf-related protein 1 DMEM Dulbecco's Modified Eagle's Media DMSO Dimethylsulfoxid DNA deoxyribonucleic acid cDNA complementary DNA dH2O distilled water, Ampuwa Dox Doxycycline DPBS Dulbecco's Phosphate-Buffered Saline dpc day post coitum Dpp4 Dipeptidylpeptidase 4 Dsh Dishevelled E embryonic day ECC embryonic carcinoma cell EDTA Ethylenediaminetetraacetic Acid III

EGC embryonic germ cell EB embryoid body EGFP enhanced Green fluorescent protein EMT epithelial-to-mesenchymal transition Eomes EpiSC epiblast stem cell ESC Esrrb Estrogen-related  ERK extracellular receptor kinase FACS Fluorescence Activated Cell Sorting FCS Fetal Calf Serum FGF Fibroblast growth factor FHF first field FoxA2 Forkhead-Box-Protein A Fwd forward GATA GATA binding protein Gbx2 Gastrulation homeobox 2 GFP Green fluorescent protein GMEM Glasgow Minimum Essential Media GS Goat Serum Gsc Goosecoid hESC human embryonic stem cell HCl Hydrochloric Acid HCN4 Hyperpolarization-Activated Cyclic Nucleotide-gated potassium channel 4 hHex Haematopoietic homeobox gene, Hex Hmbs Hydroxymethylbilane synthase HMG High-mobility group Hnf Hepatocyte nuclear factor HOS Holt-Oram syndrome HPRT Hypoxanthine-guanine phosphoribosyltransferase ICM inner cell mass Id Inhibitor of differentiation IF Immunofluorescence IV

IgG/M immunoglobulin G/M IMDM Iscove's Modified Dulbecco's Media ISH In situ hybridization iTbx3 tetracycline-controlled inducible Tbx3 expression system iPSC induced pluripotent stem cell JAK Janus kinase Krueppel-like factor 4 KDR Kinase insert domain receptor KO knockout LB Luria-Bertani Lefty Left-right determination factor Lhx1 LIM-homeobox1, LIM1 LIF Leukaemia Inhibitory Factor LIFR LIF receptor lox2272/ loxP cre recombinase cutting sites -ME -Mercaptoethanol MAPK Mitogen-activated protein kinase MEF mouse embryonic fibroblast Mef2c Myocyte enhancer factor 2C MEK Mitogen-activated protein/extracellular signal regulated kinase mESC mouse embryonic stem cell Mesp1 Mesoderm posterior 1 homolog MSC mesenchymal stem cell MTG Monothioglycerol Myh6 Myosin heavy chain 6 Myl Myosin light chain NEAA Non-Essential-Amino Acids neoR Neomycin Resistance Nkx2.5 NK2 homeobox 5 Nkx6.1 Nk6 homeobox 1 NP-40 Nonylphenolethoxylat with MO=40 NSC Oct3/4 Octamer binding 3/4, Otf3, Otf4, POU5F1 OFT outflow tract V o.N. overnight Otx2 PAGE Polyacrylamide gel electrophoresis Pax6 Paired box protein 6 PCR Polymerase chain reaction Pdx1 Pancreatic and duodenal homeobox 1 PFA Paraformaldehyde PBS Phosphate-buffered saline PGC primordial germ cell PGK Phosphoglycerokinase PI3K Phosphatidylinositide 3-kinase Pitx2 Paired-like homeodomain transcription factor 2 PLB Passive lysis buffer P/S Penicillin-Streptomycin qRT-PCR quantitative real-time polymerase chain reaction mRNA messenger RNA RI BMP receptor type I RII BMP receptor type II Rev reverse Rex1 Zfp42 RFP Red fluorescent protein RL Renilla Luciferase RLU relative luciferase unit RNA ribonucleic acid rtTA reverse tetracycline transactivator Sall4 Sal-like 4 SB421542 inhibitor of TGF subfamily, type I SCNT Somatic cell nuclear transfer SEM Standard error of the mean SHF second heart field SDS Sodium Dodecyl Sulfate SMAD Mothers against decapentaplegic homolog SRY (sex determining region Y)-box 2 Sox9 SRY (sex determining region Y)-box 9 VI

Sox17 SRY (sex determining region Y)-box 17 SSEA1 Stage-specific embryonic antigen 1 STAT3 Signal Transducers and Activators of Transcription 3 T TBE T-Box binding element TBS TRIS-buffered saline TBS-T TRIS-buffered saline+Tween TF transcription factor TGF Transforming growth factor 

Tm melting temperature TRE Tetracycline Response Element TRIS Tris-aminomethan Tbx2/3/6 T-Box transcription factor 2/3/6 TEMED Tetramethylethylendiamin UMS Ulnar Mammary Syndrome VEGF-A Vascular Endothelial Growth Factor A WB Western Blot WNT Wingless-related MMTV integration site WT wild-type

VII

1. Introduction

Pluripotency is characterised by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers and was first described by Stevens LC. He published a technique of deriving stem cells from teratocarcinomas, termed embryonic carcinoma cells (ECCs) [158], and was further able to establish a stable cell line [79]. In 1981 two independent groups [45] succeeded in establishing mouse embryonic stem cell (mESC) cultures by isolating pluripotent cells from the inner cell mass (ICM) of early mouse blastocysts and cultivating them in vitro using different techniques. They termed them ‘ESCs’ (embryonic stem cells) [45, 105]. Just less than 10 years later Thomson’s group published the establishment of ESCs lines derived from human blastocysts [172] and it was evident that there were major differences in the respective culture conditions. Since then, several seminal publications have been published revealing its increased progression and importance of stem cell research to mankind. Not surprisingly, the Nobel Prize in 2012 was given to two outstanding stem cell researchers Shin’ya Yamanaka and John Bertrand Gurdon. In 1962, J. B. Gurdon [62] delineated the transplantation of a nucleus obtained from differentiated intestinal of a donor Xenopus embryo, into an enucleated egg that finally gave rise to a normal developing tadpole. He concluded that nuclei of differentiated somatic cells contain the genetic information to ensure the differentiation programme during maturation and possess the ability to form all cell types. Later on, in 2006, S. Yamanaka [169] was able to reprogramme mouse embryonic and adult into a pluripotent state by adding four pivotal factors: Octamer binding transcription factor 3/4 (Oct3/4), SRY (sex determining region Y)-box 2 (Sox2), Krueppel-like factor 4 (Klf4) and c- in his experimental setup. Thus, the reprogrammed cells are referred to as induced pluripotent stem cells (iPSC) as they show ESC similar properties and express distinctive marker [59, 169]. The astonishing advances especially in mouse and later human ESC (hESC) research have enabled new opportunities for developmental biology, biology and cell therapies in regenerative medicine. Notably, recent years have illustrated novel facets of pluripotency factors initiating lineage differentiation [173]. In this regard they were classified depending on the promoted germ layer fate. Oct3/4, a master pluripotency factor, induces

- 1 - mesodermal lineages, thus being classified a mesendoderm-class gene, whereas Sox2 promotes neuroectoderm and was hence classified as a neuroectodermal- class gene. Notably, further crucial pluripotency factors have been classified to date, as mentioned below.

Classes of stem cells and cell potency Stem cells can generally be characterised by the following criteria: 1. the ability of unlimited proliferation and self-renewal, 2. high differentiation potential into various cell types and 3. the capability of regenerating damaged tissue [9].The broad differentiation potential into either members of their prospective lineage or any other cell of the entire organism, is called cell potency. Thus, cells of highest differentiation potential are referred to as totipotent, followed by pluripotent, multipotent, oligopotent and unipotent cells (Figure 1) [146]. Depending on the level of potency stem cells are segmented into different groups:

1.1.1 Totipotency The capacity of a cell to differentiate into any cell type including the extraembryonic tissue and most importantly upon transplantation of a single totipotent cell into pseudopregnant mother, being able to generate a complete viable organism, is called ‘totipotency’ [175]. In vivo, totipotency is represented by the zygote [175], the initial cell constituting the foundation of mammalian development and by the subsequent arising blastomeres present until the early morula stages (8-16 cell stage) (Figure 1). Besides, recent studies have described iPSCs with totipotent properties in vitro [169]. This was implemented by temporary induction of pivotal transcription factors (TFs) (Sox2, Klf4, c-Myc and Oct3/4) in mice. Please note, that this dissertation is focusing on mESCs if not other stated.

1.1.2 Pluripotency A pluripotent cell can differentiate into every cell type of the mature organism [190], albeit a full organism can’t be formed due to the lacking extraembryonic lineage. Pluripotent cells are exclusively found in the developing organism. Herein, the late stage morula, the ICM, the epiblast and primordial germ cells reveal pluripotent features [188] (Figure 1). There are two states of pluripotency, so called naive and primed. The ICM of the blastocyst and the early preimplantation

- 2 - epiblast show naive pluripotency, which is further mirrored in their in vitro counterparts, namely naive ESCs (see also below). After implantation, the epiblast undergoes morphological transformation and becomes an epitheliased cup- shaped egg cylinder. Stem cells derived from the epitheliased epiblast are no longer referred to as ESCs, but epiblast stem cell (EpiSCs). Further, there are also molecular changes as for X- inactivation in female embryos (XX) during cleavage which is the premise for exit of pluripotency towards lineage specification [61]. While in vivo these cells are rather transient, stable in vitro counterparts can be generated under the appropriate culture conditions as outlined below.

1.1.2.1 Embryonic stem cells mESCs mirror the naive state of pluripotency and are derived from the ICM of the mammalian blastocyst [175]. They are characterised by the capacity of high proliferation, chromosomal stability and continuous self-renewal potential while maintaining the ability to differentiate into cells of all three germ layers: ectoderm, endoderm, mesoderm and further into the germ lineage. Their pluripotent properties can be demonstrated in vivo through blastocyst injection. The resulting mouse chimaeras reveal the ability of contributing to all three germ lines including germ line chimaeras in case of germ line transmission [17]. Isolation techniques of mESCs: Evans and Kaufman were the first to obtain euploid pluripotent stem cells from mouse blastocysts in an in vitro culture system. They described a technique of delaying the implantation of an embryo by ovariectomy of the donor mother, combined with progesterone treatment. The intrauterine culture was isolated followed by in vitro culture, until the cells of the ICM had formed an ‘egg cylinder-like structure’. These were then co-cultured on inactivated mouse embryonic fibroblast cells (MEFs), either by Mitomycin C inactivation or ϒ-irradiation, forming clonal cell lines in the end [45]. However, G. Martin described a different technique, whereby the embryo was removed from the uterus and cultured in vitro. Subsequently, the trophoblast layer of the blastocyst was removed via immunosurgery, leaving an intact ICM [152] which was then cultivated in ECC conditioned media (CM), complemented with factors which were assumed to stimulate the division of stem cells. Shortly after, colonies had derived demonstrating pluripotent properties. Martin termed them ‘ESCs’ [105].

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Properties of mESCs: mESCs are characterised by stage-specific molecules (e.g. SSEA-1, alkaline phosphatase (APS)) and TFs (e.g. Oct3/4, Nanog). Stage- specific embryonic antigen (SSEA1) is expressed from the 8 cell stage onwards and partly in ICM cells [91], later in germ cell (PGCs) [138] and neural lineage development [40]. Further, TFs will be outlined below. Morphologically, mESCs grow in round, multilayered clusters and are able to form embryoid bodies (EBs, three-dimensional bodies). Differences between hESCs and mESCs: Morphologically naive pluripotent stem cells grow in dome-shaped compact colonies, whereas primed cells, grow in flat colonies [68]. Already based on these characteristics hESCs more closely resemble mouse EpiSCs and correlate more to the late stage epiblast in vivo. Besides, there are molecular differences between hESCs and mESCs. hESCs don’t express SSEA 1 but show high expression levels of TRA-1-60, TRA-1-81 and SSEA-3 [20]. Further, self-renewal in mESCs is mainly regulated via Leukaemia Inhibitory Factor (LIF) and Bone morphogenetic protein 4 (BMP4) whereas hESCs are dependent on basic Fibroblast growth factor (bFGF) a feature also correlating with mouse EpiSCs. LIF is redundant for maintaining pluripotency in hESCs. There are some similarities between them, for example mESCs and hESCs express Sox2, Nanog and Oct3/4 and both are able to form teratomas in vivo [189].

1.1.2.2 Other pluripotent cell types: Embryonic germ cells PGCs can be isolated from developing mammalian gonads on 10.5-11.5 dpc and are cultured as so-called EGCs under similar culture conditions as ESCs [114, 142]. Epiblast stem cells EpiSCs are mainly isolated from epitheliased postimplantation epiblasts [118] and have the ability to differentiate into all three germ layers, PGCs and further form teratomas. EpiSCs exhibit primed pluripotency, thus showing distinct differential potential from ESCs and have a much lower capacity to form blastocyst chimeras and usually do not convert to the naive stage albeit certain culture conditions can

- 4 - overcome these limitation [61, 66]. Like ESCs, EpiSCs express pivotal pluripotency factors (Oct3/4, Sox2, Nanog) [198]. Embryonic carcinoma cells ECCs are stem cells obtained from germ line tumors, referred to as teratocarcinomas, and are able to differentiate into the three primary germ layers [90, 106, 189]. They are also able to form chimeras under defined circumstances but nevertheless their presence during normal embryogenesis is not common [106, 111, 128].

1.1.3 Induced pluripotency Induced pluripotency is achieved by enforcing the combined expression of a set of TFs, resulting in cells exhibiting an embryonic-like [155]. These so called iPSCs are gaining more and more importance in regenerative medicine and especially in analysing patient specific defects. Yamanaka hypothesised, that unfertilised eggs and ESCs possess the ability to convert somatic cells into an embryo-like state. In 2006, he first published the successful generation of iPSCs in mice by retroviral transduction of four pivotal factors [169] and laid the foundation for direct reprogramming. Nonetheless, the overexpression of the TFs (Oct3/4, Sox2, Klf4, c-Myc) in somatic cells is sufficient to achieve iPSCs, yet at a low frequency. Alternatively induced pluripotency can be obtained by either Somatic cell nuclear transfer (SCNT) or Cell fusion. In SCNT the epigenome of a differentiated cell can be reset into a pluripotent state [72] by transferring a highly differentiated nucleus into a previously enucleated, unfertilised somatic cell. Subsequently, after transfer into a surrogate mother, a viable offspring (clone) emerges [19, 185]. Cell fusion has been implemented to study the plasticity of the differentiated state [72]. It takes advantage of the fact, that the phenotype of the less-differentiated partner in hybrids is dominant [164-166]. Briefly, pluripotent cells are fused with differentiated somatic cells, resulting in a somatic cell exhibiting pluripotent properties [72] and a stable karyotype [36].

1.1.4 Multipotency MSCs (mesenchymal stem cells) are multipotent, tissue specific stem cells possessing the ability of self-renewal. They are usually located in various adult

- 5 - tissues [175], thus playing an important role during tissue repair. MSCs preferentially differentiate into cells of the same lineage their tissue originates from (Figure 1). As an example haematopoietic stem cells progress by developing into blood cells, bone marrow MSCs give rise to mesodermal derivatives and neural stem cell (NSC) develop into and glia. Thus, in comparison to ESCs, their differentiation range is limited to a certain number of lineages [174]. To date, about 20 different somatic stem cell classes are known in the human body, for example in the brain, liver, haematopoietic system, skin and intestines [9]. Recent studies have demonstrated a phenomenon called transdifferentiation, describing a process where cells which are differentiating along a specific developmental lineage are able to cross germinal boundaries and derive cells from another lineage. This stunning insight shows the multidifferentiation potential of MSCs although they lineage specific commitment has already occurred [154].

Figure 1 Hierarchy of mammalian ESC development and their impact on differentiation in vivo and in vitro. The image illustrates early developmental stages of the mouse. Zygote and (8 cell stage) morula are defined as totipotent. Subsequently, the morula differentiates into the blastocyst which consists of the outer trophoblast and the ICM. After reaching the blastocyst stage the developmental potential is slightly reduced and is referred to as pluripotent. Throughout continuous development (in vivo) the cell potential is reduced and finally is determinated to one cell type. Pluripotent stem cells for experimental procedures are isolated from the blastocyst stage. Abbreviations: CNS: central nervous system, ESC: embryonic stem cell, ICM: inner cell mass.

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Regulation of pluripotency An orchestra of signalling axis precisely regulates ground state conditions (Figure 2) and acts in concert with a combination of key TFs. Notably, LIF is indispensable for sustaining mESC pluripotency in vitro [37, 159]. It`s main target is Signal Transducers and Activators of Transcription 3 (STAT3), nevertheless it also activates parallel circuits as for the PI3K- and extracellular receptor kinase (ERK)- pathway.

1.2.1 Signalling cascades impacting pluripotency Leukaemia Inhibitory Factor signalling LIF belongs to the `helical type I´ Interleukin 6 class cytokines and acts through the LIF receptor (LIFR)/gp130 receptor by activating STAT3, an essential regulator of mESC self-renewal [39]. Its impact in maintaining pluripotency in vitro has been demonstrated in several facets [122, 124]. Further, LIF plays a crucial role during embryogenesis [110]. LIF knockout (KO) mice were used to demonstrate its importance of transient expression for the implantation process of the blastocyst. Thus, female mice lacking LIF were fertile, albeit the embryo was neither able to attach to the uterus nor to develop. Notably, this phenomenon was abolished by transferring the viable blastocysts into a wild-type (WT) mouse [159]. LIF/STAT3 pathway: STAT3 belongs to the family of Signal Transducers and Activators of Transcription and acts as intracellular messengers [122, 156]. LIF strongly induces its expression levels in order to maintain ‘stemness’ in vitro by signalling through a transmembrane complex consisting of the common receptor subunit gp130 which is linked to the low-affinity LIFR [122] (Figure 2). Briefly, LIF first binds to the specific LIFR (gp190), which in turn, forms a heterodimer with the common gp130 (IL6-) signal transducing subunit. Subsequently, the formed complex activates the receptor-associated Janus kinase (JAK) which phosphorylates both subunits. Next STAT3 molecules are recruited and when bound to the receptor complex, phosphorylated by JAKs, respectively. Finally, phosphorylated STAT3 molecules dimerise and translocate into the nucleus where they activate key target genes, such as Myc [23], resulting in the promotion of self- renewal and inhibition of mesodermal and endodermal differentiation [122]. Experiments using a STAT3 mutant (STAT3F) illustrated a loss of self-renewal

- 7 - capacity towards differentiation, showing STAT3 to be an essential component of the LIF-dependent self-renewal [122]. LIF-/ERK-/PI3K-pathway: Besides LIF-STAT3 signalling, there are two other LIF signalling pathways which occur in parallel (Figure 2). PI3K has been shown to maintain self-renewal in mESCs by reducing the ubiquitin dependent degradation of -catenin via GSK3 inhibition [127]. Upon its inhibition mESCs lose their pluripotent phenotype and start to differentiate. This is due to LIF-dependent activation of the third target, namely ERK [136]. ERK belongs to the MAPK (Mitogen-activated protein kinase) pathway promoting early cell fate decision in the preimplantation embryo [134] and is present from the 2 cell until the blastocyst stage [181]. It is not completely clarified why a differentiation promoting pathway is activated while attempting to maintain self-renewal, but it is assumed that pluripotency is obtained by a balance between the pluripotent (LIF/STAT3 activation) and the differential state.

BMP-ID-pathway LIF isn’t able to maintain the self-renewal state in a serum-free culture setting and at the same time prevent ectodermal differentiation [193]. Therefore, another crucial component required for mESC culture is BMP. BMP4 (and BMP2), a known ectoderm-antagonist [97, 186], successfully replaces serum requirements resulting in maintenance of mESC pluripotency and inhibition of multi-lineage differentiation under continuous LIF stimulation [194] (Figure 2). However, in the absence of LIF, BMP induces mesodermal differentiation at the expense of the neural lineage [48]. Besides, another BMP4-dependent signalling pathway has been described to maintain the self-renewal state. Thus, BMP4 promotes mESC pluripotency via MAPK pathway (p38, ERK) inhibition [134]

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Figure 2 Signalling pathways regulating mESC pluripotency. A schematic representation of the main extrinsic pathways (LIF, BMP, WNT) regulating pluripotency in mESCs. Upon LIF-binding to its receptor, gp130 phosphorylates JAK, which in turn phosphorylates STAT3. Phosphorylated STAT3 operates as a transcription factor which sustains the pluripotency state in mESCs and further inhibits endodermal and mesodermal differentiation. LIF further inhibits GSK3 via PI3K. GSK3 stabilises the self-renewal state by reducing the ubiquitin-dependent -catenin. Further, WNT bind the Frizzled receptor which in turn forms a complex with LRP5/6 protein and signal downstream via Dsh and -catenin.  - catenin accumulates in the cytoplasm and nucleus resulting in STAT3 transcription. STAT3 is activated through JAK phosphorylation. Upon BMP4-receptor complex formation, BMP RI phosphorylates SMAD1 and SMAD 5 which then interacts with SMAD4 resulting in Id gene transcription and maintenance of the pluripotency state. RI also inhibits MAPK which in turn diminishes its negative impact on phosphorylated SMAD1. Abbreviations: BMP: Bone morphogenetic protein, Dsh: Dishevelled, ERK: extracellular receptor kinase, Id: Inhibitor of differentiation, JAK: Janus kinase, mESCs: murine embryoid stem cells. LIF: Leukaemia Inhibitory Factor, MAPK: Mitogen-activated protein kinase, Oct3/4: Octamer binding transcription factor 3/4, PI3K: Phosphatidylinositide 3-kinase, SMAD 1/4/5: Mothers against decapentaplegic homolog 1/4/5, STAT3: Signal Transducers and Activators of Transcription 3, RI: BMP receptor type I, RII: BMP receptor type II, WNT: Wingless-related MMTV integration site. Pluripotency Factors on their lineage move. Weidgang C, Seufferlein T, Kleger A, Mueller M. Stem Cells International, vol. 2016, Article ID 6838253, 16 pages, 2016. doi: 10.1155/2016/6838253; License (CC-BY-4.0); Link https://creativecommons.org/licenses/by/4.0/. Reprinted by permission from Hindawi Publishing Corporation. - 9 -

1.2.2 Core transcription factors of the pluripotency network Signalling pathways are not solely responsible for maintaining pluripotency in mESCs. Therefore pluripotency-inducing TFs have been integrated into the core regulatory network of pluripotency. They are activated by signalling pathways like LIF or by mutual interactions (Figure 6), resulting in a strong regulation of their expression levels [107]. Further, the signalling occurs in parallel pathways demonstrating its robustness. Briefly, the pluripotent state is maintained by a unique network of interacting TFs (especially Oct3/4, Sox2, Nanog, Tbx3, Klf4/5 among others) by promoting pluripotency-associated genes and further preventing exit of pluripotency by inhibiting target required for lineage differentiation. Recent publications have emphasised their role during lineage allocation illustrating their function as molecular switches to induce or repress specific gene expression programmes. We want to outline important features of the key players involved in pluripotency.

1.2.2.1 Oct3/4 Oct3/4 is a mammalian homeodomain TF of the POU (Pit-Oct-Unc) family. It is a master regulator of mESC pluripotency in vitro and in vivo. [121], hereby functioning in a complex, consisting of Nanog, Oct3/4 and Sox2 [140] and further interacting with several signalling pathways [123, 124]. It also has the ability, together with Sox2, to bind a unique region, the Oct/Sox motif. This highly conserved element is located on numerous genes (Oct3/4, Nanog, Sox2) participating in mESC pluripotency [92, 145]. Oct3/4 is also able to modulate stem cell fate depending on its expression levels [135]. This was recently illustrated by Chambers Laboratory who showed heterozygous mESCs to adequately maintain mESC pluripotency via WNT and LIF signalling. Notably, WNT signalling was reduced in homozygous Oct3/4 mESCs destabilising the pluripotency levels resulting in differentiation [83]. Interestingly, it also participates in early lineage decisions, thus guiding proper segregation of the ICM and the trophectoderm lineage. Here, Oct3/4 promotes ICM via inhibition of trophectoderm formation via the Oct3/4-Cdx2 complex, thus representing a negative regulator of Cdx2 (Caudal type homeobox 2) [162]. In the postgastrulation stage Oct3/4 promotes primitive endodermal, mesendodermal gene expression and is expressed in germ cells

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(Figure 4) [123, 131]. Oct3/4-deficient mouse embryos develop to the blastocyst stage but fail to form a pluripotent ICM thus resulting in embryonic lethality due to differentiation into the extraembryonic trophoblast lineage [121].

1.2.2.2 Sox2 Sox2 represents another crucial key player of mESC pluripotency in mESCs. It is a member of the HMG (High-mobility group) box proteins. In mESCs Sox2 preferentially interacts with Oct3/4 in a reciprocal fashion via the highly conserved Oct/Sox motif [31]. In the pregastrulation embryo, Sox2 is mainly expressed in the ICM, epiblast and trophectoderm [85]. Like Oct3/4, Sox2 is dependent on tightly regulated expression levels to sustain self-renewal. Periimplantation lethality of Sox2 KO embryos is due to absent epiblast development, further mESCs derived from these embryos are unable to maintain the self-renewal state [8]. This is due to either low or stable Oct3/4 and Nanog expression levels and at the same time low trophectoderm (e.g. Eomesodermin (Eomes)) expression [85]. Stable Oct3/4 and Nanog levels seem surprising regarding the core role of Sox2 in the pluripotency circuity. This finding could be described by autoregulatory functions which have previously been described for Oct3/4, Nanog, Klfs [80]. However, recent studies demonstrated trophectoderm lineage differentiation upon reduction of Sox2 levels [95] which has been suggested to be due to a secondary loss of Oct3/4 upon Sox2 withdrawal, thus missing trophectoderm inhibition. Sox2 heterozygous KO demonstrated severe defects in the brain [47]. This is in line with previous work demonstrating its expression in the neuroectoderm lineage and further to the germ layer upon differentiation onset (Figure 4) [8, 197].

1.2.2.3 Nanog Nanog is a homeodomain TF and essential for mESC pluripotency. Nanog acts in concert with Oct3/4 and Sox2 to maintain the complex pluripotency network of mESCs by direct binding of Oct3/4 and Sox2 through the Oct/Sox motif [140]. Interestingly, Nanog overexpression is sufficient to maintain pluripotency of mESCs independently from LIF/STAT3, albeit at a reduced self-renewal capacity level [112]. Nanog-null mice were first thought to be incapable of maintaining mESC self-renewal as mESCs lost their pluripotent identity and differentiated towards the extraembryonic endoderm lineage [112]. Recent studies, however,

- 11 - have suggested Nanog to be dispensable for self-renewal thus ascribing its stabilising role for mESC pluripotency [24]. Nanog is first expressed in the morula stage and upon differentiation gets limited to the ICM and finally epiblast (Figure 4) [92]. Here it functions during early lineage decisions by promoting ICM on behalf of trophectoderm [112]. Like Oct3/4, Nanog has been classified as a mesendodermal-class gene, however lineage specification roles for mouse are still missing [173]. Its presence during germ cell development has also been reported [24].

1.2.2.4 Klf4 Krueppel-like Factor (Klf4, previously known as gut-enriched Krueppel-like Factor (GLKF)), a zinkfinger TF, belongs to the Klf family and is also a member of the core transcriptional pluripotency circuit. Klf4 together with Klf2 and Klf5 has great impact on maintaining pluripotency, whereas LIF strongly enforces Klf4 expression and Oct3/4 directly promotes Klf2 activation, resulting in naive mESC pluripotency [65]. The precise mechanism of how Klf4 maintains self-renewal is still not clear but a recent paper revealed a functional role of Klf4, by describing chromosomal changes in the Oct3/4 in pluripotent mESCs. This was also described regarding reprogramming [182] where Klf4 is a part of the four pivotal factors essentital for iPSCs formation described by Yamanaka [169]. Recent experimental settings have revealed the inhibition of visceral endoderm and definitive endoderm (SRY (sex determining region Y)-box 17 (Sox17), GATA binding protein 6 (GATA6)) by Klf4 and mesodermal lineage genes (T, Mixl1) by Klf5 to ensure self- renewal [3] (Figure 4). Klf4 KO by LIF withdrawl induced differentitation whereas Klf4 overexpression strongly promotes pluripotency. Klf4 KO mice die postnatal due to basal membrane formation problems and tissue abnormalities within the smooth and [195].

1.2.2.5 Brief overview of other core transcription network factors The comprehension of stem cell behaviour was an illusory thought for many years. Its large complexity to maintain self-renewal seemed infinite. Dunn et al. was able to reduce the complex regulation of naive pluripotency to a simple molecular programme containing 16 interactions between 12 components (Gbx2, Esrrb, Klf2/4, Nanog, Oct3/4, Sal-like 4 (Sall4), Sox2, STAT3, Tfcp2l1) [43].

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Early mouse embryonic development in vivo Preimplantation blastocyst stage (E0 – E4.25): Briefly, an oocyte is fertilised by a sperm, generating a zygote, depicting the earliest developmental stage of a mammalian embryo. Shortly after, the zygote undergoes cleavage expeditious mitotic divisions form the morula. After the 7th cleavage the morula consists of about 128 cells and is no longer referred to as morula but blastocyst (embryonic day 3.5 (E3.5)). The latter comprises of an outer layer, the trophectoderm, surrounding a mass of pluripotent cells called ICM, both emerging from the first lineage decision [81] (Figure 3).

Figure 3 Preimplantation development of mammalian embryo. The initial embryo is formed by fusion of two haploid gametes forming the zygote. The zygote undergoes cleavage, resulting in the formation of the morula. At E3.5 the morula has experienced further cleavages and develops into the blastocyst containing the pluripotent ICM, surrounded by trophectoderm. Before implantation at E4.0 the ICM divides into the epiblast and the primitive endoderm. Abbreviations: E: embryonic day, ICM: inner cell mass, PrE: primitive endoderm, TE: trophectoderm.

Implantation (E4.0 – E4.5): Shortly before implantation the second lineage decision occurs. Here, the ICM segregates into two layers, the extraembryonic primitive endoderm and the epiblast. Previous work showed subpopulations formed within the ICM expressing either GATA6 or Nanog [28]. These subpopulations are committed to form either primitive endoderm, later giving rise to the visceral and parietal layers of the yolk sac [53], or the epiblast (primitive ectoderm), eventually giving rise to primary germ layers. Trophectoderm, the outlying tissue of extraembryonic origin, will generate the chorion and placenta (Figure 4).

Post-implantation (E4.5–E6.0): After implantation the blastocyst becomes asymmetric thereby forming a cavity within the epiblast and extending along P-D axis, forming the ‘egg cylinder’ stage. The extraembryonic tissue settles as a cup- - 13 - shaped layer of epithelial cells on the proximal pole. This process is further solidified by distal visceral endoderm moving to the anterior pole of the embryo to finally form the anterior visceral endoderm.

Figure 4 Pluripotency factor expression throughout early embryonic stages. (A) The early blastocyst consists of the ICM surrounded by trophectoderm. Pluripotency factors expressed in the ICM interact with trophectoderm genes for proper lineage development. (B) At the late blastocyst stage, the ICM develops into the epiblast, revealing slightly different gene expression patterns, and into the extraembryonic endoderm, later generating the yolk sac. Further, the trophectoderm has formed the extraembryonic ectoderm lineage which will give rise to the placenta. (C) Subsequently, after implantation the primitive ectoderm, which has origins in the epiblast, forms the four embryo-derived stem cell lineages (ectoderm, mesoderm, endoderm and germ lineage). Notably, pluripotency factors drive early embryonic decision according to their expression profile. Abbreviations: Cdx2: Caudal type homeobox 2, Eomes: Eomesodermin, ICM: inner cell mass, Esrrb: Estrogen-related receptor , Klf4/5: Krueppel-like factor 4/5, Oct3/4: Octamer binding transcription factor 3/4, Rex1: Zfp42, Sall4: Sal-like 4, Sox2: Sex determining region Y (SRY) - box 2, Tbx3: T-Box transcription factor 3. Pluripotency Factors on their lineage move. Weidgang C, Seufferlein T, Kleger A, Mueller M. Stem Cells International, vol. 2016, Article ID 6838253, 16 pages, 2016. doi: 10.1155/2016/6838253; License (CC-BY-4.0); Link https://creativecommons.org/licenses/by/4.0/. Reprinted by permission from Hindawi Publishing Corporation. - 14 -

Gastrulation (E6.5): The symmetrical embryo becomes asymmetric, later pear- shaped and is further prepatterned by regional fluctuating levels of signalling pathways along the embryonic axes, accompanied by the expression of early differentiation marker genes [7]. Around E6.25 the induction of primitive streak formation proceeds at the posterior proximal pole of the epiblast bordering on the extraembryonic ectoderm [139]. Briefly, mesoderm is formed and intercalates between ectoderm and endoderm. Mesoderm generation is induced in epiblast cells undergoing EMT (epithelial-to-mesenchymal transition) by BMP4 and Nodal- SMAD2/3 (Mothers against decapentaplegic homolog 2/3) signals. Prospective derivatives of the ectoderm lineage, the sole of the three germ layers developing independent of the primitive streak, are the neural tube, neural crest cells which contribute to the craniofacial patterning and the epidermis [52]. Mesoderm gives rise to somites forming muscle and blood vessels among others liver, pancreas, the epithelium of and digestive system originate from the endoderm [42, 147].

Progression of gastrulation: In the following, definitive endoderm and anterior mesoderm derivatives, including cardiovascular progenitors, are generated from a transient precursor cell population, referred to as mesendoderm, located in the region of the anterior primitive streak. This cell population is marked by the expression of genes including Chordin (Chrd), Eomesodermin (Eomes), Forkhead- Box-Protein A (Foxa2), Goosecoid (Gsc), and Lhx1 (LIM-homeobox1, Lim1) [167]. In due course developmental progression of the now more specified germ layers towards their derivatives will occur:

Endoderm formation (hepatic and pancreatic development): Briefly, endoderm is regarded as the innermost germ layer of the embryo. During gastrulation definitive endoderm is first detected while egressing from the primitive streak into the visceral lineage and thereby progressively displacing the visceral endoderm to the extraembryonic yolk sac [94]. At the end of gastrulation definitive endoderm forms the epithelium of the primitive gut tube. Preceding organogenesis the anterior gut tube develops into the foregut, giving rise to liver, lung, and ventral pancreas, whereas the hindgut arises from the posterior par, finally generating the large intestine. On gene expression levels Sox17 emerges very early and marks visceral endoderm and definitive endoderm. Subsequently, during definitive endoderm

- 15 - specification liver and pancreas originate. Hepatic progenitor cells express Hnf4[163], more mature cells express Albumin (Alb) [113]. Early pancreatic progenitor cells on the other hand express Hnf1 [163], whereas more specified pancreatic anlagen express Pdx1 [103], a pancreatic homeodomain TF, first expressed in cells of the posterior foregut and midgut. Heterozygous mutations display different types of diabetes, whereas homozygous Pdx1 aberrations in mice and human can result in pancreatic agenesis [126, 160].

Heart formation: Murine cardiogenesis is already initiated during gastrulation (E7.5) when mesodermal cells migrate to the antero-posterior (A-P) side of the embryo and is guided by the earliest marker of the cardiac lineage Mesoderm posterior 1 homolog (Mesp1) [89].The anterior lateral plate mesoderm then segregates into somatic and splanchnic layers. At about E7.75 cardiac progenitor cells (CPC) originating from the latter will form an epithelial structure called the crescent mesoderm [170]. CPCs, expressing NK2 homeobox 5 (Nkx2.5) [14], represent progenitors of two distinct cardiac lineages called first heart field (FHF) and second heart field (SHF). Subsequently, cells of the FHF blend at the midline forming the primordial heart tube, consisting of an anterior (arterial) outflow and a posterior (venous) inflow pole. Shortly after, between E8.25-E.10.5, cardiac looping occurs. The FHF (T-Box transcription factor 5 (Tbx5) [71], Myosin light chain (Myl2a) and T-Box transcription factor 20 (Tbx20) [191]) gives rise to the left ventricular and further left and right atria. The SHF (Islet1 [191], Myocyte enhancer factor 2C (Mef2c) [98]) remains in a longer CPC-state and contributes to the right ventricle and outflow tract (OFT) formation (Paired-like homeodomain transcription factor 2 (Pitx2)) [71, 196]. Maturation of myocardial development is marked by sarcomeric protein expressing -actinin.

The T-Box gene family T-Box factors are transcriptional regulators playing a crucial role in a wide range of developmental processes particularly in early embryonic development, cell fate decision and organogenesis. This is due to several aspects: they show specific and dynamic expression patterns during early developmental stages. Further genetic abnormalities in mice result in severe developmental and mutations in these genes have been linked to developmental syndromes in - 16 - humans [21, 71]. Their DNA-binding site is called T-Box [117] and has been identified in metazoan species, including human and mouse. T-Box genes can be classified phylogenetically by assorting them into subfamilies referring to similar sequences coding for their T-Box domain, thus providing insight into their evolutionary origin (Figure 5). Notably, studies showed that Tbx2/3 and Tbx4/5 were akin due to their high genetic analogy and their similar phenotype [26]. Interestingly, Tbx3 and Tbx2 are able to substitute for each other because of their cognate gene sets [26].

T-Box domain structure and binding mechanisms: The T-Box encodes a T-Box binding sequence, or T-domain that consists of a palindromic sequence and binds to specific sequences in the promoter of target genes. The DNA binding domain was first discovered in 1993 showing the highest affinity to the T protein, thus terming it T-Box binding element (TBE). We can distinguish between a highly conserved N-terminal harboring the DNA binding domain and a less conserved C- terminal half, functioning as a transcriptional modulation domain and showing properties [88]. Previous studies demonstrated T-binding to the consensus target site as a dimer with each monomer attaching to the same amino acids at two different DNA half-sites (T half-site) [87]. Interestingly other T-Box proteins reveal the same binding manner but prefer slightly different target sequences and half-site preferences. T-Box proteins bind the T-domain structure in the minor and major grooves [116].

1.4.1 Brief overview of the most important T-Box factors involved in early embryogenesis Brachyury (T) represents the T-Box family’s most historical member [184] and is expressed in the early primitive streak. It is required for early mesoderm formation and morphogenesis of the notochord [179]. Heterozygous mutant mice show incomplete mesoderm and axial development, demonstrating a phenotype ranging from a short tail to the malformation of sacral vertebrae. Homozygous mice die in utero at midgestation due to posterior mesodermal aberration or more specifically due to lack of the allantois [57].

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Eomesodermin (Eomes, also referred to as Tbr2), belongs to the Tbr1 subfamily. Its role during gastrulation was first described in Xenopus [144]. Eomes performs essential functions during gastrulation as for A-P axis formation, anterior visceral endoderm induction, EMT, definitive endoderm and cardiac mesoderm delamination. In later stages it induces trophoblast development and governs the specification of definitive endoderm and cardiac mesoderm [6, 35]. Heterozygous mutant mouse embryos have a nondescript phenotype whereas homozygous mutated mice die at the blastocyst stage not being able to form organised embryonic and extraembryonic structures. This is assumed to be due to defective trophectoderm development, which causes an unsuccessful uterine implantation in vivo [143].

Tbx2 belongs to the Tbx2 subfamily, depicting the structural and functional homologue of Tbx3 by sharing 90% of sequence alignment [1]. However it plays a minor role in maintaining pluripotency. Like Tbx3, Tbx2 exhibits an important role during organogenesis especially in the developing vertebrate heart, thus being expressed in the inflow tract, atrioventricular canal, inner curvatures and the OFT [70]. Further, Tbx2 is also crucial for hindlimb formation [56]. Mice with heterozygous Tbx2 mutations show no specific phenotype, whereas mice displaying homozygous null mutations depict limb abnormalities and severe cardiovascular defects leading to fetal mortality [70]. Notably, Tbx2 has also been described in murine endocrine and ductal cells of pancreas, unlike Tbx3 which has been described to be expressed in exocrine pancreas [13].

Tbx6 has been classified into the Tbx6 subfamily, thus showing little phylogenetical relationship to Tbx3. Nevertheless, Tbx6 expression profiles show a high functional relationship to Tbx3, as both are coexpressed during differentiation in vitro [130] and in vivo [64]. Intriguingly, Tbx6 is required for mesoderm specification and patterning during gastrulation and particularly plays a crucial role in specification of the posterior-paraxial mesoderm towards mesodermal fate. Heterozygous mutant show an inconspicuous phenotype, whereas homozygous mutant mice die in midgestation mainly due to vascular abnormalities [25, 27].

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Figure 5 Origin of T-Box genes in vertebrates. Developmental hierarchy of T-Box transcription factors resulting in genetically closely related genes with similar properties. Tbx3 belongs to the Tbx2 family, thus Tbx2 depicts a structural and functional homologue of Tbx3. Tbx2 and Tbx3 exhibit an important role during organogenesis, especially during cardiogenesis. Abbreviations: Eomes: Eomesodermin, T: Brachyury, Tbx: T-Box transcription factor. Reprinted by permission from Elsevier Publishers Ltd [183].

1.4.1.1 Tbx3 The murine T-Box 3 gene (Tbx3, also referred to as D5Ertd189e) was first discovered in 1994 as a family member of genes that showed in the T- domain. Tbx2, Tbx3, Tbx4 and Tbx5 were further classified into the Tbx2 subfamily due to their highly [15].The murine Tbx3 gene is situated on chromosome 5 [178] whereas the human ortholog is located on chromosome 12 [177]. At present, there are various Tbx3 orthologs known in several species such as chicken, lizard, rat, Xenopus, zebrafish [54]. Depending on alternative splicing Tbx3 exhibits various splice isoforms which to date seem to function similarly [73].

Tbx3 expression in mESCs and during early stages of embryonic development: Already at very early stages of murine embryonic development Tbx3 expression can be identified within the ICM. Here, Tbx3, together with previously identified factors namely Oct3/4, Nanog and Sox2, is able to maintain pluripotency by mediating via LIF/STAT3 signalling [78, 124]. In more detail, maintaining self- renewal is a process involving various actions running in parallel. This tightly regulated transcriptional network downstream of LIF activates Tbx3 via the PI(3)K- Akt pathway and Klf4 via the JAK-STAT3 pathway. Both Tbx3 and Klf4 are TFs - 19 - inducing Nanog and Sox2 expression resulting in Oct3/4 maintenance, the core member for pluripotency maintenance. During gastrulation of the mouse embryo, Tbx3 is expressed in mesodermal as well as endodermal progenitor lineages (yolk sac, definitive endoderm progenitor cells [30, 38]). In addition, Tbx3 together with the histone demethylase JMJD3 and Eomes are involved in endoderm formation [82]. In midgestation, Tbx3 plays an important role in cardiac chamber specification, hepatic bud expansion, and limb patterning [26, 38, 101, 108]. A minor role was identified in the craniofacial region (pharynx, thyroid) and weakly in the central nervous system [11, 168] and mesenchyme of the lung [26]. Tbx3 also modulates the formation of extraembryonic visceral endoderm by directly activating GATA6 expression [100] and further acts as a downstream activator of WNT (wingless-related MMTV integration site) signalling [133]. Tbx3 improves iPSC quality significantly especially regarding germ-line transmission and the high efficiency [67] by direct binding and activation of the Oct3/4 promoter. Regarding the strong position in the core pluripotency network its role during tumorigenesis is not surprising. Its overexpression has been associated with various as for liver tumor whereby Tbx3 is downstream of the WNT/-catenin pathway [137] . Tbx3 is also highly expressed in breast cancer by inhibiting p14ARF and [192] promotes invasive melanoma formation [141]. Tbx3 expression patterns during embryonic development correlate with phenotypic abnormalities in its mutants. Tbx3 heterozygous mice are viable and fertile but exhibit abnormal external genitalia. In contrast homozygous mutated mice demonstrate hindlimb and mammary gland abnormalities and also yolk sac defects. The latter is essential to the embryo due to embryonic haematopoiesis and maternal-fetal exchange. It has been assumed that yolk sac defects are the main cause of embryonic lethality in early stages [38]. The time point of death varies, so does the yolk sac phenotype. In the literature homozygous mutated mice die in midgestation. Some embryos survive until late gestation when they die of unknown causes. Tbx3 in humans is referred to as Ulnar Mammary Syndrome (UMS). It is likewise inherited in an autosomal dominant mode [11] and is characterised by forelimb defects and hypoplastic mammary and apocrine glands. UMS shows a high variable clinical presentation, typical symptoms being delayed puberty, deficient urogenital and tooth development, ventricular septal defects and posterior foot defects. A human homozygous Tbx3 mutant has not - 20 - been reported to date [11, 29]. Notably, the mutation site of UMS contains a locus for the Holt-Oram syndrome (HOS) which hints to an allelic similarity of UMS due to Tbx3 and HOS due to Tbx5 mutation [10]. Like UMS, HOS is also due to an autosomal dominant inheritance mode, involving similar organs [150].

Figure 6 Core transcription factors maintain pluripotency in mESCs mediated by LIF. Pluripotency in mESCs is maintained through a complex network signalling downstream of LIF. Klf4 is activated via the STAT3 pathway and Tbx3 via PI3K. Both upregulate the transcriptional activity of Sox2 and Nanog which in turn activate Oct3/4. Abbreviations: ERK: extracellular receptor kinase, Klf4: Krueppel-like factor 4,mESC: mouse embryonic stem cell, LIF: Leukaemia Inhibitory Factor, MAPK: Mitogen-activated protein kinase, Oct3/4: Octamer binding transcription factor 3/4, PI3K: Phosphatidylinositide 3-kinase, STAT3: Signal Transducers and Activators of Transcription 3, Sox2: SRY (sex determining region Y)-box 2, Tbx3: T-Box transcription factor 3.

1.4.2 Molecular mechanism and signalling during lineage allocation An evolutionarily conserved regulatory network, including WNT, TGF (Transforming growth factor ), Nodal, BMP, and FGF signalling, acts in concert with key TFs to control early lineage segregation [199]. Recent reports suggest that pluripotency-associated TFs switch promoters upon exit of pluripotency towards germ layer specification. As mentioned above Oct3/4 and Nanog promote mesodermal as well as endodermal fate and limit neuroectoderm differentiation. In contrast, Sox2 enhances neuroectoderm while restricting mesoderm and endoderm development. Accordingly, two main groups of pluripotency factors

- 21 - were defined as mesendoderm-class (e.g., Oct3/4, Nanog, Klf5) or neuroectoderm-class (e.g., Sox2, RBPJ) ESC genes [173]. Furthermore members of the T-Box transcription factor family exert highly specific and crucial functions throughout development. During gastrulation Eomes regulates the specification of definitive endoderm and cardiovascular mesoderm from the anterior primitive streak region, T is required for the expansion of the posterior mesoderm and Tbx6 specifies intermediate primitive streak derivatives [130]. Tbx3 is expressed in the ICM to impact pluripotency and in midgestation Tbx3 plays important roles in cardiac chamber specification, hepatic bud expansion and limb patterning [21, 26, 74, 101, 124].

Aim of the thesis Maintaining the pluripotent state requires a precise transcriptional profile. This complex network has been studied extensively. Key players, such as Oct3/4, Nanog and Sox2, together with many other TFs and various signalling pathways, impact this circuitry, by activating pluripotency-associated genes and inhibiting gene expression responses required for differentiation. However, upon withdrawal of stemness-inducing signals, pluripotency factors guide exit of pluripotency. Intriguingly, several pluripotency-associated genes reveal this ability. Hereby, upon onset of differentiation TFs, initially embedded in complex pluripotency promoting gene programmes undergo a molecular switch and promote differentiation gene expression levels. Recent work classified these multifaceted genes depending on their impact on the respective germ layer. Thus, TFs were classified into mesendoderm-, neuroectoderm- and extraembryonic-class genes. The TF Tbx3 belongs to the inner core of the pluripotency network. Within the embryo, it is expressed as early as the 4 cell stage and subsequently in the ICM. Later, Tbx3 is involved in early heart and liver formation. However, there is an unsolved question whether Tbx3 is involved in the regulation of early lineage decisions, regarding endodermal, ectodermal, mesodermal development. To address this question, we made use of an inducible gain of function approach in differentiating mESCs to close this gap of knowledge.

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2 Material and Methods

Material

2.1.1 Cell lines Table 1 Overview of used cell lines. Abbreviations: Dpp4: Dipeptidylpeptidase, ESC: embryonic stem cell, GFP: Green fluorescent protein, RFP: Red fluorescent protein Sox17: SRY (sex determining region Y)-box 17, Tbx3: T-Box-transcription factor. Name Acquisition A2lox.cre ESCs kindly provided by M. Kyba (species: mouse) iTbx3 tdTomato ESC iTbx3 cells lentivirally transduced with LVdtTomato construct (kindly provided by K. Hochedlinger) CgR8 cell line kindly provided by Austin Smith Sox 17-RFP reporter kindly provided by Douglas Melton: RFP is under control of the cell line endodermal Sox17 promoter T-GFP/Dppa4-RFP kindly provided by Hans-Jörg Fehling: RFP fluorescence is under reporter cell line control of the pluripotent Dpp4 promoter and further GFP is under control of the mesodermal Brachyury promoter HEK 293T cell line Clontech, Mountain View, CA, USA

2.1.2 Cell culture components/preparation

Table 2 Preparation of cell culture components. Abbreviations: dH2O: distilled water, Ampuwa, DPBS: Dulbecco's Phosphate-Buffered Saline, ESC: embryonic stem cell, FCS: Fetal Calf Serum, HCl: Hydrochloric Acid , MTG: Monothioglycerol, PFA: Paraformaldehyde.

Name Manufacturer Preparation Storage

ESC- Lonza, Basel, BS, Preparation: Inactivation was performed by placing a 500ml bottle into a qualified CH 65°C water bath for 30min. Thereafter 50ml aliquots were prepared and -20°C FCS stored at -20°C.

Stock concentration: 50mg: 2mg Mitomycin C+48mg sodium chloride. Sigma-Aldrich, St. Mitomycin C 2mg Mitomycin C was dissolved in 10ml DPBS solution was sterile- +4°C Louis, MO, USA filtered to a final concentration of 0.5mg/ml and applied to cell culture dark media at 50µl/ml.

Sigma-Aldrich, St. MTG 26µl/2ml of differentiation media Louis, MO, USA +4C

8g PFA was dissolved in 80ml dH2O and remained in 57°C water bath PFA 4%/ Sigma-Aldrich, St. until clear. Further, 4g Sucrose was dissolved in 100ml PBS. Both Sucrose 2% Louis, MO, USA solutions were mixed. pH was adjusted to 7.4 with Hydrochloric acid -20°C

(HCl) 37%

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Table 3 Overview of cell culture components. Abbreviations: -ME-Mercaptoethanol, dH2O: distilled water, DMEM: Dulbecco's Modified Eagle's Medium , DMSO: Dimethylsulfoxid , Dox: Doxycycline, DPBS: Dulbecco`s Phosphate-Buffered Saline, EDTA: Ethylenediaminetetraacetic Acid, GMEM: Glasgow Minimum Essential Medium, IMDM: Iscove's Modified Dulbecco's Media, KO- DMEM: Knockout Dulbecco's Modified Eagle's Medium, LIF: Leukaemia Inhibitory Factor, LB: Luria-Bertani, NEAA: Non-Essential Amino Acids, OptiMEM: Reduced Serum Medium, P/S: Penicillin- Streptomycin, RT: room temperature.

Name Manufacturer Concentration Solvent Storage

Ampicillin Sigma-Aldrich, St. Louis, MO, USA 10µg/ml dH2O -20°C L-ascorbic acid Sigma-Aldrich, St. Louis, MO, USA 50µg/ml media DPBS +4°C DMSO Sigma-Aldrich, St. Louis, MO, USA -20°C Dox Sigma-Aldrich, St. Louis, MO, USA 1µg/ml DPBS -20°C, dark DMEM Invitrogen, Carlsbad, CA, USA +4°C DPBS Invitrogen, Carlsbad, CA, USA +4°C

Gelatin from porcine pig Sigma-Aldrich, St. Louis, MO, USA 0.2% dH2O +4°C Glutamax Invitrogen, Carlsbad, CA, USA 1:100 -20°C GMEM Sigma-Aldrich, St. Louis, MO, USA +4°C IMDM Invitrogen, Carlsbad, CA, USA +4°C KO-DMEM Invitrogen, Carlsbad, CA, USA +4°C LIF Sigma-Aldrich, St. Louis, MO, USA 2.4ng/ml DPBS -20°C MP Biomedicals, Santa Ana, CA, LB media +4°C USA M16 Media Sigma-Aldrich, St. Louis, MO, USA +4°C -ME Sigma-Aldrich, St. Louis, MO, USA +4°C Neomycin (G-418) Sigma-Aldrich, St. Louis, MO, USA 400µg/ml DPBS RT Nodal Antibody, sc- 28913 Santa Cruz, Santa Cruz, CA, USA 3µM +4°C NEAA Invitrogen, Carlsbad, CA, USA 1:100 -20°C P/S Millipore, Billerica, MA, USA 1:100 -20°C OptiMEM Invitrogen, Carlsbad, CA, USA +4°C SB421542 (inhibitor of TGF subfamily, type I) Sigma-Aldrich, St. Louis, MO, USA 10µM -20°C Sodium Pyruvat Invitrogen, Carlsbad, CA, USA 1:100 -20°C 0.25% Trypsin/1mM EDTA Millipore, Billerica, MA, USA -20°C 0.05% Trypsin/0.53mM EDTA Millipore, Billerica, MA, USA -20°C

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2.1.2.1 Cell culture media Table 4 Cell culture media. Table illustrating the components used for cell culture media. Please note, that the applied designations for the different types of media will be retained throughout the text below. Abbreviations: β-ME: β- Mercaptoethanol, DMEM: Dulbecco's Modified Eagle's Media, ESC: embryonic stem cell, FCS: Fetal Calf Serum, DMSO: Dimethylsulfoxid, GMEM: Glasgow Minimum Essential Media, IMDM: Iscove's Modified Dulbecco's Media, KO: knockout, LIF: Leukaemia Inhibitory Factor, MEF: mouse embryonic fibroblast, NEAA: Non-Essential-Amino Acids, P/S: Penicillin- Streptomycin.

CgR8-ESC media ESC-Differentiation media Feeder-ESC media

Basalmedia GMEM IMDM KO-DMEM FCS 10% 10% 15% Glutamax 1% 1% 1% LIF 2.4 ng/ml 2.4 ng/ml

β-ME 1% 1%

NEAA 1% 1% 1% P/S 1% 1% 1% Sodium Pyruvat 1% 1%

Freezing media HEK 293T media Thaw media MEF media Basalmedia DMEM DMEM DMEM DMSO 10%

FCS 90% 10% 30% 15% Glutamax 1% 1% β-ME 1% 1% NEAA 1% 1% P/S 1% 1% 1% Sodium Pyruvat 1% 1% Vitamin C 1% 1%

2.1.3 Materials used for Lentiviral Infection  Lentiviral vector LV-tdTomato  Vectors (pMD2, psPAX2) (Addgene, Cambridge, MA, USA)  PolyFect reagent (Qiagen, Hilden, NRW, Germany)  Polybrene (Sigma-Aldrich, St. Louis, MO, USA) Stock concentration: 2µg/ml Final concentration: 2ng/ml

Solvent: dH2O Storage: -20°C

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2.1.4 Materials used for Reporter  Dual-Luciferase ® Reporter Assay System Kit (Promega, Madison, WI, USA)  Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA)  OptiMEM  Plasmids (200ng) o Brachyury-reporter (kindly provided by R. Kemler) o Eomes-reporter (kindly provided by L. Vallier) o Proximal Oct3/ 4 promotor (kindly provided by Hans Schöler) o Distal Oct3/ 4 promotor (kindly provided by Hans Schöler) o Sox17-reporter (kindly provided by J. Wells) o pFIEN Tbx3 cDNA (kindly provided by Tata Purushothama Rao) o Empty (Control)  pTK-RL (Addgene, Cambridge, MA, USA )  Transfection media components (for one 24-well) o 50µl OptiMEM o 50ng pTK-RL

2.1.5 Kits used for Plasmid Preparation  Competent cells (Invitrogen, Carlsbad, CA, USA)  PureLink HiPure Plasmid Maxiprep Kit (Invitrogen, Carlsbad, CA, USA)  LB media + competent cells (E.coli, Thermo Fischer Scientific)  Isopropanol  Ethanol 70%

2.1.6 Kits used for RNA Extraction  RNAesy Mini Kit (Qiagen, Hilden, NRW, Germany)  14,3M -ME  Ethanol 100%  Ethanol 70%

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2.1.7 Materials used for Immunofluorescence Table 5 Overview of components used for Immunofluorescence. Abbreviations: BSA: Bovine Serum Albumin, DAPI:

4′,6-Diamidin-2-phenylindol, dH2O: distilled water, FG: Gelatin from cold water fish skin, GS: Goat Serum, PBS: Phosphate- buffered saline, NH4Cl : Ammonium Chloride, RT: room temperature.

Name Manufacturer Concentration Solvent Storage

BSA Carl Roth, Karlsruhe, BW, Germany 5% PBS + 4°C DAPI Invitrogen, Carlsbad, CA, USA 1:20.000 PBS RT

GS JacksonImmunoResearch, West Grove, PA, USA 2mg/ml dH2O -20 °C

FG Sigma-Aldrich, St. Louis, MO, USA 1% dH2O + 4°C FluorSave™ Reagent Calbiochem, Gibbstown, NJ, USA + 4°C

NH4Cl Sigma-Aldrich, St. Louis, MO, USA 0.05mM PBS RT

PBS Millipore, Billerica, MA, USA 9.55g/1l dH2O RT TritonX100 Sigma-Aldrich, St. Louis, MO, USA RT

2.1.7.1 Antibodies Table 6 Primary Antibodies for Immunofluorescence. Table illustrating primary antibodies used for IF, including isotype, blocking condition, dilution, application, storage conditions and manufacturer. Abbreviations: BSA: Bovine Serum Albumin, Eomes: Eomesodermin, FG: gelatin from cold water fish skin, GS: Goat Serum, h: hour, HCN4: Hyperpolarization-Activated Cyclic Nucleotide-gated potassium channel 4, IF: immunofluorescence, IgG: immunoglobulin G, IgM: immunoglobulin M, NA: not available, Oct3/4: Octamer binding transcription factor 3/4, o.N.: overnight, Pdx1: Pancreatic and duodenal homeobox 1, RT: room temperature, SSEA1: Stage-specific embryonic antigen 1, SRY (sex determining region Y)-box 17, Tbx2/3/6: T-Box transcription factor 2/3/6. Cedarlane, Burlington, ON, Canada; Cell Signaling, Danvers, MA, USA; R&D Systems, Minneapolis, MN, USA; DSHB, Iowa City, IA, USA

Antibody Isotype Blocking condition Dilution/ Stored Manufacturer/ Application aliquots #

α-Actinin monoclonal 5% GS, 1% FG, 1:150 / - 20°C Sigma-Aldrich mouse IgG1 0.2% Triton X 1h, RT A7811

Albumin polyclonal 5% BSA, 1:100 / - 20°C Cedarlane ( FITC conjugated) rabbit IgG 0.3% Triton X 1h, RT CLFAG3140

Brachyury polyclonal 5% BSA, 1:100 / - 20°C R&D Systems goat IgG 0.3% Triton X o.N., 4°C AF2085

Eomes monoclonal 5% BSA, 1:200 / + 4°C eBioscience rat IgG2a, k 0.3% Triton X o.N., 4°C 14-4876-82

HCN4 polyclonal 5% GS, 1% FG, 1:100 / - 20°C Millipore rabbit IgG 0.2% Triton X o.N., 4°C AB5808

Oct3/4 monoclonal 5% BSA, 1:200 / + 4°C Santa Cruz mouse IgG 0.3% Triton X o.N., 4°C sc-5279 Pdx1 polyclonal 5% BSA, 1:500 / - 20°C R&D Systems goat IgG 0.3% Triton X o.N., 4°C AF2419

Sox17 polyclonal 5% BSA, 1:500 / - 20°C R&D Systems goat IgG 0.3% Triton X o.N., 4°C AF1924

SSEA1 monoclonal 5% GS, 1% FG 1:500 / + 4°C DSHB mouse IgM 1h, RT MC-480

Tbx2 NA rabbit IgG 5% BSA, 1: 1000 / + 4°C provided by 0.3% Triton X o.N., 4°C C.Goding

Tbx3 polyclonal 5% BSA, 1: 1000 / - 20°C provided by rabbit IgG 0.3% Triton X 1h, RT H. Niwa

Tbx3 polyclonal 5% BSA, 1: 1000 / - 20°C provided by (mouse embryo) rabbit IgG 0.3% Triton X o.N., 4°C H. Niwa

Tbx6 polyclonal 5% BSA, 1:500 / + 4°C Santa Cruz goat IgG 0.3% Triton X o.N., 4°C sc-17888

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Table 7 Secondary Antibodies for Immunofluorescence. Table illustrating secondary antibodies used for IF, including isotype, blocking condition, dilution, application, storage conditions and manufacturer. Abbreviations: BSA: Bovine Serum Albumin, h: hour, IF: immunofluorescence, IgG: immunoglobulin G, IgM: immunoglobulin M, min: minutes, NA: not available, RT: room temperature.

Antibody Isotype Blocking condition Dilution/ Stored Manufacturer/ Application aliquots # Alexa Flour 488 goat anti 5% BSA, 1: 500 / -20°C Invitrogen mouse IgG 0.3% Triton X 45min, RT, dark A-11001 Alexa Flour 488 donkey anti 5% BSA, 1: 500 / -20°C Invitrogen goat IgG 0.3% Triton X 45min, RT, dark A-11055 Alexa Flour 488 donkey anti 5% BSA, 1: 500 / -20°C Invitrogen rabbit IgG 0.3% Triton X 45min, RT, dark A-21206 Alexa Flour 488 goat anti 5% BSA, 1: 500 / -20°C Invitrogen rabbit IgG 0.3% Triton X 45min, RT, dark A-11008 Alexa Flour 488 donkey anti 5% BSA, 1: 500 / -20°C Invitrogen rat IgG 0.3% Triton X 45min, RT, dark A-21208 Alexa Flour 568 goat anti 5% BSA, 1: 500 / -20°C Invitrogen mouse IgG 0.3% Triton X 45min, RT, dark A-11004 Alexa Flour 568 goat anti 5% BSA, 1: 500 / -20°C Invitrogen mouse IgM 0.3% Triton X 45min, RT, dark A-21043 Alexa Flour 568 donkey anti 5% BSA, 1: 500 / -20°C Invitrogen goat IgG 0.3% Triton X 45min, RT, dark A-11057 Alexa Flour 568 donkey anti 5% BSA, 1: 500 / -20°C Invitrogen rabbit IgG 0.3% Triton X 45min, RT, dark A10042 Alexa Flour 568 goat anti 5% BSA, 1: 500 / -20°C Invitrogen rabbit IgG 0.3% Triton X 45min, RT, dark A-11011 Alexa Flour 647 goat anti 5% BSA, 1: 500 / -20°C Invitrogen mouse IgG 0.3% Triton X 45min, RT, dark A-21235 Alexa Flour 647 goat anti 5% BSA, 1: 500 / -20°C Invitrogen rabbit IgG 0.3% Triton X 45min, RT, dark A-21245

2.1.8 Materials used for Immunoblotting Table 8 Overview of materials used for Immunoblotting. Abbreviations: APS: Ammonium Persulfate, BSA: Bovine Serum Albumin, PAGE: Polyacrylamide gel electrophoresis, RT: room temperature, SDS: Sodium Dodecyl Sulfate, TEMED: Tetramethylethylendiamin, TRIS: Tris-aminomethan.

Name Manufacturer Storage Acrylamide/Bis Solution Bio Rad, Hercules, CA, USA +4°C APS Sigma-Aldrich, St. Louis, MO, USA RT BSA Serva, Heidelberg, BW, Germany +4°C AppliChem, Darmstadt, HE, Germany RT Methanol Sigma-Aldrich, St. Louis, MO, USA RT Milk powder Roth, Karlsruhe, BW, Germany RT PAGE Ruler Plus Prestained Protein Thermo Scientific, Waltham, MA, USA -20°C Ladder Protein Assay Bio Rad, Hercules, CA, USA +4°C PVDF membrane Millipore, Billerica, MA, USA RT Restore Western Blot Stripping Buffer Thermo Scientific, Waltham, MA, USA +4°C SDS Sigma-Aldrich, St. Louis, MO, USA RT SuperSignal West Dura Thermo Scientific, Waltham, MA, USA +4°C SuperSignal West Pico Thermo Scientific, Waltham, MA, USA +4°C TRIS Sigma-Aldrich, St. Louis, MO, USA RT TEMED Thermo Scientific, Waltham, MA, USA +4°C Tween 20 Sigma-Aldrich, St. Louis, MO, USA RT Western Membran Immobilon–P Millipore, Billerica, MA, USA RT - 28 -

2.1.8.1 Antibodies

Table 9 Primary Antibodies for Immunoblotting. Table illustrating primary antibodies used for Immunoblotting, including isotype, blocking condition, dilution, application, storage conditions and manufacturer. Abbreviations: h: hour, IgG: immunoglobulin G, IgM: immunoglobulin M, o.N.: overnight, RT: room temperature, SMAD2: Mothers against decapentaplegic homolog 2, TBS-T: TRIS-buffered saline+Tween.

Antibody Isotype Blocking condition Dilution/ Stored Manufacturer/ Application aliquots #

-Actin monoclonal 5 % nonfat dry milk, 1:4000/ - 20°C Santa Cruz mouse IgG TBS-T 1h RT sc-47778 Octamer binding monoclonal 5 % nonfat dry milk, 1:200/ +4°C Santa Cruz transcription factor 4 mouse IgG TBS-T o.N., 4°C sc-5279 Phospho polyclonal 5 % nonfat dry milk, 1:1000/ - 20°C Cell Signaling SMAD2 rabbit IgG TBS-T o.N., 4°C #3101 SRY (sex determining polyclonal 5 % nonfat dry milk, 1:500/ - 20°C R&D Systems region Y)-box 17 goat IgG TBS-T 1h RT AF1924 T-Box transcription polyclonal 5 % nonfat dry milk, 1:1000/ +4°C Abcam factor 3 rabbit IgG TBS-T o.N., 4°C ab66306 T-Box transcription polyclonal 5 % nonfat dry milk, 1:1000/ +4°C Santa Cruz factor 3 goat IgG TBS-T o.N., 4°C sc-17871

Table 10 Secondary Antibodies for Immunoblotting. Table illustrating secondary antibodies used for Immunoblotting, including isotype, blocking condition, dilution, application, storage conditions and manufacturer. Abbreviations: HRP: horseradish peroxidase, IgG: immunoglobulin G, min: minutes, o.N.: overnight, RT: room temperature, TBS-T: TRIS- buffered saline+Tween.

Antibody/Isotype Blocking condition Dilution/ Stored Manufacturer/ Application aliquots # Sheep anti mouse IgG 5 % nonfat dry milk, 1:4000, + 4°C GE Healthcare (HRP- conjugated ) TBS-T 45min, RT Life Sciences NA931 Rabbit anti goat IgG 5 % nonfat dry milk, 1:3000 + 4°C JacksonImmuno (HRP- conjugated ) TBS-T 45min, RT Research

305-001-001 Donkey anti rabbit IgG 5 % nonfat dry milk, 1:4000 + 4°C GE Healthcare (HRP- conjugated ) TBS-T 45min, RT Life Sciences NA934

2.1.8.2 Gels and Buffers

Table 11 Gels used for Immunoblotting. Abbreviations: APS: Ammonium Persulfate, dH2O: distilled water, TEMED: Tetramethylethylendiamin, TRIS: Tris-aminomethan, SDS: Sodium Dodecyl Sulfate.

Stacking Gel 4% Separation Gel 10%

Acrylamid 30% 650µl 3.3ml APS 25µl 50µl

dH2O 3.1ml 4.1ml TEMED 10µl 20µl TRIS 1.25ml 0.5M (pH 6.8) 2.5ml 1.5M (pH 8.8) SDS 20% 25µl 50µl

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Table 12 Buffers used for Immunoblotting. Abbreviations: ME: -Mercaptoethanol, dH2O: distilled water/Ampuwa, EDTA: Ethylenediaminetetraacetic acid, HCl: Hydrochloric Acid, NP-40: Nonylphenolethoxylat with MO=40, PAGE: Polyacrylamide gel electrophoresis, SDS: Sodium Dodecyl Sulfate TRIS: Tris-aminomethan.

Electrophoresis Buffer Laemmli Buffer RIPA Lysis Buffer SDS PAGE Buffer

900 ml dH2O 10% ME dH2O 720.6g Glycine

100ml SDS 0.05% Bromophenolblue 5mM EDTA 1500ml dH2O 20% Glycerol 10% Glycerol 151.1g TRIS 10% SDS 2.5mM Magnesium chloride 50g SDS 0.2M Tris-HCl, pH 6.8 1% NP-40 150mM Sodium Chloride 0.5% Sodium Deoxycholate pH adjusted to 0,1% SDS 7.6 with HCl 1mM Sodium Orthovanadate 50mM TRIS (pH 8)

TRIS-buffered saline Buffer Transfer Buffer TBS-T (Wash Buffer)

5000ml dH2O 45g Glycine 900ml dH2O

400g Sodium Chloride 2400ml dH2O 100ml TRIS-buffered saline Buffer 121g TRIS 600ml Methanol 1ml Tween-20 9g TRIS

2.1.9 Materials used for Chromatin Immunoprecipitation  ChIP-IT® Express Enzymatic Kit (Active Motif, Carlsbad, CA, USA)

 Ampuwa (dH2O) Storage: -20°C  -Ethanol absolute (Sigma-Aldrich, St. Louis, MO, USA) Storage: RT  Formaldehyde 1% (Sigma-Aldrich, St. Louis, MO, USA) Storage: RT  Glycerol (Sigma-Aldrich, St. Louis, MO, USA) Application : 1:1 Final concentration: 50%

Solvent: dH2O Storage: RT  Natriumacetat

Preparation: 408.24g Natriumacetat-3H2O was dissolved in

800ml H2O, pH 5.2 was adjusted with HCl Final concentration: 3M Storage: RT  Roti®-P/C/I (Phenol/Chloroform/Isoamylalkohol) pH 8

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(Roth, Karlsruhe, BW, Germany) Stored at +4°C  Sub-Q-syringe (BD Bioscience, Franklin lakes, NJ, USA)  Sonifier Cell Disrupter 250 (VWR, PA, USA)  Magnetic beads (Invitrogen, Carlsbad, CA, USA) Stored at +4°C

2.1.9.1 Antibodies Table 13 Antibodies for Chromatin Immunoprecipitation. Table illustrating antibodies used for Immunoblotting, including isotype, concentration, application, storage conditions and manufacturer. Abbreviations: IgG: immunoglobulin G, o.N.: overnight, Tbx3: T-Box transcription factor 3.

Antibody Isotype Concentration/ Stored Manufacturer/ Application aliquots #

IgG polyclonal goat IgG 3µg/ + 4°C Santa Cruz o.N., 4°C sc-2028

Tbx3 (S-17) polyclonal goat IgG 3µg/ + 4°C Santa Cruz o.N., 4°C sc- 31657

Tbx3 (A-20) polyclonal goat IgG 3µg/ + 4°C Santa Cruz o.N., 4°C sc-17871

Tbx3 polyclonal rabbit IgG 3µg/ + 4°C Invitrogen o.N., 4°C #42-4800

2.1.9.2 Polymerase chain reaction (PCR) components  Gel: Agarose Gel 1.2% Preparation: 120ml TAE, 1,44g Agarose  Loading Dye Application: 1:5 Preparation of 20ml Loading Dye . 50% Glycerin (10ml) . 0.04% Bromphenolblau (8mg) . 0.04% Xylencyanol (8mg) . Add 1xTE pH 8 Storage: +4°C  1 Kb Plus DNA Ladder (Invitrogen, Carlsbad, CA, USA) Storage: +4°C

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2.1.9.3 Quantitative real-time polymerase chain reaction (qRT-PCR) components  Ampuva  SensiMix SYBR NO-ROX Kit (Bioline, London, UK)

2.1.10 Primer Table 14 Self-designed primers for quantitative real-time polymerase chain reaction. Table illustrating self-designed primers including their function, melting Temperature and sequence. Primers were designed by the Kleger Laboratory and oligos were purchased from Biomer (Biomer, Pleasanton, CA, USA). Abbreviations: fwd: forward, rev: reverse, Tm: melting Temperature.

Abbreviations Gene Function Tm (°C) Sequence T Brachyury marker of 51,8 fwd 5' gcggacaattcatctgcttg 3' early embryogenensis 56,3 rev 5' cagtaggtgggctggcgttat 3'

Pecam Cluster of vascular marker gene 51,1 fwd 5' tactgcaggcatcggcaaa 3' differentiation 31 51,1 rev 5' gcatttcgcacacctggat 3'

Chrd Chordin marker of 53,8 fwd 5' gactgctgcaaacagtgtcc 3' early embryogenensis 53,2 rev 5' actgatgggtgccagctct 3'

Gsc Goosecoid marker of 55,9 fwd 5' cagctacttctacgggcagc 3' early embryogenensis 53,8 rev 5' ccggagacaccagtacagaa 3'

Lim Lim homeobox 1 marker of 50,5 fwd 5' aatgtaaatgcaacctgaccg 3' early embryogenensis 51,8 rev 5' aaccagatcgcttggagaga 3'

Tbx3 T-Box transcription marker of pluripotency, 53,2 fwd 5' cgg ttc cac atc gtc aga g 3' factor 3 marker of embryogenesis 52,6 rev 5' gcc att gcc agt gtc tcg 3'

Table 15 Self-designed primers for quantitative real-time polymerase chain reaction of chromatin immunoprecipitated cells. Table illustrating self-designed primers including their function, melting Temperature and Sequence. Primer oligos were designed together with Tata Purushothama Rao. Primer oligos were purchased from Biomer

(Biomer, Pleasanton, CA, USA). Abbreviations: DE: Definitive endoderm, fwd: forward, rev: reverse Tm: melting Temperature, TGF: Transforming growth factor 

Tm Abbreviations Gene Function Sequence (°C) Bone morphogenetic fwd 5' cca aga atc atg gac tgt tat tat atg BMP4 member of TGF- 55,5 protein 4 c 3' superfamily 56,3 rev 5' cat agc gct gcc tga gag tag 3'

marker of early T Brachyury 59,4 fwd 5' ccc ggc ttc tcg ccc tcc 3' embryogenensis 56,3 rev 5' agc cgt ggc tcc act tga act 3'

marker of early fwd 5' agg gaa ttc tga gta atg aaa gtg Eomes Eomesodermin 52,3 embryogenensis 3' 61 rev 5' ctg agt tcg ggt cta gtt tgg cag g 3'

Nodal Nodal marker of TGF-signalling 57,1 fwd 5' ccg cca cca ttt ctc tgt agt ct 3' 56,7 rev 5' ctc cgg aga ggc cta taa cct a 3'

Sox17 Sex determining region DE marker gene 62,5 fwd 5' gag ggt gct gag tgg ttg ctg gga 3' Y (SRY)- box 17 62,5 rev 5' cag cag cac tgt gct gtt cct cgg 3'

basic Fibroblast Growth fwd 5' aca gag aca cag aat cag aac FGF2 member of the fibroblast 56 Factor aac c 3' growth factor family 55,3 rev 5' cag tca agt tgg aag agg atg tg 3'

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Table 16 Commercial primers for quantitative real-time polymerase chain reaction. Table illustrating commercial primers acquired from Qiagen (Hilden, NRW, Germany) including their function, melting Temperature and ordering number.

Abbreviations: DE: Definitive endoderm, TGF: Transforming growth factor , Tm: melting Temperature, VE: Visceral endoderm, WNT: wingless-related MMTV integration site.

Tm Abbreviations Gene Function # (°C) Albumin Albumin hepatic marker gene 72-86 QT00115570 BMP4 Bone morphogenetic protein 4 member of TGF--family 72-86 QT00111174 Cdx2 Caudal type homeobox 2 DE and VE marker gene 72-86 QT00116739 Eomes Eomesodermin marker of early embryogenensis 72-86 QT01074332 FoxA2 Forkhead-Box-Protein A2 DE marker gene 72-86 QT00242809 GATA 4 GATA-binding factor 4 DE marker gene 72-86 QT00155400 GATA 6 GATA-binding factor 6 DE marker gene 72-86 QT00171297 Hmbs Hydroxymethylbilane synthase housekeeping gene 72-86 QT00494130 Hnf1 Hepatocyte nuclear factor 1  pancreatic marker gene 72-86 QT00103320 Hnf4 Hepatocyte nuclear factor 4  hepatic marker gene 72-86 QT00144739 Hnf6 Hepatocyte nuclear factor 6 pancreatic marker gene 72-86 QT00297815 hHex Haematopoietic homeobox gene DE marker gene 72-86 QT00118517 Isl1 Islet-1 cardiac marker gene 72-86 QT01048691 KDR Kinase insert domain receptor cardiovascular marker gene 72-86 QT00097020 Lefty1 Left-right determination factor 1 Nodal target gene 72-86 QT00127302 Lefty2 Left-right determination factor 2 Nodal target gene 72-86 QT01073821 Mesp1 Mesoderm posterior 1 homolog cardiac marker gene 72-86 QT00250446 MyH6 Myosin heavy chain 6 cardiac marker gene 72-86 QT00160902 Myl2a Myosin light chain 2a cardiac marker gene 72-86 QT01064287 Myl2v Myosin light chain 2v cardiac marker gene 72-86 QT00100198 MyoD Myogener Faktor 3 paraxial mesoderm marker gene 72-86 QT00101983 Nanog Nanog pluripotency marker gene 72-86 QT00248311 Nkx2.5 NK2 homeobox 5 cardiac marker gene 72-86 QT00124810 Nodal Nodal marker of TGF-signalling 72-86 QT00125685 Oct3/4 Octamer binding transcription factor 3/4 pluripotency marker gene 72-86 QT00109186 Pax6 Paired box protein 6 ectoderm marker 72-86 QT01052786 Pdx1 Pancreatic and duodenal homeobox 1 pancreatic marker gene 72-86 QT00102235 Paired-like homeodomain transcription Pitx2 Nodel target gene 72-86 QT00173355 factor 2 Sox2 Sex determining region Y (SRY)- box 2 pluripotency marker gene 72-86 QT00249347 Sox17 Sex determining region Y (SRY)- box 17 DE marker gene 72-86 QT00160720 Tbx2 T-Box transcription factor 2 marker of early embryogenensis 72-86 QT00165242 Tbx6 T-Box transcription factor 6 marker of early embryogenensis 72-86 QT00098861 VEGF-A Vascular Endothelial Growth Factor vascular marker gene 72-86 QT00160769 WNT3a wingless-type family, member 3A marker of WNT signalling 72-86 QT00250439

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2.1.11 Further Kits  Lenti-X Concentrator Kit (Clontech, Mountain View, CA, USA)  Mouse ES Cell Nucleofector Kit (Lonza, Basel, BS, CH)  QuantiTect SYBR Green RT-PCR kit (Qiagen, Hilden, NRW, Germany)

2.1.12 Tools and Machines Autoclave Systec, Wettenberg, HE, Germany Bacterial plates 100mm/60mm Greiner Bio-One, Kremsmünster, Austria BD FACSAria III cell sorter BD Bioscience, Franklin lakes, NJ, USA Biosphere safe seal tube 1,5ml Sarstedt, Nümbrecht, NRW, Germany Bottle-top vacuum filter system Sigma-Aldrich, St. Louis, MO, USA Cell culture T25 flascs Nunc, Wiesbaden, HV, Germany Cell culture T175 flascs Nunc, Wiesbaden, HV, Germany Cell scraper Sarstedt, Nümbrecht, NRW, Germany Cell strainer BD Bioscience, Franklin lakes, NJ, USA Cell culture dish 100mm/60mm Nunc, Wiesbaden, HV, Germany Centrifuge 4°C Eppendorf, Hamburg, HH, Germany Centrifuge RT Thermo scientific, Waltham, MA, USA Counting chamber Neubauer Laboroptik, Lancing, UK Counting chamber Thoma Laboroptik, Lancing, UK Cover glasses 22mm VWR, Darmstadt, HE, Germany Gel electrophoresis apparatus Bio Rad, Hercules, CA, USA Falcon 15ml / 50ml BD Bioscience, Franklin lakes, NJ, USA Filter system Corning, Corning, NY, USA Freezer - 20°C Liebherr, Bulle, Switzerland Freezer - 80°C Heraeus, Hanau, HE, Germany Freezing/ Cryo Tubes Roth, Karlsruhe, BW, Germany Fridge 4°C Liebherr, Bulle, Switzerland Gloves Sempermed, Wimpassing, Austria Ice machine Scotsman, Vernon Hilly, IL, USA

Incubator 37°C, 5% CO2 Heraeus Holding, Hanau, Germany Luminometer (GloMax 96) Promega, Madison, WI, USA Magnetic mixer Heidolph, Schwabach, BY, Germany Microscope Olympus, Shinjuku, Tokyo, Japan - 34 -

Microscope Axiovision software Zeiss, Oberkochen, BW, Germany Microwave Sharp, Abeno-ku, Osaka, Japan Nanodrop Thermo scientific, Waltham, MA, USA Pipetboy Brandt, Wertheim, BW, Germany Pipettes 1ml/5ml/10ml Greiner Bio-One, Kremsmünster, Austria Pipettes 2ml/25ml/50ml Thermo scientific, Waltham, MA, USA Pipette tips 10µl/200µl/1000µl Eppendorf, Hamburg, HH, Germany Power supply Bio Rad, Hercules, CA, USA Round-bottom-tubes BD Bioscience, Franklin lakes, NJ, USA RotorGene Q Systems Qiagen, Hilden, NRW, Germany qRT-PCR Tubes 0,1 ml/ 0,2ml Qiagen, Hilden, NRW, Germany Syringes 20ml/ 50ml BD Bioscience, Franklin lakes, NJ, USA Syringe filter 0,22µm Sarstedt, Nümbrecht, NRW, Germany Vortexer Bender & Hobein, Ismaning, BY, Germany Water bath Köttermann, Uetze/Hänigsen, NI,Germany 6/12 /24 /48/96 well multiwell plate Nunc, Wiesbaden, HV, Germany

Methods

2.2.1 The A2lox.cre system The A2lox.cre system consists of a recombination system located in the Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus (Figure 7A). Briefly, cre-recombinase complementary DNA (cDNA) was replaced withTbx3 cDNA via homologous cassette exchange. The reverse tetracycline transactivator (rtTA) element, transcribed from the ROSA26 gene locus, binds with Doxycycline (Dox). It is then transferred to the Tetracycline Responsive Element (TRE) where it homogenously regulates Tbx3 messenger RNA (mRNA) expression levels [77].

2.2.2 Generation of the iTbx3 cell line We generated a Tetracycline-controlled inducible expression system to create an artificial system allowing the isolated investigation of Tbx3 function, iTbx3. This part was mainly implemented by Dr. Kerstin Bauer together with Dr. Martin Müller.

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Briefly, Tbx3 cDNA was amplified from a cDNA clone derived from Image Consortium [76]. The specific cDNA was extracted, followed by a digestion process, which was also performed for the targeting vector p2loc-EGFP (enhanced Green fluorescent protein). After ligation, the plasmid was amplificated and sequenced. In the last step, the cloned cDNA (Tbx3-p2lox vector) was integrated into A2lox.cre cells using nucleofection procedure followed by a selection process using Neomycin [183].

Figure 7 Generation of iTbx3 cells. (A) Image demonstrating the original p2lox vector kindly provided by M. Kyba, including restriction map [77]. EGFP cDNA is exchanged by Tbx3 cDNA (B). A2lox.cre system: Schematic illustration of cloning strategy to generate a temporally regulated dose dependent Dox-inducible iTbx3 mESC line. The HPRT locus, before and after homologous cassette exchange leads to the replacement of the Cre-recombinase cDNA by the Tbx3 cDNA. Abbreviations: ATG: start codon, Cre: cre recombinase, complementary, cDNA: complementary deoxyribonucleic acid, Dox: Doxycycline, EGFP: enhanced Green fluorescent protein, mESC: mouse embryonic stem cell, lox2272/loxP: Cre recombinase cutting sites, neoR: Neomycin Resistance, PGK: Phospho-glycerokinase (neoR promoter), rtTA: reverse tetracycline transactivator, SEM: Standard error of the mean, Tbx3: T-Box transcription factor 3, TRE: Tetracycline Response Element. (A) Reprinted by permission from Elsevier Publishers Ltd [183], (B) kindly provided by Michael Kyba.

2.2.3 Embryonic stem culture and differentiation Thawing mESCs: Stocks were stored in liquid nitrogen. It is important not to thaw the frozen media-cell-composite completely, due to cytotoxic effects of DMSO. Therefore cells were diluted in 6ml of Thaw media, centrifuged at 1000rpm for 3min and the pellet was dissolved in 2ml of the required media and added to prepared tissue culture dishes.

Inactivation of mouse embryonic fibroblasts: MEFs were inactivated using Mitomycin C by adding the appropriate amount of Mitomycin to MEF media [34]. After sterile filtration, the media was added to the selected culture vessels for 3h at

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37°C. The inactivation media was removed from the culture dishes and the latter was thoroughly rinsed twice with 1xDPBS. In the last step MEFs were trypsinised and seeded for mESC culture at a density of 50.000/cm2.

Culture of feeder-dependent mESCs: mESCs (except CgR8s) were cultured on inactivated MEFs in a humidified incubator containing 5% CO2 at 37°C. Briefly, media changes were implemented every 24h. First the used media was completely removed from the flask, followed by a washing step with 1xDPBS. Finally pre-warmed Feeder-ESC media (37°C) was added to the cells before transferring them into the incubator. CgR8 ESCs do not require secreted factors of MEFs and were therefore constantly cultivated on 0.2% gelatin coated culture dishes in CgR8-ESC media. Flasks were coated with 0.2% gelatin 30min prior seeding.

Culturing/Passaging mESCs: mESCs grow in a strongly defined system which should be strictly adhered to, to maintain a stable cell culture. Besides regular media changes, cell density should be continuously checked on, respectively. Flasks containing mESCs were washed with 1xDPBS and a considerate volume of Trypsin/EDTA 0.25% was added. For CgR8 ESC culture Trypsin/EDTA 0.05% was used. After trypsinating for about 3-4min at 37°C, Feeder-/CgR8-ESC media was added to inactivate Trypsin. The volume (x) added, depended on the density prior splitting and the prospective mESCs density. Finally, mESCs were detached by manual force of a pipette, counted [86, 161] and plated at a density of 25.000/cm2 onto freshly prepared flasks.

Culture of HEK 293T cells: HEK 293T cells were mainly used for luciferase assay and lentiviral production. Cells were cultured in HEK 293T media and passaged every 2-3 days. Briefly, old media was replaced by 1xDPBS for washing. Next, cells were incubated in Trypsin/EDTA 0.25% for about 4min at 37°C and finally resuspended in fresh media. Cells were seeded in the desired rate (1:10) onto new tissue culture dishes.

In vitro Differentiation of mESCs: In vitro differentiation of mESCs was carried out using the hanging drop method (Figure 8). First mESCs were deprived from

- 37 - feeder cells by pre-plating. This was implemented by remaining cells in the old culture dish after trypsinisation in Feeder-ESC media for about 20min. Existing feeder cells settled to the bottom, leaving floating mESCs behind. The media was then transferred to a tube, followed by counting using the Thoma counting chamber. ESC-Differentiation media was prepared by first adding 13µl Monothioglycerol (MTG) to 1ml of ESC-Differentiation media to prepare a working stock. 1µl of the working stock solution was added to 1ml of ESC-Differentiation media. In the last step Dox was added at 1µl/ml where indicated. Media change was performed every 48h. After adding the calculated number of mESCs to the ready prepared ESC-Differentiation media, 20µl drops were placed on the lids of 10cm culture dishes filled with 10ml 1XDPBS in order to produce a humid climate. mESCs aggregated to EBs in hanging drops for 2 days. On day2, EBs were rinsed and further cultivated on non-adherent bacterial dishes. On day4 EBs (n = 11) were either harvested or alternatively plated on 0.2% gelatin coated 6-well dishes or on cover slips for further analyses, respectively and assayed at specific time points as described in the figure legends. During the differentiation, at about day8, mESCs showed morphological changes due to developmental processes. Beating clusters, visible as small definable areas of beating activity, were quantified as a read-out representing early cardiac development.

Figure 8 In vitro differentiation of mESCs. In vitro differentiation was performed by the hanging drop method. Briefly, a calculated number of mESCs was added to the ready prepared ESC-Differentiation media. 20µl drops were placed on the lids of culture dishes for 2 days. On day2, the mESCs had aggregated to EBs and were rinsed and further cultivated on non- adherent bacterial dishes. On day4 EBs were either harvested or alternatively plated on 6-well dishes or on cover slips for further analyses and assayed at specific time points as indicated. Abbreviations: ChIP: Chromatin Immunoprecipitation, EBs: embryoid bodies, ESC: embryonic stem cell, FACS: Fluorescence Activated Cell Sorting, IF: Immunofluorescence, mESCs: mouse embryonic stem cells, qRT-PCR: quantitative real-time polymerase chain reaction, WB: Western Blot.

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2.2.4 Lentivirus production One day prior transfection HEK 293T cells were split and seeded at a density of 5x106 onto 10cm culture dishes and cultured in HEK 293T media. Transfection was performed the next day on about 60-80% confluent dishes. The below described reagents were added to each dish. Transfection mixture: . 415µl DMEM . 70µl PolyFect transfection reagent . 8µg virus construct (12.5µl) . 5.5 µg psPAX2 vector DNA (6.7µl) . 2µg pMD2 vector DNA (3.3µl)  Incubated at RT for 10min before adding 1ml of fresh DMEM

The old HEK 293T media was replaced by 6.5ml DMEM. Next, the prepared transfection mixture was added drop by drop, trying to distribute the added volume across the whole dish. Then dishes were incubated for 4h at 37°C. After incubation, the transfection media was replaced by HEK 293T media. Viral supernatant was collected at 24, 48 and 72 hours after transfection, stored at +4°C for 7 days at the most.

Transduction of mESCs with expression cassette (LV tdTomato): A2lox.cre ESCs were used for transduction. mESCs were split onto 6-wells at a density of 4.5x105 and grown in Feeder-ESC media. The next day, growing media was removed and replaced by 1ml Feeder-ESC media, 20µl of Polybrene and 1ml virus supernatant per 6-well. The media was replaced every 12h by Feeder-ESC media for 3 days. Successfully transfected cells were labeled with a lentivirus expressing Red fluorescent protein (tdTomato). Transduction rate was monitored each day under fluorescence microscopy. Cells were sorted for Tomato-positive cells by FACS (Fluorescence Activated Cell Sorting).

2.2.5 Reporter plasmid & Luciferase assay mESCs were plated at a low density 48h prior transfection, thereby being about 70-90% confluent at desired time. mESCs were incubated with a transfection mixture containing the selected reporter constructs for 5min at RT. Further, 3µl

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Lipofectamin2000 and 50µl OptiMEM, which had already been incubating for 5min, were added to each well and incubated for 20min at RT. The solution was replaced by 400µl transfection media (Feeder-ESC media without P/S) for another 6h. Last, transfection media was replaced by Feeder-ESC media 6h after transfection to reduce cytotoxic effects of the transfection agents. As a transfection control, HEK 293T cells were transfected in 24-well plates with 200ng of indicated reporter plasmids and 50 ng of pTK-RL (plasmid DNA encoding Renilla luciferase) using Lipofectamine2000 reagent. The Dual-Luciferase Reporter Assay was performed according to manufacturer’s instructions. Briefly, this assay evaluates the regulated gene expression by measuring luciferase reporter activity of the two reporter genes firefly luciferase and RL (Renilla). Based on the light emission measured by the luminometer, values of firefly luciferase were normalised to the RL values. The results were represented as relative luciferase units (RLU).

2.2.6 RNA extraction Cells were washed, pelleted and lysed with 350µl RLT-Buffer. The lysate was homogenised by pipetting and transferred into a 1.5ml tube. At this stage the samples were either stored at -80°C or processed as described below. Total RNA was extracted by using the RNeasy Mini Kit recommendations of the supplier’s manuscript. The eluted RNA sample was stored at -80°C.

2.2.7 Immunocytochemistry Monolayer: Prior, mESCs were plated on a 0.2% gelatin coated cover slip and cultured until the desired time points. Briefly, Feeder-ESC media was removed, cells were washed with DPBS and fixed by incubating with PFA 4% for 15min at RT. PFA 4% was removed and mESCs were washed in DPBS, following incubation with NH4Cl for 10 min at RT. After the washing step 0.2% TritonX was added to the cells for 15min at RT to perforate the cell membrane. Regarding the following steps, cover slips were continuously placed in humid chambers and a working volume of 200µl was added. Prior antibody incubation, cells were blocked to reduce unspecific binding. After 45min primary antibodies were added. Before adding fluorescence-labeled secondary antibodies, cells were washed with DPBS. The added secondary antibodies were incubated for 45min at RT under light protection. Following washing nuclei were stained with DAPI for 5 min at RT.

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Images were captured using an upright fluorescence Zeiss Axioimager Z1 microscope and analysed using Axiovision software.

Wholemount staining of floating embryoid bodies: EBs were collected and centrifuged at 800rpm for 1min. Next, the supernatant was discarded and the EBs were incubated in PFA 4% for 15min at RT. Throughout the process tubes were placed onto a rotor to ensure continuous movement. Washing steps with a solution of DPBS + 0.3% TritonX were performed before and after adding IF antibodies. Between the washing steps the tubes were centrifuged for 3sec at 1000rpm and finally incubated in NH4Cl for 10min at RT on a rotor. After blocking procedure for 45min at RT on the rotor the primary antibody was added for 3h at +4°C in a solution containing 0.3% TritonX. Next, the fluorescence-labeled secondary antibodies were added for 45min at RT. EBs were placed in a tube containing

DAPI for nuclei staining for 5 min. In the last step, EBs were washed in dH2O and placed in 2 drops of mounting media on objective slides. Finally, a cover slip was placed on top very carefully preventing air bubble formation. Images were captured as described above.

Staining of preimplantation embryos: The staining was carried out together with Ronan Russell. Embryos were flushed at 1.5dpc and cultured in 6-well plates until 3.5dpc in M16 media. Staining was performed as previously described [120] with rabbit anti Tbx3 and mouse anti Oct3/4. All images were taken with an ApoTome fluorescence microscope.

2.2.8 Immunoblotting Cell lysate preparation: Briefly mESCs were plated on culture dishes and incubated at culture conditions for 48h-72h prior cell harvesting. On the desired day cells were placed on ice, washed twice with ice-cold DPBS, scrapped down and transferred into a tube. After centrifugation the supernatant was removed, the remaining cell pellet was incubated with RIPA lysis buffer, adding 2-3 times the volume of the cell pellet. After additional manual force the broken pellet was spun, the supernatant, containing proteins, was transferred to another tube for Bradford Protein Assay. The protein concentration was determined in relation to BSA standards. All tubes usually contained 50-100µg of protein and were normalised to

- 41 - a selected volume. Subsequently, Laemmli Buffer was added, at 1/5 of the total volume, to the tubes.

SDS-PAGE: Proteins were separated according to their molecular weight by SDS- PAGE. Briefly they are run through polyacrylamide gels, first the stacking gel and last the separation gel, located in electrophoresis buffer in a voltage-dependent environment. Usually a voltage of 130V for 1h was sufficient. Molecular size was determined by comparing the selected protein to commercial size standards.

Western Blot (WB): In order for antibodies to bind the selected proteins, they first had to be transferred onto a PVDF membrane. This requirement is achieved by electro transfer in transfer buffer, again in a voltage-dependent environment. Usually a voltage of 400mA was applied overnight. To diminish unspecific binding the membrane was blocked for 30 min at RT. Subsequently, primary antibody dilution was added as noted above, followed by washing with TBS-T and incubation with the respective secondary antibodies. After washing the membrane with TBS-T, detection was performed with either ECL or ECL+ substrates.

2.2.9 Chromatin Immunoprecipitation Preparation of cells: Chromatin Immunoprecipitation (ChIP) was performed on differentiating cells. Briefly, mESCs were first differentiated in vitro by the hanging drop method with and without Dox induction. On day4 EBs were transferred into a 15ml tube filled with DPBS and spun at 700rpm for 2min. Next EBs were separated to a single cell suspension by incubation with Trypsin/EDTA 0.25% for 1-2min and finally dissociated by manual force using a pipette. Trypsin/EDTA activity was inactivated by adding ESC-Differentiating media.

Fixation – Sonication: Method was performed by using the ChIP-IT® Express Enzymatic Kit according to manufactures protocol with modifications listed below. Cells were fixed by incubation in Formaldehyde for 7min at RT. The tube was thereby inverted several times to fix DNA-interacting proteins in all of the cells. Next, Glycine Buffer (final concentration 135mM) was added resulting in a yellow staining. Following another 7min incubation (RT) under continuous movement, 1xPBS was added. The mixture was centrifuged for 10min at 2500rpm at +4°C.

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Then the supernatant was discarded and the pellet was washed in 1xPBS. Regarding this step, it was important to completely break the pellet by vortexing to get a single-cell suspension. The tube was spun for 10min at 2500rpm at +4°C. After removing PBS, cells were resuspended in ice-cold Lysis Buffer, transferred into a 1.5ml Eppendorf tube and incubated on ice for 30min. In the meantime, cells were manually lysed with the Sub-Q-syringe and afterwards centrifuged for 10min at 5000rpm at +4°C. After removing the supernatant, the pellet was resuspended in Digestion Buffer vortexed and incubated for 5min at +37°C. An enzyme working stock was prepared by adding 1µl Enzymatic shearing cocktail to 99µl Glycerol 50%. The mixture was vortex firmly. Next, 17µl of the enzyme working stock was supplemented to the pellet and incubated for 21min at +37°C, whereas the tube was vortexed every 2min. Thereafter, EDTA was added to the tube which was then placed on ice to chill for 10min. Next, the cells were sonicated with the following settings (2 cycles of 10 pulses) (Figure 9) and after that centrifuged for 10min with 13000rpm at +4°C. This time, the supernatant containing the sheared chromatin was divided into low-binding tubes and frozen at -80°C until the concentration and size of the respective DNA was quantified. Therefore, 50µl of sheared chromatin was maintained at RT, supplemented with Ampuwa and 5M sodium chloride, and reverse cross-linked at +65°C overnight. Additionally, 11µl of unsheared chromatin, also referred to as Input, was complemented with ChIP Buffer II and 5M sodium chloride and stored at -80°C.The reverse cross-linked DNA was incubated with RNase A for 15min at 37°C. Next, Proteinase K was supplemented and incubation was prolonged for 2h at 42°C. DNA purification was performed according to supplier’s manuscript. The eluted DNA concentration was measured with Nanodrop. 10µl of a calculated concentration was then added to an agarose gel (1.2%) together with 15µl TE and 5µl of 5X Loading Dye. Finally, 10µl of DNA Ladder was added and the Electrophoresis was run for about 45min at 160V.

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Figure 9 Sonication. DNA fragmentation after 2min of enzymatic digestion and 2x10 cycles of sonication. Abbreviations: DNA: deoxyribonucleic acid.

Pre-clearing and antibody incubation: Procedure was performed according to manufactures procedures. Pre-cleared DNA was magnetically separated from the beads and transferred into a new tube. Antibodies were added as mentioned above.

Reverse Cross-linking: Reverse Cross-linking was performed according to manufactures procedures. Samples were stored at -20°C until PCR was performed. qPCR Analysis: The purified DNA and 1:10, 1:100 or 1:1000 of the respective input DNA were used as templates for qPCR using Rotor Gene RT PCR Cycler. Precipitated and different dilutions of total input DNA were analysed in triplicates after adding the following substrates: . 10µl SYBR + 1µl fwd Primer + 1µl rev Primer + 7µl Ampuwa +1µl DNA

Rotor Gene qPCR Cycler was adjusted to the following profile: Table 17 Quantitative real time polymerase chain reaction programme setup for Chromatin Immunoprecipitation. Abbreviations: min: minutes, sec: seconds Step Temperature Duration Denaturation (1x) +95°C 5min Denaturation (40x) +95°C 10sec Amplification(40x) +56°C/+60°C 30sec Melting Curve analysis ramp from +56°C/+60°C to +95°C 90sec at first step, (1°C increase per step) otherwise 5sec

End +40°C 2min

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Ct-values were first normalised to the respective input DNA and are represented as % of input. Appointed annealing temperatures: Nodal primer  56°C, Eomes and T primer  60°C, Sox17 primer  65°C.

2.2.10 Primer Primers for qPCR: As mentioned above, commercial primers were applied by QuantiTect. Self-made primers were designed by the Kleger Laboratory, Tata Purushothama Rao and purchased from Biomer. Primers for ChIP: Previous studies have demonstrated that the T-Box domains of T-Box transcription factors mostly bind to the DNA consensus sequence TC- ACACC-T. In some cases only the core sequence (ACACC) is bound, thus showing various flanking nucleotides. In order to identify whether Tbx3 binds to the putative T-Box elements located within the promoter regions of desired genes the first 2000 nucleotides located 5’ in front of the transcriptional start site of the gene of interest were analysed for the presence of putative T-Box elements. Around these identified putative Tbx binding site primer for Chip analyses were designed in that way that the resulting amplicon bearing the putative Tbx binding site was 18 to 25 base pair (bp) in size.

2.2.11 Quantitative one-step real-time polymerase chain reaction qRT-PCR was carried out with the Rotor Gene RT-PCR Q Cycler using the QuantiTect® SYBR® Green RT-PCR kit. One-step qRT-PCR contains the property of running reverse transcription and PCR in parallel. Gene expression analysis was performed by measuring the rate of fluorescent dye SYBR Green I intercalating into the minor groove of double-stranded DNA. For qRT-PCR analyses 1µl of purified RNA was reverse transcribed and amplified using the QuantiTect SYBR Green RT-PCR Master Mix.

Table 18 Substrates for quantitative real time polymerase chain reaction. Table listing the necessary substrates for qRT-PCR analyses in respect to either commercial (QuantiTect) or self-designed (Biomer) primers. Abbreviations: dH2O: distilled water, qRT-PCR: quantitative real time polymerase chain reaction, RT Mix: containing reverse transcriptase enzyme, SYBR: SYBER Green. Substrates QuantiTect (µl) Biomer (µl) Primer 2 0.5 SYBR 10 10 RT Mix 0.2 0.2

dH2O 6.8 8.3

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QuantiTect primers containing sense as well as antisense primers were added at a final concentration of 0.5µM. Self-designed primers were diluted with dH2O to a final concentration of 100pmol/µl and finally were added at a final concentration of 0.5µM. qRT-PCR pipetting preparations were performed on ice, using a pre-chilled plate. Each RNA preparation was tested for genomic DNA contamination by replacing reverse transcriptase with water. Internal standards (housekeeping gene) and samples were simultaneously amplified. qRT-PCR was performed as recommended by supplier’s manuscript. qRT-PCR conditions were adjusted to the following profile:

Table 19 Quantitative real time polymerase chain reaction programme setup. Abbreviations: min: minutes, sec: seconds

Step Temperature Duration

Reverse Transcription (1x) +50°C 10min Denaturation (1x) +95°C 5min Denaturation (40x) +95°C 10min Amplification(40x) +60°C 30sec Melting Curve analysis ramp from +60°C - +95°C, 1°C increase per step 90sec at first step, otherwise 5sec End +40°C 2min

Relative Quantification: Relative transcript expression was depicted as the ratio of target gene concentration to the housekeeping gene Hydroxymethylbilane synthase (Hmbs). Housekeeping genes show virtually stable gene expression, independent from cell cycle and other interferences. Values were calculated as follows:

c (template) Relative concentration = c ( reference)

Target gene concentration, c (template) = 10 (CP-Intersection)/ (slope) Reference concentration, c (Hmbs) = 10 (CP-Intersection)/ (slope)

Each transcript concentration was calculated by referring to the respective standard curve. This standard curve was defined by measuring samples with a known template concentration in a tenfold dilution series. Standard curve slope was defined at -3.33, intersection, log-concentration of reference RNA diluted 1:1, was defined at 15. The general principle of qPCR analysis is plotting logarithmic

- 46 - change of fluorescence amount against the number of cycles. Crossing point (CP) was set using the Rotor Gene software (Figure 10).

Figure 10 Illustration of CPs depending on amount of fluorescence. As shown gene 1 (blue, CP=21) depicts a lower CP as gene 2 (green, CP=25), illustrating a higher mRNA amount of gene 1. Abbreviations: CP= crossing point, mRNA: messenger RNA.

Melting curve analysis: Melting curves analysis is a method evaluating the dissociation property of double-stranded DNA during heating process and describes the state at which 50% of DNA is denatured. PCR product was acquired stepwise between 65°-95°C. Primer dimers, resulting in flat melting curves, were excluded.

2.2.12 Fluorescence Activating Cell Sorting analysis For FACS analysis the Sox 17-Red fluorescent protein (RFP) reporter cell line and T-GFP/Dppa4-RFP reporter cell line were used. On designated days, EBs were washed with 1XDPBS and dissociated into single cell suspension by incubation with 0.25% trypsin/EDTA. The cells were dissolved in 5% Fetal Calf Serum (FCS)/DPBS and analysed with BD FACSAria III cell sorter. All events were gated with forward-scatter (cell size) and side-scatter (cell granularity) profiles. T- GFP/Dppa4-RFP reporter cell line was sorted for GFP-positive and RFP-negative cells. Thereby, the Dpp4-RFP-positive cells, which represent pluripotent cells resisting differentiation, were able to be excluded. Regarding the Sox 17-RFP reporter cell line, RFP-positive cells were sorted. Herein, pluripotent cells were

- 47 - excluded by co-staining of the dissociated EBs with SSEA1, a surface marker labeling pluripotent cells. Sorted cells were used for further analysis containing cytospin and RNA preparation. 100,000 mESCs were sufficient for cytospin assays respectively. Briefly 100,000 mESCs were resuspended in FACS buffer (5% FCS/DPBS) and pipetted to the tubes of the cytospin. Centrifugation was set to 3min at 1000rpm, whereby cells were spun against object slides. Afterwards cells were incubated with PFA 4% and IF was implemented as described above.

2.2.13 Data Analysis and Statistics Cells were counted using the Neubauer or Thoma cell counting chamber and quantified using the AxioVision Rel. 4.8 Software [86]. Unless stated otherwise, independent experiments were performed at least three times by using mESCs of similar quality and passage. All experiments (n=3) in biological replicates. Statistical analyses were performed using the Student’s t-test or the one-way analysis of variance (ANOVA test) and subsequent post-hoc Bonferroni’s multiple comparison test. Results are demonstrated as standard error of the mean (SEM). Statistical significance was calculated with the Prism 5 software (Graph Pad) and is illustrated as follows: p<0.05 = *; p<0.01 = **; p<0.001 = ***.

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3 Results

Tbx3 expression is vigorously regulated in mouse early embryonic development Moderate Tbx3 expression has previously been indicated by published transcriptome data of preimplantation stage mouse embryos [60] beginning at the 4 cell stage, increasing towards the blastocyst stage (Figure 11A). To assess early embryonic Tbx3 expression, mRNA and protein dispersion of Tbx3 were analysed in pre-and postimplantation stage embryos. IF analyses demonstrated Tbx3 colocalisation with Oct3/4 in the ICM of E3.5 blastocysts (Figure 11B) [183]. The Arnold Laboratory supplied mRNA In situ hybridization (ISH) data on early pregastrulation stage embryos (E6.25) revealing Tbx3 expression in the proximal posterior pole of the epiblast in addition to the already reported expression in the extraembryonic structures (Figure 11C, left panel). Further, at the midgastrulation stage (E7.5) Tbx3 expression was predominantly identified in the extraembryonic visceral endoderm and in a ring of mesoderm in the proximity of the embryonic- extraembryonic intersection (Figure 11C, right panel). At late gastrulation stages (E7.75, E8.25), Tbx3 mRNA was present in the cardiac crescent and the tail region of the embryo (Figure 11D). Notably, Tbx3-reporter expression was found in E6.5 embryos (GFP expression driven by a 160kbp bacterial artificial chromosome with Tbx3 and flanking sequences kindly provided by Horsthuis [75] in extraembryonic structures and in the proximal posterior epiblast (Figure 11E). In summary, the data demonstrates that Tbx3 is vigorously regulated during early development.

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Figure 11 Tbx3 expression in the developing mouse embryo. (A) Tbx3 (blue) in preimplantation embryos plotted with other mesendoderm-class mESC genes (Oct3/4 (red), Klf5 (brown), Klf9 (pink), T (purple)). The data were obtained performing reanalyses of published data sets. (B) IF of E3.5 blastocyst for Oct3/4 (red) and Tbx3 (green). Scale bars, 20 mm. Nuclei stained in DAPI (blue). (C–E) Tbx3 expression in postimplantation mouse embryos: (C) ISH: Tbx3 mRNA expression at E6.25 (prestreak) and E7.5 (midstreak). Arrows illustrate Tbx3-positive cells within the proximo-posterior epiblast at E6.25 and mesoderm at E7.5. (D) Tbx3 mRNA expression in wholemounts of WT embryos at E7.75 and E8.25. (E) Z-section of an E6.5 Tbx3-GFP reporter embryo. Nuclei stained in DAPI (blue). Scale bars, 100 mm. Abbreviations: DAPI: 4′,6-Diamidin-2-phenylindol, GFP: Green fluorescent protein, Klf5/9: Krueppel-like factor 5/9, IF: Immunofluorescence, ISH: In situ hybridization, m: mesoderm, mESC: mouse embryonic stem cell, Oct3/4: Octamer binding transcription factor 3/4, mRNA: messenger RNA, T: Brachyury, Tbx3: T-Box transcription factor 3, WT: wild-type. In vivo data (ISH) was assessed by Arnold Laboratory. Reprinted by permission from Elsevier Publishers Ltd [183].

Tbx3 drives mESC differentiation towards mesoderm and endoderm Moderate Tbx3 mRNA expression was observed in undifferentiated mESCs (Figure 12A). During early differentiation stages (day1 and day2) Tbx3 levels were downregulated and rapidly increased with a peak on day3, thus subsequently diminishing until day6. Similar results were obtained by WB and IF (Figure 12B- 12C). Intriguingly, qRT-PCR and IF analyses exhibited elevated Tbx3 expression patterns correlating with the expression of critical lineage determining factors such as Eomes, T, Sox17, and Mesp1 (Figure 12D–12G).

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Figure 12 Tbx3 expression during mESC differentiation (A–C) Tbx3 expression during mESC differentiation using EBs: (A) qRT-PCR analysis, (B) Immunoblotting including quantification, (C) wholemount IF of Tbx3 (green) in differentiating EBs. (D–G): Temporal expression of lineage-specific markers (Eomes, T, Sox17, and Mesp1) during mESC differentiation: (D) qRT-PCR, (E–G) protein expression using IF (green). Scale bars, 20 mm. Nuclei stained in DAPI (blue). Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: d: day, DAPI: 4′,6-Diamidin-2-phenylindol, EB: embryoid bodies, Eomes: Eomesodermin, Hmbs: Hydroxymethylbilane synthase, IF: Immunofluorescence, mESC: embryonic stem cell, Mesp1: Mesoderm posterior 1 homolog, qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, SSEA1: Stage-specific embryonic antigen 1, T: Brachyury, Tbx3: T-Box transcription factor 3. Reprinted by permission from Elsevier Publishers Ltd [183].

Counting statistics at day4 of differentiation revealed Tbx3 colocalisation with the early differentiation markers Eomes (67% ± 7%), T (46% ± 2%), and Sox17 (51% ± 8%) (Figure 13A). Hence, reporter cell lines tracing mesoderm (T) and endoderm (Sox17) transcription were used to test whether Tbx3 was specifically enhanced in the subgroups of differentiating mESCs. The reporter cell lines were likewise differentiated using the hanging drop method as described above. After differentiation for 4 days EBs were dissociated and FACS-purified for T- or Sox17- positive cells. Further, remaining pluripotent cells were excluded from the analyses through the pluripotency reporter Dpp4-RFP (T-GFP/Dppa4-RFP reporter cell line) or SSEA1-GFP staining (Sox 17-RFP reporter cell line) (Figure 13B; Figure 13D). Interestingly, qRT-PCR showed enriched Tbx3 expression in cells committed

- 51 - towards the mesoderm and endoderm cell fate (Figure 13C; Figure 13E). The finding was reinforced by IF analyses confirming that Sox17- or T- positive-sorted cells co-expressed Tbx3, respectively (Figure 13F-13G). Thus, these results suggest that endogenous Tbx3 expression in differentiating mESCs correlates with mesendoderm cell fate specification.

Figure 13 Tbx3 is co-expressed with lineage-associated genes in differentiating mESCs IF shows coexpression of Tbx3 (red) with Eomes (green, upper panel row), T (green, middle panel row), Sox17 (green, lower panel row) in differentiating mESCs (day4) and quantifications of counted cells as bar graphs on the right. (B) FACS of T-GFP/DPP4-RFP knockin mESCs on day4 of differentiation and inset illustrating description of reporter alleles. (C) Expression of Tbx3 in cell populations outlined in (B). (D) FACS plot of Sox17-RFP knockin mESCs on day4 of differentiation and inset illustrating description of reporter alleles. (E) Expression of Tbx3 in cell populations outlined in (D); pluripotent cells were depleted by SSEA1 staining. (F) Tbx3 IF (magenta) of FACS-sorted T-GFP-positive and DPP4-RFP- negative cells. (G) Tbx3 IF (magenta) of Sox17-RFP-positive and SSEA1-negative cells. Scale bars, 20 mm. Nuclei stained in DAPI (blue). Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: DAPI: 4′,6-Diamidin-2-phenylindol, Dpp4: Dipeptidylpeptidase 4, Eomes: Eomesodermin, FACS: Fluorescence Activated Cell Sorting, GFP: Green fluorescent protein, Hmbs: Hydroxymethylbilane synthase, IF: Immunofluorescence, mESC: mouse embryonic stem cell, RFP: Red fluorescent protein, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, SSEA1: Stage-specific embryonic antigen 1, T: Brachyury, Tbx3: T-Box transcription factor 3. Reprinted by permission from Elsevier Publishers Ltd [183].

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Besides, Tbx3 has also been reported to improve iPSC reprogramming. In this respect, a previous study [67] used ChIP-sequencing analysis to identify pluripotency-associated genes directly regulated by Tbx3. Revaluating this data, we found that Tbx3 binding was enriched to the promoters of pluripotency promoting factors such as Klf5, Klf4 and Oct3/4 and further promoters of key lineage determining factors including SRY (sex determining region Y)-box 9 (Sox9), Nk6 homeobox 1 (Nkx6.1), and Eomes (Figure 14). This implicates that Tbx3 may play an important role in the stage of switching from pluripotency to mesendodermal lineage allocation.

Figure 14 Tbx3 directly interacts with lineage determining genes in the pluripotent state. Previously generated ChIP- sequencing data sets (Han et al., 2010) uncover the direct regulatory targets of Tbx3 during reprogramming. This data was reanalysed for Tbx3-binding of certain pluripotency genes (dark grey columns; Klf5, Klf4, Oct3/4) and lineage determining genes (light grey; Sox9, Nkx6.1, Eomes). X-axis reveals fold enrichment of certain genetic regions of the respective genes upon CHIP with a specific Tbx3-binding antibody. Abbreviations: CHIP: Chromatin Immunoprecipitation, Nkx6.1: NK6 homeobox 1, Sox9 : SRY (sex determining region Y)-box 9. Reprinted by permission from Elsevier Publishers Ltd [183].

In order to test this hypothesis, a targeted, inducible Tbx3 expression allele in mESCs was generated to allow for the temporally regulated and dose-dependent expression of Tbx3.This technique is referred to as a tetracycline-controlled inducible Tbx3 expression system (iTbx3 mESCs; Figure 15A-15C). By using the hanging drop method, effects of Tbx3 overexpression on marker genes of all three germ layers (Figure 15D) were assessed. In Tbx3-overexpressing cells, enhanced allocation towards mesoderm and endoderm progenitors was confirmed by IF for Eomes, T and Sox17 in day4 EBs (Figure 15E). On mRNA level Tbx3 overexpression resulted in upregulation of mesendodermal (Chrd, Gsc, Eomes, Lhx1 (Figure 15F), mesodermal (T, Mesp1, Nkx2.5; Figure 15G) and endodermal marker transcripts (FoxA2, Sox17, Hex1; Figure 15G). Further marker genes specific for the pregastrula epiblast (FGF5, Orthodenticle homeobox 2 (Otx2), Nanog; Figure 15I) were also upregulated after Tbx3 induction. In contrast however, ectoderm (Paired box protein 6 (Pax6); Figure 15H) and trophectoderm- specific gene expression (Cdx2, Hand1, WNT6, FGFR2; Figure 15H; Figure 15J)

- 53 - was reduced. In line with previous reports [100], marker genes of primitive endoderm, such as Hnf4, Hnf1, GATA6 and AFP were increased following Tbx3 overexpression (Figure 15K).

Figure 15 Inducible Tbx3-expression allele in mESCs drives mesoderm and endoderm specification. (A) High mRNA expression of Tbx3 in Dox-treated iTbx3 mESCs after 24 and 48h. (B-C) Tbx3 protein levels are elevated in response to Dox-treatment after 24h in iTbx3 mESCs and EBs (D) Illustration of the mESC differentiation protocol. EBs were generated over time from initially pluripotent mESCs at day0 (orange) to cells expressing markers of the three germ layers (ectoderm (blue), mesoderm (yellow), endoderm (green). (E) Wholemount IF in iTbx3 EBs on day4 for Eomes (red, left panel), T (red, middle panel) and Sox17 (red, right panel) upon Tbx3 induction. (F–H) qRT-PCR analyses demonstrating marker gene expression levels at day4 of differentiation: (F) mesendoderm, (G) mesoderm and endoderm, (H) trophoblast and ectoderm with respect to Tbx3 induction (± Dox). (I-K) Microarray expression data depicted as log2 fold for a series of lineage specific genes on day4. Tbx3-induced mESCs reveal upregulation of (I) epiblast-associated genes whereas (J) trophoblast genes are downregulated upon Tbx3-induction. (K) qRT-PCR analyses of certain marker genes representing primitive endoderm. Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: Cdx2: Caudal type homeobox 2, Chrd: Chordin, d: day, DAPI: 4`,6-Diamidin-2-phenylindol, Dox: Doxycycline, EB: embryoid bodies, Eomes: Eomesodermin, FGF5: Fibroblast growth factor 5, FoxA2: Forkhead-Box-Protein A2, Gsc: Goosecoid, hHex: Hex, Haematopoietic homeobox gene, Hmbs: Hydroxymethylbilane synthase, IF: Immunofluorescence, iTbx3: tetracycline-controlled inducible Tbx3 expression system, Lhx1: LIM-homeobox1, mESC: embryonic stem cell, Mesp1: Mesoderm posterior 1 homolog, mRNA: messenger RNA, Nkx2.5: Nk2 homeobox 5, Pax6: Paired box protein 6, qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, T: Brachyury, Tbx3: T-Box transcription factor. Microarray gene expression analysis was performed together with Dr. André Lechel. Data analysis was performed by with Prof Zenke Laboratory. Reprinted by permission from Elsevier Publishers Ltd [183]. - 54 -

To obtain a global view of Tbx3-induced mESC differentiation, genome-wide transcriptional profiling followed by gene set enrichment analysis was performed. Groups of differentially regulated genes were plotted against published sets of genes that had previously been reported to define a specific lineage committed state. As illustrated, Tbx3-induced mESCs were particularly enriched in mesoderm- and endoderm-associated genes (Figure 16A) at the expense of trophectoderm-associated genes (Figure 16B). The ectoderm lineage were not specifically regulated (Figure 16B). Also, principle component analyses and hierarchical clustering located the transcriptome of Tbx3-induced samples within the three germ layers toward definitive endoderm (Figure 16C-16D). Altogether, a subset of marker genes labelling mesoderm and endoderm are induced upon Tbx3 overexpression. Furthermore the data indicates Tbx3 acting as a regulator of the mesendoderm transcriptional programme.

Figure 16 Microarray gene expression analysis illustrating Tbx3 impact on mesoderm and endoderm Genome-wide transcriptional profiling followed by gene set enrichment analysis shows that Tbx3 selectively enriches mesoderm and endoderm (A) at the expense of trophoblast genes (B). (C) PCA positions of Tbx3-induced cells (Tbx3_d4+dox) close to endoderm. (D) Hierarchical clustering of Tbx3-expressing EBs overlaps with previously published transcriptomes from definitive endoderm [33]. Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: Dox: Doxycycline, PCA: Principal component analysis, Tbx3: T-Box transcription factor 3. Microarray gene expression analysis was performed together with Dr. André Lechel. Data analysis was performed by with Prof Zenke Laboratory. Reprinted by permission from Elsevier Publishers Ltd [183].

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Tbx3 expression promotes mature mesendodermal-derived tissues As illustrated above, Tbx3 seems to drive mesendodermal lineage segregation. Thus we assessed whether Tbx3 also promoted mesendoderm-derived tissues. We therefore overexpressed Tbx3 via prolonged Dox-treatment of mESC differentiation cultures. At late stages of differentiation, Tbx3-overexpressing EBs showed an increase in pancreatic differentiation, depicted by Hnf1 and Pdx1 mRNA levels (Figure 17A). Similar results were achieved on protein level, demonstrated by a few scattered Pdx1-positive cells under control conditions, whereas large clusters of pancreatic progenitors were detected in Tbx3- overexpressing cultures upon Dox-treatment (Figure 17B). Notably, hepatic differentiation was also induced by Tbx3 overexpression, illustrated by elevated Hnf4 and Alb mRNA levels (Figure 17C-17D). Regarding mesoderm derivatives, Tbx3 induction for the first 2 days of EB differentiation was sufficient to significantly enhance the generation of beating cardiomyocytes. mRNA levels of early cardiac- specific TFs Mesp1 and Nkx2.5 were induced respectively (Figure 15G). Further, cardiac markers representing first (Myl2a) and second heart field (Islet1) were upregulated to a similar extent (Figure 17E). However, cardiac induction was most efficient when Tbx3 was induced early (days 0–2 and 0–4) during differentiation, whereas Tbx3 induction after day4 did not further induce cardiac differentiation. This was unlike endodermal lineage allocation, where prolonged Tbx3 induction (days 0-17) enhanced endodermal lineage allocation (Figure 17A-17D). Atrial (Myl2a) and ventricular (Myl2v) markers were expressed at comparable levels (Figure 17F). We further endorsed cardiac induction by protein expression of - Actinin (Figure 17G) and evaluation of beating clusters as well as beating intensity under the respective culture conditions (Figure 17H). Solidifying previous work [108] vascular markers (Cluster of differentiation 31 (CD31), Vascular Endothelial Growth Factor A (VEGF-A), Kinase insert domain receptor (KDR)) were additionally increased, demonstrating that Tbx3 acts early during the generation of cardiovascular progenitors (Figure 17I). In summary, early Tbx3 expression during mESC differentiation promotes enhanced formation of definitive endoderm and mesoderm as well as respective later stage derivatives, namely pancreas, liver, and heart.

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Figure 17 Tbx3 expression promotes mature mesendodermal derivatives: (A–G; I) Tbx3 contributes to both pancreatic, hepatic, and cardiovascular lineage specification as assessed by qRT-PCR (A, C, E, F; I) and IF (B, D; G; Pdx1, Alb and  - Actinin all stained in green) at the indicated time points (pancreatic: Hnf1, Pdx1; hepatic: Hnf4, Alb; cardiac: Myl2a, Islet1, Myl2v, Myh6, and -Actinin; vascular: CD31, VEGF-A, KDR). All scale bars are 20 mm. Nuclei stained in DAPI (blue). (H) Assessment of cardiac beating cluster percentage (y axis) and intensity (x axis) upon different Dox-treatment regimens. Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: Alb: Albumin, d: day, CD31: Cluster of differentiation 31 ,Dox: Doxycycline, Hmbs: Hydroxymethylbilane synthase, Hnf4: Hepatocyte nuclear factor 4, Hnf1: Hepatocyte nuclear factor 1, KDR: Kinase insert domain receptor, Myh6: Myosin heavy chain 6, Myl2a: Myosin light chain 2a, Myl2v: Myosin light chain 2v, Pdx1: Pancreatic and duodenal homeobox 1, qRT-PCR: qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, VEGF-A: Vascular Endothelial Growth Factor A. Reprinted by permission from Elsevier Publishers Ltd [183].

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Tbx3 acts via a Nodal signalling dependent, non-cell-autonomous mechanism to drive mesendodermal cell fate Lineage allocation during gastrulation has been described to be dependent on secreted signalling molecules such as WNT3a, BMP4, and Nodal [199]. Thus, our microarray data was further analysed investigating a potential impact of Tbx3 on any of these three signalling pathways. Interestingly, mRNA expression levels of Nodal and its target genes Left-right determination factor 1 and Left-right determination factor 2 (Lefty1 and Lefty2) were significantly increased in Tbx3- overexpressing cells, whereas BMP4 was significantly downregulated (Figure 18A-18B). The relationship between Nodal and Tbx3 signalling was further investigated, after Nodal having been reported to be a key regulator of induction of mesendoderm both in vivo and in vitro [7]. qRT-PCR analysis showed significantly increased Nodal expression levels in Tbx3-overexpressing cells (Figure 18C) and further increased phosphorylated SMAD2 levels. SMAD2 is one of the major effectors of the activated Nodal signalling [46] (Figure 18E-18F).

Figure 18 Tbx3 activates the Nodal/SMAD2 signalling pathway by directly interacting with Nodal. (A-B) Genome-wide transcriptional profiling to identify fluctuations in developmental signalling pathways in response to Tbx3-overexpression: (A) Upregulation of Nodal within the +Dox treated sample, whereas WNT3a and BMP4 are downregulated. (B) Nodal target genes Lefty1, Lefty2 and Cer1 are upregulated in the +Dox-treated sample. (C) Increased Nodal mRNA levels in iTbx3 cells at day4 of differentiation. (D) Quantification of DNA fragment enrichment by ChIP using Tbx3-specific antibody relative to control isotype measured by qPCR using region specific Nodal primers. (E-F) Immunoblotting analysis for phosphorylated SMAD2 in day4 EBs overexpressing Tbx3 (+Dox) vs control EBs (-Dox). As illustrated Tbx3-induced EBs contain higher levels of phosphorylated SMAD2. (F) Quantification of three independent experiments from (E). Abbreviations: BMP: Bone morphogenetic protein, Cer1: Cerberus, ChIP: Chromatin Immunoprecipitation, d: day, DNA: deoxyribonucleic acid, Dox: Doxycycline, EB: embryoid bodies, Hmbs: Hydroxymethylbilane synthase, Lefty1/2: Left-right determination factor 1/2, mRNA: messenger RNA, SMAD2: Mothers against decapentaplegic homolog 2, qPCR: quantitative polymerase chain reaction, SEM: standard error of the mean, Tbx3: T-Box transcription factor 3, WNT3a: Wingless-related MMTV integration site 3a. Reprinted by permission from Elsevier Publishers Ltd [183]. - 58 -

We next analysed direct (responding to SMAD2/3 activation in the absence of protein synthesis) and indirect SMAD2/3 target genes (presence of protein synthesis) [63] and found both to be enriched upon Tbx3 induction (Figure 19A- 19B). The Tbx3-inductive effects were significantly attenuated by blocking Nodal/SMAD2 signalling (ALK4/5/6 inhibitor: SB421542) as shown by reduced expression of Eomes, Nodal, Hex1, and FoxA2 (Figure 19C). Additionally molecular mechanisms were deciphered, thus analysing Tbx3 binding on the Nodal promoter via ChIP, using Tbx3-specific antibodies. As already suggested by our mRNA data showing increased Nodal levels in Tbx3 overexpressing cultures, Tbx3 was significantly enriched on a highly conserved T-Box binding element within the promoter region of the Nodal gene (Figure 18D).

Figure 19 Mechanism of Nodal/SMAD2 pathway activation illustrated by microarray gene expression profile and Nodal blocking assay. (A-B) Tbx3 activates direct (A) and indirect (B) target genes of the Nodal/SMAD2 cascade: (A) A set of previously published genes showing direct (in absence of protein synthesis) or (B) indirect (in presence of protein synthesis) regulation upon SMAD2 phosphorylation was used for gene set enrichment analysis upon Tbx3 induction and control. (C) qRT-PCR analysis for Eomes, Nodal, Hex and FoxA2 mRNA levels in ± Dox EBs at day4 of differentiation with/without ALK4/5/7 inhibitor SB421542 (10mM), experiment performed together with R. Russell, n=2 in for biological replicates. Error bars indicate SEM. If not stated otherwise n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: ALK4/5/7 inhibitor SB421542: Inhibitor of TGF subfamily, type 1, Dox: Doxycycline, EB: embryoid bodies, Eomes: Eomesodermin, FoxA2: Forkhead-Box-Protein A, Hex: Haematopoietic homeobox gene, Hmbs: Hydroxymethylbilane synthase, mRNA: messenger RNA, SMAD2/3: Mothers against decapentaplegic homolog 2/3, qRT- PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Tbx3: T-Box transcription factor 3. Reprinted by permission from Elsevier Publishers Ltd [183]. - 59 -

Due to the inductive impact of Tbx3 on Nodal expression, and the knowledge that Nodal is likewise secreted through paracrine mechanisms, its paracrine effect on cell specification via enhancing paracrine Nodal signalling was analysed. Therefore fluorophore-labeled iTbx3 cells (expressing the fluorophore tdTomato after lentiviral transduction) and A2 lox cre cells (unlabeled control) were combined in a chimeric EB culture system. Both cell lines were mixed at the indicated ratios and Tbx3 expression was induced by adding Dox when indicated. Non-treated cells served as controls (Figure 20A). At day4 of differentiation labeled and unlabeled cells were FACS-sorted and marker genes were quantified. We found significantly increased expression levels of early mesendoderm markers, as for Lhx1 and Eomes (Figure 20B) in Tbx3-overexpressing cells but intriguingly also in cells not overexpressing Tbx3. These effects increase with rising numbers of Tbx3-overexpressing cells in the mixed EBs, respectively.

Figure 20 Generation of td-tomato-labeled iTbx3 cell line (A) Chimeric EBs were generated by mixing WT and td- tomato-labeled iTbx3 cells at the indicated ratios through the hanging drop method. After differentiation for 6 days, single cells from chimeric EBs are FACS-sorted and analysed by qRT-PCR as shown in (B): (B) qRT-PCR analyses for Lhx1 (upper panel) and Eomes (lower panel) FACS-purified as outlined in (A) for the indicated populations. High gene expression levels of Lhx1 and Eomes in both Tbx3 and WT cells correlate with higher Tbx3 expression in the origin EBs. Abbreviations: Dox: Doxycycline, EB: embryoid bodies, Eomes: Eomesodermin, Hmbs: Hydroxymethylbilane synthase, iTbx3: tetracycline- controlled inducible Tbx3 expression system, Lhx1: LIM-homeobox1, mESC: embryonic stem cell, qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Tbx3: T-Box transcription factor 3, WT: wild-type. Reprinted by permission from Elsevier Publishers Ltd [183].

Similar effects were observed when conditioned media (CM) from iTbx3 cells (treated with Dox to induce Tbx3 expression) was applied to A2 lox cre WT mESCs (Figure 21A). Therefore iTbx3 cells were differentiated using the hanging drop method as described above. At day4 of differentiation the media was - 60 - collected and used as differentiation media for WT cells. The latter were likewise differentiated, EBs were harvested and analysed at day4 showing a marked increase in the mRNA levels of Eomes, T, FoxA2, and Mesp1 (Figure 21B). The findings were confirmed on protein level revealing an increase of Eomes, T, and Sox17 expression levels (Figure 21C-21E). Non-CM-treated WT cells served as an external control and behaved in a similar way to -Dox CM (Figure 21C-21E). To confirm whether the non-cell-autonomous effect of Tbx3 holds true for late mesendoderm derivatives, WT EBs were differentiated in CM up to day4 and in normal differentiating media for the following 10 days. An increase in cardiac cells and pancreatic progenitors was noticed (Figure 22A-22C). In addition, Nodal mRNA levels were analysed to determine its role during paracrine signalling. As known, Nodal regulates its own expression but also induces negative feedback loops in which for example Lefties inhibit Nodal activity [7]. To confirm this physiological loop in CM experiments (Figure 21A), we were able to show specific upregulation of Nodal, as well as Lefty1 and Lefty2 in samples treated with CM generated from Tbx3-overexpressing cells (Figure 21F), strongly pointing to increased levels of secreted Nodal protein upon Tbx3 expression. In order to verify the requirement of secreted Nodal within our model, we applied a Nodal antibody to block secreted Nodal protein in iTbx3 cultures upon Dox-treatment. Notably, we observed a significantly reduction in the Tbx3-mediated expression of several mesendoderm-inducing genes such as Eomes, T, Sox17, and FoxA2 as well as Nodal itself (Figure 21G; Figure 22D). In summary, these experiments provide evidence that the lineage-inducing effects of Tbx3 are partly due to paracrine Nodal effects, whereas Tbx3 also alters expression of pivotal developmental factors, especially, secreted Nodal protein.

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Figure 21 Tbx3 directs cell fate by acting via non-cell-autonomous mechanisms. (A) Illustration of the treatment strategy to generate CM from iTbx3 cells and subsequent generation of EBs. (B) qRT-PCR for Eomes, T, Foxa2, and Mesp1 gene expression in differentiating WT mESCs on day4 cultured in either non-CM or +Dox-CM from iTbx3 cells. (C-E) Protein expression of differentiating WT mESCs cultured in either non-CM or CM from iTbx3 cells on day4. Notably, WT cells treated with +Dox-CM day0–4 of iTbx3 cells show a higher protein expression of the depicted markers (C) Eomes (green), (D) T (green) and (E) Sox17 (green). All scale bars are 20mm. Nuclei stained in DAPI (blue). (F) qRT-PCR analysis on day4 for Nodal, Lefty1 and Lefty2 mRNA expression levels in either non-CM or +Dox-CM from iTbx3 cells reveals its positive impact on either Nodal or its target genes. (G) Nodal, Eomes, T and Sox17 mRNA levels in untreated (-Dox) and Dox-treated (+Dox) iTbx3 cells at day4 of differentiation in ± 3mM Nodal-blocking antibody illustrate the Nodal-dependent effect of Tbx3, (n = 2) independent experiments, each in biological triplicates. Error bars indicate SEM. If not stated otherwise n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: CM: conditioned media, d: day, DAPI: 4′,6-Diamidin-2-phenylindol, Dox: Doxycycline, EB: embryoid bodies, Eomes: Eomesodermin, FoxA2: Forkhead-Box-Protein A, Hmbs: Hydroxymethylbilane synthase, IF: Immunofluorescence, iTbx3: tetracycline-controlled inducible Tbx3 expression system, Lefty1/2:Left-right determination factor1/2, mESC: embryonic stem cell, Mesp1: Mesoderm posterior 1 homolog, mRNA: messenger RNA, qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, T: Brachyury, WT: wild-type. Reprinted by permission from Elsevier Publishers Ltd [183]. - 62 -

Figure 22 Cardiac and hepatic effects of paracrine iTbx3 signalling. (A) Image illustrating the number of beating cardiomyocyte clusters counted on day14 in WT mESCs treated with either a) non-CM, b) –Dox-CM or c) +Dox-CM from iTbx3 cells (+Dox-CM treatment either day0-2 or day0-4). Higher levels of (B) the cardiac marker gene Myh6 and (C) the pancreatic progenitor marker gene Pdx1 at day14 are associated with higher Tbx3 expression levels as assessed by qRT- PCR in WT mESCs treated with either a) non-CM, b) –Dox-CM or c) +Dox-CM from iTbx3 cells (+Dox-CM treatment either day0-2 or day0-4). (D) qRT-PCR analysis for FoxA2 levels in untreated and Dox-treated iTbx3 cells at day4 of differentiation in the presence or absence of Nodal-blocking antibody (final concentration: 3μM, n=2 in for biological replicates). Error bars indicate SEM. If not stated otherwise n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: AB: antibody, CM: conditioned media, d: day, Dox: Doxycycline, FoxA2: Forkhead-Box-Protein A2, EB: embryoid bodies, Hmbs: Hydroxymethylbilane synthase, Ig: Immunoglobulin, iTbx3: tetracycline-controlled inducible Tbx3 expression system, mESC: embryonic stem cell, MyH6: Myosin heavy chain 6, Pdx1: Pancreatic and duodenal homeobox 1, qRT-PCR: quantitative real-time polymerase chain reaction, SEM: Standard error of the mean, Tbx3: T-Box transcription factor 3, WT: wild-type. Reprinted by permission from Elsevier Publishers Ltd [183].

Tbx3 directly regulates key mesendoderm inducting determinants In turn, we investigated the latter suggestion in more detail. Tbx3 actions were only partially explained by paracrine signalling mechanisms, thus its cell- autonomous effects in mediating cell lineage specification were further investigated. First, direct transcriptional activity of Tbx3 on regulating key factors for mesendoderm (Eomes), mesoderm (T) and definitive endoderm (Sox17) development was measured. Notably, the overexpression of Tbx3 led to the activation of Eomes, T and Sox17 reporter constructs (Figure 23A). The direct - 63 - binding of Tbx3 to the corresponding promoters in differentiating mESCs was confirmed by CHIP, using Tbx3-specific antibodies. Briefly, iTbx3 cells were differentiated until day4 under continuous Dox application, using the hanging drop method. On day4 EBs were harvested and ChIP was performed using either Tbx3 antibodies or the IgG antibody as an external control. Tbx3 binding was specifically increased within the promoter regions of Eomes, T, and Sox17 (Figure 23B–23D), whereas no enrichment was assessed for FGF2 (Figure 23E). In summary, Tbx3 directly and indirectly activates a mesendodermal lineage specification programme via transcriptional regulation of specific mesoderm and endoderm TFs and partly through activation of a Nodal/SMAD2 signalling pathway. Oct3/4 has been shown to promote differentiation towards mesendoderm in mESCs and is known to directly activate the Tbx3 promoter [173], thus the hypothesis was raised whether Tbx3 also regulates Oct3/4 expression levels during mESC differentiation. As demonstrated, Oct3/4 mRNA and protein expression levels were significantly increased at day2 and day4 of differentiation following Tbx3 induction (Figure 23F-23G). To further decipher the mechanism of how Tbx3 regulates Oct3/4 expression, reporter assays were performed, demonstrating an increase of Oct3/4 reporter activity upon Tbx3 overexpression (Figure 23H). The direct interaction of Tbx3 on the Oct3/4 promoter and vice versa has previously been shown [173], respectively (Figure 14). Next, an inductive effect of elevated Oct3/4 levels on mesendoderm genes was analysed. In line with previous reports [157], elevated Oct3/4 levels in mESCs enhanced the promoter activity of Eomes and Sox17. This was confirmed by overexpressing Oct3/4 during early differentiation. However, the addition of Tbx3 and thus co- expressing Oct3/4 and Tbx3, revealed a significantly elevated promoter activity of both mesodermal genes Sox17 and Eomes (Figure 23I).

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Figure 23 Tbx3 directly regulates key mesendoderm inducing determinants. (A)Illustration of luciferase reporter constructs of Eomes (left bar), T (middle bar) and Sox17 (right bar). Briefly, promoters were co-transfected into HEK 293T cells with a Tbx3-expression plasmid and evaluated for luciferase activity. (B–E) qPCR quantification of DNA fragments enrichment by ChIP using Tbx3-specific antibodies relative to a control isotype for (B) Eomes, (C) T, (D) Sox17 and (E) FGF2. (F-G) Tbx3 induction increases Oct3/4 expression levels on day4 as shown on mRNA (F) and in wholemounts on protein level (Oct3/4 green, (G)), Dox was added at indicated time points. All scale bars 20mm. Nuclei stained in DAPI (blue). (H) Schematic illustration of the Oct3/4 reporter construct with either proximal or distal Oct3/4 enhancer elements (left panel) was co-transfected into HEK 293T cells together with a Tbx3-expression plasmid and assessed for luciferase activity (right panel). (I) HEK 293T cells were co-transfected with the respective luciferase reporter constructs in the presence or absence of Oct3/4- and Tbx3-expression plasmids (left panel) and assayed for mesendodermal promoter activity of Sox17 (middle panel) and Eomes (right panel). Error bars indicate SEM. If not stated otherwise n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: Con: control, d: day, DAPI: 4′,6- Diamidin-2-phenylindol, Dox: Doxycycline, EB: embryoid bodies, Eomes: Eomesodermin, FGF2: Fibroblast growth factor 2/bFGF, Hmbs: Hydroxymethylbilane synthase, mRNA: messenger RNA, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, T: Brachyury, Tbx3: T-Box transcription factor 3. Reprinted by permission from Elsevier Publishers Ltd [183]. - 65 -

During early development Tbx3 is integrated in a complex regulatory mechanism with closely related T-Box factors So far, most of our data were based on in vitro experiments. Thus, we aimed to validate our findings in vivo. To do so, the role of Tbx3 was investigated in the developing Xenopus laevis in Collaboration with Prof. Kühl’s Laboratory. Previous studies have demonstrated that early development in Xenopus follows a similar mechanism as in higher vertebrates [55]. Thus, Tbx3 function was investigated by performing morpholino-oligonucleotide (MO)-mediated loss-of-function experiments. Regarding the requirement of Tbx3 during earlier stages of development, MO was injected into the marginal region of the two dorsal blastomeres at the 4 cell stage. Intriguingly, marker gene analyses at stage 10 and 10.5 showed the downregulation of t and gsc expression, respectively (Figure 24A-24B), thus revealing its crucial role for proper mesendoderm development and further demonstrating its function shown in mESCs. The moderate phenotype in vivo due to the loss of Tbx3 was surprising, given the fact that Tbx3 regulates central mesendodermal lineage determinants in vitro. As both mice [38] and Xenopus lack an early gastrulation phenotype we hypothesised a potential compensatory mechanisms. We therefore tested closely related T-Box factors which have been listed according to their evolutionary proximity [117] (Figure 5). The analog Tbx2 reveals structural similarities and has previously exhibited functional redundancies with Tbx3 during atrioventricular myocardial formation [149] and pancreatic development [13]. Accordingly, Tbx3 shows the highest relationship with Tbx2 and to a lesser degree with Tbx5 or Tbx4. Although the evolutionary relationship between Tbx3 and Tbx6 is not that pronounced, Tbx6, from a developmental point of view, shows the highest overlap with Tbx3 in expression in vitro [130] and in vivo [64]. Based on this knowledge, Tbx2 and Tbx6 were chosen for further analyses. Their expression levels were investigated upon Tbx3 overexpression in iTbx3 mESCs. Their expression levels comported in line with the above mentioned hypothesis upon Tbx3 overexpression. Thus, Tbx2 expression levels were almost abolished as shown by mRNA and protein levels (Figure 24C). Similar reductions were detected for Tbx6 on protein levels, but not as distinct as on mRNA levels (Figure 24C). To reinforce these findings in vivo, loss-of-function analyses in Xenopus using different combinations of MOs

- 66 - specifically targeting [32], tbx3 and tbx6 [171] were performed to analyse gastrulation in more detail. Loss of tbx3 or tbx2 individually resulted in a moderately opened blastopore at stage 13 when control MO-injected embryos already closed the blastopore, indicating a delay of gastrulation movements (Figure 24D). Notably, during ongoing development, these moderately opened blastopores closed, thus explaining the lack of any gastrulation phenotype at later stages. Only in a few cases, the phenotype was so severe that the affected embryos did not recover. Interestingly, upon co-injection of tbx2 and tbx3 MO almost all embryos revealed a gastrulation phenotype, substantiating our hypothesis indicating the functional redundancy of Tbx3 and Tbx2. The loss of Tbx6 resulted neither in an increase of pathologic phenotype nor in a delayed gastrulation movement. Taken together this body of data indicates the requirement of T-Box transcription factors for proper gastrulation movements. In line with the above mentioned hypothesis, some T-Box factors (Tbx2, Tbx3) act in a redundant fashion and are therefore capable of compensating for each other which is an important issue and an evolutionary protective mechanism during early developmental stages.

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Figure 24 Tbx3 functionally interacts with other T-box family members in directing early cell fate choices. (A-B) Injection of 40ng tbx3 MO leads to reduced t (A) and gsc (B) expression (black arrowheads). In contrast, uninjected WT and control MO injected embryos revealed a normal gene expression. Vegetal and lateral views and sagittal sections of Xenopus embryos at stages 10.5 (t expression) or stage 10 (gsc expression) are demonstrated as indicated. Scale bars are 0.5mm. (C) Tbx3 overexpression abolishes Tbx2 (green) mRNA and protein gene expression at day4 of differentiation. Tbx6 (green) expression is reduced but to a lesser extent on mRNA level. All scale bars are 20 µm. Nuclei stained in DAPI (blue). (D) Injection of tbx2 and tbx3 MO resulted in gastrulation phenotypes. The closure of the blastopore was mainly delayed in comparison to control MO injected embryos. In isolated cases, gastrulation was completely blocked. Error bars indicate SEM. n = 3 for all experiments in biological replicates.*p < 0.05; **p <0.01; ***p < 0.001. Abbreviations: Con: control, d: day, DAPI: 4′,6-Diamidin-2-phenylindol, Dox: Doxycycline, gsc: Goosecoid, Hmbs: Hydroxymethylbilane synthase, mRNA: messenger RNA, MO: morpholino, n = number of independent experiments. N = number of injected embryos analysed, SEM: Standard error of the mean, Sox17: SRY (sex determining region Y)-box 17, t: Brachyury, Tbx2/3/6: T-Box transcription factor 2/3/6. In vivo data was assessed by Kühl Laboratory. Reprinted by permission from Elsevier Publishers Ltd [183].

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All in all, this data reveals, that Tbx3 is not only required for maintaining pluripotency, but also has a great impact on promoting lineage determination via a cell-autonomous regulation of key lineage TFs, which has previously also been shown for numerous pluripotency-associated factors (Figure 25; Figure 26).

Figure 25 Spatial distribution of lineage specifying factors at gastrulation. Schematic illustration of lineage specifying transcription factors at gastrulation. Notably, Tbx3 expression patterns overlap many lineage directing genes, especially Nodal and Oct3/4, thereby suggesting a functional interplay. Abbreviations: Eomes: Eomesodermin, Mesp1: Mesoderm posterior 1 homolog, Octamer binding transcription factor 3/4, T: Brachyury, Tbx3: T-Box transcription factor 3.

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Figure 26 Spatial clustering of pluripotency-associated factors during lineage specification. Upon exit of pluripotency core pluripotency factors get committed to distinct lineages and arising tissues, thus playing a crucial role in directing early lineage decisions in mouse. This image illustrates their spatial classification in the early embryo: Neuroectoderm is mainly promoted by Gbx2 and Sox2; mesendoderm especially by Esrrb, Nanog, Oct3/4 and Tbx3. Further, the extraembryonic tissue is induced by Cdx2, Rex1, Esrrb, Sall4, Oct3/4 and Tbx3. Abbreviations: Cdx2: Caudal type homeobox 2, Esrrb: Estrogen-related receptor , Gbx2: Gastrulation brain homeobox 2, Tbx3: T-Box transcription factor 3, Oct3/4: Octamer binding transcription factor 3/4, Rex1: Zfp42, Sall4: Sal-like 4, Sox2: SRY (sex determining region Y)-box 2. Weidgang C, Seufferlein T, Kleger A, Mueller M. Stem Cells International, vol. 2016, Article ID 6838253, 16 pages, 2016. doi: 10.1155/2016/6838253; License (CC-BY-4.0); Link https://creativecommons.org/licenses/by/4.0/. Reprinted by permission from Hindawi Publishing Corporation.

Moreover, Tbx3 acts non-cell-autonomously via a direct activation of Nodal signalling, which, in turn, impacts on the specification of definitive endoderm and anterior mesoderm derivatives (Figure 27).

Figure 27 Hypothesised mechanism reflecting Tbx3 impact on guiding early lineage decisions. Schematic illustration of the proposed mechanism how Tbx3 may function during the exit of pluripotency towards early lineage commitment or rather mesendodermal specification. First, Tbx3 cell-autonomously activates T, Eomes, Sox17 and Nodal signalling players and second activates Nodal-SMAD2/3 target genes non-cell-autonomously to promote mesendodermal lineage specification, possibly involving the Tbx3-expressing cells of the extraembryonic. Abbreviations: Eomes: Eomesodermin, SMAD2/3: Mother against decapentaplegic homolog 2/3, Sox17: SRY (sex determining region Y)-box 17, T: Brachyury, Tbx3: T-Box transcription factor 3. Reprinted by permission from Elsevier Publishers Ltd [183].

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4 Discussion

Impact of Tbx3 in sustaining and exiting pluripotency Tbx3, a crucial member of the T-Box TFs, has defined roles in sustaining pluripotency of mESCs. Briefly, Tbx3 is embedded in a complex network downstream of LIF. It is activated via PI3K and interacts with the promoter of the core pluripotency members Klf4/5, Oct3/4, Nanog and Sox2 [67]. Despite their high impact on maintaining stemness, distinct TFs have previously been shown to connect the transition from pluripotency towards differentiation [173, 183]. Here, they actively inhibit lineage specific target gene expression [44, 173]. This issue was excessively approached by several research groups who dissected the mechanism of pluripotency factors in regulating early developmental processes. This is discussed in more detail below.

Lineage fate of pluripotency factors Besides its impact in maintaining the self-renewal state, Tbx3 is involved in cardiac and hepatic development [101, 149]. We therefore hypothesised whether Tbx3 may promote the exit of pluripotency towards early steps of development. Previously, studies have demonstrated Tbx3 impact on early embryonic lineage differentiation. Thus, Tbx3 supports the pluripotent state by acting as a downstream activator of WNT signalling together with LIF [133]. In pluripotent stem cells WNT ensures self-renewal by repressing lineage specific target genes [180]. Intriguingly, in the absence of LIF WNT induces lineage commitment towards visceral endoderm via Tbx3 [133, 137]. Another study demonstrated a spatial chromatin reorganisation during exit of pluripotency. This results in sensitisation of the chromatin for definitive endoderm-promoting signals, thus allowing the histone demethylase JMJD3 to transiently bind two T-Box factors Eomes and Tbx3 [82]. To clarify Tbx3 impact on mesendodermal differentiation we performed genome- wide transcriptional profiling. We were able to demonstrate, that Tbx3 induces mesendodermal differentiation non-cell autonomously by inducing the Nodal/SMAD2 signalling pathway in a SMAD2-dependent and -independent manner [183]. This was confirmed by blocking both the Nodal and the Nodal/SMAD2 pathway resulting in decreased Tbx3 inductive effects on crucial

- 71 - lineage specifiying genes (Eomes, Hex1, FoxA2, T, Sox17) [183]. Nodal, but also WNT and BMPs signalling modulate early embryogenesis by regulating gastrulation and patterning processes in the embryo [199]. We demonstrate that Tbx3 guides mesendodermal lineage differentiation via Nodal, whereas BMP and WNT signalling are downregulated. Interestingly, pluripotency and developmental processes are guided by opposing Nodal and BMP signals [84, 102]. To date it is not clear why these signalling pathways are affected in this way but this phenomenon has been demonstrated in various settings. For example it is not clear why autocrine Nodal signalling is important to modulate the BMP pathway, as BMPs are important for maintaining self-renewal in a serum-free culture setting. A diminished BMP activity may be necessary to supress mesendodermal and trophoblast lineage differentiation to sustain pluripotency in mESCs. However, in the preimplantation embryo, BMP4 is induced in the ICM of the early blastocyst. Its expression peaks in the epiblast of the E4.5 blastocyst and subsequently decreases afterwards. The decrease of BMP signalling in the postimplantation epiblast overlaps with the upregulation of Nodal/Activin signalling and subsequent target gene expression such as Lefty1 and Lefty2 [16, 84, 102]. We suggest that Tbx3 primes mESCs upon onset of differentiation by inhibiting WNT and BMP and driving Nodal signalling. These primed mESCs then become more permissive for other lineage promoting factors and receive further guidance by other mesodermal-class genes such as Oct3/4 and Nanog. We also believe that Tbx3 promotes mesendoderm formation by direct interaction with Oct3/4. It is well established that the core pluripotency factors regulate each other within complex transcriptional networks [119]. Oct3/4 is involved in maintaining pluripotency, and promotes mesendoderm differentiation [4, 104], by acting in concert with lineage specifiying factors and signalling pathways. Besides, Oct3/4 can define cell fate depending on transcription factor dynamics [132]. Cells with slower Oct3/4 kinetics are primed towards the pluripotent cell lineage, whereas cells with faster Oct3/4 kinetics mostly contribute to the extraembryonic lineage. Thus, Oct3/4 kinetics has been suggested to be more important when predicting cell lineage patterning than total TF expression levels [132]. Nevertheless, regarding the similar expression profiles of Tbx3 and Oct3/4 it was of particular interest to identify a possible relationship during the transition of pluripotency towards mesendodermal differentiation. Its direct binding to the Oct3/4 promoter and vice versa (Figure 14)

- 72 - has previously been shown and was confirmed by luciferase experiments. Therefore, we were able to achieve additive inductive effects on the mesendoderm and endoderm lineage upon Tbx3 and Oct3/4 overexpression [183]. Cell fate decisions are crucial for developmental processes. Previously a body of knowledge was gained regarding how mESC leave the pluripotent state and differentiate. Interestingly, the same pluripotency factors which maintain the self- renewal state have been discovered to guide germ layer cell fate decisions [173]. Mechanistically, differentiation signals steadily and asymmetrically regulate their protein levels and alter their binding behaviour in the genome resulting in lineage specification [173]. The study classified TFs into different classes according to the originating tissue: mesendoderm, neuroectoderm and extraembryonic lineage (Figure 26). Generally, mesendoderm-class genes inhibit neuroectoderm and vice versa to promote the appropriate lineage. Oct3/4 is the master regulator of mESC pluripotency in vitro and in vivo [121]. Also, Oct3/4 regulates trophectoderm lineage differentiation via Cdx2 inhibition [125], a crucial trophectoderm promoting factor [41]. Initially both are expressed in the morula [125] and subsequently establish distinct expression patterns. This interplay, together with Sall4, balances proper ICM and trophectoderm differentiation in the early embryo. At later stages, Oct3/4 generates a mixed population of primitive endoderm or mesendodermal differentiation [173]. Mechanistically, Oct3/4 exerts a dual action by undergoing a molecular switch between Sox2 and Sox17 during exit of pluripotency towards differentiation [157]. Briefly, Sox2 and Sox17 expression levels endow Oct3/4 with alternative functions. During the self-renewal state Oct3/4 interacts with Sox2 via the Oct/Sox motif in a reciprocal fashion to maintain the pluripotent state. The Oct/Sox motif is highly conserved and present on multiple target genes essential for pluripotency. When mESCs leave the pluripotent state, Oct3/4 switches to the Sox17 promoter thereby disrupting the pluripotent Oct/Sox-loop [5, 31] creating an endodermal environment. When comparing Oct3/4 with Tbx3 we see overlappings in expression profile and behaviour while leaving the self-renewal state. Tbx3 further guides early lineage allocation and has also been detected in heart, liver bud and pancreas, acting in a dual fashion [13, 74, 101]. We suggest a functional overlapping during exit of pluripotency, especially given the fact, that Oct3/4 is able to bind Tbx3 promoter and vice versa. Further, overexpressing Oct3/4 and in

- 73 - combination with Tbx3 significantly enhanced the Eomes and Sox17 promoter activity, thus confirming our hypothesis regarding a synergistic effect [183]. Regarding neuroectoderm-class genes, pluripotency factor Sox2 maintains the self-renewal state by mainly interacting with Oct3/4 via the Oct/Sox motif. This suggests potential co-binding with Tbx3 during self-renewal, either direct or indirect through Oct3/4. However, upon differentiation Sox2 promotes the neuroectoderm lineage [173], proposing an inhibition through mesendodermal factors, such as Tbx3 and Oct3/4. Altogether, TFs are embedded in a complex network, acting in a spatial and temporal manner to sustain pluripotency but also strongly guide exit of pluripotency towards lineage allocation. Mechanistically, TFs bind asymmetrically in regulatory regions, thereby interacting with different partners, but further studies are necessary to clarify this issue. Oct3/4, Sox2 and Tbx3 are important players orchestrating this complex stage. We show that Tbx3 is vigorously expressed during specification of the mesendoderm lineages (in vivo (Xenopus, mouse) and in vitro) and directs early embryonic development by either transcriptional activation of differentiating gene programme or modification of chromatin structure. This justifies its classification as a mesendodermal-class gene [173] .

Tbx3 in vitro data matches it’s in vivo counterpart The preimplantation expression profile of Tbx3 matches well with mesendodermal pluripotency factors such as Oct3/4 [121] and Klf5 [96] which together co-localise in the ICM of the late stage blastocyst. Around E6.0, molecular and subsequent morphological programmes initiate gastrulation, thereby moving epiblast cells towards the posterior proximal pole of the mouse embryo before a visible primitive streak has formed. [93]. This is especially due to Nodal signalling [99]. Upon gastrulation (around E6.25) the primitive streak is induced at the proximal posterior pole of the epiblast and subsequently elongates distal along the embryo (Figure 25). We show that within the gastrulating embryo Tbx3 expression patterns overlap significantly with Nodal during the formation of the cell lines at gastrulation (E6.5-E7.5) [7, 18]. Here, Tbx3 may support primitive streak formation and later gastrulation due to its direct binding and activation of the Nodal promoter. In line, we identified Tbx3 expression at the dorsal blastopore lip in Xenopus, considering the equivalent structure to the early primitive streak in mouse. Also Tbx3 enhances

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GATA6 expression levels. GATA6 is known to drive endodermal differentiation, but when overexpressed induces primitive endoderm lineage allocation [49]. Therefore, we suggest that Tbx3-expressing cells of the ExVE support Nodal- signalling in the posterior-proximal epiblast allowing the onset of gastrulation and mesendoderm formation. Notably, previous studies showed both Nodal and BMP signalling to require interactions between the epiblast and the extraembryonic tissues to support early embryonic developmental stages and direct the mammalian epiblast cells towards mesodermal and endodermal fate [176, 199]. Similarly, Tbx3 expression overlaps the expression of Brachyury [12, 115], Eomes [35], Mesp1 and Oct3/4 [115], whereas gastrulation inhibitors (Cerberus (Cer1) and Dickkopf-related protein 1 (Dkk1)) are expressed on the anterior side, respectively [22]. Most importantly, mid-streak E6.75 embryos display robust expression levels of Tbx3 in the proximal mesoderm. Previous data has identified proximal posterior epiblast cells to be already committed to a cardiac fate and Eomes expression within this region labelling the earliest cardiac mesoderm [35]. Thus, the expression pattern in streak embryos correlates well with our mESC data. Herein, Tbx3 expression coincides with critical mesendoderm markers and is specifically enriched in endoderm and mesoderm progenitors, later giving rise to pancreas, liver and heart. In summary, our in vitro data are in line with the in vivo expression pattern of Tbx3 at the posterior epiblast to support an intimate functional interaction with pluripotency and lineage core factors to specify the mesendoderm lineage.

Tbx3 loss of function and compensating mechanisms Gastrulation is a highly complex progress, ensuring the formation of the three germ layers. To ensure its proper formation a gained body of knowledge has demonstrated molecular compensatory mechanisms within the TFs or signalling pathway. This holds true for the T-Box family, as Tbx3 does not seem to be fully required during embryogenesis. Tbx3-null mice do not reveal a gastrulation phenotype. In fact, they die around developmental stage E12.5 due to cardiac and yolk sac defects [38]. Previous studies made use of monogenetic T-Box gene KO mice which displayed missing gastrulation defect, thus suggesting the existence of compensating homologous genes. T-Box genes reveal overlapping expression patterns in several developmental processes [26]. Notably, their expression profile - 75 - reflects their phylogenetic relationship. Thus Tbx2 and Tbx3, and Tbx4 and Tbx5, reveal very similar expression profiles, biochemical properties and in fact functional overlaps which have been demonstrated in both vertebrate and invertebrate embryos [129, 187]. As we know, both Tbx2 and Tbx3 share more than 90% genetic identity within the DNA binding domains. Both have previously been identified during atrioventricular canal and mammalian secondary palate formation [149] as well as during Xenopus eye development [168]. Further, Tbx2 and Tbx3 double KO mice display overall embryonic growth defects compared to single KO mice dying as early as E9.5 [109]. This is interesting as Tbx3 KO mice die at slightly later time points. We therefore suggest the amount of knocked out T- Box genes to accelerate lethality, eventually leading to a gastrulation phenotypes. Despite exhibiting overlapping expression patterns in various tissues, they also reveal distinct spatiotemporal expression profiles, hence functioning in the developing heart, mammary glands and limbs. This knowledge on the T-Box family network is highly relevant within our study, since we observe functional redundancies between various members. Notably, we were able to show significant changes in the levels of Tbx2 and Tbx6 in response to Tbx3 overexpression mESCs [183]. Regarding the fact that the Xenopus closely recapitulates early development of the mouse, we dissected the role of related T- Box family members 2, 3 and 6 for gastrulation in vivo [183]. Their combinatorial effect in Xenopus gastrulation was detected clearly, indicating a lack of compensatory mechanisms suggesting a hypogenetic development in invertebrates. Briefly in Xenopus, the loss of a single factor showed minor effects, whereas a combinatorial approach to knockdown expression of multiple T-Box TFs led to a significant delay in gastrulation in a larger number of embryos. In some occasional cases, there was a trend towards a more severe impact on gastrulation. Intriguingly, the impact of Tbx6 on gastrulation was less distinct, as it was the case for Tbx2 and Tbx3. Tbx6 mutant embryos have been characterised and subsequently shown to be crucial for correcting left/right axis determination, embryo turning and heart looping. These mice also reveal reduced Nodal levels in early development stages. These molecular and phenotype abnormalities appear similar to Tbx3 functions, as we showed Tbx3 to activate Nodal/SMAD pathway in a cell-autonomous and non-cell autonomous mechanism.

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Compensatory mechanisms have also been previously shown for secreted signalling molecules of the TGF family, which exert important roles during lineage specification. Especially one subgroup constituting of BMPs is involved in almost all vertebrate organ formations and is present in , cardiac and limb development, among others [2, 50, 148]. BMPs mediate complex in vivo functions and previous date revealed closely related BMP ligands to guide distinct cellular responses [51]. Regarding their strong impact it is obvious that compensating mechanisms have been generated evolutionary ensuring proper development. Notably, specific embryonic tissues can distinguish between signals from BMP6 and BMP7, sharing 90% identity, and BMP2 or BMP4 compared to BMP7 only sharing 60% identity, resulting in distinct tissue responses [151]. In line with T-Box data, their monogenetic KO leads to a moderate or no gastrulation defect. However, the combined ablation strategies for e.g. activin receptors or BMP subtypes [151, 153] resulted in severe gastrulation defects. At this point, we can conclude that gastrulation in vertebrates and in invertebrates is a tightly regulated process ensuring proper germ layer formation in the early embryo [58, 151].

Conclusion This study describes novel facets of Tbx3 within early cell fate determination in mouse. We propose a model in which Tbx3 orchestrates a complex network of downstream actions to direct pluripotent cells towards a mesendodermal cell fate. We have established the precise molecular mechanisms by which Tbx3 promotes early lineage allocation towards definitive endoderm and anterior mesoderm, thus acting in a dual fashion. Firstly, Tbx3 directly regulates key lineage determining TFs (cell autonomous) and secondly, we illustrate a central role for Tbx3 in a Nodal-mediated paracrine signalling pathway (non-cell autonomous). Further, we demonstrate that Tbx3 is vigorously expressed during the specification of the mesendoderm lineages in vitro and at specific sites during gastrulation onset in developing mouse and Xenopus embryos. We were further able to show an additive effect, where Tbx3 acts together with Oct3/4 to promote mesendoderm specification. Finally, we reveal a functional redundancy between Tbx3 and the closely related family member Tbx2, pointing to a complex compensatory mechanism, guaranteeing proper gastrulation. - 77 -

5 Summary

T-Box transcription factor 3 (Tbx3), a unique member of the T-Box transcription factors, has previously been identified to maintain ground state pluripotency of mouse embryonic stem cells (mESCs). In 2011, subsets of pluripotency factors, such as Nanog, Octamer binding transcription factor 3/4 (Oct3/4) and Tbx3 itself, were shown to adopt new roles in early embryonic development and were globally defined as mesendoderm-class embryonic stem cell genes. However, detailed knowledge explaining how these transcription factors orchestrate cell fate decisions remained largely elusive. Therefore this current study, we aimed to dissect the molecular mechanism elicited by Tbx3 in early mouse embryonic development. Briefly, gastrulation occurs during early embryonic development of all higher and intermediate animals. In this phase the single-layered blastula is transformed into the three-layered gastrula, and contains the three primary germ layers: ectoderm, endoderm and mesoderm. It is important to understand the distinct mechanisms regulating gastrulation, as defects in regulating factors lead to severe diseases or even result in embryonic lethality. The Ulnar Mammary Syndrome (UMS) is caused by Tbx3 mutations in humans involving abnormal forelimb, apocrine mammary gland and genital development. In our study we used a combination of loss of function and gain of function analysis, to establish novel facets of Tbx3 action in early cell fate decisions. We demonstrate that Tbx3 is vigorously expressed during mesendodermal lineage specification in differentiating mESCs and at specific sites of gastrulation onset in the developing mouse and Xenopus embryos. Interestingly, we found that Tbx3 functions in dual cell autonomous and non-cell autonomous effects. Thereby, Tbx3 directly activates core mesendoderm lineage specification factors while also inducing the Nodal/SMAD2 (Mothers against decapentaplegic homolog 2) cascade. Moreover, we show that Tbx3 acts in concert with the key pluripotency factor, Oct3/4 to promote mesendoderm specification. Lastly, we highlight complex compensatory mechanisms that are at play suggesting a functional redundancy between Tbx3 and the closely related family member Tbx2 (T-Box transcription factor 2).

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In conclusion, our data, displays a more comprehensive understanding of the role of pluripotency factors in governing the transition from pluripotency to lineage commitment. We have provided important insights into how core developmental processes such as gastrulation are tightly regulated. This knowledge might help to prevent severe gastrulation defects in the distant future. Yet more work should be performed to fully understand its complexity.

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7 Figures and Tables

Figures Figure 1 Hierarchy of mammalian ESC development and their impact on differentiation in vivo and in vitro Figure 2 Signalling pathways regulating mESC pluripotency Figure 3 Preimplantation development of mammalian embryo Figure 4 Pluripotency factor expression throughout early embryonic stages Figure 5 Origin of T-box genes in vertebrates Figure 6 Core transcription factors maintain pluripotency in mESCs mediated by LIF Figure 7 Generation of iTbx3 cells Figure 8 In vitro differentiation of mESCs Figure 9 Sonication Figure 10 Illustration of CPs depending on amount of fluorescence Figure 11.Tbx3 expression in the developing mouse embryo Figure 12 Tbx3 expression during mESC differentiation Figure 13 Tbx3 is co-expressed with lineage-associated genes in differentiating mESCs Figure 14 Tbx3 directly interacts with lineage determining genes in the pluripotent state Figure 15 Inducible Tbx3-expression allele in mESCs drives mesoderm and endoderm specification Figure 16 Microarray gene expression analysis illustrating Tbx3 impact on mesoderm and endoderm Figure 17 Tbx3 expression promotes mature mesendodermal derivatives Figure 18 Tbx3 activates the Nodal/SMAD2 signalling pathway by directly interacting with Nodal Figure 19 Mechanism of Nodal/SMAD2 pathway activation illustrated by microarray gene expression profile and Nodal blocking assay Figure 20 Generation of td-tomato-labeled iTbx3 cell line Figure 21 Tbx3 directs cell fate by acting via non-cell-autonomous mechanisms Figure 22 Cardiac and hepatic effects of paracrine iTbx3 signalling Figure 23 Tbx3 directly regulates key mesendoderm inducing determinants

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Figure 24 Tbx3 functionally interacts with other T-box family members in directing early cell fate choices Figure 25 Spatial distribution of lineage specifying factors at gastrulation Figure 26 Spatial clustering of pluripotency-associated factors during lineage specification Figure 27 Hypothesised mechanism reflecting Tbx3 impact on guiding early lineage decisions

Tables Table 1 Overview of used cell lines Table 2 Preparation of cell culture components Table 3 Overview of cell culture components Table 4 Cell culture media Table 5 Overview of components used for Immunofluorescence Table 6 Primary Antibodies for Immunofluorescence Table 7 Secondary Antibodies for Immunofluorescence Table 8 Overview of materials used for Immunoblotting Table 9 Primary Antibodies for Immunoblotting Table 10 Secondary Antibodies for Immunoblotting Table 11 Gels used for Immunoblotting Table 12 Buffers used for Immunoblotting Table 13 Antibodies for Chromatin Immunoprecipitation Table 14 Self-designed primers for quantitative real-time polymerase chain reaction Table 15 Self-designed primers for quantitative real-time polymerase chain reaction of chromatin immunoprecipitated cells Table 16 Commercial primers for quantitative real-time polymerase chain reaction Table 17: Quantitative real time polymerase chain reaction programme setup for Chromatin Immunoprecipitation Table 18: Substrates for quantitative real time polymerase chain reaction Table 19: Quantitative real time polymerase chain reaction program setup

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Acknowledgements

First of all I would like to express my sincere gratitude to my supervisor PD. Dr. Alexander Kleger. I thank you for providing me the opportunity of experiencing the exciting field of stem cell research. Thanks for your continuous support, motivation and advice. I enjoyed the time in the laboratory very much and retrospectively it was the most interesting and enjoyable time during my medical study. I especially enjoyed going to conferences, meeting interesting researchers and presenting the data I was working on. Last I want to emphasise that I feel well prepared for my current research fellowship, after having performed my thesis in your laboratory.

My sincere thanks also go to Prof. Guido Adler, Prof. Götz von Wichert and Prof. Thomas Seufferlein for giving me the opportunity of completing my medical thesis in their laboratory facilities.

I want to thank Prof. Stefan Liebau for teaching me qRT-PCR and microscopy. I also really want to thank Prof. Cornelia Brunner, as she showed me how to perform ChIP. I had a great time working with you and greatly profited from your knowledge. Thank you!

I thank my labmates Ronan Russell, Marianne Stockmann, Martin Müller and Ralf Köhntop for the long days and weekends in order to perform our experiments. We had a lot of fun in the last 5 years.

I would like to thank my family and my friends. First of all I want to express my deepest gratitude to my parents, Christine und Peter Weidgang, as I wouldn’t have been able to study medicine if I didn’t have their continuous support. I also really appreciate their comments and advice.

I especially want to thank my husband Stefan Hartmann for his patience. Stefan, thank you for encouraging me in every situation.

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I want to thank my friends Maria Rullmann and Marianne Stockmann for the longs study nights before exams and Pauline Kleger for always supporting me spiritually in the last years.

Last but not the least I want to thank the University of Ulm and the International Graduate School in Molecular Medicine.

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Curriculum vitae

Personals Name: Clair Elisabeth Hartmann (née Weidgang)

Clinics Since June 2015 Research fellow, Institute of Anaesthesiological Pathophysiology and Process Development, University Hospital of Ulm Prof. Dr. med. Dr. med. h. c. P. Radermacher Since June 2014 Anaesthesiology Residency, Department of Anaesthesiology, University Hospital Ulm Prof. Dr. med. Dr. med. h. c. M. Georgieff

Education Oct 2007 – May 2015 Medical study, University of Ulm  Third part of the elective period in Anaesthesiology, Department of Anesthesiology, University Hospital Ulm  Second part of the elective period in Thoracic Surgery, Department of Thoracic Surgery, Guy’s Hospital, London  First part of the elective period in Internal Medicine at the Kantonsspital Lucerne, Switzerland Oct 2006 - Oct 2007 Nursing occupation, Department of Septic Surgery, University Hospital Ulm Oct 2003 - Sept 2006 Graduate nurse education May 2003 - Sept 2003 Emergency medical technician training March 2003 Abitur

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Doctoral Thesis Oct 2010 – Apr 2011 Research sabbatical at the Department of Internal Medicine 1, University Hospital of Ulm

Since Jan 2010 “Tbx3 directs cell fate decision toward mesendoderm” published in Stem Cell Reports, Department of Internal Medicine 1, University Hospital of Ulm Prof. Dr. med. Thomas Seufferlein PD Dr. rer. med. Kleger

Awards and Scholarships 2010 - 2011 Scholarship of the International Graduate School in Molecular Medicine Ulm “Promotionsprogramm Experimentelle Medizin” 2008 Certificate of the achievement in the anatomy course (top 3 % of students) awarded by the Institute of Anatomy and Cell Biology, Ulm University

Ulm, 02.08.2015

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Publication list

Peer-reviewed research articles 1. Weidgang C, Seufferlein T, Kleger A, Mueller M. Titel: Pluripotency factors on their lineage move. Review, Stem Cells Int., 2016:6838253 (2016) 2. Mueller M, Schroer J, Azoitei N, Eiseler T, Bergmann W, Illing A, Lin Q, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A. Titel: A time frame permissive for Protein Kinase D2 activity to direct in mouse embryonic stem cells. Sci. Rep., 5:11742 (2015) 3. Weidgang C, Russell R, Tata P R, Kuhl S J, Illing A, Mueller M, Lin Q, Brunner C, Boeckers T M, Bauer K, Kartikasari A E, Guo Y, Radenz M, Bernemann C, Weiss M, Seufferlein T, Zenke M, Iacovino M, Kyba M, Scholer H R, Kuhl M, Liebau S, Kleger A. Titel: Tbx3 Directs Cell Fate Decision toward Mesendoderm. Stem Cell Reports, 1: 248-265 (2013) 4. Liebau S, Tischendorf M, Ansorge D, Linta L, Stockmann M, Weidgang C, Iacovino M, Boeckers T, von Wichert G, Kyba M, Kleger A. Titel: An inducible expression system of the calcium-activated potassium channel 4 to study the differential impact on embryonic stem cells. Stem Cells Int., 2011: 456815 (2011)

Poster presentations 1. Weidgang C, Tata P, Russel R, Kühl S, Muller M, von Wichert G, Schöler H, Kühl M, Arnold S, Liebau S, Kleger A. Titel: “Tbx3 directs cell fate decision toward mesendoderm” International Society for Stem Cell Research,10th Annual Meeting, Yokohama, Japan (2012) 2. Weidgang C, Liebau S, Iacovino M, von Wichert G, Kyba M and Kleger A. Titel: “An inducible expression system of the calcium activated potassium channel 4 to study the differential impact on embryonic stem cells” International Society for Stem Cell Research, Berlin, Germany (2011)

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Comment

Parts of this dissertation have been published in the following journals:

1. Tbx3 directs cell-fate decision toward mesendoderm Weidgang CE, Russell R, Tata PR, Kühl SJ, Illing A, Müller M, Lin Q, Brunner C, Boeckers TM, Bauer K, Kartikasari AE, Guo Y, Radenz M, Bernemann C, Weiß M, Seufferlein T, Zenke M, Iacovino M, Kyba M, Schöler HR, Kühl M, Liebau S, Kleger A. Stem Cell. Reports, 1: 248-265(2013) doi: http://dx.doi.org/10.1016/j.stemcr.2013.08.002

Copyright: The Authors, published by Elsevier. License: Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) License Link: https://creativecommons.org/licenses/by-nc-nd/3.0/legalcode

2. Pluripotency Factors on Their Lineage Move Weidgang C, Seufferlein T, Kleger A, Mueller M. Stem Cells International, vol. 2016, Article ID 6838253, 16 pages, 2016. doi: 10.1155/2016/6838253

Copyright: The Authors, published by Hindawi Publishing Corporation. License: Creative Commons Attribution 4.0 International (CC BY 4.0) License Link: https://creativecommons.org/licenses/by/4.0/legalcode

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