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 gene 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 base pair bFGF basic Fibroblast Growth Factor, FGF2, FGF BMP Bone morphogenetic protein BSA Bovine Serum Albumin CD31 Cluster of differentiation 31 (Pecam) Cdx2 Caudal type homeobox 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 Eomesodermin EpiSC epiblast stem cell ESC embryonic stem cell Esrrb Estrogen-related receptor ERK extracellular receptor kinase FACS Fluorescence Activated Cell Sorting FCS Fetal Calf Serum FGF Fibroblast growth factor FHF first heart field FoxA2 Forkhead-Box-Protein A Fwd forward GATA GATA binding protein Gbx2 Gastrulation brain 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 Klf4 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 neural stem cell Oct3/4 Octamer binding transcription factor 3/4, Otf3, Otf4, POU5F1 OFT outflow tract V o.N. overnight Otx2 Orthodenticle homeobox 2 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 Sox2 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 Brachyury 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 epithelium 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 fibroblasts into a pluripotent state by adding four