The role of transcription factors Sox4 and
Sox11 in mouse heart development
Die Rolle der Transkriptionsfaktoren Sox4 und Sox11 während der Herzentwicklung der Maus
Der Naturwissenschaftlichen Fakultät der Friedrich‐Alexander‐Universität Erlangen‐Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von Mandy Paul aus Bautzen
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich‐Alexander Universität Erlangen‐Nürnberg.
Tag der mündlichen Prüfung: 09.10.2014
Vorsitzende/r des Promotionsorgans: Prof. Dr. Johannes Barth Gutachter/in: Prof. Dr. Michael Wegner PD Dr. Dieter Engelkamp
“The scientist is not a person who gives the right answers, he is one who asks the right questions”
Claude Lévi‐Strauss
Meiner Familie gewidmet
I. Table of content
SUMMARY ...... I
ZUSAMMENFASSUNG...... III
1. INTRODUCTION ...... 1
1.1 CARDIAC DEVELOPMENT ...... 1
1.1.1 MORPHOLOGICAL DEVELOPMENT OF THE HEART ‐ AN OVERVIEW ...... 1
1.1.2 CARDIAC SEPTATION ...... 4
1.1.2.1 ATRIOVENTRICULAR CANAL AND ATRIAL SEPTATION ...... 4
1.1.2.2 VENTRICULAR GROWTH AND SEPTATION ...... 5
1.1.3 OUTFLOW TRACT DEVELOPMENT ...... 6
1.1.3.1 MORPHOLOGICAL DEVELOPMENT AND SEPTATION ...... 6
1.1.3.2 CONTRIBUTION OF NEURAL CREST CELLS TO OUTFLOW TRACT MORPHOLOGY ...... 8
1.1.4 OUTFLOW TRACT MALFORMATIONS ...... 9
1.2 SOX PROTEIN FAMILY ...... 12
1.2.1 SOXC PROTEINS ...... 13
1.2.1.1 SOX4 ...... 14
1.2.1.2 SOX11 ...... 15
1.2.1.3 SOX12 ...... 17
2. AIM OF THE STUDY ...... 19
3. RESULTS ...... 21
3.1 SOX4 AND SOX11 FUNCTION IN CARDIAC OUTFLOW TRACT DEVELOPMENT ...... 21
3.1.1 SOX4 AND SOX11 FUNCTION IN CARDIAC NEURAL CREST CELLS ...... 21
3.1.1.1 MACROSCOPIC ANALYSIS OF OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN CARDIAC NEURAL CREST CELLS ...... 21
3.1.1.2 DETAILED HISTOLOGICAL ANALYSIS OF OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN CARDIAC NEURAL CREST CELLS ...... 24
3.1.1.3 DETAILED HISTOLOGICAL ANALYSIS OF VENTRICULAR SEPTUM DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN CARDIAC NEURAL CREST CELLS ...... 26
3.1.1.4 APPEARANCE OF ANIMALS WITH A SPECIFIC ABLATION OF SOX4 AND SOX11 IN NEURAL CREST CELLS 27
3.1.1.5 ANALYSIS OF EARLY OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 ABLATION IN CARDIAC NEURAL CREST CELLS VIA AP2::CRE ...... 28
3.1.1.6 EFFICIENT DELETION OF SOX4 AND SOX11 IN NEURAL CREST CELLS ...... 28
3.1.2 SOX4 AND SOX11 FUNCTION IN MESODERMAL CELLS ...... 30
3.1.2.1 ANALYSIS OF EARLY OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN MESODERMAL CELLS ...... 30
3.1.2.2 ANALYSIS OF LATE OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN MESODERMAL CELLS ...... 31
3.1.3 ANALYSIS OF OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN CARDIAC NEURAL CREST AND MESODERMAL CELLS ...... 33
3.1.4 ANALYSIS OF EARLY OUTFLOW TRACT DEVELOPMENT IN MICE WITH SOX4 AND SOX11 ABLATION IN ENDOTHELIAL CELLS ...... 34
3.1.5 PROPERTIES OF CARDIAC NEURAL CREST CELLS IN THE ABSENCE OF SOX4 AND SOX11 ...... 35
3.1.6 ABLATION OF SOX4 AND SOX11 LEADS TO MORPHOLOGICAL CHANGES IN CARDIAC NEURAL CREST CELLS ...... 38
3.1.7. SOX12 EXPRESSION IN THE OUTFLOW TRACT ...... 39
3.1.8 ABLATION OF SOX4 AND SOX11 LEADS TO ALTERED MARKER GENE EXPRESSION IN CARDIAC NEURAL CREST CELLS ...... 39
3.1.8.1 IMMUNOHISTOCHEMICAL ANALYSIS OF MARKER GENE EXPRESSION OF CARDIAC NEURAL CREST CELLS IN MICE WITH SOX4 AND SOX11 ABLATION IN NEURAL CREST CELLS ...... 39
3.1.8.2 QUANTITATIVE RT‐PCR ANALYSIS OF MARKER GENE EXPRESSION OF CARDIAC NEURAL CREST CELLS IN MICE WITH SOX4 AND SOX11 ABLATION IN NEURAL CREST CELLS ...... 42
3.1.9 PUTATIVE SOX4 AND SOX11 BINDING SITES IN ADAM19 AND E‐CADHERIN PROMOTER REGIONS ..... 43
3.1.10 DIRECT BINDING OF SOX4 AND SOX11 TO NEWLY IDENTIFIED SOX BINDING SITES IN ADAM19 AND E‐ CADHERIN PROMOTER REGIONS ...... 44
3.1.11 SOX11 ASSOCIATES WITH ENDOGENOUS ADAM19 AND E‐CADHERIN PROMOTER REGION ...... 46
3.1.12 SOX4 AND SOX11 ACTIVATE ADAM19 AND E‐CADHERIN PROMOTERS ...... 49
3.2 POSTTRANSLATIONAL MODIFICATION OF SOX4 AND SOX11 ...... 51
3.2.1 PUTATIVE PHOSPHORYLATION SITES OF SOX4 AND SOX11 ...... 51
3.2.2 PHOSPHORYLATION ANALYSIS OF SOX4 AND SOX11 ...... 52
3.2.3 ANALYSIS OF ACTIVATION POTENTIAL OF PUTATIVE SOXC PHOSPHORYLATION MUTANTS ...... 54
3.2.4 ANALYSIS OF BINDING ABILITY OF PUTATIVE SOXC PHOSPHORYLATION MUTANTS ...... 55
4. DISCUSSION ...... 57
4.1 THE ROLE OF SOXC PROTEINS IN OUTFLOW TRACT DEVELOPMENT ...... 57
4.1.1 THE ROLE OF SOX4 IN OUTFLOW TRACT DEVELOPMENT ...... 57
4.1.2 THE ROLE OF SOX11 IN OUTFLOW TRACT DEVELOPMENT ...... 58
4.1.3 REDUNDANT FUNCTION OF SOX4 AND SOX11 IN OUTFLOW TRACT DEVELOPMENT ...... 59
4.1.4 THE ROLE OF SOX12 IN OUTFLOW TRACT DEVELOPMENT ...... 60
4.2 PARTICIPATION OF OTHER SOX PROTEINS IN HEART DEVELOPMENT ...... 61
4.3 THE ROLE OF SOXC PROTEINS IN OTHER TISSUES ...... 62
4.4 PROLIFERATION, MIGRATION AND APOPTOSIS OF NEURAL CREST CELLS ARE NOT AFFECTED BY SOXC DELETION ...... 63
4.5 MORPHOLOGICAL AND CYTOSKELETAL ALTERATIONS IN SOXC DEFICIENT CARDIAC NEURAL CREST CELLS ...... 63
4.6 ALTERED TRANSCRIPTION FACTOR EXPRESSION IN OUTFLOW TRACT REGIONS CONTAINING SOXC DEFICIENT CARDIAC NEURAL CREST CELLS ...... 65
4.7 ADAM19: A DIRECT TARGET OF SOXC PROTEINS ...... 66
4.8 ALTERED EMT AND MET MARKER GENE EXPRESSION IN OUTFLOW TRACT REGIONS CONTAINING SOXC DEFICIENT CARDIAC NEURAL CREST CELLS ...... 67
4.9 E‐CADHERIN: A DIRECT TARGET OF SOXC PROTEINS ...... 68
4.10 RELEVANCE IN DISEASE ...... 69
4.11 OUTLOOK ...... 70
5. MATERIALS & METHODS ...... 71
5.1 MATERIALS ...... 71
5.1.1 ORGANISM ...... 71
5.1.1.1 BACTERIAL CULTURES ...... 71
5.1.1.2 CELL LINES ...... 71
5.1.1.3 MOUSE LINES ...... 71
5.1.2 CHEMICALS AND GENERAL REAGENTS ...... 72
5.1.3 BUFFERS AND SOLUTIONS ...... 72
5.1.4 MEDIUM USED FOR BACTERIAL CULTURES ...... 74
5.1.5 OLIGONUCLEOTIDES ...... 74
5.1.5.1 OLIGONUCLEOTIDES USED FOR GENOTYPING ...... 74
5.1.5.2 OLIGONUCLEOTIDES USED FOR ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) ...... 75
5.1.5.3 OLIGONUCLEOTIDES USED FOR QUANTITATIVE REAL TIME ‐ POLYMERASE CHAIN REACTION (QRT‐PCR) ...... 75
5.1.5.4 OLIGONUCLEOTIDES USED FOR MUTAGENESIS OF SOX BINDING SITES ...... 77
5.1.5.5 OLIGONUCLEOTIDES USED FOR MUTAGENESIS OF SOX4 AND SOX11 ...... 77
5.1.5.6 OLIGONUCLEOTIDES USED FOR MUTAGENESIS OF SOX4 AND SOX11 ...... 78
5.1.6 ANTIBODIES ...... 79
5.1.6.1 PRIMARY ANTIBODIES...... 79
5.1.6.2 SECONDARY ANTIBODIES ...... 79
5.1.6.3 STAINING DYES ...... 79
5.2 METHODS ...... 80
5.2.1 MOUSE HOUSING ...... 80
5.2.2 MOUSE BREEDING ...... 80
5.2.3 MOLECULARBIOLOGICAL METHODS ...... 80
5.2.3.1 STANDARD METHODS ...... 80
5.2.3.2 ISOLATION OF GENOMIC DNA ...... 81
5.2.3.3 POLYMERASE CHAIN REACTION (PCR) ...... 81
5.2.3.4 PRIMER COMBINATIONS AND DNA FRAGMENT SIZE FOR POLYMERASE CHAIN REACTION (PCR) ...... 81
5.2.3.5 REACTION MIX FOR POLYMERASE CHAIN REACTION (PCR) ...... 82
5.2.3.6 GENOTYPING PROGRAMM ...... 83
5.2.3.7 CLONING OF DNA FRAGMENTS ...... 83
5.2.3.8 SITE‐DIRECTED MUTAGENESIS ...... 84
5.2.3.9 REVERSE TRANSCRIPTION AND RT‐PCR ...... 85
5.2.3.10 QUANTITATIVE REAL TIME ‐ PCR (QRT‐PCR)...... 86
5.2.4 HISTOLOGICAL METHODS ...... 86
5.2.4.1 TISSUE PREPARATION FOR IMMUNOHISTOLOGICAL STAINING ...... 86
5.2.4.2 TISSUE PREPARATION AND HAEMATOXYLIN – EOSIN STAINING ...... 87
5.2.4.3 IMMUNOHISTOCHEMISTRY ...... 87
5.2.4.4 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE DUTP NICK‐END LABELING (TUNEL) ...... 88
5.2.4.5 PHALLOIDIN STAINING ...... 88
5.2.4.6 QUANTIFICATIONS ...... 88
5.2.5 CELL CULTURE METHODS ...... 89
5.2.5.1 CULTIVATION OF EUKARYOTIC CELLS ...... 89
5.2.5.2 TRANSFECTION OF HEK293 CELLS FOR WHOLE CELL EXTRACTS ...... 89
5.2.5.3 TRANSFECTION OF NEURO‐2A CELLS FOR WHOLE CELL EXTRACTS ...... 89
5.2.5.4 PREPARATION OF WHOLE CELL EXTRACTS FOR ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) .... 90
5.2.5.5 PREPARATION OF WHOLE CELL EXTRACTS FOR CHROMATIN IMMUNOPRECIPITATION (CHIP) ...... 90
5.2.5.6 TRANSFECTION OF NEURO‐2A CELLS FOR LUCIFERASE ASSAY ...... 90
5.2.5.7 LUCIFERASE REPORTER GENE ASSAY ...... 91
5.2.6 CHROMATIN IMMUNOPRECIPITATION (CHIP) ...... 91
5.2.7 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) ...... 92
6. TABLE OF ABBREVIATIONS ...... 95
7. REFERENCE LIST ...... 97
PUBLICATIONS ...... 121
CURRICULUM VITAE ...... 123
DANKSAGUNG ...... 125
II. Figures
Figure 1: Stages of Mammalian Heart Development...... 2
Figure 2: Atrial Septation...... 4
Figure 3: Outflow Tract Septation...... 6
Figure 4: Overview of cardiac neural crest cell migration into the outflow tract...... 8
Figure 5: Cardiac outflow tract defects...... 10
Figure 6: Sox proteins of subgroup C...... 14
Figure 7: Early developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation...... 22
Figure 8: Late developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation...... 23
Figure 9: Early developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation...... 24
Figure 10: Late developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation...... 25
Figure 11: Development of ventricular septum in mice with neural crest specific SoxC gene ablation...... 26
Figure 12: Developmental defects in mice with neural crest specific SoxC gene ablation. .... 27
Figure 13: Deletion efficiencies of SoxC proteins in cardiac neural crest cells...... 29
Figure 14: Early developmental defects of the cardiac outflow tract in mice with mesoderm specific SoxC gene ablation...... 30
Figure 15: Late developmental defects of the cardiac outflow tract in mice with mesoderm specific SoxC gene ablation...... 31
Figure 16: Development of ventricular septum in mice with mesoderm‐ specific SoxC gene ablation...... 32
Figure 17: Developmental defects of the cardiac outflow tract in mice with mesoderm and cardiac neural crest specific SoxC gene ablation...... 34
Figure 18: Analysis of timing and migration of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 ablation...... 36
Figure 19: Analysis of proliferation and apoptosis of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 ablation...... 37
Figure 20: Analysis of the morphology of cardiac neural crest in mice with neural crest specific deletion of Sox4 and Sox11...... 38
Figure 21: Analysis of marker gene expression of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 gene ablation...... 40
Figure 22: Analysis of marker gene expression of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 gene ablation...... 41
Figure 23: In situ ‐ hybridisation of Sox12 in wildtype outflow tract regions...... 39
Figure 24: Analysis of important marker genes in the outflow tract region of mice with Sox4 and Sox11 deficiencies in cardiac neural crest...... 43
Figure 25: Analysis of Adam19 and E‐cadherin promoter regions for potential Sox binding sites...... 44
Figure 26: Analysis of potential Sox binding sites in the Adam19 promoter...... 45
Figure 27: Analysis of potential Sox binding sites in the E‐cadherin promoter...... 46
Figure 28: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions...... 47
Figure 29: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions...... 48
Figure 30: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions in whole hearts or the anterior heart pole of 13.5 dpc wildtype mice...... 48
Figure 31: Analysis of promoter activation by Sox4 and Sox11...... 49
Figure 32: Putative sites for phosphorylation of Sox4 and Sox11...... 51
Figure 33: ‐Protein Phosphatase treatment of overexpressed Sox4 and Sox11 protein...... 52
Figure 34: Western blot analysis of ‐Protein Phosphatase treated or untreated extracts from HEK293 cells over expressing Sox4 or Sox11 in wildtype or mutated versions...... 53
Figure 35: Transient transfection of HEK293 cells with wildtype or mutated Sox11...... 55
Figure 36: EMSA with oligonucleotides encompassing the Sox binding site MW‐1 (van de Wetering et al., 1993)...... 56
III. Tables
Table 1: Quantity and percentage of cardiac outflow tract malformations in mice with neural crest specific SoxC gene ablation...... 23
Table 2: Quantity and percentage of cardiac outflow tract malformations in mice with neural crest specific SoxC gene ablation...... 28
Table 3: Quantity and percentage of cardiac outflow tract malformations in mice with mesoderm specific SoxC gene ablation...... 32
Table 4: Quantity and percentage of cardiac outflow tract malformations in mice with endothelial specific SoxC gene ablation...... 35
Table 5: Oligonucleotides used for genotyping of the different mouselines ...... 74
Table 6: Oligonucleotides used for electrophoretic mobility shift assays (EMSA) additional radioactive GGG targeting not included ...... 75
Table 7: Oligonucleotides used for quantitative real time ‐ polymerase chain reaction (qRT‐ PCR) on mRNA of outflow tract tissue of the heart from 13.5 dpc old mutant and wildtype mice ...... 76
Table 8: Oligonucleotides used for mutagenesis of Sox binding sites in E‐cadherin and Adam19 promomter regions ...... 77
Table 9: Oligonucleotides used for mutagenesis of Sox4 and Sox11 phosphorylation sites, delta C and HMG‐Box and combined mutations ...... 78
Table 10: Oligonucleotides used for mutagenesis of possible phosphorylation sites in Sox4 and Sox11 or for deletion of conserved amino acids ...... 78
Table 11: Primary antibodies and dilutions used for immunohistochemistry ...... 79
Table 12: Staining dyes and their dilutions used for immunohistochemistry ...... 79
Table 13: Primer combinations used for PCR and amplified DNA fragment sizes ...... 82
Table 14: Reaction mix used for PCR specified for the analysed genes ...... 82
Table 15: Used programs for genotyping of analysed genes ...... 83
Table 16: Used chemicals and concentrations to amplify DNA fragments from plasmids or cDNA ...... 83
Table 17: Used chemical s and concentrations for site‐directed mutagenesis ...... 84
Table 18: Used chemicals and concentrations for priming of obtained mRNA ...... 85
Table 19: Used chemicals and concentrations for RT‐PCR ...... 85
Table 20: Used chemicals and concentrations for qRT‐PCR ...... 86
SUMMARY
Summary
Transcription factors Sox4 and Sox11 are part of the subgroup C of the Sox protein family. They are expressed in a broad range of tissues and cells and regulate a variety of developmental processes during embryogenesis. Analysis of constitutive knockout mice for these Sox factors revealed severe developmental defects including some concerning the outflow tract of the heart and its development. In this study Sox4 and Sox11 were deleted in a cell‐type specific manner to analyse their impact on heart development in greater detail. Deletion of Sox4 in neural crest, mesoderm or endothelium did not disturb proper outflow tract formation. In contrast, deletion of Sox11 in neural crest or mesoderm led to outflow tract defects. Whereas deletion in neural crest cells led to outflow tract malformations in only a small percentage of mutant mice, malformation rates reached 50% following Sox11 deletion in mesodermal cells. All outflow tract defects corresponded to double outlet right ventricle (DORV). Analysis of animals with a simultaneous Sox11 deletion in neural crest and mesoderm increased the phenotypic severity of the observed malformation as some mutant animals exhibited persistent truncus arteriosus (also known as arterial common trunk; CT) rather than DORV. Joint deletion of both SoxC proteins in the neural crest led to outflow tract malformations in all mutant mice. This points to an important role of Sox4 in neural crest cells that was not evident by single deletion studies. In most cases the phenotype corresponded to DORV but CT was also found. Combined SoxC deletion in neural crest cells led to an altered morphology. These cells had less filopodia‐like protrusions. Additionally, cytoskeleton and expression of adhesion molecules and extracellular matrix components were changed. Both the adhesion molecule E‐cadherin and the extracellular matrix protein Adam19 were shown to be direct target genes of SoxC transcription factors.
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ZUSAMMENFASSUNG
Zusammenfassung
Die Transkriptionsfaktoren Sox4 und Sox11 gehören zu der Untergruppe C der Sox‐ Proteinfamilie. Sie werden in vielen verschiedenen Geweben und Zellen exprimiert und regulieren eine Vielzahl von Prozessen vor allem während der Embryonalentwicklung. Untersuchungen an konstitutiven Knockout‐Mausmodellen zeigten schwerwiegende Entwicklungsdefekte bei Verlust dieser Sox‐Proteine auf. Diese betrafen auch die Ausstrombahn des Herzens. In der vorliegenden Arbeit wurden zelltypspezifische Deletionen von Sox4 und Sox11 generiert und die Folgen für die Herzentwicklung analysiert. Die Einzel‐Deletion von Sox4 in Zellen der Neuralleiste, des Mesoderms oder Endothels beeinträchtigte die Ausstrombahn des Herzens nicht. Dagegen waren nach Deletion von Sox11 in der Neuralleiste und in mesodermalen Zellen Entwicklungsdefekte der Ausstrombahn offensichtlich. Während diese nach Deletion von Sox11 in Neuralleistenzellen nur in seltenen Fällen auftraten, verursachte das Fehlen von Sox11 in mesodermalen Zellen Ausstrombahndefekte in 50% der Sox11‐ defizienten Tiere. Die Entwicklungsdefekte entsprachen einem rechten Doppelausstrom ‐ ventrikel (DORV). Wurde Sox11 allerdings in Zellen der Neuralleiste und in mesodermalen Zellen gleichzeitig deletiert, kam es in einem Anteil der untersuchten Mutanten zur Ausprägung eines persistierenden Truncus arteriosus communis (CT), bei dem es sich verglichen mit dem DORV um einen schwerwiegenderen Entwicklungsdefekt handelt. Deletion beider SoxC Proteine in Zellen der Neuralleiste führte zu Ausstrombahndefekten in allen untersuchten Mäusen. Dies deutet auf eine wichtige Funktion von Sox4 in dieser Zellpopulation hin, die bei einer Einzel‐Deletion nicht ersichtlich war. Der Phänotyp entsprach in den meisten Fällen dem DORV, aber der CT trat ebenfalls auf. Neuralleistenzellen mit SoxC Deletion zeigten eine veränderte Morphologie. Sie bildeten weniger Filopodien. Ihr Zytoskelett und die Expression von Adhäsionsmolekülen und Extrazellularmatrix‐Komponenten waren verändert. Für das Adhäsionsmolekül E‐cadherin und das Extrazellulärmatrix‐Protein Adam19 konnte gezeigt werden, dass sie direkte Zielgene der SoxC Transkriptionsfaktoren sind.
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INTRODUCTION
1. Introduction
1.1 Cardiac Development
1.1.1 Morphological Development of the Heart ‐ an overview
The heart is one of the first organs to form while the mammalian embryo develops. Its function is essential for the circulation of oxygen and nutrients and for waste removal. As the embryo grows diffusion alone is no longer sufficient to assure the necessary transport of various molecules and a circulatory system is needed. Heart development is a highly complex process involving a broad variety of morphological changes. The complex process of its construction involves the integration of different cell populations with specialised functions at distinct sites and time points in development (Vincent and Buckingham, 2010). Cardiac progenitor cells originate near the primitive streak as part of the mesoderm (Abu‐ Issa and Kirby, 2007). All cardiac progenitor cells located bilaterally in the anterior visceral mesoderm belong to the primary or first heart field (FHF) (Brand, 2003;Dunwoodie, 2007). Today, a first and a secondary heart field (SHF) are recognized. The heart fields differ in time and contribution to the development of different structures of the heart as well as in their expression profile (Dyer and Kirby, 2009;Waldo et al., 2001). While precursors of the FHF contribute to the left ventricle, precursor cells of the SHF contribute to the right ventricle, the outflow tract, sinus venosus as well as the left and right atria. During early embryonic development, the mesoderm splits into two layers, the inner splanchnic and the outer somatic layer. Cardiac progenitor cells arise from the splanchnic mesoderm which has the potential to form myocardial cells. These cells adopt a crescent shape around embryonic day 7.5. The apex of the crescent lies close to the junction between the embryonic and the extra‐embryonic tissue (Dunwoodie, 2007;Stennard and Harvey, 2005) (Figure 1, A). Around embryonic day 8.5 the cardiac crescent progenitor cells migrate ventrally and fuse into a linear heart tube at the midline (Figure 1, B). The linear heart tube is
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INTRODUCTION
composed of endothelial cells surrounded by myocardial cells which form an epithelium (Harvey, 2002).
(Stennard and Harvey, 2005)
Figure 1: Stages of Mammalian Heart Development. Whole embryos or whole hearts are shown in the top row, sections of embryos or hearts are shown in the bottom row. (A) Cardiac progenitor cells enter the mesodermal layer to form the cardiac crescent (cc) by embryonic day 7.75. (B) Heart progenitor cells move ventrally and form the linear heart tube (ht) by embryonic day 8.25. (C) The heart tube elongates and loops rightward by embryonic day 10.5. (D) Conformational changes and remodelling of the heart into a four‐chambered heart occur by embryonic day 12.5. avc: atrioventricular canal; avs: atrioventricular septum; ca: common atrium; cc: cardiac crescent; co: coelum; dm: dorsal mesocardium; dp: dorsal pericardium; e: endocardium; fg: foregut; hm: head mesoderm; ht: heart tube; ias: interatrial septum; ivs: interventricular septum; la: left atrium; lv: left ventricle; m: myocardium; ne: neural epithelium; oft: outflow tract; fe: foregut endoderm; ra: right atrium; rv: right ventricle; som: somatic mesoderm; spm: splanchnic mesoderm; tr: trabeculae.
Throughout the process, the myocardium secretes a thick extracellular matrix called cardiac jelly which forms the layer separating the myocardium from the endocardium (Kirby, 2002). Between embryonic day 8.5 to 9.5, the heartbeat is initiated and blood starts to flow in a caudal to cranial direction (Chen et al., 2010;Srivastava, 2006). For heart beat initiation and looping around embryonic day 8.5 to 10.5, new cells from the SHF are added to the tube at each end and start to differentiate (Christoffels et al., 2000). Furthermore, the already existing myocardial cells divide. At this stage, the venous inflow of the heart is located
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INTRODUCTION
caudally and the arterial outflow cranially (Dunwoodie, 2007). After elongation of the linear heart tube, it loops rightwards inside the pericardial cavity. Due to looping the caudal inflow tract including the common atrium is moved next to the cranial outflow tract (Dunwoodie, 2007) (Figure 1, C). As soon as the inflow tract is located next to the outflow tract the other segments such as atrial and ventricular chambers, atrioventricular canal and definitive outflow tract start to emerge. Atrioventricular canal and outflow tract endocardium generate cells that populate the cardiac jelly with mesenchyme. For this to happen, the endothelial cells have to undergo an epithelial to mesenchymal transition (EMT) which enables the cells to migrate to the designated areas. These mesenchymal cells together with invading neural crest cells form bulges that are referred to as endocardial cushions. The swelling endocardial cushion mesenchyme in the atrioventricular canal undergoes remodelling to form the future bicuspid (mitral) and tricuspid valves which separate atria from ventricles (Hinton and Yutzey, 2011). The maximal length of the outflow tract is reached by embryonic day 10.5. Subsequently, remodelling processes take place in the outflow tract region until embryonic day 14.5. Semilunar valves form at the angular junction of the outflow tract from the outflow tract cushions (Anderson et al., 2003). To form the atrial and ventricular compartments with their characteristic morphological and contractile skills precise information about the correct location is needed (Christoffels et al., 2000;Dunwoodie, 2007;Moorman and Christoffels, 2003;Srivastava, 2006;Waldo et al., 2001). This information is provided by transcriptional and secreted factors. Due to conformational changes and remodelling processes a four‐chambered heart is formed by embryonic day 13.5 (Figure 1, D).
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INTRODUCTION
1.1.2 Cardiac Septation
1.1.2.1 Atrioventricular Canal and Atrial Septation
After rightward looping, the atrioventricular canal emerges. Endocardial cushions are connected to the atrium and the ventricle. The cushions form and grow by expansion of the cardiac jelly followed by EMT of endocardial cells. Therefore, the opening between the atria and the ventricle narrows and forms the atrioventricular canal (AV canal). A crucial factor in development is the rightward shift of the AV canal. The blood which was so far directed only into the left ventricle is now directed into both ventricles. Hence, both atria are now connected to the ventricles (Figure 2).
(modified from www.embryologie.med.unsw.edu.au)
Figure 2: Atrial Septation. Schematic overview of atria and developing septa. Septa (septum primum, septum secundum) as well as the two openings (foramen ovale and secundum) are labelled. Both oxygenated (blue arrow) and deoxygenated blood (red arrow, left) enters the atria through the sinus venosus. Blood flows through the foramen ovale and secundum from left to right ventricle, while the septum secundum acts as an atrial valve and prevents backward flow of blood. Furthermore deoxygenated blood enters the atria via the four pulmonary veins (red arrow, right).
To build the interatrial septum, the septum primum has to form posteriorly. It is formed from myocardium that differentiates from the splanchnic mesoderm and grows towards the AC cushions and AV canal. This tissue forms a crescent shape which divides right from left atria. Due to its crescent shape the septum primum does not fully close the gap between right and left atrium. The remaining opening is necessary during embryonic development
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INTRODUCTION
because blood needs to be transported from the right to the left atrium. While the septum primum expands, the opening, called ostium primum shrinks in size. Before it is closed completely a second opening, called foramen secundum begins to appear, which is needed to maintain circulation of blood. Anteriorly to the septum secundum a thick muscular crescent shaped tissue forms. Simultaneously with the formation of the septum secundum another opening, called foramen ovale is forming. Both openings provide continuous blood circulation between the two atria. The septum primum shrinks to the size of a valve that covers foramen secundum. At the same time foramen ovale enlarges. The valve of the foramen ovale is needed for maintenance of blood circulation because it regulates opening and closing in response to pressure gradients and prevents backward flowing of blood. In embryonic development it is usually permanently open (Figure 2). At birth the pressure gradient is reversed and the foramen ovale is functionally closed (Schleich et al., 2013). With time anatomical closure of the foramen ovale follows.
1.1.2.2 Ventricular Growth and Septation
During looping the ventral surface of the heart tube becomes the outer curvature and the dorsal surface becomes the inner curvature of the heart (Schleich et al., 2013). Both curvatures have distinct functions. While the outer curvature is participating in growth of the ventricles, the remodelling processes of the inner curvature contribute to the alignment of inlet and outlet segments (Srivastava, 2006). Initially, the left ventricle develops, whereas development of the right ventricle is delayed, due to their origin from different cell lineages (Moorman et al., 2003). The myocardium of the right ventricle is derived from the SHF (Zaffran et al., 2004). Due to myocardial proliferation, the myocardial wall is growing significantly in size and the first trabeculations appear (Christoffels et al., 2000). Following growth of the ventricles further trabeculations appear and grow to larger muscular structures. The trabeculation of the ventricles is necessary for oxygenation of the myocardium, before coronary arteries are formed. On the anterior wall of the primitive ventricle a primordial muscular interventricular ridge develops. As both ventricles continue to grow, the medial walls fuse and form the interventricular septum which then separates right and left ventricles. It is composed of a greater muscular and a thinner membranous
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portion in the upper part. The muscular portion is derived from the bulboventricular ridge while the membranous portion is of neural crest origin. The membranous part of the interventricular septum connects to the endocardial cushions of the atrioventricular valves and to the aorticopulmonary septum during embryonic development to completely separate right from left ventricles (Schleich et al., 2013).
1.1.3 Outflow tract development
1.1.3.1 Morphological Development and Septation
For the outflow tract to develop the part between the conus and saccus aorticus has to elongate within the pericard. The cellular material needed for elongation is derived from cardiac neural crest cells which originate from the neural tube next to somites one to three and migrate through pharyngeal arches III, IV and VI. In addition, they contribute to the conotruncal septum which later on separates the aorta from the pulmonary trunk.
(modified from www.embryologie.med.unsw.edu.au)
Figure 3: Outflow Tract Septation. (A) Schematic representation of forming bulbar and truncal ridges of the outflow tract region. (B) Cross sections of the outflow tract prior to and post septation of the conotruncal ridges. Location of cross section is labelled by dotted lines with corresponding numbers (1‐3). LA, left atrium; LV, left ventricle, RA right atrium, RV, right ventricle.
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Due to elongation the outflow tract bends between conus and truncus. This bend marks the location of the endocardial cushions which will form the future valves of aorta and pulmonary artery. Proliferation of pharyngeal mesenchymal cells leads to bulbar ridges which are continuous in the truncus arteriosus. Cardiac neural crest cells migrate into the ridges as condensed cell columns and support the septum of the outflow tract. Important for outflow tract septation is the parallel blood flow. Oxygenated blood from the superior vena cava mainly flows through the right part of the AV canal into the right ventricle. Deoxygenated blood from the allantoic vein and the inferior vena cava passes the foramen ovale and flows through the left part of the AV canal into the left ventricle. In the outflow tract region both blood streams flow around each other in a spiral way, hereby influencing the newly forming septa. Three different septa are responsible for the outflow tract septation; the conal septum, the truncal septum and the aorticopulmonal septum. The conotruncal septa are bulbar ridges invaded by cardiac neural crest cells (Figure 3). These ridges grow into the lumen and coalesce in the middle. The aorticopulmonary septum is built by mesenchymal cells from the pharyngeal arches and forms a 180° spiral caused by the spiral blood stream. The right part of the aorticopulmonary septum connects with the lower truncal septum while the left aorticopulmonary septum fuses with the upper truncal septum. Then the upper truncal septum grows and connects with the right conal septum while the lower truncal septum fuses with the left conal septum (Figure 3). Hence, the septum of the outflow tract makes a rotation of 180° which cause the pulmonary trunk to twist around the aorta. Finally, the left conal septum connects with the interventricular septum and the right conal septum fuses with the backmost atrioventricular septum (Webb et al., 2003). The zippering effect of myocardialisation of the ridges results in fusion, which occurs in a distal to proximal direction and allows the cleavage of the aorta and the pulmonary trunk (van den Hoff et al., 1999).
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1.1.3.2 Contribution of Neural Crest Cells to Outflow Tract Morphology
Progenitors of the neural crest cells are located in the epiblast in a crescent shape (Kirby and Hutson, 2010). Later on neural crest cells originate as a stream of cells from the neural tube. For neural crest induction Wnt1 (Wingless‐type MMTV integration site family, member 1), FGF (fibroblast growth factor) and an intermediate level of BMP (bone morphogenic protein) are necessary (Sauka‐Spengler and Bronner‐Fraser, 2008). While BMP2 is only necessary for migration of neural crest cells, BMP4 and BMP7 are needed for induction of neural crest cells (Correia et al., 2007;Stuhlmiller and Garcia‐Castro, 2012). Due to reorganisation processes of their cytoskeleton and downregulation of E‐cadherin, which allows neural crest cells to detach from each other, they acquire a motile phenotype and are therefore able to leave the neural tube (Kirby and Hutson, 2010) (Figure 4). Furthermore, in migrating neural crest cells N‐cadherin is expressed and blocking its function perturbs the onset of their migration (Bronner‐Fraser et al., 1992;Hatta and Takeichi, 1986).
1. Induction and EMT
2. Initial migration
3. Pause in circumpharyngeal ridges
4. Migration into the caudal pharynx and condensation around the arch arteries
5. Migration into the cardiac outflow tract and condensation
(Kirby, 2007)
Figure 4: Overview of cardiac neural crest cell migration into the outflow tract. The cardiac neural crest cells start to migrate out of their origin in the neural tube by induction of EMT (1.). They initially migrate to somites 1 to 4 (2.) and pause with migration in the circumpharyngeal ridge (3.). From there they migrate into the caudal pharynx and condense around the arch arteries (4.). Some neural crest cells migrate further into the cardiac outflow tract and condense to build the aorticopulmonary septum (5.). Somites 1‐4 (S1‐S4); pharyngeal arches (3, 4, 6); Ao, Aorta; P, pulmonary trunk.
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Cranial crest cells migrate in three streams: cranial, middle and caudal. The majority of cardiac neural crest cells originate from the caudal stream. Those cells migrate from somite 1 to 3 into the circumpharyngeal ridges and then invade the pharyngeal arches III, IV and VI (Figure 4). Each pharyngeal arch has a unique structure and specific functions which are supported by neural crest cells. Therefore, migration of cardiac neural crest cells from the ridge to specific pharyngeal arches has to be further refined (Kirby and Hutson, 2010). As soon as cardiac neural crest cells reached the right position at their specific pharyngeal arches, a subpopulation of the cells starts to condense (Figure 4). Due to condensation of cardiac neural crest cells around the pharyngeal arches a first sheath is formed (Kirby and Hutson, 2010). Cardiac neural crest cells which do not condense to form a sheath around the pharyngeal arches migrate further and enter the distally located outflow tract cushions where they condense and form the aorticopulmonary septation complex. However, a small number of cardiac neural crest cells migrate into the proximal outflow tract (Figure 4). These cells do not form a condensed mesenchyme but migrate into the interventricular septum, form a sheath around the atrioventricular bundle and are responsible for closure of the ventricular septum (Kirby and Hutson, 2010). Furthermore, the cardiac ganglia are formed completely by cardiac neural crest cells, which includes neuronal cell bodies and supporting cells (Kirby et al., 1983;Kirby and Stewart, 1983).
1.1.4 Outflow Tract Malformations
Several cell types contribute to proper heart development. Two of the main cell lineages that contribute to outflow tract development are the SHF and the cardiac neural crest. While the second heart field contributes to myocardial and endocardial cells, the cardiac neural crest cells contribute to outflow tract septation (Snider et al., 2007;Waldo et al., 2005b). Both cell lineages reciprocally influence each other during septum formation of the outflow tract (Kodo and Yamagishi, 2011;Waldo et al., 2005a). Impaired second heart field or cardiac neural crest development results in a wide spectrum of defects in cardiac morphogenesis, particularly outflow tract malformations and misalignments with the ventricles. Amongst the malformation of the outflow tract, one finds common arterial trunk (CT), also named
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persistent arterial trunk (PTA) as well as double outlet right ventricle (DORV) and transposition of the great arteries (TGA) (Figure 5) (Neeb et al., 2013). In humans congenital heart defects represent the most common type of birth defect and appear frequently in 10 out of 1000 cases. In three out of the 10 cases early postnatal intervention is required (Dolk et al., 2011;Hoffman and Kaplan, 2002). It is estimated that approximately 30% of the congenital heart defects are caused by outflow tract defects and abnormalities of the great arteries that exit the heart (Rosamond et al., 2007). Absence of the SHF results in failure of the heart tube to elongate and loop and is embryonically lethal (Cai et al., 2003;Park et al., 2006;Prall et al., 2007). Less severe disruption results in incomplete extension of the heart tube and misalignment during cardiac septation. Atrial and atrioventricular defects result from defects in SHF development (Goddeeris et al., 2008;Hoffmann et al., 2009).
(modified from www.nlm.nih.gov/medlineplus)
Figure 5: Cardiac outflow tract defects. Overview of a normal developed heart and varieties of developmental malformations. In the normal heart (left) the aorta arises from the left and the pulmonary artery arises from the right ventricle. In a heart exhibiting a double outlet right ventricle (middle) the aorta and the pulmonary artery both arise from the right ventricle. A heart showing a common trunk (right) only one vessel arises from the right ventricle. Both defects are accompanied by a ventricular septal defect.
Cardiac neural crest cell ablation leads to a variety of cardiovascular and non‐cardiovascular defects (Nishibatake et al., 1987). Even though a variety of morphological defects can be
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caused by ablation of cardiac neural crest cells only outflow tract and conotruncal defects are seen regularly (Hutson and Kirby, 2007). Such outflow tract defects can also be caused by mutation or deletion of several genes including a number of transcription factors such as Pax3 (paired box 3), AP2 (activating enhancer binding protein 2 alpha), Insm1 (insulinoma‐associated protein 1), Hand2 (heart and neural crest derivatives‐expressed protein 2) as well as several GATA proteins (named after the consensus DNA motif 5'‐(A/T)GATA(A/G)‐3'), TBX (T‐box), Nkx (NK homeobox factor), Fox (forkhead box) and Sox (SRY box) proteins.
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1.2 Sox protein family
In 1990, Sox transcription factors were first described with the identification of the sex determining factor SRY (sex determining region on Y Chromosome) as the first member of the Sox protein family (Gubbay et al., 1990;Sinclair et al., 1990). SRY is located on the mammalian Y chromosome and contains a DNA binding domain, the so called SRY‐box or HMG‐box (high mobility group‐box). SRY and Sox proteins show high levels of conservation in the HMG domain with more than 50% amino acid identity and belong to the same subgroup within the HMG‐box superfamily. The HMG‐box superfamily is divided into two subgroups with one group comprising Sox and TCF proteins and the other group comprising HMG/UBF proteins. Whilst HMG/UBF proteins contain multiple HMG domains that bind unspecifically to DNA, Sox and TCF proteins contain exclusively one HMG domain which binds DNA in a sequence specific manner (Grosschedl et al., 1994;Laudet et al., 1993). Binding of Sox proteins to DNA occurs in the minor groove of the double helix via the A A A consensus sequence 5’‐ /T /T CAA /T G‐3’ (Harley et al., 1994;Weiss, 2001). As a consequence, DNA bends in an angle of about 70 to 85 degrees (Connor et al., 1994;Ferrari et al., 1992;Werner et al., 1995). Therefore Sox proteins are assumed to have architectural characteristics (Werner and Burley, 1997). In addition to DNA binding and bending, the HMG‐box takes part in protein‐protein interactions (Ambrosetti et al., 1997;de Santa Barbara et al., 1998;Hosking et al., 2001;Wissmuller et al., 2006). Members of the Sox protein family can be found in the whole animal kingdom. In mammals, 20 different Sox proteins have been identified. They are divided into eight subgroups (A‐H) according to HMG box sequence homology of more than 80% within each subgroup (Bowles et al., 2000;Kuhlbrodt et al., 1998;Pevny and Lovell‐Badge, 1997;Schepers et al., 2002;Wegner, 1999). Moreover, Sox proteins of the same subgroup show conserved regions outside the HMG‐Box, such as the transactivation domain which is often located in the C‐ terminal part of the protein as in SoxC and E (Kuhlbrodt et al., 1998;Pusch et al., 1998;van de Wetering et al., 1993) or centrally like in SoxF (Hosking et al., 1995). Members of the SoxD show in addition to the HMG‐Box a leucine zipper next to a glutamine rich region. Together they generate a coiled‐coil‐domain, which is necessary to mediate a homo‐ and hetero‐ dimerisation (Lefebvre et al., 1998;Takamatsu et al., 1995). Similarities can be seen in the genomic organisation of Sox genes from the same subgroup as well, e. g. mammalian family
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members of SoxB and SoxC contain only one exon, members of SoxD to SoxG in contrast contain several exons and introns (Wegner, 1999). As Sox proteins are transcription factors, they need to be translocated after translation via a nuclear localisation signal (NLS) into the nucleus. Here they activate their specific target genes and induce certain cellular programs. NLS have been described for most Sox Proteins and for SRY (Rehberg et al., 2002;Sudbeck and Scherer, 1997). The NLS of SoxB is located C‐ terminally (Malki et al., 2010). Furthermore, a nuclear export signal (NES) was identified in SoxE, SRY and SoxB, which enables the protein to be transported out of the nucleus again (Gasca et al., 2002;Malki et al., 2010;Rehberg et al., 2002). During development sequentially acting Sox proteins are necessary from an early pluripotent cell stage until terminally differentiated stages, for instance during neuronal development (Avilion et al., 2003;Bergsland et al., 2006;Bylund et al., 2003;Graham et al., 2003;Hoser et al., 2008).
1.2.1 SoxC proteins
In invertebrates like Drosophila melanogaster only one SoxC‐gene was identified (Cremazy et al., 2001;Guth and Wegner, 2008), whereas three different SoxC‐genes can be found in vertebrates. They are called Sox4, Sox11 and Sox12 (Figure 6). Members of the SoxC subgroup have only a single exon with a highly conserved N‐terminal HMG‐box(Jay et al., 1995;Kuhlbrodt et al., 1998;Maschhoff et al., 2003) and a C‐terminally located transactivation domain with a high sequence homology (Bowles et al., 2000;Schepers et al., 2002;Wegner, 1999). Due to the high similarity of Sox4, 11 and 12 these proteins are thought to have similar functions and possibly compensate for the loss of each other, when expressed in the same tissue. They show a strong overlap in their expression patterns in the developing nervous system and seem to play an important role in the neuronal maturation process (Bergsland et al., 2006;Cheung et al., 2000;Hoser et al., 2008;Jay et al., 1997). Recent studies showed that deletion of SoxC proteins in the embryonic mouse spinal cord results in a significant
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decrease of differentiated neurons with a combined increase in cell death (Bhattaram et al., 2010;Thein et al., 2010).
(modified from Bowles et al., 2000)
Figure 6: Sox proteins of subgroup C. Schematic representation of SoxC proteins, Sox4 and Sox11 in mouse (mo‐SOX4, mo‐Sox11) and Sox12 in human (hu‐Sox12). Sox24 found in trout (tr‐Sox24). SoxC proteins are composed of a single exon (no introns, ni) at the gene level. The proteins contain a N‐terminally located HMG‐Box (black rectangle) as well as a C‐ terminally located transactivation domain (TA, light grey box with red border). Highly conserved regions (white rectangle) were identified in all four proteins at N‐ and C‐terminus. In addition there is a serine rich region (light blue rhomb) detected C‐terminally in mo‐Sox4 and mo‐Sox11. Furthermore an acidic region (yellow) followed by a proline‐glutamine rich region (blue square) could be identified in mo‐Sox11. Also in hu‐Sox12 and tr‐Sox24 an acidic region was identified. Boxes on the right show legends.
1.2.1.1 Sox4
The Sox4 gene is composed of only one exon with a size of 4,9 kb (Schilham et al., 1993). The protein acts as a transactivator of transcription and its transactivation domain is located in the serine rich C‐terminal part of the protein (Figure 6). Sox4 could be detected in T‐ and B‐ lymphocytes of the thymus as well as in the gonads and in the fetal brain of the mouse (van de Wetering et al., 1993). Sox4 transcripts are found also in the mouse uterus where its expression is controlled by hormones (Graham et al., 1999;Hunt and Clarke, 1999). In the early stages of embryonic development Sox4 transcripts can be detected in the branchial arches, the trachea, the heart and in the oesophagus (Hoser et al., 2008;Schilham et al., 1996;Ya et al., 1998b). During late stages of embryonic development Sox4 is expressed in the epiphyseal cartilage of developing bones and is regulated via parathyroid hormone and its receptor (Reppe et al., 2000). Hence, Sox4 takes part in the development of chondrocytes similar to transcription factors Sox5, Sox6 and Sox9 (Sekiya et al., 2002). In addition, it has been shown that Sox4 takes part in building up bones postnatally (Nissen‐Meyer et al., 2007). Further Sox4 expression in mouse is detected in neuronal and glial precursor cells
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(Bergsland et al., 2006;Cheung et al., 2000;Hoser et al., 2007;Potzner et al., 2007) as well as the embryonic cells of the pancreas in zebrafish and mouse (Lioubinski et al., 2003;Mavropoulos et al., 2005;Wilson et al., 2005). In addition to that an over‐expression of Sox4 was found in malignant tumours like medulloblastoma (Lee et al., 2002), adenoid‐cystic carcinoma of the salivary gland (Frierson, Jr. et al., 2002) or prostate cancer (Liu et al., 2006). Ectopic overexpression of Sox4 in hepatocellular carcinoma was shown to have an anti‐ apoptotic effect (Hur et al., 2010) whereas the down regulation of Sox4 expression via RNA interference had an anti‐apoptotic effect in adenoid‐cystic carcinoma cells (Pramoonjago et al., 2006) or prostate cancer cells by inducing apoptosis (Liu et al., 2006). Furthermore, it was shown that overexpression of Sox4 is associated with diverse human cancers such as brain lung and breast cancer (Penzo‐Mendez, 2010). Recent studies show, that Sox4 is also involved in and required for TGF‐ induced EMT (Tiwari et al., 2013;Vervoort et al., 2013;Zhang et al., 2012). Schilham et al. deleted the coding region of Sox4 to analyse Sox4 function (Schilham et al., 1996;Ya et al., 1998b). The Sox4‐deficient mice died at embryonic day 14 due to malformations of the heart and histological analyses of mutant embryos revealed that proper development of the endocardial ridges into the semi lunar valves was disturbed. The development of the outlet portion of the muscular ventricular septum was impaired. As a consequence, embryos developed an arterial common trunk (CT) and died because of circulatory failure at embryonic day 14 (Schilham et al., 1996;Ya et al., 1998b).
1.2.1.2 Sox11
Besides the identification of Sox4, two other members of SoxC protein family could be detected namely Sox11 and Sox12. Sox11 shows significant similarities to Sox4. Both proteins contain a serine rich region at the carboxyterminus and an 82% homology of the last 34 amino acids. These regions contribute to their function as transcriptional activators. As shown for Sox4, Sox11 also exhibits a highly active transactivation domain (Dy et al., 2008;Hoser et al., 2008;Kuhlbrodt et al., 1998;van de Wetering et al., 1993). In addition to the carboxyterminal serine rich region, Sox11 contains also an acidic region followed by a proline‐glutamine rich area located centrally (Figure 6). The acidic region may have an
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inhibitory effect on the DNA binding ability of Sox11 in vitro and on the activation of reporter gene expression in cell culture (Wiebe et al., 2003). Furthermore, Sox11 as well as Sox4 are able to interact synergistically with POU proteins (Kuhlbrodt et al., 1998). In chicken, mouse and human Sox11 transcripts have been detected in immature neurons of the developing central and peripheral nervous system (Jay et al., 1995;Uwanogho et al., 1995), but as neuronal development progresses Sox11 expression is down‐regulated. Due to co‐expression with the microtubule associated protein doublecortin (DCX), which is expressed in postmitotic immature neurons, Sox11 was assumed to have a regulatory effect on neuronal differentiation in the adult central nervous system (Haslinger et al., 2009). In addition, Sox11 transcripts could be detected in a variety of murine organs and tissues during embryonic development, in which interactions between epithelial and mesenchymal cells are necessary. Sox11 can be found in the branchial arches, genital eminences, limbs, eyes, ears, palatal plate, lung, kidney, spleen and the epithelium of the nose, oesophagus, pancreas, stomach and the salivary glands (Hargrave et al., 1997;Jay et al., 1995). Sox11 is also required in early sympathetic ganglia for the proliferation of tyrosine hydroxylase ‐ expressing cells (Potzner et al., 2010). In contrast to Sox4 only a weak expression of Sox11 could be detected in later stages of heart development (Hoser et al., 2008). In analogy to Sox4 a high expression of Sox11 leads to malignant embryonic medulloblastomas (Lee et al., 2002). Therefore, Sox4 and Sox11 could contribute to a malignant phenotype in a similar fashion by promoting growth and proliferation of altered cells. To study Sox11 function during embryonic development in detail, a Sox11 deletion model in mouse was generated (Sock et al., 2004). Sox11 deficient mice die shortly after birth due to congenital cyanosis. By the time of birth, Sox11‐deficient mice exhibit a variety of developmental defects. Beside pulmonary insufficiency caused by hypoplasia of the lung, cardiovascular malformations lead to severe heart defects. The cause of the developmental heart defects is complex. These embryos show a ventricular septum defect (VSD). In addition, aorta and pulmonary trunk are often not divided in Sox11 deficient mice. Aside from that, some of the Sox11‐deficient mice displayed a transposition of the great arteries or double outlet right ventricle (DORV). Accompanying the complex heart defects 70% of Sox11 deficient mice show a cleft lip and palate as well as an open abdominal cavity and open eyelids. Furthermore the pancreas and stomach exhibit a hypomorphic structure, while the spleen is completely absent in Sox11‐deficient mice. Other phenotypic characteristics of
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Sox11‐deficient mice are ossification defects of the skull and skeletal malformations. In contrast, Sox11‐deficient mice show a normal neuronal development in the central and peripheral nervous system on a gross morphological level (Sock et al., 2004), which might be due to the extensive co expression of Sox4 and Sox11 in this tissue. Evidence for a cooperative function of Sox4 and Sox11 in neuronal differentiation was seen in electroporation experiments into the early neural tube of chicken. Sox4 and Sox11 are required for expression of pan neural genes like Tubulin‐ß‐III (Tubb3, Tuj1), which was identified as a possible direct target gene of SoxC proteins (Bergsland et al., 2006). Recently, Sox4 and Sox11 double‐deficient mice have been generated and analysed. Analysis of SoxC deficient mice showed that the more SoxC alleles are deleted in mouse embryos, the more severe and widespread organ hypoplasia is (Bhattaram et al., 2010). The embryos die at midgestation with a normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells (Bhattaram et al., 2010).
1.2.1.3 Sox12
Sox12 is the third member of the SoxC protein family and less well characterised as Sox4 and Sox11. When first discovered in human, it was initially referred to as Sox22 (Jay et al., 1997). Based on the high homology of Sox12 in mouse compared to Sox22 in human and on their chromosomal location it was confirmed that they are orthologues. Therefore, human Sox22 was renamed to Sox12 (Bowles et al., 2000;Jay et al., 1997;Schepers et al., 2002). Besides the HMG‐box, which is highly similar to Sox4 and Sox11 a transactivation domain could be identified C‐terminally (Jay et al., 1997;Kuhlbrodt et al., 1998;van de Wetering et al., 1993) (Figure 6). During mouse embryogenesis Sox12 shows a highly overlapping expression pattern in various tissues and the developing nervous system when compared to Sox4 and Sox11 (Hoser et al., 2008). The Sox12 expression pattern is very broad and homogenous, but expression levels are lower compared to the other SoxC proteins (Dy et al., 2008;Hoser et al., 2008). Similarly, Sox12 appears to have weaker transactivation capacity. In the adult, Sox12 is expressed in the heart, testis, pancreas and ovary (Jay et al., 1997). In contrast to the severe phenotypes seen in Sox4‐ and Sox11‐deficient mice (Schilham et al., 1996;Sock et al., 2004;Ya et al., 1998b), Sox12‐deficient mice showed normal development with no obvious
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alterations found (Hoser et al., 2008). These findings favour the idea that Sox4 and Sox11 can compensate for the loss of Sox12, whereas Sox12 alone is not capable to replace either Sox4 or Sox11 function.
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2. Aim of the Study
Transcription factors of the Sox family are expressed in a broad range of tissues and cells. It was shown that they regulate and participate in a variety of developmental processes during embryogenesis. SoxC contains the transcription factors Sox4, Sox11 and Sox12. Constitutive deletion of the genes for Sox4 or Sox11 led to altered heart development with malformations of the outflow tract ranging from DORV to CT as well as ventricular septum defect and impaired formation of the semilunar valves. Considering the high degree of homology between the two proteins and the similarity of the observed heart phenotypes in mutant animals, it seemed interesting to study their respective and joint function during heart development. For the identification of cell types in which SoxC proteins function conditional cell type specific deletion of the respective genes in the mouse seemed the method of choice. The three cell types that mainly contribute to heart formation and development are the neural crest, the mesodermal and the endothelial cell population. Mouse lines expressing Cre recombinase specifically in these different cell types combined with floxed SoxC alleles were thus to be generated in this study. Hearts of mutant embryos with various SoxC gene deletions should then be analysed morphologically and histologically at different developmental stages. Subsequently, the effects of SoxC deletion on marker gene expression, proliferation, migration and cell survival were studied by immunohistochemical methods in combination with lineage tracing techniques. Resulting hypothesis on the molecular mode of action of SoxC transcription factors should finally be investigated by the identification of SoxC target genes with relevance during outflow tract formation.
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3. Results
3.1 Sox4 and Sox11 function in cardiac outflow tract development
3.1.1 Sox4 and Sox11 function in cardiac neural crest cells
3.1.1.1 Macroscopic analysis of outflow tract development in mice with Sox4 and Sox11 ablation in cardiac neural crest cells
A Wnt1::Cre transgene was used to delete Sox4 and/or Sox11 in the cardiac neural crest precursor cell population. Since the constitutive Sox4 knockout embryos die on 14.0 days post coitum (dpc), embryos at 13.5 dpc were examined first. All of the Sox4Wnt1::Cre embryonic hearts showed a macroscopically normal outflow tract region as wildtype embryonic hearts (Table 1; Figure 7, A,D). Furthermore, most of the conditional Sox11Wnt1::Cre knockout embryos displayed a normally developed outflow tract. Only in 8% of Sox11Wnt1::Cre knockout animals an outflow tract malformation was seen (Table1; Figure 7, B,C). All mutants with an outflow tract phenotype exhibited a double outlet right ventricle (DORV), where the aorta as well as the pulmonary artery arose from the right ventricle (Figure 7, C) while in the normal developed heart the aorta arises from the left ventricle. To analyse, if there was a redundant function of Sox4 and Sox11 in cardiac neural crest cells Sox4Wnt1::CreSox11Wnt1::Cre double‐knockout (Dko) embryos were generated and examined. Interestingly, all of the analysed Dko mutant embryos displayed outflow tract malformations. Among these malformation was the more severe form of CT, which was found in addition to DORV (Table 1, Figure 7 E,F). The CT is defined as only one vessel arising from the right ventricle (Figure 7, E). To see, if there were additional defects at later stages of heart and outflow tract development, mice at 17.5 dpc were analysed. At this stage, shortly before birth, the heart is fully developed. As expected from data of earlier stages, Sox4Wnt1::Cre mutant embryonic hearts exhibited no outflow tract malformation (Figure 8, A,D). Also in Sox11Wnt1::Cre mice only a small percentage of mutant embryonic hearts showed a phenotype, which was
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concordant with the results of the earlier examined stages. All mutants with an outflow tract malformation presented with DORV (Figure 8, B,C).
Figure 7: Early developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation. Macroscopic outer appearance of the outflow tract region of wildtype (Wt) embryos and Sox4Wnt1::Cre (Sox4) and Sox11Wnt1::Cre (Sox11) as well as Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) at 13.5 dpc. Abbreviations: Ao, aorta; CT, common arterial trunk; LA, left atrium; RA, right atrium; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
As already seen in 13.5 dpc, all of the Dko mutant embryos displayed an outflow tract malformation at 17.5 dpc. Around half of these mutant embryos presented DORV (Figure 8, F) while the rest showed the more severe CT phenotype (Figure 8, D). In summary of all Dko mutant embryos analysed, 37% revealed a common arterial trunk (CT) while 63% displayed DORV (Table 1). Macroscopic examination of 17.5 dpc knockout embryos with single Sox4 or Sox11 knockout or combined Sox4 or Sox11 knockout in the cardiac neural crest cell lineage revealed no additional defects in the outflow tract region compared to earlier stages.
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Figure 8: Late developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation. Macroscopic outer appearance of the outflow tract region of wildtype (Wt) embryos and Sox4Wnt1::Cre (Sox4) and Sox11Wnt1::Cre (Sox11) as well as Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) at 13.5 dpc. Panel A, B and D shows a normal developed outflow tract region. Panel C and E pictures an outflow tract region with DORV and F a CT. Abbreviations: Ao, aorta; CT, common arterial trunk; LA, left atrium; RA, right atrium; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
Table 1: Quantity and percentage of cardiac outflow tract malformations in mice with neural crest specific SoxC gene ablation. Shown are the combined numbers (No) of Sox4Wnt1::Cre (Sox4) and Sox11Wnt1::Cre (Sox11) as well as Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) embryos at 13.5 and 17.5 dpc and the percentage of OFT defects (%) differentiated between double outlet right ventricle (DORV) and common arterial trunk (CT).
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3.1.1.2 Detailed histological analysis of outflow tract development in mice with Sox4 and Sox11 ablation in cardiac neural crest cells
To examine the consequences of SoxC gene ablation in cardiac neural crest cell lineage in detail, histological haematoxylin‐eosin stainings were performed on paraffin sections for the different genotypes and stages of development. Analysis of the outflow tract ofSox4Wnt1::Cre embryos at 13.5 dpc using serial sections showed no differences compared to wildtype outflow tract sections (data not shown). Detailed analysis of Sox11Wnt1::Cre mutant outflow tact sections provided evidence that embryos displaying an altered phenotype indeed presented with DORV (Figure 9, D‐F).
Figure 9: Early developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation. Haematoxylin‐eosin stained transverse sections of outflow tract region of wildtype (Wt), Sox11Wnt1::Cre (Sox11) and Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) embryos at 13.5 dpc. Abbreviations: Ao, aorta; CT, common arterial trunk; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
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In these Sox11Wnt1::Cre mutants the aorta arose from the right ventricle (Figure 9,D‐F) while in the wildtype sections only the pulmonary trunk arose from the right ventricle and the aorta was exclusively connected to the left ventricle (Figure 9,A‐C). Sections from the outflow tract of Dko confirmed a CT phenotype (Figure 9, G‐I). Detailed analysis of stage 17.5 dpc did not show any additional defects in mutants with a Sox4 and/or Sox11 ablation in the cardiac neural crest cell lineage compared to wildtype embryonic hearts (Figure 10, A‐F).
Figure 10: Late developmental defects of the cardiac outflow tract in mice with neural crest specific SoxC gene ablation. Haematoxylin‐eosin stained transverse sections of outflow tract region of wildtype (Wt) and Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) embryos at 17.5 dpc. Abbreviations: Ao, aorta; CT, common arterial trunk; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
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3.1.1.3 Detailed histological analysis of ventricular septum development in mice with Sox4 and Sox11 ablation in cardiac neural crest cells
In addition to the outflow tract malformation seen in constitutively deleted SoxC mutant embryos, one finds an impaired development of the endocardial ridges into semilunar or tricuspid valves and a ventricular septal defect (VSD). Hearts of constitutive Sox4 and Sox11 knockout mice always showed a ventricular septum defect as well as defects of valve development. To investigate if the same developmental defects occur in hearts of mice where Sox4 and/or Sox11 were ablated only in the cardiac neural crest cells, haematoxylin‐ eosin staining on paraffin sections was performed on mutant and wildtype embryos at 13.5 dpc. Transverse sections of Sox4Wnt1::Cre and Sox11Wnt1::Cre as well as Dko mutant embryonic hearts did not show VSD or defects of valve development (Figure 12, B‐D compared to wildtype, Figure 12, A).
Figure 11: Development of ventricular septum in mice with neural crest specific SoxC gene ablation. Hematoxylin‐eosin stained transverse sections of wildtype (Wt), Sox4Wnt1::Cre (Sox4), Sox11Wnt1::Cre (Sox11) and Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) embryos at 13.5 dpc. Abbreviations: LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Asterisk points to the ventricular septum which is fully developed, dividing the right from the left ventricle (A‐D). Scale bar: 500µm.
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3.1.1.4 Appearance of animals with a specific ablation of Sox4 and Sox11 in neural crest cells
Mutant embryos with combined ablation of Sox4 and Sox11 in the cardiac neural crest cell lineage at the age of 17.5 dpc could be clearly distinguished from their wildtype littermates. Dko embryos, especially their heads, were much smaller compared to wildtype embryos. Furthermore, mutant embryos displayed an abnormal cranio‐facial and neck region (Figure 11; A‐B). Some of the embryos with combined ablation of Sox4 and Sox11 also showed open eye lids.
Figure 12: Developmental defects in mice with neural crest specific SoxC gene ablation. Whole wildtype (Wt) (A) and Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko) (B) embryo at 17.5 dpc. Scale bar: 2mm.
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3.1.1.5 Analysis of early outflow tract development in mice with Sox4 ablation in cardiac neural crest cells via Ap2::Cre
It was shown in previous studies that Wnt1::Cre gene deletion may only be efficient in the cardiac neural crest upon or after emigration from the dorsal neural tube (Olaopa et al., 2011). If so, deletion of the Sox4 gene in cardiac neural crest cells via Wnt1::Cre might occur too late, after Sox4 function started to efficiently delete Sox4 expression. This might explain the unexpected result that no altered phenotype could be found in the hearts of these mutants. To address this problem Sox4 was deleted via an Ap2::Cre gene construct which had been reported to be active already in the premigratory neural crest (Macatee et al., 2003). The generated Sox4Ap2::Cre knockout mutant embryos showed a completely normal developed heart and outflow tract region, no outflow tract defects were found (Table 2).
Table 2: Quantity and percentage of cardiac outflow tract malformations in mice with neural crest specific SoxC gene ablation. Shown are the numbers (No) of Sox4Ap2::Cre (Sox4) embryos at 13.5 dpc and the percentage of OFT defects (%) differentiated between double outlet right ventricle (DORV) and common arterial trunk (CT).
3.1.1.6 Efficient deletion of Sox4 and Sox11 in neural crest cells
Since conditional deletion of Sox4 in neural crest cells did not show a phenotype, it was necessary to proof that the Sox4 allele was efficiently knocked out in neural crest cells when combined with Wnt1::Cre. For this analysis neural crest cells where tagged via a Rosa26stopflox‐EYFP reporter construct, which was Wnt1::Cre dependent. Therefore Wnt1::Cre activation of EYFP from the Rosa26stopflox‐EYFP allele was used to label neural crest cells. In early stages of heart development, such as 10.5 dpc, wildtype animals showed a broad expression of Sox4 or Sox11 in neural crest cells (Figure 13, A‐C) whereas in mutant animal hearts the neural crest cells did not express Sox4 or Sox11 anymore (Figure 13, D‐E). Both,
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wildtype and mutant hearts still expressed Sox4 and Sox11 in epithelial cells surrounding the heart. Therefore it can be concluded that Sox4 and Sox11 were deleted efficiently and specifically in neural crest cells of those animals (Figure 13, B‐E).
Figure 13: Deletion efficiencies of SoxC proteins in cardiac neural crest cells. Immunohistochemistry performed at 10.5 dpc with antibodies directed against Sox4 and Sox11 (red) on embryos carrying a combination of Rosa26stopflox‐EYFP and Wnt1::Cre alleles (Wt) (A‐D) or carrying the Rosa26stopflox‐EYFP on a Sox4Wnt1::Cre and Sox11Wnt1::Cre background (Mut) (D,E). Wnt1::Cre dependent activation of EYFP (green) from the Rosa26stopflox‐EYFP allele was used to label neural crest cells. Nuclei are counterstained with DAPI (blue, A). Pictures D and G were taken from the region boxed in A. Scale bars: 50 μm.
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3.1.2 Sox4 and Sox11 function in mesodermal cells
3.1.2.1 Analysis of early outflow tract development in mice with Sox4 and Sox11 ablation in mesodermal cells
For analysing the deletion of Sox4 and Sox11 in mesodermal cells in early development of the outflow tract region, Sox4fl and Sox11fl or Sox4flSox11fl allele were combined with the Nkx2.5::Cre. The resulting Sox4Nkx2.5::Cre and Sox11Nkx2.5::Cre mutants were analysed at 13.5 dpc. Unfortunately, no animals with combined deletion of Sox4and Sox11 in mesodermal cells of the heart could be obtained (Table 3). This genotype causes early embryonic lethality. Once again hearts of Sox4Nkx2.5::Cre knockout embryos showed no outflow tract defects. All mutants displayed a completely normal developed heart and outflow tract region as seen in wildtype littermates (Figure 14, A).
Figure 14: Early developmental defects of the cardiac outflow tract in mice with mesoderm specific SoxC gene ablation. Macroscopic outer appearance of the outflow tract region (A‐C) of Sox4Nkx2.5::Cre (Sox4) and Sox11Nkx2.5::Cre (Sox11) animals at 13.5 dpc and (D‐F) haematoxylin‐eosin stained serial outflow tract sections of Sox11kx2.5::Cre (Sox11) animals showing DORV in (C and D‐F). Ao, aorta; LA, left atrium; RA, right atrium; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
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For Sox11Nkx2.5::Cre knockout animals outflow tract malformations could be detected. The penetrance was higher than in Sox11Wnt1::Cre mice. Half of the hearts did not show an outflow tract malformation whereas for the other half DORV was identified (Table 3; Figure 14, B,C). Detailed histological analysis of serial sections did not show any additional defects. The haematoxylin‐eosin staining of the Sox11Nkx2.5::Cre hearts revealed DORV in all cases (Figure 14, D‐F).
3.1.2.2 Analysis of late outflow tract development in mice with Sox4 and Sox11 ablation in mesodermal cells
Since no defect was observed in outflow tract development of Sox4Nkx2.5::Cre hearts at 13.5 dpc, outflow tracts of 17.5 dpc old Sox4Nkx2.5::Cre mutants were analysed next. Sox4Nkx2.5::Cre knockout animal hearts did not display any malformation of the outflow tract at this stage of heart development (Figure 15, A). For Sox11Nkx2.5::Cre mutants the same rate of malformations of the outflow tract region could be found at 17.5 dpc as in the already examined early developmental stage. Sox11 ablation in mesodermal cells in later stages again led to DORV in almost half of all obtained mutant embryonic hearts (Figure 15, B‐C).
Figure 15: Late developmental defects of the cardiac outflow tract in mice with mesoderm specific SoxC gene ablation. Macroscopic outer appearance of the outflow tract region of Sox4Nkx2.5::Cre (Sox4) and Sox11Nkx2.5::Cre (Sox11) animals at 17.5 dpc. Abbreviations: Ao, aorta; LA, left atrium; RA, right atrium; RV, right ventricle; PT, pulmonary trunk. Scale bar: 500µm.
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Given that 13.5 dpc old Sox4Nkx2.5::CreSox11Nkx2.5::Cre embryos were not obtained, later stages of this genotype could not be analysed. Embryonic hearts of Sox11Nkx2.5::Cre knockout animals which showed an outflow tract malformation were analysed for an additional VSD. For detailed analysis, histological stainings with haematoxylin‐eosin were performed on transverse sections of paraffin embedded mutant hearts. Sections of embryonic hearts of mutants with a Sox11 deletion in mesodermal cell lineage showed no ventricular septal defect. The septum which separates the right from the left ventricle was fully developed (Figure 16, A‐B).
Figure 16: Development of ventricular septum in mice with mesoderm‐ specific SoxC gene ablation. Haematoxylin‐eosin stained transverse sections of wildtype (Wt) and Sox11Nkx2.5::Cre (Sox11) at 13.5 dpc. Abbreviations: LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Asterisk points to the ventricular septum which is fully developed, dividing the right from the left ventricle (A‐D). Scale bar: 200µm.
Table 3: Quantity and percentage of cardiac outflow tract malformations in mice with mesoderm specific SoxC gene ablation. Shown are the combined numbers (No) of Sox4Nkx2.5::Cre (Sox4), Sox11Nkx2.5::Cre (Sox11) and Sox4Nkx2.5::CreSox11Nkx2.5::Cre (Dko) embryos at 13.5 and 17.5 dpc and the percentage of OFT defects (%),double outlet right ventricle (DORV) and common arterial trunk (CT). No Dko animals were obtained.
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Since deletion of Sox11 in cardiac neural crest cells and mesodermal cells led to an outflow tract malformation, the next approach was to see what happens when Sox4 or Sox11 are deleted simultaneously in both cell types. Due to the unavailability of mutant animals with combined deletion of Sox4 and Sox11 in mesodermal cells, there was no chance to analyse the redundant function of those proteins; ideally both SoxC proteins would be deleted in cardiac neural crest and mesodermal simultaneously.
3.1.3 Analysis of outflow tract development in mice with Sox4 and Sox11 ablation in cardiac neural crest and mesodermal cells
Embryonic hearts of Sox4Wnt1::Cre;Nkx2.5::Cre animals still showed no outflow tract malformation (Figure 17, C; E). Considering the heart phenotype in constitutive Sox4 knockout animals, this was surprising. To make sure that the floxed Sox4 allele did not act differently than the constitutive null allele, Sox4 floxed animals were crossbred with animals carrying a Sox2::Cre transgene. In the resulting double transgenic animals, Sox4 should be deleted throughout the epiblast. Phenotypic examination of these mice at 13.5 dpc showed that they fully recapitulated the outflow tract phenotype of the constitutive Sox4 knockout (Figure 17, D). For mutants with Sox11 deletion in cardiac neural crest and mesodermal cells the rate of outflow tract malformations was comparable to those mutants that where Sox11 deficient only in mesodermal cells. Approximately 50% of all Sox11Wnt1::Cre;Nkx2.5::Cre knockout animal hearts showed a phenotype with an outflow tract malformation (Figure 17; A,B; E). Mutants with Sox11 ablated in cardiac neural crest and mesodermal cells however displayed a more severe phenotype. Most presented with CT (Figure 17, A,B), while selective Sox11 deletion in cardiac neural crest or in mesodermal cells led to the development of DORV.
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Figure 17: Developmental defects of the cardiac outflow tract in mice with mesoderm and cardiac neural crest specific SoxC gene ablation. Macroscopic outer appearance of the outflow tract region of mesodermal and cardiac neural crest cell specific deletion of Sox4Wnt1::Cre;Nkx2.5::Cre (Sox4) and Sox11Wnt1::Cre;Nkx2.5::Cre (Sox11) animals at 13.5 dpc (A‐C) or generalised deletion of Sox4Sox2::Cre (Sox4 Sox2::Cre) in (D). (E) Table: shown are the numbers (No) of animals with Wnt1::Cre and Nkx2.5::Cre mediated deletion of Sox4 and Sox11 and the percentage of OFT defects (%) differentiated between double outlet right ventricle (DORV) and common arterial trunk (CT). Abbreviations: Ao, aorta; LA, left atrium; RA, right atrium; RV, right ventricle; PT, pulmonary trunk. Scale bar: 200µm.
3.1.4 Analysis of early outflow tract development in mice with Sox4 and Sox11 ablation in endothelial cells
As even combined deletion of Sox4 and Sox11 in cardiac neural crest and mesodermal cells could not recapitulate the phenotype seen in constitutive knockout animals, Sox4 and Sox11 must be active in yet another cell type. To examine if Sox4 and Sox11 exerted part of their impact on heart development via their function in endothelial cells, the Tie2::Cre transgene was used to delete the genes in this cell type. The resulting Sox4Tie2::Cre and Sox11Tie2::Cre embryos never showed outflow tract malformations in the early (13.5 dpc) or the late (17.5 dpc) developmental stages analysed (Table 4). In addition, deletion of both SoxC proteins in
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endothelial cells did not lead to an outflow tract malformation in any of the obtained Sox4Tie2::CreSox11Tie2::Cre (Dko) embryos (Table 4).
Table 4: Quantity and percentage of cardiac outflow tract malformations in mice with endothelial specific SoxC gene ablation. Shown are the numbers (No) of Sox4Tie2::Cre (Sox4), Sox11Tie2::Cre (Sox11) and Sox4Tie2::CreSox11Tie2::Cre (Dko) embryos at 13.5 dpc and the percentage of OFT defects (%) differentiated between double outlet right ventricle (DORV) and common arterial trunk (CT).
3.1.5 Properties of cardiac neural crest cells in the absence of Sox4 and Sox11
The analysis of embryos with a joint deletion of Sox4 and Sox11 in cardiac neural crest cells revealed an abnormal outflow tract region in all obtained mutants. Therefore, the cardiac neural crest cells are impaired and could have defects in timing, migration, proliferation or survival. To examine these possibilities neural crest cells were genetically marked by crossing mice with a Rosa26stopflox‐EYFP allele with mice carrying a Wnt1::Cre transgene on a wildtype background, which leads to EYFP expression in neural crest cells from embryonic day 8 on. Additionally mice with a Rosa26stopflox‐EYFP allele were crossbred with mice carrying a floxed allele for Sox4 and Sox11, which lead to EYFP expression in neural crest cells where Sox4 and Sox11 is deleted. Comparing EYFP‐stained sections at 10.5 dpc of Rosa26stopflox‐EYFPWnt1::Cre (Wt) and Rosa26stopflox‐EYFPWnt1::CreSox4Wnt1::CreSox11Wnt1::Cre double knockout (Dko) animals, no difference was seen in the amount of cells that migrate to the outflow tract region (Figure 18, A‐B and Figure 19, A). Similarly, at 13.5 dpc the comparison of sections from wildtype and Dko mice did not show any difference in the amount of EYFP‐positive cells in the outflow tract region (Figure 18, C‐D and Figure 19, A). Timing of migration and the
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migration process itself were thus not affected in cardiac neural crest cell ofRosa26stopflox‐ EYFPWnt1::CreSox4Wnt1::CreSox11Wnt1::Cre embryos (Figure 19, A).
Figure 18: Analysis of timing and migration of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 ablation. Immunohistochemistry for EYFP (green) performed at 10.5 dpc (A,B) and 13.5 dpc (C,D) on embryos carrying a combination of Rosa26stopflox‐EYFP and Wnt1::Cre alleles (Wt) (A,C) or the Rosa26stopflox‐EYFPWnt1::Cre allele on a Sox4Wnt1Sox11Wnt1 background (Dko) (B,D). Nuclei are counterstained with DAPI (blue). Scale bars: 100µm.
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To label apoptotic cardiac neural crest cells EYFP immunohistochemistry was combined with TUNEL and double positive cells in the outflow tract area were counted. The amount of apoptotic cardiac neural crest cells was not changed between wildtype and mutant outflow tract regions in early or late stages (Figure 19, B). For analysis of proliferation rates of neural crest cells a co‐labelling of EYFP with a Ki67 antibody was performed and double positive cells in the outflow tract area were counted. No difference in the amount of proliferating cells between wildtype and mutant outflow tract regions was detectable in early or late developmental stages (Figure 19, C).These results showed that timing, migration, apoptosis and proliferation were not altered in the cardiac neural crest cell lineage by combined ablation of Sox4 and Sox11.
Figure 19: Analysis of proliferation and apoptosis of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 ablation. Quantification of the total number of cardiac neural crest cells (A), the TUNEL‐positive apoptotic (B) and Ki67‐ positive proliferating fraction (C) at 10.5 and 13.5 dpc in outflow tract regions of embryos carrying a combination of Rosa26stopflox‐EYFP and Wnt1::Cre alleles (Wt, black bars) or the Rosa26stopflox‐EYFPWnt1::Cre allele on a Sox4Wnt1Sox11Wnt1 background (Dko, grey bars). Data are presented as mean SEM. No statistically significant difference was observed.
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3.1.6 Ablation of Sox4 and Sox11 leads to morphological changes in cardiac neural crest cells
The morphology of cardiac neural crest cells deficient of Sox4 and Sox11 was analysed in detail. While wildtype cardiac neural crest cells migrated in single cell chains into and within the outflow tract, mutant cells no longer arranged themselves in such chain‐like formations but were randomly orientated (Figure 20, A‐B). Furthermore, cardiac neural crest cells in the wildtype were connected to each other via filopodia‐like protrusions, whereas cardiac neural crest cells in double knockouts showed fewer filopodia‐like protrusions (Figure 20, A‐B and higher magnifications). In addition, cardiac neural crest cells deficient for Sox4 and Sox11 exhibited an altered and often less compact morphology compared to wildtype cells.
Figure 20: Analysis of the morphology of cardiac neural crest in mice with neural crest specific deletion of Sox4 and Sox11. High magnification confocal images of cardiac neural crest cells (green) at 10.5 dpc taken from wildtype (Wt) (A) or age matched Sox4Wnt1Sox11Wnt1 (B) embryos. Arrowheads point to filopodia‐like protrusions. Scale bar: 15µm.
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3.1.7. Sox12 expression in the outflow tract
Since Sox12 is a member of the SoxC protein family, it was analysed if it is expressed in the outflow tract region. Sox12 transcripts can already be detected in the outflow tract region at 12.5 dpc and persists at least until 16.5dpc (Figure 23, A‐B). Quantitative RT‐PCR analysis of Sox12 expression showed no difference in expression levels when wildtype outflow tract regions (Figure 24, black bars) were compared to mutant ones (Figure 24, grey bars).
Figure 21: In situ ‐ hybridisation of Sox12 in wildtype outflow tract regions. At 12.5 dpc and 16.5 dpc Sox12 was expressed in anterior heart pole area of wildtype (Wt) hearts. Expression was found in the region of the aorta (Ao) and pulmonary trunk (PT) as well as in the endocardial cushion mesenchyme (ec) at 12.5 dpc or the valves (v) at 16.5 dpc. Scale bar: 2mm. (courtesy of Melanie Hoser)
3.1.8 Ablation of Sox4 and Sox11 leads to altered marker gene expression in cardiac neural crest cells
3.1.8.1 Immunohistochemical analysis of marker gene expression of cardiac neural crest cells in mice with Sox4 and Sox11 ablation in neural crest cells
To visualise the cytoskeleton in the cardiac neural crest cells, phalloidin staining was performed, which labels F‐actin. Cardiac neural crest cells were labelled by Wnt1::Cre
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induced EYFP expression. Wildtype cardiac neural crest cells displayed a relatively low level of phalloidin staining at 10.5 dpc. In contrast cardiac neural crest cells in mutant embryos revealed a prominent phalloidin staining (compare Figure 21, A,B and E,F).Additionally, wildtype cardiac neural crest cells were negative for NG2 at 10.5 dpc whereas many mutant cardiac neural crest cells expressed this proteoglycan (Figure 21, C,D and G,H). Immunohistochemical staining against other proteins like ‐catenin and platelet endothelial cell adhesion molecule (PECAM) did not reveal any changes during outflow tract development in early or late stages (Figure 22, compare A to C and B to D).
EYFP EYFP
Figure 22: Analysis of marker gene expression of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 gene ablation. Co‐immunohistochemistry was performed at 10.5 dpc in embryos carrying a combination of Rosa26stopflox‐EYFP and Wnt1::Cre alleles (Wt) (A‐D) or the Rosa26stopflox‐EYFPWnt1::Cre allele on a Sox4Wnt1::Cre Sox11Wnt1::Cre background (Dko) (E‐H). Antibodies directed against EYFP were used to detect cells of neural crest origin (green). Actin microfilaments were detected by fluorescently labelled phalloidin (A,B,E,F) and NG2 expression by specific antibodies (C,D,G,H) (both red). Nuclei were stained with Dapi (blue). Higher magnifications in B,D,F,H is boxed in A,C,E,G. Scale bars: 100µm.
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EYFP EYFP
Figure 23: Analysis of marker gene expression of cardiac neural crest cells in mice with neural crest specific Sox4 and Sox11 gene ablation. Co‐immunohistochemistry was performed at 10.5 dpc in embryos carrying a combination of Rosa26stopflox‐EYFP and Wnt1::Cre alleles (Wt) (A‐B) or the Rosa26stopflox‐EYFPWnt1::Cre allele on a Sox4Wnt1::Cre Sox11Wnt1::Cre background (Dko) (C‐D). Antibodies directed against EYFP were used to detect cells of neural crest origin (green). ‐catenin (A,C) and PECAM (B,D) expression was detected by specific antibodies (both red). Staining of nuclei was performed with Dapi (blue for B,D,F,H). Scale bars: 100µm.
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3.1.8.2 Quantitative RT‐PCR analysis of marker gene expression of cardiac neural crest cells in mice with Sox4 and Sox11 ablation in neural crest cells
To further prove that gene expression was changed in embryos lacking Sox4 and Sox11 in the cardiac neural crest, outflow tracts of 13.5 dpc old wildtype and mutant embryos were dissected and quantitative RT‐PCR was performed on RNA. Transcript levels were normalised to the housekeeping gene for Rpl8 (ribosomal protein L8). Several markers that were typically expressed in cardiac neural crest cells and are relevant for their development were examined. For extracellular matrix, cell communication and cytoskeletal markers, Adam9 and Adam19 (disintegrin and metalloproteinase domain‐containing protein family members 9 and 19), Cx40 and Cx43 (gap‐junction proteins connexin 40 and 43), both belonging to the connexin family alpha type subfamily, as well as NG2 and SMA (alpha smooth muscle actin) were chosen for examination. For Cx40 no changes in expression levels could be observed in mutant outflow tracts, while Cx43 was significantly increased in expression levels (Figure 24, grey bars) compared to wildtype (Figure 24, black bars). Both Adam gene family members were decreased in their expression levels in mutant outflow tracts. In particular, Adam19 was significantly decreased, while Adam9 was only slightly decreased (Figure 24). In addition, expression of transcription factors Sox10, Hand2 (heart and neural crest derivates expressed protein 2) and Foxc2 (forkhead box protein c2) were examined. In outflow tracts of mutant embryos Sox10 is slightly but significantly increased compared to wildtype outflow tract regions, while Hand2 is significantly decreased. Foxc2 showed no changes in expression levels (Figure 24). Since EMT and mesenchymal to epithelial transition (MET) is essential to form the endocardial cushion mesenchyme and the future aorticopulmonary septum, marker genes involved in these transitions were tested. Snail1 and Snail2 (snail family zinc‐finger 1 and 2), Twist1 (twist‐related protein 1) and E‐cadherin (epithelial cadherin or CDH1) were analysed. Twist1 was not changed in expression levels, while expression levels for both zing‐finger proteins Snail1 and Snail2 were significantly decreased in mutant outflow tracts (Figure 24, grey bars). For E‐cadherin a significant reduction in expression levels was observed (Figure 24, grey bars compared to black bars).
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In summary, Adam19 and E‐cadherin exhibited strikingly altered gene expression levels in mutant compared to wildtype embryonic outflow tract tissue.
Figure 24: Analysis of important marker genes in the outflow tract region of mice with Sox4 and Sox11 deficiencies in cardiac neural crest. Expression levels were determined by quantitative RT‐PCR on RNA prepared from anterior heart pole tissue of wildtype (Wt, black bars) and Sox4Wnt1::Cre Sox11Wnt1::Cre (Dko, grey bars) embryos at 13.5 dpc for several genes as indicated below the bars. Transcript levels were normalized to Rpl8 levels in the respective samples. Differences between samples were statistically significant as indicated according to student´s t‐test (*, p 0.05; *** p 0.001).
3.1.9 Putative Sox4 and Sox11 binding sites in Adam19 and E‐cadherin promoter regions
To find out, whether Adam19 and E‐cadherin were direct target genes of Sox4 and Sox11 proteins, promoter sequences were analysed in the JASPAR database to find putative consensus sites for Sox transcription factors. For the Adam19 promoter region one putative Sox binding site was identified (Figure 25, A). The sequence of the putative Adam19 Sox binding site is shown in (Figure 25, A above the ellipse). For the E‐cadherin promoter region two putative Sox binding sites were identified (Figure 25, B). The sequences of the two putative E‐cadherin Sox binding sites are shown in Figure 25, B above the ellipses.
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Figure 25: Analysis of Adam19 and E‐cadherin promoter regions for potential Sox binding sites. Schematic representation of Adam19 and E‐cadherin promoter upstream regions. Location of the short (Adam19s, Ecad s) and the long (Adam19 l, Ecad l) promoter regions used for luciferase assays as well as positions of Sox binding sites (ellipses), transcriptional start site (arrows) and control regions (Adam19 Ctrl, Ecad Ctrl, black boxes) for ChIP are indicated.
3.1.10 Direct binding of Sox4 and Sox11 to newly identified Sox binding sites in Adam19 and E‐cadherin promoter regions
To analyse if the putative Sox binding sites in the Adam19 and E‐cadherin promoter regions were recognised by SoxC proteins, electrophoretic mobility shift assays (EMSA) were performed. SiteB from the Mpz (myelin protein zero) promoter as a known Sox binding site showed a high binding affinity to Sox4 from transfected HEK293 cells, while no binding was visible for extracts from mock transfected HEK293 cells or in the absence of protein extract (Figure 26, A). The newly identified Sox site in the Adam19 promoter showed also a high binding affinity to Sox4 expressed in HEK293 (Figure 26, A). Binding of Sox4 expressed in Neuro‐2a cells was also detected (Figure 26, B) although the bands representing the Sox4/DNA complex are weaker because of lower Sox4 expression in this cell type. To further
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prove that Sox4 was bound directly to the Adam19 promoter a mutation was introduced into the Sox binding site of the Adam19 promoter. Electrophoretic mobility shift analysis confirmed that this mutation abolished Sox4 binding (Adam19mut in Figure 26).
Figure 26: Analysis of potential Sox binding sites in the Adam19 promoter. Electrophoretic mobility shift assay with oligonucleotides encompassing the potential Sox binding sites from the Adam19 promoter and the known Sox binding site SiteB from the Mpz promoter. For the Adam19 site a wildtype and a mutant version were used. Oligonucleotides were incubated in the absence (‐) or presence of extracts from mock‐transfected HEK293 or Neuro‐2a cells (Ctrl) and HEK293 or Neuro‐2a cells expressing carboxyterminally truncated Sox4 Protein (Sox4) as indicated above the lanes.
For the putative Sox binding sites in the proximal E‐cadherin promoter region binding was observed as well. Here, two binding sites named Ecad1 and Ecad2 were identified. Ecad1, located more distally to the transcription start site showed only a weak binding to Sox4 when compared to SiteB (Figure 27). Ecad2, more proximally located, bound to Sox4 with a higher affinity than Ecad1 (Figure 27) despite the fact that it conformed less well to Sox consensus binding sites. After mutation of Ecad1 and Ecad2 binding sites, Sox4 binding was abolished (Figure 27). In general, binding of Sox4 to the Adam19 promoter region appeared stronger than to the E‐cadherin promoter region (compare Figure 26, A‐B to Figure 27).
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Figure 27: Analysis of potential Sox binding sites in the E‐cadherin promoter. Electrophoretic mobility shift assay with oligonucleotides encompassing the potential Sox binding sites from the E‐cadherin promoter and the known Sox binding site SiteB from the Mpz promoter. Two wildtype sites (Ecad1, Ecad2) and two mutant versions were used (Ecad1mut, Ecad2mut). Oligonucleotides were incubated in the absence (‐) or presence of extracts from mock‐transfected HEK293 cells (Ctrl) or HEK293 cells expressing carboxyterminally truncated Sox4 protein (Sox4) as indicated above the lanes.
3.1.11 Sox11 associates with endogenous Adam19 and E‐cadherin promoter region
Studies by chromatin immunoprecipitation demonstrated that Sox11 was able to associate with the endogenous Adam19 promoter region in HEK293 cells. This was shown by the significant enrichment of this region by PCR when chromatin was precipitated with antibodies directed against Sox11 (Figure 28, A grey bars) compared to precipitates with control immunoglobulin (Figure 28, A black bars). Enrichment could only be seen for the promoter regions and not for regions more distally located from the transcriptional start site of Adam19 (Figure 28, A). Enrichment could also be demonstrated for the E‐cadherin promoter when chromatin was precipitated with Sox11 antibodies (Figure 28, B). Again there was no enrichment for more distally located control regions (Figure 28). To confirm that the endogenous Adam19 as well as the E‐cadherin promoter are bound by Sox11, chromatin immunoprecipitation was additionally performed in Neuro‐2a cells.
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Figure 28: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions. Chromatin immunoprecipitation (ChIP) was performed on chromatin from untransfected (Mock) and Sox11 transfected HEK293 cells with antibodies directed against Sox11 and control IgGs. Quantitative PCRs were carried out on immunoprecipitated chromatin to determine the relative enrichment of proximal promoters and upstream control regions from Adam19 (A) and E‐cadherin (B) gene loci (Ctrl in A,B). Differences between samples in A and B were statistically significant as indicated according to Student´s t‐test (*, p 0.05; *** p 0.001).
Again enrichment could only be seen for the Adam19 promoter regions and not for regions more distally located in the gene locus (Figure 29). Enrichment could also be demonstrated with Sox11 antibodies for the E‐cadherin promoter (Figure 29, B), but not for the control region (Figure 29). To study, if Sox11 is able to associate with endogenous Adam19 and E‐cadherin promoter in vivo, whole hearts were dissected at 13.5 dpc from wildtype embryos and used for chromatin immunoprecipitation. Sox11 associated with endogenous Adam19 and E‐cadherin promoters. Enrichment could only be seen in quantitative PCRs for the promoter regions and not for more distal regions (Figure 30, A). To be even more specific in the choice of material, anterior heart poles of 13.5 dpc old wildtype embryos were also used for chromatin immunoprecipitation. The enrichment obtained for Adam19 and E‐cadherin promoter regions with Sox11 antibodies was higher than previously seen with whole hearts (Figure 30, compare A to B).
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Figure 29: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions. Chromatin immunoprecipitation (ChIP) were performed on chromatin from untransfected and Sox11 transfected Neuro‐2a cells with antibodies directed to Sox11 and control IgGs. Quantitative PCRs were carried out on immunoprecipitated chromatin to determine the relative enrichment of proximal promoters and upstream control regions from Adam19 (A) and E‐cadherin (B) gene loci (Ctrl A,B) in precipitate from Sox11 transfected (Sox11, grey bars) over Mock transfected cells (Mock, black bars). Differences between samples in A and B were statistically significant as indicated according to student´s t‐test (*, p 0.05).
Figure 30: Analysis of Sox11 binding to endogenous Adam19 and E‐cadherin promoter regions in whole hearts or the anterior heart pole of 13.5 dpc wildtype mice. Chromatin immunoprecipitation (ChIP) was performed on chromatin from hearts (A) or from dissected anterior heart poles of 13.5 dpc wildtype animals (B) with antibodies directed against Sox11 and control IgGs. Quantitative PCRs were carried out on immunoprecipitated chromatin to determine the relative enrichment of proximal promoters and upstream control regions for Adam19 (A) and E‐cadherin (B) gene loci (Ctrl) in the Sox11 precipitate (Sox11, grey bars).
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3.1.12 Sox4 and Sox11 activate Adam19 and E‐cadherin promoters
Since Sox4 and Sox11 bind directly to Adam19 and E‐cadherin promoter regions, it was investigated subsequently, whether both proteins could activate these promoters region in luciferase assays. Transient transfections were performed in Neuro‐2a cells with luciferase reporter plasmids under control of Adam19 or E‐cadherin promoters. For transfection regulatory sequences of Adam19 were cloned in front of luciferase either in a short 274 bp version (Figure 31, A, Adam19 s) or a long 2040 bp version (Figure 31, A, Adam19 l), which included additional upstream sequences. Adam19 plasmids were then co‐transfected with either empty pCMV5 plasmid or expression plasmids for Sox4 or Sox11.
Figure 31: Analysis of promoter activation by Sox4 and Sox11. Transient transfections were performed in Neuro‐2a cells with luciferase reporters under control of the Adam19 (A) or E‐cadherin (B) promoters either in a short version encompassing the proximal promoter (274 bp for Adam19 and 238 bp for E‐cadherin) or a longer version including additional upstream sequences (approximately 2040 bp for Adam19 and 1600 bp for E‐cadherin). The Adam19 and the E‐cadherin promoters were furthermore employed in wildtype version and as variants in which Sox binding site were inactivated (Adam19 sm, Ecad sm). Empty pCMV5 expression plasmids (‐) or expression plasmids for Sox4 and Sox11 were co‐transfected as indicated below the bars. Luciferase activity obtained for reporter plasmid in the absence of ectopic transcription factor was set to 1. Fold inductions in the presence of transcription factors are presented as mean SEM. Differences between samples were statistically significant as indicated according to Student’s t‐test (*, p 0.05; **, p 0.01; ***, p 0.001).
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Sox4 increased the activity of the long version of the Adam19 promoter approximately 5‐ fold, while Sox11 activated about 9‐fold (Figure 31, A). The short version of Adam19 showed even higher activation rates for Sox11 of about 14‐fold (Figure 31, A). To further prove that activation is a direct consequence of binding of Sox4 or Sox11, transfections were additionally performed with a luciferase promoter where the Sox binding site in the Adam19 promoter was inactivated. Sox4 and Sox11 dependent activation of the Adam19 promoter was strongly diminished, when the Sox binding site in the Adam19 promoter was mutated (Figure 31, A). In the second set of reporter gene transfections the E‐cadherin promoter was used in either a short version with 238 bp (Figure 31, B, Ecad s) or a long version with 1600 bp (Figure 31, B, Ecad l), which included additional upstream sequences. E‐cadherin reporter plasmids were then co‐transfected with either empty pCMV5 plasmid or expression plasmids for Sox4 or Sox11. Sox4 increased the activity of the long version of the E‐cadherin promoter approximately 3‐fold, while Sox11 increased the activity about 6‐fold (Figure 31, B). The short version of E‐cadherin showed similar activation rates as the long version (Figure 31, B). Activation of the short version of the E‐cadherin promoter by Sox4 and Sox11 was prevented when the two identified Sox binding sites were mutated (Figure 31). Moreover, Sox11 activated luciferase expression of all tested reporter gene constructs more efficiently than Sox4.
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3.2 Posttranslational modification of Sox4 and Sox11
3.2.1 Putative phosphorylation sites of Sox4 and Sox11
Protein function often depends on posttranslational modifications. One frequent type of posttranslational modification is the phosphorylation of proteins. Using a search tool (www.phosphosite.org) putative phosphorylation sites were identified for Sox4 and Sox11 (Figure 32, A‐B). Among them for Sox4, Y123, Y126 and S244 and for Sox11 Y113, Y116 and S244 were chosen for further analysis (Figure 32) (Ballif et al., 2004;Rikova et al., 2007).
Figure 32: Putative sites for phosphorylation of Sox4 and Sox11. (A) Schematic overview of Sox4 and the putative phosphorylation sites Y123, Y126 and S244. The two tyrosines Y113 and Y116 are located in the HMG domain. (B) Schematic overview of Sox11 and the putative phosphorylation sites Y113, Y116 and S244. Both tyrosines Y123 and Y126 were located in the HMG domain.
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3.2.2 Phosphorylation analysis of Sox4 and Sox11
For a first analysis, protein extracts from HEK293 cells transfected with Sox4 or Sox11 expression plasmids were treated with ‐Protein Phosphatase and analysed side by side with untreated extracts by western blot. After phosphatase treatment molecular weights of dephosphorylated proteins are usually lowered. Western blot analysis of treated versus untreated cell extracts indicated that Sox4 and Sox11 may be phosphorylated (Figure 33). Since Sox4 as well as Sox11 were possibly phosphorylated, mutants of the putative phosphorylation sites were generated. For Sox11, the tyrosines Y113 and Y116 were changed into phenylalanine. This prevents phosphorylation of the corresponding residues (Sox11‐Tyr). Additionally, the serine S244 was exchanged to an alanine or to glutamic acid residue. Exchanging serine to alanine again prevents phosphorylation because the OH‐group is missing that serves as an acceptor for the phosphate group (Sox11‐S244A). Exchanging serine to glutamic acid in contrast mimics a state of permanent phosphorylation (Sox11‐ S244E). Some of the mutants were furthermore not only introduced into the full length Sox11 protein, but also into a C‐terminally truncated version.
Figure 33: ‐Protein Phosphatase treatment of overexpressed Sox4 and Sox11 protein. Extracts of HEK293 cells that overexpress Sox4 and Sox11 protein underwent ‐Protein Phosphatase treatment and were size fractionated by SDS‐PAGE before analysed by western blot using an antibody directed against the HMG‐box of Sox4 and Sox11. Blots show either Sox4 or Sox11 with (+) or without (‐) ‐Protein Phosphatase treatment.
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Overexpression of mutated and truncated versions of Sox11 in HEK293 cells, treatment of the resulting extracts with ‐Protein Phosphatase followed by subsequent SDS‐PAGE and western blot analysis revealed the effects of the introduced mutations. As a positive control extracts from HEK293 cells over expressing wildtype Sox11 were used. Here, Sox11 from the untreated extract showed a higher molecular weight than Sox11 from treated probes (Figure 34, A). Phosphatase treatment of Sox11‐Tyr, Sox11‐S244A and Sox11‐S244E showed the same effect (Figure 34; A). This argues that none of the mutated residues is the sole target for phosphorylation in Sox11. In contrast, no shift in molecular weight was seen after phosphatase treatment of cell extracts for Sox11‐C and Sox11‐C‐Tyr (Figure 34, A). This implies that the shortened protein did not contain phosphorylation sites.
Figure 34: Western blot analysis of ‐Protein Phosphatase treated or untreated extracts from HEK293 cells over expressing Sox4 or Sox11 in wildtype or mutated versions. (A) The following Sox11 proteins were overexpressed in HEK293 cells: wildtype, Sox11‐Tyr, Sox11‐S244A, Sox11‐S244E, Sox11‐7aaHMG, Sox11‐VQQ, Sox11‐C and Sox11‐C‐Tyr. (B) The following Sox4 proteins were overexpressed in HEK293 cells: wildtype, Sox4‐Tyr, Sox4‐S244A and Sox4‐S244E. Proteins were treated with ‐Protein Phosphatase (+) or left untreated (‐), size fractioned by SDS‐PAGE and analysed by western blot using an antibody directed against the SoxC HMG‐box domain.
The corresponding mutations were also introduced into the Sox4 protein. Residues Y123 and Y126 were changed into phenylalanine (Y123F and Y126F) and serine S244 was changed to either alanine (S244A) or glutamic acid (S244E). After mutation of the different putative phosphorylation sites ‐Protein Phosphatase treatment was performed on extracts from over‐expressing cells. Following SDS‐PAGE and western blot it was clearly visible that all Sox4
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mutants still exhibited size shifts arguing that Sox4 is phosphorylated, most likely however at other residues than the mutated ones or at multiple residues (Figure 34, B).
Sox4 and Sox11 protein variants with additional changes were generated. These were in regions conserved among SoxC proteins and possibly necessary for proper protein function. For Sox11, the first three amino acids (VQQ) following the methionine were deleted from the protein (Sox11‐VQQ). Additionally, seven amino acids (PDWCKTA) inside of the HMG‐box of SoxC proteins were deleted (Sox11‐7aaHMG). Also a truncated version was generated where the C‐terminal part immediately after the HMG‐box was deleted (Sox11‐C). Phosphatase treatment of extracts containing Sox11‐Tyr, Sox11‐S244A and Sox11‐S244E as well as Sox11‐VQQ and Sox11‐7aaHMG did not result in any changes that distinguished the mutant proteins from wild‐type full length or truncated proteins after comparable phosphatase treatment (Figure 34, A).
3.2.3 Analysis of activation potential of putative SoxC phosphorylation mutants
Mutated Sox4 and Sox11 versions were also tested for their ability to activate a Sox‐ responsive reporter plasmid in luciferase assays (Hoser et al., 2008;Kuhlbrodt et al., 1998). Transient transfections were performed in HEK293 cells. For Sox4 no changes in activation levels were observed when mutant versions were used instead of wildtype Sox4 (data not shown). For Sox11 most mutants behaved in a very similar manner as the wildtype. Sox11‐Tyr represented the only exception, as it exhibited significant lower activation levels (Figure 35).
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Figure 35: Transient transfection of HEK293 cells with wildtype or mutated Sox11. In HEK293 cells a Sox responsive reporter plasmid (pTATA‐3SX‐luc) was co‐transfected with either empty pCMV5‐HA tagged vector, wildtype Sox11 or various Sox11 mutants as indicated below the bars. Luciferase activity obtained for reporter plasmid in the absence of ectopic transcription factor was set to 1. Fold inductions in the presence of transcription factors are presented as mean SEM. Differences between samples were statistically significant as indicated according to Student’s t‐test (***, p 0.001).
3.2.4 Analysis of binding ability of putative SoxC phosphorylation mutants
To examine the binding ability of mutated SoxC proteins electrophoretic mobility shift assays (EMSA) were performed. The wildtype Sox4 and Sox11 protein bound to the Sox binding site MW‐1 previously identified by Wetering (van de Wetering et al., 1993). Compared to wildtype, Sox4‐Tyr and Sox11‐Tyr mutants exhibited a dramatically decreased binding ability. In contrast, deletion of the first seven amino acids in the HMG‐box of Sox4 (Sox4‐7aaHMG) had no impact on the binding ability to the MW‐1 probe, whereas the same mutation in the Sox11 protein (Sox11‐7aaHMG) abolished binding to the MW‐1 probe (Figure 36).
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Figure 36: EMSA with oligonucleotides encompassing the Sox binding site MW‐1 (van de Wetering et al., 1993). Oligonucleotides were incubated in the absence (‐) or presence of extracts from mock‐transfected HEK293 cells (Ctrl) or HEK293 cells expressing wildtype full length or mutated Sox4 or Sox11 proteins as indicated above the lanes.
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4. Discussion
4.1 The role of SoxC proteins in outflow tract development
4.1.1 The role of Sox4 in outflow tract development
Previous studies showed that Sox4 is essential to form a functioning heart (Schilham et al., 1996;Ya et al., 1998b). Knockout animals displayed CT and mice died at embryonic day 14 due to circulatory failure. Heart development is a highly complex process and a variety of cell types have to participate in forming the different structures. One cell type which is needed for proper heart formation is the cardiac neural crest. These cells mainly contribute to outflow tract development (Kirby and Hutson, 2010). To analyse the contribution of cardiac neural crest cells to the severe outflow tract phenotype seen in the constitutive Sox4 knockout embryos, we decided to delete Sox4 specifically in this cell lineage. Hence, the expectation was to find an outflow tract malformation in these animals. Surprisingly, no phenotype was observed in animals with an ablation of Sox4 in the cardiac neural crest cell population. The constitutive Sox4 knockout mice displayed severe outflow tract malformations, ventricular septum as well as valve defects. In contrast to that, selective ablation of Sox4 in cardiac neural crest cells failed to show any outflow tract malformation. An explanation for this unexpected finding might be that deletion of Sox4 in the cardiac neural crest cells occurred too late or was inefficient in these mutants. A previous study showed, that deletion in neural crest cells via Wnt1::Cre might only be accomplished around the stage of emigration from the neural tube (Olaopa et al., 2011). If Sox4 is necessary in cardiac neural crest cells in the premigratory stage deletion by Wnt1::Cre might happen too late. To hit the cardiac neural crest cells in the premigratory stage Sox4 was deleted using an Ap2a::Cre (Macatee et al., 2003;Olaopa et al., 2011). However, these animals did not show any outflow tract malformations either. Because the absence of a phenotype could not be attributed to an inefficient deletion of Sox4 in the cardiac neural crest cell lineage, Sox4 may function in cells other than cardiac neural cells during outflow tract development. The two main cell types that contribute to outflow tract development apart from cardiac neural crest
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cells are mesodermal cells derived from the secondary heart field and endothelial cells. So next, Sox4 was deleted in the mesodermal cell lineage using Nkx2.5::Cre. Again no malformation of the outflow tract region was detectable in animals with Sox4 deletion in the mesodermal cell lineage. Furthermore, animals with a deletion of Sox4 in endothelial cells via Tie2::Cre did not show a heart phenotype either. Since Sox4 ablation in none of the analysed cell lineages showed defective outflow tract development, Sox4 was deleted simultaneously in the cardiac neural crest cell population and mesodermal cell lineage. Astonishingly, animals still did not exhibit altered outflow tract development. To check if the floxed allele in Sox4fl animals is working correctly, these animals were crossbred with animals carrying a Sox2::Cre transgene. Sox2::Cre deletes Sox4 already in the epiblast. All obtained Sox4Sox2::Cre animals showed outflow tract malformations corresponding to malformations found in constitutive Sox4 knockout mice. This means the floxed allele in Sox4fl animals is working correctly. Our results indicate that Sox4 is not essentially required in the analysed cell lineages for proper outflow tract development. However it is possible that Sox4 is required in cells that provide fundamental cues for cardiac neural crest cells or cells of the secondary heart field that were not affected by our deletion strategy. This appears to be the most plausible explanation for the discrepancy between the phenotype seen in the constitutive and the conditional Sox4 deficient animals. Pharyngeal endoderm cells represent such a population as these cells have been shown to have an impact on outflow tract development and septation (Goddeeris et al., 2008;Park et al., 2006).
4.1.2 The role of Sox11 in outflow tract development
For Sox11 it was reported that constitutive knockout mice display severe outflow tract malformations ranging from DORV to CT in combination with VSD (Sock et al., 2004). Sox11 deficient animals die shortly after birth due to congenital cyanosis. In our study Sox11 was deleted in the cardiac neural crest cell lineage first. Here only 8% of all examined mutants displayed DORV. Due to the fact, that all constitutive Sox11 knockout embryos showed an outflow tract defect while only a small percentage of conditional Sox11
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knockout embryos showed this defect, the phenotype of the constitutive Sox11 mutants cannot be explained by Sox11 function in the cardiac neural crest cell lineage alone. This pointed to a role of Sox11 during outflow tract development in a cell lineage other than cardiac neural crest cells. It was, for instance, interesting to see whether deletion of Sox11 in the mesodermal cell lineage resembles the outflow tract phenotype of the constitutive knockout of Sox11. For this approach, animals with a deletion of Sox11 in mesodermal cell lineages were generated. Mesodermal knockout of Sox11 led to a frequency of outflow tract defects seen in 50% of all mutants. Interestingly, simultaneous Sox11 ablation in cardiac neural crest and mesodermal cells worsens the phenotype without increasing the penetrance. Whereas animals with a deletion of Sox11 in either cardiac neural crest or mesodermal cells all displayed DORV, some animals with simultaneous deletion of Sox11 in neural crest and mesodermal cells displayed the more severe phenotype of CT. The increasing severity of outflow tract defects seen in these mutants might be due to Sox11 ‐ induced signalling between the two cell lineages. Additional analyses after Sox11 ablation in the endothelial cell lineage did not reveal any outflow tract defects. This result indicates that Sox11 is not required in endothelial cells for proper outflow tract development. Since deletion of Sox11 in cardiac neural crest as well as in mesodermal cells leads to outflow tract defects while Sox4 deletion seems to have no impact on outflow tract development in these cell lineages one can conclude that Sox11 is more important in cardiac neural crest and mesodermal cells than Sox4 for outflow tract development.
4.1.3 Redundant function of Sox4 and Sox11 in outflow tract development
SoxC proteins are often co‐expressed during development and share many structural properties (Bhattaram et al., 2010;Hoser et al., 2008;Kuhlbrodt et al., 1998). A redundant function of SoxC proteins was already shown for several tissues and cell types (Bergsland et al., 2006;Bhattaram et al., 2010;Mu et al., 2012;Thein et al., 2010). So the absence of outflow tract defects in conditional Sox4 embryos might be due to the redundant function of SoxC proteins. To examine if Sox11 might be able to compensate for the loss of Sox4 in these mutants, both genes where ablated in the same cell type. Interestingly, combined deletion
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of Sox4 and Sox11 in cardiac neural crest cells led to a fully penetrant outflow tract defect in all obtained double knockout animals, ranging from DORV to CT, whereas only 8% of the mutants show an outflow tract phenotype after deletion of Sox11 alone. Unfortunately, combined deletion of Sox4 and Sox11 in mesodermal cell lineage could not be examined due to early embryonic death. Mutant embryos only survived up to embryonic day 10.5, probably because of early developmental defects in multiple other mesodermal lineages (Bhattaram et al., 2010).
The analysis of compound mutants shows that Sox11 could compensate for the loss of Sox4 function in the single knockouts while Sox4 is not capable to fully restore Sox11 function in cardiac neural crest lineage. This indicates that Sox11 is either functionally more important for cardiac neural crest development or expression is quantitatively higher than that of Sox4. At the same time, the results prove a redundant function of Sox4 and Sox11 in cardiac neural crest cells since combined loss of the two genes in this cell type increases penetrance as well as severity of the phenotype. Redundant function of SoxC proteins was found previously in the developing nervous system and many other organs (Bhattaram et al., 2010;Thein et al., 2010).
4.1.4 The role of Sox12 in outflow tract development
Considering that SoxC proteins are often co‐expressed in different tissues and cells (Bhattaram et al., 2010;Hoser et al., 2008;Kuhlbrodt et al., 1998) and are able to compensate for the loss of each other, Sox12 as the third member of the SoxC protein family also might be able to influence outflow tract development. A previous study showed that animals with a constitutive Sox12 deletion develop normally and that Sox12 expression can be found in heart tissue (Bhattaram et al., 2010;Hoser et al., 2008). Bhattharam et al. demonstrated that deletion of one allele of Sox4 and Sox11 with additional constitutive Sox12 deletion worsens the phenotype seen in outflow tract development from DORV to a CT (Bhattaram et al., 2010). This points to a role of Sox12 in outflow tract development. Although we did not find an upregulation of Sox12 expression in RT‐PCRs of animals with a combined deletion of Sox4
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and Sox11 in neural crest cells, it might be possible that Sox12 is supporting Sox11 function in Sox4 deficient cardiac neural crest cells. However, Sox12 expression alone is not capable to compensate for the loss of both SoxC proteins, since combined deletion of Sox4 and Sox11 in cardiac neural crest cells always led to morphological alterations of the outflow tract during development.
4.2 Participation of other Sox proteins in heart development
Besides Sox proteins of SoxC other Sox proteins take part in heart development, e.g. members of SoxE and F. Previous studies showed that the SoxE proteins Sox8, 9, and 10 are expressed during heart development in chicken (Montero et al., 2002). Sox9 is expressed in cushion tissue mesenchyme of the outflow tract and the endocardial cushions. Sox8 transcripts were located in the subendothelial mesenchyme, while Sox9 and a small amount of Sox10 transcripts could be detected in the subendothelial tissue of the differentiating heart septa and the leaflets of the atrioventricular valves (Montero et al., 2002). Montero et al. suggested, that SoxE genes might function as regulators of connective tissue differentiation (Montero et al., 2002) prompted by the finding that Sox9 is a direct regulator of type II Collagen and Aggrecan (Lefebvre et al., 1998). Mice with constitutive Sox9 deletion die between embryonic day 11.5 and 12.5 and exhibit hypoplastic endocardial cushions (Akiyama et al., 2004). Conditional deletion strategies for Sox9 showed that it is required in the early stage for expansion and diversification of the valve precursor cell pool following EMT while at later stages it is required for differentiation, patterning and homeostasis of mature valve structures (Lincoln et al., 2007). The SoxF proteins include the members Sox7, 17 and 18. Sox17 is essential in embryonic stem cells for the specification of cardiac mesoderm (Liu et al., 2007). The same group showed that Hhex (hematopoietically‐expressed homeobox protein HHEX) and Cer1 (Ceberus) mediate the Sox17 effects on cardiac mesoderm formation in embryonic stem cells (Liu et al., 2014). In mice the constitutive knock out of Sox17 led to an aberrant heart looping and enlarged cardinal veins. Additionally, a mild malformation of the anterior dorsal aorta was observed (Sakamoto et al., 2007). Double knockout animals for Sox17 and Sox18
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displayed more severe defects. Defects were primarily observed at sites with relatively low Sox7 expression and not at sites with high Sox7 expression suggesting that all three SoxF members perform largely redundant functions (Sakamoto et al., 2007). SoxF transcription factors furthermore cooperate with Notch during acquisition of arterial identity (Sacilotto et al., 2013). How SoxC protein function relates to other Sox proteins during heart development has not been studied in detail.
4.3 The role of SoxC proteins in other tissues
In other tissues than the heart SoxC proteins are equally essential for development. During development of the central nervous system (CNS), for instance, Sox4 and Sox11 are initially expressed in a similar but not identical pattern in early differentiating neurons. Their late expression differed more strongly (Cheung et al., 2000;Dy et al., 2008;Hargrave et al., 1997;Hoser et al., 2008;Schepers et al., 2002). Sox4 and Sox11 are upregulated in all neuronal precursor cells when they start to migrate and leave the ventricular zone of the neural tube. As soon as the precursors begin to differentiate into mature neurons they downregulate Sox4 and Sox11 (Cheung et al., 2000;Mu et al., 2012). SoxC proteins are essential for neuronal survival as apoptosis is highly increased in their absence (Thein et al., 2010). In cortical spiral neurons, Sox4 and Sox11 additionally control identity and connectivity (Shim et al., 2012;Wang et al., 2013). In the adult, Sox11 regulates hippocampal neurogenesis (Wang et al., 2013). Furthermore, Sox11 is also necessary for the development of neurons in the peripheral nervous system including sensory and symphathetic neurons (Lin et al., 2011;Potzner et al., 2010;Thein et al., 2010;Wang et al., 2013). Another precursor cell population that depends on SoxC proteins is the mesodermal cell population. In many of these progenitors, SoxC proteins act redundantly (Bhattaram et al., 2010).
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4.4 Proliferation, migration and apoptosis of neural crest cells are not affected by SoxC deletion
To examine if proliferation, migration or apoptosis are affected by SoxC ablation in cardiac neural crest cells a Wnt1::Cre dependent EYFP reporter construct was introduced to follow cardiac neural crest cells via its EYFP expression. In this study we compared two different stages of heart development and analysed Sox4 and Sox11 function. Comparing SoxC deficient cardiac neural crest cells to wildtype cells at early or late stages (10.5 or 13.5 dpc) revealed no dramatic differences. We demonstrated that Sox4 and Sox11 deficient cardiac neural crest cells emigrate normally from the neural tube and enter the outflow tract region. Also, the rates of proliferation and apoptosis were unchanged in SoxC deficient cardiac neural crest cells compared to wildtype cells. Furthermore, there were no abnormalities in the migratory phase detected so that normal numbers of cardiac neural crest cells reached the outflow tract region at appropriate times. These results show that SoxC transcription factors are not needed for generation and multiplication of cardiac neural crest cells. Also SoxC transcription factors are not required for their maintenance and migration.
4.5 Morphological and cytoskeletal alterations in SoxC deficient cardiac neural crest cells
A detailed analysis of the morphology of cardiac neural crest cells during outflow tract development revealed dramatic differences between wildtype cells and cells where Sox4 and Sox11 were deleted. Previous studies showed, that normally cardiac neural crest cells migrate in chain‐like formations (Teddy and Kulesa, 2004;Young et al., 2004). These cells usually contact each other via filopodia‐like protrusions. However, cardiac neural crest cells with a deletion of Sox4 and Sox11 maintained a more compact structure with less filopodia‐ like protrusions. As a consequence cells no longer migrate in chain‐like formations and were orientated more randomly to each other as in wildtype. It is likely that SoxC proteins are needed for maintenance of such protrusions and therefore for stable cell‐cell contacts. Whether there is an independent role for SoxC proteins in cell orientation is difficult to say,
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since random localisation might be due to the loss of cell‐cell contacts in the absence of filopodia like protrusions.
Since morphology of cardiac neural crest cells with ablated Sox4 and Sox11 was altered, extracellular matrix and cytoskeletal organisation markers were analysed. On protein level cytoskeletal markers, e. g. phalloidin staining for actin filaments, revealed that Sox4 and Sox11 deficient cardiac neural crest cells lost their migratory properties earlier than wildtype cells which may correlate with changes in migratory capability. Premature loss of the migratory capability may then go along with precocious expression of the proteoglycan NG2 that was demonstrated in this study. Such misregulation might inhibit the function of cardiac neural crest cells, as misregulated cells might fail to remodel the outflow tract properly and might not participate in formation of the aorticopulmonary septum anymore. Extracellular matrix and cytoskeletal organisation proteins have previously been shown to be essential for proper outflow tract development. Two of these proteins are Cx40 and Cx43 (gap‐junction proteins connexin 40 and 43) both belonging to the connexin family alpha type (group II) subfamily. Gap‐junction molecules allow cardiac neural crest cells to interact with each other during their migration (Snider et al., 2007). In mice, ablation of Cx40 and Cx43 resulted in heart defects (Huang et al., 1998;Li et al., 2002;Simon et al., 2004;Ya et al., 1998a). In outflow tract regions of mice with Sox4 and Sox11 deletion in neural crest cells no change of mRNA level for Cx40 expression but a significant increase for Cx43 has been detected by quantitative RT‐PCR. Cx40 is selectively expressed in atrial cells and was therefore not affected by SoxC ablation. This might explain the unchanged expression levels detected in the outflow tract region. Even though Cx43 is expressed abundantly in the heart, outflow tract malformation is restricted to the junction between right ventricle and outflow tract after its deletion (Ya et al., 1998a). The upregulation of Cx43 expression in the outflow tract region after SoxC ablation in cardiac neural crest cells might be caused by a direct or indirect effect of missing SoxC in cardiac neural crest cells. These findings give further evidence for a misregulation of markers involved in extracellular matrix and cytoskeletal organisation processes due to a lack of SoxC proteins in neural crest cells.
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4.6 Altered transcription factor expression in outflow tract regions containing SoxC deficient cardiac neural crest cells
Transcription factors necessary for proper outflow tract formation have been identified in recent years (Kodo and Yamagishi, 2011). For Hand2 and Foxc2 it was already shown that they are needed for proper outflow tract development since mutant embryos always show outflow tract malformations when Hand2 or Foxc2 were absent (Holler et al., 2010;Seo and Kume, 2006). Holler et al. show that missing Hand2 in cardiac neural crest cells leads to a downregulation of Sox11 and Adam19 (Holler et al., 2010). In our study we checked for Hand2 expression levels in outflow tracts with a Sox4 and Sox11 deletion in cardiac neural crest cells and found that it was slightly decreased compared to wildtype outflow tract regions. Therefore, Hand2 seems to act mainly upstream of SoxC transcription factors. In addition, it is possible that Hand2 expression needs SoxC for a positive feedback loop promoting or maintaining its expression. Expression of Foxc2 is not changed following SoxC ablation in cardiac neural crest cells. Korsch et al. suggested the Sox10 gene to be a candidate for influencing the development of the autonomic control of the heart which plays an important role in the generation of complex heart rate dynamics that enable an organism to adapt to stress (Korsch et al., 2001). Hence, Sox10 might have an impact on outflow tract and especially cardiac neural crest cell development. Therefore, Sox10 expression levels were analysed in outflow tract tissue of Sox4 and Sox11 double‐deficient cardiac neural crest compared to wildtype cells. In our study Sox10 was increased in expression rates in SoxC deficient cardiac neural crest cells compared to wildtype. Whether increased Sox10 expression is a direct consequence of Sox4 and Sox11 deletion in cardiac neural crest cells needs to be examined in further studies.
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4.7 Adam19: a direct target of SoxC proteins
Members of the Adam (a disintegrin and metalloprotease domain) family are membrane anchored proteins. They often function as active metalloproteinases and are involved in cell‐ cell and cell‐matrix interactions, cell migration, adhesion and signal transduction. Previous studies showed that combined deletion of Adam9 and Adam19 leads to heart defects (Horiuchi et al., 2005). Moreover it was shown for Adam19 that it takes part in outflow tract formation and remodelling (Komatsu et al., 2007;Kurohara et al., 2004;Zhou et al., 2004). Interestingly, both Adam gene family members were decreased in their expression levels in outflow tracts of embryos with SoxC deletion in cardiac neural crest cells. While Adam19 was decreased dramatically, Adam9 was mildly but still significantly decreased in outflow tract regions of mutant animals. The drastic decrease in Adam19 expression levels in outflow tract regions of animals with a combined deletion of Sox4 and Sox11 in the cardiac neural crest cells pointed to a direct effector‐target gene relationship between SoxC proteins A A and Adam19. Binding of Sox proteins to DNA occurs via the consensus sequence 5’‐ /T /T A CAA /T G‐3’ (Harley et al., 1994;Weiss, 2001). A potential binding sequence for SoxC proteins was found in the Adam19 promoter. In this study we demonstrated that Sox4 is able to bind to the Adam19 promoter in electrophoretic mobility shift assays. Furthermore, mutation of the Sox binding site abolished binding. We also showed that Sox11 is able to bind to the endogenous Adam19 promoter in chromatin immunoprecipitations. Luciferase assays with either a short or a long form of the Adam19 promoter, including the Sox binding sequence, were performed. Sox4 and Sox11 were able to activate the short as well as the long Adam19 promoter region. Sox4 activation in general was weaker compared to Sox11 activation. Previous studies already showed that Sox11 activation efficiencies with other target promoters are higher compared to Sox4 activation efficiencies (Dy et al., 2008;Hoser et al., 2008). Furthermore, the short form comprising the 274 bp proximal promoter region of Adam19 was activated to a higher extent than the 2040 bp version including additional upstream regions of the Adam19 promoter. Lower activation rates of the long Adam19 promoter region may result from repressing sequences in the upstream sequences which might interfere with SoxC binding or inhibit Adam19 transcription otherwise. To further prove direct activation of the proximal Adam19 promoter by SoxC, the Adam19 binding site was mutated in the context of the promoter. This proximal promoter
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region with mutated SoxC binding site showed diminished activation efficiencies for both SoxC proteins.
4.8 Altered EMT and MET marker gene expression in outflow tract regions containing SoxC deficient cardiac neural crest cells
Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) are essential to form the endocardial cushion mesenchyme and the future aorticopulmonary septum (Person et al., 2005). Therefore, marker genes involved in EMT or MET such as Snail1 and Snail2 as well as Twist1 and E‐cadherin were analysed. Snail1 and Snail2 are required for EMT in cardiac cushion morphogenesis whereas Twist1 is needed in cardiac neural crest cells for repressing neuronal and for controlling ectodermal versus mesodermal cell fate (Luo et al., 2001;Niessen et al., 2008;Vincentz et al., 2008;Vincentz et al., 2013). In our study, Twist1 was not changed in expression levels in mutant compared to wildtype outflow tracts. However, expression of Snail1 and Snail2 was significantly decreased in outflow tract tissue containing a SoxC ablation in cardiac neural crest cells. Hence, missing Sox4 and Sox11 expression in these cells leads to a downregulation of marker genes essential for EMT or MET. Luo et al. suggested that cadherin adhesion is essential for cell survival and for normal heart development. They also showed that E‐cadherin can functionally substitute for N‐cadherin during cardiogenesis suggesting a critical role for cadherin‐mediated cell‐cell adhesion, but not for cadherin family member‐specific signalling, at the looping stage of heart development (Luo et al., 2001). Therefore, E‐cadherin was examined as a marker for EMT and cell‐cell adhesion. We could show that the expression of E‐cadherin is dramatically reduced in Sox4 and Sox11 double‐deficient embryonic outflow tract tissue compared to wildtype. Therefore, we further focused on E‐cadherin.
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4.9 E‐cadherin: a direct target of SoxC proteins
The fact that E‐cadherin was strongly reduced in its expression level led to the assumption that there might be a direct influence of Sox4 and/or Sox11 on E‐cadherin expression in cardiac neural crest cells. Two potential binding sequences for SoxC proteins were found in the E‐cadherin promoter. In this study we demonstrated that Sox4 is able to bind to both Sox binding sites predicted in the E‐cadherin promoter in electrophoretic mobility shift assays. However, binding efficiency of Sox4 to E‐cadherin site 1 is lower than to E‐cadherin site 2. In general both Sox binding sites found in the E‐cadherin promoter region were bound by Sox4 with lower efficiencies than the one within the Adam19 promoter. Furthermore, mutation of both binding sites abolished binding of Sox4 to the E‐cadherin promoter. We also showed that Sox11 is able to bind to the endogenous E‐cadherin promoter in chromatin immunoprecipitations. Therefore, SoxC proteins are able to directly bind the E‐cadherin promoter in vivo. In reporter assays with either a short 238 bp or a long 1600 bp form of the E‐cadherin promoter in front of a luciferase gene, Sox4 and Sox11 were able to activate gene expression. However, compared to activation levels obtained with the Adam19 promoter region the activation rates were lower. In addition, Sox4 activation was less compared to Sox11 activation as seen for Adam19. To further prove activation of the proximal E‐cadherin promoter region via SoxC proteins, binding sites were mutated. The proximal promoter with mutated SoxC binding sites showed diminished activation efficiencies for both SoxC proteins. E‐cadherin is essential for cell‐cell adhesion and its downregulation is needed under physiological conditions for EMT and subsequent migration of mesenchymal cells (Kirby and Hutson, 2010). In the outflow tract of mutants with SoxC ablation in cardiac neural crest cells, E‐cadherin expression is dramatically downregulated. Additionally, for outflow tract development MET is needed after migration for the cells to form an organised epithelium.
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4.10 Relevance in disease
Congenital heart defects represent the most common type of birth defect in humans and manifest in 10 out of 1000 births. Three out of 10 cases require early postnatal intervention by surgery (Dolk et al., 2011;Hoffman and Kaplan, 2002). It is estimated that approximately 30% of the congenital heart defects are caused by outflow tract defects and abnormalities of the great arteries that exit the heart (Rosamond et al., 2007). Formation of the outflow tract results from coordinated development of cells from neural crest and mesodermal origin and it depends on the activity of many different transcription factors, including Pax3 and AP2 as well as the two Hand proteins and several Gata, Nkx, Fox and Sox proteins (Brewer et al., 2002;Conway et al., 2000;Seo et al. 2006;Holler et al. 2010). These proteins represent constituents of the transcriptional regulatory network in cardiac neural crest or second heart field derived cells that is active during outflow tract development, and their mutation may be causative for outflow tract defects. However, only few of them have been analysed with regards to their exact expression pattern and their relevance for human disease. In our study we analysed the contribution of the transcription factors Sox4 and Sox11 to the outflow tract development and formation. We could show that Sox11 is necessary for proper development of mesodermal and cardiac neural crest cells, because deletion of Sox11 in either one or both cell populations led to outflow tract defects. Furthermore, we showed that Sox4 supports Sox11 in its function. Both high‐mobility group transcription factors become essential after migration of the neural crest cells into the outflow tract. They are needed for proper differentiation and interaction of neural crest cells. In addition, they are required for interaction with the surrounding cells through regulation of cytoskeletal, cell adhesion and extracellular matrix molecules. Therefore, Sox4 and Sox11 have various functions in different cell types during outflow tract development and formation. These findings might help to understand the basis of congenital heart defects on a molecular level.
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DISCUSSION
4.11 Outlook
SoxC proteins seem to influence several genes during differentiation of cardiac neural crest cells. Besides, they seem to determine the interaction between cardiac neural crest cells with each other and with the surrounding cells. In case of heart development, especially the development and formation of the outflow tract, SoxC proteins in cardiac neural crest cells emerge later than most of the other already identified transcription factors. In further experiments it should be analysed how Sox4 and Sox11 are activated by other signals or transcription factors. Additionally, it should be analysed if SoxC proteins interact with transcription factors that are known to be important for proper outflow tract formation and development. Furthermore, the role of SoxC proteins in cell populations other than the three main contributors to heart development, e.g. the pharyngeal arch mesenchyme should be examined further since other cell populations may interact with migrating neural crest cells on their way into the outflow tract. These cell populations were not affected by our deletion strategy but may contribute to proper migration of neural crest cells and therefore play important roles in proper outflow tract formation. Additionally, a genome‐wide analysis and identification of SoxC target genes in mesodermal cells might provide new insides to the importance of SoxC on a molecular level in this cell population. Moreover, the same approach could be used for analysing SoxC target genes in neural crest cells. Comparing the results from the analysis of newly identified target genes of SoxC in mesodermal and neural crest cells might help to understand the basis of SoxC function in these cell populations. Also, Sox12 as the third member of SoxC transcription factors might play a role in proper heart and outflow tract formation, respectively. Within SoxC redundancy was shown previously. Sox12 was not examined in this study. Therefore, further studies should examine if and how Sox12 contributes to outflow tract development and formation.
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5. Materials & Methods
5.1 Materials
5.1.1 Organism
5.1.1.1 Bacterial cultures
Bacterial cultures for plasmid amplification were performed in Escherichia coli XL1 Blue (recA1, endA1, gyrA96, thi‐1, hsdR17, supE44, relA1, lac, [F’ proAB lacIq Z_M15 Tn10 (Tet r)]) from Stratagene (La Jolla, USA).
5.1.1.2 Cell lines
Transformed human embryonic kidney cells (HEK293) and Neuro‐2a cells (murine Neuroblastoma cells) derived from a spontaneous tumour in an albino strain A mouse from the American type culture collection (ATCC) were used for this work.
5.1.1.3 Mouse lines
Following genetically modified mouse lines were used: Sox4fl/fl (Penzo‐Mendez et al., 2007), Sox11fl/fl (Bhattaram et al., 2010), Wnt1::Cre (Danielian et al., 1998), Nkx2.5::Cre (Stanley et al., 2002), Ap2::Cre (Macatee et al., 2003), Tie2::Cre (Kisanuki et al., 2001), Rosa26stopflox‐EYFP (Srinivas et al., 2001), Sox2::Cre (Hayashi et al., 2002).
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5.1.2 Chemicals and general reagents
Chemicals, solutions, salts and general reagents were purchased from Carl Roth (Karlsruhe), Merck (Darmstadt) and Sigma (Munich) except when specified otherwise.
Culture media, buffers and cell culture material were purchased from Gibco/BRL (Eggenstein), Invitrogen (Karlsruhe), Serva (Heidelberg), Sarstedt (Nümbrecht), Renner (Darmstadt) and TPP (Trasadingen, Schweiz). Enzymes were purchased from Gibco/BRL (Eggenstein), MBI Fermentas (St. Leon‐Roth), New England Biolabs (Frankfurt) or Roche Diagnostics (Mannheim).
Radioactive chemicals were purchased from Hartmann Analytic GmbH (Braunschweig).
5.1.3 Buffers and solutions
Except specified otherwise, reagent‐grade water from a MilliQ deionizator (Millipore, Eschborn) was used for buffers, solutions and dilutions. For PCR reactions and the isolation of RNA nuclease‐free, DEPC treated, sterile filtered and autoclaved water obtained from Carl Roth (Karlsruhe) was used.
Cell lysis buffer 10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA before use add: DTT to a final conc. of 2 mM, leupeptin and aprotinin to a final conc. of 10 µg/ml
DNA loading buffer (10x) 50 % TE, 50 % glycerol, 0.05 % xylene cyanol, 0.05 % brom‐ phenol blue
ECL solution A 0.025 % luminal, 0.1 M Tris, pH 8.6
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ECL solution B 0.11 % p‐hydroxy‐cumaric acid in DMSO
ECL developer reagent 3 ml solution A, 60 µl solution B, 3 ml PBS, 1.8 µl H2O2 (30%)
Luciferase lysis buffer 88 mM Tris, pH 7.8, 88 mM MES, pH 7.8, 12.5 mM MgOAc, 1 mM DTT, 1% Triton X‐100, 2.5 mM ATP
10x Mobility shift buffer 100 mM Hepes, pH 8.0, 250 mM NaCl, 50 mM MgCl2, 20 mM DTT, 1 mM EDTA, 50% glycerol
Mowiol 6 g glycerol, 2.4 g Mowiol 4‐88 (Calbiochem), incubate in 6 ml
H2O for 2 h at room temperature, add 12 ml 0.2M Tris, pH 8.5, rotate o/n at 53°C, centrifuge 2x 20 min, 4000 rpm
4% paraformaldehyde (PFA) dissolve 20 g paraformaldehyde in 250 ml H2O (65°C), add dropwise 10 M NaOH, add 50 ml 10x PBS, adjust to pH 7.4, add
H2O ad 500 ml, sterile filter, store at ‐20°C
10x Phosphate buffered 1.4 M NaCl, 27 mM KCl, 100 mM Na2HPO42H2O, 18 mM saline (PBS) KH2PO4, adjust to pH 7.4
Tail lysis buffer 50 mM Tris, pH 8.0, 100 mM EDTA, pH 8.0, 0.5 % SDS
TBE buffer (10x) 0.9 M Tris, 0.9 M boric acid, 25 mM EDTA, adjust to pH 8.3
TE buffer 10 mM Tris, 0.1 mM EDTA, adjust to pH 8.0
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5.1.4 Medium used for Bacterial cultures
LB‐agar LB‐medium, 1.5 % agar
LB‐medium 1 % bacto‐trypton, 0.5 % yeast extract, 0.5 % NaCl
For selection Ampicillin (100 μg/ml) or Kanamycin (40 μg/ml) was added to the media, depending on the resistance of the desired Plasmid.
5.1.5 Oligonucleotides
All used oligonucleotides were purchased from Invitrogen (Karlsruhe).
5.1.5.1 Oligonucleotides used for genotyping
Name Primername Sequence (5’ to 3’) Primer Cre CRE tg1 ATGCTGTTTCACTGGTTATG 5’ CRE tg2 ATTGCCCCTGTTTCACTATC 3’ CreNkx2.5 for GATGACTCTGGTCAGAGATACCTG 5’ Nkx2.5 rev ACGCACTCACTTTAATGGGAAGAG 3’ Nkx2.5for GCCCTGTCCCTCGGATTTCACACC 5’ Wnt1Cre1 TAAGAGGCCTATAAGAGGCGG 5’ Wnt1Cre2 ACTAGTCTCCACTGAAGC 3’ Sox4 FP‐Sox4‐3 TGGTGGCTAAAAAAGCTACTTCG 5’ RP‐Sox4‐5 AGCGTGGAGTTTCTCCATGCC 3’ Sox11 Sox11fl+ [FP2] TTCGTGATTGCAACAAAGGCGGAG 5’ Sox11fl+ [RP4] GCTCCCTGCAGTTTAAGAAATCGG 3’ YFP R26 wt 0883 AAAGTCGCTCTGAGTTGTTAT 5’ R26 wt 0316 GGAGCGGGAGAAATGGATATG 5’ R26 YFP 4982 AAGACCGCGAAG AGTTTGTC 3’
Table 5: Oligonucleotides used for genotyping of the different mouselines
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5.1.5.2 Oligonucleotides used for electrophoretic mobility shift assay (EMSA)
Name Sequence (5’ to 3’) Primer Adam19 CGTCCACCACAAATCCCTCCTC 5’ GAGGAGGGATTTGTGGTGGACG 3’ Adam19mut CGTCCACCAGCGATCCCTCCTC 5’ GAGGAGGGATCGCTGGTGGACG 3’ Ecad1 CTAGAGGGTCAACGCGTCTATG 5’ CATAGACGCGTTGACCCTCTAG 3’ Ecad1mut CCAGGCTAGAGGCCGCGCTCGT 5’ ACGAGCGCGGCCTCTAGCCTGG 3’ Ecad2 ACCCCCTCTCAGTGGCGTCGGA 5’ TCCGACGCCACTGAGAGGGGGT 3’ Ecad2mut CACGCACCCCCTCTGCGTGGCG 5’ CGCCACGCAGAGGGGGTGCGTG 3’ SiteB CAGAGTATACAATGCCCCTTA 5’ TAAGGGGCATTGTATACTCTG 3’
Table 6: Oligonucleotides used for electrophoretic mobility shift assays (EMSA) additional radioactive GGG targeting not included
5.1.5.3 Oligonucleotides used for quantitative real time ‐ polymerase chain reaction (qRT‐PCR)
Name Sequence (5’ to 3’) Primer Actin‐1 CCT GGG CAT GGA GTC CTG 5’ Actin‐2 GGA GCA ATG ATC TTG ATC TTC 3’ Adam9‐for GCCTCTCTGCGACTAAGGTG 5’ Adam9‐rev CAGCAAGCCTCTGAGTCCAA 3’ Adam19‐for CCCCCTAAGAGTGTGGGTCCCG 5’ Adam19‐rev TGGCGATTCAACGCGCAGGT 3’ Cx40‐for GGAAAGGAAGCAGAAGGCTCGGC 5’ Cx40‐rev TGGGAGATGGGGAAGGCTTGGT 3’
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Name Sequence (5’ to 3’) Primer Cx43‐for AGTGAAAGAGAGGTGCCCAGACAT 5’ Cx43‐rev TCGGTCTGCGCCACTTTGAGC 3’ Ecadherin‐for AGCTTTTCCGCGCTCCTGCT 5’ Ecadherin‐rev AGAGGCAGGGTCGCGGTGGT 3’ Foxc2‐1for TGCCCAACTGTTACTGCCAA 5’ Foxc2‐2rev GGGCAGAAAAACAACACGGG 3’ Hand2‐for AAGGAGCGGCGCAGGACTCA 5’ Hand2‐rev CAGCCTGTCCGGCCTTTGGT 3’ Insm1‐3for TGCGTCCGGCCTGCTAGAGT 5’ Insm1‐4rev GCTCCACCGAAGCGAAGCGA 3’ NG2‐for GCCTTCACGATCACCATCCT 5’ NG2‐rev CAGGGCTCCTCTGTGTGAGA 3’ Snail1‐1for TCCGCACCCACACTGGTGAGA 5’ Snail1‐2rev GGAGGCTCTGGGCGGGTACA 3’ Snail2‐for ACTGGACACACACACAGTTAT 5’ Snail2‐rev ATGGCATGGGGGTCTGAAAG 3’ SMA‐for GCTGCTCCAGCTATGTGTGA 5’ SMA‐rev AGTGGTGCCAGATCTTTTCCA 3’ Sox10‐fwd‐38 TGGACCACCGGCACCCAGAA 5’ Sox10‐rev‐38 CGTGGGCAGAGCCACACCTG 3’ Sox12‐27 CAGTCTGGGGACAGAGTTCC 5’ Sox12‐28 GACCAGTGGAGCAGACTTGG 3’ Twist1‐for GCAGTCGCTGAACGAGGCGT 5’ Twist1‐rev ACAATGACATCTAGGTCTCCGGCCT 3’ Rpl8‐1 GTTCGTGTACTGCGGCAAGA 5’ Rpl8‐2 ACAGGATTCATGGCCACACC 3’
Table 7: Oligonucleotides used for quantitative real time ‐ polymerase chain reaction (qRT‐PCR) on mRNA of outflow tract tissue of the heart from 13.5 dpc old mutant and wildtype mice
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5.1.5.4 Oligonucleotides used for mutagenesis of Sox binding sites
Name Sequence (5’ to 3’) Ecadmutsox2‐3 AGGCTAGAGGGTGCGCGCGTCTATGCG Ecadmutsox2‐4 CGCATAGACGCGCGCACCCTCTAGCCTGGA Ecadmutsox4‐4 CGCACCCCCTCTGCGTGGCGTCGGAAC Ecadmutsox4‐4 GTTCCGACGCCACGCAGAGGGGGTGCGTGG MADDAMmut240‐for GCGTCCACCAGCGATCCCTCCTCC MADDAMmut240‐rev GGAGGAGGGA TCGCTGGTGG ACGC
Table 8: Oligonucleotides used for mutagenesis of Sox binding sites in E‐cadherin and Adam19 promomter regions
5.1.5.5 Oligonucleotides used for mutagenesis of Sox4 and Sox11
Name Sequence (5’ to 3’) Sox4‐27 AAGCACATGGCTGACTTCCCTGACTTCAAGTACCGGCCGCGAAAGAAGGTG Sox4‐28 CGGCCGGTACTTGAAGTCAGGGAAGTCAGCCATGTGCTTGAGGCGCAG Sox4‐29 CCGGCCGCCTCCTCCGCGCCGTCCAGCGCGCTGGCCACCCCC Sox3‐30 CAGCGCGCTGGACGGCGCGGAGGAGGCGGCCGGGGTGGCCGC Sox4‐33 ACGGGTGGCAAGGCGGACGACAGTGGCCACATCAAGCGGCCC Sox4‐34 GGGCCGCTTGATGTGGCCACTGTCGTCCGCCTTGCCACCCGT Sox4‐35 GGGGATCCGCCATGGTACAACAGACCAACAACGCG Sox4‐36 GACTCGAGCTACTTCACCTTCTTTCGCTCCTG Sox4‐37 GGGGATCCGCCATGACCAACAACGCGGAGAACACG Sox4‐38 CCGCTTGGAGATCTCGGCGTTGTG Sox4‐39 GCCGCCTCCTCCGAGCCGTCCAGCGCG Sox4‐40 GCCGCCTCCTCCGCGCCGTCCAGCGCG Sox11‐55 AAGCACATGGCTGATTTTCCCGACTTCAAGTACCGGCCGCGCAAAAAGCCC Sox11‐56 GCGCGGCCGGTACTTGAAGTCGGGAAAATCAGCCATGTGCTTGAGGCGCAG Sox11‐57 GCCAAGGTGCCCGCCGCCCCCACGCTCAGCAGTGCCGCCGAG Sox11‐58 ACTGCTGAGCGTGGGGGCGGCGGGCACCTTGGCCACGCTGTA Sox11‐59 GCCAAGGTGCCCGCCGAGCCCACGCTCAGCAGTGCCGCCGAG
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Name Sequence (5’ to 3’) Sox11‐60 ACTGCTGAGCGTGGGCTCGGCGGGCACCTTGGCCACGCTGTA Sox11‐61 GTGGCCCTGGATGAGAGCGACTCGGGCCACATCAAACGGCCC Sox11‐62 GGGCCGTTTGATGTGGCCCGAGTCGCTCTCATCCAGGGCCAC Sox11‐63 GGGGATCCACCATGGTGCAGCAGGCCGAGAGCTCG Sox11‐64 GGCTCGAGCTAGTCCGTCTTGGGCTTTTTGCGCGG Sox11‐65 GGGGATCCACCATGGCCGAGAGCTCGGAAGCCGAG Sox11‐66 CCTCTTGGAGATCTCGGCGTTATG
Table 9: Oligonucleotides used for mutagenesis of Sox4 and Sox11 phosphorylation sites, delta C and HMG‐ Box and combined mutations
5.1.5.6 Oligonucleotides used for mutagenesis of Sox4 and Sox11
Mutagenesis of Sox4 Sox11 Y123,126F; Sox4‐27 Sox11‐55 Y113,116F Sox4‐28 Sox11‐56 S244A Sox4‐29 Sox11‐57 Sox3‐30 Sox11‐58 S244E Sox4‐39 Sox11‐59 Sox4‐40 Sox11‐60 Sox4d7aaHMG Sox4‐33 Sox11‐61 Sox4‐34 Sox11‐62 Sox4dC Sox4‐35 Sox11‐63 Sox4‐36 Sox11‐64 Sox4dVQQ Sox4‐37 Sox11‐65 Sox4‐38 Sox11‐66
Table 10: Oligonucleotides used for mutagenesis of possible phosphorylation sites in Sox4 and Sox11 or for deletion of conserved amino acids
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5.1.6 Antibodies
5.1.6.1 Primary Antibodies
Antigen Species Dilution Supplier ‐Catenin rabbit 1:500 Cell Signaling EYFP rat 1:1.000 GERBU Biotechnik EYFP rabbit 1:2.000 Molecular Probes Ki67 rabbit 1:500 Lab Vision NG2 rabbit 1:200 Chemicon PECAM rat 1:100 Pharmingen Sox4 guinea pig 1:1.000 Pineda Sox11 giunea pig 1:1.000 Pineda
Table 11: Primary antibodies and dilutions used for immunohistochemistry
5.1.6.2 Secondary Antibodies
Secondary Antibodies conjugated to Alexa488 (dilution 1:500) or Cy3 (dilution 1:200) immunofuorescent dyes (Molecular Probes and Dianova) were used for detection.
5.1.6.3 Staining dyes
Name Dilution Supplier Phalloidin 1:5.000 Sigma‐Aldrich DAPI 1:50.000 Sigma‐Aldrich TUNEL assay Chemicon
Table 12: Staining dyes and their dilutions used for immunohistochemistry
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5.2 Methods
5.2.1 Mouse housing
Mice were housed under standard conditions according to the German Animal Welfare Act (TierSchG) in an adequate animal facility at the Institute of Biochemistry in Erlangen, Germany.
5.2.2 Mouse breeding
Mice, double‐homozygous for the floxed alleles of the Sox4 and the Sox11 genes (Sox4fl/fl, Sox11fl/fl), were intercrossed to produce double‐homozygous offspring. For the generation of cell line specific homozygous knockout mutant embryos, triple‐heterozygous mice (Sox4fl/fl, Sox11fl/fl and Cre‐positive) were crossbred with Sox4fl/fl, Sox11fl/flanimals. The breedings were checked after the first night for a post coitum vaginal protein plaque. This plaque indicated the possible pregnancy and this day was then set as 0.5 dpc (days post coitum). Analysis of the desired animals was done on embryonic day 10.5, 13.5 and 17.5 dpc.
5.2.3 Molecularbiological methods
5.2.3.1 Standard methods
Standard methods used in the laboratory, e. g. plasmid isolation, phenol‐chloroform extraction and ethanol precipitation, enzymatic digestion of DNA, ligation and transformation in E.coli were performed as described by (Ausubel F.M. et al., 2002;Sambrook J. and Russell D.W., 2001).
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5.2.3.2 Isolation of genomic DNA
DNA isolated from tail tips of adult animals or embryos older than embryonic day 13.5. For earlier stages embryonic tissue from yolk sac was used for genotyping. Tissues were lysed in 250µl Tail Lysis Buffer supplemented with 5µl Proteinase K (20µg/µl; Roth) for 1‐2 hours shaking at 1400rpm and 55°C in a thermomix (Eppendorf, Hamburg). For precipitation of DNA 200µl of Isopropanol was added. After centrifugation for 10 minutes at 13000rpm a washing step with 500µl of 70% ethanol and a subsequent centrifugation step for 5 minutes at 13000rpm were performed. DNA was dried at room temperature and disolved in 50‐400µl of water. 1µl of DNA was used as template in polymerase chain reaction (PCR).
5.2.3.3 Polymerase chain reaction (PCR)
Mice were genotyped by PCR with the primers specified in 5.1.5.1. Amplification reactions were performed in a total volume of 20 µl reaction mix (see table 5.2.3.5) in a Thermocycler (Biometra, Göttingen) according to the amplification programs specified in table 5.2.3.6 Genotypes were determined by electrophoretic separation of the amplification products supplemented with DNA loading buffer in 1% or 2% agarose gels. DNA fragments were visualized with ethidium bromide.
5.2.3.4 Primer combinations and DNA fragment size for polymerase chain reaction (PCR) mouse line primer allele amplified fragment length Ap2::Cre;Tie2::Cre; CRE tg1 Sox2::Cre CRE tg2 Cre tg 673 bp Nkx2.5::Cre CreNkx2.5 for Nkx2.5 rev wt 264 bp Nkx2.5for tg 586 bp Wnt1::Cre Wnt1Cre1 Wnt1Cre2 tg 600 bp Sox4 FP‐Sox4‐3 wt 400 bp RP‐Sox4‐5 tg 470 bp
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mouse line primer allele amplified fragment length Sox11 Sox11fl+ [FP2] wt 319 bp Sox11fl+ [RP4] tg 467 bp YFP R26 wt 0883 R26 wt 0316 wt 600 bp R26 YFP 4982 tg 320 bp
Table 13: Primer combinations used for PCR and amplified DNA fragment sizes
5.2.3.5 Reaction mix for polymerase chain reaction (PCR) reaction mix (in µl) Nkx2.5::Cre Wnt1::Cre Ap2::Cre YFP Sox4 Tie2::Cre Sox11 Sox2::Cre Buffer 10x 2 2 2 2
MgCl2 (25mM) 1.2 1.2 1.6 1.6 dNTPs (each 2.5mM) 1 1 1 1 DMSO 1 1 1 1 primer forward (40pmol/µl) 0.2 0.2 0.4 0.4 primer revers 1 (40pmol/µl) 0.2 0.2 0.4 0.4 primer revers 2 (40pmol/µl) 0.2 0.4 Taq‐DNA Polymerase 0.2 0.2 0.4 0.4 template DNA 1 1 1 1
H2O: add to 20 20 20 20
Table 14: Reaction mix used for PCR specified for the analysed genes
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5.2.3.6 Genotyping programm genotyping programm Ap2a::Cre; Tie::Cre; 4’ 94°C, 30x [30’’ 94°C,30’’ 55°C, 30’’ 72°C],10’ 72°C Sox2::Cre Nkx2.5::Cre 1’ 94°C, 35x [20’’ 94°C,20’’ 60°C, 30’’ 72°C],1’ 72°C Wnt1::Cre 1’ 94°C, 35x [30’’ 94°C,30’’ 58°C, 30’’ 72°C],1’ 72°C Sox4; Sox11 1’ 94°C, 40x [30’’ 94°C,30’’ 58°C, 30’’ 72°C],1’ 72°C YFP 3’ 94°C, 30x [30’’ 94°C,1’’ 58°C, 50’’ 72°C],2’ 72°C
Table 15: Used programs for genotyping of analysed genes
5.2.3.7 Cloning of DNA fragments
To amplify DNA fragments from plasmids or cDNA the following chemicals, concentrations and programs were used: volume chemicals
2 μl NH4SO4‐Reactionsbuffer (10x) (MBI‐Fermentas, St. Leon‐Roth)
1.6 μl MgCl2 (25 mM) 2 μl dNTPs (je 2,5 mM) 1 μl forward‐Primer (20 pM) 1 μl reverse‐Primer (20 pM) 0.5 μl Taq‐DNA‐Polymerase (MBI‐Fermentas, St. Leon‐Roth) 0 μl/1 μl DMSO 1 μl plasmid (10 ng/μl), cDNA‐ or ChIP‐Chromatin‐solution
Add 20 μl H2O
Table 16: Used chemicals and concentrations to amplify DNA fragments from plasmids or cDNA
PCR‐Program: 2‘ 95°C, 30‐40x[30‘‘ 95°C, 30‘‘ 56°‐60°C, 1‘/kb amplified fragment 72°C], 7’ 72°C
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5.2.3.8 Site‐directed Mutagenesis
For site‐directed mutagenesis the QuikChange®Lightning Site‐Directed Mutagenesis Kit (Stratagene; La Jolla, CA, USA) was used. volume chemicals 2.5 μl QCL‐Reaktionsbuffer (10x) 0.5 μl dNTPs 0.8 μl forward‐Primer (5 pM) 0.8 μl reverse‐Primer (5 pM) 0.5 μl DNA‐Polymerase 0.75 μl Quik‐Solution 2.5 μl plasmid (10 ng/μl) add 25 μl H2O
Table 17: Used chemical s and concentrations for site‐directed mutagenesis
PCR‐Programm: 2‘ 95°C, 4x[50‘‘ 95°C, 50‘‘ 50°C, 4‘ 68°C], 12x[50‘‘ 95°C, 60‘‘ 50°C, 4‘ 68°C], 10’ 68°C
PCR was followed by digestion with DpnI for 10 min at 37°C and subsequent transformation of 2 µl PCR product in E.coli XL1‐Blue.
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5.2.3.9 Reverse Transcription and RT‐PCR
2 µg of RNA was used for reverse transcription. RNA was obtained via Trizol RNA isolation and subsequent ethanol precipitation. volume chemicals 2 μg RNA 1 μl Oligo dT‐Primer (0,5 μg/μl) add 15 μl H2O
Table 18: Used chemicals and concentrations for priming of obtained mRNA
10 min 70°C followed by 3 min on ice
volume chemicals 2.5 μl RT‐Puffer für M‐MuLV‐Reverse Transkriptase (New England Biolabs, Frankfurt) 1 μl M‐MuLV‐Reverse Transkriptase (200 U/μl) (NewEngland Biolabs, Frankfurt) 2.5 μl dNTPs
5 μl H2O
Table 19: Used chemicals and concentrations for RT‐PCR
1 h 37°C, 5 min 70°C, cooling down on ice
For subsequent RT‐PCR 2µl of cDNA was diluted 1:5. Used oligonucleotides are shown in 5.1.5.3. (Table 7).
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5.2.3.10 Quantitative real time ‐ PCR (qRT‐PCR)
Relative and normalised quantifications were done in the C1000 Therma Cycler with the CFX96 Real‐Time Detection‐System (Bio‐Rad, München) and the CFX Manager2.0 Software (Bio‐Rad, München). The following volumes, chemicals and programs were used. volume chemicals 5 μl Absolute QPCR Cybr Green Capillary Mix (2x) (Abgene, Epsom, England) 0 µl/0.5 μl DMSO 0.5 μl forward‐Primer (5 pM) 0.5 μl reverse‐Primer (5 pM)
1.5 μl H2O 2 μl DNA (dilution 1:5)
Table 20: Used chemicals and concentrations for qRT‐PCR
Program: 15‘ 95°C, 40‐50x[10‘‘ 95°C, 20‘‘ 60°C, 20‘‘ 72°C], 10‘’ 95°C
5.2.4 Histological methods
5.2.4.1 Tissue preparation for immunohistological staining
Mouse embryos were obtained from staged pregnancies at 10.5 dpc, 13.5 dpc and 17.5 dpc. Pregnant dams were euthanized by cervical dislocation and embryos were isolated by Caesarean section under a stereomicroscope (Leica Microsystems,Wetzlar). For DNA isolation and genotyping yolk sacs were collected from 10.5 dpc old embryos whereas tails were collected from stage 13.5 dpc on. Embryos were fixed as a whole in 4% PFA at 4°C overnight. At 17.5 dpc head, extremities and skin were removed prior to fixation. After fixation, embryos were washed 6 times with PBS for at least 1 h on ice and transferred into 30% sucrose in PBS for one night at 4°C for cryoprotection. Embryos were frozen in TissueTek freezing medium (Jung, Nussloch) and stored at ‐80°C.
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5.2.4.2 Tissue preparation and haematoxylin – eosin staining
For haematoxylin‐eosin staining embryos were incubated overnight in 4% PFA and subsequently dehydrated. Embryos were embedded in paraffin and sectioned on a microtome at 3 µm thickness. After sectioning slides were dewaxed, rehydrated and stained in Mayer’s haematoxylin and eosin according to standard protocols.
5.2.4.3 Immunohistochemistry
Genotyped frozen embryos were transversally sectioned at 10 µm thickness on a cryotome (LeicaMicrosystems, Wetzlar). Sections were allowed to dry at room temperature for 2‐3 hand were stored at ‐80°C subsequently. For immunohistochemical staining, thawed sections were washed two times with PBS for 10 min. The tissue was permeabilised for10 min in PBS with 0.1% Triton X‐100 and washed with PBS for 10 min again. Blocking of unspecific binding was performed at room temperature for 2 h with 400 µl PBS with10% FCS and 1% BSA per slide. Primary antibodies see 5.1.6.1 (Table 11) were diluted in blocking solution. Sections were incubated over night at 4°C with 200 µl of diluted antibody per slide. Slides were washed 6x for 10 min with PBS and incubated in the dark at room temperature for 2 h with 200 µl secondary antibody diluted in blocking solution (see 5.1.6.2). After 6 times washing with PBS for 10 min in darkness, sections were stained with 10 µl DAPI (see 5.1.6.3, Table 12) in 70 ml PBS for 5 min and washed with PBS. Stained sections were mounted with 50µl Mowiol (Calbiochem/Merck Biosciences, BadSoden) per slide and stored at 4°C. Fluorescent signal was detected with an inverted fluorescence microscope Leica DMI6000 B (Leica Microsystems, Bensheim).
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5.2.4.4 Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL)
To detect apoptotic cell bodies, TUNEL was performed according to the manufacturer’s instructions with the ApopTag Red kit (Serologicals/QBiogene, Heidelberg). Tissue sections were prepared as described in 5.2.4.1. Thawed sections were washed once with PBS for 5 min were permeabilised using 0.5% Triton‐X100 in PBS for 10 min. After 5 min in PBS, slides were incubated in ethanol/glacial acetic acid (2:1) for 5 min at ‐20°C and re‐transferred into PBS for 5 min two times. The tissue was pre‐incubated on slide with equilibration buffer for 10 min at room temperature, before TdT (terminal deoxynucleotidyl transferase) enzyme solution was added. After incubation for 1 h at 37°C, the reaction was stopped by adding a stop/wash buffer for 10 min at room temperature. Slides were washed with PBS for three times, 5 min at room temperature. Rhodamine‐conjugated anti‐DIG‐antibody was added and slides were incubated for 2 h at room temperature. Subsequent 4x 5 min washing with PBS followed by nuclear DAPI staining and mounting slides in Mowiol as described previously.
5.2.4.5 Phalloidin staining
Tissue sections were prepared as described in 5.2.4.1. Sections prestained for EYFP antibody were washed once with PBS for 5 min and permeabilised with 0.5% Triton‐X100 in PBS for 10 min. After washing 10 min in PBS slides were blocked in blocking solution (400 µl PBS with10% FCS and 1% BSA per slide) for unspecific binding. Phalloidin was diluted 1:5000 in blocking solution and incubated for 30 min at room temperature.
5.2.4.6 Quantifications
Numbers of immunoreactive cells and DAPI‐positive nuclei per section were counted in relevant areas. Data were obtained for four sections of at least three different embryos for each genotype and embryonic age. Diagrams show mean values ± SEM of biological replicates. Statistical significances were determined by unpaired two‐tailed Student’s t test using GraphPad Prism6 software as stated in the corresponding figure legends.
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5.2.5 Cell culture methods
5.2.5.1 Cultivation of eukaryotic cells
HEK293 and Neuro‐2a cells were maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL, Eggenstein) containing 10% v/v foetal calf serum(FCS; Invitrogen), 100 U/ml penicillin and 10 mg/ml streptomycin. Adherent cells were trypsinised (Trypsin‐EDTA; Gibco/BRL) for propagation every 4 days.
5.2.5.2 Transfection of HEK293 cells for whole cell extracts
For protein over‐expression HEK293 cells were transfected with polyethylenimine (PEI; Sigma, Munich) one day after plating. Per 10 cm cell culture plate (Sarstedt, Nümbrecht), 6µg DNA was mixed with 30 µl PEI and 500 µl serum‐free DMEM. The mixture was thoroughly vortexed for 10 sec, incubated at room temperature for 10–15 min and added to the medium on the cells (7 ml DMEM/10% FCS). After incubation overnight, medium was changed to DMEM/10% FCS. Cells were harvested 48 h post transfection for the preparation of whole cell extracts.
5.2.5.3 Transfection of Neuro‐2a cells for whole cell extracts
For protein over‐expression Neuro‐2a cells were transfected with Superfect reagent (Qiagen, Hilden) one day after plating. Per 10 cm cell culture plate (Sarstedt, Nümbrecht), 6µg DNA was mixed with 10 µl Superfect reagent and 500 µl serum‐free DMEM. The mixture was thoroughly vortexed for 10 sec, incubated at room temperature for 10–15 min and added to the medium on the cells (7 ml DMEM/10% FCS). After incubation for 4 h, medium was changed to DMEM/10% FCS. Cells were harvested 48 h post transfection for the preparation of whole cell extracts.
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5.2.5.4 Preparation of whole cell extracts for electrophoretic mobility shift assay (EMSA)
Whole cell extracts from transfected HEK293 and Neuro‐2a cells were prepared 48 h post‐ transfection. Plates were washed 2x with PBS. Buffer A with additional freshly added DDT, aprotinin and leupeptin was added to the plates and cells were scraped off. Cells were then pipetted into a precooled tube and NP40 was added. After vortexing for 10 s 5M NaCl was added, samples were rotated for 15 min at 4 °C and subsequently centrifuged for 5 min. Supernatant was diluted in glycerine and whole cell extract was then stored at ‐80 °C.
5.2.5.5 Preparation of whole cell extracts for chromatin immunoprecipitation (ChIP)
Whole cell extracts from transfected Neuro‐2a cells were prepared 48 h post‐transfection. Cells on a 10 cm plate were fixed with 1% formaldehyde for 10 min on a shaker. 500µl of 2.5M glycine was added to the plate and incubated for another 5 min shaking. Plates were scraped off and fixed cells were put in a 1.5 ml tube for further processing. The samples were centrifuged at 2500 rpm and 4°C. Two times washing with cold PBS for 10 min shaking with centrifugation steps in between. After the last washing and centrifugation step supernatant was discarded and either pellet was prepared for further chromatin shearing steps or immediately frozen at ‐80°C.
5.2.5.6 Transfection of Neuro‐2a cells for luciferase assay
For luciferase assays, Neuro‐2a cells were transfected in triplicates with 3 µl Superfect reagent per 35mm plate (Qiagen, Hilden) according to the manufacturer’s instructions. To analyse whether Sox11 and Sox4 are able to activate the promoter regions of E‐cadherin or Adam19, Neuro‐2a cells were co‐transfected with 0.4 µg luciferase reporter plasmid containing fragments of the human E‐cadherin promoter (Ecad l (long), position ‐1502 to +98 and Ecad s (short), position ‐220 to +18 relative to the transcriptional start site, gift of J. Behrens, Erlangen) or of the human Adam19 promoter (Adam19 l (long), position ‐2009 to +31 and Adam19 s (short), position ‐243 to + 31 relative to transcription start site, gift of M.
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Kreutz, Regensburg) or versions of E‐cad s and Adam19 s with mutated Sox binding sites (Ecad sm and Adam sm). Full length Sox4 and Sox11 pCMV5 effector plasmids were as described (Kuhlbrodt et al., 1998) and the total amount of 0,25 Mg effector plasmid was kept constant using empty pCMV5 vector when needed. Cells where harvested 48h after transfection and extracts where used for luciferase assay.
5.2.5.7 Luciferase reporter gene assay
48 h post‐transfection, Neuro‐2a cells were harvested with 300 µl luciferase lysis buffer per 35mm plate. After 10 min incubation in lysis buffer, 150 µl of the extracts were assayed for luciferase activity in a luminometer (Lumat LB 9501; Berthold, Bad Wildbad). 100 µl of a
0.5mM luciferin (Serva, Heidelberg) solution in 5mM KHPO4 with a pH of 7,8was added automatically to the extracts and light emission was measured in relative light units.
5.2.6 Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) assays were performed on HEK293 and Neuro‐2a cells after transfection with Sox11 expression plasmid as described previously(Schlierf et al., 2007). Additionally, 16 whole hearts or 16 anterior heart poles from 13.5 dpc old wildtype mouse embryos were pooled and dissociated with a gentleMACSTMDissociator 3 cycles of the protein preparation program (Miltenyi Biotec). Samples were used as chromatin source. After crosslinking proteins to DNA in the presence of 1% formaldehyde, chromatin was prepared and sheared to an average fragment length of 200–500 bp using a Sonoplus HD2070 homogenizer (Bandelin). Immunoprecipitations were performed overnight at 4°C using guinea pig antiserum against Sox11 (1:500 dilution)(Hoser et al., 2007) or guinea pig preimmune serum as control coupled to protein A Sepharose beads. Quantitative PCR was performed on input and precipitated chromatin after crosslink reversal and purification.
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5.2.7 Electrophoretic mobility shift assay (EMSA)
For analysis of protein binding to DNA, oligonucleotides (see 5.1.5.2) containing Sox binding sequences were synthesized of both DNA strands with three guanines added at the 5’ end (Invitrogen, Karlsruhe). Single stranded oligonucleotides were resolved in analytical grade water (Roth, Karlsruhe) to a concentration of 1 µg/µl and hybridized in the presence of 100mM KCl by heating to 95°C for 5 min and slow subsequent cool down. Hybridized oligonucleotides were diluted to 0.1 µg/µl and labeled with32P. 100 ng double stranded oligonucleotides were incubated for 1 h in NEB2(New England Biolabs reaction buffer 2) with 1.5 µl 32P‐dCTP (10 µCi/µl; Hartmann GmbH, Braunschweig) and 1 µl Klenow enzyme (2.5 U/µl; Invitrogen, Karlsruhe) in a total volume of 50 µl. Labeled oligonucleotides were purified using QuickSpin Mini Columns(Roche Diagnostics, Mannheim) and diluted to 10000 cpm/µl as determined by measurement in a scintillation counter (Tri‐Carb 2800TR, Perkin Elmer).For complex formation of protein and DNA, 1 µl radio‐labeled oligonucleotide and 1 µl protein extract from Sox4 transfected HEK293 or Neuro‐2a cells were incubated for 20 min on ice in the presence of 2 µl Mobility shift buffer (10x), 5mM DTT, 1 µg poly‐dGdC and3 µg BSA in a total volume of 20 µl. Samples supplemented with DNA loading buffer were separated in a native 5% polyacrylamide gel (3.75 ml acrylamide40%, 400 µl APS 20%, 10 µl TEMED, 1.5 ml TBE 10x and 24 ml H2O). 120V were applied to the gel in 0.5x TBE buffer for 1 h before loading the samples. After sample loading electrophoresis was continued for 1.5–2 h. The gel was dried on a Whatmann3MM paper (Whatman; Schleicher&Schüll, Dassel) in a SE1160 gel dryer (Hoefer Scienctific Instruments). RX X‐ray films (FUJI Medical) were exposed for autoradiography at ‐80°C for 12–48 h and then developed in an X‐Omat 1000 processor film developer (Kodak).
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TABLE OF ABBREVIATIONS
6. Table of Abbreviations
Adam disintegrin and metalloproteinase domain‐containing protein AP2 activating enhancer binding protein 2 alpha AV atrioventricular BMP bone morphogenic protein CT common arterial trunk DCX doublecortin Dko double knockout DORV double outlet right ventricle EMT epithelial to mesenchymal transition FGF fibroblast growth factor FHF first heart field Fox forkhead box GATA consensus DNA motif GATA Hand heart and neural crest derivatives‐expressed protein HEK human embryonic kidney cells HMG high mobility group‐box Insm insulinoma‐associated protein MET mesenchymal to epithelial transition Nkx NK homeobox factor NLS nuclear localisation signal Pax paired box PTA persistent arterial trunk SHF second heart field Sox SRY box SRY sex determining region on Y chromosome TBX T‐box TGA transposition of the great arteries VSD ventricular septums defect Wnt1 Wingless‐type MMTV integration site family, member 1
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PUBLICATIONS
Publications
Paul MH, Harvey RP, Wegner M, Sock E. Cardiac outflow tract development relies on the complex function of Sox4 and Sox11 in multiple cell types. Cell Mol Life Sci. August 2014, Volume 71, Issue 15, pp 2931‐2945
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CURRICULUM VITAE
Curriculum Vitae
Persönliche Daten
Name Mandy Paul Geburtsdatum 03.06.1982 Geburtsort Bautzen, Deutschland Staatsangehörigkeit deutsch
Schulbildung
1989 ‐ 1993 Grundschule, Frankfurt am Main 1993 – 2002 Carl – Schurz – Gymnasium, Frankfurt am Main 2002 Erwerb der Allgemeinen Hochschulreife
Studium
2002 – 2008 Studium der Biologie, Goethe – Universität Frankfurt am Main 2006 Diplomvorprüfung 2008 Diplomhauptprüfung 2008 Diplomarbeit am Institut für klinische Neuroanatomie I. „Expressionsanalyse von Synaptopodin mRNA und Protein in definierten Hirnregionen von Synaptopodin‐EGFP transgenen Mäusen“
Promotion
2009 – 2013 Dissertation durchgeführt am Institut für Biochemie und Pathobiochemie in der Arbeitsgruppe von Elisabeth Sock unter der Leitung von Prof. Dr. M. Wegner an der Friedrich‐Alexander Universität, Erlangen ‐ Nürnberg
Thema: The role of transcription factors Sox4 and Sox11 in mouse heart development
Die Rolle der Transkriptionsfaktoren Sox4 und Sox11 während der Herzentwicklung der Maus
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DANKSAGUNG
Danksagung
Mein ganz besonderer Dank gilt Herrn Prof. Dr. Michael Wegner für die Überlassung des spannenden Themas, für die hervorragende Betreuung während der gesamten Zeit der Doktorarbeit, für die Durchsicht der Arbeit und nicht zuletzt für die Übernahme der Erstberichterstattung.
Herrn PD Dr. Dieter Engelkamp danke ich für die freundliche und bereitwillige Übernahme der Zweitberichterstattung. Bei Herrn Prof. Dr. Johann Helmut Brandstätter und Herrn Prof. Dr. Manfred Frasch bedanke ich mich für ihre Bereitschaft, das Amt des Vorsitzenden der Prüfungskommission bzw. des Prüfers bei der Verteidigung meiner Dissertation zu übernehmen.
Ein besonders großes Dankeschön geht an PD Dr. Elisabeth Sock für die immer hilfsbereite Unterstützung bei allen Fragen und Problemen, die sich während dieser Zeit ergaben und für das gründliche Korrekturlesen der Arbeit, sowie für die Hilfe und Unterstützung auch außerhalb des Laboralltags. Bei PD. Dr. Claus Stolt bedanke ich mich für die Hilfe bei Fragen jeder Art und die wissenschaftlichen Diskussionen und Anregungen.
Anna Hartwig danke ich für die Unterstützung und Hilfe bei allen technischen und methodischen Fragen des Laboralltags.
Bei Stephanie Hoffmann bedanke ich mich für die vielen schönen „Mittags“‐Pausen, besonders in der Zeit der Revision und für die Freundschaft, die sich während der Zeit im Labor entwickelt hat. Außerdem bedanke ich mich bei Judith Johnson, Amélie Wegener, Bojana Kravic, Juliane Arter, Inga Ebermann und Igor Macinkovic für die vielen schönen Stunden, die wir auch außerhalb des Labors miteinander verbracht haben. Bei Inga Ebermann, Jessica Schlaudraff und Zachary Smith möchte ich mich außerdem auch für das Korrekturlesen der Arbeit bedanken.
Den Arbeitskollegen des ganzen Lehrstuhls danke ich herzlich für die vielen lustigen Stunden im Labor und die jederzeit große Hilfsbereitschaft und Unterstützung im Laboralltag, sowie für die sehr gute Zusammenarbeit und das hervorragende Arbeitsklima.
Bei meinen Freunden möchte ich mich dafür bedanken, dass sie immer ein offenes Ohr für mich hatten und mich wieder und wieder motiviert und unterstützt haben. Außerdem möchte ich mich bei Ihnen für die Rücksichtnahme, Geduld und das Verständnis dafür bedanken, dass ich in den letzten Jahren nicht sehr viel Zeit für sie hatte.
Zum Abschluss möchte ich meiner Familie für all das danken, was sie mir im Leben ermöglicht haben, für die dauerhafte Unterstützung und den stetigen und unerschütterlichen Glauben an mich.
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