Analysis of transcriptional targets of SOX9 during embryonic heart valve development reveals a critical network of transcription factors

ii Preface.iv Table of contents.v List of tables.viii List of figures.ix List of abbreviations.xi Acknowledgements.xiii Dedication.xiiv Chapter one: Introduction.1 1.1 General introduction.1 1.2 Heart valve formation.1 1.2.1 Embryonic development of the heart valves.1 1.2.2 Composition of the adult heart valve.5 1.2.3 Major signalling events during heart valve development.6 1.3 Embryonic limb development and shared features of limb and heart valve formation.11 1.3.1 Development of the limbs.11 1.3.2 Functional similarities between the developing heart valves and limbs.12 1.4 The role of the Sex Related Y (SRY) box like 9 (SOX9) during embryonic development.13 1.4.1 SOX transcription factors.13 1.4.2 The many roles of SOX9 during development.15 1.4.3 The role of SOX9 in proliferation, EMT, and ECM.16 1.5 SOX9 is essential for the development of the heart valves and limb.18 1.6 Campomelic dysplasia.21 1.7 Congenital heart defects and heart valve disease.22 1.7.1 Congenital heart valve abnormalities.22 1.7.2 Adult heart valve disease.23 1.7.3 The involvement of SOX9 and signaling pathways in heart valve disease.24 1.8 Hypothesis and aims of the thesis project.25 Chapter two: Materials and methods.27 2.1 Mice strains and tissue dissection.27 2.2 Immunofluorescence, cell counts, in situ hybridization, and H&E staining.31 2.3 RNA isolation.33 2.4 RT-PCR, qRT-PCR, and ChIP-qPCR.34 2.5 Chromatin immunoprecipitation coupled with high-throughput sequencing.34 2.6 Bioinformatic analysis of ChIP-Seq.38 2.7 Genotyping Sox9 mutant embryos.38 vi 2.8 RNA-Seq and bioinformatic analysis.39 2.9 Cell culture, transfection, cloning and luciferase assays.41 2.10 Site directed mutagenesis.42 Chapter three: Identification and characterization of SOX9 binding sites in the developing heart valve and limb genome.43 3.1 Sox9 mRNA and protein is enriched in the mouse heart valves throughout development.43 3.2 SOX9 directly binds thousands of DNA regions in the developing heart and limb.48 3.3 SOX9 binds active promoter regions within the genome.51 3.4 Identification of potential co-factors of SOX9.53 3.4.1 DNA motif analysis identifies numerous potential co-factors of SOX9.53 3.4.2 Comparison of SOX9 ChIP-Seq with published ChIP-Seq data sets reveals new insights into potential co-factors of SOX9.58 Chapter four: Characterization of the Sox9fl/fl;VE-Cre mice.62 4.1 SOX9 negative AV mesenchyme cells are absent in Sox9 mutant valves.62 4.2 SOX9 is maintained at later stages of heart valve development in Sox9 mutants.67 4.3 Adult Sox9 mutants have ventricular hypertrophy and thickened valves leaflets.70 Chapter five: Characterization and analysis of the Sox9fl/fl;Tie2-Cre mice.75 5.1 SOX9 negative mesenchyme was detected as early as E10.5 in the AVC.75 5.2 Deletion of Sox9 was variable in the Sox9 cKO heart valves.79 5.3 Proliferation defects in the Sox9 cKO valves.83 5.4 Global transcriptional alterations in the Sox9 cKO heart valves.87 Chapter six: SOX9 has functions involved in regulation of proliferation, transcriptional networks, and ECM formation during heart valve development.94 6.1 SOX9 occupies regulatory regions of genes associated with proliferation.94 6.2 Context independent SOX9 binding regions in the AVC, limb, and HF-SCs.96 6.3 Proliferation associated target genes are both activated and repressed by SOX9.99 6.4 SOX9 targets a network of TFs known to be involved in heart development.103 6.5 Additional known roles for SOX9 in EMT and ECM organization.111 Chapter seven: Discussion.115 7.1 Thesis overview.115 7.2 SOX9 occupies the promoter and upstream regulatory regions associated with thousands of target genes including its future co-factors.116 7.3 SOX9 negative mesenchyme was absent in the Sox9fl/fl;VEC heart valves.122 7.4 Confirmation that Sox9 cKO (Sox9fl/fl;Tie2-Cre) valves have decreased size and proliferation that leads to major heart defects and embryonic death.125 7.5 Identification and regulation of SOX9 target genes in the developing heart valves.128 7.6 SOX9 directly controls genes associated with proliferation across cell types.129 7.7 SOX9 is a master regulator of a core network of TFs in heart valve development.133 7.8 Examination of other defined roles of SOX9 in EMT and ECM generation.135 vii 7.9 Concluding remarks.137 References.140 Appendices.153 Appendix I SOX9 immunostaining on E12.5 whole embryo and E10.5 heart.153 Appendix II Quality verification of the SOX9 ChIP-Seq libraries and gene associations.154 Appendix III Characteristics of SOX9 ChIP-Seq libraries.155 Appendix IV GO analysis on E12.5 AVC and limb overlapping SOX9 peaks.156 Appendix V GO analysis on E12.5 AVC SOX9 peaks.160 Appendix VI GO analysis on E12.5 limb SOX9 peaks.164 Appendix VII Co-factor analysis on E12.5 AVC SOX9 peaks using oPPOSSUM.167 Appendix VIII Co-factor analysis on E12.5 limb SOX9 peaks using oPPOSSUM.170 Appendix IX Co-factor analysis on E12.5 AVC and limb overlapping peaks using oPPOSSUM.