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The Diverse Role of Ldb1 in Cell Differentiation and Mouse Embryonic Development

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The Diverse Role of Ldb1 in Cell Differentiation and Mouse Embryonic Development The diverse role of Ldb1 in cell differentiation and mouse embryonic development Maria-Athina Mylona The studies presented in this thesis manuscript were performed in the Department of Cell Biology of the Erasmus MC in Rotterdam, The Nether- lands. The Department is a member of the Medisch Genetisch Centrum Zuid- West Nederland (MGC). The research described in this thesis was financially supported by NWO and EUTRACC. Cover: “Stem Cell Mandala” by Mara G. Haseltine Printed by Wöhrmann Print Service, Zutphen The Diverse Role of Ldb1 in Cell Differentiation and Mouse Embryonic Development De veelzijdige rol van Ldb1 in celdifferentiatie en de embryonale ontwikkeling van de muis Thesis to obtain the degree of Doctor from the Erasmus University Rotterdam by command of the rector magnificus Prof.dr. S.W.J. Lamberts and in accordance with the decision of the Doctorate Board The public defence shall be held on Wednesday 10 June 2009 at 15.45 hrs by Maria-Athina Mylona born in Kozani, Greece Doctoral Committee Promotor: Prof.dr. F.G. Grosveld Other members: Prof.dr. J.N.J. Philipsen dr. M. von Lindern dr. J. Gribnau to my grandmother... Contents List of Abbreviations 8 Scope of this thesis 9 Chapter 1 Introduction 11 Chapter 2 The deletion of the mouse Ldb1 gene leads to severe 49 embryonic developmental defects Chapter 3 Ldb1 is essential for primitive hematopoiesis and the 77 development of the hemangioblast Chapter 4 Ldb1 is involved in the regulation of neural stem cell 111 self-renewal Chapter 5 Discussion 149 Chapter 6 Summary/Samenvatting 165 Curriculum vitae 172 List of publications 173 PhD Portfolio 175 Acknowledgements 177 7 List of abbreviations DNA: Deoxyribonucleic acid RNA: Ribonucleic acid ICM: Inner Cell Mass dpc: days post coitum VE: Visceral endoderm ES cells: Embryonic Stem cells LIF: Leukemia Inhibitory Factor MEFs: Mouse Embryonic Fibroblasts SVZ: Subventricular Zone BL-CFC: Blast Colony Forming Cell PSp: Para-aortic splanchnopleura AGM: Aorta Gonads Mesonephros CFU-S: Colony Forming Unit-Spleen HSC: Hematopoietic Stem Cell TGF-β: Transforming Growth Factor-β BMP: Bone Morphogenetic Protein LTR-HSC: Long Term Repopulating Hematopoietic Stem Cell MEL: Mouse Erythroleukemia LCR: Locus Control Region HS2: Hypersensitive Site 2 NLS: Nuclear Localization Signal MLS: Mitochondrial Localization Signal CFP: Cyan Fluorescent Protein PCR: Polymerase Chain Reaction NSC: Neural Stem Cell CNS: Central Nervous System hrs: hours 8 Scope of this thesis During the development of the mouse embryo tightly regulated differentiation pathways lead to the formation of the tissues and organs of the adult animal. The role of Ldb1 during embryonic development and in particular during hematopoiesis and neural development will be the focus of this thesis. The first chapter of this thesis is a general introduction that covers the initial stages during the formation of the mouse embryo, the onset of hematopoiesis in the extra-embryonic tissues followed by the emergence of HSCs in the intra-embryonic PSp/AGM and the shift of hematopoiesis initially to the fetal liver and finally the bone marrow. In addition the chapter includes an overview of the signalling pathways and transcription factors that are involved in the regulation of hematopoiesis. Finally Ldb1 is introduced as a member of multi-protein complexes that regulate gene expression and cell differentiation and as a facilitator of long range interactions in transcription regulation. In the second chapter the phenotype of the Ldb1-/- mouse is described in detail. The deletion of Ldb1 was found to be detrimental for embryonic development as the resulting phenotype is characterized by early embryonic lethality and severe defects. Mouse expression arrays were used in order to gain insight into which development regulatory pathways and factors were misregulated in the absence of Ldb1. In the third chapter the role of Ldb1 in early hematopoietic development and the emergence of the hemangioblast was investigated in detail with the use of an Ldb1-/- ES cell line that was differentiated into embryoid bodies. Mouse expression arrays were used in order to establish which factors act downstream of Ldb1 in the regulation of hematopoiesis in the mouse embryo. In the fourth chapter the role of Ldb1 in neural development was investigated with the use of the Ldb1-/- ES cell line that was differentiated into Neural Stem Cells (NSCs). Mouse expression arrays were used to determine the position of Ldb1 in the hierarchy of factors that are involved in the emergence of the NSCs. In the fifth chapter the role of Ldb1 in cell differentiation and transcription regulation is discussed in detail and applications of the present findings into future directions are proposed. 9 Chapter 1 Introduction Introduction 1. Development of the mouse embryo The mouse has been extensively used as a model for the study of mammalian embryonic development. The selection of the animal is based on easier housing due to its small size, the large size of the produced litters (6 to 8 animals per litter), the quick gestation time of 19 to 20 days and the sexual maturity time (6-8 weeks after birth). 1.1. Pre-implantation development Ovulation in mice is initiated under hormonal stimuli on average every 4 days and leads to the preparation of the oocyte for fertilization. The nuclear membrane of the oocyte disappears and the chromosomes move to the periphery of the cell for the first meiotic division. One set of the synthesized homologous chromosomes with the surrounding cytoplasm forms the first polar body and is excluded from the cell. The other set remains at metaphase II. At the end of ovulation the oocyte is released from the ovary. Fertilization requires the successful entrance of one sperm cell into the mature oocyte through the surrounding cumulus mass of follicle cells and the zona pellucida. The incorporation of the sperm cell into the oocyte triggers a set of events to ensure that no more sperm cells will enter the oocyte [1]. Fertilization is followed by the second meiotic division and the formation and exclusion of the second polar body from the now fertilized oocyte. Haploid male and female pro-nuclei are formed around the male and female chromosomes and DNA replication takes place. The pro-nuclei move towards the centre of the oocyte and the surrounding membranes are progressively lost. The chromosomes assemble on the spindle and the first cleavage follows that leads to the two-cell stage approximately 1.5 days after fertilization. At this stage a large number of embryonic genes are switched on while there is extensive degradation of maternal mRNA [2-5]. Subsequent cleavage steps accompanied by compaction steps lead through the 4-cell stage, 8-cell stage and 16-cell stage morulae to the more compact blastocyst 3.5 days after fertilization [1]. The blastocyst consists of the surrounding trophoectoderm, which resembles epithelial tissue, the blastocoel, which is a cavity filled with fluid and the inner cell mass (ICM) from which the embryonic stem cells are derived. Approximately 4.5 days after fertilization the newly formed blastocyst moves down the oviduct and is implanted in the uterus. A group of non-polarized ICM cells generate the primitive endoderm or hypoblast and the rest of the ICM cells give rise to the primitive ectoderm or epiblast (Fig. 1). 1.2. Post-Implantation development The trophoectoderm cells of the post-implantation blastocyst follow distinct differentiation pathways depending on their position [6]. The cells that surround the blastocoel (mural trophoectoderm) give rise to the primary trophoblastic giant cells, which are large in size, haploid and contain polytene chromosomes, although in the past the were thought to be polyploid instead [7]. The blastocyst trophoectoderm cells that are next to or surround the ICM (polar trophoectoderm) are diploid and they are characterized by an increased 13 Chapter 1 proliferation rate. After implantation a group of polar trophoectoderm cells moves inside the blastocoel cavity forming a column-like structure that forms the extra-embryonic ectoderm. The primitive endoderm (hypoblast) will subsequently differentiate to parietal endoderm at 5.5 dpc and then to visceral endoderm (VE) at 6 dpc. In addition at this time point in development the epiblast cells will form an epithelial layer that surrounds the pro-amniotic cavity (Fig. 1) [8]. Polar Ectoplacental trophoectoderm cone Extra-embryonic ectoderm ICM Extra-embryonic Primitive VE endoderm Epiblast Blastocoel Parietal endoderm Mural cavity trophoectoderm Trophoblast Blastocyst giant cells 4.5 dpc 5.5 dpc Anterior Posterior Extra-embryonic VE Anterior Posterior Chorionic Chorionic ectoderm mesoderm Visceral Extra- Anterior Posterior Yolk sac cavity embryonic Anterior Allantois mesoderm Extra-embryonic neuroectoderm VE Amnionic mesoderm Extra-embryonic ectoderm Primitive Amnionic Embryonic/ streak ectoderm Extra-embryonic Hemangioblast Embryonic junction Proamniotic mesoderm cavity VE VE VE AVE Epiblast Anterior mesendoderm Node 6 dpc 6.5 dpc 7.5 dpc Figure 1: Schematic representation of the mouse embryo post-implantation development. At 4.5 dpc the blastocyst is implanted in the uterus. ES cells can be derived from the ICM at this stage. At 6.5 dpc gastrulation takes place. The primitive streak is formed followed by the formation of the embryonic and the extra-embryonic mesoderm (adapted from [68]). Gastrulation is initiated at 6.5 dpc and is characterized by an increase in cell proliferation as the primitive streak is formed from a group of epiblast cells next to the embryonic/extra- embryonic junction marking the posterior of the embryo [9]. The emergence of the primitive streak is followed by the formation of the embryonic and extra-embryonic mesoderm, which underlies the epiblast and is surrounded by the VE. The extra-embryonic portion of the mesoderm increases in volume due to the formation of lacunae or small cavities, which will eventually unite and form the exocoelom or visceral yolk sac cavity between the chorion 14 Introduction and the amnion.
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