Zeb2 regulates myogenic differentiation in pluripotent stem cells

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions

Transcriptional repressor ZEB2 promotes terminal differentiation of CD8⁺ effector and memory T cell populations during infection

ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in epithelial-mesenchymal transition-dependent tumor metastasis. Although the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is up-regulated by activated T cells, specifically in the KLRG1(hi) effector CD8(+) T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8(+) T cells after primary and secondary infection with a severe impairment in the generation of the KLRG1(hi) effector memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress Il7r and Il2 in CD8(+) T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box sites in the Zeb2 gene and that T-bet and ZEB2 regulate similar gene expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Collectively, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8(+) T cells

The role of Zeb2 in cell fate decisions during development

Cell fate decisions during embryogenesis and in proliferation and regeneration competent adult tissues and organs are orchestrated by large numbers of regulators including transcription factors, non-coding RNAs, epigenetic modifiers and longer-range genomic interactions, each downstream of extrinsic signals and their intracellular signal transduction effector proteins. Zeb2 is a DNA-binding transcription factor that steers in such context many cellular processes during early and late embryogenesis, and knowledge is emerging about its role in various adult tissues/organs as well, especially in challenge conditions like tissue organ failure. Mutations in one allele of ZEB2 in humans cause Mowat-Wilson syndrome (MOWS) characterized by severe intellectual disability and, varying from patient to patient, epilepsy, Hirschsprung disease and other defects. In the mouse Zeb2 has been show, including by our laboratory, to regulate different aspects of central and peripheral nervous system (CNS, PNS, respectively) development as well as their post-natal functions. The same applies to embryonic and adult hematopoiesis, T-cell lineage development and function, and e.g. myelinogenesis in the CNS and (re)myelination by Schwann cells in the PNS. In addition, deregulation of the levels of Zeb2, e.g.. an abnormal increase, contributes (in this case) to many cancers and correlates with bad prognosis in humans, as supported by Zeb2 overexpression mouse models. The action mechanisms by which Zeb2, in concert with many of its emerging protein partners, steers neurogenesis and gliogenesis can be studied in cultured embryonic stem cells (ESCs) and the early CNS in vivo. In human ESCs, intact levels of ZEB2 were shown to be essential for effective neuroectoderm differentiation. The goals of this PhD project were to understand where, when and how Zeb2 functions (i) during mouse brain cortex development (using an in vivo approach) and (ii) in pluripotency and subsequent differentiation of mouse ESCs (in vitro approach). Genetic inactivation of both alleles of Zeb2 in early neural progenitors (using a Nestin-Cre approach), from dorsal telencephalon (Emx1-Cre) as well as in post-mitotic (NEX-Cre) upper layer neurons in the forming cortex in the embryonic forebrain causes a shift forward in the timing of first embryonic neurogenesis followed by embryonic and early post-natal gliogenesis. This leads to the expansion of the upper layers of the cortex at the expense of the (earlier born) cells of the deeper layers. Using gene expression profiling followed by validation, which is an essential part of this work and this PhD project, we identified neurotrophin-3 (Nt3) and fibroblast growth factor-9 (Fgf9) as extrinsic factors whose levels were significantly increased in the upper layer cells of the Zeb2 knockout (KO) embryonic brain cortex as compared to control. In this way, Zeb2 regulates the generation of subsequent waves of neurogenesis (steered by Nt3 that acts on its Trk receptor complex in the progenitor cells and promotes neurogenesis) and of gliogenesis (via Fgf9 that acts on its receptors there) emanating from the Zeb2-negative progenitor cells in the cortex. Importantly, this work identified Zeb2 as the first transcription factor acting in a cell non-autonomous fashion in cortex development. In the second part of this PhD research the focus was on the role(s) and action mechanism(s) of Zeb2 in pluripotency and during differentiation of ESCs. For this, Zeb2-deficient (KO) ESCs were established. Using these novel, unique tools in combination with omics, this PhD project was able to add important new functional and mechanistic insight to previous findings. We discovered that Zeb2 is critical for exit from the epiblast state in mouse ESCs and links the pluripotency network and DNA-methylation with irreversible commitment to neural and general differentiation. In particular, we show that Zeb2 KO ESCs display impaired differentiation in embryoid bodies by stalling in an epiblast-like state. Using RNA-Seq we further conclude that Zeb2 mainly acts here as a transcriptional repressor for many genes, either directly or indirectly, in differentiating conditions. Epithelial-mesenchymal transition (EMT), pluripotency, lineage commitment and DNA-(de)methylation genes are deregulated in Zeb2 KO embryoid bodies. By using methylome analysis, we demonstrate these mutant cells cannot maintain their initially acquired DNA-methylation marks in neural-stimulating condition and do not effectively downregulate Oct4, Nanog and Tet1 in differentiation conditions. Tet1 knockdown by RNA interference partially rescues the impaired differentiation of these KO cells. Another part of the PhD project investigated, starting again from the RNA-Seq data, the neuronal inhibitory REST gene, one of the genes whose expression was not silenced in Zeb2 KO ESCs, unlike in normal ESCs. Like for the Tet1 studies, stable REST knockdown lines were established in the Zeb2 KO background. The neural differentiation capacity of such cells line was also partially restored, indicating that REST is another important Zeb2-dependent gene in ESCs. In a last set of experiments, which provide the strong basis for future structure-function analysis of Zeb2 (and hence insight into its functional domains) in this ESC system, the neurodevelopmental potential of Zeb2 Smad-binding domain (SBD) mutant cells was documented. We observed that deletion of the SBD from Zeb2 had a positive effect on neural differentiation, indicating that this Zeb2 domain, and hence possibly its interaction with Smads, co-determines Zeb2’s neural-inducing activity.Table of contents ACKNOWLEDGMENTS 3 TABLE OF CONTENTS 5 LIST OF ABBREVIATIONS 11 LIST OF FIGURES 13 LIST OF TABLES 14 SUMMARY IN ENGLISH 15 SAMENVATTING IN HET NEDERLANDS 17 1 CHAPTER 1: GENERAL INTRODUCTION 19 1.1 Early embryonic development and the first cell fate decisions 19 1.2 Embryonic stem cells 21 1.2.1 ESC discovery 21 1.2.2 States of pluripotency 22 1.2.3 Extrinsic signaling pathways in regulation of ESC states 25 1.2.4 Transcription factors relevant for acquisition and maintenance of pluripotency and their networks. 26 1.2.5 Non-coding RNAs important for stemness maintenance 30 1.2.6 Divergent epigenetic landscapes of ground, primed and differentiating ESCs. Enzymes and protein complexes that modify the epigenetic changes. 31 1.2.6.1 DNA-methylation 32 1.2.6.2 Histone signatures 33 1.2.6.3 ATP-dependent chromatin remodeling complexes 35 1.2.7 Long-range interactions 38 1.3 Neurogenesis 40 1.3.1 In vivo 40 1.3.1.1 Early neural development (E7.5-E10.0) 40 1.3.1.2 Corticogenesis 40 1.3.2 In vitro 47 1.4 Zeb2 in development 49 1.4.1 Discovery of Sip1/Zeb2 49 1.4.2 Sip1/Zeb2 protein partners 50 1.4.3 Neural phenotype of the Zeb2 conventional knockout mouse 51 1.4.4 Zeb2 conditional, cell-type specific knockouts 53 1.4.4.1 Brain development 53 1.4.4.2 Hematopoiesis 55 1.4.4.3 Melanocyte development 56 1.5 Zeb2 in disease 57 1.5.1 Mowat-Wilson syndrome 57 1.5.2 Cancer 59 2 CHAPTER 2: OBJECTIVES 61 2.1 To identify and validate Zeb2-dependent and/or candidate target genes during mouse brain cortex development 61 2.2 To develop an embryonically relevant cell culture system to assess Zeb2’s function and action mechanism 62 2.3 To identify Zeb2-dependent genes during in vitro neural differentiation using transcriptomics 62 2.4 To compare dynamic changes in DNA-methylation during Ctrl and Zeb2 KO ESC differentiation 63 2.5 To validate selected candidate direct target genes and gain insight into possible mechanisms of action of Zeb2 during ESC differentiation 63 2.6 To explore the neurodevelopmental potential of Zeb2 domain mutant and Zeb1 knock-in mouse ESC lines 64 3 CHAPTER 3: ROLE(S) OF SIP1/ZEB2 IN THE DEVELOPMENT OF THE MOUSE BRAIN CORTEX 65 3.1 Introduction 65 3.1.1 Sip1/Zeb2 levels are high in post-mitotic neocortical cells 67 3.1.2 Sip1/Zeb2 knockout mouse models 68 3.1.3 Lack of Sip1/Zeb2 causes premature generation of upper layers at the expense of deep layers..... 69 3.1.4 Sip1/Zeb2 is also crucial for appropriate control of the timing of gliogenesis 70 3.2 Methodology and Materials 72 3.2.1 Expression profiling by microarray analysis 72 3.2.2 RT-qPCR and ISH 72 3.2.3 ChIP analysis 73 3.3 Results 74 3.4 Discussion 77 4 CHAPTER 4: ROLE(S) OF ZEB2 IN PLURIPOTENCY AND DIFFERENTIATION OF EMBRYONIC STEM CELLS 81 4.1 Introduction 82 4.2 Methodology and Materials 84 4.2.1 ESC lines 84 4.2.2 ESC maintenance 84 4.2.3 Neural differentiation 84 4.2.4 General differentiation 85 4.2.5 EB dissociation and sorting of living cells 85 4.2.6 Teratoma formation assay 85 4.2.7 Morula aggregations 85 4.2.8 RNA-sequencing and data analysis 85 4.2.9 RRBS analysis 86 4.2.10 Analysis of published Tet1 binding peaks in mESCs 87 4.2.11 shRNA-mediated knockdown 87 4.2.12 ChIP analysis 87 4.2.13 Immunohistochemistry and immunofluorescence 88 4.2.14 qPCR primers 88 4.3 Results 91 4.3.1 Knockout of Zeb2 impairs ESC differentiation 91 4.3.2 Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression 95 4.3.3 Zeb2 KO ESCs stall in an epiblast-like state 98 4.3.4 Pluripotency genes are not repressed during differentiation of Zeb2KO ESCs 99 4.3.5 The Zeb2 KO embryoid bodies, subjected to neural differentiation, fail to maintain the initially acquired DNA-methylation 102 4.3.6 Failure to maintain acquired DNA-methylation during neural differentiation is associated with Tet1-binding 105 4.3.7 Tet1 knockdown in Zeb2 KO ESCs facilitates silencing of Nanog, Oct4 and Cdh1 and partially rescues the lineage differentiation phenotypes 106 4.3.8 The neuronal inhibitory gene REST is deregulated in Zeb2 KO ESCs 108 4.3.8.1 Rest knockdown in the Zeb2 KO ESCs partially rescues neural differentiation 108 4.3.9 Zeb2 KO ESCs have the capacity to differentiate into three lineages when injected into immunodeficient mice 109 4.3.10 Zeb2 action in vitro is primarly cell-autonomous 112 4.3.11 High percentage Zeb2KO-GFP/CD1 chimeric embryos show severe defects in early embryonic development 114 4.3.12 Studies on R26-based Zeb2 domain mutant lines suggest that interactions of Zeb2 with some of its protein partners may have an inhibitory function on in vitro neural differentiation 115 4.4 Discussion 120 4.4.1 Zeb2 is critical for exit from the epiblast state in ESCs and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. 120 4.4.2 Zeb2 and Rest 123 4.4.3 Cell-autonomous action of Zeb2 in vitro 123 4.4.4 Zeb2 and Smads 124 5 CHAPTER 5: GENERAL DISCUSSION 126 5.1 Does Zeb2 regulate other processes during brain development and in the adult?.. 126 5.2 Does the NuRD complex co-operate with Zeb2 in the establishment of the correct chromatin context in embryonic cells, including in Zeb2-dependent differentiation? 128 5.3 Could high levels of Tet1 increase/stabilize recruitment of Ogt in the absence of Zeb2? 130 5.4 Are Tet1 and miR-22 the new modulators of previously identified Zeb2-miR-200 feedback loop in ESCs? 132 CHAPTER 6: GLOBAL CONCLUSIONS AND PERSPECTIVES 135 LIST OF REFERENCES 139 C.V. 151nrpages: 154status: publishe

Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors

The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.status: publishe

The effect of the topmost layer and the type of bone morphogenetic protein-2 immobilization on the mesenchymal stem cell response

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein-substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased

Integrative and perturbation based analysis of the transcriptional dynamics of TGF beta/BMP system components in transition from embryonic stem cells to neural progenitors

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.status: publishe