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Nuclear Receptor Corepressor 1 (NCoR1) and Nuclear Receptor Corepressor 2 (NCoR2) are large scaffolding proteins. As a scaffolding protein, they have multiple domains, including a deacetylase activating domain (DAD) that interacts with histone deacetylase 3 (HDAC3). HDAC3 is an enzyme that functions to deacetylate histone tails, leading to gene repression. NCoR1/2 also contains multiple nuclear receptor interacting domains to assist nuclear receptors in the downregulation of gene expression. Germline deletion of NCoR1, NCoR2 and HDAC3 in mice were lethal at e15.5, e16.5 and e9.5, respectively. This, therefore, demonstrates that they are crucial in embryogenesis. To study their roles in the embryo, we used Ncor1/2 double knockout (DKO) and HDAC3 KO mouse embryonic stem cells (mESCs) as model systems. Using RNA-seq, we discovered that Ncor1/2 DKO ESCs showed upregulated Hox genes, including Cdx1, Pbx1 and Meis2. This indicates that Ncor1/2 are essential in repressing Hox gene expression in ESCs, thus preventing uncontrolled differentiation. Additionally, the Cowley lab previously showed that HDAC3 KO embryoid bodies displayed an increased endodermal marker expression, such as in Gata6, Foxa2 and Sox17. In this thesis, we utilised a novel model of the developing embryo called gastruloids. Gastruloids are 3D organoid models containing the three germ layers (ectoderm, mesoderm, and endoderm). They also display organised anterior-posterior, left-right, and dorsal-ventral body axes. Using HDAC3 KO ESCs to generate gastruloids, we also observed increased endodermal gene expression. Moreover, many of the upregulated genes were also involved in heart development. Furthermore, Hox cluster genes were downregulated in the HDAC3 KO gastruloids, suggesting that HDAC3 modulates embryogenesis by regulating the expression of endoderm, Hox, and heart developmental genes. Hence, in this study, we have identified some of the causes of mouse embryonic lethality, which were contributed to by the absence of HDAC3 and NCoR1/2.

Histone deacetylase 1 (HDAC1) functions as the catalytic core of multiprotein corepressor complexes removing acetyl marks on histone proteins, causing the compaction of chromatin. Despite this repressive function, the removal of HDAC1 and its highly homologous sister protein HDAC2 leads to the downregulation of large numbers of genes in embryonic stem cells (ESCs) and other cell types. Early embryonic development is one of the few examples where HDAC1 and 2 are not redundant as Hdac1 knockout (KO) mice embryos fail to develop past embryonic day (e) 10.5, whereas Hdac2 KO embryos survive at least until birth. The work in this thesis was conducted with the aim of further unravelling the role of HDAC1 in ESCs and early embryonic development. The proximity-dependent biotinylation (PDB) techniques, APEX2 and BioID, were used to investigate the local protein environment of HDAC1 in ESCs, also facilitating a direct comparison of the two techniques. PDB revealed that the 'neighbours' of HDAC1 are involved in many nuclear processes including DNA methylation, while there was a strong overlap in the HDAC1 proximal proteins identified by BioID and APEX2. Epiblast like stem cell (EpiLSC) differentiation was used to study what effect reduced HDAC1/2 levels have on an early stage of embryonic development (roughly e3.5-6.5), revealing the dysregulation of hundreds of genes even at this early time point. The degradation tag (dTag) system was used to allow for far more rapid HDAC1 protein degradation than is seen following conditional KO of Hdac1, revealing that the acute degradation of HDAC1 causes an increase in histone acetylation on specific sites within two hours. RNA-seq showed that there is largely an upregulation of gene expression seen 2 hours following HDAC1 degradation with downregulation becoming more prominent by 24 hours.

In the last decade, spatial organisation of the genome has been recognised as one of the important factors linked to gene expression and cell identity. Although we have come a long way in describing how chromatin folds - from the level of single nucleosomes all the way to compartments and chromosomal territories - it is still unclear what determines this structure and why it changes upon differentiation. Mouse embryonic stem cells provide an excellent system to address these questions, since upon exit from naive pluripotency their genome undergoes a dramatic structural reorganisation. The development of new fluorophores and imaging technologies has enabled researchers to observe single biological molecules, in both fixed and live cells. These approaches have already proven themselves to be extremely valuable in the chromatin field, for example by revealing the existence and dynamics of chromatin "blobs" (e.g. Ricci et al 2015, Barth et al 2020). This project employs the single-molecule microscopy methods to study chromatin structure and dynamics in ES cells and at the onset of their differentiation. Research conducted in the Laue lab suggested that heterochromatin might play a key role in the global rearrangement of chromatin as ES cells differentiate. Thus, the first part of the thesis focused on studying the role of heterochromatin protein 1β (HP1β) in this process. HP1β was found to increase in expression as cells exit from the ground state, and to change its distribution. Interestingly, this was concomitant with a decrease in both histone H3K9 and H3K27 methylation. 2D and 3D single-particle tracking in live cells was then employed to study HP1β and chromatin dynamics. By applying novel biophysical analysis methods to the data, features of HP1β diffusion not consistent with the current models of heterochromatin compartmentalisation were found, as well as evidence for non-equilibrium processes. Transient slowdown in HP1β diffusion upon differentiation was observed, potentially reflecting a change in the nuclear environment. However, analysis of small-scale chromatin movement showed no change in dynamics between the cell states. In the second part of this work, PALM- and PAINT-based approaches to image chromatin-binding proteins and DNA in 3D throughout the entire nuclear volume of fixed cells were developed. The microscopy protocol was designed to be compatible with single-cell Hi-C, enabling modeling of the genome structures of the imaged cells (Stevens et al 2017). Correlating the images and the structures will for the first time make it possible to obtain spatial sequence-specific information about the genome fold alongside protein localisation on a single-cell level. I hope that this method will shed light on the molecular functions of chromatin-binding proteins in their native context and help understand the rules of genome organisation.

Embryonic stem cells (ESCs) hold great potential for developing future therapies for a wide range of diseases. However, the mechanisms of pattern formation during embryonic development remain poorly understood. ESCs in culture self-organise to form spatial patterns of gene expression upon geometrical confinement indicating that patterning is an emergent phenomenon that results from the many interactions between the cells. Here, we applied an agent-based modelling approach to identify biologically plausible rules acting at the mesoscale within stem cell collectives that may explain spontaneous patterning. We tested different models involving differential motile behaviours including exploring effects due to neighbour interactions. We introduced a new metric, the stem cell aggregate pattern distance (SCAPD), to assess the deviation between the probabilistic experimental pattern formation (used as ground truth) and the probabilistic simulated outcome. We demonstrated our models can produce broadly realistic pattern formation (when compared to experimental data) with a quantified level of uncertainty. The best of our models improve fitness, evaluated by SCAPD, by 70% and 77% over the random models for a discoidal or an ellipsoidal stem cell confinement, respectively. Collectively, our findings provide compelling arguments that a parsimonious mechanism that involves differential motility is sufficient to explain the spontaneous patterning of the cells upon confinement. Furthermore, our work also defines a region of the parameter space that is compatible with patterning, which assists future studies in the field of cell engineering. We envisage that the novel approaches explored within this work will be applicable to many biological systems and will contribute towards facilitating progress by reducing the need for extensive and costly experiments.

The homeodomain transcription factor NANOG promotes self-renewal of embryonic stem cells (ESCs) and specification of primordial germ cells (PGCs). NANOG binds thousands of sites within the ESC genome together with OCT4, SOX2 and RNA polymerase II (RNAPII) regulating transcription of its target genes. Replacing tryptophans within the NANOG tryptophan repeat (WR) region for alanines (W10A) causes NANOG to lose its capacity to homodimerise and to interact with other proteins including RNAPII, SOX2 and RESF1. NANOGW10A mutant is also unable to sustain LIF-independent self-renewal. However, the requirements of the WR to interact with other proteins for NANOG function remains unclear. To characterise the cellular localisation of RESF1, endogenously tagged Resf1 mouse embryonic stem cell lines were produced. RESF1 is a widely expressed nuclear protein which interacts with NANOG in nuclei of ESCs. To quantify the requirements of Resf1 for ESC self-renewal and NANOG function, Resf1-/- ES cell lines were derived. In serum/LIF culture, Resf1 deletion promotes cell differentiation and decreases expression of Pou5f1, Nanog and Esrrb. Episomal expression of NANOG sustains LIF-independent self-renewal independently of Resf1. Resf1-/- ESCs are also capable to differentiate into epiblast stem cells. To investigate the role of Resf1 in PGC specification, Resf1-/- ESCs were differentiated into PGC-like cells (PGCLCs). Deletion of Resf1decreasedPGCLCspecification4-foldandpreventedefficientactivation of Blimp1, Prdm14 and Ap2γ. The effect of NANOG on OCT4, SOX2 and RNAPII chromatin binding was evaluated by inducing NANOG in Nanog-/- mouse ESCs. OCT4, SOX2 and RNAPII occupancy before and shortly after NANOG induction was measuredbyanalysingchromatinimmunoprecipitationfollowedbysequencing data sets. The k-mean clustering of Nanog binding sites revealed seven clusters with different response patterns of OCT4, SOX2 and RNAPII to NANOG. The sites where NANOG increases OCT4, SOX2 and RNAPII chromatin binding are mostly associate with the genes activated by NANOG, whereas sites where RNAPII binding decreases after NANOG induction associate with genes repressed by NANOG. To assess the requirements of the NANOG WR and the NANOG DNA binding for directing OCT4, SOX2 and RNAPII chromatin binding, NANOG variants with debilitating mutations in the WR or the homeodomain were induced in Nanog-/- ESCs and chromatin binding of NANOG, OCT4, SOX2 and RNAPII was assessed. The NANOGW10A has reduced chromatin binding at most of the NANOG binding sites. A reduced DNA binding capacity of recombinant NANOGW10A was also suggested by electrophoretic mobility shift assay. Moreover, NANOGW10A is unable to increase OCT4, SOX2 and RNAPII binding at the sites associated with gene activation. At the sites associated with gene repression, NANOGW10A induction leads to increased OCT4 binding. NANOG variant incapable to bind DNA (NANOGN51A) is unable to direct OCT4 and RNAPII binding. However, SOX2 binding temporarily decreases after NANOGN51A induction. In summary, Resf1 is not essential for ESC self-renewal and NANOG function but it is important for efficient specification of PGCLCs. NANOG directs the core transcription complex regulating self-renewal via the WR. This thesis contributes to understanding of NANOG function in ESC self-renewal and describes the function of previously uncharacterised RESF1 in ESC self-renewal and germ cell specification.

Primordial germ cells (PGCs), the precursors of gametes, are specified in early post-implantation human embryos, which are inaccessible for direct investigation. This process can however be modelled using human embryonic stem cells (hESCs) for in vitro derivation of human PGC-like cells (hPGCLCs). hESCs in conventional culture conditions have very low potential for germ cell fate; this increases significantly if the cells are first cultured in PGC competence-promoting media. To address how the PGC-competent state might be gained, I used a new computational tool - Branch-Recombinant Gaussian Processes (B-RGPs) - to identify potentially relevant pathways based on single-cell RNA sequencing of early human embryos, as well as gonadal PGCs and soma. I then tested these predictions experimentally by modulating the candidate signalling pathways in competent hESCs, followed by hPGCLC induction. I found that inhibition of FGF receptor (FGFR) or its downstream effectors (MEK and PI3K) enhanced competency for PGC fate. By contrast, premature MEK/ERK activation in hESCs resulted in aberrant expression of endoderm markers and loss of germ cell potential. Altogether the study showed that FGFR pathway is a negative regulator of cellular competence for human PGCLC fate. PGCLC specification from competent cells occurs in response to BMP signalling and is orchestrated by specific transcription factors (TFs). Emerging evidence points to many differences between TF networks in mouse and human PGCs. For example, SOX17 is essential for human germ cell fate, but is dispensable for mouse PGCs. On the other hand, a key pluripotency factor PRDM14 is crucial for mouse PGC specification, but its role in human PGC development is unclear. I designed genetic approaches to address PRDM14 involvement in human pluripotency versus PGC specification. To achieve inducible loss of function, I deleted endogenous PRDM14 in the background of doxycycline-repressible PRDM14 transgene expression. This approach, however, did not allow fast and homogenous control over protein levels. To circumvent this, I used CRISPR/Cas9 in conjunction with conditional degrons (auxin- and jasmonate-inducible degrons) for rapid and reversible degradation of the endogenous protein. Notably, PRDM14 depletion during differentiation significantly reduced the number of specified hPGCLCs, revealing its role in human PGC fate. Furthermore, RNA sequencing of mutant cells showed aberrant gene expression, with derepression of WNT signalling and somatic genes. Importantly, ectopic PRDM14 expression in mutant cells rescued hPGCLC specification and transcriptional changes, thus proving the specificity of the observed phenotype. Interestingly, loss of PRDM14 in hESCs had a distinct transcriptional effect, indicating a context-dependent role of PRDM14 in PGCs and pluripotent cells. ChIP-sequencing in hESCs and hPGCLCs identified PRDM14 binding to regulatory elements of key pluripotency and germ cell genes and motif analysis suggested its integration in the core TF circuitry in both cell types. Interestingly, the overlap between PRDM14 targets in mouse and human was limited. Altogether, this reveals a key role of PRDM14 in human germ cell fate and exemplifies the importance of complete and fast protein depletion in loss-of-function studies. Overall my research has furthered our understanding of signalling and transcriptional regulators controlling the segregation of human germ cells from soma. It also expanded the toolkit for protein studies in dynamic processes and uncovered hitherto unknown roles of PRDM14 in early human development.

The laboratory rat is an important experimental model in biomedical research. Rat embryonic stem cells (ESCs) are valuable for studying mammalian development and for facilitating germ line-modification of rat strains. However, the germline contribution of rat ESCs when injected into developing rat embryos can be variable, restricting their utility. This investigation addressed two key questions; could rat ESC differentiation in vitro be directed towards the germline, and could the proportion of cells entering the germ cell lineage be increased by manipulating the gene network of epiblast cells? The formation of the unipotent germline precursors known as primordial germ cells (PGCs) is induced by BMP4/WNT3-mediated induction of essential PGC transcription factors (TFs) BLIMP1, PRDM14 and AP2γ within the epiblast. These factors cooperate to direct a small subset of cells at the proximal posterior region of the epiblast towards a germ cell fate by re-activating expression of naïve pluripotency markers whilst upregulating PGC-specific transcription factors. To promote differentiation of rat ESCs into the PGC fate, cells were subjected to a differentiation protocol previously developed for mouse ES-to-PGC-like cell (PGCLC) differentiation, which was modified for rat ESCs. Using this protocol, a small proportion of rat cells expressing the PGC-associated surface marker CD61 exhibited enhanced expression of PGCLC gene markers, indicating that the differentiation protocol had generated a population of rat PGCLCs. To increase the efficiency of PGCLC differentiation, three strategies were implemented to manipulate the epiblast gene network to improve the proportion of rat ESCs entering the germline lineage. The first strategy involved the use of geneediting technology (CRISPR/Cas9), to incorporate a PGC gene cassette expressing a transgene copy of Blimp1 immediately downstream of the epiblast gene promotor of Brachyury. It was hypothesised that insertion of this gene cassette would drive expression of the Blimp1 transgene during the epiblast phase of rat ESC fate determination and improve the number of rat PGCLCs generated during PGCLC differentiation. Indeed, the proportion of cells expressing the PGC-associated surface marker CD61 was elevated compared to the parental unmodified cell line. Additionally, CD61+ve cells generated from cells containing the Blimp1 transgene displayed increased expression of early PGC markers. The second strategy involved the conditional expression of the three key PGC transcription factors, Blimp1, Prdm14 and Ap2γ from stably integrated doxycyclinedependant Tet-On transposon vectors. Stably transduced cells cultured in doxycycline during embryoid body and PGCLC differentiation protocols had a greater proportion of CD61+ve cells and increased PGC marker gene expression compared to the parental cell line. This was most noticeable in cells expressing Blimp1 and either of the other two PGC transcription factors (Prdm14 or Ap2γ). Finally, CRISPR/Cas9 gene editing was also used to inactivate the rat Otx2 transcription factor gene. Deletion of Otx2 in mouse ESCs has been shown to increase the proportion of PGCLCs generated during germline differentiation in vitro. Similar to exogenous PGC transcription factor expression, rat Otx2-/- knock-out cells generated a greater proportion of CD61+ve cells compared to the parental cell line when put through a PGCLC differentiation protocol. CD61+ve Otx2-/- cells also had increased PGC transcription factor expression compared to the parental cell line, suggesting the loss of Otx2 can help drive the expression Blimp1, Prdm14 and Ap2γ. In conclusion, rat ESCs subjected to a PGCLC differentiation protocol and manipulation of PGC transcription factor expression exhibited increased expression of PGC markers. However, this response was more limited than that obtained with mouse ESCs, a result which is consistent with reduced germ line transmission of rat ESCs in chimaeric rats. This difference could be due to the initial pluripotency state of rat ESCs; rat ESCs might be in a more committed primed state compared to naïve mouse ESCs, reducing their competence for germ cell lineage differentiation. Additionally, rat ESCs may require different combinations of signals or regulatory factors to ensure efficient germ cell differentiation in vitro. Further development of the cell lines and reagents established during this investigation should provide a better understanding of germ line differentiation in the rat.

Cellular reprogramming to pluripotency can be achieved by using a defined cocktail of transcription factors, converting differentiated somatic cells into induced pluripotent stem cells (iPSCs). These cells behave like embryonic stem cells (ES) and can be used to generate all cell types in the body. OCT4, SOX2, KLF4 and cMYC (OSKM) were the first set of reprogramming factors defined based on both their importance on maintaining pluripotency, and their ability to reprogram mouse and human somatic cells to iPSCs. Despite recent progress in refining the reprogramming technique, this process is still highly inefficient and often leads to partially reprogrammed cells. This is even more dramatic in human cells, where efficiency is low (∼0.001–1%) hindering the therapeutic prospects and promise of iPSCs and their clinical applications. Extensive studies have been focused in elucidating the molecular mechanisms by which the transcription factors contribute to reprogramming, but there is still a notable lack of understanding of the reprogramming process at the protein level. Particularly little is known about the protein network by which the reprogramming factors maintain pluripotency in human ES and drive pluripotency during the reprogramming process. Due to the relevance of OCT4 as a core transcription factor for both, the pluripotency network and the reprogramming process, the work present herein focused on studying OCT4 at the proteomic level. By exploiting proteomic approaches focused in on and off-chromatin bound proteins this work described for the first time the OCT4 protein interactors involved in early stages of reprogramming and during pluripotency. For the hES interactome, OCT4 binding partners involved in pluripotency maintenance were identified, in addition to a new set of chromatin associated proteins that have non-previously been described in the pluripotency context. On the other hand, during early reprogramming, OCT4 interactors included somatic transcription factors and other interacting proteins non-previously reported in the reprogramming context, such as proteins involved in cell death, development and differentiation. Further comparison of both interactomes revealed that the initial engagement of OCT4 with the somatic proteome is markedly different from that in ES, illustrating how OCT4 is able to change its chromatin-binding dynamics in order to stablish different phenotypes. Additionally, the analysis was expanded by applying the same on and off chromatin proteomic approaches to three OCT4 mutants bearing deletions of essential or non-essential reprogramming regions located in the transactivation domains (TAD). This allowed the identification of a unique set of seven proteins present in all the OCT4 with reprogramming capacity (WT or mutant). Interestingly, these were not associated with OCT4 in ES nor the deficient OCT4 mutant bearing a deletion of an essential reprogramming domain. This set of proteins included: UFD1L, RAI1, TNIP2, ETV4, XPO6, FBRSL1 and MCMBP and further functional analysis proved that they are necessary for the reprogramming process as their depletion had negative effects in its efficiency. Remarkably, the biological processes these proteins can be involved are quite varied, including nuclear export, signalling response, post-translational modifications recognition, transcriptional regulation, chromatin remodelling and cell cycle; evidencing the versatility of OCT4 involvement in different processes necessary to achieve the pluripotent state. Furthermore, the mutants’ analysis not only allowed the identification of important proteins for reprogramming, but also revealed that removing non-essential reprogramming domains caused more expansive engagement of OCT4 with both the somatic genome and the proteome. These results suggest that different OCT4 regions of the TAD domains contribute to its binding properties, being the essential domains more important for more specific interactions with the genome and more functional interactions with the proteome. Overall, the findings of this thesis helped to get a better understanding of the protein interactions of OCT4 by describing for the first time the OCT4 networks during reprogramming and pluripotency in human, while revealing that focusing in the properties of the transactivation domains can help understand new biochemical properties of OCT4. Finally, applying the same proteomic approach analysis to the remaining reprogramming factors, can further help to understand their molecular mechanisms, expand the reprogramming and pluripotency networks, characterize crucial interactors and, thus provide a better understanding of the proteomic molecular mechanisms. This with the ultimate goal of improving the efficiency and fidelity of human iPSCs reprogramming.

Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.

Bone morphogenetic protein 4 (BMP4) is a signaling protein involved in several key developmental processes, including gastrulation and neural induction. Gastrulation is an event that changes the topology of the embryo, organising a single layer or ball of cells into the three primordial germ layers: the mesoderm, endoderm and ectoderm. In the mouse embryo, gastrulation starts with a group of asymmetrically positioned cells expressing a group of genes that include T/Brachyury. These cells undergo a directed epithelial to mesenchymal transition (EMT) that gives rise to the Primitive Streak. BMP4 mutant mice fail to form a Primitive Streak or do so defectively. Neural induction is the process that gives rise to the brain, by ensuring that embryonic cells become neural tissue rather than mesoderm, endoderm, or other types of ectoderm. Inhibition of BMP4 signaling is required for neural induction to take place. Gastruloids are a 3D culture model that does not require BMP4 to undergo gastrulation-like events. This thesis describes the effects of BMP4 on gastruloids and on a 2D adherent cell culture model for gastrulation, demonstrating the interactions between BMP4, Nodal, Wnt, and Brachyury. It also shows that BMP4, although not expressed in gastruloids, can enhance certain phenotypes associated with gastrulation when added to the gastruloid culture medium. The results shown in this part of the thesis support the hypothesis that BMP4 acts as a permissive molecule, increasing the chances of establishing the Primitive Streak in the correct region of the embryo. The development of a new 3D culture model for neural induction is also described, as are the effects of BMP4 on this model. This newly-developed model for neural induction is used to demonstrate that BMP4 inhibits neural induction and that a BMP4 antagonist (Noggin) enhances neural differentiation in this system.

The process of generating the vast diversity of immunoglobulin receptors and secreted antibodies begins with the recombination of the joining (JH), diversity (DH) and variable (VH) genes in the immunoglobulin heavy chain locus. The ability to produce antibodies is restricted to the B cell lineage and is tightly regulated, starting with the temporal separation of the recombination process, in which DH-JH precedes VH-DHJH recombination. Successful recombination of both heavy and light chain loci results in the expression of an antigen receptor on the cell surface. Subsequent selection stages remove non‑functional and autoreactivity receptors from the final pool of antigen responding B cells that ultimately give rise to antibody secreting plasma cells. Understanding the complexity of the recombination processes and the diversity of the resulting antibody repertoire has been a major focus of academic and industrial research alike. Therapeutic monoclonal antibodies have seen many successful applications within the clinic and they constitute a billion-dollar industry. However, limitations therein have resulted in the emergence of antibody engineering approaches and the use of natural sources of alternative heavy chain only antibodies (HCAbs/nanobodies). The biotechnology company Crescendo Biologics has taken the highly desired characteristics of HCAbs a step further with the creation of a mouse platform capable of producing fully humanized HCAbs. The Crescendo platform presents a unique opportunity to expand our understanding of how mouse B cell development functions by exploiting the features of heavy chain only antibody production. Furthermore, the platform enables the expansion of our limited knowledge of the epigenetic mechanisms involved in the recombination of the human immunoglobulin heavy chain locus. Using flow cytometry, with dimensionality reduction analysis approaches, I investigated B cell development in the context of HCAbs. These studies revealed a previously uncharacterised developmentally intermediate B cell population. Due to ethical and availability limitations to studies of human bone marrow, the primary pre-selection human B cell repertoire has not been studied in detail. The isolation of several B cell developmental stages and the use of our novel DNA-based high-throughput unbiased repertoire quantification technique, VDJ-seq, allowed me to study recombination of the human IGH locus sequence and observe HCAb repertoire selection within the mouse environment. The adaptation of next generation sequencing techniques to antigen receptor repertoire quantification has provided an unprecedented insight into repertoire diversity and the alterations it undergoes during infection or ageing. Our VDJ-seq assay is unique in its ability to interrogate DNA recombinants. To expand its capabilities, I investigated several limitations of the technique, including mispriming and PCR/sequencing errors, and implemented experimental and bioinformatics solutions to overcome them, which included the creation of a comprehensive analysis workflow. Finally, I have developed and applied a novel network visualisation method for genome-wide promoter interaction data generated by promoter capture Hi-C. The availability of high quality human pluripotent stem cell datasets allowed me to utilise the new techniques to further our understanding of the dynamics of genome organisation during early human embryonic development. This visualisation approach will be directly applicable to understanding B cell development.

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.

During early embryonic development in mammals, the initially uniform cells of the naïve epiblast generate the structurally diverse and functionally specific tissues of the adult organism. The naïve epiblast expresses the cell-cell adhesion molecule epithelial (E-) cadherin, which is essential for early embryonic development. During subsequent neural differentiation in the ectoderm, E-cadherin becomes downregulated, and neuronal (N-) cadherin becomes upregulated in a process known as cadherin switching. Premature loss of E-cadherin activity leads to faster, more synchronous neural differentiation in embryonic stem cells by an unknown mechanism (Malaguti et al., 2013). Cadherins are mainly classed as adhesion molecules, but can also modulate cellular signalling by binding various growth factor receptors. This raises the possibility that cadherin switching may modulate signalling during early neural induction, thus affecting differentiation. This thesis investigates the mechanisms by which cadherin switching can bias differentiation decisions during neural induction in mouse embryonic stem (ES) cells . First, the spatio-temporal patterns of cadherins were investigated in the post-implantation embryo and during differentiation of pluripotent cells in vitro. The results of this analysis suggest that cadherin switching is initiated before the loss of pluripotency and co-occurs with lineage priming, consistent with the hypothesis that this process may bias cell fate decisions. Next, ES cells in which the N-cadherin gene was knocked in to the E-cadherin locus (NcKI) were studied. These cells displayed elevated levels of neural marker genes during differentiation, suggesting that forced, premature cadherin switching promotes neural differentiation. To determine whether this effect could be ascribed to the loss of E-cadherin or to the gain of N-cadherin, an inducible N-cadherin overexpressing ESC line was generated. Experiments with this cell line confirmed that N-cadherin alone can promote neural fate. Cadherin switching also occurs at a later developmental stage: during the maturation of bi-potent neuro-mesodermal progenitors (NMPs). Premature induction of N-cadherin was shown to bias immature NMPs towards neural differentiation, similarly to the effect of N-cadherin on pluripotent cells. To establish whether the pro-neural effect of N-cadherin was due to its adhesive function, a quantitative image analysis method was used to measure adhesion defects. Adhesion phenotypes did not correlate with the pro-neural effect observed in NcKI and N-cadherin overexpressing cells, suggesting that adhesion may not be the primary cause for the neural bias of these cells. Finally, the signalling effects of cadherin switching were studied. The loss of E-cadherin in E-cadherin null (EcKO) and NcKI cells was found to correlate with a drop in global and nuclear levels of β-catenin, a central component of the canonical WNT signalling pathway. However, these cells were found to retain WNT responsiveness, suggesting that any neural bias shown during cadherin switching is unlikely to be caused by altered WNT signalling. An mRNA-based signalling pathway analysis showed that cells overexpressing N-cadherin had reduced levels of FGF signalling cascade components at days three and four of neural differentiation. Subsequent experiments in NcKI cells showed that these cells express lower levels of FGF readouts during neural differentiation than WT cells, and that their pro-neural effect can be erased by adding FGF pathway ligands, or replicated by blocking the FGF pathway. These results suggest that the pro-neural effect observed in NcKI cells is caused by dampened FGF responsiveness. Taken together, the results presented in this thesis support a model in which N-cadherin promotes neural differentiation by dampening FGF signalling. This mechanism, rather than being an instructive cue for neural induction, is likely to contribute to the robustness of neural differentiation in the developing embryo.

Although methods of assisted reproduction are viable treatments for infertility, they are not always possible for azoospermic men. However, the use of stem cells may provide an alternative means of generating sperm for treatment. To date, primordial germ cell-like cells (PGCLCs) have been produced in culture from mouse and human pluripotent stem cells (PSCs). However, these cells cannot undergo later stages of spermatogenesis unless transplanted into the testes. The implication is that the development of functional sperm requires the testis niche, primarily established by Sertoli cells. In terms of a therapeutic solution, the generation of Sertoli cell-like cells (SCLCs) by PSC differentiation is one means of recreating the testis niche and inducing maturation among PGCLCs in vitro. Drawing from knowledge of gonadogenesis, this thesis demonstrates a method of generating SCLCs entirely in vitro. Mouse PSCs were sequentially induced to form intermediate mesoderm, SCLCs and steroidogenic cells by addition of growth factors BMP7 and FGF9, and small molecules TTNPB (a retinoic acid receptor agonist) and CHIR99021 (a GSK3 inhibitor). These cells were found to endogenously express transcription factors critical for male development. Over-expression of one factor in particular, Steroidogenic factor-1 (SF1), substantially increased induction efficiency. Intriguingly, conditioned medium derived from SCLCs was also capable of specifying germ cell-like cells from Epiblast-like cells (EpiLCs), possibly due to the presence of key inducer BMP4. Co-culture of SCLCs and EpiLCs resulted in the formation of 'testicular organoids' reminiscent of the seminiferous tubules. These organoids were even capable of upregulating meiotic marker Stra8 in the presence of retinoic acid, suggesting that subsequent stages of maturation may have been achieved. On the whole, this study constitutes a significant step towards the use of PSCs to recreate spermatogenesis and testicular cell interactions in vitro, and serves as a useful basis for further investigations into the role of SF1 in Sertoli cell differentiation and function.

The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development.

The mammalian epigenome is globally reprogrammed at two stages of development; this involves the erasure and re-establishment of DNA methylation by both passive and active mechanisms, including DNA repair pathways, and occurs concurrently with an increase in developmental potency. In addition to Uhrf1 and the Tet enzymes, the interplay between activation induced cytidine deaminase (AID) and the DNA repair machinery has been implicated in epigenetic reprogramming of various in vivo and in vitro systems including mouse primordial germ cells, zygotes and induced pluripotent stem cells. AID deaminates cytosine to uracil and can also deaminate methylcytosine, whereas the primary role of UNG is to maintain the integrity of the genome through erasure of uracil. In this thesis, I have aimed to investigate the role of DNA repair in demethylation. To do this I have focused on the specific role of AID and UNG in the demethylation of a static system – primed serum ESCs and a dynamic system – serum to 2i (naïve) to epiblast-like ES cells. As the role of both AID and UNG involves genomic uracil, the central theme of my thesis is the impact of accumulation of uracil on DNA methylation levels in the genome. Therefore, my first aim was to develop a quantitative method to detect low levels of genomic uracil in DNA firstly, by mass spectrometry and secondly, by whole genome sequencing. In Chapter Three, I show that the impact of deamination during DNA preparation can be minimised, such that the level of genomic ESC uracil can be accurately determined as around 12,000 uracil per genome and that, as anticipated, Ung null ESCs have almost twice the genomic uracil content of wildtype ESCs. Secondly, I address the main question which is the impact of uracil accumulation on methylation levels. In order to do this, I generate two cell lines: Ung knockout and Aid over expressing, both of which should result in an increase in genomic uracil. I demonstrate that while over expression of Aid stimulates demethylation in static system and in a dynamic demethylating system, the impact of Ung knockout is less clear. In (static) serum ESCs, loss of Ung results in hypomethylation however, in order to transition to 2i (naïve) ESCs, a process which involves demethylation of the genome, it appears the Ung is required as loss of this gene inhibits proper demethylation. As such, I conclude that UNG-mediated DNA repair functions alongside passive demethylation, by reduction of UHRF1 levels, to demethylate 2i ESCs. To probe the mechanism by which accumulation of uracil in the genome alters methylation levels, I investigate the impact of Ung KO and Aid OE on global levels of DNA damage. I show that both cell lines have a greater incidence of double strand breaks compared to a wild type cell line, and accordingly, upregulate their DNA damage response pathway and the expression of certain repair genes. I suggest that increasing genomic levels of uracil causes genomic instability and that DNA demethylation occurs as a consequence of the repair of extensive DNA damage. More broadly, I suggest that ESCs are uniquely poised, due to their heightened DNA damage response, to use uracil as an intermediate of DNA demethylation. Interestingly, I also note that the biological impact on serum ESCs of loss of Ung appears to be an increase in pluripotency.

Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.

This thesis presents the development of microfluidic devices designed to facilitate research into mouse embryonic stem cells (ESCs). ESCs are a well-studied cell, largely due to their pluripotent nature, meaning they are able to differentiate into all cell types of the body and may self-renew indefinitely in appropriate culture conditions. ESCs, along with many other lines of biological enquiry, are increasingly studied with the use of micro uidic technology which enables fine tuning of physical and chemical environments unachievable on the macro scale. Two varieties of microfluidic technology are presented in this thesis, one for high- resolution mechanical phenotyping of ESCs and the second as a novel in-chip culturing platform to study cellular transitions. Chapter 1 presents a broad introduction to ESCs and biological enquiry with microfluidics, aimed to underpin the following Chapters. Chapters 2 and 3 present self-contained projects, thus each include a motivation and introduction section more specific than that presented in Chapter 1. These Chapters also contain their own methods, results and conclusion sections. Finally, Chapter 4 presents a summary of the work performed along with an outlook of upcoming investigations. In Chapter 2, I present a microfluidic device developed and utilised in collaboration with Christophe Verstreken (Department of Physics, University of Cambridge), which has been used to apply a mechanical stress to live cells enabling measurement of their nuclear deformability. The device facilitates detection of both nucleus and cytoplasm which can then be analysed with a custom-written MATLAB code. Quantitative measurements of nuclear sizes and strains of ESCs indicated a negative Poisson ratio for nuclei of cells cultured in specific medium conditions. Furthermore, we demonstrate that the device can be used to physically phenotype at high-throughput by detecting changes in the nuclear response after treatment with actin depolymerising and chromatin decondensing agents. Finally, we show the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. The work from this chapter is presented in Hodgson et al. [1]. In Chapter 3, I present a novel microfluidic platform developed in collaboration with Prof. Austin Smith and Dr Carla Mulas (Centre for Stem Cell Research, Cambridge). The developed platform enables individual ESCs to be cultured under continued observation as they exit their pluripotent stem cell state. Each cell within the device may be extracted from the chip at any time for further investigation without disturbing other cells. Assessing the transition from the stem cell state in individual cells is paramount if we are to understand the mechanisms of pluripotency.

In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.

Mammalian cell types and tissues have diverse functional roles within an organism but can be derived by the differentiation of the embryonic stem cells (ESCs). ESCs are pluripotent cells with self-renewal properties. During development subsets of genes in ESCs are activated or silenced for manifestation of the cell type specific function. Gene expression changes occur transiently in early developmental stages, through signals received and executed by a variety of transcription factors (TFs), regulatory elements (promoters, enhancers) and epigenetic modifications of chromatin. Post-translational modifications of the histone tails are regulated by chromatin modifiers and transform the chromatin architecture. Polycomb (PcG) and Trithorax (TrxG) group proteins are the most commonly studied histone modifiers. They were first discovered as repressors (H3K27me3) and activators (H3K4me3) respectively of Homeobox (Hox) genes in Drosophila and they are conserved in mammals. Bivalent chromatin is defined as the simultaneous presence of silencing (H3K27me3) and activating (H3K4me3) histone marks and was first discovered as a feature of many developmental gene promoters of ESCs. Bivalent promoters are thought to be in a ‘poised’ state for later activation or repression during differentiation due to the presence of the two counter-acting histone modifications and a pausing variant of RNA polymerase II (RNAPII) accompanied with intermediate-low levels of expression. By integrative analysis of publicly available ChIP sequencing (ChIP-seq) datasets in murine and human ESCs, we predicted 3,659 and 4,979 high–confidence (HC) bivalent promoters in mouse and human ESCs respectively. Using a peak-based method, we acquire a set of bivalent promoters with high enrichment for developmental regulators. Over 85% of Polycomb targets were bivalent and their expression was particularly sensitive to TF perturbation. Moreover, murine HC bivalent promoters were occupied by both Polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions. HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters and a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Using the recent technology of single cell RNA sequencing (scRNA-seq) we focused on gene expression heterogeneity and how it may affect the output of differentiation. We collected single cell gene expression profiles for 32 human and 39 murine ESCs and studied the correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further characterized properties unique to genes with high CV. Highly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent promoters and showed enrichment for response to DNA damage and DNA repair. Bivalent promoters in ESCs grouped in four distinct classes of variable biological functions according to Polycomb occupancy and three RNAPII variants. To study the dynamics of epigenetic and transcription control at promoters during development, we collected ChIPseq data for two chromatin modifications (H3K4me3 and H3K27me3) and RNAPII (8WG16 antibody) as well as expression data (RNA-seq) across 8 cell types (ESCs and seven committed cell types) in mouse. Hierarchical clustering of 22,179 unique gene promoters across cell types, showed that H3K4me3 peaks are in agreement with the expression data while H3K27me3 and RNAPII peaks were not highly consistent with the hierarchical tree of gene expression. Unsupervised clustering of ChIP-seq and RNA-seq profiles has resulted in 31 distinct profiles, which were subsequently narrowed down to nine major profile groups across cell types. TF enrichment at individual clusters using ChIP sequencing data did not fully agree with the classification of 8 major profile groups. Considering all the above results, three major epigenetic profiles (active, bivalent and latent) seem to be conserved across the species and cell types in our study. These states could recapitulate only a fraction of the transcriptional information - adding other chromatin marks could enrich it - since they are seemingly unaffected by their respective expression profiles. H3K27me3 only state has low CpG density and shows stronger signatures at differentiated cell types. Transcriptional control is tighter in active than bivalent promoters and the different occupancy levels of PcG subunits and RNAPII can be reflected at the expression variance of bivalent genes, where a fraction of them are involved in developmental functions while others are more tissue-specific. Last, there is a striking similarity in the pausing patterns of RNAPII in the progenitor cell types, which suggests that RNAPII pausing is correlated with the developmental potential of the cell type. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during development.

There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.

The Ward group have previously demonstrated that E-cadherin regulates the naïve pluripotent state of mouse Embryonic Stem (mES) cells via activation of the LIF/STAT3 pathway. Loss of E-cadherin in mES cells leads to a 'primed' pluripotent phenotype characterized by LIF-independent self-renewal through ACTIVIN/NODAL signalling, similar to mouse Epiblast Stem (EpiS) and human (h)ES cells. However, the exact mechanism of E-cadherin's regulation of pluripotency in both mouse and human ES cells has remained elusive. In this study we describe a novel network modelling approach of transcriptomic analysis using gene expression changes shared between three independently derived E-cadherin negative cell lines (collectively termed ESDeltaEcad cells). Our models predicted regulation of pluripotency pathways via the co-activator protein EP300. We show that E-cadherin regulates the cell localisation and binding of EP300 to NANOG-OCT4-SOX2 DNA clusters. EP300 also functions to phosphorylate SMAD2 in the absence of E-cadherin and to inhibit pluripotency-associated transcripts in Ecad-/- mES cells. Using comparative network analysis of E-cadherin antagonism in mouse and human transcriptomes we have identified that E-cadherin plays a major role in regulating the transcriptional circuitry between naive and primed ES cell states. We show that this is likely to be mediated at least partly by positive activation of NCOA3 and ESRRB. Differential transcript expression in Ecad-/- mES cells correlates with loss of ESRRB protein expression and altered cellular localisation of NCOA3, both regulators of naive pluripotency. We have gone on to show through peptide antagonism that while E-cadherin is an important upstream regulator of genes critical in defining the naïve pluripotent state, this action is not necessarily dependent on the loss of cell-cell contact associated with E-cadherin inhibition. We present the first data that E-cadherin regulates naïve mES cell pluripotency by regulation of EP300, ESRRB and NCOA3 activity. Furthermore we have shown E-cadherin to inhibit the naïve transcriptional phenotype in hES cells. Peptides that target E-cadherin also provide a valuable tool for controlling the transition between naive and pluripotent states in mouse and human ES cells.

TGFβ signalling via Smad transcription factors is essential for axis patterning and subsequent cell fate specification during mammalian embryogenesis. However, the cellular and molecular mechanisms have been difficult to characterise in vivo due to early embryonic lethality of mouse mutants and redundant functional activities. Here I show that combined deletion of closely related Smad2 and Smad3 in mouse embryonic stem cells impairs induction of lineage specific gene expression during differentiation, while extra-embryonic gene expression is up-regulated. Preliminary data suggest that the underlying mechanism of this differentiation defect reflects the inability of Smad2/3-/- cells to establish lineage priming. Collectively, these findings identify novel downstream target genes controlled by Smad2/3 and an absolute requirement for Smad2/3 during embryonic differentiation. TGFβ signalling via Smad1 and Smad4 is essential for induction of the transcription factor Blimp1 required for primordial germ cell specification. The direct upstream regulators of Blimp1 are unknown, but T-box factors have recently been suggested to play a role. In a second project, I performed tissue- specific ablation of the T-box transcription factor Eomes as well as components of the TGFβ signalling pathway in either the visceral endoderm or the epiblast to examine tissue-specific functions for Blimp1 induction. I show that Eomes and Smad2 functions in the visceral endoderm as well as Eomes function in the epiblast are dispensable for Blimp1 induction, but rather are required to restrict Blimp1 induction to posterior epiblast cells. In contrast, epiblast-specific Smad4 or Smad1 mutants fail to robustly induce Blimp1 in the epiblast. My preliminary analysis suggests that competence to induce primordial germ cell fate is dependent on the interplay of Smad2/Eomes functions in the visceral endoderm and Smad1/4 functions in the epiblast. Collectively, this thesis provides insight into the transition from pluripotency to cell fate specification in the mammalian embryo that is impossible to obtain from human embryos in vivo.

Red blood cell (RBC) transfusion is the major treatment for patients suffering from trauma or severe anaemias, and life-long transfusion may be needed to alleviate symptoms and maintain body functioning. However, with a relatively low portion of people are donating, shortage in blood supply is becoming a life-threatening issue in the aging society. Among attempts to identify novel sources for transfusion medicine, human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are currently the most promising candidate, which is capable of producing donor-independent, pathogen-free and immunologically compatible RBCs. Currently, hESC-derived erythropoiesis in vitro is considered to mimic the very primitive yolk sac haematopoiesis, indicated by a low or absent level of β globin production and incomplete enucleation. Thus these cells are not mature enough to be used in transfusion medicine. The aim of this PhD project was to overexpress a key erythroid transcription factor, Erythroid Krüppel-like factor (EKLF or KLF1) in an inducible manner to improve the maturation of hESC-derived erythroid cells. EKLF is a member of the Krüppel-like factor family, which is characterized by three C2H2 type zinc finger motifs. EKLF expression in vivo is highly restricted to erythroid cells in yolk sac, fetal liver, spleen and the bone marrow, although recently a low-level of expression was found in haematopoietic precursors. Published reports demonstrate that EKLF can 1) activate β globin expression by binding to the CACCC box in its promoter or by altering β-like globin locus chromatin structure; 2) exert a role in MEP (common progenitor for erythrocytes and megakaryocytes) stage by favouring erythroid differentiation against megakaryocyte differentiation; 3) promote enucleation by affecting the DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island; 4) act as an instructive factor for lineage commitment towards erythroid fate in HSCs. In this project, 1) We tested and evaluated a feeder-free, serum-free differentiation system for deriving erythroid cells from hESCs; 2) We constructed constitutive and inducible EKLF expression vectors and validated them in K562 cells; 3) We generated hESC lines carrying these EKLF expression vectors and assessed their effects on erythrocyte production and maturation. We found that our differentiation system was capable of generating haematopoietic progenitors (HPCs) and erythroid cells at high efficiency. Using this differentiation system, we concluded that enhanced expression of EKLF upregulated adult β globin expression selectively, without altering expressions of other globins. This finding provides hints for the development of novel approaches to “reprogramme” hESCs towards a certain fate and overexpression of EKLF in this differentiation system may be beneficial for resolving issues in future transfusion medicine.

Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC.

Medium spiny neurons (MSNs) play an important role in locomotion. Counterbalance between two MSN subtypes, enkephalin-positive and substance P-positive MSNs, is crucial for maintaining normal movement. Preferential degeneration of enkephalinergic MSNs in early stage Huntington’s disease (HD) contributes to abnormal involuntary movement called chorea. The reasons for this selective vulnerability are unknown. In vitro differentiation of pluripotent stem cells (PSCs) to neuronal cells is considered a potential approach for modelling neurodegenerative disorders including HD. Generation of PSC-derived enkephalinergic MSNs would be an ideal tool for dissecting their preferential degeneration. However, an enkephalinergic phenotype has never been reported in PSC-derived MSNs. We, therefore, have generated a mouse embryonic stem cell (mESC) reporter line that expresses enhanced yellow fluorescent protein (EYFP) when the cells are committed to an enkephalinergic fate. Characterisation of this mESC line via chimaera generation showed that all EYFP-positive cells were also enkephalin-positive. We have then optimised an enkephalinergic neuronal differentiation protocol using this ESC line. Interestingly, we found that a combination of Wnt inhibitor Dickkopf-related protein 1 (DKK1), sonic hedgehog (Shh) and brain-derived neurotrophic factor (BDNF), commonly used in addition to basal medium for deriving MSNs from PSCs, had a detrimental effect on enkephalin expression. Absence of these three factors, surprisingly, did not reduce the potential of ESCs to become MSNs nor did it affect the electrophysiological properties of ESC-derived MSNs. Further investigation revealed that Pre-pro-enkephalin is down-regulated in the presence of exogenous DKK1 and/or Shh but not in the presence of BDNF. We, therefore, propose that addition of exogenous DKK1 and Shh is unfavourable to derive enkephalinergic MSNs from mouse ESCs. These findings could be used to derive enkephalinergic MSNs in vitro allowing the disease in a dish approach for HD modelling.

Oct4, a transcription factor belonging to the fifth class of POU proteins (POUV), plays essential roles in the maintenance of pluripotency, differentiation and the generation of induced pluripotent stem cells (iPSCs). Oct4 regulates two levels of pluripotency, which are distinguished by their gene expression profiles and epigenetic status, namely the naïve and primed state of pluripotency. Embryonic stem cells (ESCs) and embryonic germ cells (EGCs), which are isolated from inner cell mass and primordial germ cells in the embryo, respectively, are in vitro models in which the naïve state is propagated through self-renewal. Epiblast stem cells (EpiSCs) and traditional human ESCs have gene expression profiles that are closest to the post-implantation epiblast, which is closer to embryonic differentiation, and exhibit a primed state of pluripotency. As Oct4 is important for pluripotency in all these cell types, where it regulates different targets, it appears to have two distinct sets of functions, namely germ cell/naïve ESC-like activity and epiblast/primed pluripotency-like activity. Based on protein sequences and syntenic gene analysis, Oct4/POUV homologs of jawed vertebrates can be classified into two subfamilies: POU5F1 and POU5F3, which are thought to originate from a genome duplication event that occurred in a common ancestor. Most extant vertebrates have lost one of these paralogs, while a small fraction, including coelacanths, axolotls, turtles, and marsupials, retains both POUV forms. In my thesis, I investigated the gene duplication event that underlies divergence of POU5F1 and POU5F3 in both expression pattern and specialised function. In particular, I focused on species that have retained both genes and asked whether POUV functional divergence correlates with ancestral origin. To test the function of POU5F1 and POU5F3, I substituted endogenous mouse Oct4/Pou5f1 with different POUV proteins using a cell line in which endogenous Oct4 expression can be silenced with tetracycline (ZHBTc4). Results showed that POU5F1 proteins had a greater capacity to support naïve ESC pluripotency and self-renewal than POU5F3 proteins. Global transcriptome analysis of the POUV-rescued ESC lines revealed that coelacanth POU5F1 protein regulates gene expression in a similar manner to mouse Oct4, in that genes involved in stem cell maintenance, reproduction and development are upregulated in ESCs rescued by POU5F1, but not POU5F3. Coelacanth POU5F3 rescued lines, however, expressed genes involved in various cell differentiation programs, including cell adhesion (e.g. E-cadherin and N-cadherin). This suggests that POU5F3 plays a role in primed pluripotency, while POU5F1 regulates naïve pluripotency. However, there is one POU5F3 factor that rescues ESCs like Oct4, the Xenopus gene Xlpou91 (Pou5f3.1). In Xenopus, a further duplication of POU5F3 gene enabled specialization, and Xlpou91 is expressed specifically in the primordial germ cells. Xlpou25 (Pou5f3.2) exhibits epiblast-specific activities and lacks the capacity to maintain naïve ESC pluripotency, similar to other POU5F3 proteins. This functional distinction between the different Xenopus POUV paralogs enabled us to address how specific Oct4 functions (germ cell-like versus epiblast-like activity) are related to the induction of pluripotency. To address this question, mouse Oct4 was replaced by either Xlpou91 or Xlpou25 in murine cellular reprogramming using a Nanog-GFP reporter line to monitor iPSC generation. Results showed that Xlpou91 and mouse Oct4 were required at similar levels to reprogram somatic cells toward iPSCs and reprogrammed cells emerged with similar kinetics. Conversely, Xlpou25 was required at higher expression levels and the resulting iPSCs appeared at a later timepoint, while the pluripotent population in these cultures appeared to be less stable and more prone to differentiate. I found that this phenotype of enhanced differentiation in Xlpou25 reprogrammed cultures may be a product of a different set of immediate early genes induced at the first stages of differentiation. Global transcriptome analysis of the naïve ESC-like pluripotent subpopulation of these iPSC lines confirmed the capacity of all Xenopus POUVs to drive reprogramming towards the pluripotent state. However, the gene sets induced by both Xlpou91 and mouse Oct4, but not Xlpou25, were somewhat enriched for genes involved in reproduction, emphasizing the segregated role of Xlpou91 as a germ cell specific POUV protein. Lastly, I explored the evolutionary origin of these two POUV paralogs and attempted to identify a POUV-related gene in jawless vertebrate (cyclostomes). Based on in silico analysis of genomic and transcriptome databases, my collaborators and I were able to identify a single POUV gene in the Japanese/arctic lamprey, thus providing the first insight into the origin of gnathosome POUV genes.

Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex. In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.

Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture.

Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.

DNA methylation refers to an addition of a methyl group to the 5 position of the cytosine pyrimidine ring. As the best characterized epigenetic mark, DNA methylation plays an important role in a plethora of biological functions, including gene repression, genomic imprinting, silencing of retro-transposons and X chromosome inactivation. Genomic imprinting refers to the mono-allelic expression of certain genes according to their parent-of-origin. In mammals, the expression of imprinted genes is controlled by the cis-acting regulatory elements, termed imprinted control regions (ICRs). ICRs are marked by parent-of-origin-specific DNA methylation and loss of DNA methylation at ICRs also causes aberrant expression of imprinted genes. Therefore it is believed that the genomic imprinting is a DNA methylation-associated epigenetic phenomenon. As accurate expression of imprinted genes is essential for normal embryonic growth, energy homeostasis, development of the brain and behaviour and abnormal expression of imprinted genes leads to numerous clinical phenotype and human disorders, it is important to investigate how the imprinted DNA methylation is stably maintained in mammals. DNA methyltransferases (DNMTs) are the main enzymes that play a in the establishment and maintenance of imprinted DNA methylation. In primordial germ cells (PGCs), DNMT3A and DNMT3L are involved in the establishment of imprinted DNA methylation. Whereas once established, the imprinted DNA methylation is maintained by DNMT1, DNMT3A and DNMT3B, but mainly by DNMT1. In addition, some other enzymes and DNA binding proteins also play a role in this process. One of the best examples is ZFP57, which forms a complex with KAP1 and SETDB1. ZFP57 maintains imprinted DNA methylation by recognizing a methylated hexa-nucleotide and recruits DNMTs to the ICRs in mammalian embryonic stem (ES) cells. Interestingly, DNA methylation analysis combined with promoter microarrays carried out in our lab suggested that imprinted DNA methylation is absent from some of the maternal ICRs in ES cells genetically null for G9a, a histone H3 lysine 9 methylase. This indicates that G9a might also play a role in the maintenance of imprinted DNA methylation. In my work, I found that the repressive H3K9me2 and imprinted DNA methylation are absent from several analysed ICRs in embryonic stem (ES) cells genetically null for either G9a or its partner histone methyltransferase GLP. A knockdown of G9a in ES cells reproduced these observations suggesting that G9a/GLP complex is required for the maintenance of imprinted DNA methylation. I also found that neither wild type nor catalytically inactive G9a can restore the loss of imprinted DNA methylation in G9a-/- ES cells. Chromatin immunoprecipitation (ChIP) combined with bisulfite DNA sequencing showed that imprinted DNA methylation was present on the H3K9me2-marked allele indicating a direct role for G9a in maintenance of genomic imprinting. Using a pharmacological inhibitor of G9a and mutagenesis analyses, I found that G9a maintains the imprinted DNA methylation independently of its catalytic activity and recruits DNMTs to the ICRs via its ankyrin repeat domain. Dimerization of G9a with GLP is also essential for the maintenance of genomic imprinting in ES cells. In summary, in addition to establish H3K9me2, histone methyltransferases G9a and GLP also play an essential role in the maintenance of genomic methylation imprints in ES cells.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

The transition from pluripotency to differentiation is a key event in the life of all complex multicellular organisms. In the development of the mouse, the pluripotent epiblast undergoes gastrulation and gives rise to three multipotent germ layers, which will in turn form the tissues of the adult body. The events leading up to gastrulation have been extensively studied in vivo in developing embryos, and modelled in vitro making use of embryonic stem (ES) cells. Bone morphogenic protein (BMP) signalling plays a key role in these processes. BMP can in fact maintain ES cells in a self-renewing state by inhibiting their differentiation into neural ectoderm, whilst at the same time being required for the specification of mesoderm in the developing embryo (Winnier et al. 1995, Ying et al. 2003a). A key intracellular target of BMP is the transcriptional regulator Id1, which can recapitulate the effects of BMP in the preservation of ES cell pluripotency and in the inhibition of neural specification from pluripotent cells (Ying et al. 2003a). This thesis will focus on understanding the roles of this molecule in the early decisions affecting the transition from pluripotency to differentiation. In particular, I aim to study the expression pattern of Id1 in cultures of pluripotent cells, and to clarify which extracellular and intracellular molecules regulate the expression of the factor; I aim to understand how forced Id1 expression inhibits the differentiation of pluripotent cells, and whether Id1 may play a similar role in the regulation of the asynchronous exit from pluripotency observed in differentiating wild-type cells; finally, I aim to characterise the expression pattern of Id1 in the early stages of post-implantation development at the single-cell resolution, and to understand how the expression of the molecule correlates with the previously characterised expression patterns of key signalling molecules and transcription factors. The generation of a reporter ES cell line expressing the yellow fluorescent protein Venus fused to the C-terminus of Id1 allowed me to assess the expression of the factor in culture on a single-cell basis, making use of immunofluorescence and flow cytometry. I observed that expression of Id1 is reliant on active BMP signalling and low Activin/Nodal signalling, and I characterised the combinatory effects of the two pathways on Id1 expression. Furthermore, I demonstrated that high Nanog expression is incompatible with high Id1 expression in ES cell cultured in the presence of LIF and serum, which raises the possibility that Nanog may be affecting the expression of Id1 in vivo, both in pre-implantation and in post-implantation embryos. I generated ES cell lines overexpressing Id1 and observed that the factor inhibits differentiation of pluripotent cells into neural ectoderm by delaying their exit from a post-implantation epiblast-like pluripotent state, and ultimately favouring mesodermal specification. This suggests that Id1 is acting at a specific stage of differentiation and that the differentiation process itself is following a similar developmental pathway to what is observed in the peri-gastrulation stage embryo. I performed single-cell transcriptional analysis on differentiating wild-type ES cells and observed that Id1 is not expressed at an appropriate point in time to affect the asynchronous the exit from pluripotency observed in neural adherent monolayer differentiation, which suggests that other factors must be responsible for this phenomenon. Finally, I addressed the expression pattern of Id1 protein in the embryonic tissue of gastrulating mouse embryos by imaging chimaeric embryos generated using the Id1- Venus reporter ES cells. I observed that Id1 is expressed in the proximal regions of streak stage embryos; in the epiblast and migrating mesendoderm of bud stage embryos; in cardiac, lateral and allantoic mesoderm and in foregut endoderm in headfold stage embryos. These expression patterns fit with the reported expression of BMP molecules at these stages of development, and suggest that Id1 expression is primarily dependent on BMP expression in early post-implantation embryos. However, I also observed Id1 expression in a ring of cells surrounding the node in headfold stage embryos, a previously uncharacterised expression pattern not directly attributable to BMP expression. This raises the intriguing question of what is regulating Id1 expression and what roles Id1 may be playing in this key embryonic structure.

Embryonic stem (ES) cells are cell lines isolated from the embryo at a time just prior to implantation into the uterus. In the right cocktail of medium and cytokines, these cell lines can be maintained indefinitely in vitro in a self-renewing state. Initially it was assumed that these cells represented a homogeneous population however, more recently it has been shown that there are a great number of genes that are expressed heterogeneously. ES cell cultures are therefore a mix of different subpopulations, some of which have distinct functional properties including a bias or ‘lineage priming’ towards a particular cell fate. These populations are also dynamic in nature, converting from one state to another with fairly rapid kinetics. The main focus of this thesis was to gain a more in depth understanding of the mechanisms regulating heterogeneity and lineage priming in murine ES cells by asking which signalling pathways play a role in this phenomenon and how the switch between states is regulated at a transcriptional level. These questions were asked using an ES cell line containing a sensitive reporter for the endoderm marker Hex. This reporter, developed by a previous lab member, allowed the identification and separation of a population of ES cells primed towards a primitive endoderm fate. Primarily, I assessed the effect of a defined culture system (2i) on the Hex-expressing population. This culture system contains inhibitors that block FGF signalling and the Wnt pathway component GSK3. Culturing ES cells in 2i has been suggested to generate a more homogeneous culture. Here, I have shown that culturing ES cells or pre-implantation embryos in 2i did not eliminate heterogeneity but maintained them in an early state prior to lineage segregation. When ES cells were cultured in standard serum-containing medium, Hex was expressed in a mutually exclusive manner with the embryonic marker NANOG, while in 2i a subpopulation of cells coexpressed both Hex and NANOG. This population was functionally primed towards extraembryonic endoderm and trophoblast. Furthermore, these ES cells could efficiently contribute to 2-cell embryos in chimaera assays. LIF signalling promoted this population through the JAK/STAT pathway. I then asked how transcription was regulated during the switch between unprimed ES cells to those primed towards a primitive endoderm fate, as well as how regulation changes during further differentiation. To ask this, Hex positive (primed) and negative (unprimed) ES cell populations were sorted as well as a Hex positive differentiated sample. These samples were analysed by GRO-seq to determine the location, density and orientation of RNA-polymerase throughout the genome. Changes in gene expression between primed and unprimed states were regulated primarily through elongation whereas genes upregulated during differentiation were regulated at the point of de novo initiation.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1).

The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.

Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.

Polycomb complexes act as repressors of transcription at many developmentally important genes. Repression by polycomb-repressive complex 2 (PRC2) is mediated by the deposition of a tri-methylation mark on lysine 27 of the histone H3 tail (H3K27me3). The PRC2 complex consists of three core proteins: EED, SUZ12, and one of either EZH2 or EZH1, which contain the enzymatic activity. The zinc finger containing protein AEBP2 (AE1 binding protein 2) has been co-purified with the PRC2 complex. The aim of this thesis was to study the function of AEBP2. Chromatin immunoprecipitation experiments indicate that AEBP2 is enriched at the same genomic locations as H3K27me3 genomewide. Using an X inactivation model, I found that AEBP2 and H3K27me3 also co-localise by immunofluorescence upon Xist induction. Large scale immunoprecipitation followed by mass spectrometry confirmed the interaction of AEBP2 with PRC2 complex members. Interestingly, I did not detect any peptides of the Polycomblike (PCL) family proteins. These proteins have previously been shown to interact with the PRC2 complex. Their absence in the AEBP2 immunoprecipitation, together with published work demonstrating an absence of AEBP2 in PCL purifications, suggests that AEBP2 and PCL family proteins might be mutually exclusive in PRC2 complexes. I have created an embryonic stem cell line in which both alleles of Aebp2 contain a gene-trap. This cell line was used to address the suggestion that AEBP2 might use its zinc finger domain to act as a sequence-specific PRC2 recruitment factor. AEBP2-depleted cells do not show diminished recruitment of PRC2. Additionally, electromobility shift assays failed to show that AEBP2 could bind DNA. Furthermore, recruitment of AEBP2 to genomic targets depended on the presence of the core PRC2 protein EED. Together, this makes it unlikely that AEBP2 acts as a sequence-specific PRC2 recruitment factor. In keeping with the lack of effect on PRC2 enrichment, preliminary experiments have shown that AEBP2 depletion does not greatly affect levels of H3K27me3, or the expression of PRC2 target genes. The Aebp2 gene-trapped cell line will be used to investigate further functions of AEBP2. Additionally, these cells have been used for blastocyst injection to create an Aebp2 gene-trapped mouse. Future work will show the effect of AEBP2 depletion on development.