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Spatial-modulated cells of the medial entorhinal cortex (MEC) and neighbouring cortices are thought to provide the neural substrate for self-localisation behaviours. These cells include grid cells of the MEC which are thought to compute path integration operations to update self-location estimates. In order to read this grid code, downstream cells are thought to reconstruct a positional estimate as a simple rate-coded representation of space. Here, I show the coding scheme of grid cell and putative readout cells recorded from mice performing a virtual reality (VR) linear location task which engaged mice in both beaconing and path integration behaviours. I found grid cells can encode two unique coding schemes on the linear track, namely a position code which reflects periodic grid fields anchored to salient features of the track and a distance code which reflects periodic grid fields without this anchoring. Grid cells were found to switch between these coding schemes within sessions. When grid cells were encoding position, mice performed better at trials that required path integration but not on trials that required beaconing. This result provides the first mechanistic evidence linking grid cell activity to path integration-dependent behaviour. Putative readout cells were found in the form of ramp cells which fire proportionally as a function of location in defined regions of the linear track. This ramping activity was found to be primarily explained by track position rather than other kinematic variables like speed and acceleration. These representations were found to be maintained across both trial types and outcomes indicating they likely result from recall of the track structure. Together, these results support the functional importance of grid and ramp cells for self-localisation behaviours. Future investigations will look into the coherence between these two neural populations, which may together form a complete neural system for coding and decoding self-location in the brain.

Lymphocyte development and activation are critical for the generation of effective protective immunity. Innate lymphoid cells and T cells play central roles in orchestrating bespoke immune reactions by producing specialised repertoires of immuno-regulatory cytokines. ILC2 and natural killer (NK) cells are generated in the bone marrow and thymus, whilst T cell development is restricted to the thymus. Once in the tissues, these cells respond to microenvironmental cues that refine their responses, for example, via receptor-mediated cell-cell contacts, soluble cytokines and neuropeptides. The co-development of ILC2, NK cells and T cells within a 5-day window in the foetal thymus provides a tractable system for studying their genesis and differentiation. Using combinatorial multicolour transcription factor reporter mouse lines, flow cytometry and confocal imaging I investigated the temporal stages of thymic innate lymphoid cell development. I found that NK cell progenitors (NKp) were first visible at embryonic (E) day E13.5, followed by the appearance of ILC2 progenitors (ILC2p) and developing T cells. Notably, the NKp and ILC2p formed clusters in the vicinity of medullary thymic epithelial cells (mTEC). To provide greater molecular insight into this cellular niche, single-molecule RNA fluorescence in situ hybridisation (smFISH) was performed on E17.5 foetal thymus to visually identify gene expression of 100 genes with single-cell resolution. This highlighted potential foetal thymus niche factors that may support innate lymphoid cell development. In the periphery ILC2, NK cells and T cells can respond to niche-derived factors to help initiate or potentiate immunity. Recent reports have highlighted the importance of tissue-resident stromal cells and neurons in this process, especially for ILC2. By analysing bulk RNAseq gene expression data, I identified Mc5r, encoding the fifth melanocortin receptor (Mc5r), as expressed more highly by ILC2 than ILC3. Treatment of mice with the neuropeptide alpha-melanocortin-stimulating hormone (αMSH), an agonist for melanocortin receptors, induced increases in the proportions of lung NK cells and T cells. The generation of a mouse line in which Mc5r was deleted in all lymphocytes (Il7rCreMc5rfl/fl mice) confirmed that Mc5r is required for normal NK cell and T cell representation in the lung at basal state, after IL-25 or IL-33 alarmin challenge and after viral respiratory infection of mice by the Pneumonia Virus of Mice.

Circadian rhythms are ubiquitous throughout biology. In mammals, these rhythms are centrally orchestrated by the master pacemaker, the suprachiasmatic nuclei (SCN), which aligns rhythms in internal physiology with the external geophysical time. In addition, there are other brain structures outside of the SCN which are also capable of generating endogenous oscillations in gene expression and neuronal activity, and thus control the timing of diverse processes such as mood regulation, rest-activity cycles, feeding behaviour and other aspects of the brain's reward system. Interestingly, accumulating evidence suggests that smoking behaviours are also modulated by the time of day, indicating that the circadian system might be controlling the endogenous cholinergic pathways underlying these behaviours. Therefore, to investigate this possibility, the overall aim of this thesis was to explore how the time of day impacts the key brain circuitry mediating nicotine's effects, specifically within the medial habenula (MHb). We began by investigating daily variation in behavioural responses to nicotine in rats. We found that there was significant diurnal variation in nicotine-evoked changes in locomotor behaviour which appeared to be dependent on the level of arousal of the animal. These experiments support the idea that there is a rhythmic component to the circuitry underlying nicotine responses. The next aim was to explore circadian variation in cholinergic signalling at the network level. These studies focussed specifically on the MHb, a small structure of the epithalamus, which is implicated both as a critical mediator of smoking behaviours and has also been identified as a potential circadian oscillator. We explored circadian variation in mouse MHb neuronal activity and cholinergic signalling through the use of both ex- and in vivo electrophysiological approaches. Ex vivo, we found evidence of rhythmicity in spontaneous firing activity, even in the absence of SCN input, and diurnal variation in responses to nicotine. We further confirmed rhythmicity in MHb neuronal activity in vivo, and revealed that MHb cholinergic neurons integrate both circadian and photic information, which could contribute to the rhythmic drive in nicotine addiction behaviours. Finally, we investigated the molecular mechanisms driving the diurnal variation in MHb properties. We generated a diurnal transcriptomic profile of mouse MHb tissue, and whilst we did not find evidence for robust rhythms in molecular clock gene expression, we did however find a group of rhythmic transcripts which were associated with hormone signalling and mechanisms regulating synaptic plasticity that may underlie rhythms in MHb function. Overall the results presented in this thesis indicate that there is certainly rhythmic control of rodent MHb activity, response to nicotine and cholinergic output. This highlights the MHb as a potential site for diurnal modulation of goal-directed behaviours including those related to nicotine addiction, which has implications both for the treatment of nicotine addiction as well as other mood disorders which might be modulated through this structure.

The intestinal epithelium is vital for maintaining immune homeostasis. The maintenance of homeostasis requires the host immune system to be able to differentiate between healthy, homeostatic tissue and commensal microorganisms, and danger from pathogens or host tissue damage. Damaged or dying cells release endogenous molecules termed alarmins, such as ATP. ATP is a ligand for the members of the P2 family of purinergic receptors. P2X and P2Y receptors are widely expressed in immune and non-immune cell types in humans and mice, and mediate a range of normal functions, as well as inflammatory responses. However, the function of these receptors differs between macrophages and intestinal epithelial cells, and relatively little is known about ATP sensing in gut epithelium and published studies often conflict. This thesis sought to characterise mouse intestinal epithelial cell expression of the P2 family of receptors, and the downstream function of ATP signalling in these cells. The first aim investigated ATP sensitivity in epithelial cells and expression of P2X receptors. In contrast to macrophages, IECs did not upregulate Nlrp3, Il18 or Il1β in response to LPS priming and were not susceptible to P2X7-mediated cell death. P2X7 expression was not found in IECs, and while P2X4 was expressed, it likely did not contribute to ATP-induced calcium mobilization. However, an antagonist of activity downstream of P2Y suggested that ATP-induced calcium mobilization may be mediated by the P2Y family of receptors. The second aim and results chapter further charted purinergic receptor expression and focused on P2Y in IECs. IECs expressed multiple P2Y receptors, but most prominently the ATP and UTP receptor, P2Y2. Calcium mobilization assays with antagonists suggested that P2Y2 may be, at least in part, responsible for IEC responses to ATP. The final aim then sought to assess the outcome of ATP, ADP and UTP signalling in IEC. ATP stimulation significantly contributed to LPS-induced transcription of Il6 mRNA. In addition, ATP and ADP induced significant upregulation in the transcription of the chemokines Cxcl1 and Ccl2. LPS-primed IECs, stimulated with ATP, ADP and UTP, showed significantly upregulated Cxcl1, Cxcl2, Ccl2, and Cxcl10 transcription compared to control cells, although nucleotide treatment of LPS-primed cells did not significantly increase transcription compared to LPS priming alone. While LPS did induce significant increases in CXCL2 protein secretion, significant increases were not observed in CXCL1, IL6, or CCL5 in a short-term (2-hour) protein assay. ATP signalling has been linked to cell proliferation and was also shown to increase IEC proliferation in a dose-dependent manner. Taken together, these findings suggest that P2Y receptors are involved in mediating intestinal epithelial responses to ATP signalling, leading to the upregulation of pro-inflammatory cytokines and proliferation. The results in this thesis suggest that intestinal epithelial cells respond to ATP detection by promoting immune cell recruitment to sites of damage and injury, whilst promoting epithelial renewal and repair.

Adult behaviour and cerebral cell densities after maternal or post-weaning consumption of an obesogenic diet in mice Emily Jane Mort Maternal obesity during pregnancy impairs cardiometabolic health of the adult offspring in experimental animals, and is associated with behavioural and cognitive impairments in human populations including increased rates of autism, attention deficit disorder and schizophrenia. Obesity in postnatal life has also been linked with increased risk taking behaviour and depression in teenagers and adults. Identifying whether these neurological changes are due to the pre- or postnatal environment is difficult in humans but is possible in experimental animals like mice. This study examined the independent effects of inducing maternal and post-weaning obesity by feeding a high fat, high sugar diet (HFHS) on the adult behaviour and brain cell densities of mice. Under the Animal (Scientific Procedures) Act, C57BL6/J female mice were fed either a control (Con, 11%kcal fat, 7%kcal simple sugars) or HFHS diet (37.9%kcal fat, 33.0%kcal simple sugars) from 6 weeks before pregnancy and throughout pregnancy and lactation. Offspring from HFHS fed mothers were weaned onto the control diet (HFHSC) while a subset of control offspring were weaned onto the HFHS diet to create a post-weaning obesity group (CHFHS).The HFHS diet significantly increased body fat percentage of the females before pregnancy (Con 20.7±0.6% vs. HFHS 32.3±2.0%). A subset of pregnant dams were killed at day 18.5 of pregnancy for fetal tissue collection while the rest delivered naturally, with litters reduced to 6 at postpartum day 2. Behaviour of male and female 12 weeks old offspring was tested using an elevated plus maze (EPM for anxiety), open field (for anxiety and locomotion), social interaction (for social preference) and novel object recognition (for object recognition memory) tasks. At 14 weeks of age, the offspring were killed and their brain collected for regional analysis of neuron and glia densities. Behavioural and cell density data were analysis by two-way ANOVA with post hoc Sidak's multiple comparison test or by unpaired t-test, where appropriate. Data expressed as mean ±SEM.  At day 18.5 of pregnancy HFHS pups were growth restricted relative to controls, with this restriction maintained until postnatal day 7, after which HFHS offspring displayed catch up growth. Maternal consumption of the HFHS diet led to increased adiposity and reduced adrenal gland mass in adult male HFHSC offspring. CHFHS mice significantly increased body mass and adiposity while adult male CHFHS mice reduced adrenal gland mass. No difference for brain mass was seen in either fetal or adult HFHSC or CHFHS mice. Maternal and post-weaning obesity reduced anxiety within the EPM task and impaired object recognition memory. Post-weaning obesity also resulted in increased social interaction for male and female mice as well as reducing locomotion in males. Reduced Iba1+ microglia density was found within the basolateral amygdala for both HFHSC and CHFHS offspring, as well as reduced NeuN+ neuron density for HFHSC offspring and CHFHS females accompanied by increased GFAP+ astrocyte density within the perirhinal cortex for HFHSC and CHFHS females. Overall, consumption of the HFHS diet post-weaning led to a larger range of adult behavioural changes and more robust alterations to cerebral cell densities than maternal consumption alone.

The most powerful cell in the blood differentiation hierarchy is the haematopoietic stem cell (HSC). It is the only cell capable of building an entire haematopoietic system from scratch, i.e. long-term (LT) repopulating a multilineage blood system within a lethally irradiated recipient. Though primitive blood is generated at early embryonic stages, it is only from embryonic day (E)10.5 of mouse development (4-5 weeks in human gestation) onwards that the definitive adult haematopoietic system is established with the formation of LT-HSCs. These first HSCs emerge in small ventral intra-aortic haematopoietic clusters (IAHCs) in the aorta-gonad-mesonephros (AGM) region. Like with many systems, a tight regulation of gene expression by master transcription factors (TF) is what ultimately drives cell fate change. Gata2 is one such TF known to play a crucial role in mouse definitive haematopoiesis. Through the work of de Pater et al. (2013) (Chapter 1) it became clear that Gata2 is both required for the generation of the very first HSCs and also, unlike other key TFs like Runx1, the survival of HSCs. The earliest HSCs are generated through an endothelial-to-haematopoietic transition (EHT), a process where flat arterial haemogenic endothelial cells (HECs) round up into IAHCs, containing HSCs. Solaimani Kartalaei et al. (2015) (Chapter 4) discovered that Gpr56 is one of the most highly upregulated genes during EHT (in mouse) and that it is essential for HSC generation (in zebrafish). Gpr56 is also a target of the key haematopoietic 'heptad' TFs in both mouse and human blood progenitors. To examine another signalling pathway required in the embryo for HSC generation, we used a BMP responsive element (BRE)-GFP transgenic mouse to show that all AGM HSCs are BMP-activated (Crisan et al., 2015) (Chapter 6). At slightly later stages, in the foetal liver and bone marrow, HSC heterogeneity starts to appear with the presence of genetically distinct BMP- and non-BMP activated HSCs. Given the importance of the 'heptad' TFs in establishing HSC identity, we next developed a novel Gata2-Venus (G2V) reporter mouse to isolate and examine the dynamics and function of live Gata2-expressing and non-expressing cells (Kaimakis et al., 2016) (Chapter 2). Gata2 is expressed in all HSCs, however haematopoietic progenitors can be generated either Gata2-dependently or -independently, the latter being less potent and genetically distinct from the first. With Eich et al. (2018) (Chapter 3) we went further and deciphered the role of Gata2 in haematopoietic fate establishment. By using time-lapse imaging of E10.5 live aortic sections of G2V embryos, we discovered rapid pulsatile level changes in Gata2 expression, specifically in those single cells undergoing EHT. This finding indicated the highly unstable genetic state of individual cells undergoing fate change. Furthermore, aortic cells haploinsufficient for Gata2 show many fewer pulsatile and EHT events, emphasizing the importance of Gata2 levels in this process. Having defined the specific (medium) levels of Gata2(Venus) protein in the population containing the first HSCs, we used our G2V model to isolate a pure population of HSCs as they are generated for the first time in development (Vink et al., 2020) (Chapter 5). Through iterations of index-sorting, single-cell transcriptomics and functional analyses we were able to greatly enrich for HSCs (~70x compared to Eich et al. (2018)). Based on refined CD31, cKit and CD27 expression and specific physical parameters we isolated the first cells with an HSC identity and identified their precise gene expression profile. Immunohistochemistry localized these HSCs to only the smallest IAHCs. The combined effort of my research and novel tools generated to facilitate the creation of this body of work/portfolio have now led us for the first time to study the one or two cells per embryo that harbour HSC identity at the developmental time when they are beginning to be made. Now that we have cracked the genetic code of the true HSC, the field is one step closer to translating these findings and applying them to producing human clinically relevant HSCs, which will ultimately improve transplantation therapies and benefit the fight against leukaemia.

How an organism grows and develops is one of the fundamental questions in biology. A deeper understanding of developmental programmes can help to answer those questions and also generate new insights into disease and potential treatments thereof - pathways active during development often reappear in a disrupted form in disease. Over the past decade, single-cell sequencing has become one of the key technologies to generate high resolution in vivo snapshots to study developmental trajectories. After giving an overview of the current state of single cell technologies and computational methods, I continue with my work on cell cycle in mouse embryonic stem cells. Pairing fluorescence reporters with single-cell transcriptomic data I describe various ways for cell cycle inference, identify cell cycle regulatory dynamics and study the impact of genetic knockouts and other biological systems on these dynamics. The third chapter covers my computational work generating single cell atlas of developing mouse forebrain. Most importantly, a lineage tree inference allows for an unprecedented description of the switch from neurogenesis to gliogenesis identifying primed cell states and branching points. I continue with an application of the forebrain atlas comparing brain development and brain cancer. After identifying the recapitulation of developmental trajectories in a mouse model of glioblastoma I extend the analysis further onto human data. Lastly, I present a single cell multiomics atlas of mouse skin development using transcriptomics and chromatin accessibility. This multimodal approach allows for a better description of cell states during skin development and homeostasis. I propose the existence of a slow and fast differentiating lineage during skin homeostasis and a distinct switch between cell states over those trajectories as well as improve the current understanding of the growth dynamics in the various skin compartments over development.

Collective migration in epithelial tissues is required for many developmental processes, wound healing, and cancer, and is conserved across vertebrates. It is a process that has been difficult to study for 3D epithelia in vivo as the large timelapse datasets required present significant computational challenges for quantitative analysis. An important example of collective migration in an epithelial context is the migration of Anterior Visceral Endoderm (AVE) cells in the mouse embryo. From E5.5, this subgroup of cells in the visceral endoderm monolayer migrate from the distal tip, specifying the anterior-posterior axis by patterning the underlying epiblast which will later give rise to the embryonic lineage. AVE induction and migration have yet to be fully characterised and understood, and provide an attractive in vivo system to study the biological function of collective migration due to the relatively small cell numbers at this stage and the experimental accessibility of the visceral endoderm tissue. In this thesis, a new computational framework is developed to analyse and extract quantitative information from light sheet fluorescence microscopy (LSFM) 3D timelapse datasets of E5.5 mouse embryos during AVE migration. The proposed framework initially isolates the migratory cell movement, and secondly extracts the apical surface data to simplify the 3D volume to a 2D characterisation problem. Applying this computational framework on LSFM datasets from 3 fluorescent reporter mouse lines, I demonstrate the first comprehensive tracking of individual cells and cell divisions in the entire visceral endoderm, the first study of cell division angles, and the movement of cells in the underlying epiblast. Altogether these results reveal a model of AVE migration as an active process that drives large-scale tissue deformation and changes in morphology independent of cell proliferation in the VE, and presents evidence that suggests the A-P axis could be pre-defined.

Ageing is defined as a system-wide, gradual loss in overall organ and tissue function across the lifespan of an organism, and in humans is the single largest risk factor for most chronic diseases. Thanks to ongoing improvements in healthcare, human life expectancy is steadily rising, but the proportion of life spent free of chronic disease (known as healthspan) is not extending concurrently in our increasingly aged population. Socio-economic costs are growing, both in terms of healthcare spending and quality of life. A central goal of ageing research therefore is to find methods of extending healthspan. However, ageing is a complex, heterogeneous process and the underlying mechanisms of ageing and determinants of lifespan/healthspan are still not well understood. RNA regulators of gene expression are important factors in the ageing process, and I hypothesise that they may have potential to affect healthspan, or act as biomarkers of ageing. In this thesis, I have examined some of these RNA regulatory factors and their associations with ageing and lifespan in mammals. In order to do this, I assessed the expression patterns of RNA regulatory factors in two mouse models and a human cohort. In one mouse model, I found that both mRNA splicing regulatory factors and microRNAs are associated with strain-specific longevity during normal ageing, and that it is possible that these regulators play a causal role in determining strain lifespan. In the second mouse model, I showed these splicing factors to be associated with dietary restriction (a known treatment for extension of lifespan) and provided evidence that they could be mechanistically involved in the lifespan response to dietary restriction. I also showed that expression levels of these splicing factors were associated with cognitive decline and reduction in physical ability in humans. These results indicate that correct RNA regulation is a key component of the ageing process and suggests that the factors that govern these processes may represent useful future targets for healthpan intervention in ageing people.

Stressful experiences can produce a variety of emotional states, including elevated and prolonged levels of anxiety that can lead to anxiety disorders. Anxiety disorders are the most prevalent class of mental disorders worldwide; however, the neurobiological mechanisms that regulate anxiety and its disorders are still not well understood. In this context, the current project delves into downstream effects of the cleavage of EphA4 receptor by tissue plasminogen activator (tPA)/plasmin proteases; an event recently discovered by former members of our laboratory. The initial hypothesis of this project was, therefore, that tPA/plasmin-induced proteolysis of the murine EphA4 receptor is present in the mouse brain and can regulate stress-related phenomena. An initial necessary first step was to confirm the presence of the proteolytic cascade in areas relevant for the study of anxiety. In agreement with this hypothesis, I demonstrated that tPA and plasminogen co-localise with EphA4 in the GABAergic neuronal synapses of the central amygdala (CeA) through immunhistochemical techniques. In line with this discovery, the relevant literature sufficiently proves that inhibitory interneurons in the central amygdala of the mouse brain regulate anxiety-related processes by controlling the activity of downstream output cells. Specifically, those of the lateral subdivision of the central amygdala (CeL) expressing protein kinase C delta (PKCδ+) are important for aversive stimuli processing and memory. In the present work, I show that all tPA-expressing cells in CeA are also PKCδ+, which establishes a strong link between PKCδ+ cell-types and the location of an assumptive tPA/ plasmin/EphA4 cascade in areas relevant for stress-related events and anxiety- like behaviours. Conceivably, PKCδ+ (tPA) cells can regulate the properties of their downstream GABAergic synapses during stress through a cleavage of EphA4 associated with the tPA/plasmin proteolytic cascade. It is known that stressful stimuli produce the tPA-mediated conversion of plasminogen into the active enzyme, plasmin; and, as demonstrated here, plasmin would subsequently be able to cleave the tyrosine kinase receptor, EphA4. Cleavage of EphA4 has multiple neurobiological consequences. At the molecular level, I examined how shedding of EphA4 affects postsynaptic GABAergic protein-protein interactions. Here, I show that cleavage induces the dissociation of EphA4 from the GABA-receptor anchoring protein, gephyrin. The repercussions of this event are still unknown. Furthermore, this shedding can regulate the dendritic spine shape as evidenced by spine morphology experiments. Spine morphology is thought to reflect the strength and activity of a synapse whereby the excitatory or inhibitory tone of a neuron can be tuned. Moreover, consistent with a crucial role of the tPA/plasmin/EphA4 signalling cascade in anxiogenesis, EphA4 main cleaved form of EphA4 is increased after restraint stress. Accordingly, increased protein levels in the central amygdala of a plasmin-resistant variant of EphA4 (crEphA4) prevents the expression of stress-induced anxiety-like behaviours in mice, whereas the expression of a truncated EphA4 variant that mimics the cleavage by plasmin (tEphA4) increases this expression. This indicates that the cleavage of EphA4 potentially helps to modulate the expression of anxiety-like behaviours. Therefore, the present work identified a central molecular cascade that potentially controls the structure and function of GABAergic synapses downstream of CeL-PKCδ+ interneurons in the CeA and has the ability to modify the expression of anxiety-like behaviours. Additional pieces of data presented in this work indicate that the cleavage of EphA4 is affected in other brain conditions in which tPA/ plasmin cascade is involved, such as rodent models of stroke or epilepsy. Therefore, this work opens future possibilities for the study of other mechanisms regulated by tPA/plasmin/EphA4 cascade.

During early mouse embryonic development, a single cell, the fertilised egg, will give rise to a wide range of cell types that become specialised at a functional and molecular level. Gastrulation and early organogenesis are two of the most critical events during these early stages, when pluripotent cells, able to generate any cell in the embryo, proliferate and become lineage-restricted into the progenitors of the major organs. The vast amount of cell fate decisions taking place in this 48-hour window makes these stages a suitable paradigm to study cell type diversification. Nevertheless, the low cell numbers in early mouse embryos and the limited strategies to isolate homogenous cell populations have restricted the study of the transcriptional programs and regulatory processes that underlie these processes in vivo. With the advent of high-throughput single-cell genome-wide technologies, it is now possible to obtain the molecular profiles of hundreds of individual cells at once, thus opening a new window for the study of early embryogenesis. To delineate the molecular events underlying gastrulation and early organogenesis, we have therefore generated a comprehensive single-cell transcriptomic atlas of these stages. In Chapter 3, I introduce this atlas and give a general overview of the lineages that have been captured. Due to the importance of the haemato-endothelial lineages to establish the circulatory system in the embryo for appropriate oxygenation, in Chapter 4, I characterise their emergence using the atlas. My analyses uncover a rapid formation of primitive erythrocytes that do not transition through mature endothelium. Furthermore, I report the transcriptomes of megakaryocytic and myeloid progenitors as well as show that endothelial cells from different embryonic locations present distinctive transcriptional signatures. Getting a better characterisation of embryogenesis gives us a solid baseline to understand the consequences of genetic mutations. In Chapter 5, I explore the effects of disrupting the blood regulator Tal1 using mouse embryonic chimaeras and reveal that endothelial cells are transcriptionally aberrant at early organogenesis and express genes characteristic of other mesodermal lineages. Although single-cell transcriptomics unveils the molecular programs defining each cell type, studying gene expression is not enough if we want to highlight the regulatory events behind cell type diversification. Therefore, in Chapter 5, I examine the use of single-cell transcriptomics to detect RNAs at enhancers, which may represent a surrogate for enhancer activity. Due to the limitations encountered, in Chapter 7, I perform single-nucleus ATAC-seq in cells at early organogenesis, a time-point by which all major progenitors are established. Analysing the resulting chromatin accessibility maps together with subsequent in vivo validation experiments have allowed the discovery of two novel endothelial enhancers as well as a previously unrecognised role for the ETS transcription factor FEV in the establishment of haemato-endothelial lineages. In conclusion, single-cell genome-wide technologies have permitted the comprehensive characterisation of the molecular programs and regulatory events underlying gastrulation and the start of organogenesis in the early mouse embryo. Having acquired this information has not only contributed to our understanding of embryonic development, but it will also help the optimisation of in vitro differentiation protocols in the future.

This project seeks to tackle the hypothesis of neuroregulation of sebaceous glands (SGs). SGs are appendages of mammalian skin that synthesise and secrete sebum, and SG dysfunction is associated with many common dermatoses, such as acne vulgaris. Clinical evidence suggests that SGs may be regulated by the peripheral nervous system. For example, sebum secretion is reduced in cases of partial facial paresis and following botulinum toxin injections. By understanding the putative neuronal controls of SG function, potential mechanistic links may be uncovered that exist between the nervous system and SGs in cutaneous physiology and human skin disease. This project first sought to characterise the neuroanatomy of the human and mouse pilosebaceous unit at the level of the SG. While the SG is not directly innervated, nerve endings closely associate with Lrig1-expressing stem cells in the follicular epithelium of mouse pilosebaceous units. To investigate the potential functional role of nerves in regulation of SG homeostasis, a technique for surgical denervation of mouse skin was utilised. While nerves appear to be dispensable for base maintenance of SGs, hair cycle-dependent upregulation of SG size during active hair growth was attenuated following denervation. Lineage tracing of LRIG1+ stem cells in the pilosebaceous unit suggests that reduced SG size following denervation is a result of impaired contribution of intrafollicular progenitor cells to the SG.

The two defining features of mouse embryonic stem (ES) cells are self-renewal and naive pluripotency, the ability to give rise to all cell lineages in the adult body. In addition to being a unique and interesting cell type, pluripotent ES cells have demonstrated their potential for continued advancements in biomedical science. Currently, there is an improved understanding in the chemical signals and the gene regulatory network responsible for the maintenance of ES cells in the naive pluripotent state. However, less is understood about how ES cells exit pluripotency. My main aim is to study the dynamics and the factors affecting the irreversible exit from pluripotency. Expression of the reporter Rex1-GFPd2, which is inactivated upon exit from naive pluripotency, was analyzed by quantitative long-term single-cell imaging over many generations. This technique allowed chemical, physical, and genealogical information to be recorded during the transition to exit. Culture conditions that provided homogeneous populations were used in all assays and these data were validated against bulk-culture data where appropriate. Changes in real-time cell behavior were seen in cell-cell contact, motility, and cell-cycle duration. Undifferentiated ES cells form tightly joined colonies, with cells that exhibit low motility and a constant cell-cycle duration. Exit is associated with increasing cell motility, decreased cell-cell contact, and an acceleration in cell proliferation. The onset of exit is associated with a sudden and irreversible inactivation of the Rex1-GFPd2 reporter. This inactivation is asynchronous, as it occurs at different times and in different generations during ES cell differentiation. However, examination of daughter cells generated from the same mother revealed a high level of synchronicity. Further investigation revealed that high levels of correlation in cell-cycle duration and Rex1-GFPd2 expression exist between differentiating sister and cousin cells, providing strong evidence that cell potency is inherited symmetrically in cell divisions during exit $\textit{in vitro}$. How cells change fate is a fundamental question in developmental biology. Knowing the cellular dynamics during the transition out of naive pluripotency is important for harnessing the potential of ES cells and understanding how cell fate decisions are made during embryonic development. The quantification of the timing of exit from naive pluripotency coupled with identifiable changes in cellular behaviors, such as motility, cell size, and cell-cycle duration, enhances the understanding of how cell fate changes are regulated during directed differentiation.

The morphogenetic events that give rise to the early post-implantation mouse embryo (egg cylinder) have not been thoroughly studied and our knowledge is restricted to "snap-shot" descriptions of embryos recovered at different stages of implantation from the mother. A central feature of the egg cylinder is the pro-amniotic cavity, which spans the embryo and participates in formation of the extraembryonic membranes. The major aims of my PhD studies have been to reveal how this cavity is formed (Aim 1) and then how the egg cylinder grows (Aim 2). In order to address how the pro-amniotic cavity forms (Aim 1), I first characterised in detail development of the architecture of the extra-embryonic ectoderm (ExE), which has to be remodelled to permit cavity formation. My findings indicate that the ExE comprises cells in direct contact with a basement membrane and cells that lie deeper in the tissue. The ExE originates in the polar trophectoderm, a monolayer covering the epiblast of the blastocyst, which expands and undergoes invagination to form a slit-like cavity. By carrying out analyses of fixed specimens and live imaging of cultured embryos, I have found that the epiblast and ExE cavity extend towards each other through the formation and resolution of multiple rosette structures. This leads to the fusion of the ExE and epiblast cavities to form the unified pro-amniotic cavity. I show that this process is dependent on signalling cues stemming from the underlying basement membrane that activate the b1-integrin signalling pathway to regulate cell polarity, ExE tissue architecture and rosette formation. In addition to the basement membrane's role in b1-integrin signalling, it also has physical functions that I characterise in the second part of my study (Aim 2). High resolution imaging revealed that the basement membrane underlying the epiblast is highly perforated during the implantation stages. These perforations are initially evenly distributed and then accumulate asymmetrically at the future posterior part of the embryo, just prior to gastrulation. Finally, I demonstrate that remodelling of the basement membrane requires the expression of matrix metalloproteinases (MMPs) in the epiblast under the control of Nodal. The anterior visceral endoderm inhibits Nodal signalling and hence MMP inhibition in the anterior. I demonstrate that activity of the MMPs and perforations in the basement membrane are essential for embryo growth. The domain of posterior basement membrane perforations persists beyond gastrulation suggesting a potential role for these perforations in primitive streak formation and extension. Together, my studies bring new important insights into the understanding of early mouse embryo morphogenesis.

Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.

Retrotransposons comprise approximately 40% of the mouse genome. Once thought to be useless "junk" DNA, there is growing evidence that retrotransposons play crucial roles in genome evolution and gene regulation, and contribute to the transcriptome. Several studies have found functional retrotransposon transcripts in the germline and during early development, but less is known about retrotransposon transcription in adult somatic cells. Retrotransposons are also responsible for generating gene copies in mammalian genomes (retrocopies), and there are several examples of retrocopies evolving into new genes, or being transcribed as non-coding RNA. Using computational approaches, I analyse RNA-seq data to assess the contribution of retrotransposons and retrocopies to the transcriptomes of adult mouse somatic cells, using purified naive B and T lymphocytes. First, I describe the transcriptomes generated using high-quality total RNA-seq data. Second, I quantify and characterise the retrotransposon content of these transcriptomes. Finally, I identify retrocopy transcripts and assess their relationship with the genes from which they originate. I found widespread inclusion of retrotransposons in somatic cell transcriptomes. These transcripts form distinct clusters based on retrotransposon sequence, with endogenous retroviruses being particularly prevalent in retrotransposon-rich transcripts. While these clusters are consistent between cell types, the individual retrotransposons transcribed show cell-type specificity. I also find evidence that retrotransposons may facilitate gene regulation by antisense transcripts. I demonstrate that a subset of retrocopies is transcribed, and the vast majority of these form RNA complementary to their parent mRNA, with high sequence identity. Using differential expression and proteome analysis, I present evidence for post-transcriptional regulation of parent transcripts by retrocopy RNA, possibly through stabilisation of the parent RNA. I also find that while retrocopy expression is not necessarily shared between cell types or mouse strains, certain parent transcripts tend to have an expressed retrocopy in multiple contexts. Overall, this thesis presents evidence of an important role for retrotransposons and retrocopies in the adult somatic transcriptome, and sets the stage for further investigation to experimentally elucidate the functions of these transcripts.

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.

In complex organisms, each individual derives from a single fertilised ovum. This process of development is characterised by an increase in cell number and a diversification of the various cell types that are present. Development has been extensively studied not only to understand how cells commit to various lineages, but also with a view to reversing typical developmental pathways for regenerative medicine. However, studying development in vivo is challenging due to the very high degree of spatial complexity in an embryo, and the limited amount of biological material that is actually present in these systems. Recently developed single-cell RNA-sequencing technologies offer a powerful tool for studying de- velopment. They generate a rich phenotypic readout of a cell's state, providing gene-level information about what cells are doing, and allowing reconstruction of the molecular bases of developmental pathways at the appropriate scale for which developmental decisions are made. This Thesis begins with a summary of how single-cell RNA-sequencing data are generated and analysed, and how it has been used to study cell fate decision-making. The second chapter covers my work in identifying the extent of a technical confounder of high throughput sequencing experiments (barcode swapping), and how its consequences may be excluded from a subset of single-cell RNA-sequencing experiments where thousands of single cells are captured in microfluidic droplets for library preparation. I then detail the compilation of a large single-cell RNA-sequencing timecourse atlas of mouse development between embryonic days 6.5 and 8.5. I also performed analyses into the formation of the embryonic gut, which surprisingly receives contributions from extraembryonic tissue. The fourth and fifth chapters describe my investigation into the role of the genes Tal1 and T (also known as Brachyury) using chimaeric embryos. These genes are essential for proper development, and embryos die when they are entirely absent. Chimaeric embryos are composed of both knockout and wild-type cells: the wild-type cells contribute to the lineages that are compromised by the knockout, and the embryo remains healthy. The cell-intrinsic roles of these critical genes can therefore by studied in a proper in vivo context, unconfounded by the global abnormalities that would be induced by their absence from the whole embryo. Finally, I discuss the future of the field of single-cell RNA-sequencing, with a particular focus on how we can move beyond the currently employed snapshot measurements of cell states, in order to properly identify how the state of a cell affects its future behaviour.

The onset and progression of Alzheimer's disease (AD) is characterised by increasing intracellular aggregation of hyperphosphorylated tau protein and the accumulation of amyloid beta (Aβ) in the neocortex. Despite recent success in identifying genetic risk factors for AD, the transcriptional and epigenomic mechanisms involved in disease progression are not fully understood. The main aim of this project was to evaluate transcriptional and epigenomic differences associated with the development of tau and amyloid pathology. To achieve this, I used transgenic mice harbouring human tau (rTg4510) and amyloid precursor protein (J20) mutations. I profiled transcriptional and epigenomic variation in brains from rTg4510 and J20 mice, collected at four time points carefully selected to span from early to late stages of neuropathology in each model. I identified robust gene expression and methylomic changes in both models, including genes associated with familial AD from genetic studies of human patients, and genes annotated to both common and rare variants identified in genome-wide association and exome-sequencing studies of late-onset sporadic AD. I quantified neuropathological burden across multiple brain regions in the same individual mice, identifying genomic changes paralleling the development of tau pathology in rTg4510 mice and amyloid pathology in J20 mice. Furthermore, I compared gene co-expression networks identified in my rTg4510 and J20 samples to those identified in AD human brains, finding considerable overlap with disease-associated co-expression modules (or clusters of genes) identified in the human cortex. In summary, this project represents the most systematic analysis of transcriptional and methylomic variation in mouse models of tau and amyloid pathology, providing further support for an immune-response component in the accumulation of AD-associated neuropathology, and highlighting novel molecular pathways involved in AD progression.

Maternal age is a significant risk factor for adverse pregnancy outcomes, and is strongly associated with an increased risk of aneuploidy of the conceptus, as well as a significantly higher frequency of serious pregnancy complications known as the "Great Obstetrical Syndromes", including miscarriage, pre-eclampsia and fetal growth restriction. In the last 40 years average maternal age has increased considerably in many wealthy countries, and in the UK the number of babies born to women aged 35 and over is set to surpass those born to women under 25. The high incidence of aneuploidy in older mothers can be attributed to abnormalities in the oocyte and embryo, however the "Great Obstetrical Syndromes" do not appear to be related to the oocyte and may instead be linked to abnormal development of the placenta. In this thesis, I show that advanced maternal age in the mouse is associated with a drastically increased variability of developmental progression in utero, including developmental delays and growth restriction, severe embryonic abnormalities and higher resorption rates. I find that these embryonic defects are always accompanied by gross morphological and transcriptomic abnormalities in the placenta. Notably, I show that the increased risk of these complications can be rescued by transfer of embryos from aged females to a young surrogate mother, thus implicating the aged maternal uterus as the basis for embryonic and placental defects. Transcriptomic analysis of the decidua compartment in placentas from aged pregnancies revealed abnormal expression of genes involved in the decidualization process, which occurs during early pregnancy and facilitates implantation and development of the conceptus. I show that these defects are already obvious in the peri-implantation window, with endometrial stromal cells from aged females being unable to mount an adequate decidualization response due to a decline in their ability to respond to pregnancy hormones. This blunted decidualization reaction in turn may lead to abnormal development of the placenta. These age-associated decidualization defects are cell-intrinsic and can be recapitulated in vitro. The detected insufficient activation levels and abnormal intracellular distribution of phospho-STAT3, combined with highly variable progesterone receptor expression, may be possible causes of these defects. In addition, I examined the possible effects of ageing on the epigenome as a potential contributor to the decline in endometrial function. My results indicate that ageing of the uterus displays some of the common epigenetic hallmarks of tissue ageing. However, more importantly, decidual cells of aged females exhibit abnormal distributions of the histone modification H3K4me3, and are refractory to the profound DNA methylation remodeling that I find takes place during pregnancy. These age-related changes in the epigenome may underpin, or contribute to, the observed decline in uterine function during pregnancy. Understanding the mechanism underlying these epigenomic and functional changes in the ageing reproductive tract may pave the way for new therapeutic strategies to improve maternal and fetal outcomes of pregnancy in older mothers.

This dissertation describes the development and optimisation of a set of touchscreen tasks for the assessment of motivation and reinforcement processes in mice. Changes in motivational state and reinforcement sensitivity are characteristic of depressive symptoms in a number of neuropsychiatric and neurodegenerative conditions. However, the efficacy of current therapeutic strategies is limited. Thus, the development of methods for studying these processes is important both to facilitate investigation of underlying mechanisms and subsequently develop targeted therapeutics. Part 1 describes the optimisation and application of fixed and progressive ratio tasks for the assessment of motivational state in mice. First, a comparison of multiple reinforcers under fixed and progressive ratio schedules is described. Given the limitations of performance measures derived from performance across the entire session, these experiments are complemented by the use of response rate analysis, which reveals dissociations between standard measures and rate of responding. Next, these tasks are applied to a model of maternal high fat diet (hfd), revealing selective alterations in motivated behaviour. Finally, the motivational state of the TgR406W mouse model of frontotemporal dementia, a condition frequently characterised by depressive symptoms, is investigated. It is shown that the motivational state of TgR406W mice is age dependent, with apparent early apathy-like behaviour and a late disinhibition in responding. This is accompanied by deficits in attentional performance as measured by a 3-choice serial reaction time task. Thus, part 1 demonstrates the applicability of the battery of touchscreen tasks for assessment of motivation to diverse mouse models. Part 2 describes the development and validation of touchscreen tasks for the assessment of reinforcement-related choice in mice. First, it is demonstrated that C57BL/6 mice systematically discount a preferred reinforcer in a delay discounting procedure. Subsequently, choice behaviour is modified by systemic treatment of dopaminergic drugs and a reinforcer pre-feeding procedure. Next, procedures for the assessment of learning and choice, dependent on the integration of positive and negative feedback, are described. Specifically, the valence-probe visual-discrimination (VPVD) task is an adapted version of the standard visual discrimination with the addition of a probabilistic stimulus that is reinforced 50% of the time. This modification allows for assessment of the impact of positive and negative feedback on discrimination learning. In addition, a spatial within-session probabilistic reversal learning procedure was adapted for the touchscreen apparatus. The effect of serotonin 2C receptor manipulation on these tasks is presented. Serotonin 2C receptor antagonism is shown to disrupt discrimination learning and result in diminished sensitivity to positive feedback. Conversely, 2C receptor agonism results in changes in feedback sensitivity consistent with heightened sensitivity to positive feedback and diminished sensitivity to negative feedback. Taken together, these results show that these tasks can be used to interrogate reinforcement-related behavioural profiles in experimental mice. Finally, the implications of these tasks with respect to investigation of motivation, reinforcement sensitivity and adaptive decision-making in rodent models, and their relevance to MDD in humans is discussed.

Cleft palate, one of the most common birth defects worldwide, results from an alteration in the development of the palate, or palatogenesis, which can be caused by multiple genetic or environmental factors. As palatogenesis in humans and mice consists of the same morphological steps, the mouse has been extensively used to study this process. In turn, the rat is the main model to test if a specific substance can cause developmental toxicity symptoms, including cleft palate, as toxic responses in rats can generally be extrapolated to humans. A deeper understanding of the conservation of palatogenesis in mouse and rat could help to evaluate and refine the use of the rat model in human risk assessment studies for clef palate. This situation is addressed in this thesis by employing a new approach based on the comparison of palatogenesis in mouse and rat at the morphological and molecular levels. RNA-seq analysis indicated that most of the mouse and rat orthologues differentially-expressed during palatogenesis displayed similar expression patterns, thus correlating with the morphological similarity of palatogenesis in these species proved by histological analysis. However, the proportion of differentially-expressed genes was greater in mouse. Similarly, when focusing specifically on genes associated with cleft palate, most mouse and rat orthologues were found to have similar RNA-seq expression patterns, although a minor number of potentially relevant differences was identified. Further NanoString and qRT-PCR analyses supported the reproducibility of these results across different mouse and rat biological replicates. In addition, most orthologues analysed by in situ hybridization displayed similar spatial expression patterns in mouse and rat palatal shelves. In conclusion, the results presented in this thesis indicate that palatogenesis is highly conserved in mouse and rat, and support the use of the rat in human risk assessment studies for cleft palate.

Ventral body wall closure (VBW) defects are amongst the most common human congenital anomalies. They represent a wide and heterogeneous group of phenotypic defects that can present in isolation or as a component in a larger syndromic anomaly. In addition, the incidence of associated anomalies is high and reaches 75% of fetuses in some types of VBW closure defects. Nevertheless, the embryonic origin and the underlying cellular and molecular mechanisms between ventral closure defects and their associated congenital anomalies remain poorly characterised. This is in part due to the poor understanding of the physiological mechanisms that regulate the development of ventral organs and the lack of representative transgenic animal models allowing detailed in vivo analysis of defect formation. Transforming growth factor beta (TGF-ÃŽÂ2) signalling is essential for VBW closure and vascular and cardiac development. Yet, its mechanism of action and the responding cell(s) in the body wall remain largely unknown. In addition, in various cells TGF-B can induce the expression of Tagln, encoding for a cytoskeleton associated protein that enhances cell migration. No function has been ascribed to TAGLN in body wall development. I define here a role of TGF-B during a critical time window in embryonic development to fashion the ventral body wall, anterior diaphragm and parts of the circulatory system. I identify a population of TAGLN+ myofibroblasts that respond to a temporally regulated TGF-B signalling originating from the epithelium of the primary body wall. Deletion of TGF-B receptor in TAGLN+ cells leads to failure of ventral body wall closure, anterior diaphragmatic hernia, cardiac and outflow tract anomaly. Nevertheless, the descending aorta and the large aortic branches are spared. By using advanced transgenic methodology, I generated novel transgenic mouse lines that enabled me to fate map the cells that initiate the formation of important mesenchymal tissues. These studies revealed that the origin of aortic vascular smooth muscle cells can be traced back to a group of progenitor cells that reside in the wall of the dorsal aorta before the VBW closure. My studies provide intriguing evidence for spatially restricted role for TGF-B signalling in ascending but not descending aorta morphogenesis. I used a variety of techniques to characterise, analyse and quantify important mechanisms during mesenchymal and vascular development, their response to injury and repair. This thesis has been written in an alternative format, comprising the different areas which have been investigated. Collectively, the results presented here provide new insights into the role of migratory and mechanically stabilising cells in the development and maintenance of critical structures in the body and their common role in the development of concurrent congenital anomalies. A detailed understanding of the molecular signalling pathways and cells that drive VBW closure raises the hope that the related birth defects can in the future be treated by precise gene and cell therapies.

The intestinal epithelium is rapidly renewing throughout life. A population of stem cells exist within the intestinal crypt that drive rapid cell renewal and replace each other by a pattern of neutral drift. Perturbation of these dynamics through oncogenic mutation can predispose the epithelium to neoplastic transformation. Understanding the factors that govern these dynamics will give insight into the early stages of oncogenesis. Continuous clonal labelling, whereby DNA strand slippage leading to the contraction or expansion of a microsatellite during mitotic replication, can be employed to enable the detection of a single clone. Previous studies have shown that quantification of clone size over time allows inference of the functional stem cell number and stem cell replacement rate within intestinal crypts. Current continuous labelling techniques require the introduction of a transgenic microsatellite into a model system genome, such as mouse, that leads to reporter expression following mutation. This obviously precludes human studies. Alternative somatic alterations techniques used for continuous labelling in humans requires spontaneous loss of a protein, or change in methylation status, within a single clone that can act as a clonal mark. Though these techniques have given insight into the spread of mutations within the intestinal epithelium and enabled inference of adenoma clonality, the true neutrality of these changes are currently unknown. We propose that the small changes in endogenous microsatellite length will act as a neutral clonal mark within the intestinal epithelium and allow an unbiased approach to quantifying intestinal stem cell dynamics in human intestinal tissues. To overcome the many technical challenges associated with accurate measurement of microsatellite length, a stepwise approach was taken to develop a technique for the multiplexed high throughput sequencing of up to 21 native dinucleotide repeats in hundreds of single crypts. Furthermore, a novel method was developed for the quantification of clone size from data generated from the targeted re-sequencing of microsatellites in single crypts. This protocol was validated in vitro and in vivo in mouse. Furthermore, proof of principle sequencing in human crypts was performed to show that this method is suitable for larger scale quantification of intra-cryptal clone size in human tissue. This, and similar approaches, may be the only way to quantify intestinal stem cell dynamics within the healthy human colon or, dysplastic or adenomatous patient tissue. These measurements should give a unique insight into the dynamics of healthy, pre-neoplastic and neoplastic human intestinal stem cells.

Textbook accounts of vertebrate embryonic development have been based largely upon experiments on amphibian embryos, which have shown that the tissues of the trunk and tail are organised from distinct precursors that existed during gastrulation. In the mouse and chick, however, retrospective clonal analyses and transplantation experiments have demonstrated that the amniote body instead arises progressively from a population of axial precursors that are common to both the neural and mesodermal tissues of the trunk and tail. For this reason, they are known as neuro-mesodermal progenitors (NMps). Detailed studies of NMps have been precluded by their lack of a unique gene expression profile and the technical difficulties associated with isolating them from the embryo. Mouse embryonic stem cells (ESCs) provide the possibility of instead deriving them in vitro. ESCs have been used to model developmental processes, partly through large cellular aggregates known as embryoid bodies. These structures do not, however, resemble the axial organisation of the embryo and they develop in a disordered manner. This thesis presents a novel culture system of small, three-dimensional aggregates of ESCs (gastruloids) that can recreate the events of early post-implantation development, including axial elongation. Gastruloids are the first ESC-based model for axial elongation morphogenesis; this body of work characterises their development and identifies a candidate population of NMps within their elongating tissues. Additionally, this work establishes a xenotransplantation assay for testing the functional properties of in vitro-derived NMp populations in the chicken embryo and applies it to NMps from gastruloid cultures. The results of this assay show that gastruloids are a credible source of NMps in vitro and therefore offer a new experimental means to interrogate their properties. The use of gastruloids to recreate embryonic development has implications for basic research as a synthetic system and for the therapeutic derivation of other embryonic progenitors through bioengineering.

Neurodegenerative diseases are associated with extensive physical and mental debilitation, significant costs to the healthcare system, as well as great emotional and financial burden to the patients, their families and care providers. Despite progress in our understanding of the mechanisms behind neurodegenerative diseases, the vast majority are still currently untreatable. Synapses are important pathological targets in a range of disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and lysosomal storage disorders, such as Batten disease. Loss of synaptic connections and impairments in synaptic function are present in the initial stages of neurodegenerative conditions and throughout the course of disease progression. Therefore, synaptoprotective strategies are regarded as a potentially key factor in the development of effective therapies aimed at preventing or halting neurodegeneration. Despite the continuously growing body of research elucidating the molecular mechanisms that modulate synaptic function and vulnerability, the contribution of these pathways to neurodegenerative diseases is far from fully characterized. In addition, there are frequent issues regarding the applicability of the research performed using in vitro and small animal models of disease to develop therapeutic strategies for use in human patients. In the work described in this thesis, we initially validated the involvement of a selection of key synaptic targets, previously identified as regulators of synaptic degeneration in lower animal models, including mice and Drosophila, in a large animal model of neurodegenerative disease: CLN5 Batten sheep. Subsequently, we explored two of these individual synaptic protein targets in more detail (calretinin and α-synuclein), to further investigate their contribution to synaptic function and stability. Calretinin is a poorly characterized protein, primarily known for its calcium buffering capacities and high levels of expression in a subpopulation of interneurons. In this work, we show calretinin is expressed in previously unreported cell populations, including motor axons and synapses from the peripheral nervous system, and that it is enriched in synapses in vitro. Furthermore, we show calretinin responds dynamically to synaptic activity and is directly involved in neurodegenerative pathways, as demonstrated by its ability to influence the course of Wallerian degeneration and apoptotic cell death. α-synuclein plays a central role in the pathophysiology of Parkinson’s disease and contributes to the maintenance of synaptic transmission and mitochondrial function. However, questions still remain about how to effectively manipulate α- synuclein to obtain therapeutic benefits. Therefore, we sought to explore downstream targets of α-synuclein in order to uncover new pathways through which this protein may influence synaptic stability. Using proteomics on mice lacking α-synuclein and in vitro cell systems we identified sideroflexin 3 (sfxn3). We show sfxn3 is localized at the inner mitochondrial membrane and that it functions outside the main canonical pathways of mitochondria energy production. In addition, overexpression of sfxn3 in Drosophila led to a significant loss of synaptic boutons at the level of the neuromuscular junction, suggesting regulated levels of sfxn3 are important for the maintenance of synaptic connections. Altogether, the work developed in this thesis provides novel insights into pathways regulating synaptic stability and function. We not only provide evidence that the molecular targets studied are affected in a large animal model of neurodegenerative disease, and are therefore likely to be relevant to studies in human conditions, but we also uncover two new molecular targets capable of independently regulating synaptic form and function.

Patients with dysfunctional ovaries, such as those with premature ovarian insufficiency and granulosa cell tumours, do not have normal follicle development and may not respond to traditional assisted reproductive techniques. Using the reaggregated ovary (RO) technique, these patients' oocytes may be reaggregated with functional supporting cells and cultured in vitro to develop fertilisable eggs. However, current research using ROs have only used murine ovaries as a somatic cell source. In this thesis, with the aim of moving towards a clinical treatment, we assessed follicle development in ROs in vitro and progressed to using the technique with human tissues. To assess whether an older murine somatic cell source resulted in advanced follicle development, and how follicle development differed between transplanted and cultured ROs, ROs were generated using postnatal day 2 (P2) and P6 mouse ovaries. To investigate theca cell development in follicles from cultured tissue, mouse ovaries were cultured with mouse serum or encapsulated in hyaluronan hydrogels. Prior to generating and culturing chimeric human-mouse ROs (HuMoROs), competent handling and digestion of bovine cortical tissue was required. Broadly, ROs generated from both P2 and P6 exhibited similar follicle development in vitro after 14 d of culture, and follicles from cultured ROs were more developed than those from transplanted ROs. Theca cell development observed in follicles from cultured ovaries was still poorer than those from in vivo ovaries, even when ovaries were cultured in mouse serum or encapsulated in a hyaluronan hydrogel. Finally, some follicles containing potential human oocytes developed within the generated HuMoROs after 7 d of culture. These results have highlighted the potential of the RO technique as a method to generate fertilisable eggs and identified further aspects which need to be targeted in order to improve the success of the technique.

Schizophrenia is a complex mental disorder characterised by positive symptoms such as hallucinations and psychosis, negative symptoms such as avolition and anhedonia, and cognitive defects. Schizophrenia risk has a genetic component, and it is likely that this is caused by interaction between numerous genes with individually small effects. Environment also plays a role; factors associated with schizophrenia include drug use, prenatal stressors and living environment. Candidate genes linked to schizophrenia have been identified through recent GWAS of human populations with the disorder, including ANK3, TCF4 and CACNA1C. GWAS associations alone are not sufficient to identify these as definitive risk genes for the disease. In order to validate these findings, animal models (in particular the mouse) can be used to study the effect of mutations in these genes of interest. Knockouts of these genes in the mouse are lethal, so we have used the ENU mutagenesis DNA archive at MRC Harwell to screen for additional allelic variants expressing more subtle and varied behavioural phenotypes. Endophenotypes associated with schizophrenia and bipolar disorder, such as anxiety, cognitive deficits and sensorimotor gating deficits, can be characterised in these mutants and, together with molecular characterisation, this can validate these genes as risk factors. While mutations in Ank3 and Tcf4 proved to be non-functional, two mutations in Cacna1c have been associated with anxiety phenotypes and differences in EEG power spectra, as well as causing a cardiac phenotype.

In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.

The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity.

The 3Rs of reduction, refinement and replacement are the guiding principles of animal research and embedded in national and international legislation regulating the use of animals in scientific procedures. Awake imaging by MRI of rodents can offer a reduction by increasing the quality of scientific data through longitudinal imaging using less animals by avoiding a serial sacrifice design and refinement through reducing the stressful effects animals are exposed to, in comparison to existing models. Before awake imaging can become an established biomarker it must be demonstrated that pathology is traceable with comparable or an improvement on results using existing biomarkers. To validate awake imaging of rodents three study types were conducted in two different rodent species: imaging of the progression of Aspergillus fumigatus infection in the mouse lung using anaesthetized animals; analysis of stress in rats during imaging and imaging restraint; imaging of the abdomen in awake rats and a prospective study into the utility of this method for imaging the progression of Candida albicans renal infection. The first study type used an established model of invasive pulmonary aspergillosis in mice to test the utility of MRI for tracking infection in the lung parenchyma. Images of collapsed parenchyma were obtained and shown to increase as the infection progressed. Further work is required to establish this as a clinically relevant biomarker. The study type used restraint, blood collection and imaging as stressors using corticosterone levels as a surrogate of stress. Analysis of levels in blood and faeces by RIA and ELISA allowed comparison of stress during anaethetised and awake imaging for the first time. There were no differences in rat corticosterone levels during anaethetised and awake imaging indicating that awake imaging was no more stressful than currently used procedures. The third study type employed restrainers and acclimatisation to MRI scanner noise to acclimatize rats for awake abdomen imaging. Both anaethetised and awake rats were imaged with FLASH and IntraGate™ sequences. These methods were utilized in an established model of disseminated candidiasis by imaging the kidney. Comparable image quality was obtained in awake animals, with the utility of the method validated by imaging differences in renal pathology between vehicle and low and high dose treated animals. In conclusion, the first steps have been taken towards establishing awake animal imaging by MRI. The imaging is no more stressful than using an anaesthetic and was a useful biomarker in the rat abdomen and capable of tracking disease development. Further work is required to make the technique fully quantitative and automated and hence become a useful tool for monitoring progression of fungal infection and other pathology.

Human congenital heart disease (CHD) is the most common cause of non-infectious neonatal death affecting 1-2% of live births (Hoffman and Kaplan, 2002). Treatment of CHD requires major surgery and quality of life is often significantly reduced despite treatment. With the discovery of single gene mutations that cause CHD in model animals (Lyons et al., 1995), the role of genetics in CHD has become appreciated. The genetic basis of CHD is poorly understood, with different members of the same family presenting with different types of CHD (Schott et al., 1998), suggesting the causes of CHD are multifactorial. Cardiogenesis is intimately associated with the establishment of the left-right (L-R) body axis, with the two processes sharing several important transcription factors. Heart looping, in which the heart turns dextrally, is the earliest physical manifestation of L-R asymmetry. L-R patterning disorders are associated with an increased risk of CHD; heterotaxy (in which L-R asymmetry is neither normal nor mirror image) accounts for about 3% of all CHD (Zhu et al., 2006).Investigating cardiogenesis and the causes of CHD necessitates the use of animal models, typically mice, chicks, zebrafish and Xenopus. Recently a strain of mouse with a mutation in a gene essential for cardiac development was isolated from an ENU mutagenesis screen (Kile et al., 2003) using mice carrying a balancer chromosome. It has been subsequently found that the most likely candidate gene codes for the protein Prpf8, an integral component of the spliceosome. The mutation is homozygous lethal, with homozygous mice having a grossly deformed heart, developmental delay and a high incidence of heart looping reversal, indicative of a L-R patterning disorder. In depth characterisation of homozygous mutant embryos revealed defects in the morphology of the embryonic node, nodal cilia and the expression pattern of L-R axis genes. We also investigated the expression of Prpf8 during embryogenesis, and the effect that the point mutation we found in our homozygous embryos has on splicing kinetics.

Fetal growth restriction (FGR), when a fetus fails to reach its genetic growth potential, affects up to 10 % of pregnancies and is a major risk factor for both neonatal and adulthood morbidity and mortality. There are currently no treatments for FGR except for delivery of the fetus; resulting in premature delivery which, in itself, is linked to poor outcome. Therefore, the focus of current research is to examine whether therapies successfully used to treat diseases with similar aetiologies to FGR can also be used to treat FGR. Sildenafil citrate (SC), a selective phosphodiesterase-5 inhibitor, is one such candidate. With the recent announcement of the STRIDER international clinical trial for the treatment of severe FGR with SC, it is imperative to determine the efficacy and safety of SC treatment on both fetus in utero and long-term adult health. Mouse models that mimic characteristics of human FGR represent an attractive model to perform pre-clinical studies. Recent studies in mice have demonstrated that SC increased fetal and placental weight and normalised umbilical artery blood flow velocity in FGR but no studies have assessed effects of antenatal SC on offspring health. The aims of this study were to assess the effect of antenatal SC treatment on a) fetal weight b) fetal vascular reactivity b) pup viability and d) long-term effects on postnatal development/physiology in a mouse model of FGR.All experiments were performed in the placental-specific insulin-like growth factor 2 knockout mouse (Igf2 P0+/- mice) which have mixed litters of wild-type (WT) and growth restricted (P0) mice. It has been reported that SC administered in the drinking water was able to increase P0 fetal weight and thus this mouse model was chosen to assess the effects of SC on the fetus and offspring. SC was administered to pregnant dams in two regimens; orally (120 – 160 mg.kg-1) and subcutaneously (10 mg.kg-1) between E12.5 and E18.5. WT and P0 fetal abdominal aortas were isolated at E18.5 and ex vivo vascular function was assessed using wire myography. Fetal abdominal aortas demonstrated reliable and reproducible vasocontraction and vasorelaxation; there were some sex- and genotype-specific differences. SC demonstrated dose-dependent effects on fetal aortic function. Offspring from dams treated with a subcutaneous injection of SC or saline were assessed for postnatal growth (week 5 – week 12), systolic blood pressure (week 8 and week 13), glucose tolerance (week 12) and mesenteric / aortic vascular function (week 14 – week 16). These experiments demonstrated that;• A supratherapeutic concentration of antenatal SC (120 – 160 mg.kg-1) did not increase fetal weight but significantly blunted relaxation responses of fetal abdominal aortas at E18.5. • A subcutaneous injection of antenatal SC (10 mg.kg-1) did not increase fetal weight or alter fetal abdominal aortic function in mice but led to increased systolic blood pressure in both WT and P0 offspring. Additionally, glucose sensitivity was significantly reduced in female offspring from SC treated dams. In conclusion, the studies outlined in this thesis have demonstrated that antenatal SC treatment can cause alterations in fetal blood vessel function and also lead to changes in metabolic and cardiovascular function in mouse offspring. Using ex vivo wire myography, mouse fetal abdominal aortas were able to be assessed at E18.5. This methodological advance will be beneficial as it can be applied to assessing putative treatments in mice that show characteristics of human FGR. In addition, this technique will allow for investigation of the underlying mechanisms of in utero programming of adulthood cardiovascular diseases such as hypertension. Future work must focus on the mechanisms leading to increased systolic blood pressure in offspring from SC treated dams and whether such effects are noted in other animal models of FGR using a variety of SC dosing regimens. These studies will provide information with which to increase efficacy, and ensure the safety, of SC treatment in pregnancy complications.

Deciphering the mechanisms that underlie circuit function in the hippocampal formation is a key challenge for neuroscience. This region, which includes the medial entorhinal cortex (MEC), is critical for spatial learning and episodic memory in humans. Spatially modulated cells in the MEC, the grid cells, provide a topographical representation of space, but we are yet to establish the neuronal properties that underlie this or the contribution that particular cells in different regions of the MEC and hippocampus make to circuit function. This is partially because the specific targeting of the network with genetic tools is complicated by a multitude of cell types with predominantly unknown molecular profiles. To address our limited understanding of the molecular organisation of the MEC, I have characterised how the expression of genes is distributed throughout different layers of the MEC, using a custom-designed resource that facilitates analysis of in situ hybridisation data from the Allen Brain Atlas. Through simultaneous extraction of gene expression data across thousands of 2D aligned images, I reveal striking differences between layers within MEC, demonstrating that layer II contains the highest proportion of genes enriched in a single layer, whereas gene expression is very rarely confined to layer III. Of particular interest, layer II of MEC is highly enriched for Alzheimer’s disease pathway genes, providing insight into its vulnerability as one of the first brain regions to show pathology. I also identify over 1000 genes that are expressed with a dorso-ventral gradient that maps onto the topographic organisation of MEC connectivity, grid cell spatial resolution and synaptic integrative properties of cells. An intriguing group of genes that closely relate circuit activity to gene expression, the plasticity-related activity-dependent genes, often show this pattern of expression. Focussing on the activity-dependent expression of one such activity-regulated, plasticity-related gene, Arc, I provide a novel view of MEC function. During simple novel exploration, Arc expression is up-regulated to a much greater extent in the deep layers of dorsal MEC than in the grid cell-rich superficial layers. By selectively disrupting the predominant hippocampal input to dorsal MEC, which terminates in the deep layers, I show that the significance of this up-regulation is independent of hippocampal inputs. Thus, although research addressing MEC function is particularly focussed on the superficial layers, during the exploratory behaviour that potentially primes the system for representing an environment, important plasticity may be occurring at the synapses onto deep layer neurons. In summary, my investigations of baseline and activity-dependent gene expression in MEC have revealed a molecular organisation both across different layers and along a functionally relevant gradient. This may be important for specifically targeting microcircuits in MEC and for characterising how laminar and regional differences contribute to the encoding of space in the hippocampal formation.

Damage precipitating cognitive decline in Alzheimer's disease (AD) begins long before behavioural alterations become clinically apparent. At this prodromal stage, communication between networks of neurons connecting different brain regions starts to break down; setting in motion a chain of events leading to clinical AD. A significant challenge facing Alzheimer's researchers today is finding a cheap, easy-to-perform test capable of detecting prodromal AD. Such a test would afford significant benefits to patients, including a chance of early intervention. Perhaps, more importantly, it would also aid development and testing of novel therapies aimed at combating AD before it causes irreversible damage. Since oscillations in electrical field activity are important for facilitating connectivity across the brain and have been seen to alter in AD, this work studied how oscillations and regional connectivity are affected in the AD brain. Specifically, local field oscillations were recorded from the hippocampus and prelimbic cortex (regions implicated in memory formation and maintenance) in a double transgenic AD model - the TASTPM mouse. Here, periods of predominant theta activity were assessed both spontaneously, under urethane anaesthesia and following electrical induction through dorsal periaqueductal gray (dPAG) stimulation. From these recordings, spectral power and connectivity between regions was assessed using both a traditional measure of functional connectivity (inter-region correlation) and through a novel information theoretic approach measuring effective connectivity (transfer entropy).Perhaps the most prominent finding from this study was the observation that young TASTPM mice, at an age prior to overt cognitive decline or plaque deposition, showed significant alterations in measures of both functional and effective connectivity. This suggests that such measures may be used as biomarkers predictive of prodromal AD and, as such, may be used to aid development of drugs targeted towards treatment of prodromal AD.This study also uncovered a number of interesting observations concerning hippocampal/prelimbic connectivity. Firstly, although spectral power and inter-regional correlation peaked at ∼ 3Hz, information flow between these structures was strongest at ∼6Hz. This suggests that low and high-band theta activity may fulfil separate functions. Secondly, at theta frequencies, information flowed predominantly from the prelimbic cortex to the hippocampus. However, during lower frequency activity, information flowed predominantly in the opposite direction. Suggesting that separate frequency bands may be important for routing information flow between these structures. Finally, the strength of information transfer was seen to oscillate at approximately double the frequency of its carrier signal, perhaps suggesting locking of information transfer to certain phases of an underlying oscillation. Therefore, oscillations may structure information transfer by temporal windowing and frequency-locked routing; processes which can be studied using measures of effective connectivity such as transfer entropy.

Otitis media (OM) is characterised by inflammation of the middle ear and is a common cause of conductive hearing impairment that places a substantial social, medical and economic burden on healthcare systems globally. Despite the importance of the disease, the aetiology of chronic middle ear inflammatory disease remains poorly understood. The development and persistence of chronic OM is multi-factorial with a significant genetic component. A new mouse model of chronic OM, edison, was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and discovered in a recessive screen at MRC Harwell. Homozygous edison mice have craniofacial abnormalities, an emphysema-like lung phenotype and spontaneously develop a conductive hearing loss at 28 days as measured by ABR. Histological analysis shows the hearing loss is associated with the development of chronic OM in the middle ear, characterised by mucosal inflammation and highly cellular ear exudates. Similar to the Jeff and Junbo mutants, edison shows raised levels of Vegfa, Tnfα and Il-1β in middle ear fluids. A putative functional mutation was identified, resulting in a missense Leu972Pro change in a relatively unknown gene, Nischarin (Nisch). The identification of additional ENU-induced Nisch alleles, and subsequent characterisation, validated Nisch as the causative gene in edison. NISCH selectively binds ITGA5, which is thought to have a role in modulating VEGF signalling through SRC and FAK kinases. A significant genetic interaction between Nisch and Itga5 exists and impacts upon development of chronic OM. Mice heterozygous for Itga5-null and homozygous for edison alleles show a significantly increased penetrance and severity of chronic OM. Analysis of downstream pathways suggests that the edison allele is impacting upon both RAC1 and TGF-β/SMAD signalling. I also explored the potential use of the edison mouse as a model for bacterial challenge with the human otopathogen, NTHi. Similar to the Junbo infection model at MRC Harwell, the edison mouse was identified as a robust OM model for bacterial NTHi infection. The edison mouse highlights a new candidate gene for susceptibility to chronic OM and will provide further insight into the genetic pathways and pathogenic processes involved in chronic OM.

Vertebrate genomes exhibit global methylation of cytosine residues where they occur in a cytosine-guanine dinucleotide (CpG) context and this epigenetic mark is generally thought to be repressive to transcription. Punctuating this pervasive DNA methylation landscape are short, contiguous regions of non-methylated DNA which are found at two thirds of mammalian gene promoters. These non-methylated regions exhibit CpG content close to expected levels as they escape the depletion of CpGs observed across the methylated fraction of the genome. The unique nucleotide properties of these CpG island (CGI) regions enable their identification by computational prediction in mammalian genomes. Owing to a lack of high-resolution genome-wide DNA methylation profiles in non-mammalian species, these CGI predictions have often been used as a proxy for non-methylated DNA in these organisms. In contrast to mammals, CGI predictions in cold-blooded vertebrates rarely coincide with gene promoters, leading to the belief that CGls are significantly divergent between vertebrate species, and that unique promoter-associated features may have been acquired during warmblooded vertebrate evolution. This thesis is primarily concerned with the location, establishment and biological function of non-methylated islands of DNA in vertebrate genomes. To experimentally determine genome-wide profiles of non-methylated DNA, a novel biochemical technique was established called biotinylated ZF-CxxC affinity purification (Bio-CAP), and development of this method is discussed in Chapter 3. Experimental analysis of non-methylated DNA profiles in this thesis initially addresses two main questions: (1) 'How does the non-methylated DNA landscape compare genome-wide for seven vertebrates considering distinct tissue types and developmental stages?' (2) 'How are vertebrate non-methylated regions of DNA defined and interpreted in the nuclear environment?' To address the first question, non-methylated DNA was profiled by Bio-CAP sequencing across the genomes of seven diverse vertebrate species, representing all major branch points of vertebrate evolution, and the results are discussed in Chapters 4 and S. Contrary to previously held dogma, experimentally determined nonmethylated islands of DNA (NMls) constitute an ancient epigenetic feature of vertebrate gene regulatory elements. However, despite having numerous high-resolution maps of vertebrate non-methylated DNA, the means by which NMls are identified and maintained in the nuclear environment remains poorly understood. To address the second question and identify features which determine the methylation state of DNA, exogenous DNA sequences were introduced into mouse embryonic stem (ES) c~.II~. Non-methylated DNA was profiled by Bio-CAP sequencing to investigate how different features, such as sequence-specific binding motifs, chromatin architecture and nucleotide composition of a given DNA sequence impact local DNA methylation patterns. Interestingly, the majority of exogenous promoters were appropriately non-methylated in mouse ES cells, germline and somatic cells suggesting that gene promoters have retained strong signals for the nonmethylated state across millions of years of evolution (discussed in Chapter 6). During mouse embryogenesis, genome-scale DNA demethylation and remethylation events occur to remodel the epigenetic landscape and loss of DNA methylation during this time leads to embryonic lethality. To investigate the biological function of non-methylated DNA, the third question addressed in this thesis is (3) 'What is the developmental importance of non-methylated islands of DNA during vertebrate embryogenesis?' To investigate this, members of the ZF-CxxC domain-containing family of chromatin modifiers were ablated in zebrafish embryos to perturb the chromatin landscape at NMls, and therefore interfere with their function during early development (Chapter 7). Early embryonic development and patterning was disrupted in knockdown embryos, suggesting that interpretation of non-methylated DNA and placement of chromatin modifications at NMls is essential for normal zebrafish embryogenesis. Together this work sheds light on the evolutionary origins of NMls, the mechanisms involved in the recognition and establishment of nonmethylated loci and provides an insight into the function of non-methylated DNA during early embryonic development.

Matthew-Wood syndrome is a rare human birth defect condition defined by the phenotypic constellation of clinical anophthalmia, diaphragmatic hernia, pulmonary hypoplasia and cardiac defects. Matthew-Wood syndrome has a high mortality rate, with most patients dying due to respiratory insufficiency as a consequence of pulmonary hypoplasia, within the first year of life. Mutations within STRA6 are causative for Matthew-Wood syndrome. STRA6 acts as a retinol transporter for retinol bound to its physiological carrier RBP4 allowing regulated entry of retinol into the cell. A mammalian model for Matthew-Wood syndrome was not found within the literature; however a morpholino knockdown of stra6 in the zebrafish did show phenotypic features consistent with those observed in human patients. The desire to create a mammalian model of Matthew-Wood syndrome drove the work contained within this thesis. Stra6-/- mice do not represent a model for Matthew-Wood syndrome with homozygous animals being viable, found in the expected ratio and demonstrating none of the developmental abnormalities observed in human patients. Retinal defects, cataracts and persistent hyperplastic primary vitereous affect the microphthalmic eye of Stra6-/- offspring of Stra6-/- mothers fed a retinoid-free diet from plug to birth indicating that Stra6 is required for normal eye development under low-retinoid stress. The disparity in phenotype between human Matthew-Wood patients and Stra6-/- mice may be the result of functional redundancy in the mouse between Stra6 and its paralogue, Stra6.2. Stra6.2 is well conserved through evolution and is found in diverse species, including the basal eumetazoan Trichoplax. STRA6.2 has become split across its resident chromosome with an associated break in gene synteny, in humans and great apes, causing most of the gene to no longer be transcribed. However a small portion of the gene, representing the final transmembrane domain and the C-terminal intracellular tail of the protein, remains expressed in human. stra6.2 is required for normal development in the zebrafish with stra6.2 morphants being phenotypically distinguishable from control injected embryos from the 10-somite stage by a larger head-tail distance indicating an axial extension defect. stra6.2 morphants also display microphthalmia, jaw malformation, shortened and curved body axis and retinal lamination defects. stra6.2 was found to be required to prevent an excess of retinoic acid resulting in an upregulation of retinoic acid-dependent gene expression through an increase in RA synthesis by Raldh enzymes in morphants. Stra6.2-/- mice are viable and fertile and phenotypically normal, even under retinoid-stress, supporting the notion of functional redundancy. In compound knockouts, normal development and postnatal survival can be maintained by a single copy of Stra6 in Stra6+/-;Stra6.2-/- animals. Stra6.2 is less able to support normal development and survival with ~50% of Stra6-/-;Stra6.2+/- animals dying before weaning or showing reduced growth although the remaining animals are indistinguishable from their littermates. Stra6 and Stra6.2 are functionally redundant for development under normal dietary conditions in the mouse and a single copy of either is able to support development in at least 50% of animals. Stra6-/-;Stra6.2-/- mice were therefore hypothesised to be the logical mouse model of Matthew-Wood syndrome, however these mice die early in gestation between E7.5-E9.5. The early embryonic lethality in Stra6-/-;Stra6.2-/- mouse embryos compared to postnatal survival in human Matthew-Wood patients, to which they are the comparable genetic model, could be attributed to the shortened STRA6.2 remaining within the human genome. The equivalent portion of Stra6 has validated signalling motifs, which may still be active in STRA6.2, allowing development to proceed in human ‘STRA6-/-’ embryos.

This thesis describes a program of investigation carried out by the author at MRC Harwell that shows, for the first time, that the gene Zfhx3 plays a role in modulating the circadian clock. The work has used the short circuit (Sci) mouse model to assess the molecular and genetic role of Zfhx3 in circadian behaviour and visual processing. The Sci mutant was identified in an ENU mutagenesis dominant screen at MRC Harwell. The causative mutation was found in a highly conserved region of zinc finger homeobox 3 (Zfhx3), previously not known to play a role in the function of the circadian clock. This study has shown that Zfhx3 RNA is specifically expressed in the adult SCN. Additionally, co-immunoprecipitation has shown that ZFHX3 can interact with core clock proteins, CRY1, CRY2 and PER2 and the Sci mutation causes differential effects on the RNA expression of Cry1, Cry2 and Per1. Genetic interaction experiments identified an interaction between Sci and Cry2 under constant darkness and a potential interaction between Sci and Cry1 and Cry2 under constant light. Together, these data indicate that Zfhx3 has a role in modulating the core circadian oscillator, in particular through cryptochromes. A role for Zfhx3 has also been identified in visual processing and retinal function. Zfhx3 RNA was shown to be highly expressed in the ganglion, inner and outer nuclear layers of the retina. Sci mutants have increased retinal sensitivity by 20 fold, which may be attributed to a decrease in GABA neurotransmission in the retina. From this study, novel functions for Zfhx3 within the circadian and visual systems have been identified. This exemplifies that molecular components of the circadian oscillator may remain to be identified and forward genetic approaches are facilitating advancements in this field.

The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.

Mutagenesis screening in mice is an effective means of identifying essential genes in cardiovascular development. The l11Jus8 (L8) mutant mouse line was originally isolated from a region-specific N-ethyl-N-nitrosourea (ENU) chemical mutagenesis screen and exhibited an autosomal recessive mid-gestational embryonic lethal phenotype characterised by haemorrhage in the thoracic cavity, blood pooling in the heart, right ventricular dysmorphology and yolk sac vascular degeneration. Prior work mapped the L8 mutation to a ~2.77Mb region on mouse chromosome 11. The aim of this study was to further characterise the L8 mutant phenotype and identify the L8 causative mutation. Phenotypic characterisation conducted here confirmed mid-gestational lethality, haemorrhage and yolk sac vascular degeneration in L8 mutants. Histological analysis of L8 mutants demonstrated presence of fragmented cell nuclei and loss of myocardial integrity in embryonic atrial myocardium. Areas of fragmented cell nuclei did not exhibit positive staining for apoptosis. Furthermore, L8 mutants did not appear to experience typical cardiac defects in aspects including myocardial or smooth muscle differentiation, cell proliferation, ECM production, myocardial hypoplasia/hyperplasia, basement membrane components or observable aberrations in cardiac conduction. L8 mutants exhibited atypical cardiac defects including sudden cessation of heartbeat with morphological indicators of necrosis such as swelling of mitochondria and release of microparticles both from atrial myocardial cells. The L8 mutant appears to represent a novel combination of cardiac defects or novel defects with secondary cardiac phenotypes. Sequencing of the coding exons and splice junctions of 22 candidate genes within the ~2.77Mb L8 locus did not identify the causative mutation. The L8 locus was therefore further refined to a ~1.16Mb region including 20 genes. Sanger sequencing of 10 of these genes plus targeted sequence capture and SOLiD sequencing of the region did not identify a potential L8 mutation. Given the refinement of the candidate locus and advances in sequencing technology and analysis, further sequencing will likely identify the L8 mutation and confirm the cause of the embryonic lethal phenotype.

Chronic otitis media (OM) is an archetypal complex disease, which is particularly prevalent in childhood. Epidemiological data suggest high heritability for disease susceptibility, but previous genetic association studies have had methodological flaws, and none have specifically focused on chronic OM phenotypes. Mouse models represent one way to ascertain candidate loci for human association testing. A number of mouse models of middle ear inflammation have been reported, but many susceptibility loci remain undiscovered. I demonstrate that oto-endoscopy is a robust and scalable phenotyping platform for OM in the mouse, and discuss its value in new model discovery. Chronic OM is also a feature of trisomy HSA21 (Down Syndrome). Through an interrogation of the mouse library of segmental trisomy models of Down Syndrome, I identify a critical trisomic region for chronic otitis media. This region may underlie OM susceptibility in Down Syndrome, but could also contribute to disease susceptibility in non-syndromic disease. Mouse models can also be used to interrogate disease mechanisms. Our previous work has shown that the chronically inflamed middle ear is hypoxic, and that hypoxia signalling is a potential therapeutic target. Exploiting the Junbo mouse model, I demonstrate that surgical ventilation of the Junbo ear improves inflammation, and that this is associated with loss of hypoxia signalling. I present preliminary results from transcript analyses of human middle ear effusions showing marked upregulation of hypoxia signalling. A systematic review of existing mouse models suggests that the loci FBXO11, EVI1, SMAD2, and TGIF1 are good candidates genes for human association testing. I detail recruitment and collection of DNA from families in the UK where a child is undergoing grommet insertion. Association testing using a variant of the transmission disequilibrium test shows susceptibility associated with polymorphisms at FBXO11, and possibly also SMAD2 and TGIF1.

Changes in myocardial energetics have been implicated in the pathophysiology of heart failure (HF). However, the precise contribution of creatine (Cr) / phosphocreatine (PCr) / creatine kinase (CK) energy buffer and transfer remains unclear. The aim of this thesis was to study the effects on murine cardiac function of both impairment and enhancement of creatine metabolism. In order to longitudinally follow the cause and effect relationship of myocardial creatine concentration, a non-invasive method of quantification was required. Cardiac Cr levels measured in vivo by 1H-MRS were therefore compared with gold-standard invasive HPLC and found to correlate over a wide-range (r2=0.91). 1H-MRS was reproducible for measuring Cr levels in the heart, brain, and skeletal muscle. The cardiac phenotype of a novel model of creatine depletion, the AGAT-/- mouse, was characterized using in vivo MRI, 1H-MRS and LV catheterisation, under conditions of gradually reducing Cr concentrations; zero Cr; and attempted phenotype rescue with dietary Cr. For the first time in the heart, the rate of Cr turnover was quantified (~3 % per day) and demonstrated that cardiac function was preserved even when creatine levels reduced by ~70-90%. Total absence of myocardial Cr induced impairment of inotropic and lusitropic cardiac function and reduced inotropic reserve. Cardiac dysfunction was only partially rescued by replenishment of the Cr pool, suggesting this to be a consequence of long-term adaptations to chronic low Cr. Finally, we tested the hypothesis that combined elevation of myocardial creatine and ribose would be beneficial in a mouse model of chronic HF by increasing cardiac energy availability. Despite an increase in myocardial ribose concentration, this did not prevent loss of total adenine nucleotides (TAN), and there was no improvement in post-infarct LV remodeling or function. Future studies are needed to explore alternative approaches for maintaining TAN in combination with total creatine.

Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. In the mouse, sex determination occurs at around 11.5 dpc and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors. The mutually antagonistic male and female developmental pathways are regulated by many cellular and molecular processes, disruption of which can lead to disorders of sex development (DSDs). However, many of the molecular mechanisms regulating the differentiation of the two gonads are still unknown. The boygirl (byg) mutant was identified in an ENU-based forward genetic screen for embryos with gonadal abnormalities. On the C57BL/6J background, XY byg/byg homozygotes exhibited complete embryonic gonadal sex reversal. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein (MAP) kinase signalling pathway and provides the first evidence for a function of this pathway in sex determination. This thesis describes experiments aimed at investigating the cellular and molecular basis of the sex reversal phenotype associated with the XY Map3k4byg/byg mutant. Cellular characterisations revealed a defect in male-specific proliferation at 11.5 dpc, which was attributed to a defect in Sry up-regulation. Elucidation of the downstream kinases activated by MAP3K4 during sex determination was attempted, with particular focus on identifying a role for p38α MAP kinase (MAPK). Using a conditional knockout approach, the function of p38α in Steroidogenic factor-1 (Sf1)-positive somatic cells was assessed. However, specific inactivation in these cells did not affect gonad development. Conditional inactivation of Map3k4 itself in these Sf1¬-positive cells also did not disrupt gonad development, suggesting that this pathway is either initiated in a different cell lineage or at an earlier stage than deletion driven by Sf1-Cre can disrupt. Conditional inactivation of p38α in the Müllerian duct mesenchyme and ovarian granulosa cells using Amhr2-Cre did reveal a function for p38α in female fertility, but did not disrupt embryonic sexual development. Gene knockdown in organ culture was attempted to determine a role for multiple p38 MAPKs in all cell types of the gonad. Therefore, this thesis details further characterisations of a novel signalling pathway important for the expression of Sry, focussing on the role of the p38 MAPKs.

Pre-treatment with polyunsaturated fatty acids or bioactive lipid mediators has been shown to reduce neuronal injury in rodent models of focal ischaemia, but the molecular mechanisms underlying this neuroprotection are unclear. In this study, we aimed to investigate whether systemic administration of alpha linolenic acid (ALA) leads to changes in the profile of mouse brain phospholipid and bioactive lipid mediators in both mouse brain and plasma within the previously determined neuroprotection time window. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) allowed us to detect and identify 47 phospholipids in mouse cerebral cortex, including several phospholipid species not previously reported in brain lipidomic studies. These included a phosphatidylethanolamine species with m/z 720 that has been associated with retinal stem cells. No widespread changes in cerebral cortex phospholipid composition were observed following intravenous ALA. Several significant changes in lipid mediators (P<0.05 with two-way ANOVA and post hoc Dunnett's t test) were detected in ALA-treated animals compared to untreated and vehicle-injected animals. Many of the affected lipid mediators are ligands for prostanoid receptors which have been demonstrated to play a role in the development of brain injury following cerebral ischaemia, implying that changes in bioactive lipid mediators or modulation of prostanoid receptors may occur following ALA pre-treatment in mice. This study illustrates the potential of advanced lipidomic analysis as a novel tool for neurochemists.

Our understanding of ɑ and ß globin gene regulation has been advanced by the development of transgenic mouse technology. Major regulatory elements of both gene clusters have been identified. Extensive studies have shown that the genes, although co-ordinately expressed, differ in their regulation. Transgenic mice containing the ß locus control region (ßLCR) and the ß globin genes produce high level, developmentally regulated, copy number dependent expression of the genes. High level a globin gene expression can also be achieved in mice containing the α globin regulatory element, HS-40 and the α genes. However, expression levels are lower than the endogenous mouse α genes, are developmentally unstable and are copy number independent, implying that further regulatory sequences are necessary for full regulation of the cluster. The aim of this project was to identify potentially important regions in the a globin gene cluster. Mice, transgenic for a 145kb DMA fragment spanning the cluster showed higher expression levels in adults than in those previously studied. Although expression levels were developmentally stable and correlated with copy number, they did not reach those reported for ßLCRɑ constructs. A ßLCRɑ construct was examined. The results suggested that there were still unidentified regulatory elements to be found. A natural deletion which down regulated an intact α gene in vivo provided an indication as to where some of these sequences may lie. Analysis of the deleted fragment in transgenic mice implied that it might act to block the negative regulatory influence of an Alu dense region lying downstream, and hence, may have identified the 3' end of a putative a globin domain'.