Theses - Physiology, Development and Neuroscience

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Open Access
Exploring a Nuclear Insulin Receptor Signalling Pathway in Drosophila Neural Stem Cells
Brace, Maire
The insulin receptor (InR) is a central regulator of metabolism, growth, and proliferation in both humans and *Drosophila*. Integrating a range of signals, InR coordinates pleiotropic, tissue‐specific outputs. Neural stem cells (NSCs) give rise to the vast range of cell types that comprise the central nervous system (CNS) in a manner highly regulated in both space and time. With a comparatively simple CNS that nonetheless exhibits remarkable biological conservation, combined with superior genetic tractability, *Drosophila melanogaster* is an ideal model organism for studying the principles of neurogenesis and NSC biology. In *Drosophila*, insulin signalling is necessary and sufficient for the reactivation of NSCs from quiescence, a conserved state of mitotic dormancy. InR has been observed in the nucleus of a range of cell types *in vitro* for decades, but it is only more recently that the significance of this is becoming clear. Data reveal a novel nuclear arm of the insulin signalling pathway, in which InR associates with chromatin genome‐wide, strongly localizing to promoters and activating the transcription of relevant genes. This finding represents a growing paradigm shift, encompassing other receptor tyrosine kinases (RTKs), whereby direct nuclear signalling complements canonical membrane‐localised signal transduction. Given the complexity of insulin signalling and its dysregulation across an array of chronic human diseases, this nuclear pathway constitutes an exciting avenue for basic and translational science. Characterising the nuclear actions of InR might offer a more complete understanding of the longer‐term effects of insulin signalling. In the context of the CNS, it may offer a more complete understanding of NSC dynamics, which in turn may guide future therapeutic strategies for brain repair and difficult‐to‐treat malignancies. In this thesis, nuclear insulin signalling in *Drosophila* NSCs is studied via a variety of *in vivo* methods. Cyan fluorescent protein (CFP)‐tagged InR was expressed with the GAL4 system to examine its subcellular localization. InR‐CFP is present in the nucleus of *Drosophila* NSCs throughout CNS development. This localization pattern is not upheld in NSC progeny, where InR‐CFP exhibits strong membrane localisation. Next, the endogenous InR locus was tagged with green fluorescent protein (GFP) knock‐in using the CRISPR/Cas9 system. At endogenous InR expression levels, no clear nuclear localization was visible on confocal microscopy. However, the presence of nuclear InR was later confirmed with NanoDam. NanoDam, a recently published technology based on Targeted DamID, was used to profile the chromatin association of InR in NSCs *in vivo* genome‐wide. NanoDam was performed at endogenous levels of InR expression in the InR‐GFP background, and under misexpression conditions in the UAS‐*InR‐CFP* background. InR associates with chromatin genome‐wide in both genetic backgrounds. These data represent the first known finding that InR associates with chromatin in any cell type in *Drosophila*, and that InR associates with chromatin in NSCs in any organism. More genes are reproducibly and significantly bound in the endogenously tagged InR‐GFP background than the misexpression UAS‐*InR‐CFP* background, which may be due to higher expression levels in the latter background saturating physiological binding patterns and leading to more random interactions. Consistently, significantly more genes are bound in reactivating than quiescent NSCs; and consistently, InR binding is enriched in promoters. Amongst the most significantly bound genes in reactivating NSCs are genes with known roles in NSCs, signalling pathways, cell growth, and proliferation. Given the known role of canonical insulin signalling in the reactivation of quiescent NSCs, this work hypothesizes that nuclear insulin signalling contributes to this process. Exploring the transcriptomes of quiescent and reactivating NSCs with scRNA‐seq and intersecting this data with the NanoDam data revealed genes differentially expressed and bound by InR in reactivating NSCs. These constitute candidate genes that may act downstream of nuclear InR to mediate reactivation. They include genes with known roles in NSC development; genes that encode important RNA‐modifying proteins; and genes that modulate the activity of central signalling pathways known to affect NSC dynamics. Amongst these candidate genes, several stand out as being of particular biological interest. *Mettl3* encodes a conserved RNA methyltransferase. The m(6)A modification METTL3 catalyses is the most prevalent mRNA modification in vertebrates and influences mRNA stability, translational dynamics, and RNA pol II pause release. The latter is an important mechanism of enabling rapid, synchronous cell state changes, of which reactivation is a prime example. Preliminary data show that *Mettl3* knockdown is associated with a reactivation delay (Cian Doherty and Andrea Brand, unpublished), making METTL3 acting downstream of nuclear InR to facilitate reactivation an exciting model to investigate. *dachs*, a negative regulator of Hippo signalling constitutes another intriguing candidate gene; a switch between Hippo signalling and insulin signalling is vital for reactivation to occur. Dpp is an established signal for stem cell self‐renewal and prevents premature differentiation, and genes pertinent to its signalling, including the *tkv* receptor and *shn* transcription factor are also candidate genes. Overall, the findings support a model in which nuclear InR signalling serves as a point of integration of important NSC signalling pathways and orchestrates their cross‐regulation to fine‐tune the balance between pro‐quiescence and pro‐proliferation signals. The binding of a broad range of relevant genes and a resultant modest and combinatorial transcriptional activation was a picture also seen in mammalian cells. Future work will be necessary to validate the candidate genes and models proposed by this work and to elucidate the mechanisms of InR nuclear import and transcriptional activation. Some pertinent experimental avenues are suggested in the conclusions.
Open Access
Understanding the Genetic Basis of Obesity: Lessons from Man’s Best Friend
Wallis, Natalie; Wallis, Natalie [0000-0001-9543-3711]
Obesity is an increasingly prevalent and complex disorder which poses a serious threat to canine and human health. Obesity is ultimately caused by caloric excess, and results from the complex interplay between environmental, behavioural, and genetic factors. Study of human obesity has been extensive whilst that in dogs has been limited. Past data on how biological risk factors impact canine obesity have been contradictory and the genetic basis of canine obesity is poorly understood. The heritability of human obesity is estimated at 40-70% and, despite considerable study, the majority of genetic variants responsible for this heritability are yet to be uncovered. Therefore, the understanding of obesity genetics across species is incomplete. In dogs, selective breeding and population bottlenecks simplify gene mapping for complex disease, offering the chance to unpick the ‘missing heritability’ of obesity in both dogs and humans. The studies of this thesis aim to better understand the development of canine obesity and gain insight into obesity genetics across species. First, biological risk factors for obesity were investigated in a population of British pet Labrador retrievers (n = 521), an obesity-prone dog breed. Linear regression was used to assess known and novel risk factors for canine obesity. Findings demonstrated a nuanced effect of known risk factors, with neutering and age having sex-dependent effects on obesity outcome. Chocolate coat colour was discovered as a novel risk factor for Labrador obesity. Some of these risk factors were mediated through an increase in food motivation, providing valuable insight into underlying mechanisms of action in canine obesity. Subsequently, a genome wide association study (GWAS) for obesity in Labrador retrievers was conducted. Multiple obesity-associated loci were identified, and regions of interest harboured candidate genes and variants. Candidate genes were interrogated using cross-species resources, and mechanisms of action hypothesised. Similarities between canine and human obesity means genetic associations in dogs can prioritise and identify obesity genes in human genomic studies. Syntenic human regions and genes were explored through GWAS and rare variant enrichment analyses in large scale human cohorts. Canine obesity loci of large effect served to highlight human loci implicated in common obesity, including genes *CDH8*, *CARD11* and *DENND1B*. Variants in *CSNK1A1* were also found to be associated with monogenic cases of severe, early onset obesity in humans. Carriers were pursued through familial investigation and deep clinical phenotyping. Further, a canine GWAS association of large effect at the *SEMA3D* locus provides strong orthogonal evidence of its importance in energy homeostasis. Canine GWAS summary statistics were used to construct a canine polygenic risk score method for obesity, something not previously achieved in dogs. Genetic scores for obesity predicted phenotypes in related but not unrelated dog breeds. Polygenic scores also provided insight into known within-breed variation in canine obesity risk, explaining associations with coat colour. Polygenic background was shown to influence the penetrance of a well characterised canine *POMC* mutation that affects obesity risk by altering hypothalamic leptin-melanocortin signalling. Additionally, polygenic background affected how owner management practices impact obesity outcome in dogs. This thesis provides novel insight into canine and human obesity, including the identification of new obesity-related genes. An improved understanding of canine obesity risk factors is established and highlights areas for further investigation, particularly through intra-breed study. Findings demonstrate the benefits of studying complex disease in non-traditional animal models such as the dog. This work informs the treatment and prevention of obesity in both human and veterinary medicine.
Embargo
The role of polarisation in the first cell fate decision of the mouse embryo
Lamba, Adiyant
A fundamental process in embryonic development is the first cell fate decision, when cells take on distinct lineage identities for the first time. In mammals, this separates embryonic inner cell mass (ICM) from extra-embryonic trophectoderm (TE) during pre- implantation development. In the mouse, the process is classically attributed to the consequences of apical-basal polarity, which forms at the 8-cell stage: those cells which retain the apical domain after cell divisions are specified as TE, and the rest as ICM. However, more recent research has shown that early molecular heterogeneities between cells before polarisation can also bias cell fate. The existence of different models calls into question the role of polarisation in the first cell fate decision. The first part of this study focuses on reconciling the polarity and heterogeneity models. It was previously thought that polarisation occurs only at the late 8-cell stage. By studying its timing in detail, it is possible to split cells into ‘normal polarising’ (NP) cells which polarise at the late 8-cell stage, and ‘early polarising’ (EP) cells at the early 8-cell stage, the latter found in approximately 20% of embryos. Although EP cells follow the canonical polarity pathway — involving the critical factors Tfap2c, Tead4 and RhoA — they have molecular and morphological differences from NP cells and are biased towards symmetric divisions and TE fate. Blastomeres with low activity of the arginine methyltransferase CARM1 prior to polarisation are known to be biased towards TE, and inhibition of CARM1, or overexpression of its substrate BAF155, increases the frequency of early polarisation. Thus, this study proposes that early heterogeneities influence cell fate by altering the timing of polarisation. The final part of this study addresses the ability to detect polarity. Tracking polarisation over time currently requires invasive fluorescence imaging. Here, artificial intelligence is used to detect whether an embryo is polarised from unstained images, after training based on bright-field movies annotated using the corresponding fluorescence channel. The resulting model has an accuracy of 85% for detecting polarisation, significantly outperforming human volunteers trained on the same data (61% accuracy). Taken together, this study advances our understanding of polarisation and its role in the first cell fate decision, while also providing a tool for further investigation.
Embargo
Identifying novel roles for basement membrane-associated proteins in the adult Drosophila intestinal epithelium
Eldridge-Thomas, Buffy; Eldridge-Thomas, Buffy [0000-0003-4070-2827]
Maintenance of a healthy epithelium relies on the functionality of resident stem and progenitor cells, which must divide and differentiate in a way that precisely meets tissue requirements. To achieve this, cells integrate an array of information, including chemical, mechanical and adhesion-based cues, which collectively provide the instructions to direct cellular behaviour. A potential source of such signals is the basement membrane, a thin extracellular matrix which underlines the basal side of all epithelia, and to which epithelial cells adhere via various receptors. Despite their ubiquity, basement membranes are understudied and there remains much to understand regarding how their components and receptors impact epithelia. Using the adult *Drosophila* midgut as a model system to investigate epithelial stem and progenitor cell behaviour *in vivo*, I searched for novel roles for basement membrane associated proteins in the intestinal epithelium. Integrins are major receptors used by cells to bind the extracellular matrix, and they recruit a number of intracellular proteins including the conserved mechanoeffector Vinculin. I contributed to a project investigating the role of Vinculin in the intestinal epithelium, where *vinculin* mutants show increased intestinal stem cell proliferation and accelerated progenitor cell differentiation. I used scanning electron and confocal microscopy to confirm that *vinculin* mutant midguts have elevated cell numbers and developed a workflow for quantifying proliferation and differentiation. Using transmission electron microscopy, I showed that *vinculin* mutant midguts do not have an abnormal basement membrane. This corroborated other observations which suggest that, in the context of intestinal cell production and differentiation, Vinculin functions at cadherin cell-cell junctions, not at Integrin cell-matrix adhesions, specifically in enteroblast progenitors. Here, Vinculin is required to keep the progenitor in a quiescent state and to suppress division of neighbouring intestinal stem cells. This work revealed that mechanical regulation at the contact site between stem cells and their progeny is used to control cell number and enhances understanding of how mechanical signals contribute to intestinal epithelial homeostasis. In a separate project, I identified the conserved transmembrane proteoglycan Syndecan as an essential protein for intestinal stem cell maintenance. Syndecan is a basement membrane receptor, with a plethora of other intra- and extracellular binding partners, that is dysregulated in multiple human diseases. I found that RNAi-mediated depletion of Syndecan from intestinal stem cells, but not from other intestinal epithelial cell types, causes loss of these stem cells. Without Syndecan, intestinal stem cells acquire abnormal cell morphologies and display cell division-associated defects. In addition, Syndecan-depleted intestinal stem cells develop large nuclear lamina invaginations, nuclear shape changes and acquire DNA damage. Ultimately, the vast majority of Syndecan-depleted intestinal stem cells are lost from the epithelium, via a combination of apoptosis and other mechanisms. My work found that Syndecan has negligible effects on major chemical signalling pathways, and Syndecan also seems not to act via two components of the Linker of Nucleoskeleton and Cytoskeleton complex in a potential mechanotransduction pathway to the nucleus. In parallel, I sought to investigate whether Syndecan is required in other somatic stem cell types. My collaborator, Dr Chantal Roubinet, found that Syndecan depletion from *Drosophila* neural stem cells causes abnormal nuclear size and shape; abnormal mitotic nuclear envelope remodelling and delayed cell division, indicating Syndecan plays a common role in stem cell behaviour. My work newly identifies Syndecan as a regulator of intestinal stem cell maintenance and finds a connection between this transmembrane protein and nuclear properties in multiple stem cell types. In future, uncovering Syndecan’s precise mode of action and its molecular partners in this model system may help provide a new framework to delineate its function in human disease.
Embargo
Cell Fate Decisions in the Early Mammalian Embryo
Iwamoto-Stohl, Lisa
In mammals, successful pre-implantation development leads to the formation of a tri-lineage structure known as the blastocyst, consisting of the epiblast, primitive endoderm and trophectoderm, which will give rise to the new organism, the yolk sac and placenta respectively. These three lineages must be established from the totipotent zygote via two successive cell fate decisions in the appropriate sequence, position and proportion, to generate a blastocyst capable of implantation and further development. In mammals this process has long been thought to be regulative, with cell-cell interactions flexibly determining the eventual fate of cell. This is in contrast to commonly studied non-mammalian embryos in which pre-patterning of the embryo, driven by spatially localised factors, is a common feature. However, early blastomeres of mouse embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts, prior to the first cell fate decision. Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Utilizing single-cell proteomics by mass-spectrometry I show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. Fascinatingly, halved mouse zygotes already display protein asymmetries, which resembles alpha and beta blastomeres, suggesting differential proteome localisation already within zygotes. I also find that beta blastomeres may have a greater developmental potential, and give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. This provides the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation. In humans, this early period of development is prone to failure, with a third of human pregnancies estimated as being lost prior to implantation. A high incidence of aneuploidy is thought to be a major driver of pregnancy failure and understanding the behaviour of aneuploid cells is of great interest. As the blastocyst forms and implants, aneuploid cells may be eliminated through cell competition with diploid cells or show differences in their lineage segregation, impacting the composition and proportion of each lineage in the blastocyst, The mouse embryo does not have similar high intrinsic rates of aneuploidy, but reversine, a spindle assembly checkpoint inhibitor, can be used to recapitulate the human aneuploid embryo to some extent, and to observe the elimination of aneuploid cells. Here, I return to the human, utilising human embryonic stem cells and new integrated stem cell based embryo models, to characterise conserved aneuploid cell depletion in mouse and human stem cell co-cultures of diploid and aneuploid cells. Furthermore, I use stem cell lines harbouring specific aneuploidies to determine if specific aneuploidies confer differential ‘fitness’ and elimination rates. Overall my PhD has examined two questions regarding early mammalian development: 1) when do the cells of the embryo first become different to each other and how does this interplay with lineage segregation and 2) can a model of the mosaic aneuploid human embryo be generated to better understand the fate of aneuploid cells within the three lineages of the blastocyst.
Controlled Access
The role of ventromedial and dorsolateral prefrontal cortex in physiological and behavioural dysfunction in non-human primates, of relevance to mood and anxiety disorders
Banai Tizkar, Rana
The research presented in this thesis investigates the causal role of area 25 of the subcallosal anterior cingulate cortex and area 46 of the dorsolateral prefrontal (dlPFC) cortex in depression- and anxiety-like behaviour in a non-human primate, the common marmoset. Depressed patients show hyperactivity in area 25, and hyperactivity and hypoactivity of area 46 in the right and left hemispheres respectively. Moreover, these two seemingly unrelated regions, one involved in emotion and visceral activities (area 25) and the other involved in cognition (area 46), have sparse direct anatomical connections yet their negative coupling is observed in the state of disease and after successful treatment. Furthermore, levels of the stress hormone cortisol, which is elevated in depression and anxiety, are positively correlated with activity within area 25. However, the causal role of cortisol in the activity of this region is unknown although it has been shown that area 25 expresses both mineralocorticoid and glucocorticoid receptors, which suggests elevated cortisol can directly modulate activity in this region. Three subsections investigating the above, form three chapters of this thesis. Chapter three focuses on the effect of overactivation and inactivation of area 25 in two behavioural tests assessing motivational and consummatory behaviour of relevance to the symptom of anhedonia, the loss of pleasure. In chapter four the effect of the local increase of cortisol in area 25 in three domains of motivational, anticipatory, and anxiety-like behaviour is assessed. In chapter five the effect of overactivation and inactivation of area 46 within dlPFC on anxiety-like behaviour and basal cardiovascular activity is investigated under four conditions of vehicle control, left hemisphere, right hemisphere, and bilateral manipulations, in order to assess reports of asymmetry in dlPFC function. Taken together, the findings provide evidence for bidirectional effects of area 25 manipulation on motivation, and consumption, albeit with a more complex relationship with reward value for the latter. Moreover, cortisol is shown to have a causal impact on area 25 function as manifested in anxiety-like behaviour and anticipatory anhedonia. Since the effects were observed only with a very short pre-treatment time, it implicates those cellular mechanisms known to underlie the rapid effects of cortisol. These effects were observed despite the low number of subjects (n = 4), however, whether the lack of effect on cardiovascular response is due to the low power could be investigated with more subjects. Finally, the results revealed that bilateral and left hemisphere inactivation of area 46 increased anxiety-like behaviour. However, to establish the role of area 46 in the central autonomic network, further investigation is required since ANOVA showed no effect of manipulation despite the significant effect observed with the Linear Mixed Effect Model. The current interpretation of the observed results is that the effect size is small and there are high individual differences, both of which can only be addressed with an increased number of subjects. In contrast, overactivity of the right hemisphere did not increase anxiety as implicated in the depression and anxiety literature. The evidence overall points to functional asymmetry within area 46. The opposing effects of area 25 and area 46 manipulations on anxiety-like behaviour reported here support the correlatory findings in humans for the negative relationship between subcallosal and dlPFC activity in human mood and anxiety disorders. When translating these preclinical findings to the clinical domain it should be noted that the reward used here, similar to many preclinical models, is a primary reward, which has an innate value essential for homeostasis. The neural circuit underlying primary and secondary rewards may vary and requires further investigation. Hence, these findings are only applicable with regard to primary rewards when translating to human studies.
Open Access
Neocortical functional network development at the microscale: impairments and mechanisms in Rett Syndrome.
Dunn, Alexander
The brain comprises intricate neuronal circuitry arranged in complex patterns. A key challenge in studying brain network topology is that microscopic networks at the microscale cannot be studied noninvasively in humans. It is particularly challenging to study microscale networks during early development in animal models as the brain is rapidly changing in size. This could have implications for deciphering how an individual can show apparently normal development before rapid decline. The neurodevelopmental disorder, Rett Syndrome, characterised by Mecp2 deficiency, is a pertinent example of this. In the present thesis, I aimed to thoroughly characterise early neocortical functional network development at the microscale and how this is disrupted in a Rett Syndrome mouse model, before examining some of the mechanisms involved. For translatability, I also tested the viability of using a graph theoretical approach in 3D, human tissue—cerebral organoids—for future research to study neocortical network development in health and disease. Micro-electrode array recordings of primary murine cortical cultures showed that Mecp2-deficient, Rett Syndrome model networks showed impairments in spontaneous spiking and bursting activity. They showed reduced global functional network connectivity and modularity. Simulations revealed deficits in computation of the balance of connection benefits/costs. Interestingly, Mecp2-deficient network deficits were seen as early as two weeks in vitro which precedes the putative onset of behavioural decline in this Rett Syndrome model (~8 weeks). Crucially, optogenetic suppression of parvalbumin-expressing inhibitory interneurons restored spatiotemporal spiking dynamics. Finally, human cerebral organoids did show complex topology and hub node features beyond that which could be explained by high firing rates at 180 days in vitro. To conclude, neocortical microscale functional networks mirror many aspects of macroscale network development, and several network features are disrupted in Rett Syndrome. Early cell type-specific intervention may restore network activity patterns in Mecp2-deficient networks. In the future, human cerebral organoids provide a promising complementary approach to animal models for future study of complex network topology in three-dimensional networks derived from human cells. Further work is required to establish whether they adhere to key principles and patterns seen in vivo.
Embargo
The intersection of metabolism and oxygen in T cell function and anti-tumour immunity
Minogue, Eleanor
T cells, in particular CD8+ T cells, play a critical role in maintaining immune homeostasis and in eliminating cancerous cells. In recent years it has emerged that cellular metabolism is a central regulator of T cell fate and function. T cell metabolic profiles rapidly fluctuate following T cell activation and can be highly influenced by microenvironmental factors, such as oxygen. This work explores the intertwining role of metabolism and oxygen in CD8+ T cell differentiation and anti-tumour immunity, with a particular emphasis on designing novel cancer immunotherapies. This thesis comprises four results chapters, the first three of which are centred around the metabolite glutarate, an oxygen sensitive metabolite with no known immunological function prior to this work. Chapter III identifies glutarate as an endogenous regulator of T cell function, demonstrating its capacity to enhance CD8+ T cell cytotoxicity and reduce tumour growth. Chapter IV identifies glutarate as a competitive inhibitor of alpha-ketoglutarate dependent dioxygenases (αKGDDs) and explores the effect of glutarate on hypoxia-inducible factor prolyl-hydroxylases (PHDs) and on DNA and histone methylation. Chapter V focuses on the role of glutarate in cellular metabolism and reveals that glutarate controls the activity of the pyruvate dehydrogenase complex (PDHc) via the post-translational modification glutarylation. The final results chapter, Chapter VI, focuses on the role of molecular oxygen as a regulator of T cell activation and metabolic reprogramming. This chapter further explores manipulation of oxygen sensing pathways for therapeutic gain in the context of cancer. The data presented here describe glutarate as an important metabolite with significant T cell modulatory capacity whilst also highlighting the important role of molecular oxygen in T cell activation and differentiation. Furthermore, this work highlights the important interplay between metabolism and oxygenation in T cells and illustrates that harnessing these pathways can improve anti-tumour immunity, with potential clinical importance.
Open Access
Organised variations in space and time in the hippocampal representation of location
Chaudhuri Vayalambrone, Prannoy
Navigation is the process by which an agent plans and follows routes within an environment. Animals can navigate their environments in ways that do not seem to result from simply learning a set of stimulus-response associations that result in reward. Rather, their navigation is consistent with some internal representation of the animal’s location within the environment, or a “cognitive map”. In mammals, this is thought to be supported by various spatially sensitive neurons in the hippocampal formation, including grid and place cells. The cognitive map also flexibly adjusts previous representations to reflect changes in a familiar environment. Several studies have demonstrated continuous spatial and temporal adjustments in how the cognitive map represents the animal’s location. For example, in a rescaled version of a familiar environment, grid and place cells’ firing fields shift position throughout the trial depending on which wall the animal encountered last. These dynamic adjustments may reflect the process by which an animal “redraws” its cognitive map of an environment, switching between different walls as reference points as it relearns how they are arranged. Furthermore, maps are not simply used to locate oneself, but also to plan and execute routes. Accordingly, multiple studies have shown that grid and place cells often represent locations just behind/ahead of the animal, which may reflect the system recalling and planning routes through the environment. How this “time shift” varies between different cells, and throughout a trial, is unclear. This project investigates the principles that organise these dynamic adjustments in the location represented by the cognitive map. We used data from experiments in which rats foraged in four similar asymmetrically deformed enclosures, only differing in the configuration of their western wall(s). Previous work shows that grid and place cell fields near the deformed side of each enclosure shift by larger distances than those on the stable side. We look for direction-dependent changes in grid cells’ firing fields, and whether these occurred in a pattern consistent with the asymmetrical distortions observed. We used the rat’s head direction as a proxy for which cues the rat was relying on. We find that most grid patterns show statistically significant changes in phase and average firing rate based on the animal’s current head direction, with no clear systematic variation with enclosure shape. We do not find any clear spatial organisation in these direction-dependent phase shifts. This suggests that grid cell rate maps do shift depending on which cues the animal encounters, but that this is separate to the mechanism allowing grid cell maps to distort asymmetrically. We also examine the time shifts in grid and place cells’ firing in these experiments. We find that both cell types mostly fire prospectively, on average signalling places ~150 ms ahead of the animal’s current location. The size of each grid cell’s average time shift correlates with its scale. Place cells’ time shifts similarly correlate with their firing field size. Cells’ time shifts also vary over the course of each trial, tending to contract as an animal approaches the enclosure walls. We also find that cells’ optimum time shifts are related to the LFP theta phases at which they tend to fire. These findings suggest that different grid modules together represent a spectrum of locations ahead of the animal, organised by the theta cycle. To summarise, this work shows that grid cell maps appear to continuously adjust their location readout even in familiar environments; however, these do not appear to contribute to their distorted appearance in deformed enclosures. We also show that grid and place cells primarily represent space prospectively as rats explore open fields. These time shifts are organised by each cell’s spatial scale, which hints at how the anatomical organisation of cells by firing scale may be functionally significant. Time shifts are also related to the theta cycle, suggesting how memory encoding and retrieval may be organised in the hippocampal network. Overall, these analyses help isolate and explain subtle functional signals in what is normally considered noise in the cognitive map’s representation of space. This helps us understand how the brain incorporates new information about a familiar spatial context and how it processes it to plan and select appropriate actions.
Open Access
The Mechanisms Underlying Olfactory Vicarious Fear Learning in Rats
Sherman, Emily
Vicarious learning occurs when an “observer” animal, naïve to a conditioned stimulus, is in the presence of a “demonstrator” animal that has been conditioned to react to the stimulus. This project aimed to elucidate the mechanisms behind olfactory vicarious learning to an aversive cue; namely, how male rats observed and internalised the fear behaviour of other male rats. A robust olfactory fear conditioning protocol was set up, in which an otherwise neutral odour, acetophenone, was paired with foot shocks. The rats subsequently displayed freezing behaviour. In order to assess the role of dominance in the home cages, dominance hierarchies were first measured through a resource competition task in which rats competed for an appetitive resource. The conditioned demonstrator animals were placed with naïve observer animals and the conditioned odour was introduced for vicarious learning. All animals were then tested individually the next day, and freezing behaviour was measured. Observer rats successfully learned to freeze to the odour after the vicarious learning session with the demonstrator rat. To understand the mechanism behind fear transmission, pharmacological interventions and sensory modality manipulations were employed in different experiments. Rats learned to freeze to acetophenone through exposure to the odour in the presence of only a conditioned conspecific’s faeces and urine, which showed olfaction alone was sufficient for vicarious learning. Oxytocin has previously been shown to modulate social behaviour as well as fear behaviour, and the release of oxytocin was shown to reduce freezing behaviour after direct and vicarious fear conditioning. However, injecting an oxytocin receptor antagonist to block oxytocin signaling prior to contextual conditioning did not have a significant effect on freezing behaviour. To investigate its effects on olfactory vicarious learning, an oxytocin receptor antagonist was injected into an observer animal prior to vicarious learning, and it did not show a significant effect on a rat’s ability to learn from its conspecific. On the other hand, the injection of an adrenergic receptor antagonist into an observer prior to vicarious learning did significantly block vicarious learning. Ultimately, isolating fear pheromones and targeting beta-adrenergic receptors in the observer had the greatest effect on a rat’s ability to learn to freeze to an odour vicariously.
Embargo
The Role of Mouse Placental Endocrine Function on Offspring Metabolic Health
Rodgers, Amanda
During gestation, adequate nutrient partitioning between mother and fetus is required for optimal maternal and fetal outcomes. The placenta, the barrier between mother and fetus plays a key role in this partitioning; not only by transporting nutrients to support fetal growth, but also by secreting hormones, which alter the maternal metabolism. Disturbances in placental endocrine function may lead to adverse fetal outcomes and pregnancy complications, such as gestational diabetes mellitus (GDM). The Developmental Origins of Health and Disease (DOHaD) theory suggests that adversity in prenatal and early life, influence offspring health. Multiple studies have now shown that the maternal environment (GDM, diet and stress) can affect placental growth, birthweight, and programme the offspring’s risk of long-term health, including increasing risk for metabolic diseases. However, the precise role of the placenta, particularly the role of placental endocrine function, in the developmental programming of offspring metabolic health is unknown. To study this, Insulin-like Growth Factor 2 (Igf2), known to be important in controlling the formation and function of placental endocrine cells, was conditionally knocked down in the mouse placental endocrine zone (junctional zone, Jz; paternally inherited Jz-Igf2UE). The aims of this study were to; 1) evaluate how Jz-Igf2UE impacts offspring metabolic health, 2) assess the impacts of Jz-Igf2UE on offspring insulin signalling and lipid handling in the liver, skeletal muscle and adipose tissue 3) determine the effect of Jz-Igf2UE on adipose tissue respirometry, and on the gonadal adipose transcriptome. The impact of Jz-Igf2UE on offspring metabolic health was evaluated via insulin tolerance tests, metabolic cage assessments and measuring lean mass and adiposity at multiple timepoints. Insulin signalling and lipid handling in the offspring was assessed by determining the abundance of proteins and mRNAs involved in gluconeogenesis, insulin signalling and lipid metabolism in the liver, skeletal muscle and white adipose tissue, using western blotting and quantitative Polymerase Chain Reactions (qPCR). RNA sequencing and bioinformatic analysis was used to assess changes in gene expression/functional pathways in the adipose tissue, and adipose tissue respirometry was assessed by ex vivo high resolution respirometry. This study showed that Jz-Igf2UE did lead to changes in the metabolic phenotype of the offspring, as indicated by alterations in growth, adiposity, insulin handling, respiratory exchange ratio (RER) and energy expenditure. This was also accompanied by changes in the abundance of proteins and mRNA involved in gluconeogenesis, insulin signalling and lipid metabolism in offspring liver, skeletal muscle and white adipose tissue. However, these alterations were largely dependent on offspring sex and age, with female offspring showing more detrimental metabolic changes than males. These sex-dependent programmed changes in offspring metabolic physiology, in part, may be linked to the sex-specific alterations seen in the adipose tissue transcriptome. Overall, this study highlights the importance of the placental endocrine function in postnatal offspring metabolic health. Moreover, these placental defects programme sex-specific changes in offspring metabolic physiology as well as sex-specific transcriptome changes in the adipose tissue. By improving our understanding of how placental endocrine malfunction leads to the programming of offspring health, in the future we may consequently improve metabolic disease outcomes associated with developmental origins.
Embargo
Transcriptional Heterogeneity in ALS: Patients with Retrovirus-mediated Disease and Potential Cell-type Specific Therapeutic Approaches
Pasternack, Nicholas
Amyotrophic lateral sclerosis (ALS) is a universally fatal neurodegenerative disease affecting both upper and lower motor neurons. There is currently no cure for ALS and most clinical trials treat all ALS patients as a single group. The hypothesis addressed in this thesis is that ALS is a transcriptionally heterogeneous disease with distinct biological pathways converging on a similar phenotype. To address this hypothesis, a bulk RNA sequencing (RNA-seq) dataset from almost 2,000 ALS and unaffected control samples was leveraged to uncover transcriptionally distinct patient subpopulations. Using non-negative matrix factorization (NMF), an unsupervised clustering algorithm, four distinct clusters of ALS patients and controls were identified in the cortex (CTX) and spinal cord (SC). Differential expression analysis (DEA) revealed that certain loci associated with human endogenous retrovirus K (HERV-K), a type of transposable element (TE), as well as other non-TE features were dysregulated in patient samples compared to controls. In both the CTX and SC, around 20% of ALS patients had higher overall HERV-K expression than other patients and controls (high HERV-K ALS). Additionally, in both the CTX and SC, there was an NMF cluster that was enriched for high HERV-K ALS patients relative to other patients. Moreover, the high HERV-K ALS patients had unique transcriptomic signatures at the individual feature and biological pathway levels relative to other ALS patients and controls. There were four robustly upregulated HERV-K loci (1q21.3, 5q15, 8q24.3, and 11q13.4) that can encode for truncated HERV-K envelope protein (env) in high HERV-K ALS patients. These truncated peptides generally corresponded to the transmembrane domain of the full-length env, implying it is neuropathological in ALS. Moreover, the DEA results were validated using a regularised logistic regression, a type of supervised machine learning, classifier for ALS patient or control samples. Finally, the cell type specificity of these results was determined using a publicly available single nucleus RNA sequencing (snRNA-seq) dataset consisting of 23 ALS patients and 17 age- and biological sex- matched controls. Carbonic anhydrase 1, CA1, and the sulfate transporter, SLC13A4, are the most promising targets as they were the most robustly dysregulated protein coding genes across analyses and are also specifically upregulated in upper motor neurons in ALS patients. In summary, these results demonstrate that ALS is a transcriptionally heterogeneous disease, and patient subpopulation- and cell-type-specific approaches will be needed to effectively treat ALS neuropathology.
Embargo
A multi-lineage in vitro mouse embryo model derived exclusively from mouse embryonic stem cells
Lau, Yui
The mouse embryo has been a vital model to study human embryonic development and congenital diseases. As the mouse embryo implants, it undergoes drastic morphological changes to form the egg cylinder consisting of the epiblast (EPI), extraembryonic ectoderm (ExE) and visceral endoderm (VE), which give rise to the embryo proper, placenta and yolk sac respectively. By using stem cells derived from these three fundamental lineages, many *in vitro* models have been developed to recapitulate various events of post-implantation development. Using only mouse embryonic stem cells (ESCs), the gastruloid model has been demonstrated to mimic basic body axis formation and aspects of gastrulation, somitogenesis, cardiogenesis, and neurulation. However, it fails to recapitulate the spatiotemporal interplay of signalling pathways between embryonic and extraembryonic tissues. Another approach is to combine ESCs with extraembryonic stem cells (trophoblast stem cells (TSCs) and extraembryonic endoderm stem cells (XEN cells)) in ETX embryos (embryo model composed of ESCs, TSCs and XEN cells) and iETX embryos (embryo model composed of ESCs, TSCs and induced XEN cells). While these integrated models have demonstrated greater developmental potentials, one remaining complication is that TSCs used in these models are maintained in undefined culture condition and are heterogenous in developmental state, which increase the difficulty and variability of embryoid formation. The central aim of this thesis is to develop an ESC-based *in vitro* embryo model that can reconstitute the three fundamental cell lineages of the post-implantation mouse embryo. It was shown that ESCs that transiently overexpressed Cdx2 upon doxycycline induction could effectively replace TSCs to form embryo-like structures. The resulting ‘EiTiX-embryoids’ underwent development from pre-gastrulation stages to neurulation stages, developing headfolds, a beating heart structure, and extraembryonic tissues, including a yolk sac and chorion. Further single-cell RNA sequencing analysis of individual embryoids showed a robust recapitulation of cell states in both embryonic and extraembryonic lineages, with little variation in the overall gene expression programme in these cell states. Together, this thesis presented the ‘EiTiX-embryoids’ as a novel ESC-based multi-lineage *in vitro* model of mouse post- implantation development to neurulation stages. This model combines the advantages of both existing ESC-based models and integrated models and it has reached an advanced developmental stage that is previously not achieved in ESC-based models. Lastly, this model can potentially be used in drug screening or as a novel disease model by using stem cell types with gene mutations.
Embargo
The placenta in adverse maternal environments; exploring how hypoxia and food restriction limit fetal growth
Siragher, Emma
The placenta is a vital link between mother and fetus, supporting fetal growth through supply of oxygen and nutrients. However, it must also work to balance both maternal and fetal needs and adapt to the maternal environment to support successful pregnancy. Failure to adapt can impact maternal health and fetal growth, which may have long-lasting effects on the offspring. For this study, pregnant mice were exposed to either maternal inhalation hypoxia (MIH; 10% or 13% inspired oxygen), or food restriction (FR) between days 14-19 of gestation. To explore the role of the placenta in adverse conditions, the distinct functional zones of the mouse placenta, the endocrine junctional zone (JZ) and the labyrinth zone (LZ), which is responsible for transport, have been examined separately. Where possible, analyses have been performed on placentas from both male and female fetuses. Fetal weight was reduced and placental weight unchanged in both sexes under MIH and FR. Gene expression analysis of the LZ in MIH identified that altered lipid handling, peroxisome proliferator-activated receptor (PPAR) signalling, and calcium binding may contribute to reduced fetal weight. Histological analysis showed a striking increase in calcium deposition, corresponding with areas of fibrosis. In the JZ, increased glycogen deposition was observed, and dysregulation of steroidogenic genes. With FR, transcriptomic changes in the LZ included genes involved in the extracellular matrix and circadian rhythms. FR also increased glycogen deposition and altered JZ cellular composition. Despite reduced food intake in dams exposed to 10% MIH, differences in maternal physiology were observed between FR and MIH. FR dams had features of a fasting response, whereas this was not observed in hypoxic dams. Proteomic analysis identified altered abundance of proteins involved in lipid metabolism in MIH and iron handling in FR. Placentally secreted proteins were detected in maternal plasma, identifying potential biomarkers to inform on placental function, and hence fetal and maternal health. In summary, although both MIH and FR had the same overall outcome to limit fetal growth, environment-specific placental changes have been identified, underlining the integrative function of the placenta for maternal and fetal health. Future research based on this study will help to understand and characterise pathogenesis of pregnancy complications and how the placenta orchestrates both fetal and maternal health during pregnancy.
Open Access
Illuminating the Black Box: Defining and Modeling Peri-Implantation Human Embryogenesis
Weatherbee, Bailey; Weatherbee, Bailey [0000-0002-6825-6278]
Human pre-gastrulation development is a complex process where the single cell zygote transitions to the multilineage blastocyst, comprised of the epiblast (precursor of the fetus), hypoblast (precursor of the yolk sac) and trophoblast (precursor of the placenta). The blastocyst then implants into the maternal uterus and specifies body axes in preparation for gastrulation. It is thought that most pregnancies fail during the two-week period between fertilization and gastrulation. However, the inaccessibility of human embryos during implantation has limited our understanding of these stages. In this Thesis, I interrogated multiple aspects of human peri-implantation development. Utilizing human embryos cultured through implantation in vitro, I first generated a single cell transcriptomic dataset of post-implantation development. Through analysis of this dataset together with immunofluorescence and functional experiments, I characterized key events during peri-implantation development, including pluripotent state transitions within the epiblast, the role of the growth factor FGF to ensure proliferation of embryonic and extraembryonic tissues, and the emergence of a group of asymmetrically positioned hypoblast cells that express inhibitors of BMP, NODAL, and WNT signaling pathways and act as the anterior signaling center of the embryo. Next, I extended this work by generating an integrated single cell RNA sequencing atlas. Using this atlas as a guide, I characterized the role of several signaling pathways in the pre- to post-implantation transition. I find that both NODAL and BMP signaling are enriched in the hypoblast and essential to specify the anterior hypoblast. Further, NODAL and BMP are enriched in the human epiblast at pre-implantation compared to post-implantation stages, while NOTCH signaling is required for the epiblast only after implantation. These results demonstrate a crucial role for NODAL, BMP, and NOTCH in anterior hypoblast formation and imply an unanticipated switch in the roles of these pathways upon implantation in humans. Given the ethical and technical challenges of human embryo research, I have utilized stem cells as a complementary tool to study peri-implantation development. I established a human post-implantation embryo model comprised of embryonic and extraembryonic-like tissues. Combining the two types of extraembryonic-like cells generated by transcription factor overexpression with embryonic stem cells generates embryoids that contain an epiblast-like domain surrounded by extraembryonic-like tissues. These inducible human embryoids robustly generate amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP signaling. Using the modularity of this model, I identified an inhibitory role for SOX17 in anterior hypoblast specification. Modulation of the subpopulations of hypoblast-like cells impacted epiblast-like domain differentiation, highlighting functional tissue crosstalk. Overall, this Thesis begins to illuminate the ‘black box’ of human implantation.
Open Access
Spatial Transcriptome Profiling of Gastrulating Primate Embryos
Bergmann, Sophie; Bergmann, Sophie [0000-0001-6018-2844]
Implantation is the hallmark event during early pregnancy where a connection between the embryo and mother is formed. During the following phase of early postimplantation development, the pluripotent cells of the embryonic disc, the embryo proper, undergo gastrulation. This key process establishes the three germ layers, endoderm, mesoderm and ectoderm, which set the basis for all tissues emerging in the human embryo. Since postimplantation human embryos cannot be accessed routinely due to ethical and legal restrictions, early postimplantation development has been studied so far using stem cell-based in vitro models or embryo culture systems that provided valuable insights into early embryo development. However, the developmental relevance of these system could not be validated due to a lack of an early postimplantation, spatially defined in vivo reference. In my PhD project, I aimed to delineate the signalling environment of spatially defined tissues of early gastrulating embryos, using the marmoset, an established primate model, to study conserved human postimplantation embryo developmental processes. I first characterised marmoset embryos of three early postimplantation developmental stages on a morphological and molecular level, which allowed me to then perform spatially defined transcriptome profiling. Preserving the spatial coordinates of each sample processed for transcriptome analysis enabled me to integrate the transcriptome information of each sample individually into 3D embryo reconstructions. A collaborator applied gaussian process regression to establish 3D embryo transcriptomes models with continuous gene expression patterns. Using these 3D embryo transcriptomes, I identified the earliest timepoint of symmetry breaking and the onset of gastrulation, and analysed gene expression patterns associated with anterior-posterior axis formation. I defined genetic marker profiles for extraembryonic lineages such as the visceral endoderm, specifically defining markers for the anterior visceral endoderm (AVE), a signalling centre which drives symmetry breaking in the mouse. Furthermore, I defined marker profiles and analysed signalling pathways involved in the formation of the secondary yolk sac, and I explored the molecular framework and the potential origin of the extraembryonic mesoderm, which presumably forms the connecting stalk of the embryo with its mother. I further described the migration of primordial germ cells (PGCs), the precursor cells of germ cells, validating previous studies in primates, and I investigated involved signalling pathways and established novel marker profiles in primates. I analysed the formation of the primitive streak, a structure which defines the embryo midline in the posterior of the embryo proper and is established following the onset of gastrulation. I investigated signalling pathways which I extrapolated from mouse and primate literature i.e., NODAL, FGF, WNT, and BMP, and I analysed their expression patterns within the embryonic disc and its surrounding tissues. To test the signalling environment hypotheses derived from said analyses, I established in vitro marmoset embryonic stem cell-based models of the postimplantation embryonic disc and amnion, an extraembryonic membrane derived from the embryonic disc, and I tested the effects of NODAL, FGF, WNT and BMP on the in vitro models. Taken together, I found that FGF, together with NODAL, maintains pluripotency in the embryonic disc, similar to BMP and WNT inhibition, that I found in the anterior embryonic disc. WNT and BMP signalling drives mesoderm formation in the posterior, gastrulating embryonic disc. I was further able to delineate the role of NODAL, BMP and WNT in the formation of amnion. Given the versatility of the established in vitro systems, I tested the effects of these signalling pathways on a human stem cell-based system using a similar set-up, and characterised similarities and differences in amnion and mesendoderm formation. Lastly, I provided an outlook of potential future applications of my established 3D embryo transcriptomes, including as a validation tool for in vitro models, stem cell lines, or in vivo datasets.
Open Access
Traps, Tensions and Transmissions: The Unionisation of Resistance Movements against External Destabilising Forces; Design, Development and Deployment of Holographic System for Active Surveillance and Targeted Manipulation
Memon, Ahsan
Microscopic imaging of rapid biophysical processes often relies on high-contrast, high-resolution, and high-speed acquisition. However, confocal microscopes capable of such imaging lack the capacity to manipulate the sample or its surrounding environment in real-time. As a result, the possibility to non-invasively initiate, alter or halt a biochemical process during imaging is restricted. To overcome this limitation, we have established a sui generis versatile holographic system equipped with diverse photo-perturbation techniques including photo-manipulation, photo-activation, photo-ablation, optical-trapping and optogenetics, combined with the ability for active surveillance and monitoring of biological targets through confocal imaging and potential capacity for super-resolution imaging. This hybrid system is comprised of a spinning disk confocal unit, a spatial light modulator and a digital micromirror device, and is able to elucidate the dynamics of molecules, measure local forces, and re-localise or switch molecular behaviour. Here, we present the first application of this hybrid system to the study of cell shape regulation and the role of effective membrane tension in unionising actomyosin movement to resist external deformational and destabilising forces. We demonstrate that simultaneously trapping and unfolding the cell membrane, quantitatively imaging actin network dynamics and measuring cellular forces, allowing for a multi-level understanding of how the interplay of membrane tension and actin dynamics governs cell shape. Specifically, our results show a link between the movements of myosin that lead to a surge in actin concentration and cause an increase in the effective membrane tension. These results provide direct evidence for the role of physical interactions by plasma membrane in interpreting the environment that surrounds the cell to regulate and control cell dynamics and by extension cell behaviours. Furthermore, the results strongly reiterate the role of the actomyosin cortex in regulating cell shape during the transition phase and maintaining cell shape by resisting deformation during the stationary phase. More generally, our findings have the potential to expand our understanding of how mechanical properties of the cell surface are locally and globally responsible for driving cell shape changes in physiological and disease conditions. Additionally, we demonstrate the possibility of simultaneously holographic-trapping and dynamically exciting multiple independent cellular targets each located in specific areas and having different excitation and emission wavelengths, by custom developed and optimised method which utilises a single modulator to generate multi-chromatic holograms. Our programming code uses an iterative algorithm with only ten iterations to achieve a PSNR of above 35 dB, an efficiency of 96\% and a crosstalk of less than 1\% in the results. The method retains high adaptability and customisability to prioritise the quality of the reconstructed image, or the speed of the hologram generation, or improve both quality and speed at the cost of other variables based on the application specificity.
Embargo
Systemic and local signalling regulating neural stem cell proliferation
Arman, Diana
Neural stem cells (NSCs) in the brain can be reactivated to exit quiescence and generate new cells in response to injury or disease. Determining the specific signals involved in NSC reactivation is essential to understanding neural regeneration. Investigating NSCs in *Drosophila melanogaster* provides a powerful model due to ease of identification of quiescent NSCs and a plethora of genetic and molecular tools available to study the nervous system *in vivo*. During development, *Drosophila* NSCs reactivate in response to dietary amino acids sensed by the fat body, a sensor organ analogous to liver and adipose tissue in mammals. Previously unknown signals from the fat body are transmitted to the bloodbrain barrier (BBB) glia on the surface of the brain. The glia secrete insulin-like peptides, which are received by the insulin receptor on NSCs, inducing their reactivation. To identify the fat body-derived signals sent to the brain, I used Targeted DamID to generate transcriptional profiles of the fat body under fed and starved conditions. I discovered upregulation of *dpp (decapentaplegic)*, a transforming growth factor (TGFβ) secreted morphogen, after feeding. Knockdown of Dpp in the fat body severely impaired NSC reactivation. Similarly, knocking down a key receptor for Dpp, Tkv (Thickveins), in the BBB glia led to severely impaired NSC reactivation. To compare gene expression in glial subtypes under fed and starved conditions, I generated single-cell RNA sequencing datasets. I found Dpp to be upregulated in BBB glia in response to feeding and functional experiments showed that Dpp signalling in the glial niche is required for NSC reactivation. The presence of Medea transcription factor binding sites at the dIlp6 locus and impaired NSC reactivation when Medea is knocked down in the BBB glia suggest that Dpp signalling may regulate expression of dIlp6. Knockdown of Tkv in the NSCs themselves did not affect reactivation, suggesting autocrine Dpp signalling in the BBB glia in response to interorgan Dpp signalling from the fat body to the brain. The findings may help understand how to induce neural stem cell proliferation after brain damage or neurodegeneration.
Embargo
Impact of mechanical signals on human foetal lung cell differentiation
Sokleva, Vanesa
A major function of the lung is as a mechanical pump to bring oxygen-rich air into the body, allow gas exchange to occur, and remove carbon dioxide-rich air. It is increasingly recognized that physical forces, such as tension, compression, or shear, as well as passive mechanical material properties such as substrate stiffness affect lung development and play a role in many lung diseases. If the foetal breathing movements leading to amniotic fluid inhalation, and therefore the exertion of physical forces on the developing lung, are perturbed this leads to pulmonary hypoplasia and can result in neonatal morbidity and mortality. Part of the mechanism for these effects is that the sheer stress exerted by the amniotic fluid plays a role in alveolar differentiation as the pressure allows alveolar type I cells to flatten. In humans, mechanical forces and material properties are clinically important both in development, evident in prematurely delivered infants as well as in diseases such as pulmonary fibrosis, where increase in tissue stiffness leads to lung dysfunction. However, not all mechanical cues affecting lung development and disease are known, nor their mechanism of action. There are different biological materials deposited along the proximal-distal axis in the lung, such as cartilage in the trachea and smooth muscle in the bronchi, versus elastic fibres in the alveoli. Considering this, as well as the clinical relevance of stiffness in lung diseases, I hypothesised that tissue stiffness can play a role in defining cellular fate. I measured the stiffness of human embryonic lung slices over developmental time using Atomic Force microscopy-based indentation measurements and found both temporal and spatial stiffness gradients. The spatial gradient revealed that the epithelial tips of the growing airway tubes are softer than the main body of the airway tubes. This contrasted with the closely aligned blood vessels, which did not show significant changes spatially in stiffness in the timeframe of the experiment. To test the hypothesis that stiffness affects lung cell fate decisions, I grew human embryonic lung organoids in 3D hydrogels of tuneable stiffness using stiffness values derived from the in vivo measurements. Organoids grown in soft (100 Pa) gels were spherical and hollow in morphology, similar to the Matrigel-grown controls. By contrast, organoids grown in stiff gels (4 kPa) were highly folded. Moreover, organoids grown in stiff gels lost nuclear localisation of their SOX9 protein and failed to undergo alveolar differentiation, suggesting a loss of progenitor cell identity. Inhibition of Rho-kinase (ROCK) reverted both the SOX9 and alveolar differentiation phenotypes in stiff gels, showing that contractility is important for the cells to sense and respond to the surrounding stiffness. Moreover, I observed that growth of organoids at high stiffness can lead to spatial constraint. Growing organoids in soft gels with either high or low density showed that high density mimics the effects of high stiffness gels, suggesting that spatial constraint downstream of stiffness is what causes the observed phenotypes. Taken together, these results show that there are mechanically distinctive features in the embryonic human lung and that the epithelial tip cells are capable of sensing and responding to them by changing their cell fate accordingly. These findings not only contribute to a better understanding of lung development but can also have potential clinical relevance in treating diseases such as pulmonary fibrosis.
Open Access
Super-resolution Imaging of Chromatin and Functional Nuclear Architecture
Ball, Madeleine
The regulation of transcription is well understood to be linked to the three-dimensional organisation of the genome within the nucleus, however, the mechanisms through which the different levels of organisation regulate genes are poorly understood. Newly developing super-resolution imaging techniques offer an important new way to investigate structural features of the genome, however, imaging chromatin within the nucleus is traditionally very difficult due to the dense packaging with a small nuclear volume. The nuclei of Drosophila melanogaster primary spermatocytes have unique properties that can circumvent these limitations, with comparatively large nuclei with well-separated chromosome masses, and transcriptionally active Y loops that expand into the nuclear interior as clearly observable individual fibres. Therefore, during this thesis I took advantage of this model system to image both the chromatin of the Y loops and chromosome masses. A single molecule localisation microscopy technique was optimised for imaging within the nucleus, with both 3D and dual-colour capability. Then, the Y loops were imaged at super-resolution, and a specialised clustering protocol was developed for quantification. This showed that the Y loops are organised as a chain of clusters, with an average width of approximately 50 nm, and an average distance apart from each other of roughly 100 nm. The relationship between actively elongating transcription, as well as different phosphorylation states of RNA polymerase II, and the Y loop chromatin was investigated and quantified, revealing that polymerase appears adjacent to the Y loop fibres, attached via a smaller chromatin loop emanating from the clusters. The role of transcription in chromatin organisation was assessed on the Y loops through the use of transcription inhibition, and on the autosomal chromosomes through mutant fly lines, using empty-space statistics to quantify their organisation. This indicated that the Y loop chains of clusters structure was unlikely to be determined by transcription. Active and inactive histone modifications were labelled along the Y loops, which were quantified for comparative analysis, with implications for the link between chromatin state and function. The work conducted during this thesis identified novel architectures of transcriptionallyactive chromatin and so provides a foundation for understanding chromatin organisation within the nucleus, and the relationship between transcription and chromatin state.