Theses - Genetics

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  • ItemEmbargo
    A genetic interaction network of constitutive heterochromatin in Caenorhabditis elegans
    Townley, Anna
    Constitutive heterochromatin is an epigenetic compartment of animal genomes that is important for gene regulation, chromosome architecture and the maintenance of genome integrity. It is associated with the silencing of gene expression and the repression of repetitive element activity. Constitutive heterochromatin is also an important modulator of development and becomes dysregulated in human cancers. However, the mechanisms by which it is formed and functions in animals, and how it interacts with other processes in the nucleus, are not well understood. In this work, I defined a network of functional components of constitutive heterochromatin in *Caenorhabditis elegans* through conducting a set of genetic interaction screens. I began by designing a high throughput screening approach using RNAi to find genetic enhancers and suppressors of the growth and fertility defects of heterochromatin-defective mutants. Using an RNAi sub-library targeting 2309 genes encoding nuclear proteins, I then screened seven heterochromatin mutants for genetic interactors. I identified 289 enhancers and 89 suppressors, which form a highly interconnected network, with 75% interacting with more than one heterochromatin mutant. Genetic enhancers included components of chromatin modifying complexes, ubiquitination and sumoylation pathways, and transcription and RNA processing factors. Some of these factors were required for silencing of a heterochromatic reporter, suggesting roles in gene silencing. Genetic suppressors were enriched for a broad range of chromatin modifying factors, many of which are associated with active transcription. I next characterised the changes in gene and repetitive element expression shown by eight different heterochromatin mutants. As for genetic interactions, I found that shared alterations were common, and I identified candidates that may be responsible for the slow growth of some heterochromatin mutant strains. Alongside the upregulation of repetitive elements, I observed three major gene expression changes: the ectopic expression of germline genes in somatic tissues, the downregulation of metabolic pathways and the induction of diverse stress- response pathways. The promoters of some upregulated germline genes are marked by histone H3 lysine 9 dimethylation (H3K9me2), consistent with direct repression, whereas deregulation of the metabolic and stress response pathway genes appears indirect as their promoters generally lack H3K9me2. I investigated the mechanisms by which genetic suppressors rescue the growth defect of heterochromatin mutants through assessing the effect of their knockdown on gene and repetitive element expression in the *hpl-1;hpl-2* double mutant, which lacks both HP1 orthologues. The loss of diverse genetic suppressors corrected shared groups of gene and repetitive element expression alterations, including the upregulation of stress response pathways. This suggests that suppression is through the restoration of normal gene expression programmes rather than through induction of additional changes that counteract those of heterochromatin mutants. Overall, my work expands the known network of functional components of constitutive heterochromatin in the context of a developing animal. I reveal how activating and silencing chromatin modifying pathways are balanced to prevent genome deregulation. Finally, I implicate the activation of stress response pathways in the physiological defects of heterochromatin-defective animals. These results hold relevance for the understanding and potential treatment of diseases such as cancer which show defective heterochromatin function.
  • ItemEmbargo
    Investigating mitochondrial dynamics in Drosophila spermatogenesis: a focused study on the mitoball
    Rathore, Sumaera
    Mitochondria are highly dynamic organelles capable of significant reorganisation to meet cellular demands. Spermatogenesis – a complex developmental process that generates male gametes – is accompanied by substantial alterations in mitochondrial shape, quantity, and distribution. Previous studies have mainly focused on mitochondrial dynamics in post-meiotic spermatids, consequently little is known about mitochondrial dynamics during early spermatogenesis and its impact on sperm development. A potent example of this is the striking reorganization of the mitochondrial network into a large ball-like structure adjacent to the nucleus during the pre-meiotic spermatocyte stage in *Drosophila melanogaster*. This distinct cluster of mitochondria was initially observed in an electron microscopy study by Tates et al. in 1971 but has remained uncharacterized since then. Our lab termed this cluster of mitochondria the "mitoball”. The main objective of this thesis is to investigate the genetic basis of mitochondrial dynamics within the novel *in vivo* environment of the mitoball and explore its impact on sperm cell development and male fertility. Our lab showed mitoballs are conserved among many insect species and are densely packed with other organelles. To explore the function of mitoball, I investigated the role of Milton, an adaptor protein involved in the microtubule-based transport of mitochondria. Through my research, I discovered that Milton collaborates with Mitochondrial Rho (Miro), a protein located on the mitochondrial outer membrane, and Khc, a microtubule motor, to facilitate the transportation of mitochondria along microtubules and form the mitoball. By generating homozygous viable *milton* mutants, I demonstrated that a 54-amino acid region in the C-terminus of Milton is essential for mitoball formation. I further observed that flies lacking mitoballs had swollen mitochondria in their spermatocytes, reduced ATP production, altered transcriptome, and compromised male fertility. These findings indicate that the subcellular distribution of mitochondria can modulate mitochondrial morphology and function during early spermatogenesis to impact male fertility. To reveal other players regulating mitoball formation, I performed a forward genetic screen by feeding *D. melanogaster* with EMS, a mutagen that induces random mutations in the nuclear genome, and visualising mitoballs by confocal imaging. This screen identified 121 lines with abnormal mitochondrial and testis morphology. Focusing on nine EMS lines with mitoball defects, I performed whole genome sequencing and deletion mapping to identify the responsible genes that modulate mitochondrial dynamics in early spermatogenesis. I further validated the role of eight genes using RNAi knockdown and knockout mutants. This part of my research uncovered both previously known and novel regulators of mitochondrial dynamics. Overall, this thesis investigates mitochondrial dynamics in *Drosophila* spermatogenesis within the context of the mitoball and establishes the essential role of Milton for mitoball formation. Since premeiotic clustering of mitochondria is observed in various insect species, regulators of mitoball formation could be utilised as targets for inducing male sterility in genetically modified insects, offering an environmentally friendly approach to pest control. Moreover, uncovering conserved genetic factors that influence mitochondrial dynamics during spermatogenesis holds the potential to address male fertility disorders in humans, as well as neurodegenerative and metabolic diseases associated with mitochondrial abnormalities.
  • ItemOpen Access
    The regulation of tissue proportions: analysis of progenitor behavioural dynamics in response to cell ablation in the zebrafish tailbud.
    Saunders, Dillan
    In order to form a correctly patterned embryo the behaviour of cell populations and tissues must be coordinated so that different structures form with the correct relative proportions. The fundamental body plan of the vertebrate embryo is laid down in a head to tail progression known as primary body axis elongation. This begins with the differentiation and morphogenetic events of gastrulation which initially give rise to the head and anterior trunk. During this phase of development, it has been shown that the proportions of germ-layer specification scale with the number of cells in the embryo. Cells that have not formed part of the main axis at this time remain in a structure known as the tailbud. During this later phase, the tailbud progenitors undergo morphogenesis and differentiation to form the axial tissues of the posterior trunk and tail such as neural tube and somitic mesoderm. Importantly, within the tailbud certain cells, known as neuro-mesodermal competent progenitors (NMC cells/NMPs) can differentiate into either neural or mesodermal tissue. I hypothesise that the NMC progenitor population could be maintained in the tailbud during posterior body elongation in order to facilitate the correct proportioning of neural and mesodermal tissue in response to any fluctuations in more differentiated progenitor numbers. In this thesis, I utilise two-photon laser ablation to alter the functional number of progenitor cells in the neural fated region of the zebrafish tailbud and quantify the effect this has on the behaviour of the remaining progenitors. In order to investigate this in detail I developed an open source image processing pipeline for three-dimensional nuclear segmentation, point cloud registration, and the analysis of gene expression intensities and cell tracking data. I first demonstrate that the neural tube and somitic mesoderm are proportionally reduced in elongation following ablation. Then, by combining fluorescent in situ hybridisation with live imaging I show that the progress of NMC cell differentiation is robust to ablation. However, I observe that healing of the ablation causes increased convergence of neural-fated progenitors without contribution from mesodermal-fated NMC cells. This suggests, that in this case, NMC cells are unable to regulate the proportion of neural versus mesodermal differentiation. Finally, I find that there is considerable robustness in elongation following ablation of mesodermal fated progenitors. Taken together, my analyses indicate that progenitors are not the main driver of the proportionality of tissue elongation in the tail. These results are in line with growing bodies of evidence that demonstrate that tailbud progenitors are a highly dynamic group of cells, but that zebrafish body elongation is driven primarily by more anterior multi-tissue interactions.
  • ItemEmbargo
    Intergenerational and Transgenerational Effects of Endocrine Insults
    Fisher, Benjamin; Fisher, Benjamin [0000-0001-5497-9689]
    Environmental perturbation can lead to non-genetic, non-Mendelian transmission of acquired traits to descendants. Such inheritance may be intergenerational or transgenerational, the latter describing effects that persist in the absence of any exposure (either directly or of parental germ cells) to the triggering environment. Numerous reports of non-genetic inheritance ascribe it to epigenetic mechanisms, but behavioural, microbiotic, cultural, and/or ecological factors more likely play a part in many instances. During my doctoral studies, I explored inter- and transgenerational effects of developmental exposure to three groups of endocrine insults – endocrine disrupting chemicals (EDCs), synthetic glucocorticoids (sGCs), and hyperthyroidism – in two different species, humans and mice. EDCs are exogenous substances that interfere with endocrine axes to cause adverse health effects. Humans are ubiquitously exposed to EDCs and recent animal studies have suggested a role for them in non-genetic inheritance. For the first part of my thesis, I investigated prospective relationships between maternal serum concentrations of three groups of EDCs (phthalate metabolites, phenols, and parabens) in early pregnancy and male infant genital development in a human cohort study. I found associations between bisphenol A (BPA) level and offspring cryptorchidism, and between detection of n-propyl paraben and shorter anogenital distance (a marker of intrauterine androgen activity). Next, I investigated inter- and transgenerational effects of EDCs in mice by exposing pregnant/lactating females to BPA and phenotyping their patrilineal F1-F3 offspring. I observed metabolic changes in F1 and F2, but not F3, males, which instead had increased body weight; conclusions about mechanism could not be drawn. sGCs are a group of drugs that bind with high potency to glucocorticoid receptors (GRs), with pleiotropic effects. Therapeutic indications include inflammatory and autoimmune diseases, and threatened preterm birth. The latter usage results in significant fetal exposure to sGCs, which may affect the germline and impact on future generations. I therefore used the sGC dexamethasone and two novel genetic models to investigate inter- and transgenerational effects of GR activation in mice. In a patrilineal experiment, antenatal dexamethasone treatment caused impaired glucose tolerance in F1 and F2 males, and heavier internal genitalia in F2 females, but these findings were not replicated in F2 mice generated by in vitro fertilisation (IVF), suggesting a non-germline effect. The F3 phenotype was of increased body weight (males) and altered thyroid hormone levels and body composition (females). Dexamethasone treatment of oocyte donors prior to IVF led to altered body composition and heavier testes in male offspring, consistent with epigenetic inheritance. Mice expressing a constitutively active GR (ΔGR) under a germ cell- or epididymis-specific promoter had no robust phenotype, but their wild-type male offspring had improved glycaemia, with sexually dimorphic effects on body composition also observed. Intrauterine exposure to hyperthyroidism due to a maternal thyroid hormone receptor mutation has recently been associated with non-genetic patrilineal transgenerational transmission of a thyroid hormone resistance phenotype in humans. In the final part of my thesis, I used the sperm of four F2-generation men from this pedigree to determine the feasibility of assaying various epigenetic marks in cryopreserved human samples, with success for long RNAs, small RNAs, and DNA methylation; and to test the hypothesis that non-genetic transmission is associated with hypermethylation at a specific locus, for which I found no evidence. My results confirm and extend previous reports of inter- and transgenerational effects of endocrine insults, including novel findings in both humans and mice. Further studies are required to delineate underlying mechanisms and develop protective strategies.
  • ItemEmbargo
    A role for environmental epigenetics in the rapid diversification of East African cichlid fishes
    Putman, Audrey
    Phenotypic plasticity via gene-environment interaction is a central feature of regulatory biology. By definition, phenotypic plasticity involves a change in gene regulation. These gene regulatory transitions are commonly mediated by an array of epigenetic mechanisms, which exert dynamic, environmentally responsive control over the genome. In vertebrates, these mechanisms include regulation via histone posttranslational modifications, DNA methylation, and non-coding RNAs. By regulating genetic processes in response to external signals, epigenetic mechanisms have an evident role in phenotypic adaptation. Recent explorations have revealed that the eukaryotic epigenome detects and responds to a range of environmental inputs, including nutrition, temperature, and toxicant exposure. However, studies in environmental epigenetics have focused primarily on DNA methylation, leaving the role of other epigenetic mechanisms largely unexplored. This is particularly true regarding vertebrate models. Moreover, very few studies in vertebrates show robust support for the link between epigenetics and adaptive plasticity. The relevance of other epigenetic mechanisms in vertebrate gene-environment interactions remains unclear. Here, I use East African cichlid fishes—a model system for phenotypic plasticity and rapid evolution—to decipher the role of chromatin regulation in the establishment of phenotypic diversity in novel environments. To illustrate chromatin state dynamics genome-wide, I focus on four major histone modifications associated with enhancer/promoter activity and gene silencing: H3K4me3, H3K27ac, H3K4me1, and H3K27me3. I began by establishing natural epigenetic variability in a lake environment. On the basis of ecomorphological diversity and genetic similarity, four species were selected from Lake Malawi, an ecosystem containing the most extensive cichlid adaptive radiation, which houses more fish species than any other lake in the world. Using Cut&Run, I profiled genome-wide enrichment of the chosen histone modifications in adult males from each species. Incorporating a supervised model to integrate mark enrichment with gene expression, I defined a set of functional chromatin states in Malawi cichlids. Results from differential binding analysis reveal ecotype-specific regulatory variation in key metabolic and developmental pathways, reflecting trophic adaptation and regulation of alternate developmental trajectories. To date, these results present the most comprehensive characterization of chromatin features in cichlid fishes. To further dissect the influence of environment on the epigenome, I present an experiment simulating short-term nutritional adaptation in a controlled setting. Here, I supply generalist cichlid Astatotilapia calliptera with two alternate diets, formulated to mimic piscivorous and algivorous trophic regimes. Within one generation of this experiment, fish of each diet treatment developed novel craniofacial and body shape morphologies, accompanied by changes in relative eye size, brain size, pigmentation, and growth patterns. To study the epigenetic basis of these phenotypic changes, I measured chromatin landscape variation in liver from each treatment. Interestingly, the regulatory differences between treatments occur in the same pathways identified as most variable between the Malawi ecotypes, in patterns that emulate the standing epigenetic variation in the lake. In these investigations, I detect consistent patterns of epigenetic variation in conspecific treatment groups under environmental stress and between species phylogenetically separated by thousands of years. In the context of cichlid trophic expansion, I conclude that chromatin regulatory mechanisms might mediate dietary adaptation through modulation of metabolic pathways in a conserved crosstalk between genes and environment.
  • ItemEmbargo
    A neuron within a neuron: the effect of narrow axonal ER tubules on molecular movement
    Chahwala, Kishen Suresh
    Hereditary spastic paraplegias (HSP) are a group of diseases showing degeneration of lower motor axons. A common cause of HSP is the mutation of proteins that shape the tubular endoplasmic reticulum (ER). The architecture of the ER tubular network that extends through axons is therefore likely to be integral to the maintenance and survival of axons. The continuity and ubiquity of the ER network throughout neurons has earned it the term ‘a neuron within a neuron’. The reasons for its striking continuity are not fully understood, but it could potentially make the ER a channel for long-distance signalling, independent of action potentials at the plasma membrane, or motor-based transport. ER tubules are also much narrower in axons than in most other locations. This presents a potential paradox, of why a cellular structure that is physically continuous over long distances shows constrained continuity in its lumen. Narrow ER nanotubules could limit axial diffusion along the ER tubule lumen. I aimed to explore this model. I used *Drosophila* larval motor axons, in which I could use mutations in ER-shaping proteins to manipulate tubule properties and genetic tools to label and express sensors in individual motor axons in vivo. In FRAP (fluorescence recovery after photobleaching) experiments I found that wildtype ER tubules were permissive for recovery of a GFP-tagged luminal protein, despite the difficulty of visualizing the lumen by scanning EM (electron microscopy), implying that proteins can diffuse along this lumen. To test whether this lumen diameter is limiting for diffusion, I compared recovery in wildtype axonal ER tubules with that in tubules of a larger diameter, in a triple mutant lacking the ER-shaping proteins, Rtnl1, ReepA and ReepB. Luminal diffusion of GFP was significantly faster in the larger mutant ER tubules, with the time to half recovery in wider mutant axonal ER tubules approximately twice as fast as in wildtype. Therefore, the narrow ER nanotubule diameter in wildtype axons limits luminal protein diffusion. To begin to dissect the roles of the different classes of ER-shaping proteins in determining tubule diameter, I also compared the FRAP recovery rates of a single Rtnl1 mutant with wildtype, and found no difference, implying that loss of Rtnl1 alone did not increase the ability of axon ER tubules to support the rate of axial diffusion. To better understand the parameters of molecular movement along the ER nanotubule lumen, I generated larvae that expressed ER lumen markers suitable for single-molecule imaging. On sparse chemical labelling of a luminal HaloTag protein, I found two classes of single-particle movement: slower and relatively bright particles that showed largely unidirectional movement, and faster less brightly labelled particles. The slower class showed no bias in the direction of luminal movement and also appeared to move faster than microtubule-based motors. The faster class are similar to the single-molecule movements previously described in non-neuronal ER tubules. One experimental constraint on this approach was an unfavourable ratio of signal to background labelling. Therefore, I initiated a neuronal cell culture model for single-particle imaging to mitigate the challenges of larval nerve preparation. One physiologically important molecule that diffuses through the ER lumen is Ca2+, and constraints on this diffusion could have consequences for intracellular Ca2+ signalling. Ca2+ is much smaller than GFP or a HaloTag, and therefore its movement might be less constrained by small tubule diameter. I aimed to develop a strategy to test the effect of axonal ER tubule diameter on intraluminal Ca2+ diffusion. This involved making and testing two components: *Drosophila* stocks containing an ER-localised channelrhodopsin and stocks containing an axonal ER-localised Ca2+ sensor (HaloCaMP). I report on the development of these stocks. In summary, I have shown that luminal protein diffusion is constrained in axon ER nanotubules. I have developed tools that can be useful for further understanding the nature of these constraints and their physiological consequences.
  • ItemOpen Access
    On Different Uses of Abstraction in Models of Developmental Systems
    Harper-Donnelly, Giles
    Abstraction is centrally important to the success of models, mathematical and otherwise, in biology. The move beyond traditional reductionist approaches towards a more holistic, system level, understanding of development, has produced a concurrent growth in the types of abstractions used in quantitative modelling. This is accompanied with new challenges and opportunities in evaluating and integrating the evidence provided by different modelling approaches and in constructing coherent and general models of complex biological processes. In this thesis, I address some of these challenges by critically assessing current models and proposing a new conceptual framework to address system level understanding of developmental systems. The integration of results from different modelling approaches is demonstrated with a novel approach to parameter inference for differential equation modelling of gene regulatory networks in a developmental patterning system. After a brief introduction motivating the work and highlighting some of the central themes. This is followed by the five principal results chapters which are each summarized below. Chapter 1 presents a critique of some recent work modelling the dynamics of stem cell differentiation. Some problems with the parameterization of one of the models are demonstrated and provide context for a more general discussion of the challenges in accounting for model complexity during model selection. The chapter concludes with a more general discussion of model selection and the appropriate integration of the resulting statistical evidence into a broader corpus of knowledge about the system. Chapter 2 considers the use of hierarchical systems of levels of abstraction in the analysis of complex developmental systems, with a particular focus on the tripartite system of levels proposed by the neuroscientist David Marr. Marr’s levels have been highly influential in computational neuroscience since their inception in the 1970’s and this chapter argues that such a framework has the potential to play an instrumental role in our attempts to understand complex developmental systems. To this end, a novel analysis of Marr’s levels is presented laying the groundwork for their application in the following chapter. Chapter 3 presents two detailed case studies in order to demonstrate how such a conceptual framework might be applied to different types of developmental systems. The first of these case studies analyses an information-based approach to studying gap gene patterning in the Drosophila embryo. This work highlights that some of the core ideas in Marr’s approach are, often implicitly, present in the literature regarding patterning systems however without the appropriate intellectual infrastructure to support them they have either been forgotten or not used to their full potential. In order to demonstrate how the type of functional analysis presented in the previous chapter might be extended beyond information processing systems, the second case study considers work modelling branching morphogenesis in the developing mouse mammary gland. A brief reprieve from the theoretical concerns of the preceding chapters comes with Chapter 4. A stand-alone analysis of gene expression data from the Drosophila embryo, this chapter establishes the presence of artefacts which can arise in such datasets during data acquisition. The final chapter (Chapter 5) builds on some of the insights in Chapter 3 to propose a specific method for combining top-down constraints on a lower-level model a gene regulatory network controlling cell-cell communication in a two cell system. This method presents a novel approach to parameter inference for differential equation models of the gene regulatory networks involved in developmental patterning systems.
  • ItemEmbargo
    Characterisation of Mitochondrial Dynamics and Mitochondrial DNA Inheritance in Drosophila melanogaster
    Li, Andy Yu Zhi
    Mitochondria are dynamic organelles and there is mounting evidence that mitochondrial dysfunction is linked to a variety of diseases. During spermatogenesis in Drosophila melanogaster, the organisation of mitochondrial networks is altered during each stage of development. This thesis is focused on a novel mitochondrial structure, termed the mitoball, which occurs in the premeiotic spermatocytes in the 16 cell stage. The mitoball is a transient aggregation of mitochondria on one side of the cytoplasm, and little is known about the functionality and dynamics of this structure. First, I found that the mitoball is conserved in multiple insect species. Second, live imaging of the mitoball revealed that the mitoball consists of individual mitochondria which move around in the region of the mitoball. Third, mitochondrial DNA (mtDNA) replication was found to occur in the mitoball stage. Fourth, mitoballs were associated with other cellular structures like the endoplasmic reticulum network, Golgi bodies, and the fusome. Next, I used a reverse genetic screen to identify genes linked to mitochondrial trafficking along microtubules that were required for mitoball assembly: milton, miro, and khc. I also generated an X0 male and found that the loss of the Y chromosome did not affect mitoball formation. To discover new genes linked to the mitoball, I performed a whole genome forward genetic screen using EMS on the second and third chromosome. In total, 8027 EMS mutant lines were screened. Several lines with defective mitochondrial morphology were recovered and sequenced. One EMS line had no mitoballs and had an early stop codon in milton and ssp3. Further mapping of this line revealed that the early stop codon in milton was linked to the absence of mitoballs phenotype and the early stop codon in ssp3 was linked to defects in later stages. Altogether, the mitoball structure has promising characteristics as a system to study mitochondrial dynamics. Furthermore, premeiotic clustering of mitochondria may also be a hallmark of insect spermatogenesis. As a multi-copy genome, mutant mtDNA molecules often co-exist with wild-type counterparts, and the competition in transmission among these genomes is central to the inheritance and progression of mitochondrial disease. Currently, it is not possible to easily manipulate animal mtDNA for genetic studies. It is thus difficult to map sequences in mtDNA that can confer a transmission advantage to certain mitochondrial genomes. One of my projects is designed to employ irradiation and chemical based methods to generate mutant and recombinant mtDNA in order to identify and map mtDNA sequences linked to a transmission advantage. Through this project, several recombinant mitochondrial genomes were isolated and this system allows for the mapping of the mitochondrial genome through the generation of recombinant genomes.
  • ItemOpen Access
    Multi-scalar mechanisms timing axial progenitor contribution to the vertebrate body axis
    Busby, Lara
    Timing is a fundamental feature of embryogenesis: the embryos of a given species develop with reproducible timing and a precise ordering of events. The underlying mechanisms that control the absolute timing of cell state changes and morphogenetic events in development are largely not understood, and crucially can be considered at a wide range of length scales, from the integration of positional cues over time in a single cell to whole-organism events such as hormone release. A key context in which cell behaviour must be coordinated with time occurs in the development of the vertebrate posterior body, which is elaborated in a strict anterior to posterior sequence. This process occurs over a period of hours to days depending upon the species, necessitating mechanisms to ensure that cells are allocated to the body axis in a controlled manner. The prevailing model for regulation of the timing of progenitor contribution to the body axis centres around a changing Hox gene expression complement in the population (‘The Hox Clock’) that can influence the timing with which cells enter the body axis (Deschamps and Duboule, 2017; Iimura and Pourquie, 2006). In this thesis, I utilise classical experimental embryology approaches including grafting and explant culture in combination with modern techniques such as single cell transcriptomics and multiplexed in situ hybridisation to ask about the contribution of intrinsic and extrinsic influences to cell decisions during gastrulation and axis elongation. My primary experimental assay involves the heterochronic grafting of HH8 somite progenitors to a HH4 embryo. I show that HH8 somite progenitors undergo ingression through the primitive streak but are delayed relative to HH4 progenitors at the transition from ingressed to migratory mesenchyme, ultimately resulting in a more posterior axis contribution. To assess the role of Hox gene expression, I identified a set of genes with differential expression between HH4 and HH8 progenitor regions, then assayed expression of these genes in heterochronic (HH8-HH4) grafts. I found consistent support for the maintenance of the HH8 Hox profile upon grafting to the HH4 embryonic environment, suggesting that Hox regulation is population-intrinsic in this cell population. By performing grafts with variable sized pieces of donor tissue, I show that Hox profile can be uncoupled from cell behaviour in small grafts, suggesting that in addition to Hox expression, there are population size-related inputs on cell behaviour. By culturing explants on fibronectin, I show that both HH4 and HH8 MSP populations have a propensity to migrate, but HH8 populations do so with a delay and much lower tissue velocity. Finally, I present work from an additional project in which I ask about the regulation of timing upon progenitor population ablation – the surgical removal of Hensen’s node (the avian Organizer) results in the production of a morphologically normal embryo. Alexandra Neaverson and I designed and performed a single cell RNA-sequencing experiment to ask about global transcriptomic changes resulting from node ablation. We find that there are essentially no global transcriptomic changes in ablated embryos relative to control ones, and that expression of prospective neural plate marker genes is unchanged after removal of the morphological node. A possible reason for this, despite the hypothesised role for Hensen’s node in neural induction and maintenance, is that expression of many ‘node’ marker genes extends into the anterior streak, outside of the morphological node. Together, the work described here has led to a working model for timing axial progenitor contribution to the axis where population and cell intrinsic Hox gene expression (the Hox Clock) is integrated with population size related inputs. Crucially, such a model can account for the ‘streaming’ of cells from the progenitor region (asynchronous allocation of cells to the axis) in a way that previous models could not. This is multi-scalar regulation; together with the literature review presented in Chapter 1, my work leads me to an understanding of developmental timing as a highly distributed phenomenon with aspects acting at different length scales.
  • ItemEmbargo
    DNA methylation: the "stable" epigenetic mark
    Hay, Amir; Hay, Amir [0000-0001-9435-2848]
    DNA methylation is regarded as a stable epigenetic mark given its faithful maintenance across successive cell divisions. Methylation occurs at most CpG sites in mammalian genomes and is generally associated with transcriptional repression. An accepted evolutionary role for DNA methylation is to prevent the mobility of transposable elements (TEs). This thesis investigates the stability of DNA methylation in two separate contexts, particularly relating to intermediate levels of methylation. First, I characterise the properties of variably methylated TEs (VM-TEs) between individual mice. Second, I assess the fidelity of DNA methylation inheritance across cellular generations at VM-TEs, and more widely at the genome-scale, to ascertain the heritability, mechanism, and function of intermediate methylation states. My findings show that variable methylation extends beyond the boundaries of the TEs, and that all VM-TEs are enriched for binding of the transcription factor CTCF, which is inversely correlated with DNA methylation. I propose that molecular antagonism between CTCF and DNA methylation machinery influences the formation of variably methylated states in the early embryo. Within an individual mouse, VM-TEs are intermediately methylated between 10% and 90%, representing the cell population average of methylation states. The prevailing hypothesis supports the notion that methylation is established de novo by DNA methyltransferases DNMT3A/3B and then faithfully maintained by DNMT1. Hence, intermediate methylation levels likely represent stochastic de novo establishment (DNMT3A/3B) and clonal maintenance (DNMT1) within the cell population. To test this, I subcloned single cells from both mouse embryonic fibroblasts (MEFs) and embryonic stem cells (mESCs), growing them into multiple subclonal populations to assess methylation fidelity through cell divisions. This allowed me to address the degree to which a particular locus acquires intermediate methylation in the clonal population, as well as the properties and mechanism of that state. If methylation is indeed propagated faithfully, one would expect that the single-cell derived populations always exhibit one of the three symmetric methylation states: 0%, 50% or 100%. At VM-TEs, we find that the subcloned cell lines attain intermediate methylation levels that reflect the level of the parent population, which implies that the original single-cell methylation state is not faithfully maintained at these loci. Expanding the analysis genome-wide, I use a target capture bisulphite sequencing method to evaluate methylation fidelity in the subclonal cell lines more globally. I find that CpGs exhibiting intermediate methylation at the cell population level, are generally unfaithfully inherited between cell divisions and attain methylation independently of neighbouring CpGs. While faithful hypo- and hypermethylation associate with transcriptional activity, unfaithful intermediate methylation associates with transcriptionally inactive genes or intergenic regions of the genome. Finally, in DNMT3A/3B mutants, methylation is not depleted consistently at any CpG, regardless of its methylation state in the control. Therefore, DNMT1 has two functions: 1) canonical maintenance of faithful methylation and 2) as shown here, it is responsible for the acquisition of intermediate methylation states that are unfaithfully inherited between cell divisions.
  • ItemOpen Access
    Genetic diversity of Anopheles mosquitoes
    Bodde, Marilou
    This thesis presents two projects related to the genetic diversity of Anopheles mosquitoes. The first project concerns the development and application of sequence based species assignment methods for any species in the genus. The second project discusses the population structure of the major malaria vector Anopheles funestus, with a specific focus on inversions. The ANOSPP amplicon panel is a genus-wide targeted sequencing panel to facilitate large-scale monitoring of Anopheles species diversity. Combining information from the 62 nuclear amplicons present in the ANOSPP panel allows for a more sensitive and specific species assignment than single gene (e.g. COI) barcoding, which is desirable in the light of permeable species boundaries. Here, I present NNoVAE, a method using Nearest Neighbours (NN) and Variational Autoencoders (VAE), which I apply to k-mers resulting from the ANOSPP amplicon sequences in order to hierarchically assign species identity. The NN step assigns a sample to a species-group by comparing the k-mers arising from each haplotype’s amplicon sequence to a reference database. The VAE step distinguishes between closely related species, and also has sufficient resolution to reveal population structure within species. In tests on independent samples with over 80% and 16 % amplicon coverage respectively, NNoVAE correctly classifies to species level 98% of samples within the An. gambiae complex and 89% of samples outside the complex. I apply NNoVAE to nearly 10,000 new samples from Burkina Faso, Gabon, Nigeria and Uganda. In Gabon I found four putative An. arabiensis mosquitoes, a species that was not known to occur in the area. I present the ANOSPP Report Cards, the format used to communicate a summary of the ANOSPP results to the partners who contributed the samples. I also show some additional analyses which can be done using the ANOSPP sequence data, e.g. karyotyping of the 2La inversion. The MalariaGEN Vector Observatory Anopheles funestus Genomic Surveillance Project phase 1.0 dataset (vobs-funestus phase 1.0 for short) consists of whole genome sequence data for 656 samples from 13 different countries in sub-Saharan Africa. Different analysis methods, including PCA, ADMIXTURE and pairwise fixation indices, show that these samples are divided into three primary clusters: one containing specimens from the equatorial region from Ghana in the west to Kenya in the east, and another containing specimens from more southern locations, although here the split between the samples fromnear the east coast and those from near the west coast is more pronounced. There is a small third cluster, separate from the two main clusters, containing only samples from north Ghana. I identify five segregating inversions in this dataset. The positions of three of these, 3Ra, 3Rb and 2Ra, are consistent with previously published genomic coordinates. The other two likely correspond to previously described 3La and 2Rh, based on comparisons to published cytogenetic and microsatellite results. I perform in silico karyotyping for all individuals and identify four putative double recombinant individuals. I characterise the inversion frequencies in their geographical context and identify that all mosquitoes from Benin share the same homozygous inversion state on four inversions, while these states are rare in cohorts from the neighbouring countries. For 2Ra and 2Rh, I present evidence suggesting that the inverted orientation is younger and hence likely the derived one. The 3La inversion shows a pattern consistent with occasional recombination between the different inversion orientations. I consistently observe, in all inversions, a stronger differentiation between the different inversion orientations in the southern cohort compared to the equatorial cohorts, which leads me to speculate that these five inversions originated in equatorial populations.
  • ItemOpen Access
    Ancestral Paths: Redefining local genetic ancestry and its inference with application to Europeans
    Pearson, Alice
    Recently, two new approaches have transformed our understanding of human population history. Firstly, the sequencing of ancient genomes which gives us a snapshot of past genetic variation. We can therefore make inferences from observed genetic signatures present before historical events such as population bottlenecks and natural selection have obscured them from the modern gene pool. Ancient DNA has thus revealed what cannot be determined from modern genomes alone. Secondly, the development of methods that aim to reconstruct population genealogies from genetic variation data. Together with an understanding of how evolutionary processes alter genealogies, this has allowed inference of historical and ongoing processes in real world populations. The latest updates in these approaches now allow us to combine the two and infer genealogies involving both present-day and ancient individuals. In this thesis I present a new method to infer local ancestry along sample chromosomes. The method applies machine learning to tree sequences built from ancient and present-day genomes and is based on a deterministic model of population structure, within which I introduce the concept of ‘path ancestry’. I show with extensive simulation that the method is robust to a variety of demographic scenarios and generalises over model misspecification. Subsequent downstream analyses include estimating past effective population size, timing of population specific selection and the time since admixture for individuals. I apply the method to a large ancient DNA dataset covering Europe and West Eurasia to paint all sample chromosomes. I show that the inferred admixture ages are a better metric than sample ages alone for understanding movements of people across Europe in the past.
  • ItemControlled Access
    The Evolution of Drosophila Immunity
    Ding, Shuai; Ding, Shuai [0000-0002-4973-7631]
    The ubiquity of pathogens in the natural world poses a strong selection force on the evolution of Drosophila immunity. Under the pressure of this natural selection by pathogenic organisms, species diverge along the evolutionary path and depart in phenotypes. Interspecific differences in gene expression are an important contributor to this divergence where gene expression is regulated by both cis-regulatory elements, which affect nearby gene expression levels in an allele-specific fashion, and trans-regulatory elements, which affect gene expression by interacting with linked cis-elements. The relative contributions of cis- and trans-divergence can be estimated by comparing gene expression between two species and allele-specific expression within their F1 hybrids. The total interspecific divergence and the cis-divergence are captured respectively between the parents and within the hybrids, and the trans-divergence is attained by subtracting the cis from the total divergence. We investigated the transcriptional response of Drosophila simulans and D. sechellia to parasitoid wasp infection. Many genes had large expression differences between the two species from cis-regulatory divergence in uninfected and infected conditions alike. However, the divergence of immune response is overwhelmingly driven by trans, suggesting that altering trans-regulatory elements, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion. I then investigated the transcriptional differences between D. melanogaster and D. simulans responding to injuries and found that constitutive expression divergence between the two species was mostly cis-driven, and there was extensive misexpression of immune-related genes in hybrids.
  • ItemOpen Access
    Identification and characterisation of KZFPs directly regulated by p53
    Daffern, Samuel
    Approximately 50% of human DNA is composed of transposable elements (TEs), which in subsequent waves colonized our genome across evolutionary history. KRAB zinc finger proteins (KZFPs) are the largest group of DNA binding factors in the human genome and mostly target TEs, controlling chromatin accessibility by effecting epigenetic changes at their binding sites which result in a localized establishment of heterochromatin. Many of these bound TEs themselves contain binding sites for regulators of transcription and represent a huge source of genetic variation and regulatory potential by acting as enhancers or alternative promoters, influencing nearby genes. The KZFP family presents an interesting conundrum; the family is highly evolutionary dynamic, with new members appearing at every phylogenetic branch since their inception in tetrapods, yet their binding sites in TEs are conserved millions of years after their initial TE target has been inactivated. Furthermore, individual KZFPs have specific expression patterns in various somatic cell types, leading to the hypothesis that they participate in the epigenetic control of enhancers or alternative promoters derived from transposable elements, contributing to gene regulatory networks. So far, only a handful of the 350 human KZFPs have been functionally characterized, yet many of those studied have important and novel biological functions, such as ZFP57 and imprinting, with several examples of KZFPs and their TE targets demonstrably contributing to gene regulatory events. In this thesis, I describe the recently identified KZFPs which display inducible expression in response to direct trans-activation from the tumour suppressor protein p53, focussing on the KZFPs ZNF79 and ZNF561. ZNF79 presents an unusual case of a marsupial-conserved KZFP significantly older than the majority of the KZFP family at 160 million years old, yet strongly conserved across many phylogenetic branches with marsupial ZNF79. Furthermore, ZNF79 demonstrates an unusual intermediate TRIM28 binding status and a unique nucleolar localisation. In contrast, ZNF561 resembles a canonical KZFP but possesses intriguing characteristics given that its expression is induced by p53. First, its binding profile is enriched for specific LTR10 elements which can act as p53 driven enhancers. Second, it unusually binds to its own promoter and the promoter of its antisense RNA. Together, these characteristics suggest a complex regulatory pathway. By utilising CRISPR knock-out models and RNA-seq, I have identified interesting changes in the expression of genes implicated in the transcriptional and cellular response to p53 activation and in genes related to cell proliferation and survival. These results indicate that ZNF79 and ZNF561 may play a role in the transcriptional response to DNA damage and p53, identifying new preliminary networks of gene regulation. Ultimately, this work offers valuable insights into the relationships between KZFPs, evolution, and key cellular processes, providing models by which to explore these characteristics.
  • ItemOpen Access
    Genetic interactors of the SWI/SNF chromatin remodelling complex in Caenorhabditis elegans
    Lampersberger, Lisa
    Chromatin remodellers are adenosine triphosphate (ATP)-powered molecular machines that can directly alter the structure of chromatin by reshuffling or evicting nucleosomes. Thereby they control the access to DNA elements like promoters and replication origins that need to be exposed to execute essential cellular processes such as transcription and replication. SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are a family of chromatin remodellers that are conserved across eukary- otes. Mutations in subunits of SWI/SNF complexes cause a multitude of different developmental disorders including Coffin-Siris syndrome, Nicolaides-Baraitser syn- drome, and autism. However, the molecular mechanism(s) by which mutations in SWI/SNF cause developmental disorders remain unclear and there are currently no treatments that can restore normal development in humans or animal models lacking proper SWI/SNF function. In this thesis, I used a unique conditional Caenorhabditis elegans swsn-1 (SMARCC1/2 in humans) mutant model that resembles a complete loss of SWI/SNF function only when animals are exposed to a restrictive temperature. By employing chemical mutagenesis, I identified compensating mutations that can restore viability and prevent developmental defects from manifesting in these swsn-1 mutants. Firstly, I found that a specific alanine to valine mutation in the SWI/SNF core subunit snfc-5 (SMARCB1 in humans) can prevent embryonic lethality in animals harbouring the loss-of-function mutation in the SWI/SNF core subunit swsn-1. Secondly, I found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Furthermore, I showed that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Finally, using my mutant models, I established a set of approximately 1200 genes that are dysregulated in swsn-1 mutants but exhibit wild-type expression in swsn-1; snfc-5 double and swsn-1; snfc-5; ubr-5 triple mutants, suggesting that the proper regulation of these genes by the SWI/SNF complex promotes animal development. In summary, my findings establish a novel link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 might be viable therapeutic targets for the for the many developmental disorders caused by missense mutations in SWI/SNF subunits.
  • ItemOpen Access
    Cell cycle control governing built-in asymmetry underlying the spindle pole body duplication cycle in Saccharomyces cerevisiae
    Peng, Qiuran
    Self-renewing stem cell divisions typically couple the polarised orientation of the mitotic spindle with an invariant age-dependent pattern of centrosome inheritance. Self- renewal supports tissue homeostasis by providing, in a balanced manner, one daughter cell retaining stemness and another daughter cell set to differentiate. Such balance is central to avoid tumourigenesis. Age-dependent spindle pole inheritance directing the polarised orientation of the mitotic spindle was first discovered in the budding yeast Saccharomyces cerevisiae, a unicellular organism that divides asymmetrically like stem cells. Furthermore, this budding yeast is at the forefront in cell cycle studies. Thus, S. cerevisiae represents a powerful model for linking the molecular basis for age-dependent asymmetric fate with cell cycle regulation. Our group previously showed that Spc72, a component of the spindle pole body (SPB, the yeast centrosome), is the key factor priming the invariant segregation of the old SPB (the SPB present prior to duplication) into the bud and the new SPB into the mother cell. In this thesis, I present quantitative imaging analysis paired to biochemical studies that reveal Spc72 asymmetry as an intrinsic feature of SPB duplication under cyclin-dependent kinase (CDK) control. In turn, I uncovered a mechanism for Spc72 spatial partition centred on two further SPB components that bind Spc72 in a cell-cycle dependent manner governed by CDK phosphorylation. The SPB components involved in my study as well as CDK, the master regulator of the eukaryotic cell cycle, are highly conserved. It follows that my work will provide crucial insight into cell cycle control of intrinsic centrosome asymmetries with impact on stem cell self-renewing divisions and beyond.
  • ItemOpen Access
    Modelling Embryonic Haematopoiesis Using In Vitro State-of-art Gastruloid Culture
    Suen, Chun Wai
    Haematopoietic stem cell progenitor cells (HSPCs) have substantial research and therapeutic value, but the in vitro production of HSPCs from pluripotent stem cells remains elusive. A novel 3D culture method, known as a gastruloid, can mimic gastrulation in embryogenesis, which is crucial to the emergence of HSPC precursors, highlighting the potential to develop this protocol as an in vitro platform for haematopoietic research. This project aims to adapt the previously published gastruloid culture protocol and develop it into a haemogenic gastruloid protocol to produce HSPCs from mouse ES cells and model haematopoiesis which occurs at the aorta-gonad-mesonephros (AGM) region of the mouse embryo. In this project, the gastruloid culture protocol is modified to maintain a gastruloid (gastrulation organoid) for up to 216 hr by introducing a 2iLIF pretreatment and a panel of cytokines throughout the culture. Under the new culture conditions, gastruloids can generate haemogenic endothelium and pro- and pre-HSCs in sequential order, as detected by a combination of markers in flow cytometry assays (Flk-1, c-Kit, CD41 and CD45). Concomitant use of cytokines VEGF and FGF2 can facilitate the emergence of cells with the property of haemogenic endothelial cells, pro-HSCs and type 1 pre-HSCs from 96 to 144 hr. Applying Shh, Flt-3l, TPO, and SCF accordingly can promote the formation CD45+ cells, which is indicative of the formation of type 2 pre-HSCs at 192 and 216 hr. The emergence of these haematopoietic makers suggests stepwise haematopoiesis is recapitulated in the haemogenic gastruloid. The AGM-like haematopoietic clusters are observed at 192 and 216 hr under confocal imaging, further suggesting that definitive haematopoiesis likely occurs in the gastruloid under the optimised protocol. The CFC assay shows that gastruloid cells have the potential to form multipotential myeloid and lymphoid haematopoietic progenitors. The CD45+ cells from the 216 hr gastruloids can occasionally be enriched in OP9 co-culture, and they can also sometimes form committed B-cell linage progenitors with OP9 cells. The chick embryo chorioallantoic membrane (CAM) assay and mouse transplantation experiments imply that the haemogenic gastruloid cells may possess engraftment capability. Single cell SMART sequencing data confirms that gastruloids have haematopoietic gene expression profiles similar to the AGM region of the mouse embryo and captures successive progenitors with erythroid, myeloid-lymphoid and HSC-like signatures. Finally, this haemogenic gastruloid protocol has been applied to other mouse ES cell lines to generate gastruloids, and they can also recapitulate stepwise haematopoiesis. This study has successfully developed a haemogenic gastruloid protocol with a cocktail of haematopoietic cytokines. This haemogenic gastruloid is able to form AGM-like haematopoietic clusters and recapitulate stepwise definitive haematopoiesis to produce haematopoietic progenitors. In the future, this protocol could be developed to validate and reduce the use of animals in scientific research. The results from this study could also be useful preliminary data for developing a human haemogenic gastruloid protocol. Finally, this protocol could be applied as a drug screening or disease modelling tool, such as an infant leukaemia disease model.
  • ItemOpen Access
    The evolution and genetics of virus resistance in Drosophila
    Montero, Gaspar
    Parasites impose high selection pressure on host fitness and are thought to be a major selective factor that promotes the evolution of resistance in host populations. Much of the resistance is determined by genetic factors, however, it is unclear what genetic factors promote resistance to parasites. In this thesis, I used Drosophila melanogaster as a model system to study the genetic basis of resistance against RNA viruses. To understand the genetic basis of infection between different viral and Drosophila genotypes, I investigated two classic models, gene-for-gene and matching-allele models. These models consider that the outcome of the infection depends on the specific compatibility between host and parasite genotypes. Here, I demonstrate that the genetic background of flies explained substantially the resistance against the viral pathogen, which represents an exception to the genotype-by-genotype interaction models. Additionally, I developed an accessible and reproducible protocol to isolate and characterize RNA viruses from wild population of Drosophila. As a result of the protocol, two novel positive-stranded RNA viruses were isolated, La Jolla virus (Iflaviriade) and Newfield virus (Permutotetraviridae). Using RNA sequencing and a customised bioinformatics pipeline, I recovered partial viral genomes which were used to reconstructed their phylogeny. Then, I experimentally explored the impact of the newly isolated viruses on Drosophila infected with Wolbachia, a mutualistic endosymbiotic bacterium that protects the flies against RNA viruses. Furthermore, I determined the host range of these viruses infecting several Drosophila species of the Sophora group. In particular, I evaluated the potential of the novel viruses as biological control agents on the invasive species D. suzukii, one of the most important invasive pests of ripening fruits and wine production worldwide. Finally, I performed a genome-wide association analysis to investigate the genetic variation of resistance to the novel viruses using the Drosophila Genetic Reference Panel. The genome-wide analysis revealed a substantial genetic variation in resistance to the virus isolates, providing new insights into the natural genetic variation in resistance to viruses in Drosophila, and antiviral response in insects.
  • ItemOpen Access
    Testing roles of Hereditary Spastic Paraplegia (HSP) proteins in organization of axonal endoplasmic reticulum (ER) and ER-mitochondria contacts
    Ozturk, Zeynep
    The Hereditary Spastic Paraplegias (HSPs) are a group of genetically heterogeneous, neurodegenerative and neurodevelopmental diseases characterised by spasticity and lower limb weaknesses. Some known causative genes imply the importance of endoplasmic reticulum (ER) function and morphogenesis in HSPs. These genes encode ER-shaping proteins: spastin (SPG4), atlastin (SPG3A), Receptor Expression Enhancing Protein 1 (REEP1/SPG31) and reticulon (SPG12). These proteins share a common feature of one or two intramembrane hairpin domains that can recognise or drive curvature of the ER membrane. In Drosophila, removing widely expressed members of the reticulon and REEP families leads to fewer ER tubules in axons of wider diameter, although there is no widespread absence of tubules. Therefore, other proteins must be involved in shaping the tubular ER network in Drosophila axons, and a major goal of mine was therefore to identify and assess additional candidate proteins with roles in this process. One potential source of ER-shaping proteins is other HSP genes. C19orf12 (SPG43) is an HSP gene; C19orf12 reportedly localise to ER and has a predicted intramembrane domain. It is therefore a candidate for helping to shape the axonal ER network. To investigate possible roles of C19orf12 in ER structure and function, I generated transgenic flies carrying a fusion of GFP to CG3740, which showed strong localisation to axons. I also generated loss-of function mutants of the most widely expressed Drosophila ortholog of C19orf12, CG3740, using P element excision and CRISPR/Cas9. These mutants are homozygous viable, as are quadruple mutants lacking CG3740 and all the widely expressed reticulon and REEP proteins, suggesting that these 4 proteins together are not sufficient for tubular ER formation. I did not see any overt effect in ER level due to CG3740 mutation. It also did not enhance the ER fragmentation phenotype was seen in Rtnl1, ReepA, and ReepB triple mutant. Another potential source of ER-shaping proteins is proteomic studies highlighting the proteins enriched in ER tubules. I therefore, performed proteomic analysis from previously conducted proteomic studies. These analyses identified most of the known proteins with roles in ER shaping, implying that it may also be a way to identify additional proteins with similar roles. Around 15 proteins were finalised as potential candidates for an ER-shaping role and two of them appeared as strong candidates. 8 Many HSP proteins localised in ER, but some localised in mitochondria. How might proteins in two different organelles give a similar disease phenotype? I, therefore, wanted to have a way to monitor ER-mitochondria contacts in HSP mutants. I adapted a published sensor that is based on a split-GFP strategy for ER-mitochondria contacts in transgenic Drosophila. Split GFP peptides were fused with ER and mitochondria localised proteins. When two organelles are nearby, GFP is reconstituted and becomes fluorescent. In Drosophila larval axons, the reporter showed a punctate distribution similar to that of mitochondria, consistent with localisation to contact sites. The reporter also showed alteration in different diet conditions showing that it responds to different physiological conditions.